DK181310B1

DK181310B1 - Cell factories for lnt-ii production

Info

Publication number: DK181310B1
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: 2023-08-08
Also published as: WO2022243307A1; DK202170252A1; DK202170252A9

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 181310 B1 1

CELL FACTORIES FOR LNT4I PRODUCTION

FIELD

This disclosure relates to a method of producing human milk oligosaccharides (HMOs) with high titers, wherein the HMO is LNT-II. Several genetic engineering approaches have been applied to change the abundance of the HMO produced by cells that express a heterologous (-1,3-N-acetyl- glucosaminyltransferase. The strain engineering strategies to achieve this goal depend on the B- 1,3-N-acetyl-glucosaminyltransferase introduced in the host. The disclosure highlights several strain engineering strategies to enable the formation of LNT-II at high titers in the E. coli DH1 K12 host. Specifically, these strategies deal with different aspects for the design of an efficient LNT-II 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 mixture 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 at 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 to the discovery of their important functionality in human development. Besides their prebiotic properties, HMOs have been linked to additional positive effects, which expands their field of application. The health benefits of HMOs have enabled their 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 traditionally been developed for individual HMOs such as 2'-fucosyllactose, 3-fucosyllactose, lacto-

N-tetraose, lacto-N-neotetraose, 3'-sialyllactose and 6'-sialyllactose. Fermentation based

DK 181310 B1 2 processes typically utilize genetically engineered bacterial strains, such as recombinant

Escherichia coli (E. 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 to express the glycosyltransferases needed for synthesis of the desired oligosaccharides and takes advantage of the bacteria's innate pool of nucleotide sugars as HMO precursors. For example, CN 111979168 addresses the production of lactoyl-N-triose II (LNT-II) by use of a genetically engineered E. coli host, wherein genetic manipulations to the N- acetylglucosamine (GIcNAc) glycolysis pathway resulted in an increased yield of LNT-II.

At present, knowledge as to how to engineer cells to produce high titers of LNT-II, and how to select the optimal glycosyltransferases and subsequent genetic modifications which enable LNT-II production is limited, since LNT-II in many regards is considered a precursor HMO for production of more complex oligosaccharides.

SUMMARY

This disclosure 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, 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 II (LNT-II), produced, while only specific genetic modifications enhanced the LNT-II titer. This is particularly — true for cells expressing the -1,3-N-acetyl-glucosaminyltransferase, HD0466 (GenBank ID:

WP _010944479.1), where expression of a single copy enhanced the production of LNT-II over cells expressing the B-1,3-N-acetyl-glucosaminyltransferase LgtA (GenBank ID:

WP_033911473.1).

Furthermore, combination of the B-1,3-N-acetyl-glucosaminyltransferase HD0466 with a second 3- 1,3-N-acetyl-glucosaminyltransferase LgtA or PmnagT (WP_014390683.1) was capable of further enhancing the LNT-II titer. Also, combining expression of the B-1,3-N-acetyl- glucosaminyltransferases HD0466 and LgtA with overexpression of the E. coli native Lactose permease also enhanced the LNT-II titer.

This disclosure highlights ways of achieving high LNT-II titers related to strain engineering strategies. The strain engineering strategies to achieve this goal comprise the manipulation of the following genetic traits of the HMO producer cell: 1. Introduction of a specific B-1,3-N-acetyl-glucosaminyltransferase, namely HD0466 2. Introduction of a second B-1,3-N-acetyl-glucosaminyltransferase, namely LgtA and/or

PmnagT 3. Over-expression the native gene JacY 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-II, in enhanced amounts.

The enhanced amounts of LNT-II, 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 engineered cell capable of producing an HMO, wherein said cell comprises i) a heterologous B-1,3-N-acetyl-glucosaminyl-transferase 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 (a) in a suitable cell culture medium; and

C. harvesting the HMO(s) produced in step (b).

In another aspect, the present disclosure 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 B-1,3-N-acetyl-glucosaminyl-transferase, 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 B-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 ii) a native or heterologous regulatory element for controlling the expression of i).

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

DETAILED DESCRIPTION

This disclosure enables the skilled person to produce HMOs, primarily LNT-II, in enhanced amounts. In this manner, the present disclosure first of all pinpoints and compares different 3-1,3-

N-acetyl-glucosaminyl transferases that can efficiently convert lactose to LNT-II, and indicate how their expression can be balanced for reaching optimal LNT-II production.

Secondly, it shows that LNT-II production systems that simultaneously express specific combinations of B-1,3-N-acetyl-glucosaminyl transferases are more 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 /acY at levels that are higher than the normal cell physiological level can further enhance the synthesis of LNT-II in cells expressing more than one B-1,3-N-acetyl-glucosaminyl transferases — but not necessarily, when a single transferase is expressed at the same copy number.

According to the present disclosure, the E. coli DH1 K12 host can be engineered to form LNT-II at high levels by introducing the B-1,3-N-acetyl-glucosaminyl transferase HD0466 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-II levels. 5 Thus, the enhanced amounts of LNT-II, gives a more sustainable manufacturing process; valuable

HMO product in fermentation is thus done at a higher overall yield.

Exemplary methods > HD0466

In Example 1, it is demonstrated how different GIcNAcTs can be beneficially expressed at varied genomic copy numbers in the genetic background of E. coli K12 cells for high-level LNT-II production. The Example reveals the HD0466 enzyme as a novel enzyme for the in vivo production of LNT-II.

In one or more exemplary embodiments, the method for the production of LNT-II 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-acetyl-glucosaminyl-transferase 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 (a) in a suitable cell culture medium; and

C. harvesting the HMO(s) produced in step (b).

Moreover, the present disclosure shows marked gains in LNT-II titers when different 3-1,3-N- acetyl-glucosaminyl transferases are simultaneously expressed in the same production cell compared to LNT-II producers expressing a single transferase. According to the present disclosure, the most marked increase in LNT-II titers could be achieved by combined expression of the HD0466 and LgtA enzymes.

In Example 2, it is demonstrated that the pairwise expression of two different GIcNAcTs can be a more efficient approach in converting E. coli K12 cells to an efficient LNT-II cell factory than merely expressing a single GIcNACT.

HD0466/IgtA

Thus, in one or more exemplary embodiments the method for the production of LNT-II 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-acetyl-glucosaminyl-transferase 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; ii) a native or heterologous regulatory element for controlling the expression of i); and lii) a heterologous B-1,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ

ID NO: 2 [LgtA], 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 iii), b. culturing the cell according to (a) in a suitable cell culture medium; and

C. harvesting the HMO(s) produced in step (b).

HD0466/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: i) a heterologous B-1,3-N-acetyl-glucosaminyl-transferase 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; ii) a native or heterologous regulatory element for controlling the expression of i); lii) a 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 iii), b. culturing the cell according to (a) in a suitable cell culture medium; and

C. harvesting the HMO(s) produced in step (b).

Since our goal is to convert e.g., the E. coli host to a cell factory for LNT-II production, a rational genetic engineering program includes the following major focus areas: a) the introduction one or more highly active B-1,3-N-acetyl-glucosaminyl transferase for the conversion of the externally added lactose to LNT-II 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.

HD0466/L gtA/LacY

In addition, the over-expression of the JacY gene in the cells expressing more than one B-1,3-N- acetyl-glucosaminyl transferases, namely HD0466 and LgtA, can be advantageous for high-level

LNT-II 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 GIcNAcTs, namely HD0466 and LgtA, are co-expressed in the same 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 i) a heterologous B-1,3-N-acetyl-glucosaminyl-transferase 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); lii) a heterologous B-1,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ

ID NO: 2 [LgtA], 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 iii), 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, vi) a native or heterologous regulatory element for controlling the expression of v) and b. culturing the cell according to (a) in a suitable cell culture medium; and

C. harvesting the HMO(s) produced in step (b).

The LNT-II enzymes

The present disclosure demonstrates the superior activity of another enzyme, namely HD0466 from Haemophilus ducreyi, towards LNT-II 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-II, the genetically engineered cells comprise all the required enzymes to facilitate the production LNT-II. One of these enzymes may for example be i) a heterologous B-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.

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-II during culturing becomes increases, as shown in Example 2.

Heterologous B-1,3-N-acetyl-glucosaminyl-transferase

A heterologous B-1,3-N-acetyl-glucosaminyl-transferase is any protein which comprises the ability of transferring the N-acetyl-glucosamine of UDP-N-acetyl-glucosamine to lactose. The B-1,3-N- acetyl-glucosaminyl-transferase used herein does not originate in the species of the genetically engineered cell i.e. the gene encoding the B-1,3-galactosyltransferase is of heterologous origin.

