DK202170252A9

DK202170252A9 - Cell factories for lnt-ii production

Info

Publication number: DK202170252A9
Application number: DKPA202170252A
Authority: DK
Inventors: Papadakis Manos
Original assignee: Dsm Ip Assets Bv
Priority date: 2021-05-17
Filing date: 2021-05-17
Publication date: 2022-12-05
Also published as: DK181310B1; WO2022243307A1; DK202170252A1

Abstract

This invention relates to multiple genetic engineering approaches applied to enhance the formation of the human milk oligosaccharide (HMO) lacto-N-triose II (LNT-II). Several aspects towards the construction of a beneficial LNT-II cell factory have been investigated, including the LNT-II biosynthesis pathway per se, along with the cellular lactose import mechanism was investigated as to promote the cellular LNT-II production.

Description

DK 2021 70252 A9 1 CELL FACTORIES FOR LNT- PRODUCTION

FIELD This disclosure relates to a method of producing human milk oligosaccharides (HMOs) with high titers, wherein the HMO is LNT-H. Several genetic engineering approaches have been applied to change the abundance of the HMO produced by cells that express a heterologous B-1,3-N-acetyl- glucosaminyltransferase. The strain engineering strategies to achieve this goal depend on the B- 1,3-N-acstyl-glucosaminyltransferase introduced in the host. Tha disclosurs highlights several strain engineering strategies to enable the formation of LNT-H at high titers in the E. coff DHT K12 host. Specifically, these strategies deal with different aspects for the design of an efficient LNY-H — production system, which include the introduction and optimization of the pathway enabling LNT-II biosynthesis, the modification and/or introduction of a lactose and combinatorial solutions thereof.

BACKGROUND Human milk represents a complex mixdure of carbohydrates, fats, proteins, vitamins, minerals and — trace elements. The by far most predominant fraction is represented by carbohydrates, which can be further divided into lactose and more complex oligosaccharides (Human Milk Oligosaccharides, HMO). Whereas lactose is used as an energy source, the complex oligosaccharides are not metabolized by the infant. The fraction of complex oligosaccharides accounts for up to 1/10 of the total carbohydrate fraction and consists of probably more than 150 different oligosaccharides. The occurrence and concentration of these complex oligosaccharides are specific to humans and thus cannot be found in large quantities in the milk of other mammals, like for example domesticated dairy animals. To date, the structures of af least 115 HMOs have been determined, and considerably more are — probably present in human milk. HMOs have become of great interest in the last decade, due fo the discovery of their important functionality in human development. Besides their prebictic properties, HMOs have been linked to additional positive effects, which expands their field of application. The health benefits of HMOs have enabled thelr approval for use in foods, such as infant formulas and foods, and for consumer health products.

To bypass the drawbacks associated with the chemical synthesis of HMOs, several enzymatic methods and fermentative approaches have been developed. Fermentation based processes have iraditionally been developed for individual HMOs such as ?'-fucosyllactose, 3-fucosyliactose, lacto- N-tetraose, lacto-N-nectetraose, 3'-sialyllaciose and 6'-sialyllactose. Fermentation based

DK 2021 70252 A9 2 processes typically utilize genetically engineered bacterial strains, such as recombinant Escherichia coli (FE. coli}. Biotechnological production, such as a fermentation process, of HMOs is a valuable, cost-efficient and large-scale approach to HMO manufacturing. it relies on genetically engineered bacteria constructed so as fo express the glycosyltransferases needed for synthesis of the desired oligosaccharides and takes advantage of the bacteria's innate pool of nuclestide sugars as HMO precursors. At present, knowledge as to how to engineer cells to produce high titers of LNT-H, and how to select the optimal glycosyltransferases and subsequent genetic modifications which enable ENT-H production is limited, since LNT-il in many regards is considered a precursor HMO for production of more complex oligosaccharides.

SUMMARY This disclosure relates to multiple genstic engineering approaches applied to enhance the — formation of the human milk oligosaccharide (HMO) lacto-N-triose II (LNT-Hl). Several aspects towards the construction of a beneficial LNT-IF cell factory have been investigated, including the ENT-H biosynthesis pathway per se, but also a mechanism that enhances lactose import into the cell interior, altogether enhancing the LNT-II titers.

— We have observed that most genetic manipulations affected the level of lacto-N-triose fl (ENT-H), produced, whils only specific genetic modifications enhanced the LNT-H titer. This is particularly true for cells expressing the B-1,3-N-acetyl-glucosaminylransferase, HD0466 {GenBank ID: WP 010944479,1), where expression of a single copy enhanced the production of LNT-H over cells expressing the 8-1,3-N-acetyl-glucosaminyltransferase LgtA (GenBank ID: WP 033911473.1). Furthermore, combination of the B-1,3-N-acetyl-glucosaminyltransferase HDO466 with a second B- 1,3-N-acetyl-glucosaminyltransferase LgiA or PrnagT (WP 014390683.1) was capable of further enhancing the LNT-H titer. Also, combining expression of the §-1,3-N-acatyl glucosaminyliransferases HD0468 and LgtA with overexpression of the E. cof native Lactose permease also enhanced the LNTAI iter. This disclosure highlights ways of achieving high LNT-H titers related to strain engineering strategies. The strain engineering strategies to achieve this geal comprise the manipulation of the following genetic traits of the HMO producer cell:

DK 2021 70252 A9 3

1. Introduction of a specific 8-1,3-N-acetyl-glucosaminyltransferase, namely HDO466

2. Introduction of a second B-1,3-N-acetyl-glucosaminyltransferase, namely LgtA and/or PmnagT

3. Over-expression the native gene fac Y encoding the lactose permease Lacy in cells expressing the -1,3-N-acetyl-glucosaminyltransferases HD0466 and LgtA. This disclosure enables the skilled person to produce HMOs, primarily LNT-R, in enhanced amounts. — The enhanced amounts of LNT-H, gives a more sustainable manufacturing process; valuable HMOs are not discarded during the purification process and the conversion from carbon source to HMO product in fermentation is thus done at a higher overall yield. In its broadest aspect, the present disclosure relates to a method for the production of LNT-Ii, the — method comprising the steps of: a. providing a genetically enginesred cell capable of producing an HMO, wherein said cell comprises i} a heterologous -1,3-N-acetyl-glucosaminyl-transferase protein [HDO468] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1; and ii} a native or heterologous regulatory element for contralling the expression of i); and b. culturing the cell according to (a) in a suitable cell culture medium; and €. harvesting the HMO{s) produced in step {b).

In another aspect, the present disclosure relates to a genetically engineered cel comprising a) a nucleic acid sequence according to SEQ ID NO: 4 or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 4, encoding a heterologous 1-1,3-N-scetyl-glucosaminyt-transfersse,

DK 2021 70252 A9 4 b) a native or heterologous regulatory element for controlling the expression of a).

In a third aspect, the disclosure relates to a nucleic acid construct comprising a nucleic acid sequence encoding i} a heterologous 8-1,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1; and it) a native or heterologous regulatory element for contralling the expression of i}.

In a fourth aspect, the disclosure relates to use of a genetically engineered cell, or a nucleic acid construct according fo the present disclosure fo produce LNT-IL.

DETAILED DESCRIPTION This disclosure enables the skilled person to produce HMOs, primarily LNT, in enhanced amounts. In this manner, the present disclosure first of ali pinpoints and compares different B-1,3- N-acetyl-glucosaminyl transferases that can efficiently convert lactose to LNT-II, and indicate how their expression can be balanced for reaching optimal LNT production.

Secondly, it shows that ENT-H production systems that simultaneously express specific combinations of 3-1,3-N-acetyl-glucosaminyl transferases are mors productive than the ones expressing the same transferases separately at the same copy number, — Thirdly, it shows that the over-expression of the native E coli gene facY at levels that are higher than the normal cell physiological level can further enhance the synthesis of EÉNT-+II in cells expressing more than one B-1,3-N-acetyt-glucosaminyl transferases — but not necessarily, when a single transferase is expressed at the same copy number.

According to the present disclosure, the E. coli DH+ K12 host can be engineered to form LNT-H at high levels by introducing the B-1,3-N-acetyl-glucosaminyl transferase HDO468 from Haemophilus ducreyi, which can be further combined with the expression of the B-1,3-N-acetyl-glucosaminyl transferase LgtA from Neisseria meningitidis or PmnagT from Pasteurella multocida, and the over- expression of the native E£. coli JacY gene to reach even higher LNT-il levels,

DK 2021 70252 A9 3 Thus, the enhanced amounts of ENT-I, gives a more sustainable manufacturing process; valuable HMOs are not discarded during the purification process and the conversion from carbon source to HMO product in fermentation is thus done at a higher overall yield. Exemplary methods HDO466 In Example 1, it is demonstrated how different GleNAcTs can be beneficially expressed at varied genomic copy numbers in the genetic background of E. coli K12 cells for high-level ENT-I production. The Example reveals the HDO466 enzyme as a novel enzyme for the in vivo production of LNT.L In one or more exemplary embodiments, the method for the production of LNT comprises the steps of: a. providing a genetically engineered cell capable of producing an HMO, wherein said cell comprises i} a heterologous B-1,3-N-acetyt-glucosaminyl-transførase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1; and ii) a native or heterologous regulatory element for controlling the expression of i); and b. culturing the cell according to (2) in a suitable cell culture medium; and

0. harvesting the HMO{s) produced in step (b). Moreover, the present disclosure shows marked gains in LNT filters when different B-1,3-N- acetyl-glucosaminyl transferases are simultaneously expressed in the same production cell compared to LNT-II producers expressing a single transferase. According ta the present disclosure, the most marked increase in ENT-Ii titers could be achieved by combined expression of the HDG466 and LgtA enzymes.

DK 2021 70252 A9 6 In Example 2, it is demonstrated that the pairwise expression of two different GloNAcTs can be a more efficient approach in converting E. coff K72 cells to an efficient EÉNT-H cell factory than merely expressing a single GleNACT. — HDO0486/igtA Thus, in one or more exemplary embodiments the method for the production of LNT comprises the steps of: a. providing a genstically engineered cell capable of producing an HMO, wherein said cell comprises i} a heterologous p-1,3-N-acetyl-glucosaminyl-ransferase protein [RD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1;

ii} & native or heterologous regulatory element for controfling the expression of i); and iit} a heterologous [-1,3-N-acetyi-glucosaminyl-transferase protein as shown in SEQ ID NO: 2 [LgtA], or a functional homologue thereof having an amine acid sequence which is at least 80 % identical to SEQ ID NO: 2; iv) a native or heterologous regulatory element for controlling the expression of iif), b. culturing the cel according to (a) in a suitable cell culture medium; and Cc. harvesting the HMO4s) produced in step fb). HDO4686/PmnagT In one or more exemplary embodiments the method comprises the steps of: a. providing a genetically engineered cell capable of producing an HMO, wherein said cell comprises:

DK 2021 70252 A9 7 i} a heterologous B-1,3-N-acetyl-glucosaminyl-transferase protein [HDO466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1; ii} a native or heterologous regulatory element for controlling the expression of i); iif} & heterologous B-1,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 28 [PmnagT], or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 28; iv) a native or heterologous regulatory element for controlling the expression of iif), b. culturing the cell according to (2) in a suitable cell culture medium; and e. harvesting the HMOXs) produced in step fb). Since our goal is to convert e.g., the E. coli host to a cell factory for ENT-H production, a rational genetic engineering program includes the following major focus areas: a) the infroduction one or more highly active p-1,3-N-acetyl-glucosaminyl transferase for the conversion of the externally added lactose to LNT-l and b) the enhancement of lactose import into the cells. The genetic manipulation of genes involved in these cellular procedures could theoretically provide marked product yield gains. HDOSE64 gtA/LacY In addition, the over-expression of the Jac Y gene in the cells expressing more than one §-1,3-N- acetyl-glucosaminyl transferases, namely HD0468 and LgtA, can be advantageous for high-level LNT-H production, as seen in Example 3, the over-expression of the gene encoding the native lactose permease LacY can be beneficial for example when specific GloNAcTs, namely HD0466 and LgtA, are co-expressed in the sams strain.

In one or more exemplary embodiments the method comprises the steps of: a. providing a genetically engineered cell capable of producing an HMO, wherein said cell comprises

DK 2021 70252 A9 8 i} a heterologous B-1,3-N-acetyl-glucosaminyl-transferase protein [HDO466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1; and ii} a native or heterologous regulatory element for controlling the expression of i); iif} & heterologous B-1,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 2 [Lot], or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 2; iv) a native or heterologous regulatory element for controlling the expression of iif), v} a lactose permease protein as shown in SEQ ID NO: 3 [LacY], or a functional homologue thereof having an amino acid sequence which is at least 80 % identical te SEQ ID NO: 3, vi} a native or heteralogous regulatory element for controlling the expression of v) and b. culturing the cell according to (2) in a suitable cell culture medium; and

0. harvesting the HMO{s) produced in step (b). The ÉNT-H enzymes The present disclosure demonstrates the superior activity of another enzyme, namely HD0468 from Haemophilus ducreyi, towards LNT-H synthesis. Moreover, it is here shown that the combinations of different enzymes of the same type can be a highly beneficial approach for achieving high LNT-II titers. For the production of LNT-11, the genefically engineered cells comprise all the required enzymes to facilitate the production LNT-L One of these enzymes may for example be i} a heterologous 8-1,3-N-acetyl-glucosaminyt-transferase protein as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical fo SEQ ID NO: 1.

