WO2019110491A1

WO2019110491A1 - Recombinant yeast cell

Info

Publication number: WO2019110491A1
Application number: PCT/EP2018/083320
Authority: WO
Inventors: Ioannis PAPAPETRIDIS
Original assignee: Dsm Ip Assets B.V.
Priority date: 2017-12-08
Filing date: 2018-12-03
Publication date: 2019-06-13

Abstract

The present invention provides a recombinant yeast cell functionally expressing one or more heterologous nucleic acid sequences encoding:10 - a ribulose-1,5-phosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39); - a phosphoribulokinases (EC 2.7.1.19; PRK); - a catalytically inactive endonuclease (CIE); and - a constitutively expressed guide RNA; and optionally: - one or more molecular chaperones for RuBisCO,15 wherein said PRK is under control of a first promoter which is a constitutive promoter comprising PAM sequences, which PAM sequences are targeted by said guide RNA, and wherein said catalytically inactive endonuclease is under control of a second promoter, which second promoter has a CIE expression ratio(aerobic/anaerobic) of 2 or more.

Description

RECOMBINANT YEAST CELL

Field of the invention

The invention relates to a recombinant yeast cell having the ability to produce a desired fermentation product, to the functional expression of heterologous peptides in a yeast cell, and to a method for producing a fermentation product wherein said yeast cell is used.

Background of the invention

Microbial fermentation processes are applied to industrial production of a broad and rapidly expanding range of chemical compounds from renewable carbohydrate feedstocks. Especially in anaerobic fermentation processes, redox balancing of the cofactor couple NADH/NAD⁺ can cause important constraints on product yields. This challenge is exemplified by the formation of glycerol as major by-product in the industrial production of - for instance - fuel ethanol by Saccharomyces cerevisiae, a direct consequence of the need to reoxidize NADH formed in biosynthetic reactions. Ethanol production by Saccharomyces cerevisiae is currently, by volume, the single largest fermentation process in industrial biotechnology, but various other compounds, including other alcohols, carboxylic acids, isoprenoids, amino acids etc, are also currently produced in industrial biotechnological processes. Various approaches have been proposed to improve the fermentative properties of organisms used in industrial biotechnology by genetic modification. A major challenge relating to the stoichiometry of yeast-based production of ethanol, but also of other compounds, is that substantial amounts of NADH-dependent side-products (in particular glycerol) are generally formed as a by-product, especially under anaerobic and oxygen-limited conditions or under conditions where respiration is otherwise constrained or absent. It has been estimated that, in typical industrial ethanol processes, up to about 4 wt.% of the sugar feedstock is converted into glycerol (Nissen et al. Yeast 16 (2000) 463-474). Under conditions that are ideal for anaerobic growth, the conversion into glycerol may even be higher, up to about 10 %. Glycerol production under anaerobic conditions is primarily linked to redox metabolism. During anaerobic growth of S. cerevisiae, sugar dissimilation occurs via alcoholic fermentation. In this process, the NADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenase reaction is reoxidized by converting acetaldehyde, formed by decarboxylation of pyruvate to ethanol via NAD⁺-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 metabolism. Under anaerobic conditions, NADH reoxidation in 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 NAD⁺-dependent glycerol 3-phosphate dehydrogenase. Subsequently, the glycerol 3-phosphate formed in this reaction is hydrolysed by glycerol-3-phosphatase to yield glycerol and inorganic phosphate. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, which is undesired as it reduces overall conversion of sugar to ethanol. Further, the presence of glycerol in effluents of ethanol production plants may impose costs for waste-water treatment.

WO2014/129898 describes a recombinant cell functionally 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”). The PRK in the yeast of WO2014/129898 is under an galactose inducible promotor, which is not suitable on industrial scale. An object of the invention is to provide a suitable promotor for the PRK.

Summary of the invention

The present invention provides a recombinant yeast cell functionally expressing one or more heterologous nucleic acid sequences encoding:

a ribulose-1 , 5-phosphate carboxylase/oxygenase (RuBisCO, EC4.1.1.39);

a phosphoribulokinases (EC2.7.1.19; PRK);

a constitutively expressed guide RNA; and

a catalytically inactive endonuclease (CIE); and optionally:

one or more molecular chaperones for RuBisCO,

wherein said PRK is under control of a first promoter which is a constitutive promoter comprising PAM sequences, which PAM sequences are targeted by said guide RNA, and wherein said catalytically inactive endonuclease is under control of a second promoter, which second promoter has a CIE expression ratio(aerobic/anaerobic) of 2 or more.

The inventors found that, under aerobic conditions, no viable yeast cell could be obtained if a yeast cells contain RuBisCO and PRK genes and if the PRK is brought under control of strong or medium constitutive promotors, although under anaerobic conditions such yeast grow normally. When PRK is brought under control of a weak constitutive promotor, the cells were able to grow under aerobic conditions, but when such cell were use in ethanol fermentation, there was still significant amount of glycerol production. Thus, neither strong, medium strong, or weak constitutive promotors are feasible for PRK with the purpose to reduce glycerol formation using RuBisCO technology. It is essential that (industrial) ethanol yeast cells are able to grow under aerobic conditions: prior to the ethanol fermentation per se (which is conducted under anaerobic conditions) the yeast cells must be propagated, in order to produce sufficient biomass in order to reach the desired“pitch”. This propagation phase occurs mainly under aerobic conditions.

According to the invention the constitutive first promotor ensures expression of the PRK gene, unless (under aerobic conditions) the second promotor induces the catalytically inactive endonuclease which, as a complex with the guide RNA targeting the first promotor having the PAM sequences, blocks expression of the PRK. This will result in prevention of expression of the catalytically inactive endonuclease during aerobic conditions, whilst allowing expression at anaerobic conditions. This allows for the yeast to be propagated during the aerobic phase, whilst achieving significant reduction of glycerol production during the anaerobic ethanol phase.

The second promoter may be the native promoter of an ORF selected from the list consisting of YOR388C, YPL275W, YPL276W, YDR256C, YHR096C, YNL195C, YGR1 10W, YCR010C, YDL218W, YPL223C, YJR095W, YMR303C, YGR236C, YHR139C, YPR151C, YMR107W, YMR118C, YLR174W, YPL201C, YDR380W, YMR058W, YBR047W, YML054C, YLR205C, YPL147W, YDR070C, YPR001W, YER065C, YKR009C, YLL053C, and YGR256W. The catalytically inactive endonuclease may be dCas9 or dCpfl .

Description of the Figures

Fig. 1 PRK activity in cell-free extracts of IME324 (left in fig.1 ) and IMX774 (right in fig. 1 ), harvested during exponential growth phase of anaerobic shake-flask cultures in synthetic medium (20 g L¹ glucose). Values represent the averages and the standard deviations of activity when 30, 50 or 100 pi of cell-free extract were used. Data were collected from single cultures.

Fig. 2 Yields (Y) of glycerol, biomass and ethanol on glucose and the ratio of glycerol formation to biomass formation in anaerobic bioreactor batch cultures of S. cerevisiae strains IME324 and IMX774. Cultures were grown on synthetic medium containing 20 g L¹ glucose (pH 5) and sparged with a gas mixture of N2/CO2 (90%/10%). Yields and ratios were calculated from the exponential growth phase. The ethanol yield on glucose was corrected for evaporation. Values represent average and mean deviation of data from independent duplicate cultures.

Fig. 3 Sum of peak area of selected unique peptides from S. oleracea prk among samples from both strains against prk. The peak areas give an indication of protein amount.

Fig. 4 Illustration of integration of anaerobic promoter-PRK cassettes at INT1 intergenic locus. Fig. 5 Average glycerol values end of MTP batch fermentation expressed in arbitrary units (AU) for control strains IME324, IMX774, IMX765 and transformants of IMX765 with expression cassette anaerobic promoter-PRK introduced. Error bars indicate standard error of the mean.

Fig. 6 Average ethanol values expressed in arbitrary units (AU) end of MTP batch fermentation for control strains IME324, IMX774, IMX765 and transformants of IMX765 with expression cassette anaerobic promoter-PRK introduced. Error bars indicate standard error of the mean.

Table 1 - Description of the sequence listing

Detailed description of the invention

Defintitions

The term“a” or“an” as used herein is defined as“at least one” unless specified otherwise.

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. "compound", this means "at least one" of that moiety, e.g. "at least one compound", unless specified otherwise. The term‘or’ as used herein is to be understood as‘and/or’.

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 method of the invention; in particular when referring to such as compound, it includes the natural isomer(s).

The term‘fermentation’,‘fermentative’ and the like is used herein in a classical sense, i.e. to indicate that a process is or has been 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, in particular a yeast cell, and usually corresponds to an oxygen consumption of less than 5 mmol/l.h, in particular to an oxygen consumption of less than 2.5 mmol/l.h, or less than 1 mmol/l.h. More preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable. This usually corresponds to a dissolved oxygen concentration in the culture broth of less than 5 % of air saturation, in particular to a dissolved oxygen concentration of less than 1 % of air saturation, or less than 0.2 % of air saturation.

The term“yeast” or“yeast cell” refers 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, with Saccharomyces cerevisiae as the most well-known species.

