WO2023208762A2

WO2023208762A2 - Mutant yeast cell and process for the production of ethanol

Info

Publication number: WO2023208762A2
Application number: PCT/EP2023/060423
Authority: WO
Inventors: Aafke Cornelie Albertine VAN AALST; Robert MANS; Jacobus Thomas Pronk; Mickel Leonardus August Jansen
Original assignee: Dsm Ip Assets B.V.; Technische Universiteit Delft
Priority date: 2022-04-25
Filing date: 2023-04-21
Publication date: 2023-11-02
Also published as: WO2023208762A3

Abstract

A mutant yeast cell, comprising : (i) a first genetic modification for expression of a NAD+ dependent protein that functions in a first metabolic pathway converting a sugar alcohol into a fermentation product; and (ii) a second genetic modification for expression of a protein that functions in a second metabolic pathway forming a non-native redox sink. And process using such a yeast cell.

Description

MUTANT YEAST CELL AND PROCESS FOR THE PRODUCTION OF ETHANOL

Field of the invention

[001 ] The invention relates to a mutant yeast cell and to a process for the production of ethanol wherein said yeast cell is used.

Background of the invention

[002] Yeast-based fermentation processes are applied for industrial production of a broad and rapidly expanding range of chemical compounds from conventional and renewable carbohydrate feedstocks.

[003] Especially in anaerobic fermentation processes, redox balancing of the cofactor couple NADH/NAD⁺ can cause important challenges for product yields. For example, a major challenge relating to the stoichiometry of yeast-based ethanol production is that growing anaerobic cultures invariably produce glycerol as byproduct . It has been estimated that, in typical industrial ethanol processes, up to about 4 wt.% of the sugar feedstock is converted into glycerol (as described in the article by Nissen et al, 2000).

[004] Glycerol production under anaerobic conditions is primarily linked to the redox balancing mechanisms in the yeast cell. During anaerobic growth of S. cerevisiae, sugar dissimilation occurs via so- called alcoholic fermentation. In this process, the NADH formed via the NAD+-dependent glycolytic glyceraldehyde-3-phosphate dehydrogenase reaction is reoxidized by converting acetaldehyde, formed by decarboxylation of pyruvate, to ethanol via NADH-dependent alcohol dehydrogenase. The fixed stoichiometry of this redox-neutral dissimilatory pathway causes problems when a net reduction of NAD+ to NADH occurs elsewhere in the metabolism. Under anaerobic conditions, in this situation, NADH reoxidation in wild-type S. cerevisiae is strictly dependent on reduction of sugar to glycerol. Glycerol formation is initiated by reduction of the glycolytic intermediate dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (glycerol-3P), a reaction catalyzed by NADH-dependent glycerol 3-phosphate dehydrogenase. Subsequently, the glycerol 3-phosphate formed in this reaction is hydrolysed by glycerol- 3-phosphatase to yield glycerol. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae. The production of glycerol is undesired as it reduces overall conversion of sugar to a desired fermentation product such as ethanol. Further, the presence of glycerol in effluents of fermentation plants may impose costs for waste-water treatment.

[005] Several alternative reduction pathways to reoxidize the NADH produced, also referred to as alternative “redox sinks”, have been suggested and implemented into (recombinant) yeast cells.

[006] In WO 2011/010923, the NADH-related side-product (glycerol) formation in a process for the production of ethanol from a carbohydrate containing feedstock is addressed by providing a recombinant yeast cell comprising one or more recombinant nucleic acid sequences encoding an NAD+ dependent acetylating acetaldehyde dehydrogenase (EC 1.2.1.10) activity. The cell may for example lack enzymatic activity needed for the NADH dependent glycerol synthesis or the cell may have a reduced enzymatic activity with respect to the NADH dependent glycerol synthesis compared to its corresponding wild-type yeast cell.

[007] WO2014/129898 describes a recombinant cell functionally expressing heterologous nucleic acid sequences encoding for ribulose-1 ,5-phosphate carboxylase/oxygenase (EC 4.1 .1 .39; herein abbreviated as “Rubisco”), and optionally molecular chaperones for Rubisco, and phosphoribulokinase (EC 2.7.1.19; herein abbreviated as “PRK”). In addition, reference is made to the use of carbon dioxide as an electron acceptor in a recombinant chemotrophic micro-organism.

[008] Whilst the conversion of sugar by yeast cells has been extensively studied in the prior art, literature on the conversion of sugar alcohols is scarce.

[009] Jordan et al, in their article titled “Hxt13, Hxt15, Hxt16 and Hxt17 from Saccharomyces cerevisiae represent a novel type of polyol transporters”, published in Scientific Reports, srep. 23502 (2016), pages 1 to 10, describe that most S. cerevisiae strains grow on mannitol and sorbitol only after long adaptation, if at all. It is stated that Hxt13, Hxt15, Hxt16 and Hxt17 transport two major hexitols in nature, mannitol and sorbitol, with moderate affinities, by a facultative mechanism. They further refer to a study wherein, aiming to engineer S. cerevisae for fermentation of algal biomass, HXT13 and HXT17 genes, encoding hexose transporter-like proteins, as well as annotated mannitol dehydrogenase (MDH) genes DSF1 and YNR073C were found to be upregulated when yeast was adapted to grow on mannitol. Jordan et all subsequently described that in their study overexpression of the transporters HXT 13 or HXT 17 together with one of the MDH genes was necessary to confer growth of a yeast strain on mannitol.

[010] It would be an advancement in the art to provide a yeast cell and/or process for producing an ethanol that allows for an increased production of ethanol and/or a reduced production of undesired byproduct glycerol.

[011] It would also be an advancement in the art to provide a yeast cell and/or a process, that allows for the conversion of a mixed carbon source composition, comprising one or more sugars and one or more sugar alcohols, where a good or improved conversion of such sugar alcohols is obtained. The conversion of such sugar alcohols could advantageously reduce the chemical oxygen demand (COD) in the treatment of waste water from a fermentation process.

Summary of the invention

[012] The inventors have now surprising found a synergistic effect when combining the above pathways.

[013] Accordingly the invention provides a mutant yeast cell, comprising :

(I) a first genetic modification for expression of a NAD+ dependent protein that functions in a first metabolic pathway converting a sugar alcohol into a fermentation product; and

(II) a second genetic modification for expression of a protein that functions in a second metabolic pathway forming a non-native redox sink. [014] In addition the invention provides a process for the production of ethanol, the process comprising fermenting of a carbon source composition with a mutant yeast cell as described herein, wherein the carbon source composition comprises at least a sugar alcohol and wherein the process is carried out under oxygenlimited conditions or anaerobic conditions. [015] Inventors found that the above mutant yeast and process advantageously allow for an improved conversion of sugar alcohols and/or an increased amount of ethanol being retrieved from a carbon source composition comprising at least a sugar alcohol.

Brief description of the sequence listing [016] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. An overview is provided by Table 1 below.

Table 1 : Overview of sequence listings:

[017] In the context of this patent application, each of the above protein / amino acid sequences is preferably encoded by a DNA / nucleic acid sequence that is codon-pair optimized for expression in a yeast, more preferably for expression in a Saccharomyces cerevisiae yeast. [018] In order to reach an optimal expression one or more promoters may be added. Promoters may be regulated from strong to weak and may include one or more of TDH3, FBA1 , ENO2, PGK1 , TEF1 , HTA1 , HHF2, RPL8A, CHO1 , RPS3, EFT2, HTA2, ACT1 , PFY1 , CUP1 , ZUO1 , VMA6 and/or ANB1 , HEM13, YHK8, FET4, TIR4, AAC3. Description of the Figures

[019] The invention is illustrated by the following figures:

Figure 1 provides an illustration of the construction of plasmid pUDE885 of Example 1

Figure 2 provides an illustration of the construction of plasmid pUDE941 of Example 2

Figure 3 provides an illustration of the construction of yeast strain IMX2506 of Example 9

Figure 4 provides a graphic of results of the Example 11. :residual sorbitol concentration measured during pre-steady state sampling of anaerobic bioreactor chemostat cultures of S. cerevisiae strains IMX2506 (GPD1 Agpd2 pDAN1-prk cbbm Hxt15"f Sor2"f) (circles) and IME324 (GPD1 GPD2) (squares) at a dilution rate of 0.025 h^-1 on 10 g L^-1 of glucose and 10 g L^-1 of sorbitol. For IMX2506 (circles) the average sorbitol concentration and standard deviation are based on four chemostat cultures. The CO2 inflow in the ingas was switched off after 400 hours in two of the chemostat cultures of IMX2506 (closed circles), and for the other two chemostat cultures of IMX2506 (open circles), the inflow of gas stayed the same. For IME324 (squares) the average sorbitol concentration and standard deviation are based on three chemostat cultures.

Detailed description of the invention

Definitions

[020] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[021] Throughout the present specification and the accompanying claims, the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[022] The articles “a” and “an” are used herein to refer to "one or more" otherwise phrased as "at least one", i.e. to one or to more than one of the grammatical object of the article. By way of example, “an element” may mean one element or more than one element. When referring to a noun (e.g. a compound, an additive, etc.) in the singular, the plural is meant to be included. Thus, when referring to a specific moiety, e.g. "gene", this means "at least one" of that gene, e.g. "at least one gene", unless specified otherwise.

[023] When referring to a compound of which several isomers exist (e.g. a D and an L enantiomer), the compound in principle includes all enantiomers, diastereomers and cis/trans isomers of that compound that may be used in the particular aspect of the invention; in particular when referring to such as compound, it includes the natural isomer(s).

[024] Unless explicitly indicated otherwise, the various embodiments of the invention described herein can be cross-combined.

[025] The term “carbon source” refers to a source of carbon, preferably a compound or molecule comprising carbon. Preferably the carbon source is a carbohydrate. A carbohydrate is understood herein to be an organic compound made of carbon, oxygen and hydrogen. Suitably the carbon source may be selected from the group consisting of mono-, di- and/or polysaccharides, polyols, acids and acid salts. More preferably the carbon source is a compound selected from the group of glucose, arabinose, xylose, galactose, mannose, rhamnose, fructose, glycerol, sugar alcohols and acetic acid or a salt thereof.

[026] The term “sugar alcohol”, refers to a carbohydrate, suitably derived from a sugar, containing one hydroxyl group attached to each carbon atom. More preferably the sugar alcohol is a sugar alcohol comprising 5 carbon atoms (i.e. a pentose alcohol) or 6 carbon atoms (a hexose alcohol). Suitable examples of sugar alcohols include arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, idotol, inositol, isomalt, erythritol, maltitol and lactitol. Sugar alcohols can suitably be prepared by hydrogenation of sugars. For the avoidance of doubt, glycerol is not a sugar alcohol.

[027] The term “ferment”, and variations thereof such as “fermenting”, “fermentation” and/or “fermentative”, is used herein in a classical sense, i.e. to indicate that a process is or has been carried out under anaerobic conditions. An anaerobic fermentation is herein defined to be a fermentation carried out under anaerobic conditions. Anaerobic conditions are herein defined as conditions without any oxygen or in which essentially no oxygen is consumed by the yeast cell. Conditions in which essentially no oxygen is consumed suitably corresponds to an oxygen consumption of less than 5 mmol/l.h^-1 , in particular to an oxygen consumption of less than 2.5 mmol/l.h^-1, or less than 1 mmol/l.h^-1. More preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable). This suitably corresponds to a dissolved oxygen concentration in a culture broth of less than 5 % of air saturation, more suitably to a dissolved oxygen concentration of less than 1 % of air saturation, or less than 0.2 % of air saturation.

[028] The term “fermentation process” refers to a process for the preparation or production of a fermentation product.

[029] The term "cell" refers to a eukaryotic or prokaryotic organism, preferably occurring as a single cell. In the present invention the cell is a yeast cell. That is, the mutant cell is selected from the group of genera consisting of yeast.

[030] The terms “yeast” and “yeast cell” are used herein interchangeably and refer to a phylogenetically diverse group of single-celled fungi, most of which are in the division of Ascomycota and Basidiomycota. The budding yeasts ("true yeasts") are classified in the order Saccharomycetales. The yeast cell according to the invention is a yeast cell and is preferably a yeast cell derived from the genus of Saccharomyces. More preferably the yeast cell is a yeast cell of the species Saccharomyces cerevisiae.

The term “ mutant”, for example referring to a “mutant yeast”, a “mutant cell”, a “mutant micro-organism” and/or a “mutant strain”, as used herein, refers to a yeast, cell, micro-organism or strain, respectively, which in comparison to its parent, wild-type, counterpart has undergone a genetic modification, i.e. a “mutation”. The genetic modification can for example be the result of a laboratory evolutionary process or recombinant DNA technique(s). An example of a laboratory evolutionary process is adaptive evolution. Adaptive evolution is an evolutionary process whereby a population becomes better suited (adapted) to its habitat or habitats. After applying evolutionary pressure, via natural selection, appropriate mutants can be obtained. A large number of different factors, such as e.g. nutrient availability, temperature, the availability of oxygen, etcetera, can drive adaptive evolution. Preferably the mutant yeast cell is a recombinant yeast cell. Further preferences for such recombinant yeast cell are as described herein.

[031] The term "mutated" as used herein regarding proteins or polypeptides means that at least one amino acid in the wild-type or naturally occurring protein or polypeptide sequence has been replaced with a different amino acid, inserted or deleted from the sequence via mutagenesis of nucleic acids encoding these amino acids. Mutagenesis is a well-known method in the art, and includes, for example, site-directed mutagenesis by means of PCR or via oligonucleotide-mediated mutagenesis as described in Sambrook et al., Molecular Cloning-A Laboratory Manual, 2nd ed., Vol. 1-3 (1989). The term "mutated" as used herein regarding genes means that at least one nucleotide in the nucleic acid sequence of that gene or a regulatory sequence thereof, has been replaced with a different nucleotide, or has been deleted from the sequence via mutagenesis, resulting in the transcription of a protein sequence with a qualitatively of quantitatively altered function or the knock-out of that gene. In the context of this invention an “altered gene” has the same meaning as a mutated gene.

[032] The term “recombinant”, for example referring to a “recombinant yeast”, a “recombinant cell”, “recombinant micro-organism” and/or “recombinant strain” as used herein, refers to a yeast, cell, microorganism or strain, respectively, containing nucleic acid which is the result of one or more genetic modifications. Simply put the yeast, cell, micro-organism or strain contains a different combination of nucleic acid from (either of) its parent(s). To construe a recombinant yeast, cell, micro-organism or strain, recombinant DNA technique(s) and/or another mutagenic technique(s) can be used. For example a mutant yeast and/or a mutant yeast cell may comprise nucleic acid not present in the corresponding wild-type yeast and/or cell, which nucleic acid has been introduced into that yeast and/or yeast cell using recombinant DNA techniques (i.e. a transgenic yeast and/or cell), or which nucleic acid not present in said wild-type yeast and/or cell is the result of one or more mutations - for example using recombinant DNA techniques or another mutagenesis technique such as UV-irradiation - in a nucleic acid sequence present in said wildtype yeast and/or yeast cell (such as a gene encoding a wild-type polypeptide) or wherein the nucleic acid sequence of a gene has been modified to target the polypeptide product (encoding it) towards another cellular compartment. Further, the term “recombinant” may suitably relate to a yeast, cell, micro-organism or strain from which nucleic acid sequences have been removed, for example using recombinant DNA techniques.

[033] By a recombinant yeast comprising or having a certain activity is herein understood that the recombinant yeast may comprise one or more nucleic acid sequences encoding for a protein or an enzyme having such activity. Hence allowing the recombinant yeast to functionally express such a protein or enzyme. Preferably the mutant yeast, respectively the mutant yeast cell, is a recombinant yeast, respectively a recombinant yeast cell. That is, preferably the mutant yeast, respectively the mutant yeast cell, is a transgenic or transformed yeast, respectively a transgenic or transformed yeast cell.

[034] The term “transgenic” as used herein, for example referring to a “transgenic yeast” and/or a “transgenic cell”, refers to a yeast and/or cell, respectively, containing nucleic acid not naturally occurring in that yeast and/or cell and which has been introduced into that yeast and/or cell using for example recombinant DNA techniques, such as a recombinant yeast and/or cell.

[035] The term “gen” or “gene”, as used herein, refers to a nucleic acid sequence that can be transcribed into mRNAs that are then translated into protein. A gene encoding for a certain protein refers to the one or more nucleic acid sequence(s) encoding for such a protein.

[036] The term "nucleic acid" as used herein, refers to a deoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, in either single or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e. g., peptide nucleic acids). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.