DK 2021 70252 A9 9 The above enzyme can be exchanged or supplemented by others with similar functionality. Especially, SEQ ID NO: 1, which can be supplemented with SEQ ID NO: 2 and/or SEQ ID NO: 28. When supplementing with SEQ ID NO: 2 or SEQ ID NO: 28, the level of produced LNT-H during culturing becomes increases, as shown in Example 2.

Heterologous B-1,3-N-acetyl-giucosaminyl-transferase A heterologous B-1,3-N-acetyl-glucosaminyl-transferase is any protein which comprises the ability of transferring the N-acelyl-glucosamine of UDP-N-acetyl-glucosamine to lactose. The 3-1,3-N- acetyl-glucosaminyt-transferase used herein does not originale in the species of the genetically engineered cell i.e. the gene encoding the 8-1,3-galactosyltransferase is of heterologous origin. The examples below use the heterologous 8-1,3-N-acetyl-glucosaminyt-transferase HDO466, LgtA andfor PmnagT. HDO466 genes In one or more exemplary embodiments, the HD0486 gene is as shown in SEQ ID NO: 4, or a functional homologue thereof having a nucleotide sequence that is at least 70 % identical to SEQ ID NO: 4. fgtA genes In one or more exemplary embodiments, the fgfå gene is as shown in SEQ ID NO: 5 or is a functional homologue thersof having a nucleotide sequence that is at least 70 % identical to SEQ ID NO: 5. PrmnagT genes In one or more exemplary embodiments, the PmnagT gene is as shown in SEQ ID NO: 29, or a functional homelogue thereof having a nucleotide sequence that is at least 70 % identical to SEQ ID NO: 29.

In one or more exemplary embodiments, the heterologous B-1,3-N-acetyl- glucosaminyltransferases, of the present disclosure that may, upon expression, be used to produce LNT-H are shown in the below matrix.

DK 2021 70252 A9 10 | | Protein Sequence (Gene {ID Description HMO example | | (GenBank) | | LNT-H, LNT, LNnT, | | LNFPL, LNFP-I, LNFP- | | B-1,3-N- ill, LNFP-V, LNFP-VI, | igtA | WP 033911473,1 | acetylglucosaminyl | LNDFH-I, LNDFH-II, | | transferase LNDFH-IN, på.NH, F- | | piNH I, pENnH, LST a, | | LST b, LST c, DSLNT | | LNT-H, LNT, LNnT, | | LNFP-I, NFP-1i, LNFP- | | B-1,3-N- ill, LNFP-V, LNFP-VI, | HDO4686 | WP 010944479,1 | acetylglucosantinyl | LNDFHH, LNDFH-I, | | transferase LNDFH-I1, pLNH, F- | | påNH I, på.

NnH, LST a, | LST b, LST 6. DSLNT LNT-H, LNT, LNnT, LNFP-I, ENFP-Ii, LNFP- Putative 8-1,3-N- — | Il, LNFP-V_LNFP-VI, PmnagT | WP 014390683,1 | acetylglucosaminyl | LNDFH-, LNDFH-, -transferase LNDFH-IH, pLNH, F- på NH I, på NnH, LST a, LST b, LST c, DSLNT In one or more exemplary preferred embodiments, the heterologous 5-1,3-N-acetyt- glucosaminyltransferases and the HMC LNT- can be generated using the protein of the amino acid sequence SEQ ID NO: 1 [HD0466] in combination enzymes are shown in the below matrix, r | | Protein Sequence | Gene | ID Description HMO example | {GenBank} UNT-H, LNT, LNnT, B-1,3-N- LNFP-L, ENFPI, LNFP- igtA WP 033911473.1 | acetylglucosaminyl | ill, LNFP-V, LNFP-VI, »transferase LNDFHJ, LNDFH-, LNDEH-H, pl NH, F-

DK 2021 70252 A9 11 | | Protein Sequence (Gene | ID Description HMO example | | (GenBank) CT me LST b, LST ¢, DSLNT LNT-H, LNT, LNnT, LNFP-I, ÅNFP-I, LNFP- Putative 8-1,3-N- il, LNEP-V, LNFP-VI, Frmnag7? | WP_014390683.1 | acetvlglucosaminyl | LNDFH-, LNDFH-II, transferase LNDFH-IN, pl.NH, F- påNH I, pE NnH, LST a, LST 5, LST c, DSLNT Lactose permease HDO466, can provide high LNT-H liters when expressed from one or two genomic copies (Figure 1a}. The descending order of activity of the three selected GloNACTs on lactose, as it is indirectly revealed by the observed final LNT-II titers is as follows: HD0468 > PmnagT > LgtA, The LNT-H titers reached by the strains MP5 andior MP6, which express HD0466 from a different copy number, can be up to 40% or 15% higher than for strains expressing LgtA (strain MP1) or PmnagT {strain MP3), respectively.

Lactose permease is a membrane protein which is a member of the major facilitator superfamily and can be classified as a symporter, which uses the proton gradient towards the cell to transport B-galactosides such as lactose in the same direction into the call. in HMO production laciose is the molecule being decorated to produce any HMO of interest and bioconversions happen in the cell interior, Thus, there is a desire to be able to import lactose into the cell, which can mainly be done by a certain activity of lactose permease, 8.¢., the native Jac Y copy under the control of a promoter, In one or more exemplary embodiments, the lactose permease protein is as shown in SEQ ID NO: 3, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 3, In one or more exemplary embodiments, the lactose permease gene is a nucleotide sequence as shown in SEQ ID NO: 8 or is a functional homologue thereof having a nucleotids sequence that is at least 70 % identical to SEQ ID NO: 6,

DK 2021 70252 A9 12 Lactose permease over-expression As shown in Example 3, the over-expression of the lacy gene coding lactose permease is used as a genetic tool to obtain an enhanced level of LNT-H produced by the genetically engineered cell of the present disclosure. As is shown in Example 3 only the combined expression of a specific pair of 8-1,3-N-acetyl-glucosaminyltransferases and lacY over-expression results in an enhanced LNT- I} production. The genetically engineered cells disclosed herein may comprise a regulatory element for increasing the expression of the native lactose permease protein, such as but not limited to — Ribosome Binding Sites (RASs). The RBSs may for example be the Shine-Dalgarne (SD) saquence. Mutations in the Shine-Dalgarno sequence can reduce or increase translation in prokaryotes. This change is due to a reduced or increased mRNA-ribosome pairing efficiency, as evidenced by the fact that compensatory mutations in the 3'-terminal 165 rRNA sequence can restore translation.

The regulatory element for increasing the expression of the native lactose permease protein could also be a promoter. The genetically engineered cells disclosed hersin may also comprise a heterologous episomal element for increasing the expression of the native lactose permease protein. This could for example be a plasmid-borne facY gene. The increased expression of the lactose permease may be achieved by direct integration of a copy of the facY gene in the genome. In this manner, Examples 2 and 3 provide enough data to — conclude that the combined expression of HDD466 and LgiA results in higher LNT-H titers regardless of intracellular lactose levels compared to when only one of these GlcNACTs is expressed by the cel at the same copy number. importantly, this trend is unique for this GleNACT pair and it is not observed for any other GIcNACT pair that can be formed from HDO48B, LgtA and Pmnagt.

The increased expression of the lactose permease may be achieved by deleting a repressor of the lactose operon. An example of such being the Jacf gene - UniProtKB - P03023 (LACE ECOL). As shown in the examples, an additional genomic copy of the fac Y gene which encodes the lactose permease with the PgloF promoter in HD0O466 expressing cells resulfed in a decrease in produced LNT-H. Contrary, the over-expression of the facY gene by an additional genomic PglpF-driven copy

DK 2021 70252 A9 13 in HD0466- and LgtA- expressing cells resulted in an increase in produced LNT-II, as is shown in Example 3. Amino acid identity In one or more exemplary embodiments, the amino acid sequence of the proteins of the present disclosure as shown in SEQ ID NO: 1-3 and 28, is at least 80% identical to SEQ ID NO: 1-3 and 28, such as at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85 % identical, at least 86 % identical, at least 87 % identical, at least 88 % identical, at least 89 % identical, at least 90 % identical, at least 81 % identical, af least 92 % identical, at least 93 % identical, at least 84 % identical, at least 95 % identical at least 96 % identical, at least 97 % identical, at least 98 % identical, or at least 98 % identical.

Controlling the expression In the present context the term “controlling the expression” relates to gene expression where the — franscription of a gene inte mRNA and its subsequent translation into protein is controlled.

Gene expression is primarily controlled at the level of transcription, largely as a result of binding of prateins to specific sites on DNA, such as but not limited to regulatory elements.

As described above, engineering strategy can be applied in multiple ways 29 1) the copy number of the gene of interest, 2) controlling the expression of any copy of these genes at the franscriptional or the translational level using heterologous or native regulatory elements, 3) the deletion of regulators that repress the expression of key genes in the HMO production process, 4) the over-expression of regulators that activate and/or enhance the expression of key genes in the HMO production process Over-expression A variety of molecular mechanisms ensures that genes are expressed at the appropriate level and under conditions of relevance to the applied production process.

For instance, the regulation of transcription can be summarized into the following routes of influence; genetic {direct interaction af a control factor with the gene of interest), modulation and/or interaction of a control factor within the transcriptional machinery and epigenetic {non-sequence changes in DNA structure that influence transcription).

DK 2021 70252 A9 14 It is known that a reduction in gene expression below a critical threshold for any gene will result in a mutant phenotype, since such a defect essentially mimics either a partial or complete loss of function of the target gene, whereas increased expression of a native gene can be both beneficial — or disruptive to a cell or organism. Over-axpression of a gene may be achieved directly by transcriptional activators that bind to key gene regulatory sequences to promote franscription or enhancers that constitute sequence elements positively affecting transcription. Similarly, direct over-expression of a gene can be achieved by simply increasing its copy number in the genomes, or replacing its native promoter with a promoter of higher strength or even modifying the sequence controlling the binding of the corresponding miRNA fo the ribosomes, i.e. the Shine-Dalgarno sequence being present upstream of the gene's coding sequence.

Moreover, over-expression of a gene may also be achieved indirectly through the partial or full inactivation of transcriptional repressors that normally bind Key regulatory sequences around the coding sequence of the gene of interest and thereby inhibit its transeription.

The term “over-expression” that is used here may for example refer to the native gene lacY and 29 includes 1) the replacement of the native promoter of the any £. coli gene by another, stronger promoter, 2) the modification of the native Shine-Dalgamo sequence of these genes by a stronger sequence with the goal of promoting ribosomal binding, 3) the deletion of the gene encoding a direct repressor or the enhancement of the expression of a gene encoding a direct activator of the native promoter of the £. coli gene(s) of interest, 4) the increase in the copy number of the gene(s) of interest, where the gene(s) are expressed from a genomic locus other than the native locus and the expression is driven by the native or a synthetic promoter {(e.g.. PglpF), and 5) the episomal expression of the gene(s) of interest from a low (5-10 copies per cell} io a high-copy number plasmid (300-800 copies per cel).

Thus, in one or more exemplary embodiments, the over-expression of the B-1,3-N-acetyl- glucosaminyltransferase proteins and/or the lactose permease of the present disclosure is provided by increasing the copy number of the genes coding said protein(s}, andlor by choosing an appropriate element for or adding an extra genomic copy for the genes encoding the B=1,3-N- acstyl-glucosaminyktransferase protains and/or the genes encoding the lactose permease, andlor conferring a non-functional (or absent} gene product that normally binds to and repress the expression of any of the the 8-1,3-N-acetyl-glucosaminyl-transferase proteins and/or the lactose permease of the present disclosure.

DK 2021 70252 A9 15 increasing the copy number As shown in Table 1 below, the only difference among the strains is the beta-1,3-N- acetyloglucosamine transferase being expressed or the copy number of the chosen transferase.

Copy number variation is a type of structural variation: specifically, it is a type of duplication or multiglication of a considerable number of base pairs. In one or more exemplary embodiments, expression is controlled by increasing the copy number of the desired protein.

Thus, in one or more exemplary embodiments, the present disclosure relates fo a method, wherein the overexpression of the B-1,3-N-2cstyl-glucosaminyl-fransferase protein(s} and/or the lactose permease is provided by increasing the copy number of any of the genes coding for said proteins) andlor by choosing an appropriate regulatory element.

