Product-specific transporters for in vivo synthesis of human milk oligosaccharides

Abstract

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

Description

Technical Field

[0001] The present disclosure relates to the production of one or more desired human milk oligosaccharides (HMOs) and genetically engineering suitable cells that express transporters capable of exporting the desired HMOs from the cells. The genetically engineered cells described herein are useful for the production of desired HMOs. Background Art

[0002] Designing and constructing bacterial cell factories for the production of human milk oligosaccharides (HMOs) composed of 3-6 monosaccharide units is crucial to provide innovative and scalable solutions for future production of more complex HMOs.

[0003] To this end, rational strain engineering principles are often applied to bacterial host cells. These principles generally involve: a) introducing the desired biosynthetic pathway into the host; b) increasing the cellular pool of the relevant reactive sugars required as donors for the desired reaction; c) ensuring sufficient lactose in the host cell, for example through the presence of a lactose permease (e.g., LacY); and d) introducing heterologous sugar efflux transporters to export the desired newly formed heterooligosaccharides (for review, see Bych et al., 2019 Current Opinion in Biotechnology 56:130–137).

[0004] The expression of substrate-specific transporters as described in step d) in production strains has attracted increasing attention in recombinant HMO production cells, for example fermentation procedures have recently been described as well as several new sugar transporter genes encoding proteins that can promote the efflux of recombinantly produced 2'-fucosyllactose (2'-FL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) or sialylated HMOs, such as 3' sialyllactose (3' SL) (WO2010 / 142305, WO2021 / 148610, WO2021 / 148611, WO2021 / 148614, WO2021 / 148615, WO2021 / 148620, WO2022 / 219188 and WO2022 / 157213).

[0005] As can be seen from these disclosures, different transporters have different abilities to export recombinantly produced oligosaccharides. Oligosaccharide export may have higher or lower specificity for the desired oligosaccharide relative to byproduct oligosaccharides also produced during the fermentation process. Therefore, it is of interest to identify other oligosaccharide exporters with optimal specificity in order to produce higher purity, higher yield products. Summary of the Invention

[0006] In a first aspect, the present disclosure relates to a genetically engineered cell capable of producing a desired HMO, wherein the cell comprises one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases and a recombinant nucleic acid sequence encoding the transporter Edic1 or a functional variant thereof, the transporter Edic1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, the amino acid sequence of a functional variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 1, wherein expression of the transporter in the cell results in export of the desired HMO from the cell.

[0007] Preferably, the desired HMO comprises a lacto-N-triose II (LNT-II, GlcNAc(β1-3)Gal(β1-4)Glc) backbone. More preferably, the HMO is selected from lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), lacto-N-fucopentaose V (LNFP-V), and 6'-sialyllacto-N-neotetraose (LST c). In some embodiments, at least 85%, such as at least 90%, or at least 95%, of the total molar content of HMOs exported from the cell is LNnT or LNT. Preferably, less than 10% of the total molar content of HMOs exported from the cell is byproduct HMOs. In some embodiments, less than 10% of the total molar content of HMOs exported from the cell is LNT-II.

[0008] In an embodiment, the one or more glycosyltransferases include a β-1,4-galactosyltransferase or a β-1,3-galactosyltransferase, and optionally a β-1,3-N-acetylglucosaminyltransferase.

[0009] In a specific embodiment, the cells of the present disclosure are selected from Escherichia coli ( Escherichia Coli ), Bacillus subtilis ( Bacillus subtilis ), Lactobacillus lactis ( lactobacillus lactis ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Yarrowia lipolytica ( Yarrowia lipolytica )、Pichia pastoris( Pichia pastoris ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

[0010] In a second aspect, the present disclosure relates to a method for producing an HMO product, wherein the method comprises providing a genetically engineered cell according to the first aspect of the present disclosure, culturing the genetically engineered cell in a culture medium under conditions that allow production of the HMO; and optionally recovering the HMO. The HMO product produced is preferably LNnT, LNT, or a mixture of LNT and LNFP-V or LNnT and LST-c.

[0011] The third aspect of the present disclosure relates to a desired HMO, for example, an HMO selected from lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), lacto-N-fucopentaose V (LNFP-V) and 6'-sialylacto-N-neotetraose (LST c), wherein the desired HMO is produced by the method according to the second aspect of the present disclosure.

[0012] Another aspect of the present disclosure relates to a nucleic acid construct comprising a recombinant nucleic acid sequence encoding the transporter Edic1 or a functional variant thereof, the transporter Edic1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, the amino acid sequence of the functional variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity to SEQ ID NO: 1, wherein the transporter coding sequence is under the control of a promoter sequence. The present disclosure also relates to the use of the nucleic acid construct in a host cell producing an HMO comprising a GlcNAc(β1-3)Gal(β1-4)Glc backbone and at least one additional sugar moiety, such as Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc or Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Overview of LNT and LNnT Synthesis Figure 2 A) Overview of the general principle of specific product transport described by LNnT; B) Principle of product (LNnT) accumulation in the supernatant when using a specific product (LNnT) transporter.

[0014] Figure 3 Overview of the general principles underlying the combination of specific precursor (LNT-II) import and specific product (LNnT) efflux transporters.

[0015] Figure 4 Overview of the general principles underlying the combination of specific precursor (lactose) import and specific product (LNnT) efflux transporters.

[0016] Figure 5 Distribution of each HMO in the total fermentation broth (supernatant and precipitate) in millimolar terms (%) relative to the total HMO produced by the Vag strain.

[0017] Figure 6 Distribution of each HMO produced in the fermentation supernatant relative to the total HMO in the fermentation supernatant of the Vag strain in terms of millimolar (%).

[0018] Figure 7 HPLC chromatograms of supernatants isolated after fermentation of (A) Vag and (B) Edic1 strains.

[0019] Figure 8 Distribution of individual HMOs produced by the Nec strain relative to the total HMOs in millimolar (%) in the total fermentation broth (supernatant and precipitate).

[0020] Figure 9 Distribution of individual HMOs in LST-c production strains with and without edic1 in deep-well analysis. A) Distribution of LNT-II core HMOs (in milligrams per liter) in the supernatant of the strain without edic1 (no TP) relative to the total LNT-II core HMOs in the supernatant. B) Distribution of LNT-II core HMOs (in milligrams per liter) in the pellet of the strain without edic1 (no TP) relative to the total LNT-II core HMOs in the pellet. DETAILED DESCRIPTION

[0021] The present disclosure addresses the biotechnological challenge of in vivo HMO production, namely, harvesting specific oligosaccharides from the culture medium used to cultivate producer cells. The disclosure provides specific strain engineering solutions to increase and / or simplify HMO production by leveraging the potential of oligosaccharide exporters, particularly HMOs with a lacto-N-triose II (LNT-II, GlcNAc(β1-3)Gal(β1-4)Glc) backbone.

[0022] Surprisingly, the transporter Edic1 was found to efficiently transport LNT-II-based HMOs out of cells, while the HMO precursor LNT-II was exported at a low level. Example 1 of this disclosure demonstrates that in LNnT-producing cells, Edic1 has a higher specificity for LNnT than for LNT-II and pLNnH. This results in the accumulation of the byproduct HMOs LNT-II and pLNnH within the cells, while the product HMO LNnT is exported and accumulates in the fermentation medium. This is highly advantageous because the product HMOs can be largely isolated directly from the fermentation supernatant without the need for isolation from the producer cells, including without the need for cell lysis, thereby avoiding the production of intracellular byproduct HMOs (e.g., LNT-II and pLNnH). Thus, the genetically modified cells described herein provide a method in which product HMOs, such as HMOs having an LNT-II backbone, particularly LNT or LNnT, can be obtained from the supernatant without significant loss of the desired product accumulated in the cells, and with low amounts of by-products present in the fermentation supernatant. That is, the producer cells act as product-specific cellular reaction compartments and nanofilters, allowing only the desired HMOs to enter the supernatant, which has significant advantages for subsequent purification of the desired HMO product.

[0023] In other words, the genetically modified cells encompassed by the present disclosure, such as E. coli strains, express genes encoding key enzymes for HMO biosynthesis, enabling the cells to produce the desired HMOs. They also express one or more genes encoding efflux transporters (preferably Edic1), which are capable of transporting the specific HMOs from the cell to the extracellular medium. Therefore, Edic1 can also be referred to as an exporter. Furthermore, the strains described herein may also contain genes encoding transporters (also known as importers) for importing the desired molecules. The most commonly used importer in HMO-producing strains is lactose permease, which is used to import the initial substrate required for HMO production. Alternative substrate importers, such as mutant variants of the E. coli LacY protein (Table 1) and / or ABC and / or MFS transporters from Gram-positive bacteria (Table 2), are used to import precursor oligosaccharide molecules (initial substrates), such as LNT-II. These molecules can be further modified intracellularly by recombinant enzymes to produce more complex molecules, which are then exported into the culture medium by the expressed efflux transporters, greatly simplifying the purification of the desired HMOs.

[0024] The advantage of exporting specific oligosaccharides into the culture medium is that it simplifies the purification of the produced oligosaccharides, where the desired oligosaccharide can be purified directly from the fermentation supernatant. In addition, the exporter prefers to transport specific products, such as LNnT and / or LNT, rather than the precursor LNT-II or the byproducts pLNH2 or pLNnH, resulting in lower levels of byproduct HMOs in the culture medium.

[0025] Typically, byproduct HMOs are either precursors (lactose or other acceptor oligosaccharides, such as LNT-II) of the desired HMO (the desired HMO or HMO product) or the product of further modification of the desired HMO product. The fewer glycosyltransferases required to produce the desired HMO product, the fewer byproduct HMOs or other impurities are produced, thereby improving the purity of the complex HMO product. For example, if LNT is produced from lactose in a single cell, two glycosyltransferases are required: β-1,3-N-acetylglucosaminyltransferase to form LNT-II and β-1,3-galactosyltransferase to form LNT. LNT can then be further modified by introducing, for example, an α-1,2-fucosyltransferase capable of forming LNFP-I or an α-1,3-fucosyltransferase capable of forming LNFP-V from LNT. In this case, the desired HMO products are LNT and / or LNnT, while LNT-II and pLNH2 or pLNnH (hexasaccharides produced after further modification of LNT or LNnT, see Figure 1 ) may be an unwanted HMO by-product.

[0026] Generally, the genetically modified cells of the present disclosure capable of importing LNT-II only require the β-1,3-galactosyltransferase or β-1,4-galactosyltransferase expressed in the genetically modified HMO-producing cells to produce LNT and / or LNnT, and lactose is not present, thereby avoiding the production of several unwanted HMOs and other impurities (e.g., Gal-LNT and Gal-Lac) as by-products, thereby allowing partial or complete conversion of LNT-II to LNT and / or LNnT at the end of fermentation, because in this case LNT-II is the substrate feed to the genetically modified cells, and the produced LNT and / or LNnT is exported from the cells via specific heterologous efflux transporters (e.g., Edic1).

[0027] Furthermore, it has been observed that the presence of a suitable exporter (e.g., Edic1) can increase the total amount of HMOs produced by cells, particularly the yield of the desired HMO, compared to cells lacking the Edic1 transporter or transporters known in the art, as shown for example in Example 3 for LNT and in Example 4 for LNFP-V. Even if the yield of byproduct HMOs is immediately reduced, increasing HMO yield is always desirable because HMO mixtures can potentially be beneficial, or significantly increasing yield can reduce overall process costs, although this does not simplify the purification process.

[0028] There are numerous applications in the field of biotechnology to which the concepts of the present disclosure may be relevant.

[0029] In the following sections, further details regarding the components of genetically modified cells, methods of using the cells to produce products, and manufacturing the products and their applications are described in more detail.

[0030] transporter proteins The genetically modified cells according to the present disclosure comprise at least one recombinant nucleic acid sequence encoding a transporter protein (also called exporter) capable of exporting a specific HMO product.

[0031] The present disclosure provides specific strain engineering solutions for producing specific HMOs, particularly from Edwardsiella ictaluri ( Edwardsiella ictalurid ) transport protein Edic1, whose GenBank accession number is WP_015873007.1 (https: / / www.ncbi.nlm.nih.gov / protein / WP_015873007.1), is also disclosed in SEQ ID NO: 1. The putative MFS transport protein is referred to herein interchangeably as "Edic1 protein" or "Edic1 transporter" or "Edic1 exporter" or "Edic1"; the nucleic acid sequence encoding the Edic1 protein is referred to herein interchangeably as " Edic1 Coding nucleic acid / DNA" or " Edic1 gene "or" edic1 ”.

[0032] In the cells of the present disclosure, the transporter protein Edic1 from Edwardsiella ictaluri is encoded by a heterologous gene encoding a putative MFS (major facilitator superfamily) transporter protein Edic1 derived from Edwardsiella ictaluri.

[0033] The present disclosure has demonstrated that Edic1 is a product-specific transporter for oligosaccharides with an LNT-II backbone, such as LNnT and LNT, as well as LNT and further modified versions of LNT, such as sialylated and / or fucosylated LNnT, in particular LST-c, or sialylated and / or fucosylated LNT, in particular LNFP-V.

[0034] More specifically, the present disclosure relates to a genetically modified cell optimized for producing oligosaccharides, such as heterologous oligosaccharides, particularly a desired HMO or HMO product. More specifically, the genetically modified cell capable of producing a desired HMO product (e.g., an HMO having an LNT-II backbone) comprises a recombinant nucleic acid encoding a protein having at least 80%, such as 85%, such as 90%, such as 95%, or 100% sequence identity to the amino acid sequence of GenBank Accession No. WP_015873007.1 (https: / / www.ncbi.nlm.nih.gov / protein / WP_015873007.1), which is also disclosed in SEQ ID NO: 1.

[0035] Thus, in a preferred embodiment, the present disclosure relates to a genetically engineered cell capable of producing a desired human milk oligosaccharide (HMO) that is not naturally produced by the cell (heterologous), wherein the cell comprises one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases and a recombinant nucleic acid sequence encoding the transporter protein Edic1 or a functional variant thereof, said transporter protein Edic1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, the amino acid sequence of a functional variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 1, wherein expression of the transporter protein in the cell results in export of the desired HMO from the cell or in an increased production of the desired HMO compared to a cell without the exporter or with an exporter known to export the desired HMO.

[0036] In an embodiment, a cell expressing the transporter Edic1 or a functional variant thereof is capable of producing LNnT, wherein the transporter Edic1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1, the amino acid sequence of a functional variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 1, wherein the total molar content of LNnT produced by the cell is at least 85%, such as at least 88%, such as at least 89%, such as at least 90% or such as at least 91% of the total HMO content produced by the cell.

[0037] As described in Example 1, the transporter Edic1 exports most of the LNnT produced by cells to the extracellular medium, while the unwanted byproduct HMO remains inside the cells (e.g. Figure 2 A), resulting in most of the total amount of LNnT produced being present in the supernatant, while only a small portion remained within the cells (biomass fraction or precipitate), as shown in Figure 2 B. Thus, in embodiments, at least 85%, such as at least 90%, such as at least 95%, or at least 99%, of the total molar content of the desired HMO produced by the cells is exported from cells expressing the product-specific transporter Edic1. In further embodiments, at least 85%, such as at least 90%, such as at least 95%, or at least 99% of the total molar content of HMOs exported from cells expressing the product-specific transporter is LNnT. In this regard, it is highly preferred that only a small amount of the total HMOs exported from cells expressing the product-specific transporter Edic1 is byproduct HMOs, such as LNT-II or pLNnH. In embodiments, byproduct HMOs account for less than 10% of the total molar content of HMOs exported from the cells, for example, LNT-II accounts for less than 10% of the total molar content of HMOs exported from the cells. In further embodiments, pLNnH accounts for less than 0.1% of the total molar content of HMOs exported from the cells, particularly when the cells are LNnT-producing cells.

[0038] In further embodiments, genetically engineered cells expressing the product-specific transporter Edic1 described herein may express a second transporter, such as an importin, or a second exportin.