The examples below use the heterologous -1,3-N-acetyl-glucosaminyl-transferase HD0466, LgtA and/or PmnagT.

HD0466 genes

In one or more exemplary embodiments, the HD0466 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.

IgtA genes

In one or more exemplary embodiments, the /gfA gene is as shown in SEQ ID NO: 5 or is a — functional homologue thereof having a nucleotide sequence that is at least 70 % identical to SEQ

ID NO: 5.

PmnagT genes

In one or more exemplary embodiments, the PmnagT gene is as shown in SEQ ID NO: 29, or a functional homologue thereof having a nucleotide sequence that is at least 70 % identical to SEQ

ID NO: 29.

DK 181310 B1 10

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-II are shown in the below matrix.

Protein Sequence

Gene ID Description HMO example

GenBank

LNT-II, LNT, LNnT,

LNFP-I, LNFP-II, LNFP-

B-1,3-N- Il, LNFP-V, LNFP-VI,

IgtA WP 033911473.1 | acetylglucosaminyl | LNDFH-I, LNDFH-II, -transferase LNDFH-III, pLNH, F- pLNH I, pLNnH, LST a,

LST b, LST c, DSLNT

LNT-II, LNT, LNnT,

LNFP-I, LNFP-II, LNFP-

B-1,3-N- Il, LNFP-V, LNFP-VI,

HD0466 WP 010944479.1 | acetylglucosaminyl | LNDFH-I, LNDFH-II, -transferase LNDFH-III, pLNH, F- pLNH I, pLNnH, LST a,

LST b, LST c, DSLNT

LNT-II, LNT, LNnT,

LNFP-I, LNFP-II, LNFP-

Putative B-1,3-N- Il, LNFP-V, LNFP-VI,

PmnagT WP 014390683.1 | acetylglucosaminyl | LNDFH-I, LNDFH-II, -transferase LNDFH-III, pLNH, F- pLNH I, pLNnH, LST a,

LST b, LST c, DSLNT

In one or more exemplary preferred embodiments, the heterologous (3-1,3-N-acetyl- glucosaminyltransferases and the HMO LNT-II 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.

DK 181310 B1 11

Protein Sequence

Gene ID Description HMO example

GenBank

LNT-II, LNT, LNnT,

LNFP-I, LNFP-II, LNFP-

B-1,3-N- Il, LNFP-V, LNFP-VI,

LST b, LST c, DSLNT

LNT-II, LNT, LNnT,

LNFP-I, LNFP-II, LNFP-

Putative B-1,3-N- Il, LNFP-V, LNFP-VI,

LST b, LST c, DSLNT

Lactose permease

HD0466, can provide high LNT-II titers when expressed from one or two genomic copies (Figure 1a). The descending order of activity of the three selected GlcNAcTs on lactose, as it is indirectly revealed by the observed final LNT-II titers is as follows: HD0466 > PmnagT > LgtA. The LNT-II titers reached by the strains MP5 and/or 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 cell. In HMO production lactose 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, e.g., the native JacY 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.

DK 181310 B1 12

In one or more exemplary embodiments, the lactose permease gene is a nucleotide sequence as shown in SEQ ID NO: 6 or is a functional homologue thereof having a nucleotide sequence that is at least 70 % identical to SEQ ID NO: 6.

Lactose permease over-expression

As shown in Example 3, the over-expression of the /acY gene coding lactose permease is used as a genetic tool to obtain an enhanced level of LNT-II 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 B-1,3-N-acetyl-glucosaminyltransferases and JacY over-expression results in an enhanced LNT-

II 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 (RBSs). The RBSs may for example be the Shine-Dalgarno (SD) sequence. 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 16S 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 herein 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 /acY gene.

The increased expression of the lactose permease may be achieved by direct integration of a copy of the /acY gene in the genome. In this manner, Examples 2 and 3 provide enough data to conclude that the combined expression of HD0466 and LgtA results in higher LNT-II titers regardless of intracellular lactose levels compared to when only one of these GIcNAcTs is expressed by the cell at the same copy number. Importantly, this trend is unique for this GIcNAcT pair and it is not observed for any other GIcNACT pair that can be formed from HD0466, 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 /ac/ gene - UniProtKB - P03023 (LACI_ECOLI).

As shown in the examples, an additional genomic copy of the /acY gene which encodes the lactose permease with the PglpF promoter in HD0466 expressing cells resulted in a decrease in produced

LNT-II. Contrary, the over-expression of the /acY gene by an additional genomic PglpF-driven copy 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 91 % identical, at least 92 % identical, at least 93 % identical, at least 94 % identical, at least 95 % identical at least 96 % identical, at least 97 % identical, at least 98 % identical, or at least 99 % identical.

Controlling the expression

In the present context the term “controlling the expression” relates to gene expression where the transcription of a gene into 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 proteins to specific sites on DNA, such as but not limited to regulatory elements. — As described above, engineering strategy can be applied in multiple ways 1) the copy number of the gene of interest, 2) controlling the expression of any copy of these genes at the transcriptional 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

DK 181310 B1 14

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 of — 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).

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-expression of a gene may be achieved directly by transcriptional activators that bind to key gene regulatory sequences to promote transcription or enhancers that constitute sequence elements positively affecting transcription. Similarly, direct over-expression of a gene can be achieved by simply increasing its copy humber in the genome, or replacing its native promoter with a promoter of higher strength or even modifying the sequence controlling the binding of the corresponding mRNA to 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 transcription.

The term “over-expression” that is used here may for example refer to the native gene JacY and includes 1) the replacement of the native promoter of the any E. coli gene by another, stronger promoter, 2) the modification of the native Shine-Dalgarno 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 E. 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) to a high-copy number — plasmid (300-500 copies per cell).

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

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 multiplication 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 to a method, wherein the overexpression of the B-1,3-N-acetyl-glucosaminyl-transferase protein(s) and/or the lactose permease is provided by increasing the copy number of any of the genes coding for said protein(s) and/or 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 and/or synthetic nucleic acid sequences.

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

DK 181310 B1 16

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

Regulatory elements are found at transcriptional and post-transcriptional levels and further enable molecular networks at those levels. For example, at the post-transcriptional 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 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 limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, binding sites for gene regulators and enhancer sequences.

Promoters and enhancers are the primary genomic regulatory components of gene expression.

Promoters are DNA regions within 1-2 kilobases (kb) of a gene's transcription start site (TSS); they contain short regulatory elements (DNA 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 to establish cell type identity and regulate gene expression. Enhancers may act independently of their sequence context and at distances of several to 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 genes (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 it 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 one 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 to drive transcription of different genes of interest integrated into the genome of the host cell or into episomal DNA.

Native

In relation to the present disclosure, the term “native” refers to nucleic 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 it originates from the E. coli K12 strain, is not of heterologous origin and not a recombined nucleic acid sequence, with respect to the genetically 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 element(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 to the person skilled in the art.

Promoter sequences

The regulatory element or elements regulating the expression of the genes and/or nucleic acid sequence(s), may comprise one or more promoter sequence(s), wherein the promoter sequence, 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.

DK 181310 B1 18

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 nucleic regulatory element.

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 transcription factors that bind those sites can be used multiple different growth conditions and there are more than 100 genes from across the E. coli 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, PnitA, PT7, Ptac, PL, PR, PnisA, Pb, Pscr,

Pscr SD1, Pscr SD7, PgatY 70UTR, PglpF, PgipF. SD1, PgipF. SD10, PgipF. SD2, PgipF SD3,

PglpF. SD4, PgipF_SD5, PglpF_SD6, PglpF_SD7, PgipF. SD8, PgipF. SD9, PglpF. B28,

Plac 16UTR, Plac, PmgiB 70UTR and PmgiB 70UTR SD4.

In one or more exemplary embodiments, the heterologous 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, PglpF.

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 lac 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 Pmg/B 70UTR, or PmgiB 70UTR 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 gatYZABCD; tagatose-1,6-bisP aldolase promoter PgatY or variants thereof.

Pscr

In one or more exemplary embodiments, the heterologous regulatory element is Pscr or variants thereof such as but not limited to SEQ ID NO: 7.

Pscr SD1

In one or more exemplary embodiments, the heterologous regulatory element is Pscr SD1 or variants thereof such as but not limited to SEQ ID NO: 8.