Regulatory element The genetically engineered cell according to the methods described herein may comprise regulatory elements enabling the controlled overexpression of endogenous or heterologous andfør synthetic nucleic acid sequences.

In one or more exemplary embodiments, the heterologous regulatory element for controlling and increasing the expression of the -1,3-N-acetyl-glucosaminyl-transferase protein(s) and/or the lactose permease protein(s) in the method(s) described above is a promoter.

The term “regulatory element”, comprises promoter sequences, signal sequence, and/or arrays of transeription factor binding sites, which sequences affect transcription and/or translation of a nucleic acid sequence operably linked fo the regulatory element.

Regulatory elements are found at transcriptional and post-transcriptional levels and further enable molecular networks at those levels. For example, af the post-transcriptionat level, the biochemical signals controlling mRNA stability, translation and subcellular localization are processed by regulatory elements. RNA binding proteins are another class of post-transcriptional regulatory elements and are further classified as sequence elements or structural elements. Specific sequence motifs that may serve as regulatory elements are also associated with mRNA

DK 2021 70252 A9 16 modifications. A variety of DNA regulatory elements are involved in the regulation of gene expression and rely on the biochemical interactions involving DNA, the cellular proteins that make up chromatin, gene activators and repressors, and transcription factors.

In general, the transcriptional and translational regulatory sequences include, but are not Emited to, promoter sequences, fibosomal binding sites, transcriptional start and stop sequences, franslational start and stop sequences, binding sites for gene regulators and enhancer sequences. Promoters and enhancers are the primary genomic regulatory components of gens expression.

Promoters are DNA regions within 1—2 kilobases (kb) of a gene's transcription start site (TSS); they contain short regulatory elements (BNA motifs) necessary to assemble RNA polymerase transcriptional machinery. However, transcription is often minimal without the contribution of DNA regulatory elements located more distal to the TSS. Such regions, often termed enhancers, are position-independent DNA regulatory elements that interact with site-specific transcription factors io establish cell type identity and regulate gene expression. Enhancers may act independently of their sequence context and at distances of several fo many hundreds of kb from their target genes through a process known as looping. Because of these features, it is difficult to identify suitable enhancers and link them to their target genes based on DNA sequence alone.

The promoter, together with other transcriptional and translational regulatory nucleic acid sequences {also termed "control sequences”) is necessary to express a given gene or group of genas {an operon).

Identification of suitable promoter sequences that promote the expression of the specific gene of — interest is a tedious task, which in many cases requires laborious efforts. In relation to the present disclosure regulatory elements may or may not be post-translational regulators or X may or may not be translational regulators.

Thus, in one embodiment of the disclosure the regulatory element comprises one or more elements capable of enhancing the expression, of the one or more nucleic acid sequence(s) according to the present disclosure.

In that regard the regulatory element, controlling the expression of nucleic acid sequences and/or genes encoding ons or more glycosyltransferases and/or a lactose permease protein may be a promoter sequence.

In carrying out the methods as disclosed herein, different or identical promoter sequences may be used fo drive transcription of different genes of interest integrated info the genome of the host cell or into episomal DNA. Native In relation to the present disclosure, the term “native” refers to nudleic acid sequences originating from the genome of the genetically engineered cell according to the method of the disclosure. In that regard a nucleic acid sequence may be considered native if i originates from the E. coli K12 strain, is not of heterologous origin and not a recombined nucleic acid sequence, with respect to the genstically engineered cell. Heterologous regulatory element A regulatory element may be endogenous or heterologous, and/or recombinant and/or synthetic nucleic acid sequences. In the present context, the term "heterologous regulatory element” is to be — understood as a regulatory element that is not endogenous to the original genetically engineered cell described herein. The heterologous regulatory element may also be a recombinant regulatory element, wherein two or more non-operably linked native regulatory slement(s) are recombined into a heterologous and/or synthetic regulatory element. The heterologous regulatory element, may be introduced into the genetically engineered cell using methods known io the person skilled in the art Promoter sequences The regulatory element or elements regulating the expression of the genes and/or nudeic acid sequencels), may comprise one or more promoter sequence(s), wherein the promoter seguence, is operably linked to the nucleic acid sequence of the gene of interest in that sense regulating the expression of the nucleic acid sequence of the gene of interest. In one or more exemplary embodiments, the heterologous regulatory element is a promoter sequence.

In general, a promoter may comprise native, heterologous and/or synthetic nucleic acid sequences, and may be a recombinant nucleic acid sequence, recombining two or more nucleic acid sequences or same or different origin as described above, thereby generating a homologous, heterologous or synthetic nucleic promoter sequence, and/or a homologous, heterologous or synthetic nudeic regulatory element.

DK 2021 70252 A9 18 In one or more exemplary embodiments, the regulatory element of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell comprises more than one native or heterologous promoter sequence.

In one or more exemplary embodiments, the regulatory element of the genetically engineered cell comprises a single promoter sequence. In one or more exemplary embodiments, the regulatory element of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell comprises two or more regulatory elements with identical promoter sequences. In one or more exemplary embodiments, regulatory element of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell comprises two or more regulatory elements with non-identical promoter sequences. The regulatory architectures i.e., gene-by-gene distributions of transcription-factor-binding sites and identities of the franscription factors that bind those sites can be used multiple different growth conditions and there are more than 100 genes from across the E. coff genome, which acts as regulatory elements. Thus, any promoter sequence enabling transcription and/or regulation of the level of transcription, of one or more heterologous or native nucleic acid sequences that encode one or more proteins as described herein may be suitable. In one or more exemplary embodiments, the heterologous regulatory element is selected from the group consisting of PBAD, Pxyl, PsacB, PxylA, ProR, Pritå, PT7, Place, PL, PR, PnisA, Pb, Pscr, Pser SD1, Pser SD7, PgatY FOUTR, PgipF, PgipF SD1, PgipF. $D10, PgipF SD2. PglpF SD3, Pglp SD4, PglpF. SDS, PglpF SDS, PglpF. SD7, PglpF SD8, PgloF SD9, PglpF B28, Plac 18UTR, Plac, PmgiB 70UTR and PmglB 7OUTR SD4. — In one or more exemplary embodiments, the heterclogous regulatory element is selected from the group consisting of PglpF, and Plac, In a preferred exemplary embodiment, the promoter sequence comprised in the regulatory element for the regulation of the expression of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell, encompasses the gipFKX operon promoter sequence, PgipF.

DK 2021 70252 A9 19 In one or more exemplary embodiments, the promoter sequence comprised in the regulatory element for the regulation of the expression of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell, encompasses the fag operon promoter sequence, Plac.

In one or more exemplary embodiments, the regulatory element for the regulation of the expression of a recombinant gene included in the construct of the disclosure is the mgiBAC; galactose/methyl-galactoside ABC transporter periplasmic binding protein promoter PmgiB or variants thereof such as but not limited to PmgiB 70UTR, or PmglB 7OUTR SD4. In one or more exemplary embodiments, the regulatory element for the regulation of the expression of a recombinant gene included in the construct of the disclosure is the gat YZABCD, tagatose-1,6-bisP aldolase promoter PgatY or variants thereof.

Pser In one or more exemplary embodiments, the heterologous regulatory element is Pscr or varianis thereof such as but not limited to SEQ ID NO: 7. —Pser SD? In one or more exemplary embodiments, the heterologous regulatory element is Pser 8D7 or variants thereof such as but not limited to SEQ ID NO: 8. Pser 8D7 In one or more exemplary embodiments, the heterologous regulatory element is Pscr SD7 or variants thereof such as but not limited to SEQ ID NO: 9. Pgaty 70UTR In one or more exemplary embodiments, the heterologous regulatory slement is FPgatY 70UTR or variants thereof such as but not limited to SEQ ID NO: 10. PgloF In one or more exemplary embodiments, the helerologous regulatory element is PglpF or variants thereof such as but not limited to SEQ ID NO: 11.

DK 2021 70252 A9 20 It is also obvious from the data shown in Figures 1a and fb that even a single genomic copy of the HDO486 gene suffice to reach the highest LNT-I titers when any of these GleNAcTs is highly expressed in the cell. A GlcNACT is hereby defined as "highly expressed” when the host strain expresses it from at least two PglpF-driven genomic copies.

FPgipF S01 In one or more exemplary embodiments, the heterologous regulatory element is PgipF SD1 or variants thereof such as but not limited to SEQ ID NO: 12.

— Pglp SD10 In one or more exemplary embodiments, the heterologous regulatory element is PgipF SD10 or variants thereof such as but not limited io SEG ID NO: 13. PglpF SD2 — In one or more exemplary embodiments, the heterologous regulatory element is PgloF SD? or variants thereof such as but not limited fo SEQ ID NO: 14. PglpF SD3 In one or more exemplary embodiments, the heterologous regulatory element is PgipF SD3 or variants thereof such as but not limited to SEG ID NO: 15. PgloF SD4 In one or more exemplary embodiments, the heterologous regulatory element is PaipF SD4 or variants thereof such as but not limited to SEQ ID NG: 16.

Polo SD5 In one of more exemplary embodiments, the heterologous regulatory element is PgipF. SD5 or variants thereof such as but not limited to SEQ ID NO: 17.

— PglpF SD6 In one or more exemplary embodiments, the heterologous regulatory element is PgipF $D6 or variants thereof such as but not limited to SEQ ID NO: 18.

DK 2021 70252 A9 21 PglpF. SD7 In one or more exemplary embodiments, the heterologous regulatory element is FgloF SD7 or variants thereof such as but not limited to SEQ ID NO: 19, PgipF SD8 In one or more exemplary embodiments, the helerologous regulatory element is PgipF SD8 or variants thereof such as but not limited to SEQ ID NO: 20. PgipF. 5D9 In one or more exemplary embodiments, the heterologous regulatory element is PglpF SDS or variants thereof such as but not limited fo SEQ ID NO: 21. PglpF B28 In one or more exemplary embodiments, the heterologous regulatory element is PgloF B28 or variants thereof such as but not limited to SEQ ID NO: 22. PgloF B29 In one or more exemplary embodiments, the heterclogous regulatory element is PginF B29 or variants thereof such as but not limited to SEQ ID NO: 23.

Plac 16UTR In one of more exemplary embodiments, the heterologous regulatory element is Plac 18UTR or variants thereof such as but not limited to SEQ ID NO: 24. Place In one or more exemplary embodiments, the heterologous regulatory element is Flac or variants thereof such as but not limited tø SEQ ID NO: 25. PmgiB 70UTR In one or more exemplary embodiments, the heterologous regulatory element is PmgiB 70UTR or variants thereof such as but not limited to SEQ ID NO: 26.

DK 2021 70252 A9 22 PmglB 70UTR SD4 In one or more exemplary embodiments, the heterologous regulatory element is PmglB 70UTR SD4or variants thereof such as but not limited to SEQ ID NO: 27.

Episomal element The term “episomal element” refers to an extrachromosomal nucleic acid sequence, that can replicate autonomously or integrate into the genome of the genetically engineered cell. Thus, an episamal nudeic acid sequences may be a plasmid that can integrate into the chromosome of the genetically engineered cell, i.e. not all plasmids are episomal elements. In one or more exemplary embodiments, episomal nucleic acid sequences may be a plasmid that is not integrated into the chromosome. in the present context, the episomal element refers to plasmid DNA sequences that carry an expression cassette of interest, with the cassetie consisting of a promoter sequence, the coding sequence of the gene of interest and a terminator sequence. In one or more exemplary embodiments, episomal nudeic acid sequences may be a plasmid with only a part of it being integrated into the chromosome. In the present context, the expression cassette resambles the one described above but it further comprises two DNA segments that are 29 homologous to the DNA regions up- and downstream of the locus that the gene of interest is to be integrated. Repressors In one or more exemplary embodiment(s), the genetically engineered cell disclosed herein comprises a non-functional or absent gene product that normally binds to and represses the expression of the required enzymes to facilitate the production of a human milk oligosaccharide {HMO} that is LNT-L The term a non-functional {or absent} gene product that normally binds to and represses the expression driven by the regulatory element in the present context relates to DNA binding sites upstream of the coding sequence of a gene of interest and specifically at the promoter region of said gene.

DK 2021 70252 A9 23 In one or more exemplary embodiments, the cell may have a non-functional {or absent} gene product(s} that would normally bind to and repress the expression of any of the B-1,3-N-acetyl- glucasaminykransferase protein(s} and/or the lactose permease protein or regions upstream of the regulatory element for controlling the expression of any of the 8-1,3-N-acetyl-glucosaminyt transferase protein(s) and/or the lactose permease protein. Moreover, the deletion of regulators that repress the expression of key steps in the biosynthesis of ENT-H can lead to increased levels of the mRNAs and eventually to higher total HMO titers.

