WO2024175777A1

WO2024175777A1 - Product specific transporter for in vivo synthesis of human milk oligosaccharides

Info

Publication number: WO2024175777A1
Application number: PCT/EP2024/054663
Authority: WO
Inventors: Manos PAPADAKIS
Original assignee: Dsm Ip Assets B.V.
Priority date: 2023-02-24
Filing date: 2024-02-23
Publication date: 2024-08-29

Abstract

The present disclosure relates to a genetically engineered cell capable of producing a desired HMO, wherein said cell expresses a transporter protein capable of exporting of the desired HMO from said cell. The application also relates to a method of producing the desired HMO using the genetically engineered cell. Preferably, the desired HMO has an LNT-II backbone, such as for example LNT or LNnT.

Description

PRODUCT SPECIFIC TRANSPORTER FOR IN VIVO SYNTHESIS OF HUMAN MILK OLIGOSACCHARIDES

FIELD

The present disclosure relates to the production of one or more desired Human Milk Oligosaccharides (HMOs) and the genetic engineering of suitable cells expressing a transporter protein capable of exporting the desired HMOs from said cell. The genetically engineered cell described herein is used in the production of the desired HMO.

BACKGROUND

The design and construction of bacterial cell factories to produce Human Milk Oligosaccharides (HMOs) consisting of 3-6 monosaccharide units is of paramount importance to provide innovative and scalable solutions for the more complex HMO products of tomorrow.

To this direction, rational strain engineering principles are commonly applied to a bacterial host cell. Such principles usually refer to a) the introduction of a desired biosynthetic pathways to the host, b) the increase of the cellular pools of relevant activated sugars required as donors in the desired reactions, c) securing sufficient lactose in the host cell, e.g. by the presence of a lactose permease such as LacY and d) the introduction of a heterologous sugar efflux transporter to export the desired newly formed heterologous oligosaccharide (for review see Bych et al 2019 Current Opinion in Biotechnology 56:130-137).

Expression of substrate specific transporters described for step d) in the production strain have attracted growing attention in recombinant HMO-producing cells, e.g., there have recently been described fermentation procedures as well as several new sugar transporter genes encoding proteins that can facilitate the efflux of a recombinantly produced 2’-fucosyllactose (2’-FL), Lacto-N-tetraose (LNT), Lacto-N-neotetraose (LNnT) or sialylated HMOs, such as 3’sialyllactose (3’SL) (WO2010/142305, W02021/148610, WO2021/14861 1 , WO2021/148614, WO2021/148615, WO2021/148620, WO2022/219188, and WO2022/157213).

As can be seen from these disclosures different transporters have different capabilities of exporting recombinantly produced oligosaccharides. The oligosaccharide export can be more or less specific for the desired oligosaccharide versus by-product oligosaccharides that are also produced in the fermentation process. There is therefore an interest in identifying further oligosaccharide exporters with specificities that are optimal for producing purer products in higher amounts.

SUMMARY

A first aspect, the present disclosure relates to a genetically engineered cell capable of producing a desired HMO, wherein said cell comprises one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases and a recombinant nucleic acid sequence encoding a transporter protein, Edict , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence which is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , wherein the expression of said transporter protein in said cell leads to export of the desired HMO from said cell.

Preferably, the desired HMO comprises a lacto-N-triose II (LNT-II, GlcNAc(p1-3)Gal(01 -4)Glc) backbone, more preferably, the HMO is selected from the group consisting of lacto-N- neotetraose (LNnT), lacto-N-tetraose (LNT), lacto-N-fucopentaose V (LNFP-V) and 6’- sialyllacto-N-neotetraose (LST c). In embodiments, at least 85%, such as at least 90%, such as at least 95% of the total molar content of HMO exported from the cell is LNnT or LNT. Preferbly, less than 10 % of the total molar content of HMO exported from the cell is a by-product HMO. In embodiments, less than 10 % of the total molar content of HMO exported from the cell is LNT-II.

In embodiments, the one or more glycosyltransferase(s) comprises a p-1 , 4- galactosyltransferase or a p-1 ,3-galactosyltransferase and optionally a 0-1 ,3-N- acetylglucosaminyltransferase.

In particular embodiments, the cell of the disclosure is selected from the group consisting of Escherichia Coli, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

In a second aspect, the present disclosure relates to a method for producing a HMO product, wherein said method comprises providing a genetically engineered cell according to the first aspect of the disclosure, cultivating the genetically engineered cell in a culture medium under conditions permissive for the production of said HMO; and optionally recovering said HMO. The HMO product produced is preferably LNnT, LNT or a mixture of LNT and LNFP-V or LNnT and LST-c.

A third aspect of the disclosure relates to an desired HMO, such as an HMO selected from the group consisting of lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), lacto-N-fucopentaose V (LNFP-V) and 6’-sialyllacto-N-neotetraose (LST c) whein the desired HMO is produced by the method according the second aspect of the disclosure.

A further aspect of the disclosure relates to a nucleic acid construct comprising a recombinant nucleic acid sequence encoding a transporter protein, Edie 1 , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , wherein the transporter protein encoding sequence is under the control of a promoter sequence. The disclosure also relates to the use of said nucleic acid construct in a host cell producing an HMO which comprises a GlcNAc(pi-3)Gal(|31-4)Glc backbone and at least one additional saccharide moiety, such as comprising a Gal(p1-4)GlcNAc(pi-3)Gal(pi-4)Glc or Gal(p1-3)GlcNAc(p1- 3)Gal(p1 -4)Glc structure.

BRIEF DESCRIPTION OF FIGURES

Figure 1. Overview of LNT and LNnT synthesis

Figure 2. A) Overview of general principle of specific product transport depicted with LNnT and B) principle of product (LNnT) accumulation in supernatant when employing specific product (LNnT) transporter protein.

Figure 3. Overview of a general principle of combination of specific precursor (LNT-II) import and specific product (LNnT) efflux transporter.

Figure 4. Overview of a general principle of combination of specific precursor (lactose) import and specific product (LNnT) efflux transporter.

Figure 5. mMolar (%) distribution of individual HMOs from the total fermentation broth (supernatant and pellet) relative to the total HMO produced by the Vag strain.

Figure 6. mMolar (%) distribution of individual produced HMOs from the supernatant of the fermentation relative to the total HMO in the supernatant of the fermentation of the Vag strain.

Figure 7. HPLC chromatograms of the isolated supernatant following fermentation of (A) Vag and (B) Edict strains.

Figure 8. mMolar (%) distribution of individual HMOs from the total fermentation broth (supernatant and pellet) relative to the total HMO produced by the Nec strain.

Figure 9. Distribution of individual HMOs from strains producing LST-c with or without edict in a deep well assay. A) mMolar (%) LNT-II core HMO distribution in the supernatant relative to the total LNT-II core HMOs in the supernatant of a strain without edict (no TP). B) mMolar (%) LNT- II core HMO distribution in the pellet relative to the total LNT-II core HMOs in the pellet of a strain without edict (no TP).

DETAILED DESCRIPTION

The present disclosure approaches the biotechnological challenges of in vivo HMO production, namely the harvest of specific oligosaccharides from the medium used to culture the production cells. The present disclosure offers specific strain engineering solutions to increase and/or simplify the production of HMOs, especially those with a lacto-N-triose II (LNT-II, GlcNAc(|31- 3)Gal(p1 -4)Glc) backbone by exploiting the potential of oligosaccharide exporter proteins. Surprisingly, the transporter Edicl was found to be highly efficient in transporting HMOs with an LNT-II backbone out of the cell with a low export of the HMO precursor LNT-II. Example 1 in the present disclosure showed that Edicl in an LNnT producing cell, had higher specificity for LNnT over LNT-II and pLNnH, which leads to accumulation of the by-product HMOs LNT-II and pLNnH in the cell, while the product HMO, LNnT, is exported and accumulated in the fermentation medium. This is highly advantageous, since the product HMO to a large extend be isolated directly from the supernatant of the fermentation broth, without the need for isolation from the production cells, including the need for lysis of said cells, thereby avoiding the byproduct HMOs inside the cell, e.g., LNT-II and pLNnH. In consequence, the genetically modified cell described herein provides a means for a method wherein the product HMO, such as an HMO with an LNT-II backbone, in particular LNT or LNnT, can be obtained from the supernatant without significant loss of desired product accumulated in the cells, and with low amounts of byproducts present in the supernatant of the fermentation broth i.e., the production cell acts as a product specific cellular reaction chamber and nano-filter, only allowing the desired HMO into the supernatant, which is a significant advantage in the subsequent purification of the desired HMO product.

In other words, genetically modified cells, such as E. coli strains, covered by the present disclosure express genes encoding key enzymes for HMO biosynthesis, such that the cell can produce the desired HMO, along with one or more genes encoding an efflux transporter, preferably Edid , capable of transporting specific HMOs from the cell to the extracellular media. Edicl may therefore also be termed an exporter protein. Furthermore, the strains described herein may also further comprise genes encoding a transporter for importing desired molecules, also termed an importer protein. The most commonly used importer in HMO production strains is lactose permease for importing the initial substrate for producing HMOs. Alternative, substrate importers are for example a mutant variant of the E. coli LacY protein (Table 1) and/or an ABC and/or MFS transporter originating from a Gram+ bacterium (Table 2) to import a precursor oligosaccharide molecule (initial substrate), such as LNT-II, which can be further decorated by recombinant enzyme(s) within the cell to produce even more complex molecules in the cell, which are then exported by the expressed efflux transporter into the media, thereby greatly simplifying the purification process of the desired HMO.

The advantage of exporting a specific oligosaccharide into the culture media is that it enables a simplified purification of the produced oligosaccharide, wherein the desired oligosaccharide may be purified directly from the supernatant of the fermentation. In addition, the preference of the exporters for transport of specific products, such as LNnT and/or LNT over e.g., the precursor LNT-II or the by-products pLNH2 or pLNnH leads to a lesser amount of by-product HMOs in the culture media. Typically, by-product HMOs are either the precursors (lactose or other acceptor oligosaccharides, such as LNT-II) of the HMO desired to be produced (desired HMO or HMO product) or products of further modifications of the desired HMO product. The fewer glycosyltransferases that are needed to generate the desired HMO product the fewer byproduct HMOs or other impurities can be generated, and the purity of the complex HMO product will therefore increase. For example, if LNT is produced from lactose in a single cell, two glycosyl transferases are required namely, |3-1 ,3-N-acetylglucosaminyl-transferase forming LNT-II, a p-1 ,3-galactosyltransferase forming LNT, whereafter LNT may be further decorated by the introduction of e.g., an alpha-1 , 2-fucosyl-transferase capable of forming LNFP-I from LNT or by a alpha-1 , 3-fucosyl-transferase capable of forming LNFP-V from LNT. In this case the desired HMO products are LNT and/or LNnT, and LNT-II and pLNH2 or pLNnH (hexasaccharides from further decoration of LNT or LNnT, see figure 1) are likely the undesired HMO by-products.

Typically, a genetically modified cell of the present disclosure, which is capable of importing LNT-II, only requires a p-1 ,3-galactosyltransferase or a p-1 ,4-galactosyltransferase which is expressed in the genetically modified HMO producing cell in order to produce LNT and/or LNnT and no lactose will be present, thereby avoiding the production of several undesired HMOs and other impurities (e.g., Gal-LNT and Gal-Lac) as by-products, thereby allowing partial or full conversion of LNT-II to LNT and/or LNnT towards the end of fermentation, since LNT-II in this case is the substrate feed to the genetically modified cell, and the produced LNT and/or LNnT is/are exported from the cell via a specific heterologous efflux transporter, such as Edict .

Furthermore, it has also been observed that the presence of a suitable exporter, such as Edict , can increase the production of both the total amount of HMOs produced by the cell and in particular the amount of the desired HMO when compared to cells without the edict transporter or compared to transporters known in the art. This is for example shown for LNT in example 3 and LNFP-V in example 4. Increasing HMO yields is always desired even if there is an immediate decrease of by-product HMOs since mixtures of HMOs also have potential benefit or a significantly increased yield can result in a cheaper overall process despite not leading to a simplified purification process.

There are several applications of biotechnological interest, wherein the concept of the present disclosure could be relevant.

In the following sections, further details relating to elements of the genetically modified cell, the method of product using said cell as well manufactured products and their applications are described in more detail. T ransporters

The genetically modified cell according to the present disclosure comprises at least one recombinant nucleic acid sequence encoding a transporter protein capable of exporting a specific HMO product, also called an exporter protein.

The present disclosure offers specific strain engineering solutions to produce specific HMOs by exploiting the potential of exporter proteins identified herein, and in particular the transporter protein Edicl from Edwardsiella ictalurid identified with the GenBank accession

WP_015873007.1 (https://www.ncbi.nlm.nih.gOv/protein/ WP_015873007.1), also disclosed in SEQ ID NO: 1. Said putative MFS transporter protein is identified herein as “Edicl protein” or “Edicl transporter” or “Edicl exporter” or “Edicl”, interchangeably; a nucleic acid sequence encoding Edicl protein is identified herein as “Edicl coding nucleic acid/DNA” or “Edicl gene" or “edicl".

The transporter protein Edicl from Edwardsiella ictaluri is, in the cell of the present disclosure, encoded by a heterologous gene encoding the putative MFS (major facilitator superfamily) transporter protein Edicl , originating from the bacterium Edwardsiella ictaluri.

Edicl has in the present disclosure been shown to be a product specific transporter for oligosaccharides with an LNT-II backbone, such as LNnT and LNT and further decorated versions of LNT and LNT, such as sialylated and/or fucosylated LNnT, in particular LST-c or sialylated and/or fucosylated LNT, in particular LNFP-V.

More specifically, the disclosure relates to a genetically modified cell optimized to produce an oligosaccharide, such as a heterologous oligosaccharide, in particular a desired HMO or an HMO product. More specifically the genetically modified cell that can produce the desired HMO product, such as an HMO with a LNT-II backbone, comprises a recombinant nucleic acid encoding a protein having at least 80%, such as 85%, such as 90% such as 95% or 100% sequence identity to the amino acid sequence of GenBank accession WP_015873007.1 (https://www.ncbi.nlm.nih.goV/protein/ WP_015873007.1), also disclosed in SEQ ID NO: 1.

Accordingly, in preferred embodiments, the present disclosure relates to a genetically engineered cell capable of producing a desired human milk oligosaccharide (HMO) which is not naturally produced by said cell (heterologous), wherein said cell comprises, one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases, and a recombinant nucleic acid sequences encoding a transporter protein, Edicl , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , wherein the expression of said transporter protein in said cell leads to export of the desired HMO from said cell or increased production of the desired HMO as compared to a cell without the exporter or with an exporter which is already known to export the desired HMO.

In embodiments, the cell expressing a transporter protein, Edict , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , is capable of producing LNnT, wherein the total molar content of LNnT produced by said cell is at least 85%, such as at least 88%, such as at least 89%, such as at least 90%, or such as at least 91 % of the total HMO content produced by said cell.