The term“recombinant (cell)” or“recombinant micro-organism” as used herein, refers to a strain (cell) containing nucleic acid which is the result of one or more genetic modifications using recombinant DNA technique(s) and/or another mutagenic technique(s). In particular a recombinant cell may comprise nucleic acid not present in a corresponding wild-type cell, which nucleic acid has been introduced into that strain (cell) using recombinant DNA techniques (a transgenic cell), or which nucleic acid not present in said wild-type 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 wild-type (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 (cell)” in particular relates to a strain (cell) from which DNA sequences have been removed using recombinant DNA techniques.

The term“transgenic (yeast) cell” as used herein, refers to a strain (cell) containing nucleic acid not naturally occurring in that strain (cell) and which has been introduced into that strain (cell) using recombinant DNA techniques, i.e. a recombinant cell).

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.

The term“gene”, as used herein, refers to a nucleic acid sequence containing a template for a nucleic acid polymerase, in eukaryotes, RNA polymerase II. Genes are transcribed into mRNAs that are then translated into protein.

The term "nucleic acid" as used herein, includes reference 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. 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.

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.

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 14 June 2016) unless specified otherwise.

Every nucleic acid sequence herein that encodes a polypeptide also, by reference to the genetic code, 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.

The term “functional homologue” (or in short “homologue”) of a polypeptide having a specific sequence (e.g. SEQ ID NO: X), as used herein, refers to a polypeptide 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. This functionality may be tested by use of an assay system comprising a recombinant yeast cell comprising an expression vector for the expression of the homologue in yeast, said expression vector comprising a heterologous nucleic acid sequence operably linked to a promoter functional in the yeast and said heterologous nucleic acid sequence encoding the homologous polypeptide of which enzymatic activity in the yeast cell is to be tested, and assessing whether said conversion occurs in said cells. Candidate homologues may be identified by using in silico similarity analyses. A detailed example of such an analysis is described in Example 2 of W02009/013159. The skilled person will be able to derive there from how suitable candidate homologues may be found and, optionally upon codon(pair) optimization, will be able to test the required functionality of such candidate homologues using a suitable assay system as described above. A suitable homologue represents a polypeptide having an amino acid sequence similar to a specific polypeptide of more than 50%, preferably of 60 % or more, in particular of at least 70 %, more in particular of at least 80 %, at least 90 %, at least 95 %, at least 97 %, at least 98 % or at least 99 % and having the required enzymatic functionality. 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.

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.

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. LongdenJ. 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.

Global Homology Definition

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”.

Longest Identity Definition

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 labelled in the output of the program as“longest-identity”. 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).

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 asparagine-glutamine. 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.

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. 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%.

As used herein, "heterologous" in reference 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.

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 an enzyme with a specific activity can be expressed in such a eukaryotic system. In some embodiments, transformed/transfected yeast 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.

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.

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 extrachromosomal DNA which is not integrated into a microorganism's genome and is usually circular in nature.

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 non-functional in that organism. Integration of the segment comprising the gene of interest may be selected if an appropriate marker is included within that segment.

By "host cell" is meant a cell which contains a vector and supports the replication and/or expression of the vector.

"Transformation" and "transforming", as used herein, refers to the insertion of an exogenous polynucleotide 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

In one aspect, the invention relates to a recombinant yeast cell functionally expressing one or more heterologous nucleic acid sequences encoding:

a ribulose-1 , 5-phosphate carboxylase/oxygenase (RuBisCO, EC4.1.1.39);

a phosphoribulokinases (EC2.7.1.19; PRK);

a catalytically inactive endonuclease (CIE); and

a constitutively expressed guide RNA; and optionally:

one or more molecular chaperones for RuBisCO,

wherein said PRK is under control of a first promoter which is a constitutive promoter comprising PAM sequences, which PAM sequences are targeted by said guide RNA and wherein said catalytically inactive endonuclease is under control of a second promoter, which second promoter has a CIE expression ratiO(aerobic/anaerobic) of 2 or more.

It is essential that (industrial) ethanol yeast cells are able to grow under aerobic conditions: prior to the ethanol fermentation per se (which is conducted under anaerobic conditions) the yeast cells must be propagated, in order to produce sufficient biomass in order to reach the desired “pitch”. This propagation phase occurs mainly under aerobic conditions.

The inventors found that, under aerobic conditions, no viable yeast cell could be obtained if said yeast cells contain RuBisCO and PRK genes and if the PRK was brought under control of strong or medium constitutive promotors, although under anaerobic conditions such yeast grow normally. When PRK is brought under control of a weak constitutive promotor, the cells were able to grow under aerobic conditions, but when such cell were use in ethanol fermentation, there was still significant amount of glycerol production. Thus, neither strong, medium strong, or weak constitutive promotors are feasible for PRK with the purpose to reduce glycerol formation.

RNA-guided nuclease systems such as the CRISPR/Cas9 system are powerful tools that are used for genome editing and gene regulation. This tool requires the expression of the Cas9 protein and a guide-RNA (gRNA or sgRNA) that enables Cas9 to target a specific sequence of DNA. In e.g. eukaryotic host systems, the guide-RNA is often expressed from RNA polymerase III (POLIII) promoters that recruit endogenous RNA polymerase III for transcription, which is an RNA polymerase that generates guide-RNAs without a 5’ cap. Others have used RNA polymerase II (POLII) promoters in combinations with a ribozyme in order to produce guide-RNAs without a 5’ cap (uncapped RNA).

It is understood that the PAM (“Protospacer Adjacent Motif”) sequences form an integrated part of the first promotor and that these sequences are targeted by a complex of the catalytically inactive endonuclease and a suitable guide-RNA. The constitutive first promotor ensures expression of the PRK gene, unless (under aerobic conditions) the second promotor induces the catalytically inactive endonuclease which, as a complex with the guide RNA targeting the first promotor having the PAM sequences, blocks expression of the PRK. This will result in prevention of expression of the catalytically inactive endonuclease during aerobic conditions, whilst allowing expression at anaerobic conditions. A suitable guide RNA is RNA polymerase II RNA.

The second promoter has a CIE expression ratiO(aerobic/anaerobic) of 2 or more, preferably of 3 or more, 4 or more, 5 or more, 10 or more, 100 or more.

In an embodiment second promoter is the native promoter of an ORF selected from the list consisting of YOR388C, YPL275W, YPL276W, YDR256C, YHR096C, YNL195C, YGR1 10W, YCR010C, YDL218W, YPL223C, YJR095W, YMR303C, YGR236C, YHR139C, YPR151 C, YMR107W, YMR1 18C, YLR174W, YPL201 C, YDR380W, YMR058W, YBR047W, YML054C, YLR205C, YPL147W, YDR070C, YPR001W, YER065C, YKR009C, YLL053C, and YGR256W. Such promotors, and how to measure the expression ratiO(aerobic/anaerobic) are described for example in Linde at al, Journal of Bacteriology, Dec. 1999, p. 7409-7413, see e.g. Table 2 therein. In an embodiment the catalytically inactive endonuclease is dCas9 or dCpfl . The term “catalytically inactive endonuclease” is also referred to as“dead endonuclease”. See Smith et al, Genome Biology (2016) 17:45, which describe how to obtain an inactive endonuclease Cas9 (dCas9) and how to modify dCas9 to function as a transcription activator or repressor, capable of modulating gene expression in eukaryotes. Thus, dCas9 and dCpfl refers to dead or inactive endonuclease Cas9 and Cpf1 , respectively. In an embodiment PAM sequences can be used specifically targeted to the native sequence to ensure proper binding of cCas9 or dCpfl .

"Expression" refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into a protein.

In an embodiment the CIE expression ratio is determined by measuring the amount of TA protein of cells grown under aerobic and anaerobic conditions, or under conditions of different cellular NADH/NAD⁺ ratios. The amount of CIE protein can be determined by proteomics.

In yet another embodiment the level or CIE expression ratio is determined by measuring the transcription level (e.g. as amount of mRNA) of the CIE gene of cells grown under aerobic and anaerobic conditions. The skilled person knows how to determine translation levels using methods commonly known in the art, e.g. Q-PCR, real-time PCR, northern blot, RNA-seq.

Suitable constitutive promotors, as first promotors, include promotors of Saccharomyces proteins TEF1 , PGK1 , TDH3, ADH 1 , ACT1 , and TPI 1. Saccharomyces bayanus TDH3 (SbTDH3) promoter is also a suitable promotor.

As used herein "promoter" is a DNA sequence that directs the transcription of a (structural) gene.

In an embodiment, the second promoter may be a synthetic oligonucleotide. It may be a product of artificial oligonucleotide synthesis. Artificial oligonucleotide synthesis is a method in synthetic biology that is used to create artificial oligonucleotides, such as genes, in the laboratory. Commercial gene synthesis services are now available from numerous companies worldwide, some of which have built their business model around this task. Current gene synthesis approaches are most often based on a combination of organic chemistry and molecular biological techniques and entire genes may be synthesized "de novo", without the need for precursor template DNA. In an embodiment, RuBisCO is under a constitutive promotor.

The recombinant yeast cell is preferably selected from the group of Saccharomycetaceae, such as Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces beticus, Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces uvarum and Saccharomyces bayanus; Schizosaccharomyces such as Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus and Schizosaccharomyces cryophilus; Torulaspora such as Torulaspora delbrueckii; Kluyveromyces such as Kluyveromyces marxianus ; Pichia such as Pichia stipitis, Pichia pastoris or pichia angusta, Zygosaccharomyces such as Zygosaccharomyces baiiir, Brettanomyces such as Brettanomyces intermedius, Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkera Bruxellis and Dekkera anomala; Metschnikowia, Issatchenkia, such as Issatchenkia orientalis, Kloeckera such as Kloeckera apiculata; Aureobasisium such as Aureobasidium pullulans.