[0039] In an embodiment, the second exporter is selected from MFS transporters known to export HMOs.

[0040] As used herein, the term "MFS transporter" refers to a protein that facilitates the transport of oligosaccharides, preferably HMOs, across cell membranes, preferably from the cell cytoplasm to the cell culture medium, as synthesized by the genetically engineered cells described herein. Additionally or alternatively, MFS transporters may also facilitate the efflux of molecules that are not considered HMOs or oligosaccharides, such as lactose, glucose, cellular metabolites, and / or toxins.

[0041] A suitable MFS transporter can be obtained from Rosenbergia spp. Rosenbergiella nectarea ). An exemplary MFS transporter from Rosenbergia spp. contains or comprises the amino acid sequence of SEQ ID NO: 83, interchangeably referred to herein as "Nec protein" or "Nec transporter" or "Nec"; the nucleic acid sequence encoding the nec protein is referred to herein as "Nec encoding nucleic acid / DNA" or "nec gene" or "nec"; the amino acid sequence represented herein by SEQ ID NO: 83 is 100% identical to the amino acid sequence of GenBank Accession No. WP_092672081.1 and described in WO2021 / 148615. In another embodiment, the second MFS transporter has the amino acid sequence of SEQ ID NO: 83, or is a functional homolog having an amino acid sequence at least 80% identical, such as at least 85% identical, such as at least 90% identical, such as at least 95% identical, or such as at least 99% identical, to any of SEQ ID NOs: 83.

[0042] Another suitable MFS transporter can be obtained from Pantoea eutropha ( Pantoea vagans ). An exemplary Pantoea eutropha MFS transporter comprises or contains the amino acid sequence of SEQ ID NO: 5, referred to interchangeably herein as "Vag protein" or "Vag transporter" or "Vag"; the nucleic acid sequence encoding the Vag protein is referred to interchangeably herein as "Vag encoding nucleic acid / DNA" or "Vag gene" or "Vag"; the amino acid sequence set forth herein in SEQ ID NO: 5 is an amino acid sequence that is 100% identical to the amino acid sequence of GenBank Accession No. WP_048785139.1 and described in WO2021 / 148611. In a further embodiment, the second MFS transporter has the amino acid sequence of SEQ ID NO: 5, or is a functional homolog having an amino acid sequence that is at least 80% identical, such as at least 85% identical, such as at least 90% identical, such as at least 95% identical, or such as at least 99% identical to any of SEQ ID NOs: 5.

[0043] In one or more exemplary embodiments, the second transporter or the second exporter is selected from an MFS transporter from Pantoea dispersae or Rosenbergia sp., such as an MFS transporter Vag having an amino acid sequence at least 85% identical to SEQ ID NO: 5, or an MFS transporter Nec having an amino acid sequence at least 85% identical to SEQ ID NO: 83.

[0044] In a further embodiment, the LNnT producer cells expressing the product-specific transporter Edic1 as described herein further express a second transporter, in particular the MFS transporter Vag having an amino acid sequence of SEQ ID NO: 5, or a functional homolog thereof, the amino acid sequence of which is at least 80% identical, such as at least 85% identical, such as at least 90% identical, such as at least 95% identical, or such as at least 99% identical to SEQ ID NO: 5.

[0045] In an embodiment, a cell expressing the transporter Edic1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 or a functional variant thereof, the amino acid sequence of which is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identical to SEQ ID NO: 1, is capable of producing LNT, wherein the total molar content of LNT produced by the cell is at least 80%, such as at least 85%, such as at least 88%, such as at least 89%, such as at least 90% or such as at least 91% of the total content of HMOs produced by the cell.

[0046] In a further embodiment, LNT-producing cells expressing the product-specific transporter Edic1 produce less than 20% of the total molar content of HMOs expressed by the cells and less than 0.5% of pLNH2. In a further embodiment, LNT-producing cells expressing the product-specific transporter Edic1 produce at least 10% more HMOs than comparable cells expressing the Nec transporter (SEQ ID NO: 83). Preferably, LNT-producing cells expressing the product-specific transporter Edic1 produce at least 5%, such as at least 8%, more LNT than comparable cells expressing the Nec transporter and have at least 0.5% less pLNH2 than comparable cells expressing the Nec transporter.

[0047] In a further embodiment, the LNT producer cells expressing the product-specific transporter Edic1 as described herein further express a second transporter, in particular the MFS transporter Nec having an amino acid sequence of SEQ ID NO: 83, or a functional homolog thereof having an amino acid sequence at least 80% identical, such as at least 85% identical, such as at least 90% identical, such as at least 95% identical, or such as at least 99% identical to SEQ ID NO: 83. Preferably, the LNT producer cells comprising the Edic1 and Nec transporters, or functional variants thereof, produce at least 85%, such as at least 90% LNT and less than 15%, such as less than 10%, LNT-II and less than 15%, such as less than 0.5% pLNH2.

[0048] In a further embodiment, LNT producing cells expressing the product-specific transporters Edic1 and Nec, or functional variants thereof, produce at least 10% more HMO than a similar cell expressing only the Nec transporter (SEQ ID NO: 83). Preferably, LNT producing cells expressing the product-specific transporters Edic1 and Nec produce at least 15%, such as at least 20%, more LNT, and at least 3%, such as at least 5%, less LNT-II, and at least 0.5% less pLNH2 than a similar cell expressing only the Nec transporter.

[0049] In an embodiment, a cell expressing the transporter Edic1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, or a functional variant thereof, whose amino acid sequence is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, or such as at least 99% identical to SEQ ID NO: 1, is capable of producing LNFP-V. In a further embodiment, an LNFP-V producing cell expressing the product-specific transporter Edic1 as described herein produces at least 20-fold, such as at least 30-fold, such as at least 40-fold more LNT-II core HMOs than a similar cell not containing the Edic1 transporter. In particular, the level of LNFP-V is increased by at least 5-fold, such as at least 8-fold, compared to a similar cell not expressing Edic1.

[0050] In an embodiment, a cell expressing the transporter Edic1 comprising or consisting of an amino acid sequence according to SEQ ID NO: 1, or a functional variant thereof, whose amino acid sequence is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identical to SEQ ID NO: 1, is capable of producing LST-c. In a further embodiment, an LST-c producing cell expressing the product-specific transporter Edic1 as described herein exports at least 5% more LST-c to the supernatant than a cell without the Edic1 transporter.

[0051] Substrate import protein Furthermore, the genetically modified cells according to the present disclosure may further comprise at least one recombinant nucleic acid sequence encoding a transporter capable of importing HMO product precursors, such as initial substrates (also known as acceptor oligosaccharides), such as, but not limited to, lactose and / or LNT-II.

[0052] Substrate import proteins are capable of importing substrates required for HMO synthesis, such as, but not limited to, lactose or LNT-II, and can be used to increase the production of desired HMO / HMO products (e.g., LNT and / or LNnT or fucosylated or sialylated versions thereof). Figure 3 and Figure 4 The basic principle is demonstrated using the production of LNnT as a non-limiting example, where the presence of a substrate import protein is shown to directly import lactose ( Figure 4 ) or LNT-II ( Figure 3 ), thereby increasing substrate / precursor availability and thus product yield, e.g. Figure 1 The production of LNnT is shown. The presence of both an importer and a specific product exporter is particularly advantageous because it allows for optimal production of a specific HMO product in the cell while preventing product accumulation within the cell. Therefore, in some embodiments, the cell further comprises a substrate importer selected from the group consisting of a lactose importer and a lacto-N-triose-II (LNT-II) importer.

[0053] In a further embodiment, the cell of the present disclosure comprises: a. one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases; b. a recombinant nucleic acid sequence encoding the transporter Edic1 or a functional variant thereof, wherein the transporter Edic1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1, the amino acid sequence of the functional variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 1; and c. a recombinant nucleic acid sequence encoding a precursor importin, Therein, expression of the precursor import protein results in the import of the desired precursor disaccharide or oligosaccharide into the cell, and expression of the transporter Edic1 in the cell results in the export of the desired oligosaccharide (HMO product) from the cell.

[0054] Wild-type lactose permease (LacY) is known to be able to transport the disaccharide lactose from the outside of the cell into the Escherichia coli cell. Therefore, if the initial substrate for HMO production is lactose, LacY is an ideal import protein.

[0055] Mutant variants of LacY have been described as being able to transport the trisaccharide maltotriose (Olsen et al. 1993 J Bacteriol. 175(19):6269-75). In the present disclosure, these mutants are described as potential importers of trisaccharides (acceptor oligosaccharides / HMO precursor molecules) associated with HMO production, such as lacto-N-triose (LNT-II).

[0056] The genetically modified cells according to the present disclosure may further comprise, in addition to an efflux transporter (e.g., Edic1), a recombinant nucleic acid sequence encoding a transporter capable of importing an intermediate (acceptor) oligosaccharide of at least three monosaccharide units into the cell, wherein the transporter is a mutant lactose permease (LacY), as shown in Table 1.

[0057] Table 1. E. coli DH1 K12 lactose permease LacY (SEQ ID NO: 1) that can be used as input for 2'FL, 3FL, or LNT-II. ID NO: 14). In a preferred embodiment, the lactose permease variant of Table 1 has a higher affinity for LNT-II than for lactose. In a specific embodiment, the cells of the present disclosure comprise a mutant lactose permease (LacY) as shown in Table 1, wherein the lactose permease variant is selected from mut2 and mut10 as shown in Table 1.

[0058] Thus, in an embodiment, a cell of the present disclosure comprises a recombinant nucleic acid sequence encoding a mutant lactose permease (LacY), with reference to SEQ ID NO: 14, comprising His at position 236 and / or Val at position 177 and Thr at position 306. In one embodiment, the LacY variant comprises the following substitutions compared to SEQ ID NO: 14: Y236H and / or A177V and S306T, wherein the variant is at least 80% identical to SEQ ID NO: 14, such as at least 85% identical, such as at least 90% identical, such as at least 95% identical, or such as 99.5% identical to SEQ ID NO: 14.

[0059] Thus, in an embodiment, a cell of the present disclosure comprises: a. one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases; and b. a recombinant nucleic acid sequence encoding the transporter Edic1 or a functional variant thereof, wherein the transporter Edic1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1, the amino acid sequence of the functional variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 1; and c. a nucleic acid sequence encoding a lactose permease (e.g., LacY) or a functional variant thereof, said lactose permease comprising or consisting of an amino acid sequence according to SEQ ID NO: 14, wherein the amino acid sequence of the functional variant thereof is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identical to SEQ ID NO: 14, wherein expression of the transporter in the cell results in export of the desired oligosaccharide (HMO product) from the cell.

[0060] In embodiments, it may be advantageous to overexpress one or more lactose permeases, such as a native lactose permease. In one embodiment, additional copies of a nucleic acid encoding a lactose permease are recombinantly introduced into the host cell, preferably into the genome of the host cell.

[0061] According to the present disclosure, overexpression of one or more lactose permeases can enhance the production of one or more HMOs, particularly LNT or LNnT.

[0062] As used herein, "overexpression" refers to expression of a desired protein at a level greater than that naturally obtained from an endogenous copy of the nucleic acid encoding the desired protein in a cell. Overexpression can be determined by transcriptional or translational analysis, for example, by quantitative determination of mRNA levels or protein levels using any method known to those skilled in the art, such as, but not limited to, quantitative PCR or mass spectrometry. Overexpression can be achieved, for example, by replacing the wild-type promoter of an endogenous gene with a stronger promoter. Alternatively, one or more additional copies of an endogenous gene can be recombinantly introduced into the host cell, or nucleic acids that negatively regulate the desired endogenous gene can be deleted.

[0063] In other embodiments, the cells of the present disclosure comprise: a. one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases; b. a recombinant nucleic acid sequence encoding the transporter Edic1 or a functional variant thereof, wherein the transporter Edic1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1, the amino acid sequence of the functional variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 1; and c. A recombinant nucleic acid sequence encoding a LacY variant or a functional variant thereof, wherein the LacY variant comprises or consists of the amino acid sequence according to SEQ ID NO: 14, and the amino acid sequence of the functional variant thereof has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 14.

[0064] wherein the variant comprises a mutation according to Table 1, wherein expression of the LacY variant results in import of the desired precursor oligosaccharide into the cell, and expression of the transporter in the cell results in export of the desired oligosaccharide (HMO product) from the cell.

[0065] In a preferred embodiment, the LacY variant comprises or consists of the following substitutions compared to SEQ ID NO: 14: Y236H and / or A177V and S306T.

[0066] Among Gram-positive (Gram+) bacteria, especially Bifidobacterium spp. Bifidobacterium ), Roseburia ( Roseburia ) and Eubacterium ( Eubacterium ) members, alternative importins with the potential to import LNT-II have been identified.

[0067] Table 2 shows MFS transporters derived from Gram-positive or Gram-negative bacteria and ABC transporter clusters derived from Gram-positive bacteria that are capable of importing acceptor oligosaccharides of at least three monosaccharide units into cells.

[0068] Table 2. ABC transporters and MFS transporters of Gram-positive bacteria, with the expected import of the transporter indicated. ABC transporters consist of three to four genes. For ease of reference, each transporter is assigned a transporter protein IDs (TP IDs). The present disclosure relates to a genetically modified cell as described herein, further comprising a recombinant nucleic acid sequence encoding an importin. The present disclosure relates to a genetically modified cell as described herein, further comprising a recombinant nucleic acid sequence encoding an importin selected from Table 2.

[0069] The present disclosure relates to a genetically modified cell comprising a recombinant nucleic acid sequence encoding a transporter protein Edic1 or a functional variant thereof, wherein the transporter protein Edic1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1, and the amino acid sequence of the functional variant thereof has at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 1. In addition, the cell of the present disclosure may further comprise a recombinant nucleic acid sequence cluster encoding a transporter protein and / or a protein cluster capable of importing at least three units of an acceptor oligosaccharide into the cell, wherein the protein cluster is an ABC transporter protein from a Gram-positive cell. In particular, the cells of the present disclosure may further comprise an ABC transporter as listed in Table 2, in particular a transporter selected from TP ID: 8 (Blon_2177, Blon_2176, Blon_2175), TP ID 11 (BBR_0527 / lntP1, BBR_0528 / lntP2, BBR_0530 / lntS, BBR_0531), TP ID 13 (Blon_0962) or TP ID18 (BBPC_1775, BBPC_1776, BBPC_1777).

[0070] In an embodiment, the present disclosure relates to a genetically engineered cell of the present disclosure capable of producing a desired human milk oligosaccharide (HMO product), wherein the cell further comprises a recombinant nucleic acid sequence encoding a transporter capable of importing preferably three units of an acceptor oligosaccharide into the cell, wherein the protein is Blon_0962 or a functional variant thereof, Blon_0962 comprising or consisting of an amino acid sequence according to SEQ ID NO: 42, the amino acid sequence of the functional variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 42.

[0071] In an embodiment, the present disclosure relates to a genetically engineered cell of the present disclosure capable of producing a desired human milk oligosaccharide (HMO), wherein the cell further comprises a recombinant nucleic acid sequence encoding a protein cluster capable of importing preferably three units of an acceptor oligosaccharide into the cell, wherein the protein cluster is a transporter selected from TP ID: 8, 11 or 18.

[0072] In an embodiment, the present disclosure relates to a genetically engineered cell of the present disclosure capable of producing a desired human milk oligosaccharide (HMO), wherein the cell further comprises a recombinant nucleic acid sequence encoding a protein cluster capable of importing preferably three units of an acceptor oligosaccharide into the cell, wherein the protein cluster is the protein cluster of TP ID: 8, wherein TP ID: 8 comprises or consists of fragments Blon_2177, Blon_2176 and Blon_2175 (which comprise or consist of the amino acid sequence according to SEQ ID NOs: 60, 61 and 62), or a functional variant thereof, the amino acid sequence of the functional variant having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NOs: 60, 61 and 62.