Pscr SD7

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 element is PgatY 70UTR or variants thereof such as but not limited to SEQ ID NO: 10. => PglpF

In one or more exemplary embodiments, the heterologous regulatory element is PglpF or variants thereof such as but not limited to SEQ ID NO: 11.

It is also obvious from the data shown in Figures 1a and 1b that even a single genomic copy of the

HDO0466 gene suffice to reach the highest LNT-II titers when any of these GIcNAcTs 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.

PglpF. SD1

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.

PglpF. SD10

In one or more exemplary embodiments, the heterologous regulatory element is PgipF. SD10 or variants thereof such as but not limited to SEQ ID NO: 13.

PglpF SD2

In one or more exemplary embodiments, the heterologous regulatory element is PgipF. SD2 or variants thereof such as but not limited to 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 SEQ ID NO: 15. — PglpF SD4

In one or more exemplary embodiments, the heterologous regulatory element is PgipF. SD4 or variants thereof such as but not limited to SEQ ID NO: 16.

PglpF SD5

In one or 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. SD6 or variants thereof such as but not limited to SEQ ID NO: 18.

PglpF SD7

In one or more exemplary embodiments, the heterologous regulatory element is PglpF_SD7 or variants thereof such as but not limited to SEQ ID NO: 19.

PglpF SD8

In one or more exemplary embodiments, the heterologous regulatory element is PgipF. SD8 or variants thereof such as but not limited to SEQ ID NO: 20.

PglpF SD9

In one or more exemplary embodiments, the heterologous regulatory element is PgipF. SD9 or variants thereof such as but not limited to SEQ ID NO: 21.

PglpF_B28

In one or more exemplary embodiments, the heterologous regulatory element is PgipF. B28 or variants thereof such as but not limited to SEQ ID NO: 22.

PglpF B29

In one or more exemplary embodiments, the heterologous regulatory element is PglpF B29 or variants thereof such as but not limited to SEQ ID NO: 23.

Plac_16UTR

In one or more exemplary embodiments, the heterologous regulatory element is Plac_16UTR or variants thereof such as but not limited to SEQ ID NO: 24.

Plac

In one or more exemplary embodiments, the heterologous regulatory element is Plac or variants thereof such as but not limited to SEQ ID NO: 25.

PmgiB 70UTR

In one or more exemplary embodiments, the heterologous regulatory element is PmglB_70UTR or variants thereof such as but not limited to SEQ ID NO: 26.

PmgiB 70UTR SD4

In one or more exemplary embodiments, the heterologous regulatory element is

PmglB 70UTR SD4 or 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 episomal nucleic 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 cassette consisting of a promoter sequence, the coding sequence of the gene of interest and a terminator sequence.

In one or more exemplary embodiments, episomal nucleic acid sequences may be a plasmid with only a part of it being integrated into the chromosome. In the present context, the expression cassette resembles the one described above but it further comprises two DNA segments that are 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-II.

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.

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 -1,3-N-acetyl- glucosaminyl-transferase protein(s) and/or the lactose permease protein or regions upstream of the regulatory element for controlling the expression of any of the -1,3-N-acetyl-glucosaminyl- 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

LNT-II can lead to increased levels of the mRNAs and eventually to higher total HMO titers.

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 B-1,3-N-acetyl-glucosaminyl-transferase protein(s) of the present disclosure, and wherein the B-1,3-N-acetyl-glucosaminyl-transferase protein is HD0466 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 B-1,3-N-acetyl-glucosaminyl-transferase protein(s) of the present disclosure, and wherein the 3-1,3-N-acetyl-glucosaminyl-transferase protein is HD0466.

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 p-1,3-N-acetyl-glucosaminyl-transferase protein(s) and/or the lactose permease protein, and wherein the B-1,3-N-acetyl-glucosaminyl-transferase protein is

HD0466 and LgtA.

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

GIpR

GIpR belongs to the DeoR family of transcriptional regulators and acts as the repressor of the glycerol-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 (g/pR) 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 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 glpR gene refers to the inactivation of the gipR gene by complete or partial deletion of the corresponding nucleic acid sequence from the bacterial genome. The g/pR 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 deletion of the repressor gene that would otherwise reduce the transcriptional activity associated with the PgipF promoters.

In one or more exemplary embodiments, the g/pR gene is deleted.

The deletion of the glpR gene could eliminate the GlpR-imposed repression of transcription from all

PglpF promoters in the cell and in this manner enhance gene expression from all PgipF-based cassettes.

Activators

In one or more exemplary embodiment(s), the genetically engineered cell disclosed herein comprises an over-expressed gene product that enhances the expression of the gene(s) encoding the enzyme(s) required to facilitate the production of a human milk oligosaccharide (HMO) that is

LNT-II.

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 -1,3-N- acetyl-glucosaminyl-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 B-1,3-N-

acetyl-glucosaminyl-transferase protein(s) of the present disclosure, and wherein the B-1,3-N- acetyl-glucosaminyl-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 -1,3-N- acetyl-glucosaminyl-transferase protein(s) of the present disclosure, and wherein the B-1,3-N- acetyl-glucosaminyl-transferase protein is HD0466.

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 B-1,3-N-acetyl-glucosaminyl- transferase protein(s) and/or the lactose permease protein, and wherein the B-1,3-N-acetyl- glucosaminyl-transferase protein is HD0466 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 sequences, the binding recruits the RNA polymerase through direct interaction, which in turn activates the transcription of the nucleic acid sequence following the promoter sequence leading to expression of the gene of interest. Thus, over-expression of CRP may lead to an enhanced expression of a gene/nucleic 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 PgipF family. In this way, over-expression of CRP in the genetically engineered cell 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 at 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-acetyl-glucosaminyl-transferase; and ii) a native or heterologous regulatory element for controlling the expression of i).

In one or more exemplary embodiments the nucleic acid construct also comprises: lii) a nucleic acid sequence according to SEQ ID NO: 5 [IgtA], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 5, encoding a heterologous B-1,3-N-acetyl-glucosaminyl-transferase, and iv) a native or heterologous regulatory element for controlling the expression of iii).

In one or more exemplary embodiments the nucleic acid construct also comprises: v) a nucleic acid sequence according 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, 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: vii) a nucleic acid sequence according to SEQ ID NO: 29 [PmnagT], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ

ID NO: 29, encoding a heterologous B-1,3-N-acetyl-glucosaminyl-transferase; viii) 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), vii). The regulatory element(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).

Recombinant nucleic acid sequence

The nucleic acid construct can be a recombinant nucleic acid sequence. By the term “recombinant nucleic acid sequence”, “recombinant gene/nucleic acid/DNA encoding” 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 into a protein when under the control of the appropriate control sequences, i.e. a promoter sequence.

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

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

The recombinant nucleic 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 to 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 glp operon, or a promoter sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence, wherein the coding and promoter sequences are operably linked. — 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 to a transcribed sequence. For example, a promoter sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.

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

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

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 gene/coding 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 attTn7-site (Waddell C.S. and Craig N.L., Genes Dev. (1988) Feb;2(2):137-49.); methods for genomic integration of nucleic acid sequences in which recombination is mediated by the Red recombinase function of the phage A or the RecE/RecT recombinase function of the Rac prophage (Murphy, J Bacteriol. (1998);180(8):2063-7; Zhang et al., Nature Genetics (1998) 20: 123-128

Muyrers et al., EMBO Rep. (2000) 1(3): 239-243); methods based on Red/ET recombination (Wenzel et al., Chem Biol. (2005), 12(3):349-56.; Vetcher et al., Appl Environ Microbiol. (2005);71(4): 1829-35); or positive clones, i.e., clones that carry the expression cassette, can be selected e.g. by means of a marker gene, or loss or gain of gene function.

DK 181310 B1 29

Nucleic acid identity

In one or more exemplary embodiments, the present disclosure relates to a recombinant nucleic acid shown in SEQ ID NO: 4-6 or 29, or a functional homologue thereof having a sequence that is at least 70% identical to SEQ ID NO: 4-6 or 29, such as at least 71% identical, at least 72% identical, at least 73% identical, at least 74% identical, at least 75% identical, at least 76% 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 86 % identical, at least 87 % identical, at least 88 % identical, at least 89 % identical, at least 90 % identical, at least 91 % identical, at least 92 % identical, at least 93 % identical, at least 94 % identical, at least 95 % identical at least 96 % identical, at least 97 % identical, at least 98 % identical, or at least 99 % identical.