[037] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulphation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[038] The term “enzyme” refers herein to a protein having a catalytic function. Where a protein catalyzes a certain biological reaction, the terms “protein” and “enzyme” may be used interchangeable herein. When an enzyme is mentioned with reference to an enzyme class (EC), the enzyme class is a class wherein the enzyme is classified or may be classified, on the basis of the Enzyme Nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), which nomenclature may be found at http://www.chem.qmul.ac.uk/iubmb/enzyme/. Other suitable enzymes that have not (yet) been classified in a specified class but may be classified as such, are meant to be included. [039] If referred herein to a protein or a nucleic acid sequence, such as a gene, by reference to a accession number, this number in particular is used to refer to a protein or nucleic acid sequence (gene) having a sequence as can be found via www.ncbi.nlm.nih.gov/ , (as available on 1 October 2020) unless specified otherwise.

[040] Every nucleic acid sequence herein that encodes a polypeptide also includes any conservatively modified variants thereof. This includes that, by reference to the genetic code, it describes every possible silent variation of the nucleic acid. The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences due to the degeneracy of the genetic code. The term "degeneracy of the genetic code" refers to the fact that a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation.

[041] The terms “nucleotide sequence” and “nucleic acid sequence” are used interchangeably herein.

[042] The term “functional homologue” (or in short “homologue”) of a polypeptide and/or amino acid sequence having a specific sequence (e.g. “SEQ ID NO: X”), as used herein, refers to a polypeptide and/or amino acid sequence comprising said specific sequence with the proviso that one or more amino acids are substituted, deleted, added, and/or inserted, and which polypeptide has (qualitatively) the same enzymatic functionality for substrate conversion. With respect to nucleic acid sequences, the term functional homologue is meant to include nucleic acid sequences which differ from another nucleic acid sequence due to the degeneracy of the genetic code and encode the same polypeptide sequence.

[043] Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences compared. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.

[044] Amino acid or nucleotide sequences are said to be homologous when exhibiting a certain level of similarity. Two sequences being homologous indicate a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by “percent identity” or “percent similarity”, which is high or low respectively. Although disputed, to indicate “percent identity” or “percent similarity”, “level of homology” or “percent homology” are frequently used interchangeably. A comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the homology between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The algorithm aligns amino acid sequences as well as nucleotide sequences. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice,P. Longden.l. and Bleasby.A. Trends in Genetics 16, (6) pp276 — 277, http://emboss.bioinformatics.nl/). For protein sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. Other matrices can be specified. The optional parameters used for alignment of amino acid sequences are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

[045] The homology or identity is the percentage of identical matches between the two full sequences over the total aligned region including any gaps or extensions. The homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment including the gaps. The identity defined as herein can be obtained from NEEDLE and is labelled in the output of the program as “IDENTITY”.

[046] The homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as “longest-identity”.

[047] A variant of a nucleotide or amino acid sequence disclosed herein may also be defined as a nucleotide or amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the nucleotide or amino acid sequence specifically disclosed herein (e.g. in de the sequence listing).

[048] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. In an embodiment, conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagineglutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. In an embodiment, conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; lie to Leu or Vai; Leu to lie or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to lie or Leu.

[049] Nucleotide sequences of the invention may also be defined by their capability to hybridise with parts of specific nucleotide sequences disclosed herein, respectively, under moderate, or preferably under stringent hybridisation conditions. Stringent hybridisation conditions are herein defined as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and most preferably of about 200 or more nucleotides, to hybridise at a temperature of about 65°C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at 65°C in a solution comprising about 0.1 M salt, or less, preferably 0.2 x SSC or any other solution having a comparable ionic strength. Preferably, the hybridisation is performed overnight, i.e. at least for 10 hours and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridisation of sequences having about 90% or more sequence identity.

[050] Moderate conditions are herein defined as conditions that allow a nucleic acid sequences of at least 50 nucleotides, preferably of about 200 or more nucleotides, to hybridise at a temperature of about 45°C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength. Preferably, the hybridisation is performed overnight, i.e. at least for 10 hours, and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridisation of sequences having up to 50% sequence identity. The person skilled in the art will be able to modify these hybridisation conditions in order to specifically identify sequences varying in identity between 50% and 90%.

[051] "Expression" refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into a protein. “Overexpression” refers to expression of a gene by a recombinant cell in excess to its expression in a corresponding wild-type cell. Such overexpression can for example be arranged for by: increasing the frequency of transcription of one or more nucleic acid sequences, for example by operational linking of the nucleic acid sequence to a promoter functional within the recombinant cell; and/or by increasing the number of copies of a certain nucleic acid sequence. [052] The terms “upregulate”, “upregulated” and “upregulation” refer to a process by which a cell increases the quantity of a cellular component, such as RNA or protein. Such an upregulation may be in response to or caused by a genetic modification.

[053] By the term “pathway” or “metabolic pathway” is herein understood a series of chemical reactions in a cell that build and breakdown molecules.

[054] Nucleic acid sequences (i.e. polynucleotides) or proteins (i.e. polypeptides) may be homologous or heterologous to the genome of a host cell.

[055] “Homologous” with respect to a host cell, means that the nucleic acid sequence does naturally occur in the genome of the host cell or that the protein is naturally produced by that cell. Homologous protein expression may e.g. be an overexpression or expression under control of a different promoter. In the present inventions the host cell is a yeast.

[056] The term "heterologous", with respect to the host cell, means that the polynucleotide does not naturally occur in that way in the genome of the host cell or that the polypeptide is not naturally produced by that cell. Heterologous protein expression involves expression of a protein that is not naturally produced in that way in the host cell. As used herein, "heterologous" may refer to a nucleic acid or protein is a nucleic acid or protein that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.

[057] The term “heterologous expression” refers to the expression of heterologous nucleic acids in a host cell. The expression of heterologous proteins in eukaryotic host cell systems such as yeast are well known to those of skill in the art. A polynucleotide comprising a nucleic acid sequence of a gene encoding a certain protein or enzyme with a specific activity can be expressed in such a eukaryotic system. In some embodiments, transformed/transfected cells may be employed as expression systems for the expression of the enzymes. Expression of heterologous proteins in yeast is well known. Sherman, F., et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is a well-recognized work describing the various methods available to express proteins in yeast. Two widely utilized yeasts are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.

[058] As used herein "promoter" is a DNA sequence that directs the transcription of a (structural) gene. Typically, a promoter is located in the 5'-region of a gene, proximal to the transcriptional start site of a (structural) gene. Promoter sequences may be constitutive, inducible or repressible. In an embodiment there is no (external) inducer needed. [059] The term “vector” as used herein, includes reference to an autosomal expression vector and to an integration vector used for integration into the chromosome.

[060] The term "expression vector" refers to a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest under the control of (i.e. operably linked to) additional nucleic acid segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. In particular an expression vector comprises a nucleic acid sequence that comprises in the 5' to 3' direction and operably linked: (a) a yeast-recognized transcription and translation initiation region, (b) a coding sequence for a polypeptide of interest, and (c) a yeast- recognized transcription and translation termination region. “Plasmid" refers to autonomously replicating extrachromosoma I DNA which is not integrated into a microorganism's genome and is usually circular in nature.

[061] An “integration vector” refers to a DNA molecule, linear or circular, that can be incorporated in a microorganism's genome and provides for stable inheritance of a gene encoding a polypeptide of interest. The integration vector generally comprises one or more segments comprising a gene sequence encoding a polypeptide of interest under the control of (i.e. operably linked to) additional nucleic acid segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and one or more segments that drive the incorporation of the gene of interest into the genome of the target cell, usually by the process of homologous recombination. Typically, the integration vector will be one which can be transferred into the target cell, but which has a replicon which is nonfunctional in that organism. Integration of the segment comprising the gene of interest may be selected if an appropriate marker is included within that segment.

[062] By "host cell" is herein understood a cell, such as a yeast cell, that is to be transformed with one or more nucleic acid sequences encoding for one or more heterologous proteins, to construe a transformed cell, also referred to as a recombinant cell. For example, the transformed cell may contain a vector and may support the replication and/or expression of the vector.

[063] "Transformation" and "transforming", as used herein, refers to the insertion of an exogenous polynucleotide (i.e. an exogenous nucleic acid sequence) into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.

[064] By “constitutive expression” and “constitutively expressing” is herein understood that there is a continuous transcription of a nucleic acid sequence. That is, the nucleic acid sequence is transcribed in an ongoing manner. Constitutively expressed genes are always “on”.

[065] By “anaerobic constitutive expression” is herein understood that nucleic acid sequence is constitutively expressed in an organism under anaerobic conditions. That is, under anaerobic conditions the nucleic acid sequence is transcribed in an ongoing manner, i.e. under such anaerobic conditions the genes are always “on”.

[066] By "disruption" is herein understood any disruption of activity, including, but not limited to, deletion, mutation and reduction of the affinity of the disrupted gene and expression of RNA complementary to such disrupted gene. It includes all nucleic acid modifications such as nucleotide deletions or substitutions, gene knock-outs, and other actions which affect the translation or transcription of the corresponding polypeptide and/or which affect the enzymatic (specific) activity, its substrate specificity, and/or or stability. It also includes modifications that may be targeted on the coding sequence or on the promotor of the gene. A gene disruptant is a cell that has one or more disruptions of the respective gene. Native to yeast herein is understood as that the gene is present in the yeast cell before the disruption.

[067] The term “encoding” has the same meaning as “coding for”. Thus, by way of example, “one or more genes encoding a sorbitol dehydrogenase” has the same meaning as “one or more genes coding for a sorbitol dehydrogenase”.

[068] As far as genes or nucleic acid sequences encoding a protein or an enzyme are concerned, the phrase “one or more nucleic acid sequences encoding a X”, wherein X denotes a protein, has the same meaning as “one or more nucleic acid sequences encoding a protein having X activity”. Thus, by way of example, “one or more nucleic acid sequences encoding a sorbitol dehydrogenase” has the same meaning as “one or more nucleic acid sequences encoding a protein having sorbitol dehydrogenase activity”.

[069] The abbreviation “NADH” refers to reduced, hydrogenated form of nicotinamide adenine dinucleotide. The abbreviation “NAD+” refers to the oxidized form of nicotinamide adenine dinucleotide. Nicotinamide adenine dinucleotide may act as a so-called cofactor, assisting in biochemical reactions and/or transformations in a cell.

[070] The conversion of NADH into NAD+ and vice-versa is a so-called redox reaction. In reductionoxidation ("redox") reactions, electrons are transferred from a donor (i.e. a reducing agent that is being oxidized) to an acceptor (i.e. an oxidizing agent that is being reduced). Electron-transfer reactions proceed in the direction in which electrons flow from sources (reducing agents) to sinks (oxidizing agents). For example the NAD+ ions can serve as an electron sink to NADH.

[071] By a “redox sink” is herein understood a metabolic pathway that, overall, consumes or oxidizes NADH into NAD+ and/or prevents or reduces the consumption or reduction of NAD+ into NADH. A nonnative metabolic pathway is a metabolic pathway that does not occur in the corresponding wild-type cell. Hence, a non-native metabolic pathway forming a redox sink is preferably a non-native metabolic pathway that, as compared to a corresponding wild-type yeast cell, increases NADH consumption and/or decreases NAD+ consumption. By increasing NADH consumption and/or decreasing NAD+ consumption advantageously an (additional) non-native redox sink can be created within the cell.

[072] “NADH dependent” is herein equivalent to NADH specific and “NADH dependency” is herein equivalent to NADH specificity. Y1

[073] By a NADH dependent enzyme is herein understood an enzyme that is exclusively depended on NADH as a co-factor or that is predominantly dependent on NADH as a cofactor. By an “exclusive NADH dependent” enzyme is herein understood an enzyme that has an absolute requirement for NADH over NADPH. That is, it is only active when NADH is applied as cofactor. By a “predominantly NADH-dependent” enzyme is herein understood an enzyme that has a higher specificity and/or a higher catalytic efficiency for NADH as a cofactor than for NADPH as a cofactor.

The enzyme’s specificity characteristics can be described by the formula:

1 < Km NADP⁺Z Km NAD⁺ < “ (infinity) wherein K_m is the so-called Michaelis constant.

For a predominantly NADH-dependent enzyme, preferably K_mNADP⁺ / K_mNAD⁺ is between 1 and 1000, between 1 and 500, between 1 and 200, between 1 and 100, between 1 and 50, between 1 and 10, between 5 and 100, between 5 and 50, between 5 and 20 or between 5 and 10. The Km’s for the enzymes herein can be determined as enzyme specific, for NAD⁺ and NADP⁺ respectively, using know analysis techniques, calculations and protocols. These are described for instance in Lodish et al., Molecular Cell Biology 6^th Edition, Ed. Freeman, pages 80 and 81 , e.g. Figure 3-22.

For an predominantly NADH-dependent enzyme, preferably the ratio of the catalytic efficiency for NADPH/NADP+ as a cofactor (fcat/K_m)^NADP+ to NADH/NAD+ as cofactor (k_Cat/K_m)^NAD+, i.e. the catalytic efficiency ratio (kcat/K_m)^NADP+ : (kcat/K_m)^NAD+, is more than 1 :1 , more preferably equal to or more than 2:1 , still more preferably equal to or more than 5:1 , even more preferably equal to or more than 10:1 , yet even more preferably equal to or more than 20:1 , even still more preferably equal to or more than 100:1 , and most preferably equal to or more than 1000:1 .

There is no upper limit, but for practical reasons the predominantly NADH-dependent enzyme may have a catalytic efficiency ratio (fcat/K_m)^NADP+ : (fcat/K_m)^NAD+ of equal to or less than 1 .000.000.000:1 (i.e. 1 .10⁹: 1 ) .

The yeast cell

[074] The mutant yeast cell is preferably a yeast cell, or derived from a yeast cell, from the genus of Saccharomycetaceae or the genus of Schizosaccharomycetaceae.

[075] Examples of suitable yeast cells include Saccharomyces, such as Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces jurei, Saccharomyces pastorianus, Saccharomyces beticus, Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces uvarum and Saccharomyces bayanus.

[076] Examples of suitable yeast cells further include Schizosaccharomyces, such as Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus and Schizosaccharomyces cryophilus;.

[077] Other exemplary yeasts include Torulaspora such as Torulaspora delbrueckii; Kluyveromyces such as Kluyveromyces marxianus; Pichia such as Pichia stipitis, Pichia pastoris or pichia angusta; Zygosaccharomyces such as Zygosaccharomyces bailii; Brettanomyces such as Brettanomyces inter medius; Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkera bruxellensis and Dekkera anomala; Metschmkowia, Issatchenkia, such as Issatchenkia orientalis, KJoeckera such as KJoeckera apiculata; and Aureobasidium such as Aureobasidium pullulans.

[078] The yeast cell is preferably a yeast cell of the genus Schizosaccharomyces, herein also referred to as a Schizosaccharomyces yeast cell, or a yeast cell of the genus Saccharomyces, herein also referred to as a Saccharomyces yeast cell. More preferably the yeast cell is a yeast cell derived from a yeast cell of the species Saccharomyces cerevisiae, herein also referred to as a Saccharomyces cerevisae yeast cell.

[079] Preferably the yeast cell is an industrial yeast cell. The living environments of yeast cells in industrial processes are significantly different from that in the laboratory. Industrial yeast cells must be able to perform well under multiple environmental conditions which may vary during the process. Such variations include changes in nutrient sources, pH, ethanol concentration, temperature, oxygen concentration, etc., which together have potential impact on the cellular growth and ethanol production of the yeast cell. An industrial yeast cell can be understood to refer to a yeast cell that, when compared to a laboratory counterpart, has a more robust performance. That is, when compared to a laboratory counterpart, the industrial yeast cell shows less variation in performance when one or more environmental conditions selected from the group of nutrient sources, pH, ethanol concentration, temperature, oxygen concentration, are varied during fermentation. Preferably, the yeast cell is constructed on the basis of an industrial yeast cell as a parent or a host, wherein the construction is conducted as described hereinafter. Examples of industrial yeast cells are Ethanol Red® (Fermentis) Fermiol® (DSM) and Thermosacc® (Lallemand).

[080] The mutant yeast cell described herein may be derived from a parent yeast cell capable of producing a fermentation product. Preferably the parent yeast cell is an industrial yeast cell as described herein above. Preferably the yeast cell described herein is derived from a parent yeast cell having the ability to produce ethanol.

[081] Preferably the mutant yeast cell is a recombinant yeast cell. This recombinant yeast cell may be derived from any host cell capable of producing a fermentation product. Preferably the host cell is an industrial yeast cell as described herein above. Preferably the mutant yeast cell is derived from a host cell having the ability to produce ethanol.

[082] The yeast cell described herein may be derived from the parent or host cell through any technique known by one skilled in the art to be suitable therefore. Such techniques may include any one or more of adaptive evolution, mutagenesis, recombinant DNA technology (including, but not limited to, CRISPR-CAS techniques), selective and/or adaptive evolution, mating, cell fusion, and/or cytoduction between yeast strains. Suitably the one or more desired genes are incorporated in the yeast cell by a combination of one or more of the above techniques. First genetic modifications

[083] The mutant yeast cell suitably comprises a first genetic modification for, preferably constitutive, expression of a NAD+ dependent protein that functions in a first metabolic pathway converting a sugar alcohol into a fermentation product.