[0073] In an embodiment, the present disclosure relates to a genetically engineered cell of the present disclosure capable of producing a desired human milk oligosaccharide (HMO), wherein the cell further comprises a recombinant nucleic acid sequence encoding a protein cluster capable of importing preferably three units of an acceptor oligosaccharide into the cell, wherein the protein cluster is the protein cluster of TP ID: 11, wherein TP ID: 11 comprises or consists of the fragments BBR_0527 / lntP1, BBR_0528 / lntP2, BBR_0530 / lntS and BBR_0531 (which comprise or consist of the amino acid sequence according to SEQ ID NOs: 75, 76, 77 and 78), or a functional variant thereof, the amino acid sequence of the functional variant having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NOs: 75, 76, 77 and 78.

[0074] In an embodiment, the present disclosure relates to a genetically engineered cell of the present disclosure capable of producing a desired human milk oligosaccharide (HMO), wherein the cell further comprises a recombinant nucleic acid sequence encoding a protein cluster capable of importing preferably three units of an acceptor oligosaccharide into the cell, wherein the protein cluster is the protein cluster of TP ID: 18, wherein TP ID: 18 comprises or consists of fragments BBPC_1775, BBPC_1776 and BBPC_1777 (which comprise or consist of the amino acid sequence according to SEQ ID NOs: 72, 72 and 74), or a functional variant thereof, the amino acid sequence of the functional variant having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NOs: 72, 72 and 74.

[0075] Typically, the genetically modified cells lack an enzyme activity that has an acceptor oligosaccharide susceptible to degradation of at least three or four monosaccharide units.

[0076] LacY negative In one aspect, the genetically modified cells according to the present disclosure do not express a functional lactose permease. That is, the genetically modified cells are lacY negative. Specifically, the genetically modified cells do not express wild-type lactose permease, but may express one or more lactose permease mutants listed in Table 1.

[0077] The endogenous natural lactose permease (LacY) in E. coli is specific for galactose and simple galactosyl disaccharides such as lactose. lacY The gene is a very effective genetic tool that can specifically prevent the import of lactose from outside the cell into the cytoplasm, thereby ensuring that oligosaccharides with preferably more complex structures (e.g. oligosaccharides with at least 3 monosaccharide units) are imported into the cell via specific transporters and / or protein clusters as described herein that are capable of importing, for example, acceptor oligosaccharides with 3 monosaccharide units into the cell.

[0078] In this document, the term "lacY negative" is used to describe the disruption of the native lactose permease (LacY) in the genetically modified cell and does not exclude that the genetically modified cell comprises a recombinant nucleic acid sequence selected from mutant LacY (e.g., as shown in Table 1), as long as the recombinant nucleic acid sequence encodes a transporter and / or protein cluster capable of importing an acceptor oligosaccharide of at least 3 monosaccharide units into the cell.

[0079] Oligosaccharides As used herein, the term "oligosaccharide" refers to a carbohydrate polymer containing at least three monosaccharide units, i.e., tri-, tetra-, penta-, hexa-, or higher oligosaccharides. Oligosaccharides may have a linear or branched structure, comprising monosaccharide units interconnected by interglycosidic bonds. Specifically, the oligosaccharide comprises a lactose residue at the reducing end and one or more naturally occurring monosaccharides of 5-9 carbon atoms, selected from aldoses (e.g., glucose, galactose, ribose, arabinose, xylose, etc.), ketoses (e.g., fructose, sorbose, tagatose, etc.), deoxysugars (e.g., rhamnose, fucose, etc.), deoxyaminosugars (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), uronic acids, and ketoalonic acids (e.g., N-acetylneuraminic acid). Preferably, the oligosaccharide is an HMO.

[0080] Human milk oligosaccharides (HMOs) Preferred oligosaccharides of the present disclosure are human milk oligosaccharides (HMOs).

[0081] The term "human milk oligosaccharide" or "HMO" as used herein refers to complex carbohydrates found in human breast milk. HMOs have a core structure comprising a lactose unit at the reducing end, which may be extended by one or more β-N-acetyl-lactosamine units and / or one or more β-lacto-N-biosyl units, and which may be substituted with α-L-fucopyranosyl and / or α-N-acetyl-neuraminoyl (fucosyl) moieties. For example, the structure of HMOs was disclosed by Xi Chen in Advances in Carbohydrate Chemistry and Biochemistry, Vol. 72, Chapter 4, 2015.

[0082] HMOs are either neutral or acidic. Non-acidic (or neutral) HMOs contain no sialic acid residues, while acidic HMOs contain at least one sialic acid residue in their structure. Non-acidic (or neutral) HMOs can be either fucosylated or non-fucosylated.

[0083] Examples of such neutral non-fucosylated HMOs include lacto-N-triose II (LNT-II), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), p-lacto-N-neohexaose (pLNnH), p-lacto-N-hexaose (pLNH), and lacto-N-hexaose (LNH).

[0084] Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2'FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3'FL), difucosyllactose (DFL or LDFT), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucopentaose (LNFP-II), lacto-N-difucopentaose (LNFP-III ... LNDFH-III, fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-p-lacto-N-hexaose I (FpLNH-I), fucosyl-p-lacto-N-neohexaose II (F-pLNnH II), and fucosyl-lacto-N-neohexaose (FLNnH).

[0085] Examples of acidic HMOs include 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), 3-fucosyl-3'-sialyllactose (FSL), 3'-sialyllacto-N-tetraose a (LST a), fucosyl-LST a (FLST a), 6'-sialyllacto-N-tetraose b (LST b), fucosyl-LST b (FLST b), 6'-sialyllacto-N-neotetraose (LST c), fucosyl-LST c (FLST c), 3'-sialyllacto-N-neotetraose (LST d), fucosyl-LST d (FLST d), sialyllacto-N-hexaose (SLNH), sialyllacto-N-neohexaose I (SLNH-I), sialyllacto-N-neohexaose II (SLNH-II), and disialyllacto-N-tetraose (DSLNT).

[0086] In the context described herein, the desired HMO is preferably an HMO comprising at least four monosaccharide units, such as LNT and / or LNnT. Furthermore, the desired HMO of the present disclosure is preferably an HMO comprising a lacto-N-triose II (LNT-II, GlcNAc(β1-3)Gal(β1-4)Glc) backbone. The term LNT-II backbone refers to an HMO structure in which the monosaccharides N-acetylglucosamine (GlcNAc), galactose (gal), and glucose (glc) are linked in the following configuration: GlcNAc(β1-3)Gal(β1-4)Glc, and are linked to at least one additional modification (e.g., an additional monosaccharide). An HMO having an LNT-II backbone may also be referred to as an LNT-II core HMO. In the context of the present disclosure, LNT-II itself is not considered an HMO having an LNT-II backbone. Preferably, the HMO consists of or comprises the following structure: Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc or Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc. Examples of HMOs comprising an LNT-II backbone are, for example, LNT, LNnT, LNnH, pLNnH, pLNH, LNH, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, LST a, LST b, LST c, LST d, FLST a, FLST b, FLST c, FLST d, FLNH-I, FLNnH-I, SLNH, and DSLNT.

[0087] In one embodiment of the present disclosure, the desired human milk oligosaccharide (HMO) is an HMO of only four monosaccharide units, such as LNT or LNnT.

[0088] In another embodiment of the present disclosure, the desired human milk oligosaccharide (HMO) is a five-monosaccharide HMO in which LNT or LNnT has been fucosylated or sialylated. Preferred five-monosaccharide HMOs are LNFP-V and LST-c.

[0089] When producing one or more specific desired HMOs, the synthesis process may also produce unwanted HMO species, which are described herein as HMO by-products. For example, when producing the desired HMO product LNnT, the synthesis process may also produce LNT-II and pLNnH (e.g., Figure 1 ), where LNT-II and pLNnH are considered as unwanted by-product oligosaccharides. The cells of the present disclosure can be considered as a cell purification system that can achieve the separation of the desired HMO product from the unwanted by-product oligosaccharides.

[0090] Receptor / precursor molecule The genetically engineered cells according to the present disclosure comprise one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases capable of producing a desired HMO from a precursor molecule (eg, a disaccharide or oligosaccharide, such as lactose or LNT-II).

[0091] As described herein, an acceptor oligosaccharide is a molecule that can serve as a substrate for a glycosyltransferase, an enzyme that transfers a glycosyl moiety from a glycosyl donor to a precursor molecule. If the precursor molecule is the first molecule modified by the glycosyltransferase, it is sometimes referred to as an initial substrate. The initial substrate molecule is preferably a disaccharide or a trisaccharide. Depending on the complexity of the oligosaccharide produced, other acceptor molecules can be produced within the cell. For example, LNT is an acceptor molecule for further production of LNFP-I, LNFP-II, LNFP-V, and LST-a, while LNnT is an acceptor molecule for further production of LNFP-III, LNFP-VI, and LST-c. The glycosyl donor is preferably a nucleotide-activated sugar, as described in the "Glycosyl Donor-Nucleotide-Activated Sugar Pathway" section. Preferably, the acceptor sugar is a precursor for the generation of more complex HMOs, also referred to as a precursor molecule.

[0092] The acceptor sugar or acceptor oligosaccharide can be an intermediate in the present fermentation process, the end product of a separate fermentation process using separate genetically engineered cells, or an enzymatically or chemically produced molecule.

[0093] In this context, the acceptor oligosaccharides used to produce the desired HMO are preferably lactose and / or LNT-II, which can be directly imported from the production medium or, in the case of LNT-II, can be produced from the initial precursor molecule lactose (e.g., the acceptor for β-1,3-N-acetylglucosaminyltransferase) (see Figure 1), or directly into the cell via the substrate transporters described herein. Preferably, the initial precursor molecule is supplied to a genetically engineered cell capable of producing, for example, LNT-II, LNT, LNnT, or more complex HMOs from the precursor molecule. Most commonly, the initial precursor is lactose, and the genetically engineered cell is capable of producing the intermediate precursors (acceptor oligosaccharides, such as LNT-II and LNnT or LNT) intracellularly. Alternatively, if the cell is capable of importing LNT-II, the initial precursor may also be LNT-II.

[0094] As an alternative to supplying the fermentation medium with the initial precursor molecules / substrates for the production of the desired HMOs, genetically engineered cells can be further modified to produce the initial substrates intracellularly (see, for example, WO 2015 / 150328).

[0095] Glycosyltransferase The genetically engineered cells according to the present disclosure comprise at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase.

[0096] The genetically engineered cells according to the present disclosure may comprise one or more additional recombinant nucleic acids encoding one or more recombinant and / or heterologous glycosyltransferases capable of transferring a glycosyl residue from a glycosyl donor to an acceptor oligosaccharide. Preferably, the additional glycosyltransferases enable the genetically engineered cells to synthesize LNT or LNnT from precursor molecules (e.g., lactose or LNT-II). In embodiments, the genetically engineered cells described herein comprise one or more additional recombinant nucleic acids encoding one or more recombinant and / or heterologous glycosyltransferases.

[0097] The additional glycosyltransferase is preferably selected from the group consisting of galactosyltransferases and glucosaminyltransferases.

[0098] The genetically modified cell according to the present disclosure further comprises at least one recombinant nucleic acid sequence encoding at least one glycosyltransferase capable of transferring a glycosyl residue from a glycosyl donor to the acceptor oligosaccharide, thereby synthesizing a human milk oligosaccharide product having at least four monosaccharide units.

[0099] The glycosyltransferase is preferably selected from galactosyltransferase, glucosaminyltransferase, N-acetylglucosaminyltransferase and N-acetylglucosaminyltransferase. In one aspect, the glycosyltransferase is selected from the β-1,4-galactosyltransferase or β-1,3-galactosyltransferase listed herein. In an embodiment, one or more glycosyltransferases are selected from β-1,3-N-acetylglucosaminyltransferase, β-1,4-galactosyltransferase and β-1,3-galactosyltransferase. In a preferred embodiment, the genetically engineered cell comprises β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase and is capable of producing LNnT. In another preferred embodiment, the genetically engineered cell comprises β-1,3-N-acetylglucosaminyltransferase and β-1,3-galactosyltransferase and is capable of producing LNT.

[0100] Furthermore, the genetically engineered cells may comprise one or more additional glycosyltransferases selected from the group consisting of enzymes having α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3 / 4-fucosyltransferase, α-1,4-fucosyltransferase, α-2,3-sialyltransferase, and α-2,6-sialyltransferase activities.

[0101] Typically, the glycosyl donor is a nucleotide-activating sugar or oligosaccharide, for example selected from glucose-UDP-GlcNAc, GDP-fucose, UDP-galactose (UDP-gal), UDP-glucose (UDP-glc), UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-glaNAc) and CMP-N-acetylneuraminic acid (CMP-Neu5Ac), preferably using UDP-Gal and / or UDP-GlcNAc.

[0102] The glycosyl donor is preferably synthesized by endogenous or recombinant pathways in the genetically engineered cells, but can also be added exogenously to the culture medium. Preferably, the glycosyl donor is a nucleotide activating sugar that is synthesized by the host cell using existing pathways (endogenous), where the existing pathways can be modified to increase the pool of the relevant nucleotide activating sugar, or synthesized by introducing a nucleotide sequence encoding an enzyme required to produce the relevant nucleotide activating sugar in the cell.

[0103] In the present disclosure, at least one functional enzyme capable of transferring a sugar moiety from a glycosyl donor to an acceptor oligosaccharide can be selected from galT and galTK. For example, these enzymes can be used to produce LNnT or LNT, respectively, starting from LNT-II as the acceptor oligosaccharide.

[0104] In a preferred embodiment, the genetically modified cell according to the present disclosure does not contain more than two or more than three recombinant nucleic acid sequences encoding glycosyltransferases capable of transferring a glycosyl residue from a glycosyl donor to an acceptor oligosaccharide, thereby synthesizing a human milk oligosaccharide product having at least four or five monosaccharide units. The one, two or three glycosyltransferase activities are preferably selected from the following activities.

[0105] Heterologous β-1,3-N-acetylglucosaminyltransferase β-1,3-N-acetylglucosaminyltransferase is any protein that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to lactose or other acceptor molecules via a β-1,3-linkage (see Figure 1 ). Preferably, the β-1,3-N-acetylglucosaminyltransferase used herein is not derived from the species of the genetically engineered cell, that is, the gene encoding the β-1,3-N-acetylglucosaminyltransferase is of heterologous origin.

[0106] Thus, in an embodiment, the genetically engineered cell comprises one or more recombinant nucleic acid sequences encoding β-1,3-N-acetylglucosaminyltransferase.

[0107] Non-limiting examples of β-1,3-N-acetylglucosaminyltransferases are listed in Table 3. β-1,3-N-acetylglucosaminyltransferase variants may also be useful, preferably such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to the amino acid sequence of any of the β-1,3-N-acetylglucosaminyltransferases in Table 3.

[0108] Table 3. List of β-1,3-N-acetylglucosaminyltransferases In an embodiment, the genetically engineered cell comprises a recombinant nucleic acid sequence encoding a β-1,3-N-acetylglucosaminyltransferase. In one embodiment, the recombinant nucleic acid sequence encodes a β-1,3-N-acetylglucosaminyltransferase or a functional homolog thereof, wherein the β-1,3-N-acetylglucosaminyltransferase comprises or is represented by SEQ ID NO: 11 (from Neisseria meningitidis). LgtA ), the amino acid sequence of its functional homologue has at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO: 11.

[0109] To produce LNnT or LNT using lactose as a substrate, β-1,3-N-acetylglucosaminyltransferase is used to form the LNT-II precursor. In an embodiment, the genetically engineered cell comprises a β-1,3-N-acetylglucosaminyltransferase gene or a functional homolog or fragment thereof to produce the intermediate LNT-II from lactose.

[0110] Some of the following examples utilize a heterologous β-1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis designated LgtA or a variant thereof.