Sequence identity

The term “sequence identity of [a certain] %” in the context of two or more nucleic 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 90 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. http://www.ncbi.nim.nih.gov/BLAST/). This definition also applies to the complement of a test sequence and to sequences that have deletions and/or 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 et al. Nucl. Acids Res. 25, 3389 (1997). BLAST 2.2.20+ is used to determine percent sequence identity for the nucleic acids and proteins of the disclosure. Software for performing BLAST analyses is publicly available through the National

Center for Biotechnology Information (http://www.ncbi.nim.nih.gov/). Examples of commonly used sequence alignment algorithms are

CLUSTAL Omega (Hiito:/fwww.ebi ac uk/Tools/msa/clustalo/),

EMBOSS Needle (http://www eb ac uk/Toois/osafemboss nsedlev),

MAFFT (attp//mafit.chre jofalignment/servær/), or

MUSCLE (htto før ep ac. uk/Fools/msa/muscie/).

Functional homologue

A functional homologue of a protein/nucleotide as described herein is a protein/nucleotide 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 60%, 70%, 80 %, 90% or 100% compared to the functionality of the protein/nucleotide.

A functional homologue of any one of the disclosed amino 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 yield, purity, reduction in biomass formation, viability of the genetically engineered cell, robustness of the genetically engineered cell according to the disclosure, or reduction in consumables.

Genetically 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 B-1,3-N-acetyl-glucosaminyl-transferase; and b) a native or heterologous regulatory element for controlling the expression of a).

In one or more exemplary embodiments the genetically engineered cell also comprises: c) 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 B-1,3-N-acetyl-glucosaminyl-transferase; 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: 6 [Lac], 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: 29 [PmnagT], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to 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).

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

LNT-II.

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 (host cell) may be e.g., a bacterial or yeast 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 to the disclosure could be Erwinia herbicola (Pantoea 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 lentus, Bacillus cereus, and Bacillus circulans. Similarly, bacteria of the genera

Lactobacillus and Lactococcus may be engineered using the methods of this disclosure, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum,

Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuters,

Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the disclosure described herein. Also included as part of this disclosure are strains, engineered as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g.,

Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g.,

Pseudomonas fluorescens and Pseudomonas aeruginosa).

In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of E. coli, 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 more exemplary embodiments, the genetically engineered cell is B. subtilis.

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

In the 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.

Cell 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, casamino 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.

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, 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 and/or 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

HMOXs) included in both the biomass (i.e. the host cells) and cultivation media, i.e. before/without separation of the fermentation broth from the biomass. In other embodiments, the produced HMOs

DK 181310 B1 34 may be collected separately from the biomass and fermentation broth, i.e. after/following 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 a later stage after storing the fermentation broth at appropriate conditions. Recovery of the produced HMO(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.

Human milk oligosaccharide (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 at the reducing end that can be elongated by one or more beta-N-acetyl-lactosaminyl and/or one or more beta-lacto-N-biosyl units, and this core structure can be substituted by an alpha-L- fucopyranosyl and/or 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 (or neutral) HMOs can be fucosylated or non-fucosylated. Examples of such neutral non-fucosylated HMOs include lacto-N- triose 2 (LNT-II) lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH) and lacto-N-hexaose (LNH).

Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2'-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose | (LNDFH-I), 3-fucosyllactose (3'-FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucohexaose Ill (LNDFH-III), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N- — difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-para-lacto-N-hexaose (FPLNH-I), fucosyl-para-lacto-N-neohexaose II (F-pLNnH II) and fucosyl-lacto-N-neohexaose

(FLNnH). Examples of acidic HMOs include 3'-sialyllactose (3'-SL), 6-sialyllactose (6'-SL), 3- fucosyl-3'-sialyllactose (FSL), 3'-O-sialyllacto-N-tetraose a (LST a), fucosyl-LST a (FLST a), 6'-0- sialyllacto-N-tetraose b (LST b), fucosyl-LST b (FLST b), 6'-O-sialyllacto-N-neotetraose (LST c), fucosyl-LST c (FLST c), 3'-O-sialyllacto-N-neotetraose (LST d), fucosyl-LST d (FLST d), sialyl- lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose | (SLNH-I), sialyl-lacto-N-neohexaose Il (SLNH-II) and disialyl-lacto-N-tetraose (DSLNT).

In the context of the present disclosure lactose is not regarded as an HMO species.

Use of a genetically engineered cell

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 cell(s) or the nucleic acid construct(s) comprise at least one heterologous protein(s), such as but not limited to i) a heterologous B-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 optionally ii) a heterologous B-1,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID

NO: 2 or 28 or a functional homologue thereof having an amino 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 cell(s) or the nucleic acid construct(s) may comprise a native or heterologous regulatory element for controlling the expression of the B-1,3-N-acetyl-glucosaminyl- — transferase(s).

The genetically engineered cell(s) or the nucleic acid construct(s) may also comprise a native or heterologous regulatory or episomal element for increasing the expression of the lactose permease.

DK 181310 B1 36

The genetically engineered cell(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 B-1,3-N-acetyl- glucosaminyl-transferase 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 one 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-II.

Manufacturing of HMOs

To produce one or more HMOs, 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, W02017182965 or WO2017152918, 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 cultivation in larger volumes. The term “manufacturing” 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 to simple lab scale methods like shake flask cultivation, is characterized by the use of the technical system of a bioreactor (fermenter) which is equipped with devices for agitation, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.). To a large extent, the behavior of an expression system in a lab scale method, such as shake flasks, benchtop bioreactors or the deep well format described in the examples of the disclosure, does allow to predict the 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 limitations.

The culture medium may be semi-defined, i.e. containing complex media compounds (e.g. yeast extract, soy peptone, casamino acids, etc.), or it may be chemically defined, without any complex compounds. Where sucrose is used as the carbon and energy source, a minimal medium might be preferable.

Manufactured product

The strain engineering strategy of the present invention contributes to a sustainable manufacturing process for the high-level production of LNT-II, where the conversion of the provided carbon source to HMO product in fermentation is done at a high overall yield. As shown in Example 2, the concentration of the detected HMOs (in g/L) in each sample was used to calculate the % quantitative differences in the LNT-II content of the strains tested, i.e., the % differences in the

LNT-II concentrations formed by strains expressing LgtA, PmnagT or HD0466, or pairwise combinations of all three GIcNAcTSs relative to LgtA-expressing 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

HMOXs). 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 (and/or 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.

Importantly, beneficial features for the construction of efficient LNT-II cell factories described above do not provide an additive effect in a single production strain that produces any of the three

GIcNAcTs or combinations thereof. In other words, the features described in the present disclosure can be exploited only in the way presented here to 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-II formation when combined in any of these strain backgrounds.

DK 181310 B1 38

Tables

Table 1. Genotypes of the strains MP1, MP2, MP3, MP4, MP5 and MP8

Strain iD Genotype Heterslogous Protein

Sequence ID {GenBank

WP, 033911478.1

MDO x2 igtå-PgioF WP 0338114731

MDG x1 PmnagT-PalpF WP 014390683.1

WP 014390683.1

MDO x1 HDO466-PgipF WP 010944479.1

MDG x2 HDO486-PulpF WP 010844479.1 " fgtå: gene coding for B-1 3-N-scetyloglucosamine transferase ? PrmnagT: gene coding for 3-1 3-N-acetylogiugosamine transferase ¥ HDO468; gene coding for 3-1 3-N-acetylogiucosamine iransferase

Table 2. Genotypes of the strains MP2, MP4, MP8, MP7, MP8 and MP9 sow lowe 00]

MDO x2 igid-PglpF WP 033011473.

WP 014380683.

MBO x2 HDO465 -PgipF WP 3100444781

MDO xt HDO466'-PglpF x1 fgta'-Polo WP 010944479,1, WP 03391144731

WP 010944479.1, WP. 014390683.