— none or more exemplary preferred embodiments, the method according to the present disclosure comprise a cell further comprising non-functional (or absent) gene product that binds to and represses the expression of any of the B-1,3-N-acetyl-glucosaminyl-transferase protein(s) of the present disclosure, and wherein the $-1,3-N-acetyl-glucosaminyk-transferase protein is HDO486 and LgtA or PmnagT.

In one or more exemplary preferred embodiments, the method according to the present disclosure comprise a cell further comprising non-functional (or absent) gene product that binds to and represses the expression of any of the 8-1,3-N-acetyl-glucosaminyi-transferase protein(s) of the present disclosure, and wherein the $-1,3-N-acetyl-glucosaminyl-transferase protein is HD0488.

In one or more exemplary preferred embodiments, the method according to the present disclosure comprise a cell further comprising non-functional (or absent) gene product that binds to and represses the expression of any of 8-1,3-N-acetyl-glucosaminyl-transferase protein(s) and/or the lactose permease protein, and wherein the $=1,3-N-acetyl-glucosaminyi-transferase protein is HDO466 and LgtA.

In one or more exemplary embodiments, said gene product is the DNA-binding transcriptional reprassor GIpR.

GIR GIpR belongs ta the DeoR family of transcriptional regulators and acts as the repressor of the glycerch-3-phosphate regulon, which is organized in different operons. This regulator is part of the glpEGR operon, yet it can also be constitutively expressed as an independent {glo} transcription unit. In addition, the operons regulated are induced when Escherichia coli is grown in the presence of inductor, glycerol, or glycerol-3-phosphate {G3P), and the absence of glucose. in the absence of

DK 2021 70252 A9 24 inductor, this repressor binds in tandem to inverted repeat sequences that consist of 20-nucleic acid-long DNA target sites. The term “non-functional or absent” in relation to the gipR gene refers to the inactivation of the glpR gene by complete or partial deletion of the corresponding nucleic acid sequence from the bacterial genome. The gjp gene encodes the DNA-binding transcriptional repressor GIpR. In this way promoter sequences of the PgipF family are more active in the genetically engineered cell, due to delelion of the repressor gene that would otherwise reduce the franscriptional activity associated with the PolpF promoters.

In one or more exemplary embodiments, the gipR gene is deleted. The deletion of the gipR gene could eliminate the GlpR-imposed repression of transcription from all PgipF promoters in the cell and in this manner enhance gene expression from all PgipF-based casseties. Activators In one or more exemplary embodiment(s), the genetically engineered cel disclosed herein comprises an over-expressed gene product that enhances the expression of the genels) encoding the enzyme(s) required to facilitate the production of a human milk oligosaccharide (HMO) that is ENT-H. In one or more exemplary embodiments, the cel of the present disclosure may comprise an over- expressed gene product that enhances the expression of the gene(s) encoding any of the 8-1,3-M- — acetyl.glucosaminyt-transferase protein(s) and/or the lactose permease protein. In one or more exemplary embodiments, the cell of the present disclosure may comprise an over- expressed gene product that enhances the expression of the gene(s) encoding any of the 3-1,3-N- acetyl-glucosaminyl-transferase protein(s) of the present disclosure, and wherein the 3-1,3-N- acetyl-glicosaminyl-transferase protein is HD0466 and LgtA or PmnagT. In one or more exemplary embodiments, the cell of the present disclosure may comprise an over- expressed gene product that enhances the expression of the gene(s) encoding any of the B-1,3-N- acelyl-ghicosaminyl-iransferase protein(s} of the present disclosure, and wherein the 3-1,3-N- — acetyl-glucosaminyl-transferase protein is HD0466.

DK 2021 70252 A9 25 In one or more exemplary embodiments, the cell of the present disclosure may comprise an over- expressed gene product that enhances the expression of any of the 8-1,3-N-acetyt-glucosaminyt- transferase protein(s) and/or the lactose permease protein, and wherein the B-1,3-N-acetyl- glucosaminyl-transferase protein is HDO466 and LgtA.

CRP In one or more exemplary embodiments, said gene product is the cAMP DNA-binding transcriptional dual regulator CRP.

CRP belongs to the CRP-FNR superfamily of transcription factors. CRP regulates the expression of several of the E. coli genes, many of which are involved in catabolism of secondary carbon sources. Upon activation by cyclic-AMP, (cAMP) CRP binds directly to specific promoter saquences, the binding recruits the RNA polymerase through direct interaction, which in turn activates the transcription of the nucleic acid sequence following the prometer sequence leading to expression of the gene of interest. Thus, over-expression of CRP may lead fo an enhanced expression of a genefnudleic acid sequence of interest. Amongst other functions, CRP exerts its function on the PglpF promoters, where it contrary to the repressor GIpR, activates promoter sequences of the PglpF family. In this way, over-expression of CRP in the genetically engineered coll of the present disclosure, promotes expression of genes that are regulated by promoters of the PglpF family.

Thus, in one or more exemplary embodiments, the crp gene is over-expressed. Genetic engineering of GIpR and/or CRP, as suggested in the present disclosure, in 2'-FL producing strains is beneficial for the overall production of 2'-FL by these strains. Nucleic acid constructs An aspect of the present disclosure is the provision of a nucleic acid construct. Thus, in one or more exemplary embodiments the nucleic acid construct may comprise ai least i} a nucleic acid sequence according to SEQ ID NO: 4 or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 4, encoding a heterologous B-1,3-N-acetyt-glucoseminyt-transferase; and ii) a native or heterologous regulatory element for controlling the expression of i).

DK 2021 70252 A9 26 In one or more exemplary embodiments the nucleie acid construct also comprises: ili} a nucleic acid sequence according fo SEQ ID NO: 5 flgtAl, or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 5, encoding a heterologous $-1,3-N-acetyl-glucosaminyl-transferase, and iv) a native or heterologous regulatory element for controlling the expression of iii}. In one or move exemplary embodiments the nucleic acid construct also comprises: v} a nucleic acid sequence according SEQ ID NO: 6 fLacY], or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 8, to encoding a lactose permease, and vi} a native or heterologous regulatory element for controlling the expression of v). In one or more exemplary embodiments the nucleic acid construct also comprises: vi) a nucleic acid seguence according to SEQ ID NO: 29 [PmnagT], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical fo SEQ ID NO: 29, encoding a heterologous $-1,3-N-acetyl-glucosaminyl-transferase; vill) a native or heterologous regulatory element for controlling the expression of iii).

The nucleic acid construct may further comprise one or more regulatory element for controlling the expression of i), iii), v), vif}. The regulatory elementi(s} may be a native or heterologous or episomal. The nucleic acid construct may further comprise a heterologous regulatory or episomal element for increasing the expression of v) a lactose permease protein as shown in SEQ ID NO: 3, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 3. The nucleic acid construct may further comprise a non-functional {or absent) gene product that normally binds to and represses the expression of the regulatory element{s).

DK 2021 70252 A9 27 Recombinant nuclsic acid sequence The nucleic acid construct can be a recombinant nucleic acid sequence. By the ferm "recombinant nucleic acid sequence”, “recombinant gene/nucleic acid/DNA encading” or “coding nucleic acid sequence” used interchangeably is meant 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 inte 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 Send of the mRNA, a franscriptional start codon (AUG, GUG or ULKS), 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 sequence may be a coding DNA sequence e.g., a gene, or non-coding DNA sequence e.g., a regulatory DNA, such as a promoter sequence.

Accordingly, in one exemplified embodiment the disclosure relates fo a nucleic acid construct comprising a coding nucleic sequence, i.e. recombinant DNA sequence of a gene of interest, e.g. a B-1,3-N-acetyl-glucosaminyl-transferase gene, 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 Jac operon or an gip operon, of a promoier sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence, wherein the coding and promoter sequences are operably linked.

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

DK 2021 70252 A9 28 Generally, promoter sequences that are operably linked to a transcribed sequence are physically contiguous fo 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 if is an expression cassetie/carindge that is integrated in the genome of a host cell. Nucleic acid construct Accordingly, the term “nucleic acid construct” means an artificially constructed segment of nucleic acid, in particular a DNA segment, which is intended to be transplanted’ into a target cell, e.g. a bacterial cell, to modify expression of a gene of the genome or express a genefcoding DNA sequence which may be included in the construct.

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 atfTn7-site (Waddell C.S. and Craig N.L., Genes Dev. (1988) Feb; 2{2):137-49.}; methods for genomic integration of nudeic 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):2083-7; 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 af, 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.

Nucleic acid identity In one or more exemplary embodiments, the present disclosure relates to a recombinant nucleic acid shown in SEQ ID NO: 4-8 or 29, or a functional homologue thereof having a sequence that is at least 70% identical to SEQ IO NO; 4-8 or 29, such as af least 71% identical, at least 72% identical, at least 73% identical, at least 74% identical, at least 75% identical, at least 78% identical, at least 77% identical, at least 78% identical, at least 79% identical, at least 80 % identical, at least 81 % identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85 % identical, at least 88 % identical, at least 87 % identical, at least 88 % identical, at least 89 % identical, at least 90 % identical, af least 91 % identical, at least 92 %

DK 2021 70252 A9 29 identical, at least 83 % identical, at least 94 % identical, at least 95 % identical af least 96 % identical, at least 97 % identical, at least 98 % identical, or at least 88 % identical. Sequence identity The term “sequence identity of [a certain} %" in the context of two or more nudleic acid or amino acid sequences means that the two or more sequences have nucleic acids or amino acid residues in common in the given percent, when compared and aligned for maximum correspondence over a comparison window or designated sequences of nucleic acids or amino acids {i.e. the sequences have at least 80 percent (%) identity). Percent identity of nucleic acid or amino acid sequences can — be measured using a BLAST 2.0 sequence comparison algorithm with default parameters, or by manual alignment and visual inspection (see e.g. hitpi/Aww.nobi.nlm.nih.gov/BLASTY). This definition alse applies to the complement of a fest sequence and fo sequences that have deletions andfor additions, as well as those that have substitutions. An example of an algorithm that is suitable for determining percent identity, sequence similarity and for alignment is the BLAST

2.2.20+ algorithm, which is described in Altschul ef af. Nucl. Acids Res. 25, 3388 (1997). BLAST

2.2.20+ is used to determine percent sequence identity for the nucleic acids and profeins of the disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information {hitp:/fwaww.nebi nlm nih.gov/}. Examples of commonly used ssquence alignment algorithms are CLUSTAL Omega (hin fføww ebl ao ut/Toosmsafdustale), EMBOSS Needle (hip fy ebl ac uk/Tools/usa/smitoss needled), MAFFT (hitpdimalit ore jodalignmentserver), or MUSCLE (http ffernv. ebl.ao uk/Toole/swsa/musdied).

Functional homologue A functional homologue of a protein/nucleotide as described herein is a profein/nuclectide with alterations in the genetic code, which retain its original functionality. A functional homologue may be obtained by mutagenesis. The functional homologue should have a remaining functionality of at least 50%, such as 80%, 70%, 80 %, 90% or 100% compared to the functionality of the pratein/nucleotide, A functional homologue of any one of the disclosed amine acid or nucleotide sequences can also have a higher functionality. A functional homologue of any one of SEQ ID NOs: 1-29, should ideally — be able to participate in the HMO production, in terms of HMO vield, purity, reduction in biomass

DK 2021 70252 A9 30 formation, viability of the genetically engineered cell, robustness of the genetically engineered cell according to the disclosure, or reduction in consumables.

Genelically engineered cell — The present disclosure also relates to a genetically engineered cell comprising a) a nucleic acid sequence according to SEQ ID NO: 4 or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 4, encoding a heterologous 3-1,3-N-acetyl-glucosaminyt-transferase; and b) a native or heterologous regulatory element for controlling the expression of al.

In one or more exemplary embodiments the genetically engineered cell also comprises: ¢} a nucleic acid sequence according to SEQ ID NO: 5 [LgtA), or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 5, encoding a heterologous $-1,3-N-acetyl-glucosaminyl-ransferase; d) a native or heterologous regulatory element for controlling the expression of c). In one or more exemplary embodiments the genetically engineered cell also comprises: e) a nucleic acid sequence according to SEQ ID NO: 8 [LacY], or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 6 encoding a lactose permease, and f) a native or heterologous regulatory element for controlling the expression of e). In one or more exemplary embodiments the genetically engineered cell also comprises: g) a nucleic acid sequence according to SEQ ID NO: 28 [PmnagT], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical fo SEQ ID NO: 29, encoding a heterologous B-1,3-N-acetyl-glucosaminyl-transferase; h) a native or heterologous regulatory element for controlling the expression of g).