In an embodiment, the yeast cell is selected from the group of Saccharomycetaceae. In particular, good results have been achieved with a Saccharomyces cerevisiae cell. It has been found possible to use such a cell according to the invention in a method for preparing an alcohol (ethanol) wherein the NADH-dependent side-product formation (glycerol) was reduced by about 90%, and wherein the yield of the desired product (ethanol) was increase by about 10%, compared to a similar cell without RuBisCO and PRK.

The RuBisCO may in principle be selected from eukaryotic and prokaryotic RuBisCOs. The RuBisCO is preferably from a non-phototrophic organism. In particular, the RuBisCO may be from a chemolithoautotrophic microorganism. Good results have been achieved with a bacterial RuBisCO. Preferably, the bacterial RuBisCO originates from a Thiobacillus, in particular, Thiobacillus denitrificans, which is chemolithoautotrophic. The RuBisCO may be a single-subunit RuBisCO or a RuBisCO having more than one subunit. In particular, good results have been achieved with a single-subunit RuBisCO. In particular, good results have been achieved with a form-ll RuBisCO, more in particular CbbM. A suitable RuBisCO in accordance with the invention is encoded by the cbbM gene from Thiobacillus denitrificans. An alternative to this RuBisCO, is a functional homologue of this RuBisCO, in particular such functional homologue comprising a sequence having at least 80%, 85%, 90% or 95% sequence identity with the cbbM gene from Thiobacillus denitrificans. Suitable natural RuBisCO polypeptides are given in Table 2, with identity to the cbbM gene from Thiobacillus denitrificans. Another suitable RuBisCO is from Synechococcus.

Table 2: Natural RuBisCO polypeptides suitable for expression

In accordance with the invention, the RuBisCO is functionally expressed in the microorganism, at least during use in an industrial process for preparing a compound of interest.

To increase the likelihood that herein enzyme activity is expressed at sufficient levels and in active form in the transformed (recombinant) host cells of the invention, the nucleotide sequence encoding these enzymes, as well as the RuBisCO enzyme and other enzymes of the invention (see below), are preferably adapted to optimise their codon usage to that of the host cell in question. The adaptiveness of a nucleotide 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 (81:2242-51 ). An adapted nucleotide 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. . In an embodiment, the sequences which have been codon optimised for expression in the fungal host cell in question such as e.g. S. cerevisiae cells.

Preferably the functionally expressed RuBisCO 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.

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

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.

PRK 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 eukaryotic PRK originates from a plant selected from Caryophyllales , in particular from Amaranthaceae, more in particular from Spinacia. As an alternative to PRK from Spinacia a functional homologue of PRK from Spinacia may be present, in particular a functional homologue comprising a sequence having at least 70%, 75%, 80%. 85%, 90 % or 95% sequence identity with the PRK from Spinacia. Suitable natural PRK polypeptides are given in Table 3. Table 3: Natural PRK polypeptides suitable for expression with identity to PRK from Spinacia

In an embodiment the recombinant microorganism further comprises a nucleic acid sequence encoding one or more heterologous prokaryotic or eukaryotic molecular chaperones, which - when expressed - are capable of functionally interacting with an enzyme in the microorganism, in particular with at least one of RuBisCO and PRK.

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. Trends in Biochemical Sciences, August 2011 , Vol. 36, No. 8.

In an embodiment the chaperone or chaperones are from a bacterium, more preferably from Escherichia, in particular E. coli GroEL and GroEs from E. coli may in particular encoded in a microorganism according to the invention. In an embodiment, chaperones are chaperones from Saccharomyces, in particular Saccharomyces cerevisiae Hsp10 and Hsp60. 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 recombinant yeast cell may serve as a chaperone for the RuBisCO. See Zeilstra-Ryalls J, Fayet O, Georgopoulos C (1991 ). "The universally conserved GroE (Hsp60) chaperonins". Annu Rev Microbiol. 45: 301-25. doi:10.1146/annurev. mi.45.100191.001505. PMID 1683763 and Horwich AL, Fenton WA, Chapman E, Farr GW (2007). "Two Families of Chaperonin: Physiology and Mechanism". Annu Rev Cell Dev Biol. 23: 115-45. doi:10.1146/annurev.cellbio.23.090506.123555. PMID 17489689. Good results have been achieved with a recombinant 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 a sequence having at least 70%, 75%, 80%, 85%, 90 % or 95% sequence identity with GroES.

The invention allows in particular a reduction in formation of an NADH dependent side- product, especially glycerol, by up to 100%, up to 99%, or up to 90%, compared to said production in a corresponding reference strain. The NADH dependent side-product formation is preferably reduced by more than 10% compared to the corresponding reference strain, in particular by at least 20%, more in particular by at least 50%. NADH dependent side-product production is preferably reduced by 10-100%, in particular by 20-95%, more in particular by 50-90%.

In an embodiment a fermentation process is provided, wherein RuBisCO, or another enzyme capable of catalysing the formation of an organic compound from CO2 (and another substrate) or another enzyme that catalyses the function of CO2 as an electron acceptor, is used, and carbon dioxide is present in the gas mixture above the fermentation broth and/or dissolved in the fermentation broth. In a specific embodiment, the carbon dioxide or part thereof is formed in situ by the microorganism.

If desired, the method further comprises the step of adding external CC to the reaction system, usually by aeration with CO2 or a gas mixture containing CO2, for instance a CO2 /nitrogen mixture. Adding external CO2 in particular is used to (increase or) maintain the CO2 within a desired concentration range, if no or insufficient CO2 is formed in situ.

As a carbon source, in principle any carbon source that the microorganism can use as a substrate can be used. In particular an organic carbon source may be used, selected from the group of carbohydrates and lipids (including fatty acids). Suitable carbohydrates include monosaccharides, disaccharides, and hydrolysed polysaccharides (e.g. hydrolysed starches, lignocellulosic hydrolysates). Although a carboxylic acid may be present, it is not necessary to include a carboxylic acid such as acetic acid, as a carbon source.

As shown in the Examples below, the invention is in suitable for the production of an alcohol, notably ethanol. However, it is contemplated that the insight that CO2 can be used as an electron acceptor in microorganisms that do not naturally allow this, has an industrial benefit for other biotechnological processes for the production of organic molecules, in particular organic molecules of a relatively low molecular weight, particularly organic molecules with a molecular weight below 1000 g/mol. The following items are mentioned herein as embodiments of the use of carbon dioxide as an electron acceptor in accordance with the invention.

Regarding the production of ethanol, details are found herein above, when describing the yeast cell comprising PRK and RuBisCO and in the examples. The ethanol or another alcohol is preferably produced in a fermentative process.

For the production of several organic acids (carboxylates), e.g. citric acid, an aerobic process is useful. For citric acid production for instance Aspergillus niger, Yarrowia lipolytica, or another known citrate producing organism may be used.

An example of an organic acid that is preferably produced anaerobically is lactic acid. Various lactic acid producing bacterial strains and yeast strains that have been engineered for lactate production are generally known in the art. Other embodiments of the invention are now described in more detail.

In an embodiment the invention relates to the use of the recombinant yeast cell as described herein in fermentation in the biofuel industry. The recombinant yeast cell may contain genes of a pentose metabolic pathway non-native to the recombinant yeast cell and/or that allow the recombinant yeast cell to convert pentose(s). In one embodiment, the recombinant yeast cell may comprise one or two or more copies of one or more xylose isomerases and/or one or two or more copies of one or more xylose reductase and xylitol dehydrogenase genes, allowing the recombinant yeast cell to convert xylose. In an embodiment thereof, these genes may be integrated into the recombinant yeast cell genome. In another embodiment, the recombinant yeast cell comprises the genes araA, araB and araD. It is then able to ferment arabinose. In one embodiment of the invention the recombinant yeast cell comprises xylA- gene, XYL1 gene and XYL2 gene and/or XKS1- gene, to allow the recombinant yeast cell to ferment xylose; deletion of the aldose reductase (GRE3) gene; and/or overexpression of GAL2 and/or deletion of GAL80. Thus though inclusion of the above genes, suitable pentose or other metabolic pathway(s) may be introduced in the recombinant yeast cell that were non-native in the (wild type) recombinant yeast cell. According to an embodiment, the following genes may be introduced in the recombinant yeast cell by introduction into a host cell:

1 ) a set consisting of PPP-genes TAL1, TKL1, RPE1 and RKI1, optionally under control of strong constitutive promoter;

2) a set consisting of a xy/A-gene under control of strong constitutive promoter;

3) a set comprising a XKS1- gene under control of strong constitutive promoter,

4) a set consisting of the genes araA, araB and araD under control of a strong constitutive promoter

5) deletion of an aldose reductase gene

The above cells may be constructed using known recombinant expression techniques. The co-factor modification may be effected before, simultaneous or after any of the modifications 1 ) to 5). The recombinant yeast cell according to the invention may be subjected to evolutionary engineering to improve its properties. Evolutionary engineering processes are known processes. Evolutionary engineering is a process wherein industrially relevant phenotypes of a microorganism, herein the recombinant yeast cell, can be coupled to the specific growth rate and/or the affinity for a nutrient, by a process of rationally set-up natural selection. Evolutionary Engineering is for instance described in detail in Kuijper, M, et al, FEMS, Eukaryotic cell Research 5(2005) 925-934, W02008/041840 and W02009/1 12472. After the evolutionary engineering the resulting pentose fermenting recombinant yeast cell is isolated. The isolation may be executed in any known manner, e.g. by separation of cells from a recombinant yeast cell broth used in the evolutionary engineering, for instance by taking a cell sample or by filtration or centrifugation.