[084] Suitably one or more first genetic modifications may result in the, preferably constitutive, expression of merely one NAD+ dependent protein, or more than one NAD+ dependent protein, such as two or more NAD+ dependent proteins, that function in a first metabolic pathway converting a sugar alcohol into a fermentation product.

[085] Preferably the mutant yeast cell comprises one or more first genetic modifications for anaerobic constitutive expression (i.e. constitutive expression under anaerobic conditions) of one or more NAD+ dependent proteins that function in a first metabolic pathway converting a sugar alcohol into a fermentation product.

[086] Preferably the "one or more first genetic modifications for, preferably constitutive, expression of a NAD+ dependent protein that functions in a first metabolic pathway converting a sugar alcohol into a fermentation product " are chosen from the group consisting of: a) one or more first genetic modifications comprising or consisting of a genetic modification to constitutively express and/or upregulate the activity of one or more proteins having NAD+ dependent sugar alcohol dehydrogenase activity, preferably a NAD+ dependent sorbitol dehydrogenase and/or a NAD+ dependent mannitol dehydrogenase; and/or b) one or more first genetic modifications comprising or consisting of a genetic modification to downregulate the activity of one or more proteins that play a role in the glucose repression of the yeast. [087] Preferably the NAD+ dependent protein is an enzyme having the ability to convert a sugar alcohol as described herein. Preferred sugar alcohols include sorbitol and mannitol. More preferably the NAD+ dependent protein is a protein having NAD+ dependent sugar alcohol dehydrogenase activity. Most preferably the protein having NAD+ dependent sugar alcohol dehydrogenase activity is a NAD+ dependent sorbitol dehydrogenase or a NAD+ dependent mannitol dehydrogenase. Such sorbitol dehydrogenase and mannitol dehydrogenase are preferable derived from Saccharomyces cerevisiae.

[088] That is, preferably the first metabolic pathway to convert a sugar alcohol preferably comprises a NAD+ dependent sugar alcohol dehydrogenase. More preferably the NAD+ dependent protein that functions in the first metabolic pathway converting a sugar alcohol into a fermentation product is therefore such a NAD+ dependent sugar alcohol dehydrogenase. Hence, preferably the mutant yeast cell is a mutant yeast cell comprising one or more first genetic modifications for, preferably constitutive, expression of a NAD+ dependent sugar alcohol dehydrogenase.

[089] Thus, the mutant yeast cell can be a mutant yeast cell, comprising :

(I) one or more first genetic modifications for, preferably constitutive, expression of a NAD+ dependent sugar alcohol dehydrogenase; and (ii) one or more second genetic modifications for, preferably constitutive, expression of a protein that functions in a second metabolic pathway forming a non-native redox sink.

[090] The first genetic modifications may result in the constitutive expression of merely one NAD+ dependent sugar alcohol dehydrogenase, or more than one NAD+ dependent sugar alcohol dehydrogenase, such as two or more NAD+ dependent sugar alcohol dehydrogenases.

[091] Preferably the first metabolic pathway further comprises a sugar alcohol transporter and more preferably the mutant yeast cell comprises one or more genetic modifications to upregulate the activity of one or more sugar alcohol transporters.

[092] The first genetic modifications may therefore suitably include modifications for the, preferably constitutive, expression of merely one sugar alcohol transporter, or more than one sugar alcohol transporter, such as two or more sugar alcohol transporters, that function in the first metabolic pathway converting a sugar alcohol into a fermentation product. More preferably the mutant yeast cell is therefore a mutant yeast cell comprising one or more first genetic modifications for constitutive expression of a NAD+ dependent sugar alcohol dehydrogenase and optionally for constitutive expression of a sugar alcohol transporter.

[093] Preferably the one or more first genetic modifications allow for an increase of the activity, as compared to the non-modified yeast cell, of the NAD+ dependent protein(s) that function(s) in the first metabolic pathway to convert a sugar alcohol into a fermentation product. That is, preferably the mutant yeast cell is a mutant yeast cell comprising one or more first genetic modifications for increasing, as compared to the non-modified yeast cell, the activity of a NAD+ dependent sugar alcohol dehydrogenase and/or for increasing, as compared to the non-modified yeast cell, the activity of a sugar alcohol transporter. [094] Thus, the mutant yeast cell can be a mutant yeast cell, comprising :

(I) one or more first genetic modifications for increasing, as compared to the non-modified yeast cell, the activity of a NAD+ dependent sugar alcohol dehydrogenase and/or for increasing, as compared to the nonmodified yeast cell, the activity of a sugar alcohol transporter.

(II) one or more second genetic modifications for constitutive expression of a protein that functions in a second metabolic pathway forming a non-native redox sink.

[095] The activity of the NAD+ dependent sugar alcohol dehydrogenases and/or the sugar alcohol transporter can be increased in any manner known to be suitable therefore by the person skilled in the art. The mutant yeast cell can be prepared with known recombination techniques.

[096] The manners to increase activity may for example include:

- the functional expression of one or more heterologous nucleic acid sequences encoding for a NAD+ dependent sugar alcohol dehydrogenase and/or a sugar alcohol transporter; and/or

- the overexpression of one or more homologous nucleic acid sequence(s) encoding for a NAD+ dependent sugar alcohol dehydrogenase and/or a sugar alcohol transporter.

The nucleic acid sequence encoding for the protein or enzyme is herein also referred to as the “coding sequence” or the “coding nucleic acid sequence”. [097] Various means are known to those skilled in the art for expression and overexpression of proteins or enzymes in a mutant yeast cell.

[098] For example, a protein or enzyme may be overexpressed by increasing the copy number of the gene coding for the protein or enzyme in the host cell, for example by integrating additional copies of the gene in the host cell's genome, by expressing the gene from an episomal multicopy expression vector or by introducing a episomal expression vector that comprises multiple copies of the gene.

[099] In addition or as an alternative thereto, overexpression of a protein or enzyme in the mutant yeast cell may be achieved by using a, preferably heterologous, promoter. Such a promotor may suitably be nonnative to the nucleic acid sequence coding for the protein or enzyme to be overexpressed, i.e. a promoter that is heterologous to the coding nucleic acid sequence to which it is operably linked. Although such a promoter preferably is heterologous to the coding nucleic acid sequence to which it is operably linked, it is still possible for the promoter itself to be homologous, i.e. endogenous to the host cell. For example, the promoter may be a promoter that is normally operably linked to another nucleic acid sequence within the cell.

[100] Preferably the heterologous promoter is capable of producing a higher steady state level of the transcript comprising the coding nucleic acid sequence (or is capable of producing more transcript molecules, i.e. mRNA molecules, per unit of time) than is the promoter that is native to the coding nucleic acid sequence. Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters.

[101] The coding nucleic acid sequence used for overexpression of the proteins or enzymes mentioned above may preferably be homologous to the host cell. However, coding nucleic acid sequences that are heterologous to the host may also be used.

[102] In addition or as an alternative to the above, the mutant yeast may comprises a genetic modification to downregulate one or more genes that have a role in glucose repression. More preferably, the mutant yeast may comprise a genetic modification to downregulate the activity of a homologous gene encoding a TUP1 protein and/or CYC8 protein of the yeast.

[103] Examples of such a TUP1 protein include:

- the protein of SEQ ID NO: 47, or

- a functional homologue thereof comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of respectively SEQ ID NO: 47; or

- a functional homologue comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 47; wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 47.

[104] Examples of such a CYC8 protein include:

- the protein of SEQ ID NO: 48, or

- a functional homologue thereof comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of respectively SEQ ID NO: 48; or

- a functional homologue comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 48; wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 48.

[105] Overexpression of a protein or enzyme, when referring to the production of such a protein or enzyme in a mutant yeast cell, suitably means that the protein or enzyme is produced at a higher level of specific enzymatic activity as compared to the unmodified host cell under identical conditions. Preferably the enzymatically active protein (or proteins in case of multi-subunit enzymes) is produced in greater amounts, or rather at a higher steady state level as compared to the unmodified host cell under identical conditions. More preferably the mRNA coding for the enzymatically active protein is produced in greater amounts, or again rather at a higher steady state level as compared to the unmodified host cell under identical conditions. In the mutant yeast cell, the above NAD+ dependent sugar alcohol dehydrogenase is preferably overexpressed by a factor of at least 1 .1 , at least 1 .2, at least 1 .5, at least 2, at least 5, at least 10 or at least 20 as compared to a strain which is genetically identical except for the genetic modification causing the overexpression. It is to be understood that these levels of overexpression may apply to the steady state level of the enzyme's activity, the steady state level of the enzyme's protein as well as to the steady state level of the transcript coding for the enzyme.

Sugar alcohol dehydrogenase

[106] By a "sugar alcohol dehydrogenase" is herein understood a protein having sugar alcohol dehydrogenase activity and by a "NAD+ dependent sugar alcohol dehydrogenase" is herein understood a NAD+ dependent protein having sugar alcohol dehydrogenase activity. For example, by a "sorbitol dehydrogenase" is herein understood any protein having sorbitol dehydrogenase activity. By a "mannitol dehydrogenase" is herein understood any protein having mannitol dehydrogenase activity.

[107] A "NAD+ dependent protein having sugar alcohol dehydrogenase activity" can herein also be referred to as a "protein having NAD+ dependent sugar alcohol dehydrogenase activity", a "sugar alcohol dehydrogenase protein", a "sugar alcohol dehydrogenase enzyme", or simply a "NAD+ dependent sugar alcohol dehydrogenase" or "sugar alcohol dehydrogenase".

[108] Preferably the sugar alcohol is a sugar alcohol having equal to or more than 5 carbon atoms, more preferably equal to or more than 6 carbon atoms. Examples of suitable sugar alcohols include arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, idotol, inositol, isomalt, maltitol and lactitol. Examples of suitable sugar alcohol dehydrogenases include arabitol dehydrogenase, xylitol dehydrogenase, ribitol dehydrogenase, mannitol dehydrogenase, sorbitol dehydrogenase, galactitol dehydrogenase, fucitol dehydrogenase, idotol dehydrogenase, inositol dehydrogenase, isomalt dehydrogenase, maltitol dehydrogenase and lactitol dehydrogenase.

[109] As explained previously, glycerol is not considered to be a sugar alcohol and the sugar alcohol is not glycerol. The sugar alcohol dehydrogenase is therefore not glycerol dehydrogenase.

[110] Most preferably the sugar alcohol is a sugar alcohol comprising 6 carbon atoms (also referred to as a “C6 sugar alcohol”). Most preferred sugar alcohols are sorbitol and mannitol. Most preferred NAD+ dependent sugar alcohol dehydrogenases are NAD+ dependent sorbitol dehydrogenase and NAD+ dependent mannitol dehydrogenase. Preferably the sugar alcohol is not xylitol and/or preferably the sugar alcohol dehydrogenase is not xylitol dehydrogenase. Sugar alcohols comprising 5 carbon atoms (also referred to as "C5 sugar alcohols"), respectively sugar alcohol dehydrogenases for such C5 sugar alcohols are less preferred.

[111] More preferably the sugar alcohol dehydrogenase is:

- a NAD+ dependent sorbitol dehydrogenase chosen from the group consisting of NAD+ dependent sorbitol dehydrogenase 1 (SOR1), preferably having an amino acid sequence of SEQ ID NO: 9, and NAD+ dependent sorbitol dehydrogenase 2 (SOR2), preferably having an amino acid sequence of SEQ ID NO: 11 ; or

- a NAD+ dependent mannitol dehydrogenase chosen from the group consisting of NAD+ dependent mannitol dehydrogenase 1 (MAN1), preferably having an amino acid sequence of SEQ ID NO: 13, and NAD+ dependent mannitol dehydrogenase 2 (MAN2), preferably having an amino acid sequence of SEQ ID NO: 15; or

- a functional homologue of any of the above, preferably a functional homologue comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of respectively SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13 or SEQ ID NO: 15; or a functional homologue comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13 or SEQ ID NO: 15, wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13 or SEQ ID NO: 15.

[112] The nucleic acid sequence encoding the sugar alcohol dehydrogenases as listed above can be an exogenous or heterologous nucleic acid sequence or endogenous or native nucleic acid sequence. When the mutant yeast cell comprises a nucleic acid sequence encoding an exogenous or heterologous sugar alcohol dehydrogenase, one or more endogenous or native nucleic acid sequence(s) encoding one or more native sugar alcohol dehydrogenases may be deleted or disrupted.

[113] More preferably the mutant yeast cell is a mutant yeast cell comprising:

- a nucleic acid sequence encoding for any of the above mentioned sugar alcohol dehydrogenases; and/or

- a nucleic acid sequence of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16; and/or

- a nucleic acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the nucleic acid sequence of respectively SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16; and/or

- a nucleic acid sequence having one or several substitutions, insertions and/or deletions as compared to the nucleic acid sequence of respectively SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16, wherein more preferably the nucleic acid sequence has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 nucleic acid substitutions, insertions and/or deletions as compared to the nucleic acid sequence of respectively SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.

[114] The first genetic modifications can be present for example in the form of:

- an exogenous nucleic acid sequence as described in the preceding paragraph, suitably in one or more copies, and/or

- an exogenous promoter operably linked to an endogenous nucleic acid sequence as described in the preceding paragraph, and/or

- multiple copies of an endogenous nucleic acid sequence as described in the preceding paragraph.

[115] The mutant yeast cell can thus suitably be a mutant yeast cell, comprising : one or more first genetic modifications for constitutive expression, and preferably overexpression, of a nucleic acid sequence chosen from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16; or a nucleic acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with such nucleic acid sequence; or a nucleic acid sequence having one or several substitutions, insertions and/or deletions as compared to such nucleic acid sequence, wherein more preferably the nucleic acid sequence has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 nucleic acid substitutions, insertions and/or deletions as compared to the nucleic acid sequence of respectively SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.

[116] Preferably the NAD+ dependent protein is sorbitol dehydrogenase and/or the mutant yeast cell comprises one or more first genetic modifications for constitutive expression or overexpression of a sorbitol dehydrogenase, for example in the form of:

- an exogenous nucleic acid sequence encoding for a sorbitol dehydrogenase, suitably in one or more copies, and/or

- an exogenous promoter operably linked to an endogenous nucleic acid sequence encoding for a sorbitol dehydrogenase, and/or

- multiple copies of an endogenous nucleic acid sequence encoding for a sorbitol dehydrogenase.

[117] In one embodiment the mutant yeast cell comprises an exogenous gene or exogenous nucleic acid sequence coding for an NAD+ dependent sorbitol dehydrogenase selected from the group consisting of:

- NAD+ dependent sorbitol dehydrogenase as obtained or derived from Bacillus megaterium, Bacillus subtilis, Candida boidinii, Candida sp. HA 167, Deinococcus geothermalis, Gluconobacter oxydans, Komagataella pastoris, Malus domesticus, Pseudomonas sp., Pyrus pyrifolia, Rhodobacter sphaeroides, Saccharomyces bayanus, Saccharomyces kudriavzevii, Saccharomyces paradoxus, Scheffersomyces stipites, Solanum lycopersicum, Zea mays; and

- functional homologues of such NAD+ dependent sorbitol dehydrogenase comprising an amino acid sequence with at least 50 %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity with one or more of such aforementioned NAD+ dependent sorbitol dehydrogenase; and

- functional homologues of such NAD+ dependent sorbitol dehydrogenase comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of one or more of such aforementioned NAD+ dependent sorbitol dehydrogenase, wherein preferably the amino acid sequence of any of the above functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to such aforementioned NAD+ dependent sorbitol dehydrogenase.

[118] A sorbitol dehydrogenase having an amino acid sequence as listed in SEQ ID NO: 11 is especially preferred. SEQ ID NO: 11 shows the amino acid sequence of a highly preferred NAD+ dependent sorbitol dehydrogenase protein, i.e. sorbitol dehydrogenase 2 (SOR2), encoded by a nucleic acid sequence from Saccharomyces cerevisiae. SEQ ID NO 12 shows a suitable nucleic acid sequence encoding for this highly preferred amino acid sequence. Other preferred sorbitol dehydrogenase proteins include functional homologues of this protein, preferably functional homologues comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 11 or functional homologues comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of SEQ ID NO: 11 , wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to the amino acid sequence of SEQ ID NO: 11 .

[119] Examples of suitable sorbitol dehydrogenase proteins and their origin are given in Table 2 below, with reference to their sequence identity with the amino acid sequence of SEQ ID NO:11 .