As described in example 1 , the transporter Edici exports most of the LNnT produced by the cell into the extracellular medium, while the unwanted by-product HMOs stays inside said cell (illustrated in Figure 2A), thereby resulting in most of the total LNnT produced I present in the supernatant, while only small fraction is maintained inside the cells (biomass fraction or pellet) as illustrated in Figure 2B. Accordingly, in embodiments, at least 85%, such as at least 90%, such as at least 95%, such as at least 99% of the total molar content of desired HMO produced by said cell is exported from the cell expressing the product specific transporter Edici . In further embodiments, at least 85%, such as at least 90%, such as at least 95%, such as at least 99% of the total molar content of HMO exported from the cell expressing a product specific transporter is LNnT. In that regard it is highly preferable that only a minor content of the total HMO exported from the cell expressing the product specific transporter Edici is by-product HMO, such as LNT-II or pLNnH. In embodiments, less than 10 % of the total molar content of HMO exported from the cell is a by-product HMO, such as less than 10 % of the total molar content of HMO exported from the cell is LNT-II. In further embodiments, less than 0.1 % of the total molar content of HMO exported from the cell is pLNnH. In particular when the cell is an LNnT producing cell.

In further embodiments, the genetically engineered cell, expressing the product specific transporter Edici described herein may express a second transporter protein, such as an importer protein or a second exporter protein.

IN embodiments the second exporter is selected from MFS transporters known to export HMOs.

The term “MFS transporter” in the present context means, a protein that facilitates transport of an oligosaccharide, preferably, an HMO, through the cell membrane, preferably transport of an HMO/oligosaccharide synthesized by the genetically engineered cell as described herein from the cell cytosol to the cell medium. Additionally, or alternatively, the MFS transporter may also facilitate efflux of molecules that are not considered HMO or oligosaccharides, such as lactose, glucose, cell metabolites and/or toxins. One suitable MFS transporter protein can be obtained from Rosenbergiella nectarea. One exemplary MFS transporter protein from Rosenbergiella nectarea contains or comprises the amino acid sequence of SEQ ID NO: 83 and is identified herein as “Nec protein” or “Nec transporter” or “Nec”, interchangeably; a nucleic acid sequence encoding nec protein is identified herein as “Nec coding nucleic acid/DNA” or “nec gene” or “nec”; The amino acid sequence identified herein as SEQ ID NO: 83 is the amino acid sequence that is 100 % identical to the amino acid sequence having the GenBank accession ID WP_092672081.1 and described in WO2021/148615. In a further embodiment the second MFS transporter has the amino acid sequence of SEQ ID NO: 83 or is a functional homologue having an amino acid sequence which at least 80% identical, such as at least 85% identical, such as at least 90 % identical, such as at least 95 % identical or such as at least 99 % identical to any one of SEQ ID NO: 83.

Another suitable MFS transporter protein can be obtained from Pantoea vagans. One exemplary MFS transporter protein from Pantoea vagans contains or comprises the amino acid sequence of SEQ ID NO: 5 is identified herein as “Vag protein” or “Vag transporter” or “Vag”, interchangeably; a nucleic acid sequence encoding vag protein is identified herein as “Vag coding nucleic acid/DNA” or “vag gene” or “vag”; The amino acid sequence identified herein as SEQ ID NO: 5 is the amino acid sequence that is 100 % identical to the amino acid sequence having the GenBank accession ID WP_048785139.1 and described in WO2021/148611 . In a further embodiment the second MFS transporter has the amino acid sequence of SEQ ID NO: 5 or is a functional homologue having an amino acid sequence which at least 80% identical, such as at least 85% identical, such as at least 90 % identical, such as at least 95 % identical or such as at least 99 % identical to any one of SEQ ID NO: 5.

In one or more exemplary embodiments, the second transporter protein the second exporter is selected from an MFS transporter from Pantoea vagans or from Rosenbergiella nectarea, such as the MFS transporter Vag with an amino acid sequence that is at least 85 % identical to SEQ ID NO: 5 or the MFS transporter Nec with an amino acid sequence that is at least 85 % identical to SEQ ID NO: 83.

In further embodiments, a LNnT producing cell, expressing the product specific transporter Edicl described herein additionally express a second transporter protein, in particular the MFS transporter Vag with an amino acid sequence of SEQ ID NO: 5, or a functional homologue thereof having an amino acid sequence which at least 80% identical, such as at least 85% identical, such as at least 90 % identical, such as at least 95 % identical or such as at least 99 % identical to SEQ ID NO: 5.

In embodiments, the cell expressing a transporter protein, Edicl , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , is capable of producing LNT, wherein the total molar content of LNT produced by said cell is at least 80%, such as at least 85%, such as at least 88%, such as at least 89%, such as at least 90%, or such as at least 91% of the total HMO content produced by said cell.

In further embodiments, a LNT producing cell, expressing the product specific transporter Edic1 produce less than 20% of LNT-II and less than 0.5% pLNH2 of the total molar content of HMO expressed from the cell. In further embodiments, a LNT producing cell, expressing the product specific transporter Edid produce at least 10% more HMO than a similar cell expressing the Nec transporter (SEQ ID NO: 83). Preferably, the LNT producing cell expressing the product specific transporter Edid produce at least 5%, such as at least 8% more LNT and at least 0.5% less pLNH2 than a similar cell expressing the Nec transporter.

In further embodiments, a LNT producing cell, expressing the product specific transporter Edid described herein additionally express a second transporter protein, in particular the MFS transporter Nec with an amino acid sequence of SEQ ID NO: 83 or a functional homologue thereof having an amino acid sequence which at least 80% identical, such as at least 85% identical, such as at least 90 % identical, such as at least 95 % identical or such as at least 99 % identical to SEQ ID NO: 83. Preferably, an LNT producing cell, comprising both the Edid and the Nec transporter or functional variants thereof, produce at least 85%, such as at least 90% LNT and less than 15% such as less than 10% LNT-II and less than 15 such as less than 0.5% pLNH2 of the total HMO produced by the cell.

In further embodiments, a LNT producing cell, expressing the product specific transporter Edid and the Nec transporter, or functional variants thereof, produce at least 10% more HMO than a similar cell expressing only the Nec transporter (SEQ ID NO: 83). Preferably, the LNT producing cell expressing the product specific transporter Edid and Nec produce at least 15%, such as at least 20% more LNT and at least 3%, such as at least 5% less LNT-II and at least 0.5% less pLNH2 than a similar cell expressing only the Nec transporter.

In embodiments, the cell expressing a transporter protein, Edid , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , is capable of producing LNFP-V. In further embodiments, a LNFP-V producing cell, expressing the product specific transporter Edid described herein produce at least 20 fold, such as at least 30 fold, such as at least 40 fold more LNT-II core HMOs than a similar cell without the Edid transporter. In particular the LNFP-V levels are increased by at least 5 fold, such as at least 8 fold compared to a similar cell not expressing Edid . In embodiments, the cell expressing a transporter protein, Edict , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , is capable of producing LST-c. In further embodiments, a LST-c producing cell, expressing the product specific transporter Edict described herein export at least 5% more LST-c to the supernatant than a cell without the Edict transporter.

Substrate importers

In addition, the genetically modified cell according to the present disclosure may also comprise at least one recombinant nucleic acid sequence encoding a transporter protein capable of importing an HMO product precursor, such as an initial substrate, also referred to as an acceptor oligosaccharide, such as, but not limited to lactose and/or LNT-II.

Substrate importers, capable of importing substrates for HMO synthesis, such as, but not limited to lactose or LNT-II, may be used to improve the production of the desired HMO/HMO product, such as LNT and/or LNnT or fucosylated or sialylated versions thereof. The underlying principle is depicted in figures 3 and 4 for production of LNnT as non-limiting examples, wherein the presence of a substrate importer is shown to directly import lactose (fig. 4) or LNT-II (fig. 3), which increases the substrate/precursor availability and hence the product production of e.g., LNnT as depicted in figure 1 . The presence of both an importer and a specific product exporter is particularly favorable, since this allows for optimal conditions for the production of the specific HMO product in the cell, while avoiding product accumulation in the cell. Thus, in embodiments, the cell further comprises a substrate importer selected from a lactose importer and a lacto-N- triose-ll (LNT-II) importer.

In further embodiments, the cell of the present disclosure comprises, a. one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases, b. a recombinant nucleic acid sequences encoding a transporter protein, Edict , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , and c. a recombinant nucleic acid sequences encoding a precursor import protein, wherein the expression of the precursor import protein leads to import of desired precursor disaccharide or oligosaccharides into said cell and the expression of said transporter protein Edict in said cell leads to export of the desired oligosaccharide (HMO product) from said cell. Lactose permease (LacY) is known in its wild-type form to transport the disaccharide lactose from the cell exterior into the E. coll cell, and is therefore a desired importer protein if the initial substrate for the HMO production is lactose.

Mutated variants of LacY have been described to be capable of transporting the trisaccharide maltotriose (Olsen et al 1993 J Bacteriol.175(19):6269-75). In the present disclosure, these mutants are described as potential importers of trisaccharides (acceptor oligosaccharides/HMO precursor molecules) of relevance in the HMO production, e.g., lacto-N-triose (LNT-II).

The genetically modified cell according to the present disclosure may in addition to the efflux transport protein, such as Edid , also comprise a recombinant nucleic acid sequence encoding a transporter protein capable of importing an intermediate (acceptor) oligosaccharide of at least three monosaccharide units into said cell, wherein said transporter protein is a mutated lactose permease (LacY) as shown in table 1.

Table 1. List of exemplary mutants of the E. coll DH1 K12 lactose permease LacY (SEQ ID NO: 14) that could be useful for the import of 2’FL, 3FL or LNT-II.

In preferred embodiments, the lactose permease variants of table 1 have higher affinity for LNT- II compared to lactose. In particular embodiments, the cell of the present disclosure comprises a mutated lactose permease (LacY) as shown in table 1 , wherein a lactose permease variant is selected from the group consisting of mut2 and mut10 as shown in table 1. Accordingly, in embodiments, the cell of the present disclosure comprises a recombinant nucleic acid sequence encoding a mutated lactose permease (LacY), comprises a His at position 236, and/or an Vai at position 177 along with a Thr at position 306, with reference to SEQ ID NO: 14. In one embodiment, the LacY variant comprises the following substitutions Y236H and/or, A177V and S306T as compared to SEQ ID NO: 14, wherein the variant has at least 80% identity to SEQ ID NO: 14, such as at least 85% identity, such as at least 90% identity, such as at least 95% identity, or such as 99.5% identity to SEQ ID NO: 14.

Accordingly, in embodiments, the cell of the present disclosure comprises, a. one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases, and b. a recombinant nucleic acid sequences encoding a transporter protein, Edicl , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , and c. a nucleic acid sequences encoding a lactose permease, such as LacY, comprising or consisting of an amino acid sequence according to SEQ ID NO: 14, or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 14, wherein the expression of said transporter protein in said cell leads to export of the desired oligosaccharide (HMO product) from said cell.

In embodiments it may be an advantage to overexpresses one or more lactose permease(s), such as the native lactose permease. In one embodiment an additional copy of the nucleic acid encoding the lactose permease is recombinantly introduced into the host cell, preferably into the genome of the host cell.

The overexpression of one or more lactose permease(s) according to the present disclosure can enhance the production of one or more HMO(s), in particular LNT or LNnT.

By “overexpression” is in the present context meant that the expression level of a desired protein is higher than what is obtained naturally from the endogenous copy of the nucleic acid encoding the desired protein in the cell. Overexpression may be determined by transcriptional or translational analysis, of for instance, quantitative determination of mRNA levels or protein levels in any of the methods known to the person skilled in the art, such as but not limited to, quantitative PCR or mass spectrometry. The overexpression can for example be achieved by exchanging the wild type promoter of the endogenous gene with a stronger promoter.

Alternatively, one or more additional copies of the endogenous genes can be recombinantly introduced into the host cell, or nucleic acids resulting in negative regulatory effects on the desired endogenous gene can be deleted.

In other embodiments, the cell of the present disclosure comprises, a. one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases, b. a recombinant nucleic acid sequences encoding a transporter protein, Edict , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , and c. a recombinant nucleic acid sequences encoding a LacY variant comprising or consisting of an amino acid sequence according to SEQ ID NO: 14, or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 14 wherein said variant comprises a mutation according to table 1 wherein the expression of the LacY variant leads to import of desired precursor oligosaccharides into said cell and the expression of said transporter protein in said cell leads to export of the desired oligosaccharide (HMO product) from said cell.

In a preferred embodiment the LacY variant comprises or consist of the following substitutions Y236H and/or, A177V and S306T as compared to SEQ ID NO: 14.

Alternative importer proteins with the potential to import LNT-II have been identified in Grampositive (Gram+) bacteria, and in particular in members of the Bifidobacterium, Roseburia and Eubacterium species.

Table 2 shows MFS-transporter proteins of gram-positive or gram-negative origin and ABC- transporter protein clusters of gram-positive origin, capable of importing an acceptor oligosaccharide of at least three monosaccharide units into a cell.

Table 2. ABC- and MFS-transporters from gram-positive bacteria with an indication of the substrate oligosaccharide the transporter is expected to import. The ABC transporters are composed of three to four genes. For ease of reference each transporter has been given a transporter ID (TP ID).

The present disclosure relates to a genetically modified cell as described herein, further comprising a recombinant nucleic acid sequence encoding an importer protein. The present disclosure relates to a genetically modified cell as described herein further comprising a recombinant nucleic acid sequence encoding an importer protein selected from table 2. The present disclosure relates to a genetically modified cell comprising a recombinant nucleic acid sequence encoding a transporter protein, Edict , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 . In addition, the cell of the present disclosure may also comprise a cluster of recombinant nucleic acid sequences encoding a transporter protein and/or a cluster of proteins capable of importing an acceptor oligosaccharide of at least three units into said cell, wherein said cluster of proteins is an ABC transporter from a gram-positive cell. In particular, the cell of the present disclosure may also comprise an ABC transporter as listed in table 2, in particular a transporter selected from the group consisting of TP ID: 8 (Blon_2177, Blon_2176, Blon_2175), TP ID 1 1 (BBR_0527/lntP1 , BBR_0528/lntP2, BBR_0530/lntS, BBR_0531), TP ID 13 (Blon_0962) or TP ID 18 (BBPC_1775, BBPC_1776, BBPC_1777).

In embodiments, the disclosure relates to a genetically engineered cell of the present disclosure, capable of producing a desired human milk oligosaccharide (HMO product), wherein said cell further comprises a recombinant nucleic acid sequences encoding a transporter protein capable of importing an acceptor oligosaccharide of preferably three units into said cell, wherein said protein is Blon_0962, comprising or consisting of an amino acid sequences according to SEQ ID NO: 42, or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 42.

In embodiments, the disclosure relates to a genetically engineered cell of the present disclosure capable of producing a desired human milk oligosaccharide (HMO), wherein said cell further comprises, a recombinant nucleic acid sequences encoding a cluster of proteins capable of importing an acceptor oligosaccharide of preferably three units into said cell, wherein said cluster of proteins is a transporter selected from the group consisting of TP ID: 8, 11 or 18.

In embodiments, the disclosure relates to a genetically engineered cell of the present disclosure capable of producing a desired human milk oligosaccharide (HMO), wherein said cell further comprises, a recombinant nucleic acid sequences encoding a cluster of proteins capable of importing an acceptor oligosaccharide of preferably three units into said cell, wherein said cluster of proteins is the of TP ID: 8, wherein TP ID: 8 comprise or consists of the fragments Blon_2177, Blon_2176 and Blon_2175, comprising or consisting of an amino acid sequences according to SEQ ID NO: 60, 61 and 62, or functional variants thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 60, 61 and 62.