In an embodiment, the recombinant yeast cell is marker-free. As used herein, the term "marker" refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker. Marker-free means that markers are essentially absent in the recombinant yeast cell. Being marker-free is particularly advantageous when antibiotic markers have been used in construction of the recombinant yeast cell and are removed thereafter. Removal of markers may be done using any suitable prior art technique, e.g. intramolecular recombination.

In one embodiment, the industrial recombinant yeast cell is constructed on the basis of an inhibitor tolerant host cell, wherein the construction is conducted as described hereinafter. Inhibitor tolerant host cells may be selected by screening strains for growth on inhibitors containing materials, such as illustrated in Kadar et al, Appl. Biochem. Biotechnol. (2007), Vol. 136-140, 847- 858, wherein an inhibitor tolerant S. cerevisiae strain ATCC 26602 was selected.

The recombinant yeast cell further may comprise those enzymatic activities required for conversion of pyruvate to a desired fermentation product, such as ethanol, butanol (e.g. n-butanol, 2-butanol and isobutanol), lactic acid, 3 -hydroxy- propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, fumaric acid, malic acid, itaconic acid, an amino acid, 1 ,3- propane-diol, ethylene, glycerol, a b-lactam antibiotic or a cephalosporin.

In an embodiment, the recombinant yeast cell is derived from an industrial recombinant yeast cell. An industrial cell and industrial recombinant yeast cell may be defined as follows. The living environments of (recombinant yeast cell) cells in industrial processes are significantly different from that in the laboratory. Industrial recombinant yeast cells must be able to perform well under multiple environmental conditions which may vary during the process. Such variations include change in nutrient sources, pH, ethanol concentration, temperature, oxygen concentration, etc., which together have potential impact on the cellular growth and ethanol production of Saccharomyces cerevisiae. Under adverse industrial conditions, the environmental tolerant strains should allow robust growth and production. Industrial recombinant yeast cell strains are generally more robust towards these changes in environmental conditions which may occur in the applications they are used, such as in the baking industry, brewing industry, wine making and the biofuel ethanol industry. In one embodiment, the industrial recombinant yeast cell is constructed on the basis of an industrial host cell, wherein the construction is conducted as described hereinafter. Examples of industrial yeast cell (S. cerevisiae) are Ethanol Red® (Fermentis) Fermiol® (DSM) and Thermosacc® (Lallemand).

The recombinant yeast cells according to the invention are preferably inhibitor tolerant, i.e. they can withstand common inhibitors at the level that they typically have with common pretreatment and hydrolysis conditions, so that the recombinant yeast cells can find broad application, i.e. it has high applicability for different feedstock, different pretreatment methods and different hydrolysis conditions. In an embodiment the recombinant yeast cell is inhibitor tolerant. Inhibitor tolerance is resistance to inhibiting compounds. The presence and level of inhibitory compounds in lignocellulose may vary widely with variation of feedstock, pretreatment method hydrolysis process. Examples of categories of inhibitors are carboxylic acids, furans and/or phenolic compounds. Examples of carboxylic acids are lactic acid, acetic acid or formic acid. Examples of furans are furfural and hydroxy- methylfurfural. Examples or phenolic compounds are vannilin, syringic acid, ferulic acid and coumaric acid. The typical amounts of inhibitors are for carboxylic acids: several grams per liter, up to 20 grams per liter or more, depending on the feedstock, the pretreatment and the hydrolysis conditions. For furans: several hundreds of milligrams per liter up to several grams per liter, depending on the feedstock, the pretreatment and the hydrolysis conditions. For phenolics: several tens of milligrams per liter, up to a gram per liter, depending on the feedstock, the pretreatment and the hydrolysis conditions.

In an embodiment, the recombinant yeast cell is a cell that is naturally capable of alcoholic fermentation, preferably, anaerobic alcoholic fermentation. A recombinant yeast cell preferably has a high tolerance to ethanol, a high tolerance to low pH (i.e. capable of growth at a pH lower than about 5, about 4, about 3, or about 2.5) and towards organic and/or a high tolerance to elevated temperatures.

In an embodiment one or more genes of the non-oxidative branch of the pentose phosphate pathway of the recombinant yeast of the invention are overexpressed, and/or a glycerol-3- phosphate dehydrogenase (GPD) gene is deleted or disrupted. In another embodiment a glycerol- 3-phosphate dehydrogenase (GPD) gene is deleted or disrupted. In yet another embodiment one or more genes of the non-oxidative branch of the pentose phosphate pathway of the recombinant yeast of the invention are overexpressed and a glycerol-3-phosphate dehydrogenase (GPD) gene is deleted or disrupted. The GPD gene may be a GPD1 and/or a GPD2 gene. Both GPD1 and GPD2 genes may be deleted or disrupted, although it is preferred that GPD2, but not GPD1 is deleted or disrupted. The GPD gene encodes for an enzyme having at least EC number 1.1.1.8. WO201 1/010923 describes methods to delete or disrupt a glycerol-3-phosphate dehydrogenase. In an embodiment the one or more genes of the pentose phosphate pathway that is overexpressed encodes for an enzyme selected from the list of a transaldolase (EC 2.2.1 .2), a transketolase (EC 2.2.1 .1 ), a ribose-5-phosphate isomerase (EC 5.3.1.6) and a D-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1 ). In another embodiment the one or more genes of the pentose phosphate pathway that is overexpressed is selected from the list of TAL1 , TAL2, NQM1 , TKL1 , TKL2, RPE1 and RKI1.

The invention further provides a process for the production of an organic compound, in particular ethanol comprising:

fermenting a composition comprising a fermentable carbohydrate in the presence of a recombinant yeast according to the invention, thereby forming the organic compound; and

recovering the organic compound.

In said process the glycerol yield is preferably at least 5%, at least 10% or at least 10%, at least 20% or at least 30% lower than that of a process with the corresponding wild-type recombinant yeast cell. In an embodiment of such process, the ethanol yield is not increased or decreased, compared to that of a process with the corresponding wild-type recombinant yeast cell.

In an embodiment the composition comprising a fermentable carbohydrate is a biomass hydrolysate. By "hydrolysate" is meant a polysaccharide that has 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 One such biomass hydrolysate may be a lignocellulosic biomass hydrolysate. Lignocellulose herein includes hemicellulose and hemicellulose parts 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, 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. 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.

In another embodiment such composition is a pre-treated cornstover hydrolysate. Another preferred composition is a corn fiber hydrolysate, which is optionally pre-treated. Pretreatment may be done with conventional methods, e.g. contacting with cellulases, for instance cellobiohydrolase(s), endoglucanase(s), beta-glucosidase(s) and optionally other enzymes, The conversion with the cellulases may be executed at ambient temperatures or at higher temperatures, at a reaction time to release sufficient amounts of sugar(s). The result of the enzymatic hydrolysis is hydrolysis product comprising C5/C6 sugars, herein designated as the sugar composition.

In yet another embodiment such composition is a starch hydrolysate, such as a corn starch hydrolysate. In an embodiment the fermentable carbohydrate is obtained from starch, lignocellulose, and/or pectin.

The starch, lignocellulose, and/or pectin may be contacted with an enzyme composition, wherein one or more sugar is produced, and wherein the produced sugar is fermented to give a fermentation product, wherein the fermentation is conducted with a recombinant yeast of the invention.

The process is particularly useful when glycerol is fed externally to the process, such as crude glycerol from transesterification-based biodiesel production or recirculation of backset, which is then taken up and converted to ethanol by the claimed recombinant yeast.

The fermentation process may be an aerobic or an anaerobic fermentation process. An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than about 5, about 2.5 or about 1 mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable), and wherein organic molecules serve as both electron donor and electron acceptors. In the absence of oxygen, NADH produced in glycolysis and biomass formation, cannot be oxidised by oxidative phosphorylation. To solve this problem many microorganisms use pyruvate or one of its derivatives as an electron and hydrogen acceptor thereby regenerating NAD⁺.

Thus, in an embodiment, anaerobic fermentation process pyruvate is used as an electron (and hydrogen acceptor) and is reduced to fermentation products such as ethanol, butanol, lactic acid, 3 -hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, malic acid, fumaric acid, an amino acid and ethylene.

The fermentation process is preferably run at a temperature that is optimal for the cell. Thus, for most recombinant yeast cells, the fermentation process is performed at a temperature which is less than about 50°C, less than about 42°C, or less than about 38°C. For recombinant yeast cell or filamentous fungal host cells, the fermentation process is preferably performed at a temperature which is lower than about 35, about 33, about 30 or about 28°C and at a temperature which is higher than about 20, about 22, or about 25°C.