DK 2021 70252 A9 31 A "genetically engineered cell” as used herein is understood as a cell which has been transformed or transfected, by a recombinant nucleic acid sequence. Accordingly, a "genetically engineered cell” is in the present context understood as a host cell which has been transformed or transfected by a recombinant nucleic acid sequence.

In one or more exemplary embodiments, the HMO produced by the genetically engineered cell is ENT-H. The genetically engineered cell is preferably a prokaryotic 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 {hast cell) may be e.g., a bacterial or veast cell. in one preferred embodiment, the genetically engineered cell is a bacterial cell.

Bacterial host cells 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 fo the disclosure could be Erwinia herbicola (Panfoea agglomerans), Citrobacter freundii, 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 lertus, Bacillus cereus, and Bacillus circudans. 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 salfvarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus erispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bactenal species for the disclosure described herein. Also included as part of this disclosure are strains, engineered as described hare, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus fhermophiles), Bifidobacterium (e.g.

Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporofactobacillus

DK 2021 70252 A9 32 spp.. Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas {e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa). In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of E. cofi, C. glutamicum, L. lactis, B. subtilis. S. lividans, P. pastoris, and S. cerevisiae. In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of B. subtilis, S. cerevisiae and E. coli. In one or move exemplary embodiments, the genetically engineered cell is 8. subtilis. In one or more exemplary embodiments, the genetically engineered cell is &. cerevisiae. In one or more exemplary embodiments, the genetically engineered cell is E. coli.

In one or more exemplary embodiments, the disclosure relates to a genetically engineered cell, wherein the cell is derived from the E. coli K12 strain. Culturing Inthe present context, culturing refers to the process by which cells are grown under controlled conditions, generally outside their natural environment, thus a method used to cultivate, propagate and grow a large number of cells. The terms culturing and fermentation are used interchangeably.

Celt culture medium In the present context, a growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms, cells, or small plants. The medium comprises an appropriate source of energy and may comprise compounds which regulate the cell cycle. The culture medium may be semi-defined, i.e. containing complex media compounds (e.g. yeast extract, soy peptone, casaming acids, etc.), or it may be chemically defined, without any complex compounds. Exemplary suitable medias are provided in experimental examples. In one or more exemplary embodiments, the culturing media is minimal media.

DK 2021 70252 A9 33 In one or more exemplary embodiments, the culturing media is supplemented with one or more anergy and carbon sources selected form the group condaining glycerol, sucrose, glucose and fructose. — 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 one or more exemplary embodiments, the culturing media is supplemented with glycerol, sucrose and/or glucose.

In one or more exemplary embodiments, the culturing media is supplemented with glycerol andfor glucose. In one or more exemplary embodiments, the culturing media is supplemented with sucrose and/or — glucose. In one or more exemplary embodiments, the culturing media is supplemented with glycerol and/or sucrose. Harvesting The term “harvesting” in the context relates to collecting the produced HMO(s) following the termination of fermentation. In one or more exemplary embodiments it may include collecting the HMO(s) included in both the biomass (i.e. the host cells) and culfivation media, i.e. before/without separation of the fermentation broth from the biomass. In other embodiments, the produced HMOs may be collected separately from the biomass and fermentation broth, i.e. after/foliowing the separation of biomass from cultivation media {i.e. fermentation broth). 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 s later stage after storing the fermentation broth at appropriate conditions. Recovery of the produced HMG{s) from the remaining biomass (or total fermentation) include extraction thereof from the biomass (i.e. the production cells). — After recovery from fermentation, HMO(s) are available for further processing and purification.

DK 2021 70252 A9 34 Human milk ofigosaccharide {HMO} In the context of the disclosure, the term “oligosaccharide” means a saccharide polymer containing a number of monosaccharide units. in some embodiments, preferred oligosaccharides are saccharide polymers consisting of three or four monosaccharide units, i.e. trisaccharides or — tetrasaccharides.

Preferable 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 af the reducing end that can be elongated by one or more beta-N-acetyt-factosaminyl and/or one or more beta-lacto-N-biosyl units, and this core structure can be substituted by an alpha-L- fucopyranosyl andfor an alpha-N-acetyl-neuraminyl {sialyl} moiety.

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 før neutral) HMOs can be fucosylated or non-fucosylated.

Examples of such neutral non-fucosylated HMOs include lacto-N- triose 2 (LNT-) lacto-N-tetraose (LNT), lacto-N-neotetraose (LNAT), lactc-N-nechexacse (LNnH), para-lacto-N-neohexaose (pi NnH), paradacio-N-hexaose (pLNH} and lacto-N-hexaose (LNH). Examples of neutral fucasylated HMOs include 2'-fucøsyllactose (2-FL), lacto-N-fucopentaose |

— {LNFP-}, lacto-N-difucohexaose | (LNDFH-D), 3-fucosyllactose (3'-FL), difucosyllactose (DFL), lacto-N-fucopentaose II {LNFP-il}, lacto-N-fucopentaose il (LNFPJM, lacto-N-difucohexaose IN (LNDFHN), fucosyi-lacto-N-hexaose II (FENH-D, lacto-N-fucopentaose V {LNFP-V}, lacto-N- difucohexaose H (LNDFHD, fucosyl-lacta-N-hexaose I (FLNH-), fucosyl-para-lacto-N-hexsose I (FpLNH-1}, fucosyl-para-lacto-N-neohexaose Ii (F-pLNnH 11) and fucosyt-lacto-N-neohexaose

{FLNnH). Examples of acidic HMOs include 3'-sialyllactose (3-SL), 6'-sialyllactose (6'-SL), 3- fucosyl-3'-sialyllactose (FSL), 3-0-sialykacto-N-tetraose a (LST a), fucosyt-LST a (FLST a), 6'-0- sialyllacto-N-tefraose b (LST b), fucosyt-LST b fFLET b), 6-O-sialyllacto-N-neotetravse {LST 0), fucosyl-LST c (FLST c), 3'-0-sialylacto-N-neotetraose (LST d), fucosyl4 ST d (FLST d), stalyt- lacto-N-hexaose (SLNH), sialyklacto-N-nechexasose | (SLENHVD), sialyl-lacto-N-neohexaose H

39 — (SLNH4B and disialyl-lacto-N-tetraose (DSLNT). In the context of the present disclosure lactose is not regarded as an HMO species.

DK 2021 70252 A9 35 Use of a genelically engineered ceil The disclosure also relates to any commercial use of the genetically engineered cell{s) or the nucleic acid construct(s) disclosed herein. The genetically engineered cells) or the nucleic acid construct{s) comprise at least one heterologous protein(s), such as but not limited to i} a heterologous 8-1,3-N-acetyl-glucosaminyt-transferase protein as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1; and optionally i) a heterologous 3-1,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 2 or 28 or a functional homologue thereof having an amine acid sequence which is at least 80 % identical to any one of SEQ ID NO: 2 or 28. The genetically engineered cell{s} or the nucleic acid construct(s) may further comprise a lactose permease protein as shown in SEQ ID NO: 3, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 3. The genetically engineered celi{s) or the nucleic acid constructfs) may comprise a native or heterologous regulatory element for controlling the expression of the 8-1,3-N-acetyl-glucosaminyt- transferssel(s). The genetically engineered cels) or the nucleic acid construct{s) may also comprise a native or heterologous regulatory or episomal element for increasing the exprassion of the lactose permease. The genetically engineered celi{s) or the nucleic acid construct{s) may comprise a non-functional (or absent) gene product that normally binds to and represses the expression of the p-1,3-N-acetyl- glucosaminyl-fransferase and/or the lactose permease.

In one or more exemplary embodiments, the genetically engineered cell or the nucleic acid construct is used in the manufacturing of ane or more HMOs. In another exemplified embodiment, the genetically engineered cell and/or the nucleic acid construct according to the disclosure, is used in the manufacturing of LNT-H.

DK 2021 70252 A9 36 Manufacturing of HMOs To produce one or more MMOs, the genetically engineered cells as described herein are cultivated according to the procedures known in the art in the presence of a suitable carbon and energy source, e.g. glucose, glycerol or sucrose, and a suitable acceptor, e.g. lactose or any HMO, and the produced HMO blend is harvested from the cultivation media and the microbial biomass formed during the cultivation process. Thereafter, the HMOs are purified according to the procedures known in the art, e.g. such as described in WO2015188834, WO2017182965 or WQ2017152918, and the purified HMOs are used as nutraceuticals, pharmaceuticals, or for any other purpose, e.g. for research.

Manufacturing of HMOs is typically accomplished by performing cultvation in larger volumes. The term "manufsciuring” and "manufacturing scale” in the meaning of the disclosure defines a fermentation with a minimum volume of 5 L culture broth. Usually, a “manufacturing scale” process is defined by being capable of processing large volumes of a preparation containing the product of interest and yielding amounts of the HMO product of interest that meet, e.g. in the case of a therapeutic compound or composition, the demands for clinical trials as well as for market supply. In addition to the large volume, a manufacturing scale method, as opposed fo simple lab svale methods like shake flask cultivation, is characterized by the use of the technical system of a bioreactor (formenter) 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 hehavior of an expression system in a lab scale method, such as shake flasks, benchiop bioreactors or the deep well format described in the examples of the disclosure, does allow to predict the behavior of that system in the complex environment of a bioreactor.

With regard to the suitable cell medium used in the fermentation process, there are no imitations. The culiure medium may be semi-defined, i.e. containing complex media compounds {e.g. yeast extract, soy peptone, casaming acids, etc.), or it may be chemically defined, without any complex compounds, Where sucrose is used as the carbon and energy source, a minimal medium might be preferable.

Manufactured product The strain enginesring strategy of the present invention contributes to a sustainable manufacturing process for the high-level production of ÉNT-I, where the conversion of the provided carbon — source to HMO product in førmentation is done at a high overall vield. As shown in Example 2, the concentration of the detected HMOs (in g/L) in each sample was used to calculate the % guantitative differences in the ENT content of the strains tested, i.e., the % differences in the

DK 2021 70252 A9 37 LNT-H concentrations formed by strains expressing LgtA, PmnagT or HDO466, or pairwise combinations of all three GIcNACTs relative to LgtA-exprassing cells.

The term “manufactured product” according to the use of the genetically engineered cell or the nucleic acid construct refer to the one or more HMOs intended as the one or more product HMMO(s). The various products are described above.

Advantageously, the methods disclosed herein provides both a decreased ratio of by-product to product and an increased overall yield of the product (andfor HMOs in total}. This, less by-product formation in relation to product formation facilitates an elevated product production and increases efficiency of both the production and product recovery process, providing superior manufacturing procedure of HMOs.

The manufactured product may be a powder, a composition, a suspension, or a gel comprising one — or more HMOs, Impostantly, beneficial features for the construction of efficient ÅNT-HI cell factories described above do not provide an additive effect in a single production strain that produces any of the three GleNACTs or combinations thereof. in other words, the features described in the present disclosure — can be exploited only in the way presented here fo provide the desired positive effect on LNT-II titers, Moreover, two given modifications that are proven beneficial for different strain backgrounds should not be expected to enhance LNT-H formation when combined in any of these strain backgrounds.

Tables Table 1. Genolypes of the strains MP1, MP2, MP3, MP4, MPS and MP8 | Strain IO Genotype Heterologous Protein | Sequence ID | GenBank WP, 033911473.1 WP, 033911473.1 WP, 014390683.1 WP, 014390683.1 WP, 010944479.1 WP 010944479.1

DK 2021 70252 A9 38 ! fgtA: gene coding for B-1,3-N-acetylogtucasamine transferase 2 PrnagT: gene coding for B-1,3-N-acetyloglucosamine transferase ? HDO466: gene coding for B-1,3-N-acetyloglucosamine transferase Table 2. Genotypes of the strains MP2, MP4, MP8, MP7, MP8 and MPS svane | cent V0 I] ” fgtA: gene coding for B-1,3-N-acetyjoglucosamine transferase ? PmnagT: gene coding for B-1,3-N-acetylogiucosamine fransferase ? HDQ4686: gene cading for 8-1, 3-N-acetylogtucosamine transfarase Table 3 - Genolypes of the strains MP2, MP10, MP4, MP11, MP6, MP12, MP7, MP13, MP8, MP14, MP9 and MP15.

skan |, , ; | facY*-PgipF NP 434877.1 | x1 tacY*-PalpF NP_414877.1

DK 2021 70252 A9 39 MBO, x1 PmnagT2-PolpF, x? IgA’ -PgloF, x1 WP 074390683.7, WP 033911473.1, fac Y-PglpF NP 414877.1 ' JgtA: gene coding for B-1.3-N-acetyloglucosamine transferase 2 PmnagT: gene coding for B-1,3-N-acelylogiucosamine transferase 3 HDO468: gene coding for §-1,3-N-acelyloglucosamine transferase fLacY: gene encoding for the lactose permease protein

GENERAL It should be understood that any feature and/or aspect discussed above in connections with the methods according to the disclosure apply by analogy to the engineered cell, the nucleic acid constructs and/or use described herein, The terms fermentation and culiuring are used interchangeably. The terms Lacto-N-triose, LNT- LNT RB, LNTZ and LNT 2, are used interchangeably, The following figures and examples are provided below to illustrate the present disclosure. They are intended to be illustrative and are not to be construed as limiting in any way,

DK 2021 70252 A9 40

BRIEF DESCRIPTION OF THE FIGURES Figure 1 Final LNT-H titers reached by cells expressing different GloNAcTs at different genomic copy numbers. (a) ENT-H titers for strains that bear a genomic copy of the PrminagT (strain MP3) or — HD0488 (strains MP5 and MP8) genes, shown relative to the final ENT-I titers of igtA-expressing cells {strain MP1}. The reference level {given as 100%) is shown for strain MP1. (b) LNT} titers for sirains that bear two genomic copies of the PminagT (strain MP4) or HD0466 (strain MP8) genes, shown relative to the final ÅNT-I titers of igfd-expressing cells (strain MP2). The reference level {given as 100%) is shown for strain MP2.