MDC x7 Prinsg F-PgipF. x1 tgtå'-PgloF WP 014390683.1, WP 033911473.1 —'igtå: gene coding for 8-1, 3-N-acetylogiucosamine transferase ? PrmnagT: gene coding for 5-1, 3-N-acetylogiucosamine transferase ? HDOA466: gene coding for 8-1,3-N-acetylogiucosamine transferase

Table 3 - Genotypes of the strains MP2, MP10, MP4, MPT1, MP6, MPT2, MP7, MP13, MPS, i5 MP4, MPS and MP15. stone | emo

MBO x2 igtA'-PglpF WP 033911473.1

MP10 MDO x2 IgtA’-PgipF x1 lacY*-PgipF WP 033911473.1 NP 414877.1

MDO x2 PmnagT?-PgloF WP 014390683.1

MP11 MDO x2 PmnagT?-PglpF x1 lacY'-PgloF WP 014390683.1 NP 414877.1

MDO x2 HD0466°-PgipF WP 010944479.1

MP12 MDO x2 HD04663-PglpF x1 lacY*-PgipF WP 010944479.1 NP 414877.1

MDO x1 HD0466?-PgipF x1 IgtA'-PgipF WP 010944479.1 WP 033911473.1

MP13 MDO x1 HD0466?-PglpF x1IgtA!-PgipF x1 WP 010944479.1 WP 033911473.1 lacY*-PgipF NP 414877.1

MDO x1 PmnagT?-PgipF x1 HD0466%-PglpF | WP 014390683,1 WP 010944479.1

MP14 MDO x1 PmnagT?-PgipF x1 HD0466?-PgipF WP_014390683.1 WP_010944479.1 x1 lacY'-PgloF NP 414877.1

MDO x1 PmnagT?-PglpF x1 IgtA!-PglpF WP 014390683.1 WP 033911473.1

MP15 MDO x1 PmnagT?-PgipF x1 IgtA’-PgipF x1 WP_014390683.1 WP_033911473.1 lacY*-PgipF NP 414877.1 ' IgtA: gene coding for B-1,3-N-acetyloglucosamine transferase 2 PmnagT: gene coding for B-1,3-N-acetyloglucosamine transferase 3 HD0466: gene coding for B-1,3-N-acetyloglucosamine transferase 4 LacY: 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 culturing are used interchangeably.

The terms Lacto-N-triose, LNT-II, LNT II, LNT2 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.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1

Final LNT-II titers reached by cells expressing different GIcNAcTs at different genomic copy numbers. (a) LNT-II titers for strains that bear a genomic copy of the PmnagT (strain MP3) or

HDO0466 (strains MP5 and MP8) genes, shown relative to the final LNT-II titers of /gtA-expressing cells (strain MP1). The reference level (given as 100%) is shown for strain MP1. (b) LNT-II titers for strains that bear two genomic copies of the PmnagT (strain MP4) or HD0466 (strain MP6) genes, shown relative to the final LNT-II titers of IgtA-expressing cells (strain MP2). The reference level (given as 100%) is shown for strain MP2.

Figure 2

Final LNT-II titers reached by cells expressing a single GICNACT or a pairwise combination of

GIcNACTs at the same genomic copy number. LNT-II titers are shown for strains that bear two genomic copies of a single GIcNAcT (LgtA/strain MP2 or PmnagT/strain MP4 or HD0466/strain

MP6) and for strains bearing one copy of each of two GIcNAcTs (LgtA & HD0466/strain MP7 or

PmnagT & HD0466/strain MP8 or PmnagT & LgtA/strain MP9). The reference level (given as 100%) is shown for strain MP2.

Figure 3

Final LNT-II titers reached by cells expressing different GIcNAcTs with and without the native MFS transporter LacY. LNT-II titers for strains that bear two genomic PglpF-driven copies of gene(s) encoding GIcNAcT(s) and a single PglpF-driven copy of the JacY gene (strains MP10, MP11,

MP12, MP13, MP 14, MP 15), shown relative to the corresponding final LNT-II titers of each

GIcNAcT-expressing cells (strains MP2, MP4, MP6, MP7, MP8, MP9). The reference level (given as 100%) is shown for strains MP2, MP4, MP6, MP7, MP8, MP9.

Figure 4

Final LNT-II titers reached by cells bearing beneficial modifications as described in the present disclosure. LNT-II titers for strains that bear a genomic PglpF-driven copy of the IgtA and HD0466 genes and of the lacY gene (strains MP13), shown relative to the final LNT-II titers of cells that only express a PglpF-driven copy of the IgtA and HD0466 genes (strain MP7). The reference level (given as 100%) is shown for strain MP7.

DK 181310 B1 41

SEQUENCE ID'S

SEQ ID NO: 1 [HD0466 protein]

MTTLVSVLICAYNVEKYIDECLNAVIAQTYKNLEIIVVNDGSTDGTLAKLRQFEAKDPRVKIIDNIVNQ

GTSKSLNIGIQYCQGEIIARTDSDDIVDIHWIETLMRELDNSPETIAISAYLEFLAEKGNGSKLSRSRK

HGKNAENPISSEAISQRMLFGNPVHNNVALVRRKVFSEYGLRFDPDYIHAEDYKFWFEVSKLGKM

RTYPKALVKYRLHATQVSSAYNQKQRSIAKKIKREAISHYLQQYGIQLPEKLTIHDLFSIFSPQIELSL

TVANKQELFWSLATSLSEYHFRDLLKIYSLDIFHQLSFKYKKRIFRKFLLPNRYPSVI

SEQ ID NO: 2 [LgtA protein]

MQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLEILIVDDGSTDGTLAIAKDFQKRDSRIKILAQAQ

NSGLIPSLNIGLDELAKSGMGEYIARTDADDIAAPDWIEKIVGEMEKDRSIIAMGAWLEVLSEEKDG

NRLARHHRHGKIWKKPTRPEDIADFFPFGNPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYD

VSKLGRLAYYPEALVKYRLHANQVSSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIK

AVAYELLEKHLPEEDFERARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKN

R

SEQ ID NO: 3 [Lac protein]

MYYLKNTNFWMFGLFFFFYFFIMGAYFPFFPIWLHDINHISKSDTGIIFAAISLFSLLFQPLFGLLSDK

LGLRKYLLWIITGMLVMFAPFFIFIFGPLLQYNILVGSIVGGIYLGFCFNAGAPAVEAFIEKVSRRSNF

EFGRARMFGCVGWALCASIVGIMFTINNQFVFWLGSGCALILAVLLFFAKTDAPSSATVANAVGAN

HSAFSLKLALELFRQPKLWFLSLYVIGVSCTYDVFDQQFANFFTSFFATGEQGTRVFGYVTTMGEL

LNASIMFFAPLIINRIGGKNALLLAGTIMSVRIIGSSFATSALEVVILKTLHMFEVPFLLVGCFKYITSQF

EVRFSATIYLVCFCFFKQLAMIFMSVLAGNMYESIGFQGAYLVLGLVALGFTLISVFTLSGPGPLSLL

RRQVNEVA

SEQ ID NO: 4 [HD0466 gene]

ATGACCACACTGGTTAGCGTTCTGATTTGTGCCTATAACGTGGAAAAATACATCGATGAATGTC

TGAATGCAGTTATTGCCCAGACCTATAAAAACCTGGAAATTATCGTTGTGAATGATGGTAGCAC

CGATGGCACCCTGGCAAAACTGCGTCAGTTTGAAGCAAAAGATCCGCGTGTTAAAATCATCGA

TAACATTGTTAATCAGGGCACCAGCAAAAGCCTGAATATTGGTATTCAGTATTGTCAGGGCGA

AATTATTGCACGTACCGATTCAGATGATATCGTGGATATTCATTGGATCGAAACCCTGATGCGT

GAACTGGATAATAGTCCGGAAACCATTGCAATTAGCGCCTATCTGGAATTTCTGGCCGAAAAA

GGTAATGGTAGCAAACTGAGCCGTAGCCGTAAACATGGTAAAAATGCAGAAAATCCGATTAGC

DK 181310 B1 42

AGCGAAGCAATTAGCCAGCGTATGCTGTTTGGTAATCCGGTTCATAACAATGTGGCACTGGTT

CGTCGTAAAGTGTTTAGCGAATATGGTCTGCGTTTTGATCCGGATTATATTCATGCCGAGGATT

ACAAATTTTGGTTCGAAGTGAGCAAACTGGGTAAAATGCGTACCTATCCGAAAGCGCTGGTTA

AATATCGTCTGCATGCAACCCAGGTTAGCAGCGCATATAATCAGAAACAGCGTAGCATTGCCA

>AAAAAATCAAACGTGAAGCCATCAGCCATTATCTGCAGCAGTATGGCATTCAGCTGCCGGAAA

AACTGACCATTCATGACCTGTTTAGCATTTTTAGTCCGCAGATTGAACTGAGCCTGACCGTTG

CAAATAAACAAGAACTGTTTTGGAGCCTGGCAACCAGCCTGAGCGAATATCATTTTCGTGATC

TGCTGAAAATCTACAGCCTGGATATTTTTCATCAGCTGAGCTTCAAATACAAAAAGCGCATCTT

TCGCAAATTTCTGCTGCCGAATCGTTATCCGAGCGTTATT

SEQ ID NO: 5 [IgtA gene]