In embodiments, the disclosure relates to a genetically engineered cell of the present disclosure capable of producing a desired human milk oligosaccharide (HMO), wherein said cell further comprises, a recombinant nucleic acid sequences encoding a cluster of proteins capable of importing an acceptor oligosaccharide of preferably three units into said cell, wherein said cluster of proteins is the of TP ID: 11 , wherein TP ID: 1 1 comprise or consists of the fragments BBR_0527/lntP1 , BBR_0528/lntP2, BBR_0530/lntS and BBR_0531 , comprising or consisting of an amino acid sequences according to SEQ ID NO: 75, 76, 77 and 78, or functional variants thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 75, 76, 77 and 78.

In embodiments, the disclosure relates to a genetically engineered cell of the present disclosure capable of producing a desired human milk oligosaccharide (HMO), wherein said cell further comprises, a recombinant nucleic acid sequences encoding a cluster of proteins capable of importing an acceptor oligosaccharide of preferably three units into said cell, wherein said cluster of proteins is the of TP ID: 18, wherein TP ID: 18 comprise or consists of the fragments BBPC_1775, BBPC_1776 and BBPC_1777, comprising or consisting of an amino acid sequences according to SEQ ID NO: 72, 72 and 74, or functional variants thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 72, 72 and 74.

Typically, the genetically modified cell lacks enzymatic activity liable to degrade the acceptor oligosaccharide of at least three or four monosaccharide units.

LacY negative

In one aspect, the genetically modified cell according to the present disclosure does not express a functional lactose permease. I.e., the genetically modified cell is lacY negative. In particular the genetically modified cell does not express the wild-type lactose permease, but may express one or more of the lactose permease mutants in table 1 .

The E. coli endogenous native lactose permease (LacY) has specificity towards galactose and simple galactosyl disaccharides like lactose. The disruption of the endogenous native lacY gene in E.coli is thus a highly sufficient genetic tool to specifically hinder the import of lactose from the cell exterior into the cytoplasm and thus for ensuring that preferably oligosaccharides with a more complex structure, such as oligosaccharides of at least 3 monosaccharide units are imported into said cell by means of the herein described specific transporter protein and/or a cluster of proteins capable of importing an acceptor oligosaccharide of e.g. 3 monosaccharide units into said cell.

In the present context, the term “lacY negative” is used to describe the disruption of the native lactose permease (LacY) in the genetically modified cell and does not exclude that the genetically modified cell comprises a recombinant nucleic acid sequence that is selected from the group consisting of mutated LacY (e g., as shown in table 1), as long as that recombinant nucleic acid sequence encodes a transporter protein and/or a cluster of proteins capable of importing an acceptor oligosaccharide of at least 3 monosaccharide units into said cell.

Oligosaccharides

In the present context, the term “oligosaccharide” means a sugar polymer containing at least three monosaccharide units, i.e., a tri-, tetra-, penta-, hexa- or higher oligosaccharide. The oligosaccharide can have a linear or branched structure containing monosaccharide units that are linked to each other by interglycosidic linkages. Particularly, the oligosaccharide comprises a lactose residue at the reducing end and one or more naturally occurring monosaccharides of 5-9 carbon atoms selected from aldoses (e.g., glucose, galactose, ribose, arabinose, xylose, etc.), ketoses (e.g., fructose, sorbose, tagatose, etc.), deoxysugars (e.g. rhamnose, fucose, etc.), deoxy-aminosugars (e.g. N-acetyl-glucosamine, N-acetyl-mannosamine, N-acetyl- galactosamine, etc.), uronic acids and ketoaldonic acids (e.g. N-acetylneuraminic acid). Preferably, the oligosaccharide is an HMO. Human milk oligosaccharides (HMOs)

Preferred oligosaccharides of the disclosure are human milk oligosaccharides (HMOs).

The term “human milk oligosaccharide" or "HMO" in the present context means a complex carbohydrate found in human breast milk. The HMOs have a core structure comprising a lactose unit at the reducing end that can be elongated by one or more beta-N-acetyl- lactosaminyl and/or one or more beta-lacto-N-biosyl unit, and this core structure can be substituted by an a-L-fucopyranosyl and/or an a-N-acetyl-neuraminyl (fucosyl) moiety. HMO structures are e.g., disclosed by Xi Chen in Chapter 4 of Advances in Carbohydrate Chemistry and Biochemistry 2015 vol 72.

HMOs are either neutral or acidic. In this regard, the non-acidic (or neutral) HMOs are devoid of a sialyl residue, and the acidic HMOs have at least one sialyl residue in their structure. The non- acidic (or neutral) HMOs can be fucosylated or non-fucosylated.

Examples of such neutral non-fucosylated HMOs include lacto-N-triose II (LNT-II) lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), para-lacto-N- neohexaose (pLNnH), para-lacto-N-hexaose (pLNH) and lacto-N-hexaose (LNH).

Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2’FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3’FL), difucosyllactose (DFL or LDFT), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N- difucohexaose III (LNDFH-III), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl- para-lacto-N-hexaose I (FpLNH-l), fucosyl-para-lacto-N-neohexaose II (F-pLNnH II) and fucosyl- lacto-N-neohexaose (FLNnH).

Examples of acidic HMOs include 3’-sialyllactose (3’SL), 6’-sialyllactose (6’SL), 3-fucosyl-3’- sialyllactose (FSL), 3’-sialyllacto-N-tetraose a (LST a), fucosyl-LST a (FLST a), 6’-sialyllacto-N- tetraose b (LST b), fucosyl-LST b (FLST b), 6’-sialyllacto-N-neotetraose (LST c), fucosyl-LST c (FLST c), 3’-sialyllacto-N-neotetraose (LST d), fucosyl-LST d (FLST d), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I (SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II) and disialyl-lacto-N-tetraose (DSLNT).

In the context described herein, the desired HMOs are preferably HMOs that comprise at least 4 monosaccharide units, such as LNT and/or LNnT. In addition, the desired HMOs of the present disclosure are preferably HMOs which comprise a lacto-N-triose II (LNT-II, GlcNAc(p1-3)Gal(pi- 4)Glc) backbone. By the term LNT-II backbone is meant that in the structure of the HMO the following monosaccharides, N-acetylglucosamine (GIcNAc), galactose (gal) and glucose (glc) are linked in the following configuration, GlcNAc(p1-3)Gal(pi-4)Glc, and are present in connection with at least one additional modification, such as an additional monosaccharide. HMOs with an LNT-II backbone may also be termed LNT-II core HMOs.lN the context of the present disclosure, LNT-II itself is not considered to be an HMO with a LNT-II backbone. Preferably, the HMO consist of or contains the following structure Gal(pi-4)GlcNAc(pi- 3)Gal(p1 -4)Glc or Gal(p1-3)GlcNAc(pi-3)Gal(pi-4)Glc. Examples of HMOs comprising LNT-II backbone are e.g., LNT, LNnT, LNnH, pLNnH, pLNH, LNH, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, LST a, LST b, LST c, LST d, FLST a, FLST b, FLST c, FLST d, FLNH-I, FLNnH-l, SLNH, and DSLNT.

In one embodiment of the present disclosure, the desired human milk oligosaccharide (HMO) is an HMO of only four monosaccharide units, such as LNT or LNnT.

In another embodiment of the present disclosure, the desired human milk oligosaccharide (HMO) is an HMO of five monosaccharide units, where LNT or LNnT has been fucosylated or sialylated. Preferred, HMOs of five monosaccharides are LNFP-V and LST-c.

In the production of one or more specific desired HMOs, the synthesis may also result in unwanted HMOs species, described herein as HMO by-products. In example, in the production of LNnT as the desired HMO product, the synthesis may also give rise to both LNT-II and pLNnH (as shown in figure 1), wherein LNT-II and pLNnH are considered as unwanted byproduct oligosaccharides. The cell of the present disclosure may be considered as a cellular purification system, which enables the separation of the desired HMO product and the undesired by-product oligosaccharides.

An acceptor/precursor molecule

A genetically engineered cell according to the present disclosure comprises one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases, which enables the production of a desired HMO form a precursor molecule, such as a di- or oligosaccharide, e.g., lactose or LNT-II.

As described herein, an acceptor oligosaccharide is a molecule that can act as a substrate for a glycosyltransferase capable of transferring a glycosyl moiety from a glycosyl donor to the precursor molecule. The precursor molecule is also sometimes termed the initial substrate if it is the first molecule to be decorated by the glycosyltransferase. The initial substrate molecules is preferably a di- or trisaccharide. Depending on the complexity of the oligosaccharide produced, further acceptor molecules can be produced inside the cell, LNT is for example the acceptor molecule for the further production of e.g., LNFP-I, LNFP-II, LNFP-V and LST-a and LNnT is the acceptor molecule for the further production of e.g., LNFP-III, LNFP-VI and LST-c. The glycosyl donor is preferably a nucleotide-activated sugar as described in the section on “Glycosyl-donor - nucleotide-activated sugar pathways”. Preferably, the acceptor saccharide is a precursor for making a more complex HMO and can also be termed the precursor molecule. The acceptor saccharide or acceptor oligosaccharide can be either an intermediate product of the present fermentation process, an end-product of a separate fermentation process employing a separate genetically engineered cell, or an enzymatically or chemically produced molecule.

In the present context, said acceptor oligosaccharide for the production of the desired HMO is preferably lactose and/or LNT-II, which is either imported directly from production media or, in the case of LNT-II can either be produced from the initial precursor molecule lactose (e.g., acceptor for the |3-1 ,3-N-acetyl-glucosaminyl-transferase) (see figure 1) or imported directly into the cell via a substrate transporter as described herein. The initial precursor molecule is preferably fed to the genetically engineered cell, which is capable of producing e.g., LNT-II, LNT, LNnT or more complex HMOs from the precursor molecule. Most often, the initial precursor is lactose, and the genetically engineered cell is capable of producing the intermediate precursors (acceptor oligosaccharides, e.g. LNT-II and LNnT or LNT) inside the cell. Alternatively, the initial precursor may also be LNT-II if the cell is capable of importing this.

As an alternative to feeding the initial precursor molecule/substrate for the production of the desired HMO to the fermentation medium, the genetically modified cell may be further engineered to produce the initial substrate inside the cell (see for example WO2015/150328).

Glycosyltransferases

The genetically engineered cell according to the present disclosure comprises at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase.

The genetically engineered cell according to the present disclosure may comprise one or more further recombinant nucleic acids encoding one or more recombinant and/or heterologous glycosyltransferase capable of transferring a glycosyl residue from a glycosyl donor to an acceptor oligosaccharide. Preferably, the additional glycosyltransferase(s) enables the genetically engineered cell to synthesize LNT or LNnT from a precursor molecule, such as lactose or LNT-II. In embodiments, the genetically engineered cell described herein, comprises one or more further recombinant nucleic acid encoding one or more recombinant and/or heterologous glycosyltransferase.

The additional glycosyltransferase is preferably selected from the group consisting of, galactosyltransferases and glucosaminyltransferases.

The genetically modified cell according to the present disclosure further comprises at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase capable of transferring a glycosyl residue from a glycosyl donor to said acceptor oligosaccharide to synthesize a human milk oligosaccharide product having at least four monosaccharide units.

The glycosyltransferase is preferably selected from the group consisting of galactosyltransferases, glucosaminyltransferases, N-acetylglucosaminyl transferases and N- acetylglucosaminyl transferases. In one aspect, the glycosyltransferase is selected from the beta-1 ,4-galactosyltransferases or beta-1 ,3-galactosyltransferases listed herein. In embodiments, the one or more glycosyltransferases are selected from the group consisting of -

1 .3-N-acetylglucosaminyltransferase, p-1 ,4-galactosyltransferase and p-1 ,3- galactosyltransferase. In preferred embodiments the genetically engineered cell comprises a p-

1 .3-N-acetylglucosaminyltransferase and p-1 ,4-galactosyltransferase and is capable of producing LNnT. In another preferred embodiments the genetically engineered cell comprises a p-1 ,3-N-acetylglucosaminyltransferase and p-1 ,3-galactosyltransferase and is capable of producing LNT.

Additionally, genetically engineered cell may comprise one or more further glycosyltransferase selected from the group of enzymes having the activity of an a-1 ,2-fucosyltransferase, a-1 ,3- fucosyltransferase, a-1 ,3/4-fucosyltransferase, a-1 ,4-fucosyltransferase a-2,3-sialyltransferase, a-2,6-sialyltransferase.

Typically, the glycosyl donor is a nucleotide-activated sugar or an oligosaccharide, such as selected from the group consisting of glucose-UDP-GIcNAc, GDP-fucose, UDP-galactose (UDP-gal), UDP-glucose (UDP-glc), UDP-N-acetylglucosamine (UDP-GIcNAc), UDP-N- acetylgalactosamine (UDP-glaNAc) and CMP-N-acetylneuraminic acid (CMP-Neu5Ac), preferably UDP-Gal and/or UDP-GIcNAc is used.

Said glycosyl donor is preferably synthesized by endogenous or recombinant pathways in the genetically engineered cells, but can alternatively by exogenously added to the culture medium. Preferably, the glycosyl donor is a nucleotide-activated sugar which is synthesized by the host cell either using an already existing pathway (endogenous), which may be modified to increase the pool of the relevant nucleotide-activated sugar or by introducing nucleotide sequences encoding for enzymes needed to produce the relevant nucleotide-activated sugar within the cell.

In the present disclosure, the at least one functional enzyme capable of transferring a saccharide moiety from a glycosyl donor to an acceptor oligosaccharide can be selected from the list consisting of galT and galTK. These enzymes can for example be used to produce LNnT or LNT, respectively, starting from LNT-II as acceptor oligosaccharide.

In a preferred embodiment the genetically modified cell according to the present disclosure does not comprise more than two or more than three recombinant nucleic acid sequences encoding a glycosyltransferase capable of transferring a glycosyl residue from a glycosyl donor to said acceptor oligosaccharide to synthesize a human milk oligosaccharide product having at least four or five monosaccharide units. The one or two or three glycosyltransferase activities are preferably selected from the activities described below. Heterologous /3-1,3-N-acetyl-glucosaminyl-transferase

A p-1 ,3-N-acetyl-glucosaminyl-transferase is any protein which comprises the ability of transferring the N-acetyl-glucosamine of UDP-N-acetyl-glucosamine to lactose or another acceptor molecule, in a beta-1 ,3-linkage (see figure 1). Preferably the p-1 ,3-N-acetyl- glucosaminyl-transferase used herein does not originate in the species of the genetically engineered cell, i.e., the gene encoding the p-1 ,3-N-acetyl-glucosaminyl-transferase is of heterologous origin.

Accordingly, in embodiments, the genetically engineered cell comprises one or more recombinant nucleic acid sequence(s) encoding a p-1 ,3-N-acetyl-glucosaminyltransferase.

Non-limiting examples of p-1 ,3-N-acetyl-glucosaminyltransferases are given in table 3. p-1 ,3-N- acetyl-glucosaminyltransferase variants may also be useful, preferably such variants are at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identical to the amino acid sequence of any one of the p-1 ,3-N-acetyl-glucosaminyltransferase in table 3.

Table 3. List of p-1 ,3-N-acetyl-glucosaminyltransferase

In embodiments, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding a p-1 ,3-N-acetyl-glucosaminyltransferase. In one embodiment, the recombinant nucleic acid sequence encoding a p-1 ,3-N-acetylglucosaminyltransferase comprises or consists of the amino acid sequence of SEQ ID NO: 11 (LgtA from N. meningitidis) or a functional homologue thereof with an amino acid sequence with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 11.