Figure 2 Final LNT-H titers reached by cells expressing a single GleNACT or a pairwise combination of GleNÅETs at the same genomic copy number. LNTHI titers are shown for strains that bear two genomic copies of a single GloNACT (LgtA/strain MP2 or PmnagT/strain MP4 or HD0486/strain MP8) and for strains bearing one copy of sach of two GlcNAcTs {LgtA & HD0466/strain MPT or Pmnagt & HD0486/strain MP8 or PmnagT & LgtA/strain MP8). The reference level {given as 100%) is shown for strain MP2, Figure 3 28 — Final LNT-H titers reached by cells expressing different GlcNAcTs with and without the native MFS transporter Lacy. LNT-H titers for strains that bear two genomic PgipF-driven copies of gene(s) encoding GlcNAcTis) and a single PglpF-driven copy of the facY gene (strains MP10, MP11, MP2, MP13, MP14, MP415), shown relative to the corresponding final LNT-IE titers of each GleNAcT-expressing celis (strains MP2, MP4, MPS, MP7, MP8, MP9). The reference level (given as 100%) is shown for strains MP2, MP4, MP8, MP7, MP8, MP2. Figure 4 Final LNT-H titers reached by cells bearing beneficial modifications as described in the present disclosure. LNT-H titers for strains that bear a genomic PgipF-driven copy of the lgtå and HDO466 genes and of the Jac Y gene (strains MP13), shown relative to the final LNT titers of cells that only express a PglpF-driven copy of the igtad and HD(0466 genes (strain MP7}. The reference level (given as 100%) is shown for strain MP7.

DK 2021 70252 A9 41 SEQUENCE I'S SEQ ID NO: 1 [HD0466 protein]

MTTLVSVLICAYNVEKYIDECLNAVIAQTYKNLEIVVNDGS TDG TLAKLRQFEAKDPRVKIDNIVNG GTSKSLNIGIQYCOGENARTDSDDIVDIHWIETLMRELDNSPETIAISAYLEFLAÆKGNGSKLSRSRK HGKNAENPISSEAISQRMLFGNPVHNNVALVRRKVFSEYGLRFDPDYIHAEDYKFWFEVSKLGKM RTYPKALVKYRLHATOVSSAYNOKORSIAKKIKREAISHYLQOYGIQLPEKLTIHDEFSIFSPOIEL SE

TVANKOELFWSLATSLSEYHFRDLLKIYSLDIFHOLSFKYKKRIFRKFLLPNRYPSVI SEQ ID NO: 2 [LgtÅ protein] — MOPLVSVLICAYNVEKYFAQSLAAVVNQTWRNIEILIVDDGSTDGTLAIAKDFOKRDSRIKILAOAQ

NSGLIPSLNIGLDELAKSGMGEYIAR TDADDIAAPDWIEKIVGEMEKDRSHAMGAWLEVLSEEKDG NRLARHHRHGKIWKKPTRPEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYD VSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIOKTARNDFLOSMGFKTRFDSLEYRQIK AVAYELLEKHLPEEDFERARRFLYQCFKR TD TLPAGAWL DFAADGRMRRLFTLRQYFGILHRLLKN

R SEQ ID NO: 3 [Lacy protein]

MYYLKNTNFWMFGLEFFFYFFIMGAY FPEFPIWLHDINHISKSD TGHFAAISLFSLLFQPLFGLLSDK LGLRKYLLWITGMLVMFAPFFIFIFGPLLQYNILVGSIVGGIYLGFCFNAGAPAVEAFIEKVSRRSNF —EFGRARMFGCVOWALCASIVGIMFTINNOFVFWLGSGCALILAVLLFFAKTDAPSSATVANAVGAN HSAFSLKLALELFROPKLWFLSLYVIGVSCTYDVFDQOFANFFTSFFATGEQGTRYFGYVTTMGEL LNASIMFFAPLIINRIGGKNALLLAGTIMSVRIIGSSFATSALEVVILKTLHMFEVPFLLVGCFKYITSOF EVRFSATIYLVCFCFFKOLAMIFMSVLAGNMYESIGFQGAYLVLGLVALGFTLISVFTLSGPGPLSLL

RRQVNEVA SEQ ID NO: 4 [HD0466 gene]

ATGACCACACTGGTTAGCGTTCTGATTTGTGCCTATAACGTGGAAAAATACATCGATGAATGTC TGAATGCAGTTATTGCCCAGACCTATAAAAACCTGGAAATTATCGTTGTGAATGATGGTAGCAC CGATGGOACCCTGGCAAAACTGCGTCAGTTTGAAGCAAAAGATCCGCGTGTTAAAATCATCGA — TAACATTGTTAATCAGGGCACCAGCAAAAGCCTGAATATTGGTATTCAGTATTGTCAGGGCGA AATTATTGCACGTACCGATTCAGATGATATCGTGGATATTCATTGGATCOAAACCCTGATGCGT GAACTGGATAATAGTCCGGAAACCATTGCAATTAGCGCCTATCTGGAATTTCTGGCCGAAAAA GGTAATGGTAGCAAACTGAGCCGTAGCCGTAAACATGGTAAAAATGCAGAAAATCCGATTAGC

DK 2021 70252 A9 42

AGCGAAGCAATTAGCCAGCGTATGCTGTTTGGTAATCCGGTTCATAACAATGTGGCACTGGTT CGTCGTAAAGTGTTTAGCGAATATGGTCTGCGTTTTGATCCGGATTATATTCATGCCCGAGGATT ACAAATTTTGGTTCGAAGTGAGCAAACTGGGTAAAATGCGTACCTATCCOAAAGCGCTGGTTA AATATCGTCTGCATGCAACCCAGGTTAGCAGCGCATATAATCAGAAACAGCGTAGCATTGCCA AARAAATCAAACGTGAAGCCATCAGCCATTATCTGCAGCAGTATGGCATTCAGCTGCCSGAAA AACTGACCATTCATGACCTGTTTAGCATTTTTAGTCCGCAGATTGAACTGAGCCTGACCGTTG CAAATAAACAAGAACTGTTTTGGAGCCTGGCAACCAGCCTGUAGCGAATATCATTTTCGTGATC TGCTOAAAATCTACAGCCTGGATATTTTTCATCAGCTGAGCTTCAAATACAAAAAGCOCATCTT

TCGCAAATTTCTGCTGCCGAATCGTTATCCGAGCGTTATT SEQ ID NO: 5 [lgtå gene]

ATGCAACCGCTGGTCTCCGTGCTGATCTGTGCTTACAATGTGGAAAAATACTTCGCCCAATCG CTGGCCGCAGTCGTGAATCAAACGTGOCGCAACCTGGAAATTCTGATCGTGGATGACGGCAG TACCGATGGTACGCTGGCGATCGCCAAAGATTTTCAGAAACGTGACTCCCGOCATTAAAATCCT GGCACAGGCTCAAAACAGTGGCCTGATTCCGTOCCTGAATATCGGTCTGGATGAACTGGCGA AAAGTGGCATGGGTGAATATATCGCACGCACCGATGCTGATGACATTGCGGCCCCGGACTGO ATTGAAAAAATCGTCGGCGAAATGGOAAMMAGATCGTAGCATTATCGCGATGGGTGCCTGGCT GGAAGTGCTGTCTGAAGAAAMAAGATGGCAATCGTCTGGCACGCCATCACCGTCATGGTAAAA

TCTGGAAAAAACCGACGCGTCCGGAAGATATTGCCGACTTTTTCCCGTTTGOCAACCCGATTOC > ACAACAATACCATGATCATGCGTCGCTCAGTTATTGATGGCGGTCTGCGCTATAATACGGAAC

GTGATTGGOGCGGAAGACTATCAGTTCTGGTACGATGTCTCGAAACTGGGTCGCCTGGCGTAT TACCCGGAAGCCCTGGTGAAATATCGTCTGCATGCCAACCAAGTTAGCTCTAAATACTCTATC CGCCAACACGAAATTGCACAGGGCATCCAAAAAACCOCTCGTAATGATTTTCTGCAGTCAATG

GGTTTTAAAACOCGCTTCGACTOCGCTGGOAATATCGTCAAATTAAAGCGGTTOCCTACGAACTG > CTGGAAAAACATCTGCCGGAAGAAGATTTTGAACGCGCGCGTOGCTTTCTGTATCAGTGCTTC

AAACGTACCGACACGCTGCCGGCAGGTGCTTGGCTOGATTTCGCAGCTGACGGTCGCATOC

GTCGCCTGTTTACCCTGCGTCAATACTTCGGCATTCTGCACCGCCTGCTGAAAAACCGTTAA SEQID NO: 8 flacY gene]

ATGTACTATTTAAAAAACACAAACTTTTGGATGTTCGGTTTATTOTTITICTTTTACTTTIITTATO ATGGGAGCCTACTTCCCGOTTTTFCCCGATTTGGCTACATGACATCAACCATATCAGCAAAAGT GATACGGGTATTATTTTTGCCGCTATTTCTCTGTTCTCOCTATTATTCCAACCGCTGTTIOGTC TGCTTCTGACAMACTCGGOCTCCGCAAATACCTGCTGTGGATTATTACCGGCATGTTAGTGA — TØTITGCOCCGTTCTITATTTTTATCTTCGOGOGCOCACTGTTACAATACAACATTTTAGTAGGATC

DK 2021 70252 A9 43

GATTGTTGGTGGTATTTATCTAGGCTTTTGTTTTAACGCCGGTGOCGCCAGCAGTAGAGGCATT TATTGAGAAAGTCAGCCGTCGCAGTAATTTCGAATTTGGTCGCGCGCGGATGTTTGGCTGTGT TSGCTGGLCGCTGTGTGCCTCGATTGTCGGCATCATGTTCACCATCAATAATCAGTTTGTTTT

CTGGCTGGGCTCTGGTTGTGCACTCATCCTCGCCGTTTTACTCTTTTTCGCCAAMACGGATGC n—GCCCTCTTCTGCCACGGTTGCCAATGCGGTAGGTGCCAACCATTCGGCATTTAGCCTTAAGCT

GGCACTGGAACTGTTCAGACAGCCAAAACTGTGGTTTTTGTCACTGTATGTTATTGGCGTTTC CTSCACCTACGATGTITTTGACCAACAGTTTGCTAATTTCTTTACTTOGTTCTTTGCTACCGGT GAACAGGGTACGCGGGTATTTGGCTACGTAACGACAATGGGCGAATTACTTAACGCCTCGAT TATGTTCTTTGCGCCACTGATCATTAATCGCATCGOGTGGGAAAAACGCCCTGCTGCTGOCTGG — CACTATTATGTCTGTACGTATTATTGGCTCATCGTTCGCCACCTCAGCGCTGGAAGTGGTTATT CTGAAAACGCTGCATATGTTTGAAGTACCGTTCCTGCTGGTGGGCTGCTTTAAATATATTACCA GCCAGTTTGAAGTGCGTTTTTCAGCGACGATTTATCTGGTCTGTTTCTGCTTCTTTAAGCAACT GGCGATGATTTTTATGTCTGTACTGGCGGGCAATATGTATGAAAGCATCGGTTTCCAGGGCGC

TTATCTGGTGCTGGGTCTGGTGGCGCTGGGCTTCACCTTAATTTCCGTGTTCACGCTTAGCGG — CCCCGGCCCECTTTCCCTGCTGCGTCGTCAGGTGAATGAAGTOCGCTTAA SEQ ID NO: 7 [Pscr}

GGTTAACGGCCCACTTTOCTGGCGACATCACAATTCTTAAACCGGTTTAGCAATTTTTATTTTO ACCGCGTTACCGACATGTTTACCATATCAACTAAACCGGTTTAGCAAACATTAGCACACTCACT

GATTTACCTTTGGATGTCACCAAC SEQ ID NO: 8 [Pscr SD1]

