HMO Production

Abstract

The inventive concept relates to a genetically modified cell capable of producing oligosaccharides, preferably HMOs, comprising a recombinant nucleic acid encoding a putative transporter protein of the MFS superfamily; and a method of producing oligosaccharides (one or more), preferably HMOs, using said cell.

Description

Technical Field

[0001] The present invention relates to the field of recombinant production of biomolecules in host cells. More specifically, the present invention relates to methods for recombinant production of human milk oligosaccharides (HMOs) using genetically modified cells expressing major facilitator superfamily (MFS) proteins. Background Art

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component in human milk and are highly resistant to enzymatic hydrolysis. Therefore, a large portion of HMOs are essentially undigested and unabsorbed, which allows them to pass through the colon. In the colon, HMOs can serve as substrates for shaping the intestinal ecosystem by selectively stimulating the growth of specific sugar-decomposing bacteria. This selectivity is seen as beneficial for both infants and adults, as strains of bifidobacteria are seen as having a positive effect on intestinal health (Chichlowski M. et al., (2012) J. Pediatr. Gastroenterol. Nutr. 5: 251-258; Elison E. et al., (2016) Brit J. Nutr, 116: 1356-1368).

[0003] Besides their prebiotic properties, HMOs have additional positive effects, which expand their field of application (Kunz C. et al., (2014) Food Oligosaccharides: Production, Analysis and Bioactivity, 1st edition, pp. 5-20, Editors: Moreno J. and Luz Sanz M., John Wiley & Sons, Ltd).

[0004] The obvious health benefits of HMO have led to its approval for use in foods, such as infant formula, and consumer health products. The biotechnological production of HMO is a valuable, cost-effective way to manufacture HMO on a large scale. It relies on constructed genetically engineered bacteria to express the glycosyltransferases required to synthesize the desired oligosaccharides, and utilizes the inherent nucleotide sugar pool of bacteria as HMO precursors. The latest developments in the biotechnological production of HMOs make it possible to overcome certain inherent limitations of bacterial expression systems. For example, bacterial cells producing HMOs can be genetically modified to increase the limited intracellular nucleotide sugar pool in bacteria (WO2012112777), to increase the activity of enzymes involved in HMO production (WO2016040531), or to promote the secretion of synthesized HMOs into the extracellular medium (WO2010142305, WO2017042382). In addition, the expression of the target gene in the recombinant cell can be regulated by using a specific promoter or other gene expression regulator, such as recently described in WO2019123324.

[0005] The advantage of the method described in WO2010142305 and WO2017042382 is that it allows for a reduction in the metabolic burden on the producing cells through high levels of recombinant gene expression, for example using the methods of WO2012112777, WO201604531 or WO2019123324. This approach has attracted increasing attention in the engineering of recombinant HMO producing cells, for example, several new sugar transporter genes have recently been described that encode proteins and fermentation processes that facilitate the efflux of recombinantly produced 2'-fucosyllactose (2'-FL), the most abundant HMO in human milk (WO2018077892, US201900323053, US201900323052). However, there is currently no algorithm that can pinpoint the correct transporter capable of effluxing different recombinantly produced HMO structures among the numerous bacterial proteins predicted to have transport functions in multiple protein accessions (e.g., UniProt), because the structures / factors that define the substrate specificity of sugar transporters remain understudied and remain highly unpredictable. Summary of the invention

[0006] Identification of new and efficient sugar efflux transporters specific for different recombinantly produced HMOs and development of recombinant cells expressing said proteins would facilitate large-scale industrial HMO manufacturing.

[0007] The present invention provides a recombinant cell capable of producing human milk oligosaccharides (HMOs), wherein the cell expresses a heterologous gene encoding a putative MFS (major facilitator superfamily) transporter derived from the bacterium Rosenbergiella nectarea. More specifically, the present invention relates to a genetically modified cell optimized for the production of oligosaccharides, in particular HMOs, comprising a recombinant nucleic acid encoding a protein having at least 80% sequence similarity to the amino acid sequence of SEQ ID NO: 1. The amino acid sequence identified herein as SEQ ID NO: 1 is an amino acid sequence having 100% identity to the amino acid sequence having GenBank accession ID WP_092672081.1 (https: / / www.ncbi.nlm.nih.gov / proteinlWP_092672081.1). The MFS transporter having the amino acid sequence of SEQ ID NO: 1 is identified herein as interchangeably “Nec protein” or “Nec transporter” or “Nec”; the nucleic acid sequence encoding the Nec protein is identified herein as “Nec encoding nucleic acid / DNA” or “nec gene” or “nec”.