In an embodiment the organic compound made with the process of the invention is selected from the group consisting of ethanol, n-butanol, 2-butanol, isobutanol, lactic acid, 3-hydroxy- propionic acid, acrylic acid, acetic acid, succinic acid, fumaric acid, malic acid, itaconic acid, maleic acid, citric acid, adipic acid, an amino acid, such as lysine, methionine, tryptophan, threonine, and aspartic acid, 1 ,3-propane-diol, ethylene, glycerol, a b-lactam antibiotic and a cephalosporin, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, including biofuels and biogas or organic polymers, and an industrial enzyme, such as a protease, a cellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, an oxidoreductases, a transferase or a xylanase.

For the recovery of the organic product existing technologies can be used. For different fermentation products different recovery processes are appropriate. Existing methods of recovering ethanol from aqueous mixtures commonly use fractionation and adsorption techniques. For example, a beer still can be used to process a fermented product, which contains ethanol in an aqueous mixture, to produce an enriched ethanol-containing mixture that is then subjected to fractionation (e.g., fractional distillation or other like techniques). Next, the fractions containing the highest concentrations of ethanol can be passed through an adsorber to remove most, if not all, of the remaining water from the ethanol. In an embodiment in addition to the recovery of fermentation product, the yeast may be recycled. The following non-limiting examples are intended to be purely illustrative.

EXAMPLES

Example 1 : Expression of DAN1p-prk in a RuBisCO expressing strain from the CEN.PK lineage

Maintenance of strains

Strains originating from the CEN.PK lineage were used in this example (Table 4) [1 ,2].

Cultures were propagated in synthetic medium [3], supplemented with 20 g L^_1 glucose. Propagation of E. coli DH5a stock cultures was performed 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 or 50 pg mL^-1 kanamycin. Frozen stocks were prepared by addition of glycerol (30 % v/v final concentration) to growing cultures and subsequent storage at -80°C.

Table 4. S. cerevisiae strains used in this example.

Plasmid and cassette construction

A list of plasmids used in this example is given in Table 5. CRISPR/Cas9 genome editing was used to perform genetic modifications in all constructed strains [5]. Unique CRISPR/Cas9 sequences targeting GPD1, GPD2, SGA1 or -2 were identified using a publicly available list [5]. For markerless deletion of GPD1 and GPD2, plasmid pUDR240 was constructed. The plasmid backbone was PCR amplified using primer combination 5793-5793 (double-binding) and pROSIO as template. The plasmid insert, containing the expression cassettes coding for the unique 20-bp gRNA sequences targeting GPD1 and GPD2, was obtained using primer combination 6965-6966 and plasmid pROSI O as template. For markerless genomic integration of gene cassettes, plasmids expressing unique gRNAs targeting the SGA1 locus or the intergenic region X-2 [6] were constructed. The plasmid backbones of puDR1 19 and pURD164 were obtained by PCR amplification using the primer combination 5792-5980 and plasmids pMEL1 1 and pMEL10, respectively, as templates. The plasmid inserts of pUDR1 19 and pUDR164, containing the expression cassettes coding for the unique 20-bp gRNA sequences targeting SGA1 and X-2 respectively, were obtained by PCR amplification using the primer combinations 5979-7023 for SGA1 and 5979-7374 for X-2 and plasmids pMEL1 1 and pMEL10, respectively, as templates. Phusion® Hot Start II High Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA, USA) was used for construction of plasmids and expression cassettes in all cases, according to the manufacturer’s guidelines. The assembly of plasmids pUDR1 19, pUDR164 and pUDR240 was performed in vitro using the Gibson Assembly® Cloning kit (New England Biolabs, Ipswich, MA, USA) following the supplier’s guidelines. The assembly was enabled by homologous sequences present at the 5’ and 3’ ends of the PCR-amplified plasmid backbones and inserts. In each case, 1 ul of the Gibson-assembly mix was used for E. coli DH5a transformation by electroporation, performed in a Gene PulserXcell Electroporation System (Biorad, Hercules, CA, USA). Correct assembly of plasmids was confirmed by diagnostic PCR (Dreamtaq®, Thermo Scientific) or restriction digestion. The constructed plasmids pUDR1 19, pUDR164 and pUDR240 were isolated from transformed E. coli cultures using a Sigma GenElute Plasmid kit (Sigma-Aldrich, St. Louis, MO, USA) and used for transformation of S. cerevisiae.

A yeast expression cassette of cbbM was obtained by PCR amplification using plasmid pBTWW002 as template and primer combination 7549-7550. The resulting fragment was ligated to a pJET/1.2 blunt vector (Thermo-Scientific) following the supplier’s protocol and cloned to E. coli. The resulting plasmid was used as PCR template to generate integration cbbM cassettes, using primer combinations 7074-7075 (integration at the SGA1 locus along with prk, groES, groEL), 7548- 6285, 6280-6273, 6281-6270, 6282-6271 , 6284-6272, 6283-6275, 6287-6276, 6288-6277, 6289- 7075 (multiple-cbbM-copy integration at the SGA1 locus). The expression cassettes of cbbM were genetically identical, except for different overhangs present at the 5’ and 3’ ends of the fragments to allow for in vivo homologous recombination. Yeast expression cassettes of groEL and groES were obtained using plasmids pUD232 and pUD233 as templates and primer combinations 7076- 7077 and 7078-7079 respectively. The genomic sequences corresponding to the constitutive promoters of LYS1, UBC6, YEN1 and the anaerobically active promoter of DAN1 [7] were obtained by PCR amplification with primer combinations 7082-7083, 7292-7294, 7293-7295 and 7930-7931 respectively, using genomic DNA of IMX585 as template. In the case of integration at the X-2 locus, primer combination 7933-7295 was used for amplification of the YEN1 promoter region. The terminator of PGK1 was obtained by PCR amplification with genomic DNA of IMX585 as template using primer combinations 7084-7085 (integration at the SGA1 locus along with cbbM, groES, groEL) and 7084-7934 (individual integration of prk at the X-2 locus). The ORF of prk was obtained by PCR amplification using primer combinations 7080-7081 ( LYS1p cassette construction), 7296- 7081 ( UBC6p cassette construction), 7297-7081 ( YEN1p cassette construction), 7932-7081 (DAN1p cassette construction) and plasmid pUDE046 as template. The various primer combinations resulted in pr/c-ORF fragments with homologous overhangs to the different promoter sequences and the terminator of PGK1. The expression cassettes LYS1p-prk-PGK1t, UBC6p-prk- PGK1t and YEN1p-prk-PGK1t were assembled in vitro using fusion PCR by combining the respective promoter/ prk/PGKIt fragments as templates and primer combinations 7082-7085, 7292- 7085 and 7293-7085 respectively, in the case of aimed integration at the SGA1 locus of strain IMX675 (along with a KIURA3 fragment, cbbM cassete and groEL, groES chaperones. When prk cassettes were integrated individually (integration at the X-2 locus), the complete expression cassettes ( YEN1 p-prk-PGK1t and DAN1p-prk-PGK1t) was assembled by in vivo homologous recombination after transformation to yeast and correct assembly was verified by diagnostic PCR. Primer combination 7086-7087 was used to obtain a KIURA3 fragment using plasmid pUG72 as template.

Table 5. Plasmids used in this example.

Strain construction

The lithium-acetate transformation protocol was used for yeast transformations [11]. Transformation mixtures were plated on synthetic medium agar plates [3] (2 % Bacto Agar, BD, Franklin Lakes, NJ, USA), supplemented with 20 g L^_1 glucose in the case of transformations performed with puDR164 and pUDR240. In transformations performed with plasmid pUDR119, the agar plates were prepared as described previously [12]. For the construction of strain IMX765 uracil was additionally supplemented to the agar plates (150 mg L ¹) (Sigma-Aldrich). Confirmation of the desired genotypes in each case was performed by diagnostic colony PCR. Recycling of pUDR240 was performed using 5-fluoorotic acid (Zymo Research, Irvine, CA, USA) counter-selection, following the supplier’s guidelines. Recycling of pUDR1 19 was performed as described previously [12]. Strain IMX675 was constructed by co-transformation of the double-gRNA-expressing, GPD1/GPD2 targeting plasmid pUDR240 and the repair-oligonucleotide combinations 6967-6968 and 6969-6970 to the Cas9-expressing strain IMX581 (after plasmid recycling from the correct mutant). The expression cassettes LYS1p-prk-PGK1t, UBC6p-prk-PGK1t and YEN1p-prk-PGK1t were respectively co-transformed to strain IMX675 along with a single copy of the cbbM cassette, groEL, groES, the URA3 fragment and the gRNA-expressing, SGA 1-targeting plasmid pUDR1 19. Overhangs present at the 5’ and 3’ ends of the molecules were designed to allow for complete assembly of the pathways in the SGA1 locus. Strain IMX765 was obtained by co-transformation of pUDR1 19, 9 copies of the expression cassette of cbbM and the expression cassettes of groEL and groES to IMX581 (after plasmid recycling from the correct mutant). Overhangs present at the 5’ and 3’ ends of the molecules allowed for in vivo assembly of the entire construct (1 1 fragments) and integration in the SGA1 locus. Strain IMX774 was obtained by transformation of strain IMX765 with the gRNA-expressing, X-2 targeting plasmid pUDR164 and the DAN1p, prk ORF, PGK1t fragments which were assembled in vivo into the complete construct and subsequently integrated in the X-2 locus. Strain IMX773 was obtained by transformation of strains IMX765 with pUDR164 and the YEN1p, prk ORF, PGK1t fragments which were similarly assembled in vivo and subsequently integrated in the X-2 locus. The control strain IME324 was obtained by transformation of IMX581 with the empty vector p426- TEF.