ATGCAACCGCTGGTCTCCGTGCTGATCTGTGCTTACAATGTGGAAAAATACTTCGCCCAATCG

CTGGCCGCAGTCGTGAATCAAACGTGGCGCAACCTGGAAATTCTGATCGTGGATGACGGCAG

TACCGATGGTACGCTGGCGATCGCCAAAGATTTTCAGAAACGTGACTCCCGCATTAAAATCCT

GGCACAGGCTCAAAACAGTGGCCTGATTCCGTCCCTGAATATCGGTCTGGATGAACTGGCGA

AAAGTGGCATGGGTGAATATATCGCACGCACCGATGCTGATGACATTGCGGCCCCGGACTGG

ATTGAAAAAATCGTCGGCGAAATGGAAAAAGATCGTAGCATTATCGCGATGGGTGCCTGGCT

GGAAGTGCTGTCTGAAGAAAAAGATGGCAATCGTCTGGCACGCCATCACCGTCATGGTAAAA

TCTGGAAAAAACCGACGCGTCCGGAAGATATTGCCGACTTTTTCCCGTTTGGCAACCCGATTC

ACAACAATACCATGATCATGCGTCGCTCAGTTATTGATGGCGGTCTGCGCTATAATACGGAAC

GTGATTGGGCGGAAGACTATCAGTTCTGGTACGATGTCTCGAAACTGGGTCGCCTGGCGTAT

TACCCGGAAGCCCTGGTGAAATATCGTCTGCATGCCAACCAAGTTAGCTCTAAATACTCTATC

CGCCAACACGAAATTGCACAGGGCATCCAAAAAACCGCTCGTAATGATTTTCTGCAGTCAATG

GGTTTTAAAACGCGCTTCGACTCGCTGGAATATCGTCAAATTAAAGCGGTTGCCTACGAACTG

CTGGAAAAACATCTGCCGGAAGAAGATTTTGAACGCGCGCGTCGCTTTCTGTATCAGTGCTTC

AAACGTACCGACACGCTGCCGGCAGGTGCTTGGCTGGATTTCGCAGCTGACGGTCGCATGC

GTCGCCTGTTTACCCTGCGTCAATACTTCGGCATTCTGCACCGCCTGCTGAAAAACCGTTAA

SEQ ID NO: 6 [lacY gene]

ATGTACTATTTAAAAAACACAAACTTTTGGATGTTCGGTTTATTCTTTTTCTTTTACTTTTTTATC

ATGGGAGCCTACTTCCCGTTTTTCCCGATTTGGCTACATGACATCAACCATATCAGCAAAAGT

GATACGGGTATTATTTTTGCCGCTATTTCTCTGTTCTCGCTATTATTCCAACCGCTGTTTGGTC

TGCTTTCTGACAAACTCGGGCTGCGCAAATACCTGCTGTGGATTATTACCGGCATGTTAGTGA

TGTTTGCGCCGTTCTTTATTTTTATCTTCGGGCCACTGTTACAATACAACATTTTAGTAGGATC

DK 181310 B1 43

GATTGTTGGTGGTATTTATCTAGGCTTTTGTTTTAACGCCGGTGCGCCAGCAGTAGAGGCATT

TATTGAGAAAGTCAGCCGTCGCAGTAATTTCGAATTTGGTCGCGCGCGGATGTTTGGCTGTGT

TGGCTGGGCGCTGTGTGCCTCGATTGTCGGCATCATGTTCACCATCAATAATCAGTTTGTTTT

CTGGCTGGGCTCTGGCTGTGCACTCATCCTCGCCGTTTTACTCTTTTTCGCCAAAACGGATGC

GCCCTCTTCTGCCACGGTTGCCAATGCGGTAGGTGCCAACCATTCGGCATTTAGCCTTAAGCT

GGCACTGGAACTGTTCAGACAGCCAAAACTGTGGTTTTTGTCACTGTATGTTATTGGCGTTTC

CTGCACCTACGATGTTTTTGACCAACAGTTTGCTAATTTCTTTACTTCGTTCTTTGCTACCGGT

GAACAGGGTACGCGGGTATTTGGCTACGTAACGACAATGGGCGAATTACTTAACGCCTCGAT

TATGTTCTTTGCGCCACTGATCATTAATCGCATCGGTGGGAAAAACGCCCTGCTGCTGGCTGG

> CACTATTATGTCTGTACGTATTATTGGCTCATCGTTCGCCACCTCAGCGCTGGAAGTGGTTATT

CTGAAAACGCTGCATATGTTTGAAGTACCGTTCCTGCTGGTGGGCTGCTTTAAATATATTACCA

GCCAGTTTGAAGTGCGTTTTTCAGCGACGATTTATCTGGTCTGTTTCTGCTTCTTTAAGCAACT

GGCGATGATTTTTATGTCTGTACTGGCGGGCAATATGTATGAAAGCATCGGTTTCCAGGGCGC

TTATCTGGTGCTGGGTCTGGTGGCGCTGGGCTTCACCTTAATTTCCGTGTTCACGCTTAGCGG

CCCCGGCCCGCTTTCCCTGCTGCGTCGTCAGGTGAATGAAGTCGCTTAA

SEQ ID NO: 7 [Pscr]

GGTTAACGGCCCACTTTGCTGGCGACATCACAATTCTTAAACCGGTTTAGCAATTTTTATTTTC

ACCGCGTTACCGACATGTTTACCATATCAACTAAACCGGTTTAGCAAACATTAGCACACTCACT

> GATTTACCTTTGGATGTCACCAAC

SEQ ID NO: 8 /Pscr SD1]

GGTTAACGGCCCACTTTGCTGGCGACATCACAATTCTTAAACCGGTTTAGCAATTTTTATTTTC

ACCGCGTTACCGACATGTTTACCATATCAACTAAACCGGTTTAGCAAACATTAGCACACTCACT

> GATTTACCCAAATTCGAAACAGCT

SEQ ID NO: 9 /Pscr SD7]

GGTTAACGGCCCACTTTGCTGGCGACATCACAATTCTTAAACCGGTTTAGCAATTTTTATTTTC

ACCGCGTTACCGACATGTTTACCATATCAACTAAACCGGTTTAGCAAACATTAGCACACTCACT

GATTTACCCAAGAGCAAAACAGCT

DK 181310 B1 44

SEQ ID NO: 10 [PgatY_70UTR]

CGGCAACCTATGCCTGATGCGACGCTGAAGCGTCTTATCATGCCTACATAGCACTGCCACGT

ATGTTTACACCGCATCCGGCATAAAAACACGCGCACTTTGCTACGGCTTCCCTATCGGGAGG

CCGTTTTTTTGCCTTTCACTCCTCGAATAATTTTCATATTGTCGTTTTTGTGATCGTTATCTCGA

TATTTAAAAACAAATAATTTCATTATATTTTGTGCCTACAAGCATCGTGGAGGTCCGTGACTTTC

ACGCATACAACAAACATTAACCAAGGAGGAAACAGCT

SEQ ID NO: 11 [PglpF]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

— CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAAGGAGGAAACAGCT

SEQ ID NO: 12 [PglpF. SD1]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

> GTGACTTTCACGCATACAACAAACATTAACCAAATTCGAAACAGCT

SEQ ID NO: 13 [PglpF_SD10]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

— TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAACTGAGAAACAGCT

SEQ ID NO: 14 [PgipF. SD2]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

DK 181310 B1 45

ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAAGCGCAAAACAGCT

SEQ ID NO: 15 [PgipF. SD3]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAAGAACAAAACAGCT

SEQ ID NO: 16 [PgipF. SD4J

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAACTAGGAAACAGCT

SEQ ID NO: 17 [PgipF. SDS]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAACCGAGAAACAGCT

SEQ ID NO: 18 [PglpF_SD6]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAAGAGCTAAACAGCT

DK 181310 B1 46

SEQ ID NO: 19 [PgipF. SD7]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

>ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAAGAGCAAAACAGCT

SEQ ID NO: 20 [PgipF. SD8]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

— CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAAGAGAAAAACAGCT

SEQ ID NO: 21 [PglpF_SD9]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

ATATTTCTCGTTTTTGCTCGTTAACGATAAGTTTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAAAGGAAAAACAGCT

SEQ ID NO: 22 [PgipF. B28]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

— TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

ATATTTCTCGTTTTTGCTCGTTAACGATTTAATTACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAAGGAGGAAACAGCT

SEQ ID NO: 23 [PgipF. B29]