GGTTAACGGCCCACTTTGCTGGCGACATCACAATTCTTAAACCGGTTTAGCAATTTTTATTTTC

ACCGCGTTACCGACATGTTTACCATATCAACTAAACCGGTTTAGCAAACATTAGCACACTCACT —GATTTACCCAAATTCGAAACAGCT SEQ ID NO: 8 [Pscr_SD7]

GGTTAACGGCCCACTTTGCTGGCGACATCACAATTCTTAAACCGGTTTAGCAATTTTTATTTTC ACCGCGTTACCGACATGTTTACCATATCAACTAAACCGGTTTAGCAAACATTAGCACACTCACT GATITACCCAAGAGCAAAACAGCT

DK 2021 70252 A9 44 SEQ ID NO: 10 [PgatY_70UTR]

CGGCAACCTATGCCTGATGCGACGCTGAAGUGTCTTATCATGCCTACATAGCACTGCCACGT ATGTTTACACCGCATCCGGCATAAAAACACGCGCACTTTGCTACGGCTTCCCTATCGGGAGG CCGTTTTITTGCCTTTCACTCCTCGAATAATTTTCATATTGTCGTTTTTGTGATCGTTATCTCGA — TATTTAAAMACAAATAATTTCATTATATTTTGTGCCTACAAGCATCGTGGAGGTCCGTGACTTTC

ACGCATACAACAAACATTAACCAAGGAGGAAACAGCT SEQ ID NO: 11 [PglpF]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGOGTGACATAATCCA — CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCU

GTGACTTTCACGCATACAACAAACATTAACCAAGGAGGAAACAGCT SEQ ID NO: 12 [PgipF. SD1]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGOGTGACATAATCCA CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCU

GTGACTTTCACGCATACAACAAACATTAACCAAATTCGAAACAGCT SEQ ID NO: 13 [PgipF. SD10]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA — TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCUC

GTGACTTTCACGCATACAACAAACATTAACCAACTGAGAAACAGCT SEQ ID NO: 14 [PgipF. SD2] nm GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATOCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA TCAGATGGAATAAATGGCGOGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

DK 2021 70252 A9 45

ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCO

GTGACTTTCACGCATACAACAAACATTAACCAAGCGCAAAACAGCT SEQ ID NO: 15 [PglpF_SD3] — GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCO

GTGACTTTCACGCATACAACAAACATTAACCAAGAACAAAACAGCT SEQ ID NO: 16 [PglpF_SD4]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG — ATATTTCTCGTITTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAACTAGGAAACAGCT SEQ ID NO: 17 [PglpF_SD5]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA — CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCO

GTGACTTTCACGCATACAACAAACATTAACCAACCGAGAAACAGCT — SEQIDNO: 18 [PolpF. SD6]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG ATATTTCTCGTTTTTØCTCGTTAACGATAAGTTTACAGCATGOCCTACAAGCATCGTGGAGGTCO — GTGACTTTCACGCATACAACAAACATTAACCAAGAGCTAAACAGCT

DK 2021 70252 A9 46 SEQ ID NO: 19 [PglpF_SD7]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCUTGOGTGACATAATCCA CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG > ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAAGAGCAAAACAGCT SEQ ID NO: 20 [PgipF. SD8)

GTGACTTTCACGTATACAACAAACATTAACCAAGAGAAASACAGCT SEQ ID NO: 21 [PgipF. SD9)

GTGACTTTCACGCATACAACAAACATTAACCAAAGGAAAAACAGCT SEQ ID NO: 22 [PglpF_B28]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA — TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG ATATTTCTCGTTTTTGCTCGTTAACGATTTAATTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAAGGAGGAAACAGCT SEQ ID NO: 23 [PgipF. 829] nm GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

DK 2021 70252 A9 47

ATATTTCTCGTTTTTGCTCGTTAACGATCAGAATACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAAGGAGGAAACAGCT SEQ ID NO: 24 [Plac 18UTR)

TGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTT

GTGTGGAATTGTGAGCGGATAACAATTTCAAGGAGGAAACAGCT SEQ ID NO: 25 [Plac]

ATGCGCAAATTGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGG — CTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATG ATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGGTACCTAAGGAGGA

AACAGCT SEQ ID NO: 28 [PmgiB_TOUTR]

TGCGTCGCCATTCTGTCGCAACACGCCAGAATGCGGCGGCGATCACTAACTCAACAAATCAG GCGATGTAACCGCTTTCAATCTGTGAGTGATTTCACAGTATCTTAACAATGTGATAGCTATGAT TGCACCGTGCCTACAAGCATCGTGGAGGTCCGTGACTTTCACGCATACAACAAACATTAACCA

AGGAGGAACAGCT SEQID NO: 27 [PmgiB_7O0UTR_SD4]

TGCGTCGCCATTCTGTCGCAACACGCCAGAATGCGGCCGCGATCACTAACTCAACAAATCAG GCGATGTAACCGCTTTCAATCTGTGAGTGATTTCACAGTATCTTAACAATGTGATAGCTATGAT TGCACCGTGCCTACAAGCATCGTGGAGGTCCGTGACTTTCACGCATACAACAAACATTAACCA

ACTAGGAAACAGCT SEQ ID NO: 28 [PmnagT protein]

MENKPLVSVLICAYNVEKYIEECINAVINQTYKNLEHIVNDGSSDNTYFLLKKLAEKDNRIKILNFNNHI GISALNEGLKEIAGEYIARTDSDDITKPDWIEKILTOMONDPKRAMGSYLTVLSEENNGSVLANHRK

NKVEWKNPLEHKDIVEKMLFGNPIHNNSMVMRSEIYTKYHLIYDPDYHYAEDYKFWLEVSRIGKLA n— NYPESLVYYRLHRNQTSSIHNSQQEINGKKLRLQALNYYLKDLGIDYQLPEKFLFKDIALLQEIFYER

GMFRENHRRIIYECYLSLGEYNYKDIYYFLINKNNFLSIKDKFKHKKYLRPDKYSSTY

DK 2021 70252 A9 48 SEQ ID NO: 29 [PminagT gene]

ATGGAAAATAAACCGCTGGTTAGCGTTCTGATTTGCGCCTATAATGTGGAAAAATACATCGAA GAATGCATCAACGCCGTTATTAACCAGACCTATAAARACCTGGAAATCATCATTOGTGAATGATG GCAGCAGCGATAACACCTATTTTCTGCTGAAAAAACTGGCCGAAAAAGACAACCGTATCAAGA TCCTGAACTTCAACAACCATATTGGOCATTATTAGCGCACTGAATGAAGGCCTGAAAGAAATTG CCGGTGAATATATTGCACGTACCGATTCAGATGATATCACCAAACCGGATTGGATCGAAAAAA TTCTGACCTGTATGCAGAACGACCCGAAAATTATCOCAATGOGTAGCTATCTGACCGTTCTGA GCGAAGAAAATAATGGTAGCGTGCTGGCCAATCACCATAAAAACAAAGTGGAATGGAMAAAACC

CGCTGOGAACATAAAGATATCGTOGAAAAAATGCTGTTTGGOCAACCCGATTCATAATAACAGCA 19 TGGTTATGCGCAGCGAGATCTATACCAAATATCACCTGATTTATGATCCGGATTATCATTATGC

CBAGGACTATAAATTCTGGCTGGAAGTTAGCCGTATTGGTAAACTGGOCAAATTATCCGGAAAG CCTGGTTTATTATCGTCTGCATCGTAATCAGACCAGCAGCATTCATAATTCCCAGCAAGAAATC AACGGTAAAAAACTGCGTCTGCAGGCACTGAACTATTATCTGAAAGATCTGGGCATTGATTAT

CAGCTGCCGGAAAAATTTCTGTTCAAAGATATTGCACTGCTGCAAGAGATCTTTTATGAACGTG 13 — GTATGTTCCGCGAAAACATTATTCGTCGCATTATCTATGAGTGCTATCTGAGCCTGGGCGAGT

ATAATTACAAAGATATCTACTACTTCCTGATCAACAAAMAACAACTTTCTGAGCATCAAAGACAAA TTCAAAATCATCAAAAAATACCTGCGTCCGGACAAATATAGCAGCACCTATTAA

EXAMPLES Example 7 — Heterologous enzymes appropriate for the construction of efficient LNT-Il production systems Description of the genotype of strains tested in deep well assays Based on the previously reported platform strain MDO”), the modifications summarised in the table below, ware made to obtain the fully chromosomal strains MP1, MPZ, MP3, MPS, MP8 and MP4. The strains can produce the trisaccharide HMO LNT-H. Each of the six strains expresses a single beta-1,3-N-acetyloglucosamine transferase (GIcNACT) selected from the group consisting of {a} LgiA from Neisseria meningitidis {GenBank ID: WP_033011473.1), (a) PronagT from Pasteurella multocida (GenBank ID; WP 214390883.1), or (0) HDO466 from Haemophilus ducreyi {GenBank ID: WP_010844479.1). The only difference between the strain pairs, MP1-MP2, MP3-

DK 2021 70252 A9 49 MP4 and MP5-MP8, is the GloNAcT being expressed. The copy number of the GlcNACT in each strain pair is the only difference among the strains of the pair. For example, the strain MP6 bears two copies of the HDO4S88 gene while the strain MPS bears just one PgipF-driven copy of this gene.

In the present Example, it is demonstrated how different GlcNAcTs can be beneficially expressed at varied genomic copy numbers in the genetic background of E. coli KT2 cells for high-level ENT-H production. The Example reveals the HDU466 enzyme as a novel enzyme for the in wvo production of LNT As shown in table 1, the only difference among the strains is the beta-1,3-N-acetyloglucosamine transferase being expressed or the copy number of the chosen transferase. Description of the applied deep well assay protocol for strain characterization — The strains disclosed in the present example were screened in 96 deep well plates using a 4-day protocol. During the first 24 hours, preculiures were grown to high densities 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 supplemented with magnesium sulphate, thiamine and glucose. The precultures were incubated for 24 hours at 34 °C and 1000 rpm shaking and then further transferred to a new basal minimal medium (BMM, pH 7,5) in order to start the main culture, The new BMM was supplemented with magnesium sulphate, thiamine, a bolus of 20 % glucese solution (50 ul per 100 ml) and a bolus of 10 % lactose solution (5 mil per 100 ml). Moreover, 50 % sucrose solution was provided as carbon source, accompanied by the addition of sucrose hydrolase (invertase), so that glucose was — released at a rate suitable for C-limited growth. The main cultures were incubated for 72 hours at 28 °C and 1000 rpm shaking. For the analysis of total broth, the 96-well plates were boiled at 100°C, subsequently centrifuged, and finally the supernatants were analysed by HPLC. For supernatant samples, the initial 309 centnfugation of microtiter plates was followed by the removal of 0.1 ml. supematant for direct analysis by HPLC. For pellet samples, the cells were initially washed, then dissolved in deionized water and centrifuged. Following centrifugation, the pellets were analysed for HMO content in the cell interior after resuspension, boiling, centrifugation and analysis of the final supematant.

DK 2021 70252 A9 50 Results of the deep well assays Strains were characterized in deep well assays and samples were collected from the total broth.

All samples were analysed for HMO content by HPLC following the 72-hour protocol described above.

The concentration of the detected HMOs {in g/L} in each sample was used to calculate the % — guantitative differences in the LNT-II content of the strains tested, i.e., the % differences in the

LNT-H concentrations of PmnagT- or HD0466-expressing cells relative to JgtA-expressing cells.

As revealed by the analysis of total samples in deep-well cultures, apart from the LgtÅ and PrnnagT enzymes that have been previously applied for the production of various LNTH-core

HMOs, a novel GIcNACT, namely HD04686, can provide high LNT-H titers when expressed from one or two genomic copies (Figure 1a}. The descending order of activity of the three selected GlcNAGTs on lactose, as it is indirectly revealed by the observed final LNT-li fiters is as follows: HDO486 > PmnagT > LgtA.

The LNT-H titers reached by the strains MP5 andfor MPG, which express HDO486 from a different copy number, can beup to 40% or 15% higher than for strains expressing LgtA (strain MP1) or PmnagT (strain MP3), respectively.

It is also obvious from the data shown in Figures 1a and 1b that even a single genomic copy of the HD0466 gene suffice to reach the highest LNT titers when any of these GlcNAcTs is highly expressed in the cell.

A GIcNACT is hereby defined as "highly expressed” when the host strain expresses it from at least two PglpF-driven genomic copies.