[0111] Heterologous β-1,3-galactosyltransferase β-1,3-Galactosyltransferase is any protein that transfers the galactose of UDP-galactose to the N-acetylglucosamine moiety of an acceptor molecule via a β-1,3-linkage (see Figure 1 In some embodiments, the cells of the present disclosure comprise a β-1,4-glycosyltransferase and, optionally, a β-1,3-N-acetylglucosaminyltransferase. Preferably, the β-1,3-galactosyltransferase used herein is not derived from a genetically engineered cell species, i.e., the gene encoding the β-1,3-galactosyltransferase is of heterologous origin. As described herein, the acceptor molecule is an acceptor carbohydrate, such as LNT-II or a more complex HMO structure.

[0112] The following examples use a heterologous β-1,3-galactosyltransferase named GalTK or a variant thereof to produce LNT.

[0113] β-1,3-Galactosyltransferase can be obtained from any of a variety of sources, for example, from Helicobacter pylori. galTK gene (homologous to GenBank protein accession number BD182026.1), or from Escherichia coli 055:H7 Wlq gene (GenBank accession number WP_000582563.1), or from Helicobacter pylori jhp0563 gene (GenBank accession number AEZ55696.1).

[0114] In one embodiment, the recombinant nucleic acid sequence encodes a β-1,3-galactosyltransferase or a functional homolog thereof, wherein the β-1,3-galactosyltransferase comprises or consists of the amino acid sequence of SEQ ID NO: 12 (galTK from Helicobacter pylori), and the amino acid sequence of its functional homolog is at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% identical to SEQ ID NO: 12.

[0115] To produce LNT from the LNT-II precursor, a β-1,3-galactosyltransferase is required. In one embodiment, the genetically modified cell comprises a β-1,3-galactosyltransferase gene or a functional homolog or fragment thereof.

[0116] In a further embodiment, the genetically engineered cell described herein comprises a β1,3-N-acetylglucosaminyltransferase or a functional homolog thereof having an amino acid sequence according to SEQ ID NO: 11, wherein the amino acid sequence of the functional homolog is at least 80% identical to SEQ ID NO: 11; and a β-1,3-galactosyltransferase or a functional homolog thereof having an amino acid sequence according to SEQ ID NO: 12, wherein the amino acid sequence of the functional homolog is at least 80% identical to SEQ ID NO: 12.

[0117] The following are examples of genetically modified strains described herein that possess specific combinations of glycosyltransferases that allow for the production of LNT using either lactose or LNT-II as initial substrates.

[0118] In one embodiment, LgtA from Neisseria meningitidis is used in combination with galTK from Helicobacter pylori to produce LNT using lactose as the initial substrate.

[0119] In one embodiment, LNT is produced using galTK from Helicobacter pylori and LNT-II as the initial substrate.

[0120] Heterologous β-1,4-galactosyltransferase β-1,4-Galactosyltransferase is any protein that transfers the galactose of UDP-galactose to the N-acetylglucosamine moiety of an acceptor molecule via a β-1,4-linkage (see Figure 1 Preferably, the β-1,4-galactosyltransferase used herein is not derived from a genetically engineered cell species, i.e., the gene encoding the β-1,4-galactosyltransferase is of heterologous origin. The receptor molecule described herein is a receptor carbohydrate, such as LNT-II or a more complex HMO structure.

[0121] The following examples use heterologous β-1,4-galactosyltransferase GalT or its variants to produce LNnT. Therefore, in an embodiment, the genetically engineered cells contain one or more recombinant nucleic acid sequences encoding β-1,4-galactosyltransferase.

[0122] Non-limiting examples of β-1,4-galactosyltransferases are provided in Table 4. β-1,4-galactosyltransferase variants may also be useful, preferably such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to the amino acid sequence of any of the β-1,4-galactosyltransferases in Table 4.

[0123] Table 4. List of β-1,4-glycosyltransferases In the embodiments described herein, the β-1,3-N-acetylglucosaminyltransferase is from Neisseria meningitidis, and the β-1,3-galactosyltransferase and / or the β-1,4-galactosyltransferase are each from Helicobacter pylori.

[0124] In one embodiment, the recombinant nucleic acid sequence encodes a β-1,4-galactosyltransferase or a functional homolog thereof, wherein the β-1,4-galactosyltransferase comprises or consists of the amino acid sequence of SEQ ID NO: 13 (galT from Helicobacter pylori), and the amino acid sequence of its functional homolog has at least 80%, such as at least 85%, such as at least 90%, such as at least 95% or such as at least 99% sequence identity with SEQ ID NO: 13.

[0125] In order to produce LNnT from the LNT-II precursor, a β-1,4-galactosyltransferase is required. In one embodiment, the genetically engineered cell comprises a β-1,4-galactosyltransferase gene or a functional homologue or fragment thereof.

[0126] In an embodiment, the genetically engineered cells described herein comprise a β-1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis and a β-1,4-galactosyltransferase from Helicobacter pylori. In a further embodiment, the β-1,3-N-acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 11, or a functional homolog thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 11, and the β-1,4-galactosyltransferase has an amino acid sequence according to SEQ ID NO: 13, or a functional homolog thereof having an amino acid sequence at least 80% identical to SEQ ID NO: 13.

[0127] In one embodiment, LgtA from Neisseria meningitidis is used in combination with galT from Helicobacter pylori to produce LNnT using lactose as the initial substrate.

[0128] In one embodiment, LNnT is produced using galT from Helicobacter pylori and LNT-II as the initial substrate.

[0129] In one embodiment, LgtA from Neisseria meningitidis is used in combination with LgtB from Neisseria meningitidis to produce LNnT using lactose as an initial substrate.

[0130] In one embodiment, LNT-II is used as the initial substrate to produce LNnT using LgtB from Neisseria meningitidis.

[0131] α-1,2-fucosyltransferase An α-1,2-fucosyltransferase is a protein that catalyzes the transfer of fucose from a donor substrate (e.g., GDP-fucose) to an acceptor molecule via an α-1,2 linkage. Preferably, the α-1,2-fucosyltransferase used herein is not derived from a genetically engineered cell species, i.e., the gene encoding the α-1,2-fucosyltransferase is of heterologous origin. Table 4 lists non-limiting examples of α-1,2-fucosyltransferases. α-1,2-fucosyltransferase variants may also be useful, preferably such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, identity to one of the α-1,2-fucosyltransferases listed in Table 13.

[0132] Table 13. List of α-1,2-fucosyltransferases α-1,3-fucosyltransferase α-1,3-fucosyltransferase refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate (e.g., GDP-fucose) to an acceptor molecule via an α-1,3-linkage. Preferably, the α-1,3-fucosyltransferase used herein is not derived from a genetically engineered cell species, i.e., the gene encoding the α-1,3-fucosyltransferase is of heterologous origin. Table 5 provides non-limiting examples of α-1,3-fucosyltransferases. α-1,3-fucosyltransferase variants may also be useful, preferably such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, identity to one of the α-1,3-fucosyltransferases listed in Table 14.

[0133] Table 14. List of α-1,3-fucosyltransferases α-1,3 / 4-fucosyltransferase α-1,3 / 4-fucosyltransferase refers to a glycosyltransferase that catalyzes the transfer of fucose from a donor substrate (e.g., GDP-fucose) to an acceptor molecule via an α-1,3- or α-1,4-linkage. Preferably, the α-1,3 / 4-fucosyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding the α-1,3 / 4-fucosyltransferase is of heterologous origin. Table 6 provides non-limiting examples of α-1,3 / 4-fucosyltransferases. α-1,3 / 4-fucosyltransferase variants may also be useful, preferably such variants have at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, identity to one of the α-1,3 / 4-fucosyltransferases in Table 15.

[0134] Table 15. List of α-1,3 / 4-fucosyltransferases α-2,3-sialyltransferase α-2,3-sialyltransferase refers to a glycosyltransferase that catalyzes the transfer of sialic acid from a donor substrate (e.g., CMP-N-acetylneuraminic acid) to an acceptor molecule via an α-2,3-linkage. Preferably, the α-2,3-sialyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding the 2,3-sialyltransferase is of heterologous origin. Table 7 provides non-limiting examples of α-2,3-sialyltransferases. α-2,3-sialyltransferase variants may also be useful, preferably such variants having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, identity to an α-2,3-sialyltransferase in Table 16.

[0135] Table 16. List of α-2,3-sialyltransferases α-2,6-sialyltransferase α-2,6-sialyltransferase refers to a glycosyltransferase that catalyzes the transfer of sialic acid from a donor substrate (e.g., CMP-N-acetylneuraminic acid) to an acceptor molecule via an α-2,6-linkage. Preferably, the α-2,6-sialyltransferase used herein is not derived from a species of genetically engineered cells, i.e., the gene encoding the 2,6-sialyltransferase is of heterologous origin. Table 8 provides non-limiting examples of α-2,6-sialyltransferases. α-2,6-sialyltransferase variants may also be useful, preferably such variants having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, identity to an α-2,6-sialyltransferase in Table 17.

[0136] Table 17. List of α-2,6-sialyltransferases Glycosyl donor-nucleotide activated sugar pathway When implementing the disclosed method, a glycosyltransferase-mediated glycosylation reaction is preferably performed, wherein an activated sugar nucleotide serves as the glycosyl donor. Activated sugar nucleotides typically have a phosphorylated glycosyl residue attached to the nucleoside. Specific glycosyltransferases only accept specific sugar nucleotides. Therefore, the following activated sugar nucleotides are preferably involved in the glycosyl transfer: glucose-UDP-GlcNAc, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine (UDP-GlcNAc), GDP-fucose, and CMP-N-acetylneuraminic acid (CMP-Neu5Ac).

[0137] The genetically engineered cells described herein may comprise one or more pathways for producing a nucleotide activating sugar selected from the group consisting of glucose-UDP-GlcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, and CMP-N-acetylneuraminic acid.

[0138] In one embodiment of the present disclosure, the genetically engineered cells are capable of producing one or more of the above-mentioned activated sugar nucleotides via a de novo pathway. In this regard, the activated sugar nucleotides are produced by the cells in a stepwise reaction sequence starting from a simple carbon source (such as glycerol, sucrose, fructose, or glucose) under the action of enzymes involved in the de novo biosynthetic pathway of the corresponding sugar nucleotide (for a review of monosaccharide metabolism, see, for example, HH Freeze and AD Elbein: Chapter 4: Glycosylation Precursors, in Essentials of Glycobiology, 2nd Edition (Eds. A. Varki et al.), Cold Spring Harbour Laboratory Press (2009)).

[0139] The enzymes involved in the de novo biosynthesis pathway of activated sugar nucleotides may exist naturally in cells or be introduced into cells through gene technology or recombinant DNA technology, all of which are common knowledge to those skilled in the art.

[0140] In another embodiment, genetically engineered cells can utilize recycled monosaccharides for sugar nucleotides. In a recycling pathway, the monosaccharides produced by degrading oligosaccharides are phosphorylated by kinases and converted to nucleotide sugars by pyrophosphorylases. The enzymes involved in this process can be heterologous or native to the host cell.

[0141] For the production of fucosylated HMOs, the de novo GDP-fucose synthesis pathway is crucial to ensure sufficient GDP-fucose. The colanic acid gene cluster of Escherichia coli encodes selected enzymes involved in the de novo GDP-fucose synthesis ( gmd 、 wcaG 、 wxya 、 wcal 、 manB 、 manC ), and one or more genes downstream of GDP-L-fucose responsible for the production of the exopolysaccharide colanic acid (the major oligosaccharide of the bacterial cell wall) (e.g., wcaJ) can be deleted to prevent the conversion of GDP-fucose to colanic acid.

[0142] To produce sialylated oligosaccharides / HMOs, the genetically modified cells contain a biosynthetic pathway for the production of a sialic acid sugar nucleotide, such as CMP-N-acetylneuraminic acid, as a glycosyl donor for the sialyltransferase introduced into the genetically engineered cells. The genetically engineered cells can be provided, for example, by providing exogenous UDP-GlcNAc 2-epimerase (e.g., neuC from Campylobacter jejuni (GenBank AAK91727.1) or equivalents (e.g., (GenBank CAR04561.1), Neu5Ac synthase (e.g., (GenBank CAR04561.1) from Campylobacter jejuni), or Neu5Ac synthase (e.g., (GenBank CAR04561.1) from Campylobacter jejuni). neuB (GenBank AAK91726.1) or equivalent (e.g. Flavobacterium limnosediminis Sialic acid synthase (GenBank WP_023580510.1) and / or CMP-Neu5Ac synthetase (e.g., neuA from Campylobacter jejuni (GenBank AAK91728.1) or equivalents (e.g., Vibrio brasiliensis ( Vibrio brasiliensis ) CMP-sialic acid synthase, GenBank WP_006881452.1) has the ability to synthesize sialic acid.

[0143] β-galactosidase Host cells suitable for HMO production, such as E. coli, may contain an endogenous β-galactosidase gene or an exogenous β-galactosidase gene. For example, E. coli contains an endogenous lacZ For the purposes of the present disclosure, when producing HMOs, if lactose is used as the initial substrate for producing the desired HMOs, it is preferred that the genetically engineered cells do not express functional β-galactosidase to avoid lactose degradation. In an embodiment, lacZThe gene can be inactivated by completely or partially deleting the corresponding nucleic acid sequence from the bacterial genome, or by causing a mutation in the gene sequence, resulting in its inability to be transcribed, or even if transcribed, the transcript cannot be translated, or even if translated into a protein (i.e., β-galactosidase), the protein does not have the corresponding enzymatic activity. In this way, HMO-producing bacteria accumulate a larger intracellular lactose pool, which is beneficial for HMO production.

[0144] genetically engineered cells As used herein, the terms "genetically modified cells" and "genetically engineered cells" are used interchangeably. As used herein, "genetically modified cells" refer to cells whose genetic material has been altered by human intervention using genetic engineering techniques, such as, but not limited to, transformation or transfection, for example, using heterologous polynucleotide sequences, Crisper / Cas editing, and / or random mutagenesis. In one embodiment, the genetically engineered cells have been transformed or transfected with a recombinant nucleic acid sequence.

[0145] The genetically engineered cells are preferably prokaryotic cells, such as microbial cells. Suitable microbial cells that can be used as host cells include yeast cells, bacterial cells, archaeal cells, algae cells and fungal cells.

[0146] The genetically engineered cell (host cell) can be, for example, a bacterial cell or a yeast cell. In a preferred embodiment, the genetically engineered cell is a bacterial cell.

[0147] host cells In an embodiment, the engineered cell is a microorganism. The genetically engineered cell is preferably a microbial cell, such as a prokaryotic cell or a eukaryotic cell. Suitable microbial cells that can be used as host cells include bacterial cells, archaeal cells, algae cells, and fungal cells.

[0148] The genetically engineered cells can be, for example, bacteria or yeast cells. In a preferred embodiment, the genetically engineered cells are bacterial cells.