[120] In addition to the examples of sorbitol dehydrogenase proteins listed in Table 2, other examples of suitable sorbitol dehydrogenase proteins are the sorbitol dehydrogenase proteins identified in:

- Sinorhizobium melilotil (SmoS) (as described in the article of Kohlmeier et al., titled " Characterization of Sorbitol Dehydrogenase SmoS from Sinorhizobium meliloti 1021", published in BioRxiv (2019), as 689042 (2019), herewith incorporated by reference);

- Pseudomonas mandelii (as described in the article of DangThu et al., titled "Molecular cloning and biochemical characterization of a NAD-dependent sorbitol dehydrogenase from cold-adapted Pseudomonas mandelii", published in FEMS Microbiology Letters, vol. 368(2), (2021), as fnaa222, herewith incorporated by reference);

- Rhodobacter sphaeroides (as described in the article of Philippsen et al., titled "Structure of zinc- independent sorbitol dehydrogenase from Rhodobacter sphaeroides at 2.4 A resolution", published in Acta Crystallogr D Biol Crystallogr., vol. 61 (2005), pages 374-379, herewith incorporated by reference);

- P. mandelii strain isolated from potato fields (as described in the article of Formusa et al. , titled " Genome sequence of Pseudomonas mandelii PD30", published in Genome Announc., vol. 2(4) (2014), as e00713- 4, herewith incorporated by reference);

- Bradyrhyzobium japonicum (as described in the article by Fredslund et al., titled "Structural characterization of the thermostable Bradyrhizobium japonicum D-sorbitol dehydrogenase" , published in Acta Cryst., F Struct Biol Communications, vol. 72 (2016), pages 846-852, herewith incorporated by reference);

- Arabidopsis thialiana (as described in the article by Aguayo et al, titled "Sorbitol dehydrogenase is a cytosolic protein required for sorbitol metabolism in Arabidopsis thaliana", published in Plant science, vol. 205 (2013), pages 63-75, herewith incorporated by reference); and

- Aspergillus niger, wherein both a sorbitol dehydrogenase (sdhA) and a L-arabitol dehydrogenase (LadA) were identified. Suitably a single amino acid change (Y318F) in LadA was found to increase the affinity for D-sorbitol. ( as described in the article of Rutten et al., titled "A single amino acid change (Y318F) in the L- arabitol dehydrogenase (LadA) from Aspergillus niger results in a significant increase in affinity for D- sorbitof', published in BMC microbiology, vol. 9(1), (2009), pages 1-7, and in the article of Koivistoinen et al., titled "Sorbitol dehydrogenase of Aspergillus niger, SdhA, is part of the oxido -reductive d-galactose pathway and essential for d-sorbitol catabolism" FEBS letters, 586(4), (2012), pages 378-383, both herewith incorporated by reference.) Table 2: Examples of suitable sorbitol dehydrogenase proteins with % identity as compared to Saccharomyces cerevisiae s288c SOR2, compiled using BLASTP.

[121 ] The above exemplified proteins are similar to the exemplified SOR1 and/or SOR 2 and can be used in the present invention in a manner alike to SOR1 and/or SOR2. All exemplified proteins above can be combined with the other preferences and embodiments as described herein for sorbitol dehydrogenase, including those described for SOR 1 and/or SOR2.

[122] In addition or in the alternative, the NAD+ dependent protein can be mannitol dehydrogenase and/or the mutant yeast cell can comprise one or more first genetic modifications for constitutive expression or overexpression of a mannitol dehydrogenase, for example in the form of: - an exogenous nucleic acid sequence encoding for a mannitol dehydrogenase, suitably in one or more copies, and/or - an exogenous promoter operably linked to an endogenous nucleic acid sequence encoding for a mannitol dehydrogenase, and/or

- multiple copies of an endogenous nucleic acid sequence encoding for a mannitol dehydrogenase.

[123] In one embodiment the mutant yeast cell comprises an exogenous gene coding for an NAD+ dependent mannitol dehydrogenase selected from the group consisting of:

- NAD+ dependent mannitol dehydrogenase as obtained or derived from Saccharina japonica, Thermotoga maritima, Thermotoga neapolitana, Aspergillus fumigatus, Streptomyces lavendulae, Brevi bacterium flavum, Gluconobacter oxydans, Rhodobacter sphaeroides, Lactobacillus brevis, Leuconostoc mesenteroides, Pseudomonas sp.; and

- functional homologues of such NAD+ dependent mannitol dehydrogenase comprising an amino acid sequence with at least 50 %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% amino acid sequence identity with one or more of such aforementioned NAD+ dependent mannitol dehydrogenase; and

- functional homologues of such NAD+ dependent mannitol dehydrogenase comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of one or more of such aforementioned NAD+ dependent mannitol dehydrogenase, wherein preferably the amino acid sequence of any of the above functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to such aforementioned NAD+ dependent mannitol dehydrogenase.

[124] A mannitol dehydrogenase having an amino acid sequence as listed in SEQ ID NO: 15 is especially preferred. SEQ ID NO: 15 shows the amino acid sequence of a NAD+ dependent suitable mannitol dehydrogenase protein, i.e. mannitol dehydrogenase 2 (MAN2) encoded by a nucleic acid sequence from Saccharomyces cerevisiae. SEQ ID NO 16 shows an optimized nucleic acid sequence encoding for this amino acid sequence. Other suitable mannitol dehydrogenase proteins include functional homologues of this protein, preferably functional homologues comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 15; or functional homologues comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of SEQ ID NO: 15, wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to the amino acid sequence of SEQ ID NO: 15. Sugar alcohol transporter

[125] In addition to a NAD+ dependent sugar alcohol dehydrogenase, the mutant yeast cell may preferably comprise one or more transporters suitable for the transport of a sugar alcohol into the mutant yeast cell. By a "sugar alcohol transporter" is thus herein understood to be a protein capable of transporting the sugar alcohol into the mutant yeast cell.

[126] Preferences for the sugar alcohols are as described above. Such sugar alcohols may suitably be transported by a pentose transporter or hexose transporter, dependent on the type of sugar alcohol. Preferably the sugar alcohol is a sugar alcohol comprising 6 carbon atoms (also referred to as a “C6 sugar alcohol”). Such a C6 sugar alcohol is preferably transported by a hexose transporter. Hence, preferably the sugar alcohol transporter is a hexose transporter. The above exemplified sorbitol and/or mannitol are preferably transported by a hexose transporter into the mutant yeast cell.

[127] Suitable sugar alcohol transporters include those as mentioned by Jordan et al. Preferably the sugar alcohol transporter is a hexose transporter chosen from the group consisting of HXT13, HXT15 and HXT17.

[128] Preferred hexose transporters include hexose transporters chosen from the group consisting of:

- HXT13, preferably having an amino acid sequence of SEQ ID NO: 17;

- HXT15, preferably having an amino acid sequence of SEQ ID NO: 19; and

- functional homologues of these, preferably a functional homologue comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of respectively SEQ ID NO: 17 or SEQ ID NO: 19; and/or a functional homologue comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 17 or SEQ ID NO: 19, wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to the amino acid sequence of respectively SEQ ID NO: 17 or SEQ ID NO: 19.

[129] The nucleic acid sequence encoding the hexose transporters as listed above can be an exogenous or heterologous nucleic acid sequence or an endogenous or native nucleic acid sequence. When the mutant yeast cell comprises a nucleic acid sequence encoding an exogenous or heterologous hexose transporter, one or more endogenous or native nucleic acid sequence(s) encoding one or more native hexose transporters may be deleted or disrupted.

[130] More preferably the mutant yeast cell is a mutant yeast cell comprising:

- a nucleic acid sequence encoding for any of the above mentioned hexose transporters; and/or

- a nucleic acid sequence of SEQ ID NO: 18, or SEQ ID NO: 20; and/or - a nucleic acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the nucleic acid sequence of respectively SEQ ID NO: 18 or SEQ ID NO: 20; and/or

- a nucleic acid sequence having one or several substitutions, insertions and/or deletions as compared to the nucleic acid sequence of respectively SEQ ID NO: 18 or SEQ ID NO: 20, wherein more preferably the nucleic acid sequence has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 nucleic acid substitutions, insertions and/or deletions as compared to the nucleic acid sequence of respectively SEQ ID NO: 18 or SEQ ID NO: 20.

[131] Jordan et al. found that HXT13 and HXT15 have moderate affinity for sorbitol, while HXT13, HXT15 and HXT17 exhibit high affinity for mannitol. The inventors of the present invention have found that the hexose transporter HXT13 can advantageously be combined with sorbitol dehydrogenase to render exceptionally good results.

[132] SEQ ID NO: 17 shows the amino acid sequence of a hexose transporter HXT13. Other suitable hexose transporters include functional homologues of this protein, preferably functional homologues comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 17; or functional homologues comprising an amino acid sequence having one or several substitutions, insertions and/or deletions as compared to the amino acid sequence of SEQ ID NO: 17, wherein more preferably the amino acid sequence of such functional homologues has no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 75, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10 or no more than 5 amino acid substitutions, insertions and/or deletions as compared to the amino acid sequence of SEQ ID NO: 17.

[133] As hexose transporters are highly conserved amongst the Saccharomyces species, functional homologues are very likely to be found in other Saccharomyces species including for example Saccharomyces paradoxus. Examples of suitable hexose transporters and their origin are given in Table 3 below, with reference to their sequence identity with the amino acid sequence of SEQ ID NO:17.

[134] In addition to the examples of hexose transporters identified in Saccharomyces species as listed in Table 3, other examples of suitable hexose transporters in n-Saccharomyces species are the ones identified in:

- KJuyveromyces marxianus (as described in the article by Varela et al., titled " Polymorphisms in the LAC12 gene explain lactose utilisation variability in KJuyveromyces marxianus strains", published in FEMS Yeast Research, vol. 17, (2017), as fox021 , herewith incorporated by reference); - Torulaspore delbrueckii (as described in the article by Pacheco et al. titled "Hexose transport in Torulaspora delbrueckii: identification of Igt1, a new dual-affinity transporter", FEMS yeast research, vol. 20(1), (2020) as foaa004, herewith incorporated by reference);

- Yarrowia lipolytica, (as described in the article by Lazar et al., titled "Characterization of hexose transporters in Yarrowia lipolytica reveals new groups of Sugar Porters involved in yeast growth", published in Fungal Genetics and Biology vol. 100, (2017), pages 1-12, herewith incorporated by reference); and/or

- Candida glycerinogenes (as described in the article by Liang et al., titled "Identification and characterization from Candida glycerinogenes of hexose transporters having high efficiency at high glucose concentrations", published in Applied Microbiology and Biotechnology vol.102, (2018) pages 5557-5567, herewith incorporated by reference).

[135] Alternatively or in addition, polyol/H⁺ symporters can be used for the transport of sugar alcohols, such as for example sorbitol and/or mannitol. Hence, the sugar alcohol transporter can also be a polyol/H⁺ symporter. In such polyol/proton symporters, transport is coupled. Connecting these polyol/proton symporters to the sugar alcohol dehydrogenase can advantageously result in a lower energy use and an increase in yield.

[136] In a preferred embodiment, the mutant yeast cell comprises one or more heterologous nucleic acid sequences encoding a hexose transporter and/or polyol/H+ symporter, whilst one or more native nucleic acid sequence(s) encoding one or more native hexose transporters and/or polyol/H+ symporter may be deleted or disrupted. Polyol/H⁺ symporters that can be suitable for the transport of sugar alcohols, such as sorbitol and/or mannitol, are those characterized in S. cerevisiae and those identified in other yeasts and/or plants, such as :

- Debaryomyces hansenii (as described in the article by Pereira et al, titled "Characterization of new Polyol/H+ Symporters in Debaryomyces hansenif', published in PLosONE, vol. 9(2), (2014), as e88180, herewith incorporated by reference), of which more preferred are: the D. hansenii polyol/H+ symporters identified therein for D-sorbitol/D-mannitol/ribitol/D-arabitol/D-galactitol (DhSyH , Symporter Polyols 1) and for D-sorbitol/D-mannitol/ribitol/D-arabitol (DhSyl2, Symporter Polyols 2);

- Prunus cerasus (as described in the article by Gao et al. titled "Cloning, Expression, and Characterization of Sorbitol Transporters from Developing Sour Cherry Fruit and Leaf Sink Tissues", published in Plant Physiology, vol. 131 (4), (2003), pages 1566-1575, herein incorporated by reference);

- Malus domestica (as described in the article by Watari et al., titled "Identification of sorbitol transporters expressed in the phloem of apple source leaves", published in Plant Cell Physiology, vol. 45(8) (2004), pages 1032-1041 , herein incorporated by reference); and/or

- Aradopsis thailiana (as described in the article by Klepek, et al, titled "Arabidopsis POLYOL TRANSPORTERS, a New Member of the Monosaccharide Transporter-Like Superfamily, Mediates H+- Symport of Numerous Substrates, Including myo-lnositol, Glycerol, and Ribose", published in The Plant Cell, 17(1) (2005), pages 204-218 , herein incorporated by reference) . Other suitable Polyol/H⁺ symporters include functional homologues of the above proteins, for example functional homologues comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity therewith.

Table 3: alternative hexose transporters (proteins) for expression with % identity as compared to

Saccharomyces cerevisiae Hxt13, compiled using BLASTP

Second genetic modifications [137] The mutant yeast cell further comprises a second genetic modification for, preferably constitutive, expression of a protein that functions in a second metabolic pathway forming a non-native redox sink.

[138] Preferably, these one or more second genetic modifications are one or more second genetic modifications for the functional expression of one or more heterologous nucleic acid sequences encoding for one or more NADH dependent proteins that function in a second metabolic pathway to convert NADH to NAD+. Several examples of such second metabolic pathways exist, as illustrated further below.

[139] Preferably the mutant yeast cell comprises one or more second genetic modifications for anaerobic constitutive expression of one or more NADH dependent proteins that function in a second metabolic pathway to convert NADH to NAD+. [140] Preferably the "one or more second genetic modifications for constitutive expression of a protein that functions in a second metabolic pathway forming a non-native redox sink" are chosen from the group consisting of: a) one or more second genetic modifications comprising or consisting of:

- a heterologous nucleic acid sequence encoding a protein comprising phosphoketolase activity (EC 4.1 .2.9 or EC 4.1 .2.22, PKL); and/or

- a heterologous nucleic acid sequence encoding a protein having phosphotransacetylase (PTA) activity (EC 2.3.1 .8); and/or

- a heterologous nucleic acid sequence encoding a protein having acetate kinase (ACK) activity (EC 2.7.2.12); b) one or more second genetic modifications comprising or consisting of:

- a heterologous nucleic acid sequence encoding for a protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity; and/or

- a heterologous nucleic acid sequences encoding for a protein having phosphoribulokinase (PRK) activity; and,

- optionally, a heterologous nucleic acid sequence encoding for one or more molecular chaperones for the protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity. and/or c) one or more second genetic modifications comprising or consisting of: a heterologous nucleic acid sequence encoding a protein comprising NADH dependent acetylating acetaldehyde dehydrogenase activity.

[141 ] For example WO2014/081803 describes a recombinant microorganism expressing a heterologous phosphoketolase, phosphotransacetylase or acetate kinase and bifunctional acetaldeyde-alcohol dehydrogenase and WO2015/148272 describes a recombinant S. cerevisiae strain expressing a heterologous phosphoketolase, phosphotransacetylase and acetylating acetaldehyde dehydrogenase. Also WO2018172328A1 describes recombinant cell may comprise one or more (heterologous) genes coding for an enzyme having phosphoketolase activity. The phosphoketalase (PKL) routes described in WO2014/081803, WO2015/148272 and WO2018172328A1 , all incorporated herein by reference, provide preferred metabolic pathways to convert NADH to NAD+ and the NADH dependent phosphoketolase described therein is a preferred NADH dependent protein for application in the current invention.

[142] A suitable example of the mutant yeast cell according to the invention is therefore a mutant yeast cell, comprising:

(I) one or more first genetic modifications for constitutive expression of a NAD+ dependent protein that functions in a first metabolic pathway converting a sugar alcohol into a fermentation product, such as for example one or more first genetic modifications for constitutive expression of a NAD+ dependent sugar alcohol dehydrogenase; and

(ii) one or more second genetic modifications comprising or consisting of: - a heterologous nucleic acid sequence encoding a protein comprising phosphoketolase activity (EC 4.1 .2.9 or EC 4.1 .2.22, PKL); and/or

- a heterologous nucleic acid sequence encoding a protein having acetate kinase (ACK) activity (EC 2.7.2.12);

[143] Another example is provided by the protein with NADH dependent acetylating acetaldehyde dehydrogenase activity as illustrated in US8795998B, also incorporated herein by reference.

[144] Another suitable example of the mutant yeast cell according to the invention is therefore a mutant yeast cell, comprising:

(II) one or more upregulated nucleic acid sequences encoding a protein comprising NADH dependent acetylating acetaldehyde dehydrogenase activity, such as for example listed under EC number EC 1.2.1.10.