For the production of LNnT or LNT from lactose as substrate, the LNT-II precursor is formed using a p-1 ,3-N-acetylglucosaminyltransferase. In embodiments, the genetically engineered cell comprises a p-1 ,3-N-acetylglucosaminyltransferase gene, or a functional homologue or fragment thereof, to produce the intermediate LNT-II from lactose.

Some of the examples below use the heterologous p-1 ,3-N-acetyl-glucosaminyl-transferase named LgtA from Neisseria meningitidis or a variant thereof. Heterologous p-1, 3-galactosyltransferase

A p-1 , 3-galactosyltransferase is any protein that comprises the ability of transferring the galactose of UDP-Galactose to a N-acetyl-glucosaminyl moiety to an acceptor molecule in a p- 1 ,3-linkage (see figure 1). In embodiments, the cell of the present disclosure comprises a p-1 , 4- glycosyltransferase and optionally a p-1 ,3-N-acetylglucosaminyltransferase. Preferably, a p-1 , 3- galactosyltransferase used herein does not originate in the species of the genetically engineered cell i.e., the gene encoding the p-1 , 3-galactosyltransferase is of heterologous origin. As described herein the acceptor molecule, is an acceptor saccharide, e.g., LNT-II, or more complex HMO structures.

The examples below use the heterologous p-1 , 3-galactosyltransferase named GalTK or a variant thereof, to produce LNT. p-1 ,3-galactosyltransferases can be obtained from any one of a number of sources, e.g., the galTK gene from H. pylori as described, (homologous to GenBank protein Accession

BD182026.1) or the WbgO gene from E. coli 055:H7 (GenBank Accession WP_000582563.1) or the jhp0563 gene from H. pylori (GenBank Accession AEZ55696.1).

In one embodiment, the recombinant nucleic acid sequence encoding a p-1 ,3- galactosyltransferases comprises or consists of the amino acid sequence of SEQ ID NO: 12 (galTK from H. pylori) or a functional homologue thereof with an amino acid sequence with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% identity to SEQ ID NO: 12.

To produce LNT form an LNT-II precursor, a p-1 , 3-galactosyltransferase is needed. In one embodiment, the genetically modified cell comprises a p-1 , 3-galactosyltransferase gene, or a functional homologue or fragment thereof.

In further embodiments, the genetically engineered cells described herein comprises a p1 ,3-N- acetylglucosaminyltransferase with an amino acid sequence according to SEQ ID NO: 11 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 11 and a p-1 , 3-galactosyltransferase with an amino acid sequence according to SEQ ID NO: 12, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 12.

Below are examples of genetically modified strains described herein with specific combinations of glycosyl transferases that will lead to production of LNT using lactose or LNT-II as initial substrate.

In one example, LgtA from Neisseria meningitidis is used in combination with galTK from Helicobacter pylori to produce LNT starting from lactose as initial substrate. In one example, galTK from Helicobacter pylori is used to produce LNT starting from LNT-II as initial substrate.

Heterologous p-1 ,4-galactosyltransferase

A p-1 ,4-galactosyltransferase is any protein that comprises the ability of transferring the galactose of UDP-Galactose to a N-acetyl-glucosaminyl moiety to an acceptor molecule in a p - 1 ,4-linkage (see figure 1). Preferably, a p-1 ,4-galactosyltransferase used herein does not originate in the species of the genetically engineered cell i.e., the gene encoding the p-1 ,4- galactosyltransferase is of heterologous origin. In the context described herein the acceptor molecule, is an acceptor saccharide, e.g., LNT-II, or more complex HMO structures.

The examples below use the heterologous p-1 ,4-galactosyltransferase GalT, or a variant thereof, to produce LNnT. Accordingly, in embodiments, the genetically engineered cell comprises one or more recombinant nucleic acid sequence(s) encoding a p-1 ,4- galactosyltransferase.

Non-limiting examples of p-1 ,4-galactosyltransferases are provided in table 4. p-1 ,4- galactosyltransferases variants may also be useful, preferably such variants are at least 80%, such as at least 85%, such as at least 90, such as at least 95% identical to the amino acid sequence of any one of the p-1 ,4-galactosyltransferases in table 4.

Table 4. List of p-1 ,4-glycosyltransferases

In embodiments described herein the p-1 ,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis, and the p-1 ,3-galactosyltransferase and/or p-1 ,4-galactosyltransferase is from Helicobacter pylori from Helicobacter pylori, respectively.

In one embodiment, the recombinant nucleic acid sequence encoding a p-1 ,4- galactosyltransferases comprises or consists of the amino acid sequence of SEQ ID NO: 13 (galT from H. pylori) or a functional homologue thereof with an amino acid sequence with at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, or such as at least 99% sequence identity to SEQ ID NO: 13.

To produce LNnT form an LNT-II precursor, a p-1 ,4-galactosyltransferase is needed. In one embodiment, the genetically engineered cell comprises a p-1 ,4-galactosyltransferase gene, or a functional homologue or fragment thereof.

In embodiments, the genetically engineered cells described herein comprises a p-1 ,3-N- acetylglucosaminyltransferase from Neisseria meningitidis and a p-1 ,4-galactosyltransferase is from Helicobacter pylori. In further embodiments, the p1 ,3-N-acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 1 1 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 1 1 and the (3-1 ,4- galactosyltransferase has an amino acid sequence according to SEQ ID NO: 13, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 13.

In one example, LgtA from Neisseria meningitidis is used in combination with galT from Helicobacter pylori to produce LNnT starting from lactose as initial substrate.

In one example, galT from Helicobacter pylori is used to produce LNnT starting from LNT-II as initial substrate.

In one example, LgtA from Neisseria meningitidis is used in combination with LgtB from Neisseria meningitidis to produce LNnT starting from lactose as initial substrate.

In one example, LgtB from Neisseria meningitidis is used to produce LNnT starting from LNT-II as initial substrate.

Alpha-1 ,2-fucosyltransferase

An a-1 ,2-fucosyltransferase is a protein that comprises the ability to catalyze the transfer of fucose from a donor substrate, for example, GDP-fucose, to an acceptor molecule in an alpha- 1 ,2-linkage. Preferably, an alpha-1 , 2-fucosyltransferase used herein does not originate in the species of the genetically engineered cell i.e., the gene encoding the alpha-1 , 2- fucosyltransferase is of heterologous origin. Non-limiting examples of alpha-1 , 2- fucosyltransferase are given in table 4. Alpha-1 ,2-fucosyltransferase variants may also be useful, preferably such variants are at least 80%, such as at least 85%, such as at least 90, such as at least 95% identical to one of the alpha-1 , 2-fucosyltransferase in table 13.

Table 13. List of a-1 ,2-fucosyltransferase

Alpha-1 ,3-fucosyltranferase

An alpha- 1 ,3-fucosyltranferase refer to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate for example, GDP-fucose, to an acceptor molecule in an alpha- 1 ,3-linkage. Preferably, an alpha- 1 ,3-fucosyltransferase used herein does not originate in the species of the genetically engineered cell i.e., the gene encoding the alpha-1 , 3- fucosyltransferase is of heterologous origin. Non-limiting examples of alpha-1 , 3- fucosyltransferase are given in table 5. Alpha-1 ,3-fucosyltransferase variants may also be useful, preferably such variants are at least 80%, such as at least 85%, such as at least 90, such as at least 95% identical to one of the alpha-1 , 3-fucosyltransferase in table 14.

Table 14. List of a- 1 ,3-fucosyltransferase

Alpha-1 ,3/4-fucosyltransferase

An alpha- 1 ,3/4-fucosyltransferase refer to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate for example, GDP-fucose, to an acceptor molecule in an alpha- 1 ,3- or alpha 1 ,4- linkage. Preferably, an alpha-1 ,3/4-fucosyltransferase used herein does not originate in the species of the genetically engineered cell i.e., the gene encoding the alpha- 1 ,3/4-fucosyltransferase is of heterologous origin. Non-limiting examples of alpha-1 ,3/4- fucosyltransferase are given in table 6. alpha-1 , 3/4-fucosyltransferase variants may also be useful, preferably such variants are at least 80%, such as at least 85%, such as at least 90, such as at least 95% identical to one of the alpha-1 , 3/4-fucosyltransferase in table 15.

Table 15. List of a- 1 ,3/4-fucosyltransferase

Alpha-2, 3-sialyltransferase

An a-2, 3-sialyltransferase refer to a glycosyltransferase that catalyzes the transfer of sialyl from a donor substrate for example, CMP-N-acetylneuraminic acid, to an acceptor molecule in an alpha-2, 3-linkage. Preferably, an alpha-2, 3-sialyltransferase used herein does not originate in the species of the genetically engineered cell i.e., the gene encoding the 2, 3-sialyltransferase is of heterologous origin. Non-limiting examples a-2, 3-sialyltransferase are given in table 7. a-2, 3- sialyltransferase variants may also be useful, preferably such variants are at least 80%, such as at least 85%, such as at least 90, such as at least 95% identical to one of the a-2, 3- sialyltransferase in table 16.

Table 16. List of a-2, 3-sialyltransferase

Alpha-2, 6-sialyltransferase

An alpha-2, 6-sialyltransferase refer to a glycosyltransferase that catalyzes the transfer of sialyl from a donor substrate for example, CMP-N-acetylneuraminic acid, to an acceptor molecule in an alpha-2,6- linkage. Preferably, an alpha-2, 6-sialyltransferase used herein does not originate in the species of the genetically engineered cell i.e., the gene encoding the 2, 6-sialyltransferase is of heterologous origin. Non-limiting examples a-2, 6-sialyltransferase are given in table 8. a- 2, 6-sialyltransferase variants may also be useful, preferably such variants are at least 80%, such as at least 85%, such as at least 90, such as at least 95% identical to one of the a-2, 6- sialyltransferase in table 17. Table 17. List of a-2,6-sialyltransferase

Glycosyl-donor - nucleotide-activated sugar pathways

When carrying out the method of this disclosure, preferably a glycosyltransferase mediated glycosylation reaction takes place in which an activated sugar nucleotide serves as glycosyl- donor. An activated sugar nucleotide generally has a phosphorylated glycosyl residue attached to a nucleoside. A specific glycosyl transferase enzyme accepts only a specific sugar nucleotide. Thus, preferably the following activated sugar nucleotides are involved in the glycosyl transfer: glucose-UDP-GIcNAc, UDP-galactose, UDP-glucose, UDP-N- acetylglucosamine, UDP-N-acetylgalactosamine (UDP-GIcNAc), GDP-fucose and CMP-N- acetylneuraminic acid (CMP-Neu5Ac).

The genetically engineered cell described herein can comprise one or more pathways to produce a nucleotide-activated sugar selected from the group consisting of glucose-UDP- GIcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N- acetylgalactosamine, GDP-fucose and CMP-N-acetylneuraminic acid.

In one embodiment of the current disclosure, the genetically engineered cell is capable of producing one or more activated sugar nucleotides mentioned above by a de novo pathway. In this regard, an activated sugar nucleotide is made by the cell under the action of enzymes involved in the de novo biosynthetic pathway of that respective sugar nucleotide in a stepwise reaction sequence starting from a simple carbon source like glycerol, sucrose, fructose or glucose (for a review for monosaccharide metabolism see e.g. H. H. Freeze and A. D. Elbein: Chapter 4: Glycosylation precursors, in: Essentials of Glycobiology, 2nd edition (Eds. A. Varki et al.), Cold Spring Harbour Laboratory Press (2009)).

The enzymes involved in the de novo biosynthetic pathway of an activated sugar nucleotide can be naturally present in the cell or introduced into the cell by means of gene technology or recombinant DNA techniques, all of them are parts of the general knowledge of the skilled person.

In another embodiment, the genetically engineered cell can utilize salvaged monosaccharides for sugar nucleotide. In the salvage pathway, monosaccharides derived from degraded oligosaccharides are phosphorylated by kinases, and converted to nucleotide sugars by pyrophosphorylases. The enzymes involved in the procedure can be heterologous ones, or native ones of the host cell. For the production of fucosylated HMOs, the de novo GDP-fucose pathway is important to ensure presence of sufficient GDP-fucose. The colanic acid gene cluster of Escherichia coli encodes selected enzymes involved in the de novo synthesis of GDP-fucose (gmd, wcaG, wcaH, weal, manB, manC), whereas one or several of the genes downstream of GDP-L-fucose such as wcaJ, which are responsible for the production of the extracellular polysaccharide colanic acid, a major oligosaccharide of the bacterial cell wall, can be deleted to prevent conversion of GDP-fucose to colanic acid.

For production of sialylated oligosaccharides/HMOs the genetically modified cell comprises a biosynthetic pathway for making a sialate sugar nucleotide, such as CMP-N-acetylneuraminic acid as glycosyl-donor for the sialyltransferase introduced into the genetically engineered cell. The genetically engineered cell may for example comprise a sialic acid synthetic capability through provision of an exogenous UDP-GIcNAc 2-epimerase (e.g.,neuC of Campylobacter jejuni (GenBank AAK91727.1) or equivalent (e.g., (GenBank CAR04561.1), a Neu5Ac synthase e.g.,neuB of C. jejuni (GenBank AAK91726.1) or equivalent, (e.g., Flavobacterium limnosediminis sialic acid synthase, GenBank WP_023580510.1), and/or a CMP-Neu5Ac synthetase (e.g.,neuA of C. jejuni (GenBank AAK91728.1) or equivalent, (e.g., Vibrio brasiliensis CMP-sialic acid synthase, GenBank WP_006881452.1).

P-galactosidase

A host cell suitable for HMO production, e.g., E. coli, may comprise an endogenous p- galactosidase gene or an exogenous p-galactosidase gene, e.g., E. coli comprises an endogenous lacZ gene (e.g., GenBank Accession Number V00296 (Gl:41901)). For the purposes of the disclosure, when producing an HMO, it is preferred that the genetically engineered cell does not express a functional p-galactosidase to avoid the degradation of lactose if lactose is used as the initial substrate for producing the desired HMO. In embodiments the lacZ gene may be inactivated by a complete or partial deletion of the corresponding nucleic acid sequence from the bacterial genome, or the gene sequence is mutated in the way that it is not transcribed, or, if transcribed, the transcript is not translated or if translated to a protein (i.e., p-galactosidase), the protein does not have the corresponding enzymatic activity. In this way the HMO-producing bacterium accumulates an increased intracellular lactose pool which is beneficial for the production of HMOs.

The genetically engineered cell

In the present context, the terms “a genetically modified cell” and "a genetically engineered cell” are used interchangeably. As used herein ‘‘a genetically modified cell” is a cell whose genetic material has been altered by human intervention using a genetic engineering technique, such a technique is for example but not limited to transformation or transfection e.g., with a heterologous polynucleotide sequence, Crisper/Cas editing and/or random mutagenesis. In one embodiment the genetically engineered cell has been transformed or transfected with a recombinant nucleic acid sequence.

The genetically engineered cell is preferably a prokaryotic cell, such as a microbial cell. Appropriate microbial cells that may function as a host cell include yeast cells, bacterial cells, archaebacterial cells, algae cells, and fungal cells.

The genetically engineered cell (host cell) may be e.g., a bacterial or yeast cell. In one preferred embodiment, the genetically engineered cell is a bacterial cell.

Host cells

In embodiments, the engineered cell is a microorganism. The genetically engineered cell is preferably a microbial cell, such as a prokaryotic cell or eukaryotic cell. Appropriate microbial cells that may function as a host cell include bacterial cells, archaebacterial cells, algae cells and fungal cells.