[0149] Regarding bacterial host cells, there are no limitations in principle; they can be eubacteria (Gram-positive or Gram-negative) or archaea, as long as they allow for the insertion of target genes through genetic manipulation and can be cultured for large-scale production. Preferably, the host cells have properties that allow high cell density culture. Non-limiting examples of bacterial host cells suitable for the recombinant industrial production of HMOs in the present disclosure can be Enterobacteriaceae ( Enterobacterales ) orders, preferably members of the genus Escherichia ( Escherichia ), more preferably Escherichia coli ( E. coli ) species. Other examples of suitable host cells include Erwinia herbicola ( Erwinia herbicola ) (Pantoea agglomerans ( Pantoea agglomerans))、Citrobacter freundii( Citrobacter freundii ), Campylobacter spp. ( Campylobacter sp )、Pantoea citrifolia( Pantoea citrea )、Carrot soft rot pectin bacteria( Pectobacterium carotovorum ) or Xanthomonas campestris ( Xanthomonas campestris Bacillus bacteria, including Bacillus subtilis ( Bacillus subtilis ), Bacillus licheniformis ( Bacillus licheniformis ), Bacillus coagulans ( Bacillus coagulans ), Thermophilic Bacillus ( Bacillus thermophilus )、Bacillus laterosporus( Bacillus laterosporus ), Bacillus megaterium ( Bacillus megaterium ), Bacillus mycoides ( Bacillus mycoides ), Bacillus pumilus ( Bacillus pumilus ), Bacillus lentus ( Bacillus lentus ), Bacillus cereus ( Bacillus cereus ) and Bacillus circulans ( Bacillus circulans Similarly, the methods of the present disclosure can be used to isolate Lactobacillus spp. Lactobacillus ) and Lactococcus spp. ( Lactococcus ) bacteria, including but not limited to Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus salivarius ( Lactobacillus salivarius )、Lactobacillus plantarum( Lactobacillus plantarum )、Lactobacillus helveticus( Lactobacillus helveticus ), Lactobacillus delbrueckii ( Lactobacillus delbrueckii ), Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus bulgaricus ( Lactobacillus bulgaricus )、Lactobacillus crispatus( Lactobacillus crispatus ), Lactobacillus gasseri ( Lactobacillus gasseri )、Lactobacillus casei( Lactobacillus casei ), Lactobacillus reuteri ( Lactobacillus reuteri ), Lactobacillus jensenii ( Lactobacillus jensenii ) and Lactococcus lactis ( Lactococcus lactis Streptococcus thermophilus ( Streptococcus thermophiles ) and Propionibacterium freudenreichii ( Proprionibacterium freudenreichii ) are also bacterial species suitable for use in the disclosure herein. Also useful species as part of this disclosure are strains engineered as described herein from the genus Enterococcus ( Enterococcus ) (e.g., Enterococcus faecium ( Enterococcus faecium ) and Enterococcus thermophilus (Enterococcus thermophiles ) ), Bifidobacterium spp. ( Bifidobacterium ) (e.g., Bifidobacterium longum ( Bifidobacterium longum ), Bifidobacterium infantis ( Bifidobacterium infantis ) and Bifidobacterium bifidum ( Bifidobacterium bifidum ))、Sporolactobacillus( Sporolactobacillus spp. ), Micromonospora ( Micromomosporaspp. ), Micrococcus ( Micrococcus spp .), Rhodococcus ( Rhodococcus spp. ) and Pseudomonas ( Pseudomonas ) (e.g., Pseudomonas fluorescens ( Pseudomonas fluorescens ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa )).

[0150] Non-limiting examples of fungal host cells suitable for recombinant industrial production of heterologous products include yeast cells, e.g. Komagataella 、Kluyveromyces ( Kluyveromyces )、Yarrowia ( Yarrowia )、Pichia pastoris( Pichia ), Saccharomyces ( Saccaromyces ), fission yeast ( Schizosaccharomyces ) or Hansenula ( Hansenula ), or from Aspergillus spp. ( Aspargillus ), Fusarium spp. ( Fusarium ) or Trichoderma ( Thricoderma ) of filamentous fungi.

[0151] In one or more exemplary embodiments, the genetically engineered cells are selected from the group consisting of Escherichia, Bacillus, Lactobacillus, Corynebacterium ( Corynebacterium ) and Campylobacter spp.

[0152] In one or more exemplary embodiments, the genetically engineered cell is selected from Escherichia coli, Bacillus subtilis, Lactobacillus lactis, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

[0153] In one or more exemplary embodiments, the genetically engineered cell is Bacillus subtilis.

[0154] In one or more exemplary embodiments, the genetically engineered cell is Saccharomyces cerevisiae or Pichia pastoris.

[0155] In one or more exemplary embodiments, the genetically engineered cell is Escherichia coli.

[0156] In one or more exemplary embodiments, the present disclosure relates to a genetically engineered cell, wherein the cell is derived from Escherichia coli K-12 strain or DE3.

[0157] Recombinant nucleic acid sequence The present disclosure relates to a genetically engineered cell comprising a recombinant nucleic acid sequence encoding the transporter protein Edic1 or a functional variant thereof, wherein the transporter protein Edic1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1, the amino acid sequence of the functional variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 1, wherein the transporter protein coding sequence is under the control of a promoter sequence.

[0158] As used herein, the terms "recombinant nucleic acid sequence," "recombinant gene / nucleic acid / nucleotide sequence / DNA encoding..." or "coding nucleic acid sequence" are used interchangeably to refer to an artificial nucleic acid sequence (i.e., produced in vitro using standard laboratory methods for preparing nucleic acid sequences) that comprises a set of consecutive, non-overlapping triplets (codons) that can be transcribed into mRNA and translated into protein under the control of an appropriate control sequence, i.e., a promoter sequence.

[0159] The boundaries of the coding sequence are generally determined by the ribosome binding site, the transcription start codon (AUG, GUG, or UUG), and the translation stop codon (UAA, UGA, or UAG), located 5' upstream of the open reading frame at the mRNA end. Coding sequences may include, but are not limited to, genomic DNA, cDNA, synthetic, and recombinant nucleic acid sequences.

[0160] The term "nucleic acid" includes RNA, DNA and cDNA molecules. It is understood that due to the degeneracy of the genetic code, a large number of nucleic acid sequences encoding a particular protein may be generated.

[0161] The recombinant nucleic acid sequence can be a coding DNA sequence, such as a gene, or a non-coding DNA sequence, such as regulatory DNA, such as a promoter sequence or other non-coding regulatory sequence.

[0162] The recombinant nucleic acid sequence can also be heterologous. "Heterologous" as used herein refers to a polypeptide, amino acid sequence, nucleic acid sequence or nucleotide sequence that is foreign to a cell or organism, i.e., a polypeptide, amino acid sequence, nucleic acid molecule or nucleotide sequence that does not naturally occur in the cell or organism.

[0163] The present disclosure also relates to a nucleic acid construct comprising a coding nucleic acid sequence (i.e., a recombinant DNA sequence of a target gene (e.g., a transporter gene encoding Edic1)) and a non-coding regulatory DNA sequence (e.g., a promoter DNA sequence, such as a recombinant promoter sequence derived from a promoter sequence of the lac operon or the glp operon, or a promoter sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence), wherein the coding sequence and the promoter sequence are operably linked. It is understood that an endogenous or wild-type promoter sequence operably linked to a coding nucleic acid sequence to which it is not naturally linked is a recombinant promoter sequence.

[0164] The term "operably linked" refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. It refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter sequence is operably linked to a coding sequence if it stimulates or modulates transcription of the coding sequence in an appropriate host cell or other expression system.

[0165] Typically, promoter sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, ie, they are cis-acting.

[0166] In one exemplary embodiment, the nucleic acid construct of the present disclosure may be part of a vector DNA; in another embodiment, the construct is an expression cassette / cartridge that is integrated into the host cell genome.

[0167] Thus, the term "nucleic acid construct" refers to an artificially constructed nucleic acid fragment, particularly a DNA fragment, intended to be inserted into a target cell (e.g., a bacterial cell) to modify the expression of genes in the genome or the expression of genes / coding DNA sequences that may be contained in the construct. In some embodiments, the nucleic acid construct is a plasmid or expression cassette that is suitable for integration into the genome of the target cell / host cell.

[0168] Integration of the target nucleic acid construct (expression cassette) contained in the construct into the bacterial genome can be achieved by conventional methods, for example by using a linear cassette comprising flanking sequences homologous to a specific site on the chromosome, as described for the attTn7 site (Waddell CS and Craig NL, Genes Dev. (1988) Feb; 2(2): 137-49.); methods for genomic integration of nucleic acid sequences mediated by recombination via the Red recombinase function of bacteriophage λ 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 carrying the expression cassette, can be selected by marker genes, loss or gain of gene function, etc.

[0169] In one or more exemplary embodiments, the present disclosure relates to a recombinant nucleic acid sequence comprising a nucleic acid sequence as set forth in SEQ ID NO: 6, or a nucleic acid sequence having at least 70% identity to SEQ ID NO: 6, such as at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or 100% identity to SEQ ID NO: 6. The nucleic acid sequence set forth in SEQ ID NO: 6 encodes a protein, Edic1, having an amino acid sequence as set forth in SEQ ID NO: 1.

[0170] Preferably, the heterologous element, such as the Edic1 transporter, substrate importer and / or glycosyltransferase encoding sequence, is under the control of a promoter sequence selected from the promoter sequences having the nucleic acid sequence shown in Table 5.

[0171] Table 5 - Selected promoter sequences Promoter activity was assessed in the LacZ assay described below, using the PglpF promoter as a positive reference in the same assay. To compare activity between different assays, activity was calculated relative to the PglpF promoter, and ranges represent results from multiple assays.

[0172] The promoter can be of heterologous origin, can be a native promoter of a genetically engineered cell, or can be a recombinant promoter composed of a combination of heterologous and / or native elements.

[0173] One approach to increasing product yields may be to manipulate the production of an enzyme activity required for the production of the product, such as a glycosyltransferase or an enzyme involved in the glycosyl donor biosynthetic pathway.

[0174] One approach to this goal may be to increase the strength of the promoter driving expression of the desired enzyme. Promoter strength can be assessed using the lacZ enzyme assay, where β-galactosidase activity is measured as described above (e.g., see Miller JH et al., 2001). Experiments in molecular genetics , Cold Spring Harbor Laboratory Press, NY, 1972). Briefly, cells were diluted in Z buffer and permeabilized with sodium dodecyl sulfate (0.1%) and chloroform. The LacZ assay was performed at 30°C. After preheating the sample, 200 µl of o-nitrophenyl-β-galactosidase (4 mg / ml) was added to initiate the assay and 500 µl of 1 M Na2CO3 was added when the sample turned slightly yellow. Subsequently, the amount of o-nitrophenol released was determined by the change in optical density at 420 nm. Specific activity was expressed in Miller units (MU) [A420 / (min ml A600)] indicates. Regulatory elements with an activity greater than 10,000 MU are considered strong regulatory elements, regulatory elements with an activity less than 3,000 MU are considered weak regulatory elements, and regulatory elements between 3,000 MU and 10,000 MU are considered moderate regulatory elements. An example of a strong regulatory element is the PglpF promoter, which has an activity of approximately 14,000 MU; an example of a weak promoter is Plac, which has an activity of approximately 2300 MU under IPTG induction. In a preferred embodiment, expression of the nucleic acid sequence is controlled by a strong promoter selected from SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 23, and 24.

[0175] In an embodiment, expression of the nucleic acid sequence described herein is controlled by the PglpF (SEQ ID NO: 27) or Plac (SEQ ID NO: 36) promoter or PmglB_UTR70 (SEQ ID NO: 24) or PglpA_70UTR (SEQ ID NO: 25) or PglpT_70UTR (SEQ ID NO: 26) or variants thereof (e.g., the promoters shown in Table 5, in particular the PglpF_SD4 variant of SEQ ID NO: 22 or the Plac_70UTR variant of SEQ ID NO: 18, or the PmglB_70UTR variants of SEQ ID NOs: 15, 16, 17, 19, 20, 21, 23 and 24). Further suitable variants of the PglpF, PglpA_70UTR, PglpT_70UTR and PmglB_70UTR promoter sequences are described in WO2019 / 123324 and WO2020 / 255054, respectively (incorporated herein by reference).

[0176] In a preferred embodiment, the recombinant nucleic acid sequence is under the control of one or more promoters selected from PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 27, 36, 24, 25 and 26, respectively) and variants thereof.

[0177] Sequence identity As used herein, the term "sequence identity" describes the dependency of the pairwise comparisons between two amino acid sequences or between two nucleotide sequences (i.e., between a candidate sequence (e.g., sequence of the present invention) and a reference sequence (e.g., prior art sequence)). For purposes disclosed herein, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo / . Biol. 48: 443-453) implemented in the Needle program of the EMBOSS package (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later (available at https: / / www.ebi.ac.uk / Tools / psa / embossneedle / ). The parameters used are gap open penalty 10, gap extension penalty 0.5, and EBLOSUM62 (EMBOSS version 30 BLOSUM62) substitution matrix. The output of Needle labeled "identity" (obtained using the -nobrief option) is used as the percent identity. In general, sequence identity can be calculated as follows: (identical residues × 100) / (length of the aligned region).

[0178] For the purposes disclosed herein, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or higher. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and a DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of the Needle label "identity" (obtained using the -nobrief option) is used as percent identity. Sequence identity can generally be calculated as follows: (identical deoxyribonucleotides × 100) / (length of the alignment region).

[0179] Functional homologs Functional homologs or functional variants of the protein / nucleic acid sequences described herein are protein / nucleic acid sequences that have had their genetic code altered but retain their original function. Functional homologs can be obtained by mutagenesis or can be naturally occurring variants from the same or other species. Compared to the functionality of the protein / nucleic acid sequence, functional homologs should have at least 50% residual functionality, e.g., at least 60%, 70%, 80%, 90%, or 100%.

[0180] Functional homologs of any disclosed amino acid or nucleic acid sequence may also have enhanced functionality. Functional homologs of any amino acid sequence or recombinant nucleic acid disclosed herein should ideally be able to participate in the production of a desired HMO by exporting the desired HMO product out of the cell, participating in the synthesis of a desired HMO or a precursor for producing the same HMO, or facilitating the input of a substrate for HMO production, such as an acceptor oligosaccharide of at least three monosaccharide units, and optimally, simultaneously increasing the yield of the desired HMO, or by reducing the formation of byproducts, reducing biomass formation, increasing the viability of the engineered cell, increasing the robustness of the engineered cell according to the disclosure, or facilitating the reduction of consumables required for production.

[0181] Uses of genetically engineered cells or transporters The present disclosure also relates to any commercial use of the transporter Edic1, genetically engineered cells, or nucleic acid constructs disclosed herein, such as, but not limited to, methods for producing a desired HMO (HMO product). Therefore, the present disclosure also relates to the use of the nucleic acid constructs according to the present disclosure in host cells that produce HMOs comprising a GlcNAc(β1-3)Gal(β1-4)Glc backbone and at least one additional glycosyl moiety, such as a Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc structure.

[0182] In other embodiments, the genetically engineered cells and / or nucleic acid constructs described herein are used to produce HMOs, preferably HMOs with a lacto-N-triose II (LNT-II, GlcNAc(β1-3)Gal(β1-4)Glc) backbone, such as LNT and / or LNnT.

[0183] In an exemplary embodiment, the genetically modified cells and / or nucleic acid constructs according to the present disclosure are used to produce one or more HMOs, wherein the HMO is selected from lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), lacto-N-fucopentaose V (LNFP-V) and 6'-sialylacto-N-neotetraose (LST c).

[0184] In one or more exemplary embodiments, genetically engineered cells and / or nucleic acid constructs are used to produce LNTs.

[0185] In one or more exemplary embodiments, genetically engineered cells and / or nucleic acid constructs are used to produce LNnT.

[0186] In one or more exemplary embodiments, genetically engineered cells and / or nucleic acid constructs are used to produce LNFP-V.

[0187] In one or more exemplary embodiments, genetically engineered cells and / or nucleic acid constructs are used to produce a mixture of LNT and LNFP-V.

[0188] In one or more exemplary embodiments, genetically engineered cells and / or nucleic acid constructs are used to produce a mixture of LNT, LNFP-V, and LNDFH-II.

[0189] In one or more exemplary embodiments, genetically engineered cells and / or nucleic acid constructs are used to produce LST-c.

[0190] In one or more exemplary embodiments, genetically engineered cells and / or nucleic acid constructs are used to produce a mixture of LNnT and LST-c.

[0191] In one or more exemplary embodiments, genetically engineered cells and / or nucleic acid constructs are used to produce a neutral, non-fucosylated HMO selected from the group consisting of LNT, LNnT, LNH, pLNnH, LNnH, and pLNH-I.

[0192] In one or more exemplary embodiments, the genetically engineered cells and / or nucleic acid constructs are used to produce an HMO composed of four monosaccharide units, such as an HMO selected from LNT and LNnT, preferably LNnT.