[145] WO2014/129898 describes a recombinant cell functionally expressing heterologous nucleic acid sequences encoding for ribulose-1 ,5-phosphate carboxylase/oxygenase (EC 4.1 .1 .39; herein abbreviated as “Rubisco”), and optionally molecular chaperones for Rubisco, and phosphoribulokinase (EC 2.7.1.19; herein abbreviated as “PRK”). With the recombinant cell as described in WO2014/129898 it was possible to reduce or even eliminate NADH-dependent side-product synthesis by functionally expressing the recombinant protein in a yeast cell, using carbon dioxide as a substrate. This pathway is preferred for application in the current invention.

[146] In a preferred embodiment the mutant yeast cell according to the invention is therefore a mutant yeast cell, comprising:

(II) one or more second genetic modifications comprising or consisting of:

- optionally, a heterologous nucleic acid sequence encoding for one or more molecular chaperones for the protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity, More preferably the mutant yeast cell comprises one or more upregulated heterologous nucleic acid sequences encoding for a protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity; one or more upregulated heterologous nucleic acid sequences encoding for a protein having phosphoribulokinase (PRK) activity; and, optionally, one or more upregulated heterologous nucleic acid sequences encoding for one or more molecular chaperones for the protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity

[147] Without wishing to be bound by any kind of theory it is believed that the NAD+ dependent sugar alcohol dehydrogenase, such as NAD+ dependent sorbitol dehydrogenase and/or NAD+ dependent mannitol dehydrogenase can advantageously be used to moderate and/or balance any overactivity of the ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco).

[148] The protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity is herein also referred to as “Rubisco enzyme”, “Rubisco protein” or simply “Rubisco”. Preferences for the Rubisco protein and the nucleic sequences encoding for such are as described in WO2014/129898, incorporated herein by reference.

[149] The Rubisco protein may suitably be selected from the group of eukaryotic and prokaryotic Rubisco proteins. The Rubisco protein is preferably from a non-phototrophic organism. For example, the Rubisco protein may be from a chemolithoautotrophic microorganism. Good results have been achieved with a bacterial Rubisco protein. Preferably, the Rubisco protein originates from a Thiobacillus, in particular, Thiobacillus denitrificans, which is chemolithoautotrophic.

[150] The Rubisco protein may be a single-subunit Rubisco protein or a Rubisco protein having more than one subunit. Preferably the Rubisco protein is a single-subunit Rubisco protein. Good results have been obtained with a Rubisco protein that is a so-called form-ll Rubisco protein. Especially good results were achieved with a Rubisco protein encoded by a cbbM gene, also referred to as CbbM.

[151] A preferred Rubisco protein is the Rubisco protein encoded by the cbbM gene from Thiobacillus denitrificans. SEQ ID NO: 1 shows the amino acid sequence of a suitable Rubisco protein, encoded by the cbbM gene from Thiobacillus denitrificans.

[152] Other suitable Rubisco proteins include functional homologues of this Rubisco protein encoded by the cbbM gene from Thiobacillus denitrificans, preferably functional homologues comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 1. Examples of suitable Rubisco polypeptides and their origin are given in Table 1 of WO2014/129898, incorporated herein by reference, and in Table 4 below, with reference to the sequence identity with the amino acid sequence of SEQ ID NO:1. Table 4: Natural Rubisco polypeptides suitable for expression

[153] Still further suitable Rubisco proteins include the highly active Rubisco proteins as described by Davidi D., et al. in their article titled " Highly active rubiscos discovered by systematic interrogation of natural sequence diversity", published in the The Embo Journal (2020) Vol. 39, e104081 . Such suitable Rubisco proteins may include the form II Rubisco protein of Gallionella sp. (for example with an kcat of 22.2 s-1 and a kM of 276 uM) and the form II Rubisco protein of Hydrogenovibrio marinus (for example with a kcat of 15.6 s-1 and a kM of 162 uM).

[154] As indicated above, the Rubisco protein is suitably functionally expressed in the mutant yeast cell, at least during use in a fermentation process.

[155] To increase the likelihood that the Rubisco protein is expressed at sufficient levels and in active form in the transformed (recombinant) host cells of the invention, the nucleic acid sequence encoding the Rubisco protein and/or the nucleic acid sequence encoding other proteins as described herein (see below), are preferably adapted to optimise their codon usage to that of the host cell in question. The adaptiveness of a nucleic acid sequence encoding an enzyme to the codon usage of a host cell may be expressed as codon adaptation index (CAI). The codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes in a particular host cell or organism. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1 , with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li , 1987, Nucleic Acids Research 15: 1281- 1295; also see: Jansen et al., 2003, Nucleic Acids Res. 31_(8):2242-51). An adapted nucleic acid sequence preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9. Preferably, the sequences have been codon optimized for expression in the fungal host cell in question, such as for example Saccharomyces cerevisiae cells.

[156] The nucleic acid sequence encoding the Rubisco protein and/or the nucleic acid sequence encoding other proteins as described herein (see below) may be present in one or more copies. Preferably the nucleic acid sequence encoding the Rubisco protein and/or the nucleic acid sequence encoding other proteins as described herein (see below) is present in multiple copies, more preferably in the range from equal to or more than 2 to equal to or less than 20 copies, most preferably in the range from equal to or more than 3 to equal to or less than 15 copies.

[157] Preferably the functionally expressed Rubisco protein has an activity, defined by the rate of ribulose-1 ,5-bisphosphate- dependent ¹⁴C-bicarbonate incorporation by cell extracts of at least 1 nmol.min- ¹.(mg protein)^-1 , in particular an activity of at least 2 nmol. min^-1.(mg protein)^-1 , more in particular an activity of at least 4 nmol. min^-1. (mg protein)^-1. The upper limit for the activity is not critical. In practice, the activity may be about 200 nmol. min^-1. (mg protein)^-1 or less, in particular 25 nmol. min^-1. (mg protein)^-1 , more in particular 15 nmol. min^-1. (mg protein)^-1 or less, e.g. about 10 nmol. min^-1. (mg protein)^-1 or less. The conditions for an assay for determining this Rubisco activity are as found in the Examples (e.g. Example 4) of WO2014/129898, incorporated herein by reference.

[158] The protein having phosphoribulokinase (PRK) activity is herein also referred to as “PRK enzyme”, “PRK protein” or simply “PRK”. Preferences for the PRK protein and the nucleic sequences encoding for such are as described in WO2014/129898, incorporated herein by reference.

[159] A functionally expressed phosphoribulokinase (PRK, (EC 2.7.1.19)) according to the invention is capable of catalyzing the chemical reaction (I):

ATP + D-ribulose 5-phosphate ^ADP + D-ribulose 1,5-bisphosphate (I)

Thus, the two substrates of this enzyme are ATP and D-ribulose 5-phosphate; its two products are ADP and D-ribulose 1 ,5-bisphosphate.

[160] The PRK protein belongs to the family of transferases, specifically those transferring phosphorus- containing groups (phosphotransferases) with an alcohol group as acceptor. The systematic name of this enzyme class is ATP:D-ribulose- 5-phosphate 1 -phosphotransferase. Other names in common use include phosphopentokinase, ribulose-5-phosphate kinase, phosphopentokinase, phosphoribulokinase (phosphorylating), 5-phosphoribulose kinase, ribulose phosphate kinase, PKK, PRuK, and PRK. This enzyme participates in carbon fixation. The PRK can be from a prokaryote or a eukaryote. Good results have been achieved with a PRK originating from a eukaryote. Preferably the PRK protein originates from a plant selected from Caryophyllales , in particular from Amaranthaceae, more in particular from Spinacia.

[161] A preferred PRK protein is the PRK protein from Spinacia. SEQ ID NO: 3 shows the amino acid sequence of such PRK protein from Spinacia.

[162] Other suitably PRK proteins include functional homologues of the PRK protein from Spinacia, preferably functional homologues comprising an amino acid sequence sequence having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO:3. Suitable natural PRK polypeptides are given in Table 5. [163] Examples of suitable PRK polypeptides and their origin are given in Table 2 of WO2014/129898, incorporated herein by reference, and in Table 5 below, with reference to the sequence identity with the amino acid sequence of SEQ ID NO:3.

Table 5: Natural PRK polypeptides suitable for expression with identity to PRK from Spinacia

[164] The nucleic acid sequences encoding for the PRK protein may be under the control of a promoter (the "PRK promoter") that enables higher expression under anaerobic conditions than under aerobic conditions. Examples of such promoters are described in WO2017/216136A1 and WO2018/228836, both herein incorporated by reference. More preferably such promoter has a PRK expression ratio anaerobic/aerobic of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more or 50 or more. Further preferences are as described in WO2018/228836, incorporated herein by reference.

[165] As indicated above, the nucleic acid sequence encoding the PRK protein may be present in one or more copies. Preferably the nucleic acid sequence encoding the PRK protein is present in multiple copies, more preferably in the range from equal to or more than 2 to equal to or less than 20 copies, most preferably in the range from equal to or more than 3 to equal to or less than 15 copies.

[166] Optionally, the mutant yeast cell further comprises one or more nucleic acid sequences encoding for one or more molecular chaperones for the protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity.

[167] Suitably such molecular chaperones are also referred herein as “chaperone protein”, “chaperonin” or simply “chaperone”. Preferences for the chaperones and the nucleic sequences encoding for such are as described in WO2014/129898, incorporated herein by reference.

[168] Preferably the mutant yeast cell comprises one or more heterologous nucleic acid sequences encoding for one or more molecular chaperones for the protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity.

[169] Chaperonins are proteins that provide favorable conditions for the correct folding of other proteins, thus preventing aggregation. Newly made proteins usually must fold from a linear chain of amino acids into a three-dimensional form. Chaperonins belong to a large class of molecules that assist protein folding, called molecular chaperones. The energy to fold proteins is supplied by adenosine triphosphate (ATP). A review article about chaperones that is useful herein is written by Yebenes (2001); “Chaperonins: two rings for folding”; Hugo Yebenes et al. T rends in Biochemical Sciences, August 2011 , Vol. 36, No. 8.

[170] The chaperone or chaperones may be prokaryotic chaperones or eukaryotic chaperones. In addition, the chaperones may be homologous or heterologous. For example, the mutant yeast cell may comprises one or more nucleic acid sequence encoding one or more homologous or heterologous, prokaryotic or eukaryotic, molecular chaperones, which - when expressed - are capable of functionally interacting with an enzyme in the mutant yeast cell, in particular with at least one of Rubisco and PRK.

[171] Suitably the chaperone or chaperones are derived from a bacterium, more preferably from Escherichia, in particular E. coli. Preferred chaperones are GroEL and GroEs from E. coli. Other preferred chaperones are chaperones from Saccharomyces, in particular Saccharomyces cerevisiae Hsp10 and Hsp60.

[172] If the chaperones are naturally expressed in an organelle such as a mitochondrion (examples are Hsp60 and Hsp10 of Saccharomyces cerevisiae) relocation to the cytosol can be achieved e.g. by modifying the native signal sequence of the chaperonins. In eukaryotes the proteins Hsp60 and Hsp10 are structurally and functionally nearly identical to GroEL and GroES, respectively. Thus, it is contemplated that Hsp60 and Hsp10 from any mutant yeast cell may serve as a chaperone for the Rubisco. This is described for example by Zeilstra-Ryalls et al., "The universally conserved GroE (Hsp60) chaperonins", published in Annu Rev Microbiol. (1991) vol.45, pages 301-25; and Horwich et al., "Two Families of Chaperonin: Physiology and Mechanism", published in Annu. Rev. Cell. Dev. Biol. Vol. 23, pages 115-45, both herewith incorporated by reference.

[173] Good results have been achieved with a mutant yeast cell comprising both the heterologous chaperones GroEL and GroES. As an alternative to GroES a functional homologue of GroES may be present, in particular a functional homologue comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of GroES. SEQ ID NO:7 provides an amino acid sequence of GroES. Examples of suitable natural chaperones polypeptide homologous to GroES are given in Table 6.

Table 6: Natural chaperones homologous to GroES polypeptides suitable for expression

[174] As an alternative to GroEL a functional homologue of GroEL may be present, in particular a functional homologue comprising an amino acid sequence having at least 40 %, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 98% or at least 99% sequence identity with the amino acid sequence of GroEL.

SEQ ID NO:5 provides an amino acid sequence of GroEL. Suitable natural chaperones polypeptides homologous to GroEL are given in Table 7.

Table 7: Natural chaperones homologous to GroEL polypeptides suitable for expression

[175] Preferably a 10 kDa chaperone from Table 6 is combined with a matching 60kDa chaperone from Table 7 of the same organism genus or species for expression in the mutant yeast cell.

[176] For instance: >gi|189189366|ref|XP_001931022.11:71-168 10 kDa chaperonin [Pyrenophora tritici- repentis] expressed together with matching >gi|189190432|ref|XP_001931555.11 heat shock protein 60, mitochondrial precursor [Pyrenophora tritici-repentis Pt-1C-BFP], All other combinations from Table 6 and 7 similarly made with same organism source are also available to the skilled person for expression. Furthermore, one may combine a chaperone from Table 6 from one organism with a chaperone from Table 7 from another organism, or one may combine GroES with a chaperone from Table 7, or one may combine GroEL with a chaperone from Table 6.

[177] As illustrated by the above, this invention thus also provides a recombinant yeast cell, comprising:

(I) one or more first genetic modifications for, preferably constitutive, expression, preferably under anaerobic conditions, of one or more NAD+ dependent proteins that function in a first metabolic pathway to convert a sugar alcohol into a fermentation product; and (ii) one or more second genetic modifications for decreasing, as compared to the wild-type yeast cell, the concentration of NADH in the cell. Deletion or disruption of glycerol 3-phosphate phosphohydrolase and/or glycerol 3-phosphate dehydrogenase

[178] Suitably, the yeast cell may further comprise a deletion or disruption of one or more endogenous nucleotide sequence encoding a glycerol 3-phosphate phosphohydrolase gene and/or encoding a glycerol 3-phosphate dehydrogenase gene.

[179] Preferably enzymatic activity needed for the NADH-dependent glycerol synthesis in the yeast cell is reduced or deleted. The reduction or deletion of the enzymatic activity of glycerol 3-phosphate phosphohydrolase and/or glycerol 3-phosphate dehydrogenase can be achieved by modifying one or more genes encoding a NAD-dependent glycerol 3-phosphate dehydrogenase (GPD) and/or one or more genes encoding a glycerol phosphate phosphatase (GPP), such that the enzyme is expressed considerably less than in the wild-type or such that the gene encodes a polypeptide with reduced activity. Such modifications can be carried out using commonly known biotechnological techniques, and may in particular include one or more knock-out mutations or site-directed mutagenesis of promoter regions or coding regions of the structural genes encoding GPD and/or GPP. Alternatively, yeast strains that are defective in glycerol production may be obtained by random mutagenesis followed by selection of strains with reduced or absent activity of GPD and/or GPP. S. cerevisiae GPD1, GPD2, GPP1 and GPP2 genes are shown in WO2011010923, and are disclosed in SEQ ID NO: 24-27 of that application.

[180] Therefore the mutant yeast cell preferably comprises one or more genetic modifications for decreasing or inhibiting the activity of glycerol-3-phosphate dehydrogenase (GPD) and/or glycerol-3- phosphate phosphatase (GPP).

[181] Preferably at least one gene encoding a GPD and/or at least one gene encoding a GPP is entirely deleted, or at least a part of the gene is deleted that encodes a part of the enzyme that is essential for its activity. In particular, good results have been achieved with a S. cerevisiae cell, wherein the open reading frames of the GPD1 gene and of the GPD2 gene have been inactivated. Inactivation of a structural gene (target gene) can be accomplished by a person skilled in the art by synthetically synthesizing or otherwise constructing a DNA fragment consisting of a selectable marker gene flanked by DNA sequences that are identical to sequences that flank the region of the host cell's genome that is to be deleted. In particular, good results have been obtained with the inactivation of the GPD1 and GPD2 genes in Saccharomyces cerevisiae by integration of the marker genes kanMX and hphMX4. Subsequently this DNA fragment is transformed into a host cell. Transformed cells that express the dominant marker gene are checked for correct replacement of the region that was designed to be deleted, for example by a diagnostic polymerase chain reaction or Southern hybridization.