[0008] The present invention demonstrates that the use of HMO-producing recombinant cells expressing Nec proteins results in very significant improvements in the HMO manufacturing process associated with fermentation and purification of the HMOs. The recombinant cells and methods for HMO production disclosed herein provide higher yields of total produced HMOs, lower byproduct formation or byproduct to product ratios, lower biomass formation per fermentation, and facilitate recovery of HMOs during downstream processing of the fermentation broth.

[0009] Surprisingly, it was found that the expression of the DNA sequence encoding Nec in different HMO producing cells was associated with the accumulation of certain specific HMOs in the extracellular medium and other HMOs inside the producing cells, as well as an increase in the total HMO production. Surprisingly, it was found that the increased efflux of the produced HMOs was characteristic of HMOs consisting of three or four monosaccharide units, i.e. the HMOs were trisaccharides and tetrasaccharides, such as 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3-sialyllactose (3'-SL), 6-sialyllactose (6'-SL), lacto-N-triose 2 (LNT-2), lacto-N-neotetraose (LNnT) and lacto-N-tetraose (LNT), but not for larger oligosaccharide structures, such as pentasaccharides and hexasaccharides, which accumulated inside the producing cells. Surprisingly, it was also found that in the corresponding HMO production cells expressing nec, the total production of major HMOs, such as 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-triose (LNT II) and lacto-N-tetraose (LNT) was also increased, while in these cells, the production of byproducts, such as difucosyllactose (DFL), lacto-N-fucopentaose (LNFP V) or p-lacto-neohexaose-I (pLNH-I) was correspondingly often reduced, and the byproduct oligosaccharides typically accumulated in the production cells. In addition, it was very unexpected that the expression of the Nec protein in HMO production cells led to a reduction in the formation of biomass during fermentation and to healthier cell cultures, which was reflected in the reduction in the number of dead cells at the end of the fermentation, which made the manufacturing process more efficient because more product was produced per biomass unit.

[0010] Thus, a first aspect of the present invention relates to a genetically modified cell capable of producing one or more HMOs, wherein the cell comprises a protein encoding SEQ ID NO: 1 ( Figure 6 ) or a recombinant nucleic acid of a protein or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity with SEQ ID NO: 1, preferably at least 85% identity, more preferably at least 90% identity.

[0011] A second aspect of the present invention relates to a nucleic acid construct comprising a nucleic acid sequence encoding an MFS transporter protein, wherein the nucleic acid sequence encoding the protein has at least 70%, such as at least 80%, such as at least 85%, such as at least 95%, such as at least 99% sequence identity with SEQ ID NO: 2, and also relates to a genetically modified cell comprising the nucleic acid construct, which is Escherichia coli.

[0012] In one aspect, the nucleic acid construct comprises a nucleic acid sequence(s) encoding a MFS transporter, wherein the nucleic acid sequence is at least 70% identical to SEQ ID NO:2.

[0013] A third aspect of the present invention relates to a method for producing one or more oligosaccharides, the method comprising the steps of:

[0014] (i) providing a genetically modified cell capable of producing HMO, wherein the cell comprises a recombinant nucleic acid encoding a protein of SEQ ID NO: 1 or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity to SEQ ID NO: 1;

[0015] (ii) culturing the cell according to (i) in a suitable cell culture medium to express the recombinant nucleic acid;

[0016] (iii) harvesting the one or more HMOs produced in step (ii).

[0017] The present invention also relates to the use of a genetically modified cell or a nucleic acid construct comprising a heterologous nucleic acid sequence encoding a major facilitator superfamily (MFS) protein in the production of one or more human milk oligosaccharides (HMOs), wherein the nucleic acid sequence has at least 70% sequence identity with the sequence SEQ ID NO: 2.

[0018] As described above, during the cultivation of genetically modified cells capable of producing one or more HMOs, these cells contain nucleic acids encoding Nec transporters, surprisingly, it was found that the corresponding one or more HMOs were produced in high yields, while the formation of by-products and biomass was reduced. This facilitates the recovery of HMOs during downstream processes, for example, the overall recovery and purification process can contain fewer steps and the overall purification time can be shortened.

[0019] These effects of increasing product yield and facilitating product recovery make the present invention superior to prior art disclosures.

[0020] Further aspects and advantageous features of the invention will be described and illustrated in detail below by way of non-limiting embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 shows the relative production of 2'-FL by modified E. coli with and without overexpression of the MFS transporter of SEQ ID NO: 1 ( Figure 1A ), the relative distribution of 2'-FL inside and outside the cell ( Figure 1B ), the relative ratio of DFL to 2'-FL ( Figure 1C ) and relative optical density ( Figure 1D ).

[0022] Figure 2The relative distribution of 3-FL inside and outside the cell in modified E. coli with and without overexpression of the MFS transporter of SEQ ID NO: 1 is shown.