Cultivation media and analytical methods

Physiological characterization of S. cerevisiae strains was performed in anaerobic batch cultivations in 2-L bioreactors (Applikon, Delft, The Netherlands), with 1-L working volume. Salt solutions were sterilized by autoclaving at 120°C for 20 min. Glucose solutions were autoclaved separately at 1 10°C for 20 min and subsequently added to the sterile salt solutions. All fermentations were performed in synthetic medium [3] (20 g L¹ glucose), supplemented with sterile solutions of the anaerobic growth factors ergosterol (10 mg L¹) and Tween 80 (420 mg L¹), as well as with 0.2 g L¹ sterile antifoam C (Sigma-Aldrich). Anaerobic conditions were maintained by sparging of a gas mixture of N₂ / CO₂ (90%/10%, <10 ppm oxygen) at a rate of 0.5 L min ¹ and culture pH was maintained at 5 by automatic addition of 2M KOH. All cultivations were performed at a stirrer speed of 800 rpm and at a temperature of 30°C. Oxygen diffusion in the bioreactors was minimized by equipping them with Norprene tubing and Viton O-rings. Pre-culture shake flask cultivations were performed aerobically in 500-mL flasks containing 100 ml_ synthetic medium (20 g L ¹ glucose). Initial flask pH was adjusted to 6 by addition of KOH. Cultures were grown at 30°C and shaken at 200 rpm. In each case, pre-culture flasks were inoculated from frozen S. cerevisiae stock cultures. After incubation for 8-12 h, cultures from these flasks were used to inoculate fresh pre-culture flasks for bioreactor inoculum propagation. Bioreactors were inoculated to a starting OD66O of ca. 0.2. Off-gas analysis, biomass dry weight measurements, HPLC analysis of culture supernatants and correction for ethanol evaporation in bioreactor experiments were performed as described previously [13]. Optical density was determined at 660 nm, using a Libra S1 1 spectrophotometer (Biochrom, Cambridge, UK). Yields of products in each cultivation were calculated from samples taken at mid-exponential phase (minimum of five samples), as described previously [14]. For the calculation of the degree of reduction (electron) balances in performed fermentations, reported degree of reduction values for biomass, CO2, NhV and extracellular metabolites (glucose, ethanol, glycerol, succinate, pyruvate, lactate, acetate) were used [15]. For determination of in vitro enzymatic activity of PRK, cells from exponentially growing, anaerobic shake-flask cultures in synthetic-medium were harvested and cell-free extracts were prepared as previously described [16]. The harvesting and sonification buffer contained 100 mM Tris-HCI, 20 mM MgCl_2'6H₂0 and 5mM DTT (pH 8.2). The PRK assay contained 50 mM Tris-HCI (pH 8.2), 40 mM KCI, 10 mM MgCl_2'6H₂0, 0.15 mM NADH, 1 mM ATP, 3 mM phosphoenolpyruvate, 1 mM 1 ,4- dithiothreitol, 5 U of pyruvate kinase (EC 2.7.1.40), 6 U of L-lactate dehydrogenase (EC 1.1.1.27) and 30, 50 or 100 pi cell-free extract in 1 ml total volume. Reactions were started by addition of D- ribulose-5-phosphate (2.5 mM final concentration) and PRK activity was measured at 30°C on a Hitachi 100-60 spectrophotometer by monitoring NADH oxidation at 340 nm over time. Protein content determination in cell-free extracts was performed as previously described (Lowrey protein assay) [17].

Physiological characterization of strains

Expression of cbbM and prk in S. cerevisiae has previously been shown to result in decreased formation of the by-product glycerol under anaerobic conditions that are relevant for industrial ethanol production [10]. However, in this previous research, prk was expressed under the control of the galactose-inducible GAL 1-promoter. The requirement for the presence of galactose and low levels of glucose are a drawback of the use of this promoter. This example investigates the expression of prk under the control of different promoters in a strain that co-expresses RuBisCO. Expression cassettes were constructed based on three constitutive promoter sequences of varied expression strengths, with LYS1p being the strongest and YEN1p being the weakest, as well as a weak promoter that is only active in anaerobic conditions ( DAN1 ) [7]. Initially, transformations were performed with prk cassettes under the control of LYS1p, UBC6p and YEN1p and copies of cbbM, groEL, groES, according to [10]. It was not possible to obtain correct mutant colonies in the case where the LYS1p-prk-PGK1t cassette was used. In the case where UBC6p-prk-PGK1t was used, colonies were obtained but growth was inconsistent and severely impacted, and a stable strain was not obtained, see Table 7. However, in the cases where YEN1p-prk-PGK1t (weakest constitutive promoter tested) it was possible to obtain correct mutant colonies (Table 7). These results could be an indication that high & medium-constitutive expression of prk in S. cerevisiae under aerobic conditions (biomass propagation phase) results in inhibitory effects and are in agreement to data available in literature [10]. Based on the above, a multi-copy, cbb/W-expressing strain was constructed with prk under the control of YEN1p (IMX773, low-prk-expression strain). Additionally, a strain was constructed in which a DAN1 p-prk-PGK1t cassette was used instead. The promoter of DAN1 (or any other similar promoter) is of particular interest, because it is active in the process conditions of bioethanol production (anaerobic conditions in this case) and does not require the use of specific carbon sources (like GAL1p does) or any other change to the commonly used production process. This promoter (or any other similar promoter) should alleviate the toxicity of pr/c-expression under aerobic conditions (no transcription). The strain was designated IMX774 (high-pr/c- expression strain). Figure 1 shows the PRK activity in cell-free extracts of exponentially growing shake-flask cultures on synthetic medium containing 20 g L¹ glucose. Left: IME324, Right: IMX774. To determine whether the promoter of DAN1 could drive the expression of prk in S. cerevisiae, PRK enzymatic activity determination was performed in vitro, using cell-free extracts of anaerobically-grown cultures of strains IME324 (reference) and IMX774 ( 9*cbbM , DAN1p-prk). PRK activity in IMX774 was ca. 0.8 pnnol (mg protein) ¹ min ¹ (Fig 1 ). To quantitatively analyze the impact of expression of the introduced RuBisCO pathway, strains IME324 (reference), IMX773 (' 9*cbbM , YEN1p-prk) and IMX774 ( 9*cbbM , DAN1p-prk) were compared in glucose-grown, anaerobic batch cultures in bioreactors. The engineered strains IMX773 and IMX774 grew at 82% and 61 %, respectively, of the specific growth rate of the reference strain IME324. The glycerol yield on glucose of strains IMX773 and IMX774 was 0.098 and 0.058 g/g, respectively. This corresponds to 96% of the glycerol yield for IMX773 compared to IME324 indicating a limited impact of the RuBisCO pathway in a design in which prk is expressed under control of the weak constitutive promoter YEN1p (Table 6). In contrast, for IMX774 a 43% decrease in glycerol yield compared to the reference strain IME324 was observed (Table 6, Fig 2). Furthermore, the ratio of glycerol production per g biomass formed of strain IMX774 decreased by 41 % compared to the reference strain, whereas for IMX773 this was maintained to 97% of IME324 levels indicating a limited effect. A decrease of glycerol production can be expected when NAD⁺ is being regenerated via the RuBisCO pathway. Therefore, these findings are in agreement with results reported elsewhere on galactose-grown RuBisCO-expressing strains [10] and show that the effect of the pathway on galactose-grown cultures can be replicated in glucose-grown ones, when prk is expressed under the control of the promoter of DAN1 but not under control of a weak constitutive promoter such as YEN1p. Even more, prk expression in IMX765 under control of stronger constitutive promoters did not even yield viable colonies, indicating cellular toxicity as a result of transformation of these prk specific expression cassettes. Strains IME324 and IMX774 showed an ethanol yield on glucose of 0.356 and 0.409 g/g respectively (corrected for evaporation). This means that the combination of the decrease in glycerol production, CO2 fixation via the RuBisCO pathway and decrease in biomass yield of the engineered, RuBisCO-expressing strain IMX774 (Fig 2) resulted in a ca. 14% increase in the ethanol yield on glucose in the experiments performed in this example. Table 6. Maximum specific growth rate (m), yields (Y) of glycerol, biomass and ethanol on glucose and the ratio of glycerol formation to biomass formation in anaerobic bioreactor batch cultures of S. cerevisiae strains IME324, IMX773 and IM3X774.

*Degree of reduction balances are given for each individual experiment of independent duplicate cultures. Balances calculated over the exponential sampling phase. Averages and standard deviation values of the balances over the sampling points are given.

Cultures were grown on synthetic medium containing 20 g L¹ glucose (pH 5) and sparged with a gas mixture of N2/CO2 (90%/10%). Yields and ratios were calculated from the exponential growth phase. The ethanol yield on glucose was corrected for evaporation. Values represent average and mean deviation of data from independent duplicate cultures.