[182] Thus, in the yeast cells of the invention, glycerol 3-phosphate phosphohydrolase activity in the cell and/or glycerol 3-phosphate dehydrogenase activity in the cell is advantageously reduced. PPP-qenes

[183] The mutant yeast cell may further advantageously comprise one or more genetic modifications that increases the flux of the pentose phosphate pathway. In particular, the genetic modification(s) may lead to an increased flux through the non-oxidative part of the pentose phosphate pathway. A genetic modification that causes an increased flux of the non- oxidative part of the pentose phosphate pathway is herein understood to mean a modification that increases the flux by at least a factor of about 1 .1 , about 1 .2, about 1 .5, about 2, about 5, about 10 or about 20 as compared to the flux in a strain which is genetically identical except for the genetic modification causing the increased flux. The flux of the non-oxidative part of the pentose phosphate pathway may be measured by growing the modified host on xylose as sole carbon source, determining the specific xylose consumption rate and subtracting the specific xylitol production rate from the specific xylose consumption rate, if any xylitol is produced. However, the flux of the non-oxidative part of the pentose phosphate pathway is proportional with the growth rate on xylose as sole carbon source, preferably with the anaerobic growth rate on xylose as sole carbon source. There is a linear relation between the growth rate on xylose as sole carbon source (p_max) and the flux of the non-oxidative part of the pentose phosphate pathway. The specific xylose consumption rate (Q_s) is equal to the growth rate (p) divided by the yield of biomass on sugar (Yxs) because the yield of biomass on sugar is constant (under a given set of conditions: anaerobic, growth medium, pH, genetic background of the strain, etc.; i.e. Q_s = p/ Yxs). Therefore the increased flux of the non-oxidative part of the pentose phosphate pathway may be deduced from the increase in maximum growth rate under these conditions unless transport (uptake is limiting).

[184] One or more genetic modifications that increase the flux of the pentose phosphate pathway may be introduced in the host cell in various ways. These including e.g. achieving higher steady state activity levels of xylulose kinase and/or one or more of the enzymes of the non-oxidative part pentose phosphate pathway and/or a reduced steady state level of unspecific aldose reductase activity. These changes in steady state activity levels may be effected by selection of mutants (spontaneous or induced by chemicals or radiation) and/or by recombinant DNA technology e.g. by overexpression or inactivation, respectively, of genes encoding the enzymes or factors regulating these genes.

[185] In a preferred host cell, the genetic modification comprises overexpression of at least one enzyme of the (non-oxidative part) pentose phosphate pathway. Preferably the enzyme is selected from the group consisting of the enzymes encoding for ribulose- 5- phosphate isomerase, ribulose- 5-phosphate epimerase, transketolase and transaldolase. Various combinations of enzymes of the (non-oxidative part) pentose phosphate pathway may be overexpressed. E.g. the enzymes that are overexpressed may be at least the enzymes ribulose- 5-phosphate isomerase and ribulose-5-phosphate epimerase; or at least the enzymes ribulose- 5-phosphate isomerase and transketolase; or at least the enzymes ribulose-5-phosphate isomerase and transaldolase; or at least the enzymes ribulose-5-phosphate epimerase and transketolase; or at least the enzymes ribulose- 5- phosphate epimerase and transaldolase; or at least the enzymes transketolase and transaldolase; or at least the enzymes ribulose- 5-phosphate epimerase, transketolase and transaldolase; or at least the enzymes ribulose- 5-phosphate isomerase, transketolase and transaldolase; or at least the enzymes ribulose- 5-phosphate isomerase, ribulose- 5-phosphate epimerase, and transaldolase; or at least the enzymes ribulose- 5-phosphate isomerase, ribulose-5-phosphate epimerase, and transketolase. In one embodiment of the invention each of the enzymes ribulose- 5- phosphate isomerase, ribulose- 5-phosphate epimerase, transketolase and transaldolase are overexpressed in the host cell. More preferred is a host cell in which the genetic modification comprises at least overexpression of both the enzymes transketolase and transaldolase as such a host cell is already capable of anaerobic growth on xylose. In fact, under some conditions host cells overexpressing only the transketolase and the transaldolase already have the same anaerobic growth rate on xylose as do host cells that overexpress all four of the enzymes, i.e. the ribulose-5-phosphate isomerase, ribulose- 5- phosphate epimerase, transketolase and transaldolase. Moreover, host cells overexpressing both of the enzymes ribulose- 5-phosphate isomerase and ribulose-5- phosphate epimerase are preferred over host cells overexpressing only the isomerase or only the epimerase as overexpression of only one of these enzymes may produce metabolic imbalances.

[186] The enzyme "ribulose 5-phosphate epimerase" (EC 5.1.3.1) is herein defined as an enzyme that catalyses the epimerisation of D-xylulose 5-phosphate into D-ribulose 5- phosphate and vice versa. The enzyme is also known as phosphoribulose epimerase; erythrose-4-phosphate isomerase; phosphoketopentose 3-epimerase; xylulose phosphate 3-epimerase; phosphoketopentose epimerase; ribulose 5-phosphate 3- epimerase; D-ribulose phosphate-3-epimerase; D-ribulose 5-phosphate epimerase; D- ribulose-5-P 3-epimerase; D-xylulose-5-phosphate 3-epimerase; pentose- 5-phosphate 3- epimerase; or D-ribulose-5-phosphate 3-epimerase. A ribulose 5-phosphate epimerase may be further defined by its amino acid sequence. Likewise a ribulose 5-phosphate epimerase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a ribulose 5-phosphate epimerase. The nucleotide sequence encoding for ribulose 5-phosphate epimerase is herein designated RPE1.

[187] The enzyme "ribulose 5-phosphate isomerase" (EC 5.3.1.6) is herein defined as an enzyme that catalyses direct isomerisation of D-ribose 5-phosphate into D-ribulose 5-phosphate and vice versa. The enzyme is also known as phosphopentosisomerase; phosphoriboisomerase; ribose phosphate isomerase; 5-phosphoribose isomerase; D- ribose 5-phosphate isomerase; D-ribose- 5-phosphate ketol-isomerase; or D-ribose-5- phosphate aldose-ketose-isomerase. A ribulose 5-phosphate isomerase may be further defined by its amino acid sequence. Likewise a ribulose 5-phosphate isomerase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a ribulose 5-phosphate isomerase. The nucleotide sequence encoding for ribulose 5- phosphate isomerase is herein designated RKI1.

[188] The enzyme "transketolase" (EC 2.2.1.1) is herein defined as an enzyme that catalyses the reaction: D-ribose 5-phosphate + D-xylulose 5-phosphate <-> sedoheptulose 7-phosphate + D- glyceraldehyde 3-phosphate and vice versa. The enzyme is also known as glycolaldehydetransferase or sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glycolaldehydetransferase. A transketolase may be further defined by its amino acid. Likewise a transketolase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a transketolase. The nucleotide sequence encoding for transketolase is herein designated TKL1.

[189] The enzyme "transaldolase" (EC 2.2.1.2) is herein defined as an enzyme that catalyses the reaction: sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate <-> D-erythrose 4-phosphate + D- fructose 6-phosphate and vice versa. The enzyme is also known as dihydroxyacetonetransferase; dihydroxyacetone synthase; formaldehyde transketolase; or sedoheptulose-7- phosphate :D- glyceraldehyde-3 -phosphate glyceronetransferase. A transaldolase may be further defined by its amino acid sequence. Likewise a transaldolase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a transaldolase. The nucleotide sequence encoding for transketolase from is herein designated TAL1.

Further genetic modifications:

[190] In addition to the above described genetic modifications, the deletion of the aldose reductase (GRE3) gene; and/or overexpression of GAL2 and/or deletion of GAL80 may be advantageous. Such a deletion and/or overexpression can suitably be carried out as described in WO2011131667A1 , and is incorporated herein by reference.

Co-factors

[191 ] Preferably the mutant yeast cell further comprises suitable co-factors to enhance the activity of the above mentioned proteins. For example, the recombinant yeast cell may comprise zinc, zinc ions or zinc salts and/or one or more pathways to include such in the cell.

Fermentation process

[192] The invention further provides a process for the production of a fermentation product, the process comprising fermenting of a feed with a recombinant yeast cell as described above, wherein the feed comprises a source of NAD+ cofactor.

Preferably the source of NAD+ cofactor is a sugar alcohol. The process is preferably a process for the production of a fermentation product comprising fermenting of a feed with a mutant yeast cell as described above, wherein the feed comprises a sugar alcohol. More preferably the process is a process for the production of ethanol, the process comprising fermenting of a carbon source composition with a mutant yeast cell as described above, wherein the carbon source composition comprises at least a sugar alcohol. Preferably the process is carried out under oxygen-limited conditions or anaerobic conditions. Preferably the sugar alcohol is mannitol or sorbitol. [193] In another preferred embodiment the sugar alcohol is derived from a sugar by a process comprising a preceding or simultaneous hydrogenation of a sugar-containing feed by an inorganic, organic or biological catalyst.

[194] More preferably the feed comprises a sugar alcohol, such as mannitol and/or sorbitol, in combination with a sugar, such as glucose, arabinose, xylose and/or galactose. Therefore, preferably the process is a process for the production of ethanol, the process comprising fermenting of a carbon source composition with a mutant yeast cell as described above, wherein the carbon source composition comprises a sugar and a sugar alcohol and wherein both sugar and sugar alcohol are converted into ethanol.

[195] If the feed comprises a sugar alcohol and a sugar, the feed preferably comprises such sugar alcohol and sugar in a weight ratio of sugar alcohol to sugar in the range of 1000:1 to 1 :1000, more preferably 100:1 to 1 :100. Preferably the sugar alcohol is present in a higher weight percentage (wt %) than the sugar.

[196] Preferably the percentage sugar, based on the total weight of sugar and sugar alcohol in the feed, lies in the range from more than 0.001 wt % to less than or equal than 50 wt %, more preferably in the range from more than 0.01 wt % to less than or equal to than 49 wt%, still more preferably in the range from more than 0.1 wt% to less than or equal to 45 wt% and most preferably in the range from more than 1 wt% to less than or equal to 40 wt%.

[197] Conveniently the percentage sugar alcohol, based on the total weight of sugar and sugar alcohol in the feed, can be equal to or more than 0.1 wt%, more conveniently equal to or more than 1 wt%, still more conveniently equal to or more than 5 wt%, even more conveniently equal to or more than 10 wt% and most conveniently equal to or more than 20 wt%. Preferably the percentage sugar alcohol, based on the total weight of sugar and sugar alcohol together, is equal to or more than 50 wt%, more preferably equal to or more than 70 wt%, even more preferably equal to or more than 90 wt% and still more preferably equal to or more than 95 wt%. There is no maximum amount of sugar alcohol. However, for practical purposes, the percentage sugar alcohol, based on the total weight of sugar and sugar alcohol in the feed, can be equal to or less than 99.99 wt%, equal to or less than 99.9 wt %, equal to or less than 99 wt%, equal to or less than 95 wt% or equal to or less than 90 wt%. Most preferably the feed comprises 100% sugar alcohols, such as for example sorbitol and/or mannitol. That is, preferably the process is carried out in the absence of glucose, arabinose, xylose, galactose and/or any other sugars.

[198] In addition to the source of NAD+ cofactor, the feed suitably comprises one or more (additional) fermentable carbon sources. The fermentable carbon source preferably comprises or is consisting of one or more fermentable carbohydrates. More preferably, the fermentable carbon source comprises one or more mono-saccharides, disaccharides and/or polysaccharides. For example, the fermentable carbon source may comprise one or more carbohydrates selected from the group consisting of glucose, fructose, sucrose, maltose, xylose, arabinose, galactose, mannose and trehalose. The fermentable carbon source, preferably comprising or consisting of one or more carbohydrates, may suitably be obtained from starch, celulose, hemicellulose lignocellulose, and/or pectin. Suitably the fermentable carbon source may be in the form of a, preferably aqueous, slurry, suspension, or a liquid. [199] The concentration of fermentable carbohydrate, such as for example glucose, during fermentation is preferably equal to or more than 80g/L. That is, the initial concentration of glucose at the start of the fermentation, is preferably equal to or more than 80 g/L, more preferably equal to or more than 90 g/L, even more preferably equal to or more than 100 g/L, still more preferably equal to or more than 110 g/L, yet even more preferably equal to or more than 120 g/L, equal to or more than 130 g/L, equal to or more than 140 g/L, equal to or more than 150 g/L, equal to or more than 160 g/L, equal to or more than 170 g/L, or equal to or more than 180 g/L. The start of the fermentation may be the moment when the fermentable fermentable carbohydrate is brought into contact with the recombinant cell of the invention.

[200] The fermentable carbon source may be prepared by contacting starch, lignocellulose, and/or pectin with an enzyme composition, wherein one or more mono-saccharides, disaccharides and/or polysaccharides are produced, and wherein the produced mono-saccharides, disaccharides and/or polysaccharides are subsequenty fermented to give a fermentation product.

[201] In one embodiment the fermentable carbohydrate is, or is comprised by a biomass hydrolysate, such as a corn stover or corn fiber hydrolysate. Such biomass hydrolysate may in its turn comprise, or be derived from corn stover and/or corn fiber.

[202] By a "hydrolysate" is herein understood a polysaccharide-comprising material (such as corn stover, corn starch, corn fiber, or lignocellulosic material, which polysaccharides have been depolymerized through the addition of water to form mono and oligosaccharide sugars. Hydrolysates may be produced by enzymatic or acid hydrolysis of the polysaccharide-containing material.

[203] A biomass hydrolysate may be a lignocellulosic biomass hydrolysate. Lignocellulose herein includes hemicellulose and hemicellulose parts of biomass. Also lignocellulose includes lignocellulosic fractions of biomass. Suitable lignocellulosic materials may be found in the following list: orchard primings, chaparral, mill waste, urban wood waste, municipal waste, logging waste, forest thinnings, short-rotation woody crops, industrial waste, wheat straw, oat straw, rice straw, barley straw, rye straw, flax straw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane, corn stover, corn stalks, corn cobs, corn husks, switch grass, miscanthus, sweet sorghum, canola stems, soybean stems, prairie grass, gamagrass, foxtail; sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, algae (including macroalgae and microalgae), trees, softwood, hardwood, poplar, pine, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn hobs, corn kernel, fiber from kernels, products and by-products from wet or dry milling of grains, municipal solid waste, waste paper, yard waste, herbaceous material, agricultural residues, forestry residues, municipal solid waste, waste paper, pulp, paper mill residues, branches, bushes, canes, corn, corn husks, an energy crop, forest, a fruit, a flower, a grain, a grass, a herbaceous crop, a leaf, bark, a needle, a log, a root, a sapling, a shrub, switch grass, a tree, a vegetable, fruit peel, a vine, sugar beet pulp, wheat midlings, oat hulls, hard or soft wood, organic waste material generated from an agricultural process, forestry wood waste, or a combination of any two or more thereof. Algae, such as macroalgae and microalgae have the advantage that they may comprise considerable amounts of sugar alcohols such as sorbitol and/or mannitol. Lignocellulose, which may be considered as a potential renewable feedstock, generally comprises the polysaccharides cellulose (glucans) and hemicelluloses (xylans, heteroxylans and xyloglucans). In addition, some hemicellulose may be present as glucomannans, for example in wood-derived feedstocks. The enzymatic hydrolysis of these polysaccharides to soluble sugars, including both monomers and multimers, for example glucose, cellobiose, xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose, galacturonic acid, glucuronic acid and other hexoses and pentoses occurs under the action of different enzymes acting in concert. In addition, pectins and other pectic substances such as arabinans may make up considerably proportion of the dry mass of typically cell walls from non-woody plant tissues (about a quarter to half of dry mass may be pectins). Lignocellulosic material may be pretreated. The pretreatment may comprise exposing the lignocellulosic material to an acid, a base, a solvent, heat, a peroxide, ozone, mechanical shredding, grinding, milling or rapid depressurization, or a combination of any two or more thereof. This chemical pretreatment is often combined with heat-pretreatment, e.g. between 150-220°C for 1 to 30 minutes.

[204] The fermentation process can be carried out in a continuous mode, a batch mode or in a semibatch or fed-batch mode. Preferably the fermentation process is carried out in a batch mode.

Alternative process

[205] As explained above, it is advantageous to have a process for the production of ethanol, the process comprising fermenting of a carbon source composition with a mutant yeast cell as described herein, wherein the carbon source composition comprises at least a sugar alcohol, and more preferably the carbon source composition comprises at least a sugar, such as glucose, and a sugar alcohol, such as sorbitol or mannitol. Preferably the yeast overexpresses a transporter, such as HXT15, and a sorbitol dehydrogenase, such as SOR2 or a mannitol dehydrogenase, such as MAN2. The sugar, such as glucose, and the sugar alcohol, such as sorbitol or mannitol, can then conveniently be both converted into ethanol. Preferably such glucose is provided in excess conditions. Preferably the process is carried out under oxygen-limited conditions or anaerobic conditions. In addition, preferably the process is carried out in a batch mode.

[206] In addition, an alternative process is provided herein. Without wishing to be bound by any kind of theory it is believed that under specific conditions, such as for example under carbon limited conditions in a fed-batch process, a carbon source composition comprising at least a sugar, such as glucose, and a sugar alcohol, such as sorbitol or mannitol, can even be converted without the overexpression of a transporter, such as HXT15, and a sorbitol dehydrogenase, such as SOR2, or a mannitol dehydrogenase, such as MAN2.

[207] Preferably such as process is carried out in a reactor wherein fresh medium is continuously added, whilst yeast and left-over nutrients and products are continuously removed.