The genetically engineered cell may be e.g., a bacterial or yeast cell. In one preferred embodiment, the genetically engineered cell is a bacterial cell.

Regarding the bacterial host cells, there are, in principle, no limitations; they may be eubacteria (gram-positive or gram-negative) or archaebacteria, as long as they allow genetic manipulation for insertion of a gene of interest and can be cultivated on a manufacturing scale. Preferably, the host cell has the property to allow cultivation to high cell densities. Non-limiting examples of bacterial host cells that are suitable for recombinant industrial production of an HMO(s) according to the disclosure could be member of the Enterobacterales order, preferably of the genus Escherichia, more preferably of the species E. coli. Other examples of suitable host cell are Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Campylobacter sp, Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans. Similarly, bacteria of the genera Lactobacillus and Lactococcus may be engineered using the methods of this disclosure, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the disclosure described herein. Also included as part of this disclosure as useful species are strains, engineered as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa).

Non-limiting examples of fungal host cells that are suitable for recombinant industrial production of a heterologous product are e.g., yeast cells, such as Komagataella, Kluyveromyces, Yarrowia, Pichia, Saccaromyces, Schizosaccharomyces or Hansenula or from a filamentous fungus of the genera Aspargillus, Fusarium or Thricoderma.

In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of Escherichia sp., Bacillus sp., lactobacillus sp., Corynebacterium sp. and Campylobacter sp.

In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

In one or more exemplary embodiments, the genetically engineered cell is B. subtilis.

In one or more exemplary embodiments, the genetically engineered cell is S. Cerevisiae or P pastoris.

In one or more exemplary embodiments, the genetically engineered cell is Escherichia coli.

In one or more exemplary embodiments, the disclosure relates to a genetically engineered cell, wherein the cell is derived from the E. coli K-12 strain or DE3.

A recombinant nucleic acid sequence

The present disclosure relates to a genetically engineered cell comprising a recombinant nucleic acid sequence encoding a transporter protein, Edict , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80 %, such as at least 85 %, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , wherein the transporter protein encoding sequence is under the control of a promoter sequence.

In the present context, the term “recombinant nucleic acid sequence”, “recombinant gene/nucleic acid/nucleotide sequence/DNA encoding” or "coding nucleic acid sequence" is used interchangeably and intended to mean an artificial nucleic acid sequence (i.e. produced in vitro using standard laboratory methods for making nucleic acid sequences) that comprises a set of consecutive, non-overlapping triplets (codons) which is transcribed into mRNA and translated into a protein when under the control of the appropriate control sequences, i.e., a promoter sequence.

The boundaries of the coding sequence are generally determined by a ribosome binding site located just upstream of the open reading frame at the 5’end of the mRNA, a transcriptional start codon (AUG, GUG or UUG), and a translational stop codon (UAA, UGA or UAG). A coding sequence can include, but is not limited to, genomic DNA, cDNA, synthetic, and recombinant nucleic acid sequences.

The term "nucleic acid" includes RNA, DNA and cDNA molecules. It is understood that, as a result of the degeneracy of the genetic code, a multitude of nucleic acid sequences encoding a given protein may be produced.

The recombinant nucleic acid sequence may be a coding DNA sequence e.g., a gene, or noncoding DNA sequence e.g., a regulatory DNA, such as a promoter sequence or other noncoding regulatory sequences.

The recombinant nucleic acid sequence may in addition be heterologous. As used herein "heterologous" refers to a polypeptide, amino acid sequence, nucleic acid sequence or nucleotide sequence that is foreign to a cell or organism, i.e., to a polypeptide, amino acid sequence, nucleic acid molecule or nucleotide sequence that does not naturally occurs in said cell or organism.

The disclosure also relates to a nucleic acid construct comprising a coding nucleic sequence, i.e. recombinant DNA sequence of a gene of interest, e.g., the transporter protein gene encoding Edict , and a non-coding regulatory DNA sequence, e.g., a promoter DNA sequence, e.g., a recombinant promoter sequence derived from the promoter sequence of the lac operon or the glp operon, or a promoter sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence, wherein the coding and promoter sequences are operably linked. It is understood that an endogenous or wild type promoter sequence that is operably linked to a coding nucleic acid sequence to which it is not naturally linked is a recombinant promoter sequence.

The term “operably linked” refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. It refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. E.g., a promoter sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.

Generally, promoter sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.

In one exemplified embodiment, the nucleic acid construct of the disclosure may be a part of the vector DNA, in another embodiment, the construct it is an expression cassette/cartridge that is integrated in the genome of a host cell.

Accordingly, the term “nucleic acid construct” means an artificially constructed segment of nucleic acids, in particular a DNA segment, which is intended to be inserted into a target cell, e.g., a bacterial cell, to modify expression of a gene of the genome or expression of a gene/coding DNA sequence which may be included in the construct. In embodiments the nucleic acid construct is a plasmid or an expression cassette suitable for being integrated in the genome of a target/host cell.

Integration of the nucleic acid construct of interest comprised in the construct (expression cassette) into the bacterial genome can be achieved by conventional methods, e.g. by using linear cartridges that contain flanking sequences homologous to a specific site on the chromosome, as described for the attTn7-site (Waddell C.S. and Craig N.L., Genes Dev. (1988) Feb;2(2):137-49.); methods for genomic integration of nucleic acid sequences in which recombination is mediated by the Red recombinase function of the phage A or the RecE/RecT recombinase function of the Rac prophage (Murphy, J Bacteriol. (1998) ;180(8) :2063-7; Zhang et al., Nature Genetics (1998) 20: 123-128 Muyrers et al., EMBO Rep. (2000) 1 (3): 239-243); methods based on Red/ET recombination (Wenzel et al., Chem Biol. (2005), 12 (3): 349-56.;

Vetcher et al., Appl Environ Microbiol. (2005);71 (4):1829-35); or positive clones, i.e., clones that carry the expression cassette, can be selected e.g., by means of a marker gene, or loss or gain of gene function.

In one or more exemplary embodiments, the present disclosure relates a recombinant nucleic acid sequence comprising a nucleic acid sequence as illustrated in SEQ ID NO: 6, or a nucleic acid sequence which at least 70% identical to SEQ ID NO: 6, such as at least 75% identical, at least 80 % identical, at least 85 % identical, at least 90 % identical, at least, at least 95 % identical, at least 98 % identical, or 100 % identical to SEQ ID NO: 6. The nucleic acid sequence presented in SEQ ID NO: 6 encodes the protein Edicl with the amino acid sequence presented in SEQ ID NO: 1.

Preferably, the heterologous elements, e.g., the Edicl transporter, substrate importer and/or glycosyltransferase encoding sequence is under the control of a promoter sequence selected from promotor sequences with a nucleic acid sequence as identified in Table 5.

Table 5 - Selected promoter sequences

*The promoter activity is assessed in the LacZ assay described below with the PglpF promoter run as positive reference in the same assay. To compare across assays the activity is calculated relative to the PglpF promoter, a range indicates results from multiple assays

The promoter may be of heterologous origin, native to the genetically engineered cell or it may be a recombinant promoter, combining heterologous and/or native elements.

One way to increase the production of a product may be to regulate the production of the desired enzyme activity used to produce the product, such as the glycosyltransferases or enzymes involved in the biosynthetic pathway of the glycosyl donor.

Increasing the promoter strength driving the expression of the desired enzyme may be one way of doing this. The strength of a promoter can be assessed using a lacZ enzyme assay where p- galactosidase activity is assayed as described previously (see e.g., Miller J.H. Experiments in molecular genetics, Cold spring Harbor Laboratory Press, NY, 1972). Briefly the cells are diluted in Z-buffer and permeabilized with sodium dodecyl sulfate (0.1 %) and chloroform. The LacZ assay is performed at 30°C. Samples are preheated, the assay initiated by addition of 200 pl ortho-nitro-phenyl-p-galactosidase (4 mg/ml) and stopped by addition of 500 pl of 1 M Na₂CO₃ when the sample had turned slightly yellow. The release of ortho-nitrophenol is subsequently determined as the change in optical density at 420 nm. The specific activities are reported in Miller Units (MU) [A420/(min*ml*A600)]. A regulatory element with an activity above 10,000 MU is considered strong and a regulatory element with an activity below 3,000 MU is considered weak, what is in between has intermediate strength. An example of a strong regulatory element is the PglpF promoter with an activity of approximately 14.000 MU and an example of a weak promoter is Plac which when induced with IPTG has an activity of approximately 2300 MU. In preferred embodiments, the expression of said nucleic acid sequences are under control of a strong promoter selected from the group consisting of SEQ ID NOs 15, 16, 17, 18, 19, 20, 21 , 23 and 24.

In embodiments the expression of said nucleic acid sequences described herein is under control of a PglpF (SEQ ID NO: 27 or Plac (SEQ ID NO: 36 promoter or PmglB_UTR70 (SEQ ID NO: 24) or PglpA_70UTR (SEQ ID NO: 25) or PglpT_70UTR (SEQ ID NO: 26) or variants thereof such as promoters identified in Table 5, in particular the PglpF_SD4 variant of SEQ ID NO: 22 or Plac_70UTR variant of SEQ ID NO: 18, or PmglB_70UTR variants of SEQ ID NO: 15, 16, 17, 19, 20, 21 , 23 and 24. Further suitable variants of PglpF, PglpA_70UTR, PglpT_70UTR and PmglB_70UTR promoter sequences are described in or WO2019/123324 and W02020/255054 respectively (hereby incorporated by reference).

In preferred embodiments, the recombinant nucleic acid sequences individually are under the control of one or more promoters selected from the group consisting of PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 27, 36, 24, 25 and 26 respectively) and variants thereof.

Sequence identity

The term "sequence identity" as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e., a candidate sequence (e.g., a sequence of the invention) and a reference sequence (such as a prior art sequence) based on their pairwise alignment. For purposes disclosed herein, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277,), preferably version 5.0.0 or later (available at https://www.ebi.ac.uk/Tools/psa/emboss needle/). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The output of Needle labelled "identity" (obtained using the -nobrief option) is used as the percent identity. Generally sequence identity may be calculated as follows: (Identical Residues x 100)/(Length of Aligned region).

For purposes disclosed herein, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1 970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), 10 preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labelled "identity" (obtained using the -nobrief option) is used as the percent identity. Generally sequence identity may be calculated as follows: (Identical Deoxyribonucleotides x 1 OO)/(Length of Aligned region).

Functional homologue

A functional homologue or functional variant of a protein/nucleic acid sequence as described herein is a protein/nucleic acid sequence with alterations in the genetic code, which retain its original functionality. A functional homologue may be obtained by mutagenesis or may be natural occurring variants from the same or other species. The functional homologue should have a remaining functionality of at least 50%, such as at least 60%, 70%, 80 %, 90% or 100% compared to the functionality of the protein/nucleic acid sequence.

A functional homologue of any one of the disclosed amino acid or nucleic acid sequences can also have a higher functionality. A functional homologue of any one of the amino acid sequences, or a recombinant nucleic acid as disclosed herein, should ideally be able to participate in the production of the desired HMDs, in terms of export of the desired HMO product out of the cell, participate in the synthesis of the desired HMO or precursors for producing same HMO, or facilitate the import of substrate for the HMO production, such as a acceptor oligosaccharide of at least three monosaccharide units, optimally, while also improving the production of the desired HMO, or by reducing the by-product formation, reduction in biomass formation, increasing the viability of the genetically engineered cell, improving the robustness of the genetically engineered cell according to the disclosure, or facilitate the reduction in consumables needed for the production.

Use of a genetically engineered cell or transporter

The disclosure also relates to any commercial use of the transporter Edicl , genetically engineered cell(s) or the nucleic acid construct(s) disclosed herein, such as, but not limited to, in a method for producing a desired HMO (HMO product). Thus, the present disclosure also relates to the use of a nucleic acid construct according to the present disclosure, in a host cell producing an HMO which comprises a GlcNAc(p1-3)Gal(|31-4)Glc backbone and at least one additional saccharide moiety, such as a Gal(p1-4)GlcNAc(p1-3)Gal(pi -4)Glc structure.

In additional embodiments, the genetically engineered cell and/or the nucleic acid construct described herein is used in the manufacturing of HMOs. Preferably, in the manufacturing of HMOs with a lacto-N-triose II (LNT-II, GlcNAc(pi-3)Gal(|31-4)Glc) backbone, such as LNT and/or LNnT.

In an exemplified embodiment, the genetically modified cell and/or the nucleic acid construct according to the disclosure, is used in the manufacturing of one or more HMO(s), wherein the HMOs are selected from the group consisting of lacto-N-neotetraose (LNnT), lacto- N-tetraose (LNT), lacto-N-fucopentaose V (LNFP-V) and 6’-sialyllacto-N-neotetraose (LST c). In one or more exemplary embodiments, the genetically engineered cell and/or the nucleic acid construct is used in the manufacturing of LNT.

In one or more exemplary embodiments, the genetically engineered cell and/or the nucleic acid construct is used in the manufacturing of LNnT.

In one or more exemplary embodiments, the genetically engineered cell and/or the nucleic acid construct is used in the manufacturing of LNFP-V.

In one or more exemplary embodiments, the genetically engineered cell and/or the nucleic acid construct is used in the manufacturing of a mixture of LNT and LNFP-V.

In one or more exemplary embodiments, the genetically engineered cell and/or the nucleic acid construct is used in the manufacturing of a mixture of LNT, LNFP-V and LNDFH-II.

In one or more exemplary embodiments, the genetically engineered cell and/or the nucleic acid construct is used in the manufacturing of LST-c.

In one or more exemplary embodiments, the genetically engineered cell and/or the nucleic acid construct is used in the manufacturing of a mixture of LNnT and LST-c.

In one or more exemplary embodiments, the genetically engineered cell and/or the nucleic acid construct is used in the manufacturing of a neutral non-fucosylated HMO selected from the group consisting of LNT, LNnT, LNH, pLNnH, LNnH and pLNH-L

In one or more exemplary embodiments, the genetically engineered cell and/or the nucleic acid construct is used in the manufacturing of an HMO consisting of four monosaccharide units, such as an HMO selected from the group consisting of LNT and LNnT, preferably LNnT.

A method for producing human milk oligosaccharides (HMOs)

The present disclosure also relates to a method for producing a desired human milk oligosaccharide (HMO), said method comprising culturing a genetically modified cell according to the present disclosure. In embodiments, said method comprises, a. providing a genetically engineered cell according to the present disclosure, cultivating the genetically engineered cell in a culture medium under conditions permissive for the production of said HMO; and optionally c. recovering said HMO. Preferably, said desired HMO is recovered from the supernatant of the cultivation after production of said HMO.

The present disclosure thus relates to a method for producing a human milk oligosaccharide (HMO), said method comprising culturing of a genetically engineered cell capable of producing a desired human milk oligosaccharide (HMO), wherein said cell comprises, a. one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases, and b. a recombinant nucleic acid sequences encoding a transporter protein, Edicl , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , wherein the expression of said transporter protein in said cell leads to export of the desired oligosaccharide from said cell.

In embodiments, said desired HMO comprises an GlcNAc(p1-3)Gal(p1-4)Glc backbone and at least one additional saccharide moiety, e.g., Gal(|31 -3)GlcNAc (p1-3)Gal (|31-4)Glc or Gal(p1- 4)GlcNAc (p1-3)Gal (p1-4)Glc. In embodiments the desired HMO is selected from the group consisting of lacto-N-neotetraose (LNnT), lacto- N-tetraose (LNT), lacto-N-fucopentaose V (LNFP-V) and 6’-sialyllacto-N-neotetraose (LST c). Preferably, the desired HMO produced by said method is LNnT or LNT.