Table 7. Aerobic growth properties and glycerol reduction

Example 2: S. oleracea prk protein expressed exclusively under anaerobic conditions in IMX774

Shake flask cultivation strains

IME324 and IMX774 were cultivated in duplicate in mineral medium (according to Luttik et al., 2000) supplemented with 20 g L¹ glucose and 0.05 g L¹ uracil in shake flasks under aerobic and anaerobic conditions. After overnight aerobic propagation on YePhD, 75 mg L¹ of yeast was inoculated to the above described medium in either a 100 ml_ shake flask filled with 25 mL medium closed afterwards with a cotton plug to recreate aerobic cultivation conditions, or a 25 mL shake flask filled with 25 mL medium (leaving limited head space for aeration) closed afterwards with a water lock to recreate conditions which shortly after inoculation and closing off become anaerobic in the vessel. After 24 hours of cultivation at 32°C and 250 rpm (for aerobic cultures) and 150 rpm (for anaerobic cultures), 10 OD600 units/ mL of cells were harvested from each of the eight shake flasks by centrifugation and cells were washed with ice cold demineralized water. Cell pellets were stored at -80 °C for further processing.

Protein extraction and proteomics

Frozen cells were lysed using mechanical based disruption approach via VK05 glass beads and Precellys 24 homogeniser (Bertin Technologies) in the environment of cold Methanol (Sigma). Protein concentration of the disrupted cell suspension was measured using the Qubit 2.0 fluorometer (Invitrogen, Life Technologies). Two hundred fifty ug of total protein was taken from each methanol suspension and 10 ug BSA was spiked to all the samples for quality control. Proteins were extracted from the disrupted cell suspension using chloroform (Sigma) and 20% TCA (Sigma). The obtained protein pellet was dissolved in 100 mM NH4HC03 buffer at pH 7 (Sigma) to a final concentration of 0.5 ug/uL. Proteins were reduced through the addition of 5 ul of 500 mM Tris(2- carboxyethyl)phosphine hydrochloride solution (TCEP, sigma) and incubated at 55 °C for 30 minutes in a thermocycler to facilitate disulfide reduction. Alkylation was performed through the addition of 5 ul of 550mM iodoactamide and incubated at 25°C in the dark for 30 minutes. Proteolysis was carried out overnight in a thermomixer at 37°C with Trypsin Gold (Promega) at an enzyme to substrate ration of 1 :25, which specifically cleaves C-terminally of Lysine and Arginine. Tryptic digests were analyzed on an Ultimate3000 coupled to a QExactive Plus (Thermo Scientific). Gradient elution of peptide was performed on a C18 (Acquity UPLC CSH C18 Column, 130A, 1.7 pm, 2.1 mm X 100 mm). Twenty uL of injected peptides were separated with a gradient of mobile phase A (99.9% water and 0.1 % formic acid; VWR) to 20% B (99.9% acetonitrile and 0.1 % formic acid; VWR) over 20 minutes, and to 50% B over 30 minutes, for a final length of 60 minutes. Data acquisition on the qExactive MS was carried out using a data-dependent method. The top 15 precursors were selected for tandem-MS/MS (MS2) analysis after HCD fragmentation. Full MS scans covering the mass range of 400 to 1600 were acquired at a resolution of 70,000 (at m/z 200), with a maximum fill time of 75 milliseconds, and an automatic gain control (AGC) target value of 3e6. MS2 scans were acquired at a resolution of 17,500 (at m/z 200), with a maximum fill time of 75 milliseconds, and an AGC target value of 1 e5. An isolation window of 2.0 m/z with a fixed first mass of 1 10.0 m/z was applied in all experiments. HCD fragmentation was induced with a normalized collision energy of (NCE) of 27 for all peptides. Charge state exclusion was set to ignore unassigned 1 charge. Isotope exclusion was enabled and peptide match was preferred. All LC- MS/MS results were searched against the S. cerevisiae protein database to which the amino acid sequences of the heterologous introduced enzymes were manually added, using Sequest HT on Proteome Discover 1.4 Sequest HT (Thermo Fisher Scientific, San Jose, CA, USA). The cleavage preference of trypsin was used, allowing up to 2 missed cleavages (C-Term K/R restrict P). Dynamic modifications were set to carbamidomethyl (C), deamidation (N/Q) and oxidation (M). Precursor mass tolerance was set to 10 ppm and fragment mass tolerance 0.6 Da. Following peptide identification, their q-values were calculated based on target decoy approach with a 1 % false discovery rate (FDR) and filtered in the Percolator.

Analysis results

Around 1000 unique proteins were identified in each sample. To quantify the amount of S. oleracea PRK, among all samples, most abundant unique peptides were selected. Peak areas of all peptides were summed up. By normalizing against beta-actin (S. cerevisiae Actl p) amount determined by LC-MS/MS, this value finally gave an indication concerning the protein amount among all samples. As shown in Figure 3, under both aerobic and anerobic conditions no unique prk peptides are detected for negative control strain IME324 which does not express prk. For IMX774 prk peptides are detected solely under anaerobic conditions indicating that the DAN1p is only inducing prk expression. under anaerobic conditions.

Example 3: Expression of S. oleracea prk under control of more anaerobically upregulated promoters results in glycerol reduction in IMX765

Introduction of S. oleracea prk expressed under control of the anaerobically upregulated DAN1 promoter was found to be beneficial to the reduction of glycerol byproduct formation and ethanol yield improvement (Example 1 ). In constrast to DAN1p, prk introduction with other, constitutively active promoters ( UBC6 , LYS1, YEN1) placed upstream of S. oleracea prk did not yield viable, correct transformants ( UBC6 , LYS1) or did not show an impact on glycerol reduction ( YEN1p ) by implementing the RuBisCO pathway. This led us to the conclusion that anaerobically induced / derepressed prk is beneficial to RuBisCO pathway flux and results in ethanol yield improvement by reduction of glycerol. DAN1 is regulated by repressor ROX1 which is absent in anaerobic conditions thereby relieving repression on the DAN1 promoter. More such promoters are regulated by ROX1 which display a differential expression pattern being expressed at a higher level under anaerobic conditions than under aerobic conditions (Kwast et al., 1998). In this example, several of these promoters were placed upstream of S. oleracea prk and introduced to strain IMX765 after which resulting strains were subjected to anaerobic growth cultivations. In Table 8, the promoters tested in Example 3 are listed.

Table 8. Tested promotors

Material and Methods

Expression cassette construction

The promoters (listed in Table 8; SEQ ID NOs: 91 and 93-101 ) and terminator, namely

Sc_PGK1.ter (SEQ ID NO: 102) sequences were synthesized at DNA2.0 (Menlo Park, CA 94025, USA). The S. oleracea prk ORF sequence (Sole_PRK.orf) was obtained by PCR amplification using primer combinations DBC-15631 (SEQ ID NO: 103) and DBC-15632 (SEQ ID NO: 104) using pUDE046 as template. The promoter, ORF and terminator sequences were recombined by using the Golden Gate technology, as described by Engler et al (2011 ) and references therein. The expression cassettes were cloned into a standard subcloning vector. The promoters listed in Table 8 were ligated to Sole_PRK ORF and Sc_PGK1 terminator resulting in expression cassettes listed in Table 9. Table 9: List of expression (promoter-ORF-terminator) cassettes resulting from Golden Gate Cloning of containing promoter variants, S. oleracea PRK ORF and S. cerevisiae PGK1 terminator; on the 5’ end and 3’end connector sequences compatable to neighbouring pathway brick are listed.

Strain construction

Approach The followed strain construction approach is described in patent application PCT/EP2013/056623 and PCT/EP2016/050136. PCT/EP2013/056623 describes the techniques enabling the construction of expression cassettes from various genes of interest in such a way, that these cassettes are combined into a pathway and integrated in a specific locus of the yeast genome upon transformation of this yeast. PCT/EP2016/050136 describes the use of a CRISPR-Cas9 system for integration of expression cassettes into the genome of a host cell, in this case S. cerevisiae. In the construction of IMX765 a S. pyogenes Cas9 expression cassette was already integrated at the CAN1 locus. Upon introduction of an in vivo assembled gRNA-expressing plasmid and repair DNA fragments the intended modifications were made. Firstly, an integration site in the yeast genome was selected. DNA fragments of approximately 500 bp of the up- and downstream parts of the integration locus were amplified by PCR using primers introducing connectors to the generated PCR products. These connectors (50 bp in size) allow for correct in vivo recombination of the pathway upon transformation in yeast. Secondly, the genes of interest, are amplified by PCR, incorporating a different connector (compatible with the connector on the of the neighbouring biobrick) at each flank. Upon transformation of yeast cells with the DNA fragments, in vivo recombination and integration into the genome takes place at the desired location. This technique facilitates parallel testing of multiple genetic designs, as one or more genes from the pathway can be replaced with (an)other gene(s) or genetic element(s), as long as that the connectors that allow for homologous recombination remain constant and compatible with the preceding and following biobrick in the design (patent application PCT/EP2013/056623). gRNA expression plasmid

Integration site: the expression cassettes were targeted at the INT1 locus. The INT1 integration site is a non-coding region between NTR1 (YOR071 c) and GYP1 (YOR070c) located on chromosome XV of S. cerevisiae. The guide sequence to target INT1 was designed with a gRNA designer tool (https://www.dna20.com/eCommerce/cas9/input). The gRNA expression cassette (as described by DiCarlo et al., Nucleic Acids Res. 2013; pp.1-8) was ordered as synthetic DNA cassette (gBLOCK) at Integrated DNA Technologies (Leuven, Belgium) (INT1 gBLOCK; SEQ ID NO: 105). In vivo assembly of the gRNA expression plasmid is then completed by co-transforming a linear fragment derived from yeast vector pRN599. pRN599 is a multi-copy yeast shuttling vector that contains a functional kanMX marker cassette conferring resistance against G418. The backbone of this plasmid is based on pRS305 (Sikorski and Hieter, Genetics 1989, vol. 122, pp.19- 27), including a functional 2 micron ORI sequence and a functional kanMX marker cassette (SEQ ID NO: 106).