[208] WO2014/129898 describes a recombinant yeast cell, in particular a transgenic yeast cell, functionally expressing one or more recombinant, in particular heterologous, nucleic acid sequences encoding ribulose-l,5-biphosphate carboxylase oxygenase (Rubisco) and phosphoribulokinase (PRK). WO2014/129898 further describes a method for preparing an alcohol, organic acid or amino acid, comprising fermenting a carbon source, in particular a carbohydrate with such a yeast cell, thereby forming the alcohol, organic acid or amino acid, wherein the yeast cell is present in a reaction medium.

[209] WO2014/129898 further describes anaerobic chemostat cultivation with 12.5 g/l glucose and 12.5 g/l galactose as the carbon source. In passing, WO2014/129898 describes the optional presence of a heterologous nucleic acid sequence encoding a xylitol dehydrogenase from a (naturally) autotrophic organism, but WO2014/129898 does not describe any actual conversion of any carbon source composition comprising at least a sugar, such as glucose, and a sugar alcohol, such as sorbitol or mannitol, under carbon limited circumstances and does not recognize such a possibility.

[210] The invention therefore also provides a process for the production of ethanol, the process comprising fermenting of a carbon source composition with a mutant yeast cell, wherein the carbon source composition comprises at least a sugar, such as glucose, and a sugar alcohol, such as sorbitol or mannitol, wherein the process is carried out in a fed-batch mode or otherwise under carbon-limited conditions. The mutant yeast cell preferably comprises one or more, preferably recombinant, more preferably heterologous, nucleic acid sequences encoding ribulose-l,5-biphosphate carboxylase oxygenase (Rubisco) and phosphoribulokinase (PRK). In addition, the mutant yeast cell may conveniently comprise a sugar alcohol dehydrogenase, such as a sorbitol dehydrogenase, such as SOR2, or a mannitol dehydrogenase, such as MAN2, where such alcohol dehydrogenase may or may not be overexpressed. In addition the mutant yeast cell may comprise a transporter, such as HXT15, which may or may not be overexpressed.

[211] The process is preferably carried out under carbon-limited conditions. That is, the process is preferably carried out under circumstances where the feed of sugar, preferably the feed of glucose, is preferably limited to equal to or less than 60 grams per liter, more preferably equal to or less than 50 grams per liter, still more preferably equal to or less than 40 grams per liter, even more preferably equal to or less than 30 grams per liter and even still more preferably equal to or less than 20 grams per liter. The feed of sugar alcohol, preferably sorbitol, mannitol or a combination thereof, is preferably limited to equal to or less than 60 grams per liter, more preferably equal to or less than 50 grams per liter, still more preferably equal to or less than 40 grams per liter, even more preferably equal to or less than 30 grams per liter, even still more preferably equal to or less than 20 grams per liter and most preferably equal to or less than 10 grams per liter.

[21 ] Similar to the earlier described processes, the alternative process is preferably carried out with a a carbon source composition comprising sugar alcohol and sugar in a weight ratio of sugar alcohol to sugar in the range of 1000:1 to 1 :1000, more preferably 100:1 to 1 :100.

[213] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[214] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way EXAMPLES

Construction of Strains

The CEN.PK lineage of S. cerevisiae laboratory strains (as described in the article by Entian K-D, Kotter P , titled "25 yeast genetic strain and plasmid collections", Stansfield I, Stark MJR (eds.), published in Methods in Microbiology, vol. 36: Academic Press, (2007), pages 629-66) was used to construct and evolve all yeast strains used in this example. An overview of these strains is provided in Table 8. The specific construction of the strains is described below. All yeast genetic modifications were performed using CRISPR/Cas9-based genome editing (as described by Mans R., van Rossum H.M., Wijsman M., Backx A., Kuijpers N.G.A., van den Broek M., Daran-Lapujade P., Pronk J.T., van Maris A.J.A. and Daran J-M. G., in their article titled "CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae", published in FEMS Yeast Research, vol. 15, (2015), fov004). All yeast strains were grown in 2% w/v glucose synthetic medium (3.0 g L^-1 KH2PO4, 0.5 g L^-1 MgSO4'7H2O, 5.0 g L^-1 (NH4)2SO4, 1.0 ml L^-1 trace elements, 1.0 mL L^-1 vitamin solution (as described in the article by Verduyn, C., Postma, E., Scheffers, W. A., & Van Dijken, J. P. , titled “Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation” published in Yeast, volume 8(7), (1992), pages 501-517) until they reached end exponential phase, then sterile glycerol was added up to ca. 30% v/vto prepare frozen stocks.

[215] All cultures were stored in aliquots of 1 ml at -80°C.

[216] E. coliXL-1 blue stock cultures were grown in LB medium (5 g L-1 Bacto yeast extract, 10 g L-1 Bacto tryptone, 5 g L-1 NaCI), supplemented with 100 pg mL-1 ampicillin. Frozen stocks were prepared by addition of glycerol (30% v/v final concentration).

[217] All cultures were stored in aliquots of 1 ml at -80° C.

Construction of plasmids and cassettes

[218] PCR amplification for construction of plasmid fragments and yeast integration cassettes was performed with Phusion High Fidelity DNA Polymerase (commercially obtainable from Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s guidelines (Thermo ScientificPhusion High-Fidelity DNA Polymerase Product Information Sheet, 2018), using 30 cycles.

[219] Primers were ordered DST-purified, unless indicated otherwise.

[220] The fragment size of the obtained DNA fragments was determined with the help of GeneRuler DNA Ladder Mix (commercially available from Thermo Fisher Scientific Inc, Waltman, MA, USA), according to the manufacturer’s guidelines.

[221] The DNA fragments comprising 500 basepairs or more, were separated by gel electrophoresis on 1% (w/v) TopVision agarose gels (commercially obtainable from Thermo Fisher Scientific, Waltham, MA, USA) with help of Serva DNA stain G (commercially available from SERVA Electrophoresis GmbH, Heidelberg, Germany) staining, in line with manufacturer’s guidelines, in a TAE buffer (commercially obtainable from Thermo Fisher Scientific, Waltham, MA, USA), comprising Tris-acetate buffer (pH 8.0) with

1 mM EDTA, at 100 Volt for 30 minutes.

[222] The DNA fragments comprising less than 500 basepairs were separated by gel electrophoresis on

2 % (w/v) Topvision agarose gels with help of Serva DNA stain G (commercially available from SERVA Electrophoresis GmbH, Heidelberg, Germany) staining, in line with manufacturer’s guidelines, in a TBE buffer (commercially obtainable from Thermo Fisher Scientific, Waltham, MA, USA), comprising Tris-borate buffer (pH 8.3) with 1 mM EDTA, at 100 Volt for 30 minutes.

[223] The amplified DNA fragments were purified using a GeneJET PCR purification kit (GeneJET PCR Purification Kit (commercially obtainable from ThermoFisher, Waltmann, USA) according to the manufacturer’s guidelines. All purified DNA fragments were stored at -20°C.

[224] An overview of the plasmids used to construct the strains is provided in Table 9. An overview of the primers used is provided in Table 10.

Table 8: S. cerevisiae strains used in these examples

Table 9: Plasmids used in these examples

Table 10 : Primers used in these examples

Example 1 : Construction of plasmid PUDE885 comprising a pACT1-tCPS1 empty vector

[225] For the construction of plasmid pUDE885, first intermediate plasmid pUD968 was created.

[226] Plasmid pUD968 was created from plasmid p426-TEF (commercially obtainable from Addgene). Plasmid p426-TEF was amplified using desalted primer pairs 15514/10901 and 15515/7388 to obtain two DNA fragments (amplification carried out according Phusion High Fidelity DNA Polymerase manufacturer’s guidelines as indicated above) as illustrated in Figure 1. The first DNA fragment comprised a nucleotide sequence for the URA3 marker (a gene derived from chromosome V in Saccharomyces cerevisiae). The second DNA fragment comprised the “2 mu ori” nucleotide sequence (a 2micron origin of replication for propagation in S. cerevisiae) and the “AmpR” nucleotide sequence (i.e. AmpR - Beta lactamase gene encoding ampicillin resistance pBBR322 ori - pBR322 origin of replication for propagation in E. coli). Correct fragment sizes were verified by gel electrophoresis on a TopVision Agrose gel 1% according to manufacturer’s guidelines as indicated above. After verification the DNA-fragments were purified using the GeneJET PCR purification kit according to manufacturer’s guidelines as indicated above.

[227] The purified fragments were digested by restriction endonucleases Kpnl and Pfol (both commercially obtainable from Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s guidelines and ligated using T4 DNA ligase (commercially obtainable from Thermo Fisher Scientific, Waltham, MA, USA) to create plasmid pUD968, according to manufacturer’s guidelines.

[228] To propagate plasmid pUD968, plasmid pUD968 was first transformed into E.coli XL1-Blue cells (commercially obtainable from Agilent, Santa Clara, USA) and plated on LB-ampicillin and incubated overnight at 37°C. A single colony was used to inoculate LB-ampicillin liquid medium and incubated overnight at 37°C. The GeneJET Plasmid Miniprep Kit (commercially available from Thermo Fisher Scientific Inc. , Waltman, MA, USA) was used to isolate the plasmid DNA (of the plasmid pUD968) from the E.coli according to manufacturer’s instructions. Correct assembly of the plasmid can be verified either by diagnostic PCR or restriction assay. The isolated plasmid pUD986 is stored at -20°C.

This plasmid pUD968 was linearized with restriction endonuclease Kpnl (commercially obtainable by Thermo Scientific as indicated above). Next, the ACT1 promoter (pACT1) and CPS1 terminator (tCPS1) sequences were amplified using primers 15548/15549 and 15550/15551 respectively, using CEN.PK.113- 7D as template. Genomic DNA of pACT1 and tCPS1 was isolated from CEN.PK.113.7D using the method as described in the protocol by Looke, et al., titled "Extraction of genomic DNA from yeasts for PCR-based applications", published in Biotechniques, vol. 50, ., (2011) pages 325-328, herewith incorporated by reference, (further referred to as Looke et al. (2011))

[229] After linearization of plasmid pUD968, the pACT1 and tCPS1 DNA sequences were assembled onto the backbone of linearized pUD968 using Gibson assembly using NEBuilder 2x HIFI DNA assembly master mix (NEBuilder® HIFI DNA Assembly Master Mix | Gene Assembly | NEB, commercially obtainable from New England Biolabs Inc) according to manufacturer’s guidelines, as illustrated in Figure 1. The resulting plasmid was called pUDE885.

[230] Plasmid pUD885 was first transformed into E.coli XL1-Blue cells (commercially obtainable from Agilent, Santa Clara, USA) and plated on LB-ampicillin and incubated overnight at 37°C. A single colony was used to inoculate LB-ampicillin liquid medium and incubated overnight at 37°C for plasmid propagation. The GeneJET Plasmid Miniprep Kit (commercially available from Thermo Fisher Scientific Inc. , Waltman, MA, USA) was used to isolate the plasmid DNA (of the plasmid pUD885) from the E.coli according to manufacturer’s instructions. Correct assembly of the plasmid can be verified either by diagnostic PCR or restriction digestion. The isolated plasmid pUD885 is stored at -20°C.

[231] An illustration of the above process is provided in Figure 1 .

Example 2: Construction of plasmid pUDE941 comprising a pACT-SOR2-tCPS1 fragment

[232] This example 2 describes how SOR2, a gene encoding sorbitol dehydrogenase 2 from CEN- PK.113-7D, was cloned between promoter ACT1 and terminator CPS1 on plasmid pUDE885 prepared in example 1 .

[233] To this end, primers 16709/16710 with nucleotides homologous to the open reading frame of SOR2 and to the flanking regions of pACT1 and tCPS1 were used. The genomic DNA for the SOR2 DNA fragment was isolated from CEN.PK.113.7D using the method as described by Looke et al. (2011). Phusion PCR (Phusion High-Fidelity DNA Polymerase (2 U/pL), n.d.) was used to amplify the SOR2 DNA fragment, according to manufacturer’s guidelines (Thermo ScientificPhusion High-Fidelity DNA Polymerase Product Information Sheet, 2018) using 30 cycles and an annealing temperature of 57 °C. Correct fragment sizes were verified by gel electrophoresis on a TopVision Agrose gel 1 % according to manufacturer’s guidelines as indicated above. After verification, the SOR2 DNA fragment was purified using a GeneJET PCR purification kit (GeneJET PCR Purification Kit) according to manufacturer’s guidelines as indicated above.

[234] Plasmid pUDE885 was linearized by restriction endonuclease Kpnl. After linearization of pUD885, the purified SOR2 DNA fragment was assembled onto the backbone of linearized pUD885 by Gibson assembly using NEBuilder 2x HIFI DNA assembly master mix (NEBuilder® HiFi DNA Assembly Master Mix | Gene Assembly | NEB, commercially obtainable from New England Biolabs Inc) according to manufacturer’s guidelines, as illustrated in Figure 2.

[235] The resulting plasmid was named pUDE941. Plasmid pUDE941 was used as PCR template to obtain the cassette of the pACT1-SOR2 CPS1 fragment using primers (16715/16716)

Example 3: Preparation repair fragments for pTEF1, ORF of HXT15 and tCYCf

[236] HXT15 was integrated flanked by the promoter pTEF1 and the terminator tCYCf. Primers with nucleotides homologous to the ORF of HXT15 and to the flanking regions of pTEF1 and tCYCf were used (16705/16706). The genomic DNA for the HXT15 DNA fragment was isolated from CEN.PK.113.7D using the method as described by Looke et al. (2011). Phusion PCR (Phusion High-Fidelity DNA Polymerase (2 U/gL), n.d.) was used to amplify the HXT 15 DNA fragment, according to manufacturer’s guidelines (Thermo ScientificPhusion High-Fidelity DNA Polymerase Product Information Sheet, 2018) using 30 cycles and an annealing temperature of 57 °C. For in vivo homologous recombination of the pTEF1-HXT15ACYC1 , pTEF1 was amplified using Phusion PCR (Phusion High-Fidelity DNA Polymerase (2 U/gL), n.d.) with p426-TEF as template and with primer sets containing homologous nucleotides to the upstream sequence of the X-2 integration site and the HXT15 ORF (16711/17031). tCYCf was amplified using Phusion PCR (Phusion High-Fidelity DNA Polymerase (2 U/gL), n.d. with primers sets containing overlap with the HXT15 ORF and synthetic homologous recombination sequence A (Kuijpers et al. 2013) (17032/16712) and p426-TEF as template. Synthetic homologous recombination sequence A (SHR-A) was used to create identical overhangs for in vivo homologous recombination of pTEF1-HXT15-tCYC1 to pACT1-SOR2ACPS1. After PCR amplification of the three fragments, the correct fragment sizes were verified by gel electrophoresis on a TopVision Agrose gel 1% according to manufacturer’s guidelines as indicated above. After verification, the three DNA fragments (pTEF1, HXT15 and tCYCf) were purified using a GeneJET PCR purification kit (GeneJET PCR Purification Kit) according to manufacturer’s guidelines as indicated above.

Example 4: Construction of Plasmid pUDR538

[237] This example describes how plasmid pUDR538 was constructed. Plasmid pUDR538 is a pROS12- derived plasmid (Mans et al., 2015). The protocol provided in the supplementary materials of the publication of Mans et al. (2015) was followed for the construction of pUDR538. The pROS12 backbone was amplified using primer combination 5793-5793 (double binding) and the plasmid insert (containing the gRNA sequence for X-2) was amplified with primers 10866/10866 (double binding). Example 5: Construction of strain IMX2411

[238] Example 5 describes the construction of yeast strain IMX2411 from IMX581. IMX581 is a CEN.PK113-5D -based, Cas9-expressing strain used for subsequent CRISPR-Cas9-mediated genome modifications as described by Mans et al., 2015. pACT1-SOR2-tCPS (as constructed in example 2), pTEF1, HXT15 and t CYC 7 (as constructed in example 3)

[239] The intergenic region X-2 of yeast strain IMX581 was used for integration of pACT1-SOR2-tCPS (as constructed in example 2), pTEF1, HXT15 and tCYC1 (as constructed in example 3). Integration into X-2 was found to lead to stable expression of the integrated gene, without interfering with native genes (Mikkelsen et al. 2012). Plasmid pUDR538 was used to target this integration site (as constructed in example 4).

[240] Strain IMX2411 was obtained by co-transformation of pUDR538 together with 4 DNA fragments encoding pACT1-SOR2-tCPS (as constructed in example 2), pTEF1, HXT15 and tCYC7 (as constructed in example 3) into IMX581 , according to the lithium-acetate transformation protocol (Gietz and Woods 2002). Transformants were selected on solid YPD medium (10 gl_^-1 Bacto yeast extract, 20 gl_^-1 Bacto peptone, 20 gL^-1 glucose and 20 gL^-1 agar) supplemented with 200 mgL^-1 hygromycin B. Confirmation of the desired genotype was performed by diagnostic colony PCR using Dreamtaq polymerase (Thermo scientific), following the manufacturer’s instructions.