[0193] Method for producing human milk oligosaccharides (HMOs) The present disclosure also relates to a method for producing a desired human milk oligosaccharide (HMO), comprising culturing a genetically modified cell according to the present disclosure. In one embodiment, the method comprises: a. providing a genetically engineered cell according to the present disclosure, culturing the genetically engineered cell in a culture medium under conditions that allow production of the HMO; and optionally c. recovering the HMO. Preferably, after production of the HMO, the desired HMO is recovered from the culture supernatant.

[0194] Accordingly, the present disclosure relates to a method for producing human milk oligosaccharides (HMOs), the method comprising culturing genetically engineered cells capable of producing the desired human milk oligosaccharides (HMOs), wherein the cells comprise: a. one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases, and b. a recombinant nucleic acid sequence encoding the transporter Edic1 or a functional variant thereof, wherein the transporter Edic1 comprises or consists of the amino acid sequence according to SEQ ID NO: 1, the amino acid sequence of the functional variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 1, wherein expression of the transporter in the cell results in export of the desired oligosaccharide from the cell.

[0195] In an embodiment, the desired HMO comprises a GlcNAc(β1-3)Gal(β1-4)Glc backbone and at least one additional sugar moiety, such as Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc or Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc. In an embodiment, the desired HMO is selected from lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), lacto-N-fucopentaose V (LNFP-V), and 6'-sialyllacto-N-neotetraose (LST c). Preferably, the desired HMO produced by the method is LNnT or LNT.

[0196] The disclosed methods are unique in that culturing the genetically engineered cells described herein can produce desired HMOs, with the majority of the desired HMOs present in the fermentation supernatant, while retaining the majority of unwanted HMO byproducts within the cells, thereby enabling more efficient purification of the desired HMO product. Thus, in embodiments, after culturing the cells as described herein, at least 90%, for example, at least 95%, of the total molar HMO content in the culture supernatant is LNnT. Optimally, all of the product produced during the culturing process is the desired product, but this is not always possible, and unwanted byproducts may also be produced within the cells. Therefore, it is particularly noteworthy that the disclosed methods can produce LNnT at least 88%, 89%, 90%, or, for example, at least 91% of the total HMO content produced by the cells. Because of such high yields of the desired product HMO, it is particularly advantageous when less than 10% of the total molar HMO content in the culture supernatant is byproduct HMOs (e.g., LNT-II). Preferably, at the end of culturing according to the methods, less than 0.1% of the total molar HMO content in the culture supernatant is pLNnH.

[0197] Thus, in embodiments, the molar ratio of LNnT:LNT-II produced in the method is at least 20:1, such as at least 25:1, such as at least 30:1, or such as at least 33:1.

[0198] In further embodiments, the molar ratio of LNnT:pLNnH produced in the method is at least 12:1, such as at least 25:1, such as at least 30:1, or such as at least 33:1, or such as at least 37:1.

[0199] In a further embodiment, at least 88%, such as at least 90% or such as at least 92% of the total molar content of HMOs produced in the culturing step according to the method is LNnT.

[0200] In a further embodiment, less than 12% of the total molar content of HMO produced in the culturing step according to the method is by-product HMO, such as less than 10% or such as less than 8% is LNT-II and pLNnH.

[0201] In a further embodiment, less than 7% of the total molar content of HMOs produced in the culturing step according to the method is pLNnH.

[0202] In further embodiments, the molar ratio of LNnT:pLNnH produced in the culturing step according to the method is at least 12:1, such as at least 15:1, such as at least 20:1 or such as at least 21:1.

[0203] In further embodiments, the molar ratio of LNnT:LNT-II produced in the culturing step according to the method is at least 20:1, such as at least 25:1, such as at least 30:1, or such as at least 33:1.

[0204] Of particular note, the molar ratio of LNnT:LNT-II in the supernatant (as shown in Example 1 for Edic1-expressing cells) exceeded 30:1, compared to approximately 7:1 for the prior art transporter Vag. Therefore, cells expressing Edic1 and producing LNnT were superior in specifically transporting the produced LNnT out of the cell. Furthermore, only trace amounts of pLNnH were detected in the fermentation supernatant.

[0205] Thus, in embodiments, after culturing the cells according to the methods, the molar ratio of LNnT:LNT-II in the fermentation supernatant is at least 20:1, such as at least 25:1, such as at least 30:1, such as at least 35:1, or such as at least 37:1.

[0206] In further embodiments, after culturing the cells according to the methods, the molar ratio of LNnT:pLNnH in the fermentation supernatant is at least 350:1, such as at least 500:1, such as at least 1000:1, or such as at least 2000:1.

[0207] In a further embodiment, after culturing the cells according to the method, less than 0.1% of the total molar content of the fermentation broth supernatant is pLNnH.

[0208] In another embodiment, the method of the present disclosure relates to culturing a genetically engineered cell disclosed herein capable of producing LNT, wherein at least 80%, such as at least 85%, or such as at least 90%, of the total molar content of HMOs produced in the culturing step according to the method is LNT.

[0209] In another embodiment, the methods of the present disclosure involve culturing a genetically engineered cell disclosed herein that is capable of producing a mixture of LNT and LNFP-V.

[0210] In another embodiment, the methods of the present disclosure involve culturing a genetically engineered cell disclosed herein that is capable of producing a mixture of LNnT and LST-c.

[0211] Cultivation / Fermentation Cultivation, cultivation, or fermentation ("fermenting," "fermentation") in a controlled bioreactor (used interchangeably herein) typically consists of: (a) a first phase of exponential cell growth in a carbon-supplied medium; and (b) a second phase of cell growth in a carbon-limited medium, where the carbon source is continuously added along with an acceptor oligosaccharide (e.g., lactose), allowing the formation of HMO products during this phase. Carbon (sugar) limitation refers to the phase of the fermentation process where the growth rate is kinetically controlled by the carbon (sugar) concentration in the culture broth, which in turn is determined by the rate at which carbon is added to the fermentor (sugar feed rate).

[0212] The terms "manufacturing," "manufacturing scale," "large-scale production," or "large-scale fermentation" are used interchangeably and, for the purposes of this disclosure, define fermentations with a minimum volume of 100 L, e.g., 1,000 L, 10,000 L, 100,000 L, or 200,000 L of culture broth. Generally, a "manufacturing-scale" process is defined as one capable of processing large quantities to produce sufficient quantities of the desired HMO product to meet the requirements of, for example, toxicity testing, clinical trials, and market supply in the case of therapeutic compounds or compositions. In addition to their large size, manufacturing-scale processes, compared to simpler laboratory-scale methods such as shake flask cultures, are characterized by the use of bioreactor (fermenter) technology systems equipped with agitation, aeration, nutrient feed, and monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, backpressure, etc.). To a large extent, the behavior of the system expressed in laboratory-scale methods, such as the shake flask, benchtop bioreactor, or deep-well formats described in the examples of this disclosure, allows for prediction of the system's behavior in the complex environment of a bioreactor.

[0213] There are no restrictions on suitable cell culture media for use during fermentation. The culture medium can be semi-defined, i.e., containing complex culture media compounds (e.g., yeast extract, soy peptone, casamino acids, etc.), or it can be chemically defined, devoid of any complex compounds. The carbon source can be selected from glucose, sucrose, fructose, xylose, and glycerol. In one or more exemplary embodiments, the culture medium is supplemented with one or more energy and carbon sources selected from glycerol, sucrose, and glucose. In other embodiments, lactose is added during culturing of the genetically engineered cells as a substrate for HMO formation.

[0214] In one or more exemplary embodiments, the culture medium comprises sucrose as the sole carbon and energy source. In further embodiments, at least one energy source is added to the culture medium. In embodiments, the at least one energy source is preferably selected from the group consisting of glucose, sucrose, fructose, xylose, glycerol, and combinations thereof. In one or more exemplary embodiments, the genetically engineered cells comprise one or more heterologous nucleic acid sequences encoding one or more heterologous polypeptides that enable the genetically engineered cells to utilize sucrose as the sole carbon and energy source.

[0215] In one embodiment, a precursor molecule for synthesizing the desired HMO is added during the culture of the genetically engineered cells as a substrate for HMO formation. In a further embodiment, lactose and / or LNT-II are added during the culture of the genetically engineered cells as a substrate for the formation of the desired HMO. In a preferred embodiment, lactose is added during the culture of the genetically engineered cells as a substrate for the formation of the desired HMO.

[0216] In one or more exemplary embodiments, the genetically engineered cell comprises a PTS-dependent sucrose utilization system further comprising the scrYA and scrBR operons described in WO 2015 / 197082 (incorporated herein by reference).

[0217] After carrying out the disclosed methods, the desired HMOs can preferably be collected from the supernatant of the cell culture or fermentation broth in a conventional manner. Preferably, the desired HMOs are collected from the supernatant, and at least 90%, such as at least 95%, such as at least 97%, of the desired HMOs produced in the method are collected, leaving only a small amount, such as less than 10%, such as less than 5%, such as less than 3%, of the desired HMOs produced by the cells in the precipitate fraction.

[0218] Recovery / Harvest Human milk oligosaccharides (HMOs) can be recovered from the entire fermentation broth, including the biomass / cells and the supernatant. Alternatively, the HMOs can be recovered from the supernatant or the biomass after separation of the biomass from the culture medium / supernatant.

[0219] In a preferred embodiment, human milk oligosaccharides (HMOs) can be recovered directly from the culture medium after export into the culture medium.

[0220] The terms "recovery" and "harvesting" are used interchangeably herein. Both "recovery" and "harvesting" refer to the collection of produced HMOs from the culture / fermentation broth after fermentation is complete. Example 1 demonstrates that the introduction of Edic1 into LNnT-producing cells results in a method for producing LNnT in which substantially all of the LNnT produced by the cells is exported from the cells into the cell culture medium. In one or more exemplary embodiments, harvesting comprises collecting the desired HMOs directly from the culture medium, i.e., after separation of the culture medium from the biomass. Therefore, it is preferred that the desired HMOs be harvested directly from the supernatant of the fermentation broth after separation of the biomass from the fermentation medium.

[0221] The separation of cells from the culture medium can be carried out using any method well known to those skilled in the art, such as centrifugation or filtration of any suitable type. The separation of cells from the culture medium can be carried out immediately after the fermentation broth is harvested, or at a later stage when the fermentation broth is stored under appropriate conditions.

[0222] After recovery from fermentation, the desired HMOs are available for further processing and purification.

[0223] HMOs can be purified according to methods known in the art, such as those described in WO2017 / 152918, WO2017 / 182965, or WO2015 / 188834. Purified HMOs can be used as nutraceuticals, pharmaceuticals, or for other purposes, such as research.

[0224] manufactured products The term "manufactured product" refers to a composition of one or more HMOs, or a mixture of HMOs, that is intended to become one or more product HMOs. Examples of requirements for marketing LNnT can be found, for example, in GRAS notification 895 and Example 2. Preferably, the product HMO is produced by the methods described herein using the genetically engineered cells described herein. In preferred embodiments, the manufactured product is LNnT. Therefore, the present disclosure also relates to LNnT, an HMO produced by the methods described herein. For example, Examples 1 and 2 demonstrate that when LNnT is produced according to the present disclosure, the level of the byproduct pLNnH can be significantly reduced.

[0225] The manufactured product may be a powder, composition, suspension or gel comprising one or more HMOs.

[0226] project 1. A genetically engineered cell capable of producing an HMO, wherein the cell comprises: a. one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases, and b. a recombinant nucleic acid sequence encoding the transporter Edic1 or a functional variant thereof, wherein the transporter Edic1 comprises or consists of the amino acid sequence according to SEQ ID NO: 1, the amino acid sequence of the functional variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 1, Wherein expression of said transporter in said cell results in export of the desired HMO from said cell.

[0227] 2. The genetically engineered cell according to item 1, wherein the HMO comprises a lacto-N-triose II (LNT-II, GlcNAc(β1-3)Gal(β1-4)Glc) backbone.

[0228] 3. The genetically engineered cell according to item 2, wherein the lacto-N-triose II backbone comprises at least one additional sugar moiety, such as a galactose (gal) moiety.

[0229] 4. The genetically engineered cell according to item 3, wherein the galactose (gal) moiety is in a β1-4 or β1-3 configuration.

[0230] 5. The genetically engineered cell according to any of the preceding items, wherein the LNT-II backbone is selected from lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), lacto-N-fucopentaose V (LNFP-V) and 6'-sialylacto-N-neotetraose (LST c).

[0231] 6. The genetically engineered cell according to any of the preceding claims, wherein at least 85%, such as at least 90%, such as at least 95% of the total molar content of HMOs produced by the cell is LNnT.

[0232] 7. The genetically engineered cell according to any of the preceding claims, wherein less than 15% of the total molar content of HMO produced by the cell is by-product HMO, for example, less than 15% of the total molar content of HMO produced by the cell is LNT-II.

[0233] 8. The genetically engineered cell according to any of the preceding claims, wherein less than 10% of the total molar content of HMO produced by the cell is by-product HMO, for example, less than 10% of the total molar content of HMO produced by the cell is LNT-II and pLNnH.

[0234] 9. The genetically engineered cell according to any preceding claim, wherein at least 85%, such as at least 90%, such as at least 95% of the total molar content of HMOs exported from the cell is LNnT.

[0235] 10. The genetically engineered cell of any preceding claim, wherein less than 10% of the total molar content of HMOs exported from the cell is by-product HMOs, for example, less than 10% of the total molar content of HMOs exported from the cell is LNT-II and pLNnH.

[0236] 11. The genetically engineered cell according to any one of items 1 to 5, wherein at least 80%, such as at least 85%, such as at least 90%, of the total molar content of HMOs produced by the cell is LNT.

[0237] 12. The genetically engineered cell according to any preceding claim, wherein the one or more glycosyltransferases comprise β-1,4-galactosyltransferase or β-1,3-galactosyltransferase, and optionally β-1,3-N-acetylglucosaminyltransferase.

[0238] 13. The genetically engineered cell according to item 12, wherein the β-1,3-N-acetylglucosaminyltransferase is derived from Neisseria meningitidis, and the β-1,4-galactosyltransferase is derived from Helicobacter pylori.

[0239] 14. The genetically engineered cell according to item 12, wherein the β-1,3-N-acetylglucosaminyltransferase is derived from Neisseria meningitidis, and the β-1,3-galactosyltransferase is derived from Helicobacter pylori.

[0240] 15. The genetically engineered cell according to any one of item 12 or 13, wherein the 1,3-N-acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 11 or a functional homolog thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 11, and the β-1,4-galactosyltransferase has an amino acid sequence according to SEQ ID NO: 13 or a functional homolog thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 13.

[0241] 16. The genetically engineered cell according to any one of item 12 or 14, wherein the 1,3-N-acetylglucosaminyltransferase has an amino acid sequence according to SEQ ID NO: 11 or is a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 11, and the β-1,3-galactosyltransferase has an amino acid sequence according to SEQ ID NO: 12 or is a functional homologue thereof, the amino acid sequence of which is at least 80% identical to SEQ ID NO: 12.

[0242] 17. The genetically engineered cell according to any of the preceding items, wherein the cell further comprises one or more additional glycosyltransferases selected from the group consisting of α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3 / 4-fucosyltransferase, α-2,3-sialyltransferase, and α-2,6-sialyltransferase.

[0243] 18. A genetically engineered cell according to any of the preceding items, wherein the cell further comprises a recombinant nucleic acid sequence encoding a second transporter.

[0244] 19. The genetically engineered cell of item 18, wherein the second transporter is an importin or a second exportin.

[0245] 20. The genetically engineered cell of item 18 or 19, wherein the second exporter is selected from MFS transporters known to export HMOs.

[0246] 21. The genetically engineered cell of items 18 to 20, wherein the second exporter is selected from an MFS transporter from Pantoea eutropha or Rosenbergia rapa, such as an MFS transporter vag having an amino acid sequence at least 85% identical to SEQ ID NO: 5, or an MFS transporter Nec having an amino acid sequence at least 85% identical to SEQ ID NO: 83.