Example 2— Combination of heterologous befa-1,3-N-acetylogiucosamine transferases is highly beneficial for LNT titer enhancement

Description of the genotype of strains MP5196 and MP5211 tested in deep well assays Based on the previously reported platform strain {‘MDQ"), the modifications summarised in table 2, were made to obtain the fully chromosomal strains MP2, MP4, MP8, MP7, MP8 and MP9. The strains can produce the trisaccharide HMO LNT-L Each of the six strains bears in total two PglpF- driven copies of a single or two bela-1,3-N-acetyloglucosamine transferases (GlcNAcTs) selected from the group consisting of (a) LgtA from Neisseria meningitidis (GenBank ID: WP 033911473.1), {a} PmnagT from Pasteurella multocida (GenBank ID: WP 014390683.1), or {c) HDO468 from Haemophilus ducreyi (GenBank ID: WP 010944479,1). Thus, all strains express one or two different GlIoNAcTs from a total of 2 genomic copies with the only difference among the strains being the selected GloNAcTs.

DK 2021 70252 A9 51 In the present Example, it is demonstrated that the pairwise expression of two different GleNAcTs can be a more efficient approach in converting E. coli K12 cells to an efficient LNT-H cell factory than merely expressing a single GlcNAcT.

As shown in table 2 below, the only difference among the strains is the GlcNAcT(s) being expressed, while the total copy number of the chosen transferase(s) is the same for every strain. Description of the applied deep well assay protocol for strain characterization The strains disclosed in the present example were screened in 98 deep well plates using a 4-day protocol. During the first 24 hours, precultures were grown to high densities and subsequently transferred fo 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 supplemented with magnesium sulphate, thiamine and glucose. The precultures were incubated — for 24 hours at 34 °C and 1000 rpm shaking and then further transferred to a new basal minimal medium (BMM, pH 7,5) in order to start the main culture. The new BMM was supplemented with magnesium sulphate, thiamine, a bolus of 20 % glucose solution (50 ul per 100 mi) and a bolus of 10 % lactose solution (5 ml per 100 ml). Moreover, 50 % sucrose solution was provided as carbon source, accompanied by the addition of sucrose hydrolase (invertase), so that glucose was released at a rate suitable for C-limited growth. The main cultures were incubated for 72 hours at 2B “C and 1000 rpm shaking. For the analysis of total broth, the 96-well plates were bolled at 100°C, subsequently centrifuged, and finally the supernatants were analysed by HPLC. For supematant samples, the initial centrifugation of microtiter plates was followed by the removal of 0.1 ml. supematant for direct analysis by HPLC. For pellet samples, the cells were initially washed, then dissolved in deionized water and centrifuged. Following centrifugation, the pellets were analysed for HMO content in the cell interior after resuspension, boiling, centrifugation and analysis of the final supernatant.

Results of the deep well assays Strains were characierized in deep well assays and samples were collected from the total broth. All samples were analysed for HMO content by HPLC following the 72-hour protocol described above. The concentration of the detected HMOs (in g/l) in each sample was used to calculate the % guantitative differences in the LNT content of the strains tested, i.e., the % differences in the

DK 2021 70252 A9 52 ENT«H concentrations formed by strains expressing LgtA, PmnagT or HDO486, or pairwise combinations of all three GlcNAGTs relative to LgtA-expressing cells. At physiological intracellular lactose concentrations (Le., wild-type expression of the lacy gene coding the lactose permease}, higher LNT titers can be achieved when HD0486 is co-expressed with either of the two other GIGNACTSs, namely PmnagT and LgtA (strains MP7 and MP8) rather than when HDO466 (strain MPB), PmnagT (strain MP4} or LgiA (strain MP2} alone are expressed at the same copy number (Figure 2). Specifically, strains bearing a copy of HDO486 and a copy of one of the two other GlcNAcTs {strains MPT and MP8) show higher liters than strains bearing two copies of any of the three ClcNAcTs (strains MP2, MP4 or MP8) or the strain that co-expresses 19 LgiA and PmnagT (strain MP9) at the same GIcNACT copy number (Figure 2). Example 3 — Genetic manipulation of the native MFS transporter LacY can provide superior LNT-I1 production systems depending on the beta-1,3-N-acetyloglucosamine transferase(s) being expressed — Description of the genotype genotype of strains tested in deep well assays Based on the previously reported platform strain ("MDO”), the modifications summarised in table 3, were made to obtain a number of fully chromosomal strains. The strains can produce the frisaccharide HMO LNT-il. Each of these strains bears in total two PglpF-driven copies of a single or two beta-1,3-N-acetyloglucosamine transferases (GlcNAcTs) selected from the group consisting of (a) LgtA from Neisseria meningitidis (GenBank ID: WP 033911473.1), (a) PmnagT from Pasteurella multocida (GenBank ID: WP 014390683.1), or (c) HDOABS from Haemophilus ducreyi {GenBank ID: WP 010944479.1). Thus, all strains express one or wo GleNACTs from a total of 2 genomic copies with the strains differing in the identity of the selected GlcNAcTs. Moreover, apart from the identity of GIcNAcTs, the strains can differ in regard to the expression of the E. coli KT2 lactose permease LacY {GenBank ID: NP_414877.1) (Table 3). In the present Example, it is demonstrated that the over-expression of the gene encoding the native lactose permease LacY can be beneficial only when specific GlcNAcTs, namely HDO466 and ÅgtÅ, are co-expressed in the same strain.

In this manner, Examples 2 and 3 provide enough data to conclude that the combined expression of HDOMEE and LgtA results in higher LNT-II titers regardless of intracellular lactose levels compared to when only one of these GlcNAcTs is expressed by the cell at the same copy number. Importantly, this trend is unique for this GlcNACT pair and it is not observed for any other GloNAcT — pair.

DK 2021 70252 A9 53 Description of the applied deep well assay protecol for strain characterization 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 wers grown to high densities and subsequently fransferred ta 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 supplemented with magnesium sulphate, thiamine and glucose.

The precultures were incubated for 24 hours at 34 °C and 1000 rpm shaking and then further transferred to a new basal minimal medium (BMM, pH 7,5) in order to start the main culture.

The new BMM was supplemented with magnesium sulphate, thiamine, a bolus of 20 % glucose solution (50 ul per 100 mi) and a bolus of 10 % lactose solution (5 ml per 100 ml). Moreover, 50 % sucrose solution was provided as carbon source, accompanied by the addition of sucrose hydrolase (invertase), so that glucose was released at a rate suitable for C-limtted growth.

The main cultures were incubated for 72 hours at 28°C and 1000 rpm shaking.

For the analysis of total broth, the 96-well plates were boiled at 100°C, subsequently centrifuged, and finally the supematants were analysed by HPLC.

For supematant samples, the initial centrifugation of microtiter plates was followed by the removal of 0.1 mL supematant for direct analysis by HPLC.

For pellet samples, the celis were initially washed, then dissolved in deionized water and centrifuged.

Following centrifugation, the pellets were analysed for HMO content in the cell interior after resuspension, boiling, centrifugation and analysis of the final supernatant.

Results of the deep well assays Strains were characterized in deep well assays and samples were collected from the total broth.

The concentration of the detected HMOs {in g/L) in each sample was used to calculate the % quantitative differences in the LNT content of the strains tested, i.e., the % differences in the LNT-H concentrations formed by strains expressing one of the three GloNAcTs, namely LgtA, PmnagT or BDO468, or pairwise combinations of ali three GlcNAcTs relative to similar strains that also express the native transporter Lacy.

As revealed by 72-hour experiments in 96-well plates and as shown in Figure 3, higher LNT levels {approximately 10%) can be reached when HDO0468 is co-expressed with LgtA at saturating levels of intraceliular lactose (i.e. when the facY gene is over-expressed} (strain MP 13) than at physiological fac Y levels (strain MP7). This effect is even more obvious when the strains MP7 and MP13 have heen tested in 24-well assays, with the strain MP13 reaching up to 30% higher LNT-H

DK 2021 70252 A9 54 titers than the strain MP7 (Figure 4). Notably, the beneficial effect of the combined expression of HDO466 and LgiA and lacy over-expression is solely restricted to this pair of GleNAcTs (Figure 3).

Claims

DK 2021 70252 A9 55 CLAIMS

1. A method for the production of LNT-H, the method comprising the steps of: a. providing a genetically engineered cell capable of producing an HMO, wherein said cell comprises i} a heterologous 8-1,3-N-acetyl-glucosaminyt-transferase protein [HDO466] as shown in SEQ ID NG: 1 or a functional homalogue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NG: 1; and i) a native or heterologous regulatory element for controlling the expression of i); and b. culturing the cell according fo (a) in a suitable cell culture medium; and Cc. harvesting the HMO(s) produced in step (b).

2. A method according fo claim 1, wherein the cell further comprises, iii} a heterologous 8-1,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 2 [IgtA], or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 2; and iv} a native or heterologous regulatory element for controlling the expression of iii}.

3. A method according fo claim 2, wherein the cell further comprises v} a lactose permease protein as shown in SEQ ID NO: 3 [LacY], or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 3, and vi} a native or heterologous regulatory element for controlling the expression of v).

4. A method according fo claim 3, wherein the gene encoding the lactose permease protein is over-expressed. [all the mentioned ways as in the IDR]

DK 2021 70252 A9 56

5. A method according to any of the preceding claims, the heterologous regulatory element for controling and increasing the expression of i), iil) andfør v) is a promoter.

6. A method according to any of the preceding claims, wherein the promoter sequence is selected from the group consisting of PBAD, Pxyl, PsacB, PxylA, ProR, Pith, PT7, Ptac, PL, PR, PnisA, Pb, PqatY 7OUTR, PglpF, PgipF_SD1, PglpF_8D10, PgipF_SD2, PglpF_SD3, PglpF_SD4, PgipF SDS. PglpF SD6, PgloF SD7, PgipF SD8, PgipF SD9, PglpF B28, PgloF 829, Plac 18UTR, Plac, PmgiB 7OUTR and PmgiB 70UTR SD4. — 7. A method according fo any of the preceding daims, wherein the promoter sequence is selected from the group consisting of PgipF and Plac.

8. A genetically engineered cell comprising a) a nucleic acid sequence according to SEQ ID NO: 4, or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 4, encoding a heterologous B-1,3-N-acetyl-glucosaminyi-transferase, b) a native or heterologous regulatory element for controlling the expression of a). 9 A cell according to claim 8, wherein the cell further comprises ¢} a nucleic acid sequence according to as shown in SEQ ID NO: 5 [IgtA], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical fo SEQ ID NO: 5, encoding a heterologous $-1,3-N-acetyl-glucosaminyt-transferase; d) a native or heterologous regulatory element for controlling the expression of ¢).

10. A cell according to claim 9, further comprising e) a nucleic acid sequence according to as shown in SEQ ID NO: 8 [LacY], or a functional homologue thereof having a rucleic acid sequence which is at least 70 % identical to SEQ ID NO: 8, encoding a lactose permease, and f) a native or heterologous regulatory element for controlling the expression of e).

DK 2021 70252 A9 57

11. A genetically engineered cell according to any of claims 10, wherein the lactose permease protein of e) is over-expressed.

12. A genetically engineered cell according to any of claims 8-11, wherein the regulatory element for controlling the expression is a promoter.

13. A genetically engineered celf according to any of claims 8-12, wherein the promoter sequence is selected from the group consisting of PBAD, Pxyl, PsacB, PxylA, PropR, PnitA, PT7, Plac, PL, PR, PnisA, Pb, Pgaty 70UTR, PgipF, PglpF. SD1. PglpF. SD10, PglpF. SD2, PglpF_SD3, PglpF SD4, PglpF SDS, PglioF SDS, PgipF SD

7, PglpF SDB, PglpF: SD9, PalpF B28, Plac 168UTR, Plac, PmgiB 70UTR and PmgiB 7OUTR SD+4.

14. A genetically engineered cell according to any of claims 8-13, wherein the promoter sequence is selected from the group consisting of PgipF and PglpF: B28.

15. A genetically engineered cell according to any of claims 8-14, wherein the glpR gene of the genetically engineered cell, encoding the DNA-binding transcriptional repressor GIpR, is deleted.

16. A genetically engineered cell according to any of claims 8-15, wherein the cell is Escherichia coli

17. A nucleic acid construct comprising a nucleic acid sequence encoding i} a heterologous 8-1,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1; and it) a native or heterologous regulatory element for controlling the expression of i).

18. A nucleic acid construct according to claim 17, further comprising

DK 2021 70252 A9 58 ili} a nucleic acid sequence according fo SEQ ID NO: 5 {IgtA}, or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NG: 5, encoding & heterologous B-1,3-N-acetykglucosaminyl-transferase; iv) a native or heterologous regulatory element for controlling the expression of ii).

18. A nucleic acid construct according to claim 18, further comprising v} a nucleic acid sequence according to SEQ ID NO: 6 [LacY], or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 6, encoding a lactose permease, and vi) a native or heterologous regulatory element for controlling the expression of v).

20. Use of a genetically engineered cell according to any of claims 8-16, or a nucleic acid construct — according to any of claim 17-18 for the production of LNT-I.