Example 6: Construction of strain IME611

[241] Reference strain IME611 was obtained by transforming p426-TEF(empty) into IMX2411 , according to the lithium-acetate transformation protocol (Gietz and Woods 2002). Transformations were plated on solid synthetic medium (3.0 g L’¹ KH2PO4, 0.5 g L’¹ MgSO4'7H₂O, 5.0 g L’¹ (NH₄)2SO4, 1.0 ml L’¹ trace elements, 1 .0 mL L^-1 vitamin solution (Verduyn, Postma, Scheffers, & Van Dijken, 1992), 20 gL^-1 agar and 20 gL^-1 glucose) . SMD plates were used, as these do not contain uracil. Growing the transformants on these plates selects for the uracil marker present on p426-TEF(empty). Confirmation of the desired genotype was performed by diagnostic colony PCR using Dreamtaq polymerase (Thermo scientific), following the manufacturer’s instructions.

Example 7: preparation repair fragment pTEF1-HXT15-tCYC1

[242] Genomic DNA of IMX2411 was used as PCR template to obtain the repair fragment pTEF1-HXT15- tCYC1. Genomic DNA was isolated from IMX2411 using the method described by Looke et al. (2011). pTEF1-HXT15-tCYC1 was amplified with flanks to SHR-A and the upstream sequence of the X-2 integration site using PCR Phusion PCR (Phusion High-Fidelity DNA Polymerase (2 U/pL), n.d.) with genomic DNA of IMX2411 as template and primer pair 16711/16712. After PCR amplification of the DNA, the correct fragment size was verified by gel electrophoresis on a TopVision Agrose gel 1% according to manufacturer’s guidelines as indicated above. After verification, the DNA fragment pTEF1-HXT15-tCYC1 was purified using a GeneJET PCR purification kit (GeneJET PCR Purification Kit) according to manufacturer’s guidelines as indicated above

Example 8: Construction of strain IMX1489

[243] The construction of strain IMX1489 was carried out as described by Papapetridis et al. in their article titled “Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield”, published in Biotechnol Biofuels (2018), pages 1 to 17.

[244] Co-transformation of the two fragments of the GPD2-targ eting CRISPR plasmid (pROS11 backbone) and non-oxidative pentose-phosphate pathway integration cassettes, prepared as described by Papapetridis et al, to strain IMX581 yielded a strain IMX1472. The RuBisCO/PRK-expressing strain IMX1489 was obtained by co-transformation of pUDR103, the pDAN1 , prk-ORF, tPGK1 sequences, 9 copies of the expression cassette of cbbm and the expression cassettes of groEL and groES (14 fragments), prepared as described by Papapetridis et al, to strain IMX1472 (integration at the SGA1 locus, GPD2-targeting CRISPR plasmid recycled).

Example 9: Construction of strain IMX2495

[245] Example 9 describes the construction of strain IMX2495 from strain IMX1489 (as constructed in example 8). The intergenic region X-2 of yeast strain IMX1489 was used for integration of pACT1-SOR2- tCPS (as constructed in example 2) and pTEF1-HXT15-tCYC1 (as constructed in example 7). Integration into X-2 was found to lead to stable expression of the integrated gene, without interfering with native genes (Mikkelsen et al. 2012). Plasmid pUDR538 was used to target this integration site (as constructed in example 4).

[246] Strain IMX2495 was obtained by co-transformation of pUDR538 (example 4) together with the 2 repair fragments encoding pACT1-SOR2-tCPS1 (example 2) and pTEF1-HXT15 CYC1 (example 7) into IMX1489 according to the lithium-acetate transformation protocol (Gietz and Woods 2002). Transformants were selected on solid YPD medium (10 gL^-1 Bacto yeast extract, 20 gL^-1 Bacto peptone, 20 gL^-1 glucose and 20 gL^-1 agar) supplemented with 200 mgl_^-1 hygromycin B. Confirmation of the desired genotype was performed by diagnostic colony PCR using Dreamtaq polymerase (Thermo scientific), following the manufacturer’s instructions.

Example 10: construction of strain IMX2506

[247] Strain IMX2506 was obtained by transforming p426-TEF(empty) into IMX2495, according to the lithium-acetate transformation protocol (Gietz and Woods 2002). Transformations were plated on solid synthetic medium (3.0 g L^-1 KH2PO4, 0.5 g L^-1 MgSO4'7H2O, 5.0 g L^-1 (NH4)2SO4, 1.0 ml L^-1 trace elements, 1.0 mL L^-1 vitamin solution (Verduyn, Postma, Scheffers, & Van Dijken, 1992), 20 gL^-1 agar and 20 gL^-1 glucose) . SMD plates were used, as these do not contain uracil. Growing the transformants on these plates selects for the uracil marker present on p426-TEF(empty). Confirmation of the desired genotype was performed by diagnostic colony PCR using Dreamtaq polymerase (Thermo scientific), following the manufacturer’s instructions.

[248]

Example 11 : Anaerobic chemostat experiments

[249] Anaerobic chemostat cultures were performed in 2-L bioreactors (Applikon, Delft, The Netherlands). A continuous feed flow of medium was supplied to the bioreactor containing Synthetic medium (3.0 g L’¹ KH2PO4, 0.5 g L’¹ MgSO₄-7H₂O, 5.0 g L’¹ (NH₄)₂SO₄, 1.0 ml L’¹ trace elements, 1.0 ml_ L^-1 vitamin solution (Verduyn, Postma, Scheffers, & Van Dijken, 1992)) was supplemented with 10 gl_^-1 glucose and 10 gl_^-1 sorbitol, the anaerobic growth factors Tween 80 (420 mgL^-1) and ergosterol (10 mgL- ¹), and 0.2 gl_^-1 antifoam C (sigma Aldrich). The inflow of medium was set to a flow rate of 0.025 Ltr¹. A working volume of 1-L was ensured by a level sensor which controls the effluent pump. The pH was kept constant at 5.0 by automatic addition of 2 M KOH. A gas mixture of N2/CO2 (90/10%) was used to ensure anaerobic conditions and supply CO2 to ensure activity of RuBisCO. The gasflow was set at 0.5 L min^-1 and a stirrer speed of 800 rpm was used. The outlet gas was cooled to 4°C to minimize evaporation and the bioreactor was kept at a temperature of 30°C. Oxygen diffusion was minimized by the use of Norprene tubing and Viton O-rings. Bioreactor inocula were generated in 500 ml_ shakeflasks containing 100 ml_ synthetic medium containing 20 gl_^-1 glucose.

[250] The cultures were inoculated from frozen stock cultures and grown at 30 °C, 200 rpm, under atmospheric air for 15-18 h. These cultures were used to inoculate pre-cultures flaks, which were grown to mid-exponential phase (ODeeo of 3-5) and used to start the bioreactor with an ODeeo of 0.2-0.3. The initial batch phase preceding the continuous cultivation, was performed on synthetic medium supplemented with 20 gl_^-1 glucose, the anaerobic growth factors Tween 80 (420 mgL^-1) and ergosterol (10 mgL^-1), and

0.2 gl_^-1 antifoam C (sigma Aldrich).

[251] For dry weight determination, dry nitrocellulose filters with pore size of 0.45 pm (Gelman Science, Ann Arbor, Ml, USA) were used. The filter was weighed and washed with demineralized water, before adding 10 ml_ of culture to it. After washing the filter again with demineralized water, the filter was dried in a microwave for 20 minutes at 350 W and weighed again.

[252] Metabolite concentrations were determined from supernatants obtained by centrifugation of the culture samples. Supernatants were analysed by high-performance liquid chromatography (HPLC) Agilent Infinity 1260 series (Agilent technologies, santa Clara, ca, USA) with a Bio-Rad Aminex HPX-87H column at 60°C, eluted with 5 mM H2SO₄ at a flow rate of 0.6ml. min^-1.

[253] Detection of glucose, sorbitol, glycerol and ethanol was by means of a refractive-index detector (RID) (Agilent G1362A) and organic acids were detected by a dual-wavelength absorbance detector (Agilent G1314F). Metabolite concentrations in steady-state cultures were analysed after rapid quenching of the cultures samples using cold-steel beads. In the calculations, ethanol evaporation was corrected for using the volume-dependent ethanol evaporation constant of 0.008 h’¹. Therefore, the actual working volume of the chemostat was determined at the end of the run by weighing the broth. The results are illustrated in Table 11 and Table 12.

Table 11 : Sorbitol concentrations measured at different timepoints in S. cerevisiae cultures IME611 and IMX2506 in anaerobic chemostats on 10 gl_~¹ of glucose and 10 gl_~¹ of sorbitol at pH 5 and a dilution rate of 0.025 h~¹.

n.d. = not determined.

Tablet 2: Metabolite concentrations of anaerobic chemostat cultures measured during steady state sampling in the reactor (OUT) and in the medium inflow (IN) of S. cerevisiae strains IME324 and IMX2506.

*the ethanol concentration in this table is not corrected for evaporation yet.

Example 12: Anaerobic batch experiments

[254] Anaerobic batch cultures were performed in 2-L bioreactors (Applikon, Delft, The Netherlands) with a starting volume of 1 L. Cultures were grown on Synthetic medium (3.0 g L^-1 KH2PO4, 0.5 g L^-1 MgSO4'7H2O, 5.0 g L^-1 (NH4)2SO4, 1.0 ml L^-1 trace elements, 1.0 mL L^-1 vitamin solution (Verduyn, Postma, Scheffers, & Van Dijken, 1992)), supplemented with 20 gL^-1 glucose and 30 gL^-1 sorbitol, the anaerobic growth factors Tween 80 (420 mgL^-1) and ergosterol (10 mgL^-1), and 0.2 gL^-1 antifoam C (sigma Aldrich). The pH was kept constant at 5.0 by automatic addition of 2 M KOH. A gas mixture of N2/CO2 (90/10%) was used to ensure anaerobic conditions and supply CO2 to ensure activity of RuBisCO. The gasflow was set at 0.5 L min^-1 and a stirrer speed of 800 rpm was used. The outlet gas was cooled to 4°C to minimize evaporation and the bioreactor was kept at a temperature of 30°C. Oxygen diffusion was minimized by the use of Norprene tubing and Viton O-rings. Bioreactor inocula were generated in 500 mL shakeflasks containing 100 mL synthetic medium containing 20 gL^-1 glucose.

[255] The cultures were inoculated from frozen stock cultures and grown at 30 °C, 200 rpm, under atmospheric air for 15-18 h. These cultures were used to inoculate pre-cultures flaks, which were grown to mid-exponential phase (ODeeo of 3-5) and used to start the bioreactor with an ODeeo of 0.15-0.30.

[256] For dry weight determination, dry nitrocellulose filters with pore size of 0.45 pm (Gelman Science, Ann Arbor, Ml, USA) were used. The filter was weighed and washed with demineralized water, before adding 10 mL of culture to it. After washing the filter again with demineralized water, the filter was dried in a microwave for 20 minutes at 350 W and weighed again.

[257] Metabolite concentrations were determined from supernatants obtained by centrifugation of the culture samples. Supernatants were analysed by high-performance liquid chromatography (HPLC) Agilent Infinity 1260 series (Agilent technologies, Santa Clara, ca, USA) with a Bio-Rad Aminex HPX-87H column at 60°C, eluted with 5 mM H2SO4 at a flow rate of 0.6ml. min^-1.

[258] Detection of glucose, sorbitol, glycerol and ethanol was by means of a refractive-index detector (RID) (Agilent G1362A) and organic acids were detected by a dual-wavelength absorbance detector (Agilent G1314F). In the calculations, ethanol evaporation was corrected for using the volume-dependent ethanol evaporation constant of 0.008 fr¹ . Therefore, the sampled volume was determined for each sample point and the final volume at the end of the batch was measured by weighing the broth, to be able to determine the actual volume inside of the reactor at different points in time. The results are illustrated in Table 13, Table 14, Table 15 and Table 16. Table 13: Metabolite concentrations and corresponding broth volumes at different timepoints during duplicate batch cultures of IME324 in anaerobic bioreactors, pH 5.

'the ethanol concentration in this table is not corrected for evaporation yet.

Table 14: Metabolite concentrations and corresponding broth volumes at different timepoints during duplicate batch cultures of IME611 in anaerobic bioreactors, pH 5.

'the ethanol concentration in this table is not corrected for evaporation yet.

Table 15: Metabolite concentrations and corresponding broth volumes at different timepoints during duplicate batch cultures of IMX1489 in anaerobic bioreactors, pH 5

'the ethanol concentration in this table is not corrected for evaporation yet.

Table 16: Metabolite concentrations and corresponding broth volumes at different timepoints during duplicate batch cultures of IMX2506 in anaerobic bioreactors, pH 5.

'the ethanol concentration in this table is not corrected for evaporation yet.

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Claims

1 . A mutant yeast cell, comprising :

(ii) a second genetic modification for expression of a protein that functions in a second metabolic pathway forming a non-native redox sink.

2. The mutant yeast cell according to claim 1 , wherein the mutant yeast cell comprises a genetic modification to constitutively express and/or upregulate the activity of a protein having NAD+ dependent sugar alcohol dehydrogenase activity.

3. The mutant yeast cell according to claim 2, wherein the protein having NAD+ dependent sugar alcohol dehydrogenase activity is a NAD+ dependent sorbitol dehydrogenase or a NAD+ dependent mannitol dehydrogenase.

4. The mutant yeast cell according to claim 2 or 3, wherein the protein having NAD+ dependent sugar alcohol dehydrogenase activity is a sorbitol dehydrogenase chosen from the group consisting of sorbitol dehydrogenase 1 (SOR1) and sorbitol dehydrogenase 2 (SOR2).

5. The mutant yeast cell according to claim 2 or 3, wherein the protein having NAD+ dependent sugar alcohol dehydrogenase activity is a mannitol dehydrogenase chosen from the group consisting of mannitol dehydrogenase 1 (MAN1) and mannitol dehydrogenase 2 (MAN2).

6. The mutant yeast cell according to anyone of claims 1 to 5, wherein the mutant yeast comprises a genetic modification to downregulate the activity of a protein that plays a role in the glucose repression of the yeast.

7. The mutant yeast cell according to anyone of claims 1 to 6, wherein the mutant yeast comprises a genetic modification to downregulate the activity of the homologous TUP1 gene and/or CYC8 gene of the yeast.

8. The mutant yeast cell according to any one of claims 1 to 7 , wherein the mutant yeast cell comprises a genetic modification to upregulate the activity of one or more sugar alcohol transporters.

9. The mutant yeast cell according to claim 8, wherein the sugar alcohol transporter is a hexose transporter chosen from the group consisting of HXT 13, HXT 15 and/or HXT 17.

10. The mutant yeast cell according to any one of claims 1 to 9, wherein the mutant yeast cell comprises:

- a heterologous nucleic acid sequence encoding a protein comprising phosphoketolase activity (EC 4.1 .2.9 or EC 4.1.2.22, PKL); and/or

- a heterologous nucleic acid sequence encoding a protein having phosphotransacetylase (PTA) activity (EC 2.3.1.8); and/or

- a heterologous nucleic acid sequence encoding a protein having acetate kinase (ACK) activity (EC 2.7.2.12).

11 . The mutant yeast cell according to any one of claims 1 to 9, wherein the mutant yeast cell comprises a heterologous nucleic acid sequence encoding a protein comprising NADH dependent acetylating acetaldehyde dehydrogenase activity (EC 1.2.1.10).

12. The mutant yeast cell according to any one of claims 1 to 9, wherein the mutant yeast cell comprises:

- optionally, a heterologous nucleic acid sequence encoding for one or more molecular chaperones for the protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity.

13. The mutant yeast cell according to claim 12, wherein the mutant yeast cell comprises:

- a heterologous nucleic acid sequence encoding for a protein having ribulose-1 ,5-biphosphate carboxylase oxygenase (Rubisco) activity; and

- a heterologous nucleic acid sequences encoding for a protein having phosphoribulokinase (PRK) activity, wherein the phosphoribulokinase is under control of a promoter (the “PRK promoter”) which has a PRK expression ratio anaerobic/aerobic of 2 or more; and,

14. The mutant yeast cell according to any one of claims 1 to 13, wherein the mutant yeast cell comprises one or more genetic modifications for decreasing or inhibiting the activity of glycerol-3-phosphate dehydrogenase (GPD) and/or glycerol-3-phosphate phosphatase (GPP).

15. The mutant yeast cell according to any one of claims 1 to 14, wherein the mutant yeast cell is a yeast cell from the genus Saccharomyces.

16. A process for the production of ethanol, the process comprising fermenting of a carbon source composition with a mutant yeast cell according to any one of claims 1 to 15, wherein the carbon source composition comprises at least a sugar alcohol and wherein the process is carried out under oxygen-limited conditions or anaerobic conditions.

17. The process according to claim 16, wherein the carbon source composition comprises a sugar and a sugar alcohol and wherein both sugar and sugar alcohol are converted into ethanol.