[0247] 22. The genetically engineered cell of item 21, wherein the cell produces LNnT and the second transporter is a vag transporter.

[0248] 23. The genetically engineered cell of item 21, wherein the cell produces LNT and the second transporter is a Nec transporter.

[0249] 24. The genetically engineered cell of item 23, wherein less than 15% of the total molar content of HMO produced by the cell is by-product HMO, for example, less than 15% of the total molar content of HMO produced by the cell is LNT-II.

[0250] 25. The genetically engineered cell according to any preceding claim, wherein the cell further comprises a nucleic acid sequence encoding a recombinant lactose permease.

[0251] 26. The genetically engineered cell according to item 25, wherein the lactose permease is lacY and its amino acid sequence is as shown in SEQ ID NO: 14, or it is a functional homolog thereof, and the amino acid sequence of the functional homolog has at least 80% identity with SEQ ID NO: 14.

[0252] 27. The genetically engineered cell according to any one of items 1 to 23, wherein the cell comprises a recombinant nucleic acid sequence encoding an importin selected from Table 1 or Table 2, preferably, the importin imports LNT-II as the initial substrate for HMO production.

[0253] 28. The genetically engineered cell according to any one of the preceding items, wherein the recombinant nucleic acid sequences are each controlled by a promoter selected from the group consisting of PglpF, Plac, PmglB_70UTR, PglpA_70UTR and PglpT_70UTR (SEQ ID NOs: 27, 36, 24, 25 and 26, respectively) and variants thereof.

[0254] 29. The genetically engineered cell according to item 28, wherein the promoter is a strong promoter selected from SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27.

[0255] 30. The genetically engineered cell according to any one of the preceding items, wherein the engineered cell is selected from Escherichia coli, Bacillus subtilis, Lactobacillus, Corynebacterium glutamicum, Yarrowia lipolytica, Pichia pastoris and Saccharomyces cerevisiae.

[0256] 31. A method of producing an HMO, wherein the method comprises: a. Providing a genetically engineered cell according to any one of items 1 to 30; b. culturing the genetically engineered cells in a culture medium under conditions that allow production of the HMO; and c. Optionally recovering the HMO.

[0257] 32. The method of item 31, wherein the method comprises culturing the genetically engineered cell in the presence of an energy source selected from the group consisting of glucose, sucrose, fructose, xylose, and glycerol.

[0258] 33. The method of item 31 or 32, wherein the HMO is recovered from the total culture broth of the culture after production of the HMO.

[0259] 34. The method according to items 31 to 32, wherein the HMO is recovered from the culture supernatant after production of the HMO.

[0260] 35. The method of any one of items 31 to 34, wherein the HMO product comprises a lacto-N-triose II (LNT-II, GlcNAc(β1-3)Gal(β1-4)Glc) backbone.

[0261] 36. The method according to any one of items 31 to 35, wherein the HMO product is selected from lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), lacto-N-fucopentaose V (LNFP-V) and 6'-sialylacto-N-neotetraose (LST c).

[0262] 37. The method of any one of items 31 to 36, wherein the HMO product is LNnT.

[0263] 38. The method according to any one of items 31 to 37, wherein at least 85%, such as at least 88%, such as at least 90% or such as at least 92% of the total molar content of HMOs produced in the culturing step according to the method is LNnT.

[0264] 39. The method according to any one of items 31 to 38, wherein of the total molar content of HMO produced in the culturing step according to the method, less than 12% is by-product HMO, such as less than 10% or such as less than 8% LNT-II and pLNnH.

[0265] 40. The method according to any one of items 31 to 39, wherein less than 7% of the total molar content of HMOs produced in the culturing step according to the method is pLNnH.

[0266] 41. The method of items 37 to 40, wherein the molar ratio of LNnT:pLNnH produced in the culturing step according to the method is at least 12:1, such as at least 15:1, such as at least 20:1, or such as at least 21:1.

[0267] 42. The method of any one of items 37 to 41, wherein the molar ratio of LNnT:LNT-II produced in the culturing step according to the method is at least 20:1, such as at least 25:1, such as at least 30:1, or such as at least 33:1.

[0268] 43. The method according to any one of items 31 to 37, wherein at least 90%, such as at least 95%, of the total molar content of HMOs in the culture supernatant is LNnT.

[0269] 44. The method according to any one of items 31 to 43, wherein less than 10% of the total molar content of HMO in the culture supernatant is by-product HMO, such as less than 10% LNT-II and pLNnH.

[0270] 45. The method according to any one of items 31 to 44, wherein less than 0.1% of the total molar content of HMOs in the culture supernatant is pLNnH.

[0271] 46. ​​The method of any one of items 37 to 45, wherein the method produces a supernatant having a molar ratio of LNnT:pLNnH of at least 350:1, such as at least 500:1, such as at least 1000:1, or such as at least 2000:1.

[0272] 47. The method of any one of items 37 to 46, wherein the method produces a supernatant having a molar ratio of LNnT:LNT-II of at least 20:1, such as at least 25:1, such as at least 30:1, such as at least 35:1, or such as at least 37:1.

[0273] 48. The method of any one of items 37 to 47, wherein at least 70% of the total molar content of total carbohydrates is LNnT.

[0274] 49. The method according to items 37 to 48, wherein less than 6% of the total molar content of total carbohydrates is pLNnH and less than 4% of the total molar content of total carbohydrates is LNT-II.

[0275] 50. The method according to any one of items 31 or 36, wherein at least 80%, such as at least 85% or such as at least 90% of the total molar content of HMOs produced in the culturing step according to the method is LNT.

[0276] 51. The method of any one of claims 31 or 36, wherein a mixture of LNT and LNFP-V is produced.

[0277] 52. The method of any one of claims 31 or 36, wherein a mixture of LNnT and LST-c is produced.

[0278] 53. The method according to any one of items 50 to 52, wherein less than 15% of the total molar content of HMOs produced in the culturing step according to the method is by-product HMOs, such as less than 15% LNT-II.

[0279] 54. The method according to any one of items 31 to 53, wherein during the cultivation of the genetically engineered cells, lactose and / or LNT-II are added as substrates for HMO formation.

[0280] 55. An HMO produced by the method according to any one of items 31 to 54.

[0281] 56. A nucleic acid construct comprising a recombinant nucleic acid sequence encoding the transporter Edic1 or a functional variant thereof, wherein the transporter Edic1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1, the amino acid sequence of the functional variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or such as at least 99% identity with SEQ ID NO: 1, wherein the transporter coding sequence is under the control of a promoter sequence.

[0282] 57. The nucleic acid construct according to item 56, wherein the nucleic acid construct is a plasmid or an expression cassette suitable for integration into a genome.

[0283] 58. Use of the nucleic acid construct according to item 56 in a host cell producing an HMO comprising a GlcNAc(β1-3)Gal(β1-4)Glc backbone and at least one additional glycosyl moiety, such as a Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc or a Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc structure.

[0284] sequence This application contains a Sequence Listing in both text and electronic formats, which is incorporated herein by reference.

[0285] A summary of the SEQ ID NOs used in this application is given in Table 18.

[0286] Table 18 - Sequences in this application Example method Unless otherwise indicated, nucleic acid manipulation, transformation, and expression all utilize standard techniques, vectors, regulatory sequence elements, and other expression system components known in the art of molecular biology. Such standard techniques, vectors, and components can be found in Ausubel et al. (eds.), Current Protocols in Molecular Biology (1995) (John Wiley&Sons); Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning (1989) (ColdSpring Harbor Laboratory Press, NY); Berger&Kimmel, Methods in Enzymology 152: Guide to Molecular Cloning Techniques (1987) (Academic Press); Bukhari et al (eds.), DNA Insertion Elements, Plasmids and Episomes (1977) (Cold Spring Harbor Laboratory Press, NY); Miller, JH Experiments in molecular genetics (1972.) (Cold spring Harbor Laboratory Press, NY).

[0287] The embodiments described below are only used to illustrate the present disclosure and are not intended to limit the present disclosure in any way.

[0288] Strain The strain (genetically engineered cell) constructed in this application is based on Escherichia coli K-12 DH1, and its genotype is: Fˉ, ʎ-, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44 The E. coli K-12 DH1 strain was additionally modified to generate an MDO strain with the following modifications: lacZ : 1.5 kbp deletion, lacA : 0.5 kbp deletion, nanKETA : 3.3 kbp deleted, melA : 0.9 kbp deleted, wxya : 0.5 kbp deletion, mxDV : 0.5 kbp missing and gmd Gene upstream insertion Plac promoter.

[0289] Methods for inserting a gene of interest into the E. coli genome are well known to those skilled in the art. Gene cassettes can be inserted into the E. coli chromosome using gene gorging techniques (see, for example, Herring and Blattner 2004 J. Bacteriol. 186:2673-81 and Warming et al. 2005 Nucleic Acids Res. 33(4):e36) in combination with specific selectable marker genes and screening methods.

[0290] This MDO strain was further engineered to generate an LNnT-producing strain by chromosomally integrating a β-1,3-GlcNAc transferase (LgtA from Neisseria meningitidis, homologous to NCBI accession number WP_033911473.1, as shown in SEQ ID NO: 11) and a β-1,4-galactosyltransferase (GalT from Helicobacter pylori, homologous to GenBank ID WP_001262061.1, as shown in SEQ ID NO: 13), both under the control of the PglpF promoter (SEQ ID NO: 27). This strain was designated the LNnT strain. To increase LNnT production and export in cells, the putative transporters listed in Table 6 were added to the LNnT strain, resulting in the strains described in Table 7.

[0291] Table 6. List of transporters tested within the framework of the present disclosure. The sequences used in this application may be truncated at the N-terminus or C-terminus compared to the GenBank sequences.

[0292] 1 Previously disclosed in WO2022 / 157213 as a putative LNnT or LNT transporter 2 LNT and LNnT transporters previously disclosed in WO2021 / 148611 3 2'FL, 3FL, LNT-II and LNT transporters previously disclosed in WO2021 / 148615 Codon-optimized DNA sequences encoding the respective transporters were genomically integrated into the LNnT strain.

[0293] The genotypes of the background strain (MDO), LNnT strain, and transporter-expressing strains are listed in Table 7 .

[0294] Table 7. Genotypes of transporter-expressing strains used in this example. 1 lgtA – three genomic insertions encoding β-1,3-N-acetylglucosaminyltransferase (SEQ ID NO: 11) The gene copy is controlled by the PglpF promoter (SEQ ID NO: 27).

[0295] 2 galT – a genomic insertion encoding the gene for β-1,4-galactosyltransferase (SEQ ID NO: 13), Under the control of the PglpF promoter (SEQ ID NO: 27).

[0296] 3 galTK – Two genomic insertions encoding β-1,3-galactosyltransferase (SEQ ID NO: 12) Therefore, it is controlled by the PglpF promoter (SEQ ID NO: 27).

[0297] 4 scrYA, scrBR - PTS-dependent sucrose utilization system, consisting of two operons: from Klebsiella pneumoniae scrYA and from Salmonella enterica subsp. enterica serovar Typhimurium) scrBR, as described in WO2015 / 197082, is driven by the Pscr-PglpF_SD1 dual promoter. (SEQ ID NO: 87+38) and Pscr promoter (SEQ ID NO: 87).

[0298] 5 lacY – an additional genomic integrated copy of lacY (SEQ ID NO: 14), driven by the PglpF promoter (SEQ ID NO: 27).

[0299] 6 The nec gene, encoding a heterologous major facilitator superfamily (MFS) transporter (SEQ ID NO: 84), is regulated by PglpF promoter (SEQ ID NO: 27).

[0300] 7 futA_mut2 – two independent sequences encoding a FutA α-1,3-fucosyltransferase variant (SEQ ID NO: 85). The genomic copy is under the control of the PglpF promoter (SEQ ID NO: 27).

[0301] 8 neuA is a gene encoding CMP-Neu5Ac synthase from Campylobacter jejuni (GenBank AAK91728.1). The genomic copy is under the control of the PglpF promoter (SEQ ID NO: 27).

[0302] 9 neuB is a gene encoding Neu5Ac synthase from Campylobacter jejuni (GenBank AAK91726.1). Group copy, under the control of the PglpF promoter (SEQ ID NO: 27).

[0303] 10 neuC encodes the gene encoding Campylobacter jejuni UDP-GlcNAc 2-epimerase (GenBank AAK91727.1). One genomic copy of the gene is under the control of the PglpF promoter (SEQ ID NO: 27).

[0304] 11 HAC1268 - a genomic insertion encoding an α-2,6-sialyltransferase (SEQ ID NO: 86) Therefore, it is controlled by the PglpF promoter (SEQ ID NO: 27).

[0305] Deep well assay The deep-well assay in this example was performed according to the original description by Lv et al. (Bioprocess Biosyst Eng 20 (2016) 39:1737-1747) and optimized for the purposes of the present invention. More specifically, the strains disclosed in this example were screened using a 4-day protocol in 96-well deep-well plates. Within the first 24 hours, the preculture was grown to high density (OD600 of 5) and then transferred to a medium permissive for inducible gene expression and product formation. Specifically, on day 1, a fresh preculture was prepared using basal minimal medium (BMM) (pH 7.0) supplemented with magnesium sulfate (0.12 g / L), thiamine (0.004 g / L), and glucose (5.5 g / L). The composition of BMM was as follows: NaOH (1 g / L), KOH (2.5 g / L), KH2PO4 (7 g / L), NH4H2PO4 (7 g / L), citric acid (0.5 g / L), and trace mineral solution (5 mL / L). Trace mineral stock solution contains: ZnSO~7H~O 0.82 g / L, citric acid 20 g / L, MnSO4 H2O 0.98 g / L, FeSO4 7H2O 3.925 g / L, CuSO4 5H2O 0.2 g / L. The pH of the minimal medium was adjusted to 7.0 with 5 N NaOH and autoclaved. The preculture was incubated at 34°C with shaking at 700 rpm for 24 hours before being transferred to 2 mL of fresh BMM (pH 7.5) to initiate the main culture. The new BMM was supplemented with magnesium sulfate (0.12 g / L), thiamine (0.02 g / L), a bolus of glucose solution (0.1-0.15 g / L), and a bolus of lactose solution (5-20 g / L). In addition, a 20% maltodextrin stock solution (19-20 g / L) was provided as a carbon source, and a specific hydrolase, glucoamylase, was added to release glucose at a rate suitable for carbon-limited growth, similar to a typical fed-batch fermentation process. The main culture was incubated at 28°C with shaking at 700 rpm for 96 hours. To analyze the supernatant fraction, the 24-well plate was centrifuged, the supernatant was collected, and then subjected to HPLC analysis. After removing the supernatant, to analyze the precipitate fraction, sterile MQ water was added to each well of the 24-well plate, then boiled at 100°C, followed by centrifugation, and finally the supernatant was subjected to HPLC analysis.

[0306] Fermentation Escherichia coli strains were cultured in 250 mL fermenters (Ambr250 HT bioreactor system, Sartorius) starting with 100 mL of minimal culture medium consisting of 30 g / L sucrose and minimal medium containing H₃PO₄, MgSO₄ · 7H₂O, KOH, citric acid, trace element solution, and thiamine. Dissolved oxygen levels were maintained at 20% using a cascade control system, initially providing agitation and then airflow at 1200 rpm (maximum 4500 rpm) and 1 VVM (maximum 3 VVM). The pH was maintained at 6.8 by titration with 8.5% NH₄OH solution. The culture was initiated with a 2% (v / v) inoculum from a preculture containing 10 g / L sucrose, NH₄H₂PO₄, KH₂PO₄, MgSO₄ · 7H₂O, KOH, NaOH, citric acid, trace element solution, and thiamine. After sucrose in the basal minimal medium was depleted, a feeding solution containing sucrose, MgSO4 x 7H2O, KOH, H3PO4, citric acid, antifoam, and trace mineral solution was continuously added to the fermentor at a rate to maintain carbon-limiting conditions. The temperature was initially 33°C but was linearly decreased to 27°C over a 5-hour period 20 hours after the start of feeding. Lactose was added as a 25% lactose monohydrate solution via repeated bolus injections after the start of feeding and every 32 hours thereafter to prevent lactose from becoming rate-limiting. Cell growth, metabolic activity, and metabolic status were tracked by online measurements of agitation, dissolved oxygen tension, reflectivity, NH4OH base addition, O2 uptake rate, and CO2 evolution rate. Samples were collected throughout the fermentation and the concentrations of HMO products, lactose, and other minor byproducts were determined using high-performance liquid chromatography (HPLC).