The particular of the method of the present disclosure is that the cultivation of the genetically engineered cell of as disclosed herein, enables production of a desired HMO where most of the desired HMO is found in the supernatant of the fermentation broth, while maintaining the most of the undesired HMO by-products within said cell, thus enabling a more efficient purification of the desired HMO product. Accordingly, in embodiments, following cultivation of said cell as described herein, at least 90%, such as at least 95%, of the total molar content of HMO in the supernatant of the culture is LNnT. Optimally, all of the product produced by during said cultivation is the desired product, nevertheless this is not always possible and undesired byproducts will also be generated within said cell. Accordingly, it is of particular interest that the method disclosed herein produces at least 88%, 89%, 90% or such as at least 91% LNnT of the total HMO content produced by said cell. As such high production of the desired product HMO is obtained it is especially favourable when less than 10 % of the total molar content of HMO in the supernatant of the culture is a by-product HMO, such as LNT-II. Preferably, by the end of the cultivation according to the method, less than 0.1 % of the total molar content of HMO in the supernatant of the culture is pLNnH.

Accordingly, in embodiments, the molar ratio of LNnT:LNT-ll produced in said method is at least 20: 1 , such as at least 25:1 , such as at least 30:1 , or such as at least 33:1.

In further embodiments, the molar ratio of LNnT:pLNnH produced in said method is at least 12:1 , such as at least 25: 1 , such as at least 30: 1 or such as at least 33: 1 , or such as at least 37:1.

In further embodiments at least 88%, such as at least 90% or such as at least 92% of the total molar content of HMO produced in the culturing step according to said method is LNnT. In further embodiments less than 12% of the total molar content of HMO produced in the culturing step according to said method is a by-product HMO, such as less than 10%, or such as less than 8% of LNT-II and pLNnH.

In further embodiments, less than 7% of the total molar content of HMO produced in the culturing step according to said method is pLNnH.

In further embodiments, the molar ratio of LNnT:pLNnH produced in the culturing step according to said method is at least 12:1 such as at least 15:1 , such as at least 20:1 , or such as at least 21 :1.

In further embodiments, the molar ratio of LNnT:LNT-ll produced in the culturing step according to said method is at least 20:1 , such as at least 25:1 , such as at least 30:1 , or such as at least 33:1.

Of particular interest is the molar ratio of LNnT:LNT-ll in the supernatant, which as shown in Example 1 for Edie 1 expressing cells, is more than 30: 1 , which for the prior art transporter Vag was found to be about 7:1. Thus, the cells producing LNnT while expressing Edicl , was much better at specifically transporting the produced LNnT out of the cell. In addition, only trace amounts of pLNnH were found in the supernatant of the fermentation broth.

Accordingly, in embodiments, the molar ratio of LNnT:LNT-ll in the supernatant of the fermentation broth following cultivation of said cell according to said method is at least 20:1 , such as at least 25:1 , such as at least 30:1 , such as at least 35:1 , or such as at least 37:1.

In further embodiments, the molar ratio of LNnT:pLNnH in the supernatant of the fermentation broth following cultivation of said cell according to said method is at least 350: 1 , such as at least 500:1 , such as at least 1000:1 , or such as at least 2000:1.

In further embodiments, less than 0.1% of the total molar content in the supernatant of the fermentation broth is pLNnH following cultivation of said cell according to said method.

In another embodiment the method of the present disclosure relates to the cultivation of the genetically engineered cell as disclosed herein which enables production of LNT wherein at least 80%, such as at least 85% or such as at least 90% of the total molar content of HMO produced in the culturing step according to said method is LNT.

In another embodiment the method of the present disclosure relates to the cultivation of the genetically engineered cell as disclosed herein which enables production of a mixture of LNT and LNFP-V.

In another embodiment the method of the present disclosure relates to the cultivation of the genetically engineered cell as disclosed herein which enables production of a mixture of LNnT and LST-c. Culturing/ferm enting

Culturing, cultivating, or fermenting or fermentation (used interchangeably herein) in a controlled bioreactor typically comprises (a) a first phase of exponential cell growth in a culture medium ensured by a carbon-source, and (b) a second phase of cell growth in a culture medium run under carbon limitation, where the carbon-source is added continuously together with the acceptor oligosaccharide, such as lactose, allowing formation of the HMO product in this phase. By carbon (sugar) limitation is meant the stage in the fermentation where the growth rate is kinetically controlled by the concentration of the carbon source (sugar) in the culture broth, which in turn is determined by the rate of carbon addition (sugar feed-rate) to the fermenter.

The terms “manufacturing” or “manufacturing scale” or “large-scale production” or “large-scale fermentation”, are used interchangeably and in the meaning of the disclosure defines a fermentation with a minimum volume of 100 L, such as WOOL, such as 10.000L, such as 100.000L, such as 200.000L culture broth. Usually, a “manufacturing scale” process is defined by being capable of processing large volumes yielding amounts of the HMO product of interest that meet, e.g., in the case of a therapeutic compound or composition, the demands for toxicity tests, clinical trials as well as for market supply. In addition to the large volume, a manufacturing scale method, as opposed to simple lab scale methods like shake flask cultivation, is characterized by the use of the technical system of a bioreactor (fermenter) which is equipped with devices for agitation, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.). To a large extent, the behaviour of an expression system in a lab scale method, such as shake flasks, benchtop bioreactors or the deep well format described in the examples of the disclosure, does allow to predict the behaviour of that system in the complex environment of a bioreactor.

With regards to the suitable cell medium used in the fermentation process, there are no limitations. The culture medium may be semi-defined, i.e., containing complex media compounds (e.g., yeast extract, soy peptone, casamino acids, etc.), or it may be chemically defined, without any complex compounds. The carbon-source can be selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol. In one or more exemplary embodiments, the culturing media is supplemented with one or more energy and carbon sources selected form the group containing glycerol, sucrose and glucose. In additional embodiments, lactose is added during the cultivation of the genetically engineered cells as a substrate for the HMO formation.

In one or more exemplary embodiments, the culturing media contains sucrose as the sole carbon and energy source. In further embodiments, at least one energy source is added to the culture medium. In embodiments, the at least one energy source is preferably selected from the group consisting of glucose, sucrose, fructose, xylose, glycerol and combinations thereof. In one or more exemplary embodiments, the genetically engineered cell comprises one or more heterologous nucleic acid sequence encoding one or more heterologous polypeptide(s) which enables utilization of sucrose as sole carbon and energy source of said genetically engineered cell.

In embodiments, the precursor molecule for the synthesis of the desired HMO is added during the cultivation of the genetically engineered cells as a substrate for the HMO formation. In further embodiments, lactose and/or LNT-II is added during the cultivation of the genetically engineered cells as a substrate for the formation of the desired HMO. In a preferred embodiment lactose is added during the cultivation of the genetically engineered cells as a substrate for the formation of the desired HMO.

In one or more exemplary embodiments, the genetically engineered cell comprises a PTS- dependent sucrose utilization system, further comprising the scrYA and scrBR operons as described in WO2015/197082 (hereby incorporated by reference).

After carrying out the method of this disclosure, the desired HMO can preferably be collected from the supernatant of the cell culture or fermentation broth in a conventional manner.

Preferably, the collection of the desired HMO from the supernatant, collects at least 90 %, such as at least 95%, such as at least 97% of the desired HMO produced in said method, leaving only minor amounts, such as less than 10%, such as less than 5% such as less than 3% of the desired HMO produced by said cell within the pellet fraction.

Retrieving/Harvesting

The human milk oligosaccharide (HMO) can be retrieved from the entire fermentation broth including the biomass/cells and the supernatant. Alternative the HMO can be retrieved from either the supernatant or the biomass, following fractionation of the biomass and the culture medium/supernatant.

In a preferred embodiment the human milk oligosaccharide (HMO) is retrievable directly from the culture medium following export into the culture medium.

In the present context, the term “retrieving’’ is used interchangeably with the term “harvesting”. Both “retrieving” and “harvesting” in the context relate to collecting the produced HMO(s) from the culture/broth following the termination of fermentation. In example 1 it was shown that the introduction of Edic1 into the cells producing LNnT enabled a method for producing LNnT wherein almost all of the LNnT produced by the cells were exported from the cell into the cell culture medium. In one or more exemplary embodiments the harvesting comprise collecting the desired HMO(s) directly from the cultivation media, i.e., after separation of the culture medium from the biomass. Accordingly, the desired HMO is preferably harvested directly form the supernatant of the fermentation broth following separation of the biomass and the fermentation medium.

The separation of cells from the medium can be carried out with any of the methods well known to the skilled person in the art, such as any suitable type of centrifugation or filtration. The separation of cells from the medium can follow immediately after harvesting the fermentation broth or be carried out at a later stage after storing the fermentation broth at appropriate conditions.

After recovery from fermentation, the desired HMO(s) are available for further processing and purification.

The HMOs can be purified according to the procedures known in the art, e.g., such as described in WO2017/152918, WO2017/182965 or WO2015/188834. The purified HMOs can be used as nutraceuticals, pharmaceuticals, or for any other purpose, e.g., for research.

Manufactured product

The term “manufactured product” refers to the one or more HMOs intended as the one or more product HMO(s), or composition of a mixture of HMOs. An example of the requirements to marketed LNnT can e.g., be seen in GRAS notification 895 and example 2. Preferably, the product HMOs is produced by a method described herein using a genetically engineered cell described herein. In preferred embodiments, the manufactured product is LNnT. Accordingly, the present disclosure also relates to an LNnT HMO produced by the method described herein. In example, it is shown in Examples 1 and 2 that the level of the by-product pLNnH can be greatly reduced when LNnT is produced according to the present disclosure.

The manufactured product may be a powder, a composition, a suspension, or a gel comprising one or more HMOs.

ITEMS

1 . A genetically engineered cell capable of producing an HMO, wherein said cell comprises, a. one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases, and b. a recombinant nucleic acid sequences encoding a transporter protein, Edicl , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence which is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , wherein the expression of said transporter protein in said cell leads to export of the desired HMO from said cell. 2. The genetically engineered cell according to item 1 , wherein said HMO comprises a lacto-N- triose II (LNT-II, GlcNAc(pi -3)Gal(pi-4)Glc) backbone.

3. The genetically engineered cell according to item 2, wherein lacto-N-triose II backbone comprises at least one additional saccharide moiety such as a galactose (gal) moiety.

4. The genetically engineered cell according to item 3, wherein galactose (gal) moiety is in a p1 -4 or p1 -3 configuration.

5. The genetically engineered cell according to any of the preceding items, wherein the with LNT-II backbone is selected from the group consisting of lacto-N-neotetraose (LNnT), lacto- N-tetraose (LNT), lacto-N-fucopentaose V (LNFP-V) and 6’-sialyllacto-N-neotetraose (LST c).

6. The genetically engineered cell according to any one of the preceding items, wherein at least 85%, such as at least 90%, such as at least 95% of the total molar content of HMO produced by the cell is LNnT.

7. The genetically engineered cell according to any one of the preceding items, wherein less than 15 % of the total molar content of HMO produced by the cell is a by-product HMO, such as less than 15 % of the total molar content of HMO produced by the cell is LNT-II.

8. The genetically engineered cell according to any one of the preceding items, wherein less than 10 % of the total molar content of HMO produced the cell is a by-product HMO, such as less than 10 % of the total molar content of HMO produced by the cell is LNT-II and pLNnH.

9. The genetically engineered cell according to any one of the preceding items, wherein at least 85%, such as at least 90%, such as at least 95% of the total molar content of HMO exported from the cell is LNnT.

10. The genetically engineered cell according to any one of the preceding items, wherein less than 10 % of the total molar content of HMO exported from the cell is a by-product HMO, such as less than 10 % of the total molar content of HMO exported from the cell is LNT-II and pLNnH.

1 1 . The genetically engineered cell according to any one of items 1 to 5, wherein at least 80%, such as at least 85%, such as at least 90% of the total molar content of HMO produced by the cell is LNT.

12. The genetically engineered cell according to any one of the preceding items, wherein the one or more glycosyltransferases comprises a p-1 ,4-galactosyltransferase or a p-1 ,3- galactosyltransferase and optionally a p-1 ,3-N-acetylglucosaminyltransferase. 13. The genetically engineered cell according to item 12 wherein the p-1 ,3-N- acetylglucosaminyltransferase is from Neisseria meningitidis and the p-1 ,4- galactosyltransferase is from Helicobacter pylori.

14. The genetically engineered cell according to item 12 wherein the p-1 ,3-N- acetylglucosaminyltransferase is from Neisseria meningitidis and the p-1 ,3- galactosyltransferase is from Helicobacter pylori.

15. The genetically engineered cell according to any one of items 12 or 13, wherein the 1 ,3-N- acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 11 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 11 and the p-1 ,4-galactosyltransferase is has an amino acid sequence according to SEQ ID NO: 13, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 13.

16. The genetically engineered cell according to any one of items 12 or 14, wherein the 1 ,3-N- acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 11 , or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 11 and the p-1 ,3-galactosyltransferase is has an amino acid sequence according to SEQ ID NO: 12, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 12.

17. he genetically engineered cell according to any of the preceding items, wherein the cell further comprises one or more additional glycosyltransferases selected from the group consisting of alpha-1 ,2-fucosyltranferase, alpha- 1 ,3-fucosyltranferase, alpha-1 , 3/4- fucosyltransferase, alpha-2, 3-sialyltransferase and alpha-2, 6-sialyltransferase.

18. The genetically engineered cell according to any one of the preceding items, wherein the cell further comprises a recombinant nucleic acid sequence encoding a second transporter protein.

19. The genetically engineered cell according to item 18, wherein the second transporter protein is either an importer protein or a second exporter protein.

20. The genetically engineered cell according to item 18 or 19, wherein the second exporter is selected from MFS transporter known to export HMDs.

21 . The genetically engineered cell according to item 18 to 20, wherein the second exporter is selected from an MFS transporter from Pantoea vagans or from Rosenbergiella nectarea, such as the MFS transporter vag with an amino acid sequence that is at least 85 % identical to SEQ ID NO: 5 or the MFS transporter Nec with an amino acid sequence that is at least 85 % identical to SEQ ID NO: 83. 22. The genetically engineered cell according to item 21 , wherein the cell is producing LNnT and the second transporter is the vag transporter.

23. The genetically engineered cell according to item 21 , wherein the cell is producing LNT and the second transporter is the Nec transporter.

24. The genetically engineered cell according to item 23, wherein less than 15 % of the total molar content of HMO produced by the cell is a by-product HMO, such as less than 15 % of the total molar content of HMO produced by the cell is LNT-IL

25. The genetically engineered cell according to any of the preceding items, wherein the cell further comprises a nucleic acid sequence encoding a recombinant lactose permease.

26. The genetically engineered cell according to item 25, wherein the lactose permease is lacY and has an amino acid sequence according to SEQ ID NO: 14, or a functional homologue thereof with an amino acid sequence that is at least 80 % identical to SEQ ID NO: 14.

27. The genetically engineered cell according to any of items 1 to 23, wherein the cell comprises a recombinant nucleic acid sequence encoding an importer protein selected from table 1 or table 2, preferably said importer protein imports LNT-II as the initial substrate for the HMO production.