GCGGCACGCCTTGCAGATTACGGTTTGCCACACTTTTCATCCTTCTCCTGGTGACATAATCCA

CATCAATCGAAAATGTTAATAAATTTGTTGCGCGAATGATCTAACAAACATGCATCATGTACAA

TCAGATGGAATAAATGGCGCGATAACGCTCATTTTATGACGAGGCACACACATTTTAAGTTCG

DK 181310 B1 47

ATATTTCTCGTTTTTGCTCGTTAACGATCAGAATACAGCATGCCTACAAGCATCGTGGAGGTCC

GTGACTTTCACGCATACAACAAACATTAACCAAGGAGGAAACAGCT

SEQ ID NO: 24 [Plac 16UTRJ]

TGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTT

GTGTGGAATTGTGAGCGGATAACAATTTCAAGGAGGAAACAGCT

SEQ ID NO: 25 [Plac]

ATGCGCAAATTGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGG

CTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATG

ATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGGTACCTAAGGAGGA

AACAGCT

SEQ ID NO: 26 [PmgiB 70UTR]

TGCGTCGCCATTCTGTCGCAACACGCCAGAATGCGGCGGCGATCACTAACTCAACAAATCAG

GCGATGTAACCGCTTTCAATCTGTGAGTGATTTCACAGTATCTTAACAATGTGATAGCTATGAT

TGCACCGTGCCTACAAGCATCGTGGAGGTCCGTGACTTTCACGCATACAACAAACATTAACCA

AGGAGGAAACAGCT

SEQ ID NO: 27 [PmgiB 70UTR SD4J

TGCGTCGCCATTCTGTCGCAACACGCCAGAATGCGGCGGCGATCACTAACTCAACAAATCAG

GCGATGTAACCGCTTTCAATCTGTGAGTGATTTCACAGTATCTTAACAATGTGATAGCTATGAT

TGCACCGTGCCTACAAGCATCGTGGAGGTCCGTGACTTTCACGCATACAACAAACATTAACCA

ACTAGGAAACAGCT

SEQ ID NO: 28 [PmnagT protein]

MENKPLVSVLICAYNVEKYIEECINAVINQTYKNLEINIVNDGSSDNTYFLLKKLAEKDNRIKILNFNNHI

GIISALNEGLKEIAGEYIARTDSDDITKPDWIEKILTCMQNDPKIIAMGSYLTVLSEENNGSVLANHHK

NKVEWKNPLEHKDIVEKMLFGNPIHNNSMVMRSEIYTKYHLIYDPDYHYAEDYKFWLEVSRIGKLA

NYPESLVYYRLHRNQTSSIHNSQQEINGKKLRLQALNYYLKDLGIDYQLPEKFLFKDIALLQEIFYER

GMFRENIIRRIIYECYLSLGEYNYKDIYYFLINKNNFLSIKDKFKIIKKYLRPDKYSSTY

DK 181310 B1 48

SEQ ID NO: 29 [PmnagT gene]

ATGGAAAATAAACCGCTGGTTAGCGTTCTGATTTGCGCCTATAATGTGGAAAAATACATCGAA

GAATGCATCAACGCCGTTATTAACCAGACCTATAAAAACCTGGAAATCATCATTGTGAATGATG

GCAGCAGCGATAACACCTATTTTCTGCTGAAAAAACTGGCCGAAAAAGACAACCGTATCAAGA

TCCTGAACTTCAACAACCATATTGGCATTATTAGCGCACTGAATGAAGGCCTGAAAGAAATTG

CCGGTGAATATATTGCACGTACCGATTCAGATGATATCACCAAACCGGATTGGATCGAAAAAA

TTCTGACCTGTATGCAGAACGACCCGAAAATTATCGCAATGGGTAGCTATCTGACCGTTCTGA

GCGAAGAAAATAATGGTAGCGTGCTGGCCAATCACCATAAAAACAAAGTGGAATGGAAAAACC

CGCTGGAACATAAAGATATCGTGGAAAAAATGCTGTTTGGCAACCCGATTCATAATAACAGCA

TGGTTATGCGCAGCGAGATCTATACCAAATATCACCTGATTTATGATCCGGATTATCATTATGC

CGAGGACTATAAATTCTGGCTGGAAGTTAGCCGTATTGGTAAACTGGCAAATTATCCGGAAAG

CCTGGTTTATTATCGTCTGCATCGTAATCAGACCAGCAGCATTCATAATTCCCAGCAAGAAATC

AACGGTAAAAAACTGCGTCTGCAGGCACTGAACTATTATCTGAAAGATCTGGGCATTGATTAT

CAGCTGCCGGAAAAATTTCTGTTCAAAGATATTGCACTGCTGCAAGAGATCTTTTATGAACGTG

GTATGTTCCGCGAAAACATTATTCGTCGCATTATCTATGAGTGCTATCTGAGCCTGGGCGAGT

ATAATTACAAAGATATCTACTACTTCCTGATCAACAAAAACAACTTTCTGAGCATCAAAGACAAA

TTCAAAATCATCAAAAAATACCTGCGTCCGGACAAATATAGCAGCACCTATTAA

EXAMPLES

Example 1 — Heterologous enzymes appropriate for the construction of efficient LNT-II 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, were made to obtain the fully chromosomal strains MP1, MP2, MP3, MP5, MP6 and

MP4. The strains can produce the trisaccharide HMO LNT-II. Each of the six strains expresses a single beta-1,3-N-acetyloglucosamine transferase (GIcNACcT) 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) HD0466 from Haemophilus ducreyi (GenBank ID: WP_010944479.1). The only difference between the strain pairs, MP1-MP2, MP3-

DK 181310 B1 49

MP4 and MP5-MP6, is the GIcNAcT being expressed. The copy number of the GIcNACT in each strain pair is the only difference among the strains of the pair. For example, the strain MP6 bears two copies of the HD0466 gene while the strain MP5 bears just one PglpF-driven copy of this gene.

In the present Example, it is demonstrated how different GIcNAcTs can be beneficially expressed at varied genomic copy numbers in the genetic background of E. coli K12 cells for high-level LNT-II production. The Example reveals the HD0466 enzyme as a novel enzyme for the in vivo production of LNT-II.

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, precultures 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 % glucose solution (50 ul per 100 mL) 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 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 centrifugation of microtiter plates was followed by the removal of 0.1 mL supernatant 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.

DK 181310 B1 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 % quantitative differences in the LNT-II content of the strains tested, i.e., the % differences in the

LNT-II concentrations of PmnagT- or HD0466-expressing cells relative to /gtA-expressing cells.

As revealed by the analysis of total samples in deep-well cultures, apart from the LgtA and

PmnagT enzymes that have been previously applied for the production of various LNTII-core

HMOs, a novel GIcNAcT, namely HD0466, can provide high LNT-II titers when expressed from one or two genomic copies (Figure 1a). The descending order of activity of the three selected

GIcNACTSs on lactose, as it is indirectly revealed by the observed final LNT-II titers is as follows:

HD0466 > PmnagT > LgtA. The LNT-II titers reached by the strains MP5 and/or MP6, which express HD0466 from a different copy number, can beup to 40% or 15% higher than for strains expressing LgtA (strain MP1) or PmnagT (strain MP3), respectively.

HD0466 gene suffice to reach the highest LNT-II titers when any of these GIcNAcTs 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 beta-1,3-N-acetyloglucosamine transferases is highly beneficial for LNT-II titer enhancement

Description of the genotype of strains MP5196 and MP5211 tested in deep well assays

Based on the previously reported platform strain ('MDO”), the modifications summarised in table 2, were made to obtain the fully chromosomal strains MP2, MP4, MP6, MP7, MP8 and MP9. The strains can produce the trisaccharide HMO LNT-II. Each of the six strains bears in total two PglpF- driven copies of a single or two beta-1,3-N-acetyloglucosamine transferases (GIcNAcTs) 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) HD0466 from

Haemophilus ducreyi (GenBank ID: WP_010944479.1). Thus, all strains express one or two different GIcNAcTs from a total of 2 genomic copies with the only difference among the strains being the selected GIcNACTs.

DK 181310 B1 51

In the present Example, it is demonstrated that the pairwise expression of two different GIcNAcTs can be a more efficient approach in converting E. coli K12 cells to an efficient LNT-II cell factory than merely expressing a single GIcNACT.