Transformation of IMX765 with specified DNA fragments upon assembly comprising anaerobic promoter PRK cassette

Strain IMX765 was transformed with the following fragments resulting in the assembly of anaerobic promoter PRK as depicted in Figure 4: 1 ) a PCR fragment (5 -INT1 ) generated with primers BoZ-783 (SEQ ID NO: 107) and DBC- 19944 (SEQ ID NO: 108) with genomic DNA of strain CEN.PK1 13-7D as template;

2) a PCR fragment (Anaerobic.pro-PRK) generated with primers DBC-5799 (SEQ ID NO: 109 and DBC-5800 (SEQ ID NO: 1 10) using either one of the plasmids listed in Table 5 as template;

3) a PCR fragment generated with primers DBC-19947 (SEQ ID NO: 1 1 1 ) and DBC-19949 (SEQ ID NO.1 12) using genomic DNA of strain CEN.PK1 13-7D as template; this PCR resulted in the 1.2 kb marker cassette conD-l/RA3-conE ( URA3 marker flanked by connectors D and E).

4) a PCR fragment (3 -INT1 ) generated with primers DBC-19946 (SEQ ID NO: 1 13) and BoZ- 788 (SEQ ID NO: 1 14) using genomic DNA of strain CEN.PK1 13-7D as template;

5) a PCR fragment (BB-599) generated with primers DBC-13775 (SEQ ID NO: 1 15) and DBC- 13776 (SEQ ID NO: 1 16) using pRN599 (SEQ ID NO: 106) as template;

6) a PCR fragment (gRNA-INT1 ) generated with primers DBC-13773 (SEQ ID NO: 1 17) and DBC-13774 (SEQ ID NO: 1 18) using INT1 gRNA (SEQ ID NO: 105) as template;

Transformants were selected on mineral medium (according to recipe Luttik et al., 2000, Journal of Bacteriology 182, 24: 501-517) supplemented with 1.5% bactoagar supplemented with 20 g L¹ glucose and 0.2 mg G418 ml_ ¹. Diagnostic PCR was performed to confirm the correct assembly and integration at the INT1 locus of the PRK expression cassettes.

Microtiterplate batch fermentation experiment

Six to nine correct transformants per transformation design and controls strains IME324, IMX765 and IMX774 in nine fold were inoculated to 240mI mineral medium (according to Luttik et al. , 2000) supplemented with 20 g L^-1 glucose and 0.05 g L^-1 uracil in microtiterplate format. Inoculated microtiterplates were cultivated for 2 days at 30°C. Subsequently, grown cultures on agar medium were transferred to liquid 350mI mineral medium (according to Luttik et al. , 2000) supplemented with 20 g L^-1 glucose and 0.05 g L^-1 uracil with the aid of a pintool to the second microtiterplate for biomass propagation. The microtiterplates were sealed with a gas permeable seal enabling aerobic cultivation conditions and were incubated at 32°C, shaking at 750 rpm at 80% humidity for 2 days. After 2 days, 5mI grown liquid culture was transferred to 270mI mineral medium (according to Luttik et al. , 2000) supplemented with 20 g L^-1 glucose and 0.05 g L^-1 uracil to the third microtiterplate for main fermentation. A high volume of 270 mI leaving little head space volume in the well and sealing microtiterplates with aluminium seal allowed for screening mainly under anaerobic conditions. Cultures were grown for 48 hours at 32°C, 250 rpm at 80% humidity. After 48 hours samples were taken in order to measure residual glucose, glycerol and ethanol.

Analysis of glucose, ethanol, and glycerol

For the quantification of glucose, ethanol and glycerol, 150 mI of the supernatant sample was transferred accurately into a suitable vial. Subsequently 100 mI internal standard solution, containing maleic acid (20 g/l), EDTA (40 g/l), DSS (4, 4-dimethyl-4-silapentane-1 -sulfonic acid) (0.5 g/L), adjusted to pH 6.40 with NaOH, in D2O was added. The sample was further diluted with 400 pi D2O. 1 D ¹H NMR spectra of the clear solution were recorded on a Bruker Avance III HD spectrometer, operating at a proton frequency of 500MHz, equipped with a He-cooled cryo probe, using a pulse program with water suppression (ZGCPPR), solvent suppression power of 8Hz, at a temperature of 300K, 90 degree excitation pulse was used and acquisition time of 2.0 seconds and a relaxation delay of 5 seconds. The number of scans was set at 8, dummy scans were not used. The analyte concentrations [in g/L] were calculated based on the following signals (d relative to DSS):

• Glucose: a-H1 glucose signal (d, 5.21 ppm, 0.38 H, J =4Hz)

• Ethanol: (t, 1 .17 ppm, 3H, J =7 Hz)

• Glycerol H 1/H3 signals: (dd, 3.55 ppm, 2H, J =7 Hz, 12 Hz)

• The signal used for the standard:

• Maleic acid: (s, 6.05 ppm, 2H)

Results microtiterplate fermentation experiment

Glucose was completely exhausted in the medium by all replicates of transformants and control strains. The average glycerol levels (expressed as arbitrary units) detected for the transformants with different anaerobic promoter - PRK cassettes and control strains IME324, IMX765 and IMX774 are listed in Table 9 and illustrated in Figure 5. In this MTP fermentation set up, again IMX774 displayed reduced glycerol levels (87%, reduction of 13%) compared to reference strain IME324 as shown in Example 1. Transformants of cDAN1 , which are a reconstruction of IMX774, showed a similar glycerol reduction (85% of IME324 glycerol levels) as IMX774. All tested anaerobic promoter designs displayed reduced glycerol levels compare to IME324. Also one can appreciate designs with anaerobic promoters resulting in transformants with on average even larger reductions of glycerol levels than cDAN1 , such as cTIR3, cHEM13, cAAC3, and cANB1 yielding on average 76%, 68%, 82%, and 80%, respectively, of IME324 glycerol levels. As can be observed in Table 10 and Figure 6 these reductions in glycerol yield were reflected in at least similar, often higher ethanol titers compared to IME324 as seen for cDAN1 (102%), cTIR3 (103%), cHEM13 (104%), cAAC3 (101 %), and cANB1 (103%).

Table 10. Average glycerol yields expressed as arbitrary units per g glucose of

transformants per design anaerobic promoter design and reference strains IME324, IMX774, IMX765 (transformation control) determined in microtiterplate fermentation experiment on mineral medium (according to Luttik) supplemented with 20 g L ¹ glucose and 0.05 g L ¹ uracil. N.A., not applicable.

Table 11. Average ethanol yields expressed as arbitrary units per g glucose of transformants per design anaerobic promoter design and reference strains IME324, IMX774, IMX765

(transformation control) determined in microtiterplate fermentation experiment on mineral medium (according to Luttik) supplemented with 20 g L¹ glucose and 0.05 g L¹ uracil. N.A., not applicable.

j j ! ! !

Example 4. Overexpression of PPP genes and deletion of GPD2 gene.

Genes of the non-oxidative branch of the pentose-phosphate pathway (TKL1 , NQM1 , TKL1 , TKL2, RPE1 , RKI1 ) were overexpressed by transforming IMX774 with expression cassettes of the abovementioned genes under control of constitutive promoters as described in pending European Patent Application EP16194660.3. The expression casettes were integrated at the GPD2 locus by co-transforming the guide RNA expression plasmid with GPD2 targeting sequence, thereby abbrogating the coding sequence of gpd2. The resulting strain was named IMX1443. Strain IMX1443 was compared with IME324 and IMX774 in a batch fermentation experiment as described in Example 1. Results are listed in Table 12.

Table 12. Results using yeast strain with PPP genes and deletion of GPD2 gene

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Claims

1. A recombinant yeast cell functionally expressing one or more heterologous nucleic acid sequences encoding:

- a ribulose-1 , 5-phosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39);

a phosphoribulokinases (EC 2.7.1.19; PRK);

a catalytically inactive endonuclease (CIE); and

a constitutively expressed guide RNA; and optionally:

one or more molecular chaperones for RuBisCO,

2. Recombinant yeast cell of claim 1 wherein the second promoter is the native promoter of an ORF selected from the list consisting of YOR388C, YPL275W, YPL276W, YDR256C,

YHR096C, YNL195C, YGR110W, YCR010C, YDL218W, YPL223C, YJR095W, YMR303C, YGR236C, YHR139C, YPR151C, YMR107W, YMR118C, YLR174W, YPL201C, YDR380W, YMR058W, YBR047W, YML054C, YLR205C, YPL147W, YDR070C, YPR001W, YER065C, YKR009C, YLL053C, and YGR256W.

3. Recombinant yeast cell of claim 1 or 2 wherein the catalytically inactive endonuclease is dCas9 or dCpfl .

4. A recombinant yeast cell according to any of the previous claims whererin the RuBisCO is under a constitutive promotor.

5. A process for the production of an organic compound, in particular ethanol, comprising:

- fermenting a composition comprising a fermentable carbohydrate in the presence of a recombinant yeast according to any one of the previous claims, thereby forming the organic compound; and

recovering the organic compound.