28. The genetically engineered cell according to any of the preceding items, wherein recombinant nucleic acid sequences individually are under the control of a promoter selected from the group consisting of PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 27, 36, 24, 25 and 26, respectively) and variants thereof.

29. The genetically engineered cell according to item 28, wherein the promoter is a strong promoter selected from the group consisting of SEQ ID NOs 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 and 27.

30. The genetically engineered cell according to any of the preceding items, wherein said engineered cell is selected from the group consisting of Escherichia Coli, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

31 . A method for producing a HMO wherein said method comprises, a. providing a genetically engineered cell according to any one of items 1 to 30, b. cultivating the genetically engineered cell in a culture medium under conditions permissive for the production of said HMO; and c. optionally recovering said HMO. 32. The method according to any one of items 31 , wherein the method comprises cultivating the genetically engineered cell in the presence of an energy source selected from the group consisting of glucose, sucrose, fructose, xylose and glycerol.

33. The method according to item 31 or 32 , wherein said HMO is recovered from the total broth of the cultivation after production of said HMO.

34. The method according to item 31 to 32 , wherein said HMO is recovered from the supernatant of the cultivation after production of said HMO.

35. The method according to any one of items 31 to 34, wherein said HMO product comprises a lacto-N-triose II (LNT-II, GlcNAc(p1-3)Gal(p1 -4)Glc) backbone.

36. The method according to any one of items 31 to 35, wherein said HMO product is selected from the group consisting of lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), lacto-N- fucopentaose V (LNFP-V) and 6’-sialyllacto-N-neotetraose (LST c)

37. The method according to any one of items 31 to 36, wherein the HMO product is LNnT.

38. The method according to any one of items 31 to 37, wherein at least 85%, such as at least 88%, such as at least 90% or such as at least 92% of the total molar content of HMO produced in the culturing step according to said method is LNnT.

39. The method according to any one of items 31 to 38, wherein less than 12% of the total molar content of HMO produced in the culturing step according to said method is a by-product HMO, such as less than 10%, or such as less than 8% of LNT-II and pLNnH.

40. The method according to any one of items 31 to 39, wherein less than 7% of the total molar content of HMO produced in the culturing step according to said method is pLNnH.

41 . The method according to item 37 to 40, wherein the molar ratio of LNnT:pLNnH produced in the culturing step according to said method is at least 12:1 such as at least 15:1 , such as at least 20:1 , or such as at least 21 :1 .

42. The method according to any one of items 37 to 41 , wherein the molar ratio of LNnT:LNT-ll produced in the culturing step according to said method is at least 20:1 , such as at least 25:1 , such as at least 30:1 , or such as at least 33:1.

43. The method according to any one of items 31 to 37, wherein at least 90%, such as at least 95%, of the total molar content of HMO in the supernatant of the culture is LNnT.

44. The method according to any one of items 31 to 43, wherein less than 10% of the total molar content of HMO in the supernatant of the culture is a by-product HMO, such as less than 10% of LNT-II and pLNnH. The method according to any one of items 31 to 44, wherein less than 0.1% of the total molar content of HMO in the supernatant of the culture is pLNnH. The method according to any one of items 37 to 45, wherein the molar ratio of LNnT:pLNnH in the supernatant produced in said method is at least 350:1 , such as at least 500:1 , such as at least 1000: 1 , or such as at least 2000: 1 . The method according to item 37 to 46, wherein the molar ratio of LNnT:LNT-ll in the supernatant produced in said method is at least 20:1 , such as at least 25:1 , such as at least 30:1 , such as at least 35:1 , or such as at least 37:1 . The method according to item 37 to 47, wherein at least 70%, of the total molar content the total carbohydrates is LNnT. The method according to item 37 to 48, wherein less than 6% of the total molar content the total carbohydrates is pLNnH and less than 4% of the total molar content the total carbohydrates is LNT-II. The method according to any one of items 31 or 36, wherein at least 80%, such as at least 85% or such as at least 90% of the total molar content of HMO produced in the culturing step according to said method is LNT. The method according to any one of claims 31 or 36, wherein a mixture of LNT and LNFP-V is produced. The method according to any one of claims 31 or 36, wherein a mixture of LNnT and LST-c is produced. The method according to any one of items 50 to 52, wherein less than 15% of the total molar content of HMO produced in the culturing step according to said method is a by-product HMO, such as less than 15% of LNT-II. The method according to any one of items 31 to 53, wherein lactose and/or LNT-II is added during the cultivation of the genetically engineered cells as a substrate for the HMO formation. An HMO produced by the method according to any one of items 31 to 54. A nucleic acid construct comprising a recombinant nucleic acid sequence encoding a transporter protein, Edicl , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99 % identical to SEQ ID NO: 1 , wherein the transporter protein encoding sequence is under the control of a promoter sequence. 57. The nucleic acid construct according to item 56, wherein the nucleic acid construct is a plasmid or an expression cassette suitable for being integrated in the genome.

58. Use of a nucleic acid construct according to item 56, in a host cell producing an HMO which comprises a GlcNAc(p1-3)Gal(p1-4)Glc backbone and at least one additional saccharide moiety, such as a Gal(pi-4)GlcNAc(pi-3)Gal(pi-4)Glc or Gal(pi-3)GlcNAc(pi-3)Gal(pi- 4)Glc structure.

SEQUENCES

The current application contains a sequence listing in text format and electronical format which is hereby incorporated by reference. An overview of the SEQ ID NOs used in the present application can be found in table 18.

Table 18 - sequences in the application

EXAMPLES

Methods

Unless stated otherwise, standard techniques, vectors, control sequence elements, and other expression system elements known in the field of molecular biology are used for nucleic acid manipulation, transformation, and expression. Such standard techniques, vectors, and elements can be found, e.g., in: Ausubel et al. (eds.), Current Protocols in Molecular Biology (1995) (John Wiley & Sons); Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) (Academic Press); Bukhari et al. (eds.), DNA Insertion Elements, Plasmids and Episomes (1977) (Cold Spring Harbor Laboratory Press, NY); Miller, J.H. Experiments in molecular genetics (1972.) (Cold spring Harbor Laboratory Press, NY)

The embodiments described below are selected to illustrate the disclosure and are not limiting the disclosure in any way.

Strains

The strains (genetically engineered cells) constructed in the present application were based on Escherichia coli K-12 DH1 with the genotype: F", A~, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. Additional modifications were made to the E. coli K-12 DH1 strain to generate the MDO strain with the following modifications: lacZ: deletion of 1.5 kbp, lacA deletion of 0.5 kbp, nanKETA: deletion of 3.3 kbp, melA deletion of 0.9 kbp, wcad. deletion of 0.5 kbp, mdoH'. deletion of 0.5 kbp, and insertion of Plac promoter upstream of the gmd gene.

Methods of inserting gene(s) of interest into the genome of E. coli are well known to the person skilled in the art. Insertion of genetic cassettes into the E. coli chromosome can be done using gene gorging (see e.g., Herring and Blattner 2004 J. Bacteriol. 186: 2673-81 and Warming et al 2005 Nucleic Acids Res. 33(4): e36) with specific selection marker genes and screening methods.

This MDO strain was further engineered to generate an LNnT producing strain by chromosomally integrating a beta-1 ,3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI Accession nr. WP_033911473.1 and shown as SEQ ID NO: 11) and a beta-1 ,4-galactosyltransferase (GalT from Helicobacter pylori, homologous to GenBank ID WP_001262061.1 and shown as SEQ ID NO: 13) both under the control of a PglpF promoter (SEQ ID NO: 27), this strain is named the LNnT strain. To increase the LNnT production and export from the cell, the LNnT strain was supplemented with a putative transporter from table 6, resulting in the strains described in table 7. Table 6. List of transporters tested in the framework of the present disclosure.

the sequences used in the present application may be truncated at the N- or C-terminal as compared to the GenBank sequence.

Previously disclosed as a putative LNnT or LNT transporter in WO2022/157213 Previously disclosed LNT and LNnT transporter in WO2021/14861 1

Previously disclosed 2’FL, 3FL, LNT-II and LNT transporter in WO2021/148615

Codon optimized DNA sequences encoding individual transporters were genomically integrated into the LNnT strain.

The genotypes of the background strain (MDO), LNnT strain and the transporter-expressing strains are provided in Table 7.

Table 7. Genotypes of the transporter expressing strains, used in the present examples.

¹lgtA - three genomically inserted copies of a gene encoding f3-1,3-N-acetyl-g'ucosaminyltransferase (SEQ ID NO: 11) under control of the PglpF promoter (SEQ ID NO: 27). ^zgalT - one genomically Inserted gene encoding (3-1 ,4-Galactosyltransferase (SEQ ID NO: 13) under control of the PglpF promoter (SEQ ID NO: 27).

³ galTK - two genomically inserted gene encoding (3-1 ,3-Galactosyltransferase (SEQ ID NO: 12) under control of the PglpF promoter (SEQ ID NO: 27).

⁴scrYA, scrBR - PTS-dependent sucrose utilization system, comprising two operons scrYA from Klebsiella pneumoniae and scrBR from Salmonella enterica subsp enterica serovar Typhimurium as described in WQ2015/197082 under the control of the Pscr-PglpF_SD1 dual promoter (SEQ ID NO: 87+38) and Pscr promoter (SEQ ID NO: 87), respectively. ^slacY - additional genomically integrated copy of lacY (SEQ ID NO: 14) under control of the PglpF promoter (SEQ ID NO: 27).

⁶nec gene coding for a heterologous major facilitator superfamily (MFS) transporter of SEQ ID NO: 84, under the control of the PglpF promoter (SEQ ID NO: 27).

⁷futA_mut2 - two independent genomic copies of the gene encoding a FutA a-1,3-fucosyl-transferase variant of SEQ ID NO: 85 under control of the PglpF promoter ( SEQ ID NO: 27)

⁸ neuA one genomic copy of a gene encoding the CMP-Neu5Ac synthetase from Campylobacter jejuni (GenBank AAK91728.1) under control of the PglpF promoter (SEQ ID NO: 27)

⁹ neuB one genomic copy of a gene encoding the Neu5Ac synthase from Campylobacter jejuni (GenBank AAK91726. 1 ) under control of the PglpF promoter (SEQ ID NO: 27)

¹⁰ neuC one genomic copy of a gene encoding the UDP-GIcNAc 2-epimerase from Campylobacter jejuni (GenBank AAK91727.1) under control of the PglpF promoter (SEQ ID NO: 27).

¹¹HAC1268 - one genomically inserted gene encoding a-2,6-sialyltransferase (SEQ ID NO: 86) under control of the PglpF promoter (SEQ ID NO: 27)

Deep well assay

Deep Well Assays in the current examples were performed as originally described to Lv et al (Bioprocess Biosyst Eng 20 (2016) 39:1737 — 1747) and optimized for the purposes of the current invention. More specifically, the strains disclosed in the present example were screened in 96 deep well plates using a 4-day protocol. During the first 24 hours, precultures were grown to high densities (OD600 up to 5) and subsequently transferred to a medium that allowed induction of gene expression and product formation. More specifically, during day 1 , fresh precultures were prepared using a basal minimal medium (BMM) (pH 7,0) supplemented with magnesium sulphate (0.12 g/L), thiamine (0.004 g/L) and glucose (5.5 g/L). Basal Minimal medium had the following composition: NaOH (1 g/L), KOH (2.5 g/L), KH2PO4 (7 g/L), NH4H2PO4 (7 g/L), Citric acid (0.5 g/l), trace mineral solution (5 mL/L). The trace mineral stock solution contained; ZnSO~*7H~O 0.82 g/L, Citric acid 20 g/L, MnS04*H2O 0.98 g/L, FeSO4*7H2O 3.925 g/L, CuSO4*5H2O 0.2 g/L. The pH of the Basal Minimal Medium was adjusted to 7.0 with 5 N NaOH and autoclaved. The precultures were incubated for 24 hours at 34 °C and 700 rpm shaking and then further transferred to 2 mL of a new BMM (pH 7,5) to start the main culture. The new BMM was supplemented with magnesium sulfate (0.12 g/L), thiamine (0.02 g/L), a bolus of glucose solution (0.1-0.15 g/L) and a bolus of lactose solution (5-20 g/L) Moreover, a 20 % stock solution of maltodextrin (19-20 g/L) was provided as carbon source, accompanied by the addition of a specific hydrolytic enzyme, namely glycoamylase, so that glucose was released at a rate suitable for carbon-limited growth and similar to that of a typical fed-batch fermentation process. The main cultures were incubated for 96 hours at 28 °C and 700 rpm shaking. For the analysis of the supernatant fraction, the 24 well plates were centrifuged and the supernatant fraction was collected and subsequently analyzed by HPLC. After the removal of the supernatant fraction and for the analysis of the pellet fraction, sterile MQ water was then added in each well of the 24 well plates, which were then boiled at 100°C, subsequently centrifuged, and finally the supernatants were analysed by HPLC.

Fermentation

The E. coli strains were cultivated in 250 mL fermenters (Ambr250 HT Bioreactor system, Sartorius) starting with 100 mL of minimal culture medium consisting of 30 g/L sucrose and a minimal medium comprised of H3PO4, MgSC x 7H2O, KOH, citric acid, trace element solution and thiamine. The dissolved oxygen level was kept at 20% by a cascade control of first agitation and then airflow starting at 1200 rpm (up to max 4500 rpm) and 1 WM (up to max 3 WM). The pH was kept at 6.8 by titration with 8.5% NH₄OH solution. The cultivations were started with 2% (v/v) inoculums from pre-cultures comprised of 10 g/L sucrose, NH4 H2PO4, KH2PO4, MgSO4 x 7H₂O, KOH, NaOH, citric acid, trace element solution and thiamine. After depletion of the sucrose contained in the basal minimal medium, a feed solution containing sucrose, MgSO₄ x 7H2O, KOH, H3PO4, citric acid, antifoam and trace mineral solution was continuously added to the fermenter at a rate that maintained carbon-limiting conditions. The temperature was initially at 33°C but was dropped to 27°C with a 5-hour linear ramp initiated 20 hours after the start of the feed. Lactose was added as repeated bolus additions of 25% lactose monohydrate solution after feed start and then every 32 hours to keep lactose from becoming a rate limiting factor. The growth, metabolic activity and metabolic state of the cells was followed by on-line measurements of agitation, dissolved oxygen tension, reflectance, NH₄OH base addition, O₂ uptake rate and C0₂ evolution rate. Throughout the fermentations, samples were taken to determine the concentration of HMO products, lactose and other minor by-products using HPLC.

Example 1 - Production of LNnT by fermentation

Genetically engineered cells expressing individual transporter proteins were subjected to fermentation and their ability to enhance the production of LNnT, while also reducing the LNT-II and pLNnH by-product levels were assessed.

Some of the desired advantages of introducing heterologous LNnT transporters are: 1) an enhanced production of LNnT, 2) enhanced export of LNnT from the cells to the supernatant, 3) reduced by-product formation and 4) reduced export of by-product HMOs to the supernatant. A higher amount of exported LNnT and a reduction in production and/or transport of by-products, such as LNT-II and pLNnH is preferable since this allows for initial separation of LNnT from the by-products directly in the cultivation step, which greatly simplifies the subsequent purification process following the fermentation.

To evaluate the capabilities of the heterologous transporters described herein the strains were tested in a fermentation process, wherein the strains were fermented as described in the “Method” section above.