As shown in table 2 below, the only difference among the strains is the GIcNAcT(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 96 deep well plates using a 4-day protocol. During the first 24 hours, precultures 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 % glucose solution (50 ul per 100 mL) 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 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 centrifugation of microtiter plates was followed by the removal of 0.1 mL supernatant 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

DK 181310 B1 52

LNT-II concentrations formed by strains expressing LgtA, PmnagT or HD04686, or pairwise combinations of all three GIcNAcTSs relative to LgtA-expressing cells.

At physiological intracellular lactose concentrations (i.e., wild-type expression of the /lacY gene coding the lactose permease), higher LNT-II titers can be achieved when HD0466 is co-expressed — with either of the two other GIcNAcTs, namely PmnagT and LgtA (strains MP7 and MP8) rather than when HD0466 (strain MP6), PmnagT (strain MP4) or LgtA (strain MP2) alone are expressed at the same copy number (Figure 2). Specifically, strains bearing a copy of HD0466 and a copy of one of the two other GIcNAcTs (strains MP7 and MP8) show higher titers than strains bearing two copies of any of the three GIcNAcTs (strains MP2, MP4 or MP8) or the strain that co-expresses

LgtA 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-II 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 trisaccharide HMO LNT-II. Each of these strains bears in total two PglpF-driven copies of a single or two beta-1,3-N-acetyloglucosamine transferases (GIcNAcTs) 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) HD0466 from Haemophilus ducreyi (GenBank ID: WP_010944479.1). Thus, all strains express one or two GIcNAcTs from a total of 2 genomic copies with the strains differing in the identity of the selected GIcNAcTs. Moreover, apart from the identity of GIcNACTs, the strains can differ in regard to the expression of the E. coli K12 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 GICNAcTs, namely HD0466 and LgtA, are co-expressed in the same strain.

In this manner, Examples 2 and 3 provide enough data to conclude that the combined expression of HD0466 and LgtA results in higher LNT-II titers regardless of intracellular lactose levels compared to when only one of these GIcNAcTs is expressed by the cell at the same copy number.

Importantly, this trend is unique for this GICNACT pair and it is not observed for any other GIcNACT pair.

DK 181310 B1 53

Description of the applied deep well assay protocol for strain characterization

Results of the deep well assays

LNT-II concentrations formed by strains expressing one of the three GIcNAcTs, namely LgtA,

PmnagT or HD0466, or pairwise combinations of all three GIcNAcTs 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-II levels (approximately 10%) can be reached when HD0466 is co-expressed with LgtA at saturating levels of intracellular lactose (i.e. when the JacY gene is over-expressed) (strain MP13) than at physiological /acY levels (strain MP7). This effect is even more obvious when the strains MP7 and

MP13 have been tested in 24-well assays, with the strain MP13 reaching up to 30% higher LNT-II titers than the strain MP7 (Figure 4). Notably, the beneficial effect of the combined expression of

HD0466 and LgtA and /acY over-expression is solely restricted to this pair of GIcNAcTs (Figure 3).

Claims

PATENT CLAIMS

1. A method for producing LNT-II, which method comprises the following steps: a) providing a genetically engineered bacterial or yeast cell capable of producing an HMO, which cell is capable of importing lactose into the cell and comprising i) a heterologous B-1,3-N-acetylglucosaminyltransferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homolog thereof having an amino acid sequence 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 a) in a suitable cell culture medium, and c) harvesting the HMO(s) produced in step b).

2. The method according to claim 1, wherein the cell further comprises iii) a heterologous B-1,3-N-acetylglucosaminyltransferase protein as shown in SEQ ID NO: 2 [LgtA] or a functional homolog thereof having an amino acid sequence of at least 80 % identical to SEQ ID NO: 2, and iv) a native or heterologous regulatory element for controlling the expression of iii).

3. Method according to claim 2, wherein the cell further comprises v) a lactose permease protein as shown in SEQ ID NO: 3 [LacY] or a functional homolog thereof, which has an amino acid sequence that 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. Method according to claim 3, wherein the gene encoding the lactose permease protein is overexpressed.

— 5. Method according to any one of the preceding claims, in that the heterologous regulatory element for controlling and increasing the expression of i), iii) and/or v) is a promoter.

Method according to any one of the preceding claims, wherein the promoter sequence is selected from the group consisting of PgatY 70UTR, PglpF, PgipF. SD1, PgipF. SD10, PgipF. SD2, PglpF. SD3, PgipF SD4, PglpF_SD5, PgipF. SD6, PglpF. SD7, PgipF. SD8, PgloF. SD9, PglpF. B28, PglpF. B29, Plac 16UTR, Plac, PmgiB 70UTR and PmgiB 70UTR SD4.

Method according to any one of the preceding claims, wherein the promoter sequence is PglpF (SEQ ID NO: 11), PmgiB 70UTR (SEQ ID NO: 26) or Plac (SEQ ID NO: 25) or a variant of PgipF (SEQ ID NO: 11), PmgiB 70UTR (SEQ ID NO: 26) or Plac (SEQ ID NO: 25).

8. Genetically engineered bacterial or yeast cell that is | capable of importing lactose into the cell, and comprising a) a nucleic acid sequence according to SEQ ID NO: 4, or a functional homologue thereof, having an amino acid sequence at least 70% identical to SEQ ID NO: 4, which encodes a heterologous -1,3-N-acetylglucosaminyltransferase, b) a native or heterologous regulatory element for controlling the expression of a).

9. Cell according to claim 8, which cell further comprises c) a nucleic acid sequence according to SEQ ID NO: 5 [LgtA], or a functional homolog thereof, which has an amino acid sequence that is at least 70% identical to SEQ ID NO: 5, which encodes a heterologous -1,3-N-acetylglucosaminyltransferase, d) a native or heterologous regulatory element for controlling the expression of c).

10. Cell according to claim 9, which further comprises e) a nucleic acid sequence according to SEQ ID NO: 6 [LacY], or a functional homologue thereof, which has an amino acid sequence that is at least 70% identical to SEQ ID NO: 6, which encodes for a lactose permease, and f) a native or heterologous regulatory element for controlling the expression of e).

11. Genetically engineered cell according to claim 10, wherein the lactose permease protein following e) is overexpressed.

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

13. A genetically engineered cell according to any one of claims 8-12, wherein the promoter sequence is selected from — the group consisting of PgatY 70UTR, PglpF, PgipF SD1, PgipF SD10, PgipF SD2, PgipF SD3, PglpF. SD4, PgipF_SD5, PglpF_SD6, PglpF_SD7, PgipF. SD8, PgipF. SD9, PglpF. B28, Plac 16UTR, Plac, PmgiB 70UTR and PmgiB 70UTR SD4.

14. Transgenic cell according to any one of claims 8-13, wherein the promoter sequence is PglpF (SEQ ID NO: 11), PmgiB 70UTR (SEQ ID NO: 26) or Plac (SEQ ID NO: 25) or a variant of PgipF (SEQ ID NO: 11), PmgiB 70UTR (SEQ ID NO: 26) or Plac (SEQ ID NO: 25).

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

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

17. Use of a nucleic acid construct comprising a nucleic acid sequence encoding i) a heterologous B-1,3-N-acetylglucosaminyltransferase protein as shown in SEQ ID NO: 1 or a functional homolog thereof having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, and ii) a native or heterologous regulatory element for controlling the expression of i), which regulatory element is selected from the group consisting of PgatY 70UTR, PglpF, PgipF_SD1, PglpF_SD10, PgipF. SD2, PgipF. SD3, PglpF. SD4, PglpF. SDS, PglpF_SD6, PglpF SD7, PglpF. SD8, PglpF. SD9, PglpF B28, Plac 16UTR, Plac, PmgiB 70UTR and PmgiB 70UTR SD4 for the production of LNT-II.

18. Use of a nucleic acid construct according to claim 17, further comprising lii) a nucleic acid sequence according to SEQ ID NO: 5 [LgtA], or a functional homologue thereof, which has an amino acid sequence that is at least 70% identical to SEQ ID NO: 5 , which encodes a heterologous -1,3-N-acetylglucosaminyltransferase, iv) a native or heterologous regulatory element for controlling the expression of iii).

19. Use of 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, which has an amino acid sequence that is at least 70% identical to SEQ ID NO: 6 , which encodes 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 one of claims 8-16 for the production of LNT-IL