[0307] Example 1 - Production of LNnT by fermentation Genetically engineered cells expressing each transporter were fermented and evaluated for their ability to increase LNnT production while reducing the levels of LNT-II and pLNnH byproducts.

[0308] Some of the expected advantages of introducing a heterologous LNnT transporter include: 1) increased LNnT production; 2) increased LNnT export from the cells to the supernatant; 3) reduced byproduct formation; and 4) reduced byproduct HMO export to the supernatant. Exporting more LNnT and reducing the production and / or transport of byproducts (e.g., LNT-II and pLNnH) is preferred because it allows for the initial separation of LNnT from byproducts during the culture step, greatly simplifying post-fermentation purification.

[0309] To evaluate the capabilities of the heterologous transporters described herein, the strains were tested in fermentation as described above in the "Methods" section.

[0310] The total HMO content (precipitate and supernatant) produced by fermentation of LNnT production strains was analyzed, and the results are shown in Table 8 and Figure 5 Figure 5 shows the percentage of HMOs produced by each strain relative to the total amount of HMOs produced by the Vag strain.

[0311] Table 8. HMO production by each strain relative to the total HMO production by Vag strain As can be seen in Table 8, the total amount of HMO produced by strains expressing the Blon_2475, MdfA_CM, and MdfA_YR transporters relative to Vag was lower, at 35%, 47%, and 55%, respectively. This lower total amount was distributed to lower relative amounts of LNnT (30%, 41%, and 48%, respectively), slightly lower relative amounts of pLNnH (3%, 4%, and 5%, respectively), and lower relative amounts of LNT-II (2%, 2%, and 3%, respectively).

[0312] In contrast, the Edic1 strain was able to produce almost the same total amount of HMOs as the Vag strain, but the distribution of individual HMOs was different, with the relative amount of LNnT increased to 92%, compared to 82% produced by the Vag-expressing strain, and the relative amounts of pLNnH and LNT-II were lower, at 4% and 3%, respectively, compared to 11% and 8%, respectively, produced by the Vag strain.

[0313] To further evaluate the content of different HMOs produced by each strain, the HMO composition profile of each strain was calculated as a percentage of the total HMOs produced by each strain, as shown in Table 9.

[0314] Table 9. HMO composition profile of each strain relative to the total HMO produced by each strain. From the distribution of each HMO relative to the total HMO production in each strain in Table 9 , it can be seen that the total byproduct formation in the strains containing the Edic1, MdfA_CM, MdfA_YR, and Blon_2475 transporters was lower compared with the byproduct formation in the Vag strain, which resulted in a larger proportion of LNnT in the total HMO produced by the cells.

[0315] The results in Tables 8 and 9 indicate that Edic1 is superior to Vag in LNnT production, both in terms of yield (Table 8) and relative byproduct formation in cells (Table 9).

[0316] Tables 8 and 9 above analyze the distribution of HMOs in the total fermentation broth (precipitate and supernatant) during the fermentation. However, since this effect is achieved by adding transporters to the cells, it is also interesting to examine the distribution of HMOs in the supernatant only to determine how much of the produced LNnT ends up in the supernatant. The supernatants from fermentations of strains Vag and Edic1 were analyzed, as shown in Figure 5. Figure 6 Table 10 summarizes the relative amounts of each HMO in the supernatant of Edic1 and Vag strains relative to the total amount of HMOs exported by each strain.

[0317] Table 10. Individual HMOs extracted from the supernatant relative to the total HMOs produced by each strain. was determined from a single fermentation, whereas Edic1 is the average of two replicate fermentations.

[0318] HPLC chromatograms of the supernatants isolated from the two strains showed Figure 7 The pLNnH peak (9.153 min) is missing from the HPLC chromatogram of strain B (Edic). In addition, it can be seen that the LNT-II peak of strain Edic1 is smaller than that of strain Vag ( Figure 7 A).

[0319] The HMO distribution in Table 10 clearly demonstrates that the Edic1 strain is more specific in transporting the produced HMOs, as only 2.6% of the HMOs in the supernatant were LNT-II, and no pLNnH was found in the supernatant. Therefore, the Edic1 transporter has a low affinity for LNT-II and pLNnH, whereas the Vag strain exhibited a high concentration of 13.2% of the total byproduct HMOs in the supernatant. This demonstrates that LNnT-producing strains expressing the Edic1 transporter can produce highly pure LNnT in the supernatant fraction, which can be directly recovered from the fermentation broth.

[0320] also, Figure 6 The results showed that when the amount of each HMO in the supernatant was calculated relative to the total HMO in the supernatant of the Vag strain (% mM), the total HMO in the supernatant of the Edic1 strain was 2.6% higher than that of the Vag strain, and that of the LNnT strain was 13% higher than that of the Vag strain.

[0321] Another way to describe the distribution of the HMO by-product produced is by the ratio of the product produced (LNnT) to the HMO by-product produced.

[0322] Therefore, using the results in Tables 9 and 10 , the ratios of LNnT to the different byproduct HMOs (LNT-II and pLNnH) were calculated, either from the total culture fluid or from the separated supernatant, as shown in Table 11 .

[0323] Table 11. Ratio of product HMOs to by-product HMOs relative to the total amount of HMOs produced by each strain. The data were determined from a single fermentation, and the remaining data are the average results of two fermentations.

[0324] nd = not determined.

[0325] The ratios of LNnT to the byproduct HMOs LNT-II and pLNnH in Table 11 clearly demonstrate that Edic1 has a higher relative amount of LNnT in the total fermentation broth compared to Vag, MdfA_CM, MdfA_YR, and Blon_2475, with an LNnT:LNT-II ratio of 34:1 and an LNnT:pLNnH ratio of 22:1 for Edic1. These ratios are even higher when only the supernatant is examined, indicating that Edic1 prefers LNnT over LNT-II and pLNnH for transport.

[0326] Comprehensive this embodiment (Table 8-11 and Figure 5 and Figure 6 ), indicating that Edic1 is the preferred transporter expressed in LNnT-producing cells, where Edic1 is able to produce high-purity LNnT with highly preferred by-product properties without affecting LNnT yield, and LNnT can be collected directly from the fermentation broth supernatant, thereby greatly simplifying the purification process.

[0327] Example 2 - LNnT product and by-product distribution in commercial products The requirements set out in the product specifications define the limits for by-products in the final product (purified fermentation broth). Therefore, for a product like LNnT, it is desirable for the LNnT produced during fermentation to be essentially free of HMO by-products. This can have a positive impact on the cost of the final product by reducing the need for extensive post-fermentation (downstream) purification to remove unwanted by-products, significantly simplifying the production process and reducing costs.

[0328] To meet the specifications for commercially available non-crystalline LNnT, as outlined in GRAS Notice 895 and other regulations, the product must contain at least 80% LNnT. Furthermore, the specification stipulates a maximum lactose content of 10%, a maximum pLNnH content of 5%, and a maximum LNT-II content of 3% in the final product (see Table 13).

[0329] In Table 12, the content of HMO was calculated as a percentage of all carbohydrates in the total fermentation broth after fermentation in Example 2.

[0330] Table 12. Relative distribution of HMOs and major by-products in the total broth after fermentation. As shown in Table 12, by the end of fermentation in the strain expressing Edic1, LNnT accounted for 71% of the total carbohydrates, while in the Vag strain, LNnT accounted for 64% of the total carbohydrates. Furthermore, the Edic1 strain produced only 2% LNT-II and 5% pLNnH, which met the specifications of GRAS Notice 895.

[0331] In particular, the reduction of pLNnH has a high cost-saving potential because it is removed by chromatography, which is a time-consuming and expensive process. Figure 7 As shown, the pLNnH content can be reduced by separating the supernatant from the total fermentation broth. Therefore, the production of LNnT by strains carrying the Edic1 transporter has the potential to increase the productivity of the entire downstream purification process, especially the chromatography step, because the column flow rate can be increased when the amount of pLNnH to be removed is reduced.

[0332] The results in Table 12 clearly demonstrate the superiority of Edic1 for further purification, as the major byproducts, LNT-II and pLNnH, were within the specifications approved by regulatory authorities.

[0333] Example 3 - Production of LNT by fermentation Genetically engineered cells capable of producing LNT and expressing a specific transporter were fermented and evaluated for the production of LNT and the byproduct HMOs LNT-II and pLNH2.

[0334] The genotypes of the tested strains are shown in Table 6. Two replicate fermentations of the strains were performed as described above in the Methods section, with the following changes: 15 g / l sucrose was contained in 100 ml of the starting medium, and 25% lactose was added repeatedly every 16 h.

[0335] Since the Nec transporter was previously demonstrated in WO2021 / 148615, we chose this strain as a reference strain in order to improve the output and overall yield of LNT.

[0336] p-LNH2 (p-lacto-N-neohexaose-2) is an isomer / analog of p-lacto-N-hexaose naturally occurring in human milk. p-LNH2 is not officially an HMO, but because it is a common byproduct of LNT cell production, we include it in the analysis of total HMOs produced by the cells.

[0337] The total HMO content (precipitate and supernatant) produced by the fermentation of LNT production strains was analyzed, and the results are shown in Tables 19 and Figure 8 As shown, the HMO produced by each strain relative to the total HMO produced by the Nec strain is listed (percentage, %).

[0338] Table 19. HMO production by each strain relative to the total HMO production by Nec strain These data indicate that the Edic1 transporter increased the strain's total HMO production by at least 13%. When Edic1 was present alone, overall HMO production increased both LNT and the precursor / byproduct HMO LNT-II. Notably, the combination of Edic1 and the Nec transporter resulted in a further small increase in total HMO production compared to the reference strain harboring only the Nec transporter, but with a significant increase in LNT and a decrease in LNT-II.

[0339] Example 4 - Production of LNFP-V by fermentation Genetically modified cells capable of producing LNFP-V (with or without the Edic1 transporter) were fermented and evaluated for their effects on the production of LNFP-V and the LNT-II-based byproduct HMOs LNT, LNFP-II, and LNDFH-II.

[0340] The genotypes of the test strains are shown in Table 6. Two replicate fermentations were performed as described in the "Methods" section above, with the following modifications: The H₃PO₄ in the starting minimal medium was replaced with NH₄H₂PO₄ and KH₂PO₄, and NaOH was added. The MgSO₄·7H₂O, KOH, and H₃PO₄ in the feed solution were replaced with (NH₄)₂SO₄. The fermentation temperature was initially set at 33°C but was linearly decreased to 25°C over a period of 3 hours, starting 15 minutes after the start of the feed. Lactose was added as a single addition of a 25% lactose monohydrate solution 45 hours after the start of the feed.

[0341] The total HMO content (precipitate and supernatant) produced by fermentations of the LNFP-V production strains was analyzed to determine the presence of HMOs with an LNT-II backbone. The results are shown in Table 20, which lists the percentage (%) of each HMO produced by each strain relative to the total amount of LNT-II backbone HMOs produced by the LNFP-V strain without the transporter.

[0342] Table 20. HMO production by each strain relative to total HMO production by the LNFP-V strain without the transporter These data indicate that the Edic1 transporter enhances overall LNT-II core HMO production by 4000%. In particular, LNT production, as well as LNFP-V and LNDFH-II production, were significantly increased compared to a strain lacking the Edic1 transporter.

[0343] If one wishes to produce an HMO mixture containing LNT and a large number of the complex HMOs LNFP-V and LNDFH-II, Edic1 has been shown to be a beneficial transporter.

[0344] Example 5 - Production of LST-c in deep hole testing Genetically engineered cells capable of producing LST-c with and without the Edic1 transporter were compared to assess the role of Edic1 in LST-c production.

[0345] The genotypes of the tested strains are shown in Table 6. The strains were analyzed in duplicate using the deep well assay described above in the Methods section.

[0346] The total HMO content (precipitate and supernatant) in the deep well assay of the LST-c production strain was analyzed to determine the presence of HMO with the LNT-II backbone. The results are shown in Tables 21 and Figure 9 A and 9B, which lists the percentage (%) of each HMO produced by each strain relative to the total amount of LNT-II backbone HMO produced by the LST-c strain without the transporter.

[0347] Table 21. HMO production by each strain relative to total HMO production by the LST-c strain without the transporter These data demonstrate that the Edic1 transporter is able to export LST-c into the supernatant, increasing its relative level by 8% and increasing the total LNT-II backbone HMO content in the supernatant from 32% to 40%. As shown in Example 1, it was also shown that upon introduction of the Edic1 transporter into cells, the amount of LNnT remaining in the cell pellet (pellet) was significantly reduced, decreasing from 23% of the total LNT-II backbone HMO content in the cell pellet to 8%. Compared to cells without exporters, LNnT levels in the pellets of cells expressing Edic1 were reduced, but LNnT content in the supernatant fraction of these cells was not increased. Furthermore, cells expressing Edic1 produced and exported more LST-c than cells without exporters, a finding consistent with the aforementioned decrease in LNnT content in the pellets of Edic1-expressing cells without a concomitant increase in LNnT content in the supernatant.

Claims

1. A genetically engineered cell capable of producing an HMO having a lacto-N-triose II (LNT-II, GlcNAc(β1-3)Gal(β1-4)Glc) backbone, wherein the cell comprises: a. one or more recombinant nucleic acid sequences encoding one or more glycosyltransferases; and b. a recombinant nucleic acid sequence encoding a transporter protein Edic1 or a functional variant thereof, wherein the transporter protein Edic1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1, wherein the amino acid sequence of the functional variant thereof has at least 80% identity with SEQ ID NO: 1, Wherein expression of said transporter in said cell results in export of the desired HMO from said cell.

2. The genetically engineered cell according to claim 1, wherein the HMO having an LNT-II backbone is selected from lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), lacto-N-fucopentaose V (LNFP-V) and 6'-sialylacto-N-neotetraose (LST c).

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

4. The genetically engineered cell of any one of the preceding claims, wherein less than 15% of the total molar content of HMOs produced by the cell is by-product HMOs, for example, less than 15% of the total molar content of HMOs produced by the cell is LNT-II.

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

6. The genetically engineered cell according to any one of the preceding claims, wherein the engineered cell is selected from Escherichia coli ( Escherichia Coli ), Bacillus subtilis ( Bacillus subtilis ), Lactobacillus lactis ( lactobacillus lactis ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Yarrowia lipolytica ( Yarrowia lipolytica )、Pichia pastoris( Pichia pastoris ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

7. A method for producing an HMO product, wherein the method comprises: a. Providing a genetically engineered cell according to any one of claims 1 to 6, b. culturing the genetically engineered cells in a culture medium under conditions that allow production of the HMO; and c. Optionally recovering the HMO.

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

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

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

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

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

13. The method according to any one of claims 7 to 12, wherein less than 15% of the total molar content of HMOs produced in the culturing step according to the method are by-product HMOs, such as less than 15% LNT-II.

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

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

16. A nucleic acid construct comprising a recombinant nucleic acid sequence encoding a transporter protein Edic1 or a functional variant thereof, wherein the transporter protein Edic1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1, wherein the amino acid sequence of the functional variant thereof has at least 80% identity with SEQ ID NO: 1, wherein the transporter protein coding sequence is under the control of a recombinant promoter sequence.

17. Use of the nucleic acid construct according to claim 16 in a host cell producing an HMO comprising a GlcNAc(β1-3)Gal(β1-4)Glc backbone and at least one additional sugar moiety, such as a Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc or a Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc structure.

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