The total HMO content (pellet and supernatant) from the fermentation of the LNnT producing strains were analysed and the results are shown in Table 8 and figure 5 for the individual HMOs (in percentage, %) produced by each strain relative to the total HMO amount produced in the Vag strain.

Table 8. HMOs produced by the strains relative to the total HMO produced in the Vag strain

As can be seen in table 8 strains expressing the Blon_2475, MdfA_CM and MdfA_YR transporters produce an overall lower total amount of HMOs relative to Vag, at 35%, 47% and 55% respectively, which is allocated to a lower relative amount of LNnT (30 %, 41% and 48 % respectively), a slightly lower amount of the pLNnH (3%, 4% and 5% respectively) and a lower relative amount of LNT-II (2%, 2% and 3%, respectively).

In comparison, the Edid strain was capable of producing an almost identical total amount of HMOs as the Vag strain, but with a shifted distribution of the individual HMOs, increasing the relative amount of LNnT to 92% compared to the 82% produced by the Vag expressing strain, and a lower relative amount of the pLNnH and LNT-II at 4% and 3 % respectively compared to the 11 % and 8%, respectively produced by the Vag strain.

To further evaluate the content of the different HMOs produced by each strain, the HMO profile of each individual strain was calculated in percentage, %mM of the total HMO produced by the individual strain as shown in table 9.

Table 9 HMO profile of each individual strains relative to the total HMO produced by the individual strain.

When looking at the distribution of individual HMOs relative to the total HMO production in the individual strains in table 9, it can be seen that the strains with Edicl , MdfA_CM, MdfA_YR and Blon_2475 transporters all have a lower total by-product formation, compared to the by-product formation in the Vag strain, which in turn leads to a larger fraction of the total amount of HMO produced by the cell being LNnT.

The results presented in table 8 and 9 accordingly indicates that Edicl is superior to Vag for LNnT production both in terms of amount (table 8) and in terms of relative by-product formation in the cell (table 9).

Table 8 and 9 above analysed the HMO distribution in the total broth (pellet and supernatant) from the fermentation. However, since the effect is achieved by adding transporters to the cells it is also interesting to access the HMO distribution in the supernatant only, to determine how much of the produced LNnT ends up in the supernatant. The supernatants were analysed from the fermentations with the Vag and Edicl strains and are shown in figure 6, where exported HMO is shown relative to the total amount of HMO exported from the Vag strain. Table 10 summarizes the relative amount of each of the individual HMOs in the supernatant for the Edicl and Vag strains relative to total amount of HMO exported from the individual strain.

Table 10. Individual HMO amounts retrieved from the supernatant relative to the total HMO produced by the individual strain.

★Determined from a single fermentation, while Edicl is the average of two replicated fermentations.

HPLC chromatograms of the isolated supernatants of the two strains illustrate the absence of the pLNnH peak (9.153 min) in the HPLC chromatogram in figure 7B (Edie strain). In addition, it can also be seen that the LNT-II peak is smaller in the Edicl strain compared to the Vag strain (Figure 7A).

When looking at the distribution of HMOs according to table 10, it is clear that the Edicl strain is more specific in its transport of the produced HMOs, in that only 2.6 % of the HMO present in the supernatant was LNT-II and in addition no pLNnH was found in the supernatant. Hence it appears the Edicl transporter has lower affinity for LNT-II and pLNnH, whereas in the Vag strain the total by-product HMOs in the supernatant sums up to 13.2%. Accordingly, it can be seen that an LNnT producing strain wherein the Edicl transporter is expressed produces highly pure LNnT in the supernatant fraction which may be recovered directly form the fermentation broth.

Furthermore, figure 6 illustrates than when the amount of individual HMOs in the supernatant are calculated relative to the total HMO (%mM) in the supernatant from the Vag strain, the Edicl strain produce 2.6% more total HMO in the supernatant than the Vag strain and 13% more LNnT in the supernatant than the Vag strain.

Another way to picture the by-product profile of the produced HMOs, is through the ratio of produced product (LNnT) and the produced HMO by-products.

Accordingly, using the results in table 9 and 10 the ratios of LNnT to the different by-product HMOs (LNT-II and pLNnH) was calculated, either from the total broth or from the isolated supernatant, as shown in table 1 1 .

Table 11. Ratios of product HMO to by-product HMO relative to the total amount of HMO produced by the individual strain.

★Determined from a single fermentation, while remaining data average results from two fermentations. n.d. = not determined.

The ratio of LNnT to the by-product HMOs LNT-II and pLNnH shown in table 11 clearly show that Edicl has a higher relative content of LNnT in total fermentation broth compared to Vag, MdfA_CM, MdfA_YR and Blon_2475, with a ratio of 34:1 for LNnT:LNT-ll and a ratio of 22:1 for LNnT:pLNnH for Edid . When only looking at the supernatant, the ratios are even higher, suggesting that Edid has a preference for LNnT over LNT-II and pLNnH in terms of transport.

Taken together the results presented in this example (tables 8-1 1 and figure 5 and 6) shows that Edid is the preferred transporter to be expressed in the LNnT producing cells, wherein Edid enables production of highly pure LNnT, with a highly preferable by-product profile while not compromising the overall amount of LNnT produced and wherein LNnT may be collected directly from the supernatant of the fermentation broth, thus greatly simplifying the purification process.

Example 2 -LNnT product and by-product distribution in commercialized product

The requirements set by the product specification defines the boundaries for by-products in the final product (purified fermentation broth). Accordingly, for a product such as LNnT it is highly preferable that the LNnT produced in the fermentation is largely free of by-product HMOs since this can positively influence the cost of the final product by reducing the need for extensive postfermentation (down-stream) purification to remove unwanted by-products, and thus simplifies the production process and cost immensely.

To be inside the specification of marketed non-crystalized LNnT set by e.g., GRAS Notice 895, the product must have a LNnT content of at least 80% of the total product. In addition, the specification limits the lactose content to maximum 10%, the pLNnH content maximum 5 % and the LNT-II content to maximum 3%, in the final product (see table 13).

In table 12 the amount of HMOs is calculated as percentage of all carbohydrate in the total broth after fermentation in example 2.

Table 12. Relative distribution of HMOs and primary by-products in total broth after fermentation.

From table 12 it can be seen that LNnT already constitutes 71 % of the total carbohydrates at the end of fermentation of the strains expressing Edid , whereas in the Vag strains LNnT amounts to 64% of total carbohydrates. In addition, the Edid strain only produces 2% LNT-II and 5% pLNnH, which is within the specification of GRAS notice 895.

In particular the reduction of pLNnH has a high potential for cost saving, since this is removed by chromatography, which is a time consuming and expensive process. As shown in figure 7, the level of pLNnH may be reduced by isolation of the supernatant form the total broth. LNnT produced from a strain with an Edid transporter therefore potentially allows for capacity improvement of the overall down-stream purification process and in particular the capacity of the chromatography step can be improved since flow-throughput of the column can be increased when the amount of pLNnH that needs to be removed is decreased.

The results shown in table 12 clearly shows the benefit of Edid in relation to the further purification, as the main by-products LNT-II and pLNnH are within the specification of what has been regulatorily approved.

Example 3 - Production of LNT by fermentation

Genetically engineered cells capable of producing LNT and expressing selected transporter proteins were subjected to fermentation. The effect on production of LNT as well as the byproduct HMOs LNT-II and pLNH2 was assessed.

The genotypes of the tested strains are shown in table 6. The strains were fermented in replicates of 2 as described in the “Method” section above with the following changes, the 100 ml starting culture medium contained 15 g/l of sucrose and the repeated bolus additions of 25% lactose was done every 16 hours.

Since the Nec transporter has previously been shown in WO2021/148615, to improve the export and overall production of LNT, this strain was chosen as the reference strain. para-LNH2 (para-lacto-N-neohexaose-2), is an isomer/analogue of para-lacto-N-hexaose naturally present in human milk, para-LNH2 it is not officially an HMO, however since it is a commonly observed by-product in the cellular production of LNT we included it in the analysis of the total HMO produced by the cell.

The total HMO content (pellet and supernatant) from the fermentation of the LNT producing strains were analysed and the results are shown in Table 19 and figure 8 for the individual HMOs (in percentage, %) produced by each strain relative to the total HMO amount produced in the Nec strain.

Table 19. HMOs produced by the strains relative to the total HMO produced in the Nec strain

From these data it can be seen that the Edid transporter improves the overall HMO production in the strain by at least 13%. When Edid is present alone the overall HMO increase is observed both for LNT and the precursor/by-product HMO LNT-II. Interestingly, the combination of the Edid and Nec transporter result in a slightly further increase in the total HMO, but with a significant improvement of the LNT amount and a decrease in the LNT-II amount compared to the reference strain with the Nec transporter.

Example 4 - Production of LNFP-V by fermentation

Genetically engineered cells capable of producing LNFP-V with and without the Edict transporter protein were subjected to fermentation. The effect on production of LNFP-V as well as the LNT-II based by-product HMOs LNT, LNFP-II and LNDFH-II was assessed.

The genotype of the tested strains is shown in table 6. The strains were fermented in replicates of 2 as described in the “Method” section above with the following changes, the H3PO4 in the starting minimal medium was substituted with NH4H₂PO4, KH₂PO₄, and additional NaOH was added. In the feed solution MgSO₄ x 7H₂O, KOH, H₃PO₄was substituted with (NH4)₂SO₄. The temperature of the fermentation was initially at 33°C but was dropped to 25°C with a 3-hour linear ramp initiated 15 minutes after the start of the feed. Lactose was added as a bolus addition of 25% lactose monohydrate solution 45 hours after feed start.

The total HMO content (pellet and supernatant) from the fermentation of the LNFP-V producing strains were analysed for the presence of HMOs with an LNT-II backbone and the results are shown in Table 20 for the individual HMOs (in percentage, %) produced by each strain relative to the total amount of LNT-II backbone HMOs produced in the LNFP-V strain without a transporter.

Table 20. HMOs produced by the strains relative to the total HMO produced in the LNFP-V strain without a transporter

From these data it can be seen that the Edict transporter improves the overall production of LNT-II core HMOs by 4000%. In particular the amount of LNT produced by the cell, but also LNFP-V and LNDFH-II were increased significantly compared to the strain without the Edict transporter.

If it is desired to produce a mixture of HMOs containing LNT and significant amounts of the complex HMOs LNFP-V and LNDFH-II, Edict has been shown to be a beneficial transporter.

Example 5 - Production of LST-c in Deep well assays

Genetically engineered cells capable of producing LST-c with and without the Edict transporter protein were compared to assess the effect of Edict in the production of LST-c.

The genotype of the tested strains is shown in table 6. The strains were analyzed in duplicate using the Deep well assay as described in the “Method” section above. The total HMO content (pellet and supernatant) from the Deep well assay of the LST-c producing strains were analysed for the presence of HMOs with an LNT-II backbone and the results are shown in Table 21 and figures 9A and 9B for the individual HMOs (in percentage, %) produced by each strain relative to the total amount of LNT-II backbone HMOs produced in the LST-c strain without a transporter.

Table 21. HMOs produced by the strains relative to the total HMO produced in the LST-c strain without a transporter

From these data it can be seen that the Edicl transporter is capable of exporting LST-c into the supernatant, in that the relative amount in the supernatant is increased by 8% from 32% to 40% of the total amount of LNT-II backbone HMOs present in the supernatant. As shown in example 1 it can also be seen that significantly less LNnT stays in the cell (pellet) when the Edicl transporter is introduced into the cell, in that the cellular amount of LNnT is reduced from 23% to 8% of the total amount of LNT-II backbone HMOs present in the cell pellet. Compared to cells that do not express an exporter, a reduction in the LNnT levels is observed for the pellet of Edicl -expressing cells that is not accompanied by an increase in LNnT levels that are detected in the supernatant fraction of such cells. Simultaneously, Edicl -expressing cells produce and export more LST-c than cells that do not express an exporter, a fact that is well-aligned with the above-mentioned drop in LNnT levels in the pellet without a concomitant increase in the LNnT levels in the supernatant in Edicl -expressing cells.

Claims

1 . A genetically engineered cell capable of producing an HMO with a lacto-N-triose II (LNT-II, GlcNAc(pi-3)Gal(pi-4)Glc) backbone, wherein said cell comprises, a. one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases, and b. a recombinant nucleic acid sequences encoding a transporter protein, Edicl , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 , wherein the expression of said transporter protein in said cell leads to export of the desired HMO from said cell.

2. The genetically engineered cell according to claim 1 , wherein the HMO with LNT-II backbone is selected from the group consisting of lacto-N-neotetraose (LNnT), lacto-N- tetraose (LNT), lacto-N-fucopentaose (LNFP-V) and 6’-sialyllacto-N-neotetraose (LST c).

3. The genetically engineered cell according to claim 1 or 2, wherein at least 85% of the total molar content of HMO produced by the cell is LNnT or LNT.

4. The genetically engineered cell according to any one of the preceding claims, wherein less than 15 % of the total molar content of HMO produced by the cell is a by-product HMO, such as less than 15 % of the total molar content of HMO produced by the cell is LNT-II.

5. The genetically engineered cell according to any one of the preceding claims, wherein the one or more glycosyltransferases comprises a p-1 ,4-galactosyltransferase or a p-1 ,3- galactosyltransferase and optionally a p-1 ,3-N-acetylglucosaminyltransferase.

6. The genetically engineered cell according to any of the preceding claims, wherein said engineered cell is selected from the group consisting of Escherichia Coli, Bacillus subtilis, lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

7. A method for producing a HMO product, wherein said method comprises, a. providing a genetically engineered cell according to any one of claims 1 to 6, b. cultivating the genetically engineered cell in a culture medium under conditions permissive for the production of said HMO; and c. optionally recovering said HMO.

8. The method according to any one of claims 7, wherein the HMO product is selected from the group consisting of lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), lacto-N- fucopentaose V (LNFP-V) and 6’-sialyllacto-N-neotetraose (LST c).

9. The method according to any one of claims 7 or 8, wherein at least 85%, such as at least 90% or such as at least 92% of the total molar content of HMO produced in the culturing step according to said method is LNnT.

10. The method according to any one of claims 7 or 8, wherein at least 80%, such as at least 85% or such as at least 90% of the total molar content of HMO produced in the culturing step according to said method is LNT.

1 1 . The method according to any one of claims 7 or 8, wherein a mixture of LNT and LNFP-V is produced.

12. The method according to any one of claims 7 or 8, wherein a mixture of LNnT and LST-c is produced.

13. The method according to any one of claims 7 to 12 , wherein less than 15% of the total molar content of HMO produced in the culturing step according to said method is a byproduct HMO, such as less than 15% of LNT-II.

14. The method according to any one of claims 7 to 13, wherein lactose and/or LNT-II is supplied during the cultivation of the genetically engineered cells as a substrate for the HMO formation.

15. An HMO product produced by the method according to any one of claims 7 to 14.

16. A nucleic acid construct comprising a recombinant nucleic acid sequence encoding a transporter protein, Edicl , comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 , or a functional variant thereof with an amino acid sequence that is at least 80%identical to SEQ ID NO: 1 , wherein the transporter protein encoding sequence is under the control of a recombinant promoter sequence.

17. Use of a nucleic acid construct according to claim 16, in a host cell producing an HMO which comprises a GlcNAc(pi-3)Gal(pi-4)Glc backbone and at least one additional saccharide moiety, such as a Gal(p1 -4)GlcNAc(p1 -3)Gal(p1-4)Glc or a Gal(p1-3)GlcNAc(p1-3)Gal(p1 - 4)Glc structure.