[0023] Figure 3 Relative LNT2 concentration percentages (%) in total, supernatant and pellet samples are shown for strains MP4002 and MP4039. While both strains showed optimal expression of the glycosyltransferase gene IgtA, only MP4039 expressed the heterologous transporter gene nec.

[0024] Figure 4 Relative LNT and byproduct concentration percentages (%) in total, supernatant and pellet samples are shown for strains MP4473 and MP4537. While both strains showed optimal expression of the glycosyltransferase genes IgtA and galTK, only MP4537 expressed the heterologous transporter gene nec.

[0025] Figure 5 The percentage (%) of strains MP2789 and MP4597 relative to the sum of the concentrations of LNFP-I, 2'-FL, LNT and HMO in the total sample is shown.While both strains showed optimal expression of the glycosyltransferase genes IgtA, galTK and futC, only MP4597 expressed the heterologous transporter gene nec.

[0026] Figure 6 The amino acid sequence of the Nec protein is shown (SEQ ID NO: 1). DETAILED DESCRIPTION

[0027] In the following, embodiments of the present invention will be described in further detail. Unless explicitly stated otherwise, each specific variation of a feature can be applied to other embodiments of the present invention.

[0028] In general, unless explicitly defined or otherwise stated, all terms used herein should be interpreted according to their ordinary meaning in the technical field and apply to all aspects and embodiments of the present invention. Unless explicitly stated otherwise, references to "a / an / the" [cell, sequence, gene, transporter, step, etc.] should be openly interpreted as referring to at least one example of the cell, sequence, gene, transporter, step, etc. Unless explicitly stated, the steps of any method disclosed herein do not have to be performed in the exact order disclosed.

[0029] The present invention generally relates to genetically modified cells for the efficient production of oligosaccharides and the use of the genetically modified cells in methods for producing oligosaccharides. In particular, the present invention relates to genetically modified cells capable of synthesizing oligosaccharides, preferably heterologous oligosaccharides, in particular human milk oligosaccharides (HMOs). Therefore, the cells of the present invention are modified to express a set of recombinant nucleic acids necessary for the cell to synthesize one or more HMOs (which enable the cell to synthesize one or more HMOs), such as genes encoding one or more enzymes having the following glycosyltransferase activity. The oligosaccharide-producing recombinant cells of the present invention are further modified to contain a heterologous recombinant nucleic acid sequence, preferably a DNA sequence encoding a putative MFS (major facilitator superfamily) transporter protein derived from the bacterium Rosenbergiella nectarea. More specifically, the present invention relates to a genetically modified cell optimized for the production of one or more specific oligosaccharides, in particular one or more specific HMOs, comprising a recombinant nucleic acid encoding a protein having at least 80% sequence similarity, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% sequence similarity to the amino acid sequence of SEQ ID NO: 1 ( Figure 6 ). The amino acid sequence identified herein as SEQ ID NO: 1 is an amino acid sequence that has 100% identity with the amino acid sequence having GenBank Accession ID: WP_092672081.1.

[0030] Thus, a first aspect of the present invention relates to a genetically modified cell capable of producing one or more HMOs, wherein the cell comprises a recombinant nucleic acid encoding a protein of SEQ ID NO: 1 or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity to SEQ ID NO: 1. In this context, the term "functional homologue" means a protein having an amino acid sequence of 80%-99.9% identity to SEQ ID NO: 1 and having a function that is beneficial to achieving at least one beneficial effect of the present invention, such as increasing the total HMO production of the host cell, promoting the recovery of the produced HMO(s), HMO production efficiency and / or the viability of the HMO producing cell.

[0031] The MFS transporter having the amino acid sequence of SEQ ID NO: 1 is identified herein interchangeably as “Nec protein” or “Nec transporter” or “Nec”; the nucleic acid sequence encoding the Nec protein is identified herein as “Nec encoding nucleic acid / DNA” or “nec gene” or “nec”.

[0032] The term "major facilitator superfamily (MFS)" refers to a large and unusually diverse family of secondary active transporters that are responsible for transporting a range of different substrates, including sugars, drugs, hydrophobic molecules, peptides, organic ions, etc. The specificity of sugar transporters is highly unpredictable, and the identification of novel transporters that are specific for, for example, oligosaccharides requires unburdened laboratory experiments (for more details, see the review by Reddy VS et al., (2012), FEBS J. 279(11): 2022-2035). The term "MFS transporter" in this context means a protein that facilitates the transport of oligosaccharides (preferably HMOs) across the cell membrane, preferably transporting HMOs / oligosaccharides synthesized by the host cell from the cell cytosol to the cell medium, preferably HMOs / oligosaccharides containing three or four sugar units (e.g., 2'-FL, 3-FL, LNT-2, LNT, LNnT, 3'-SL or 6'-SL). Additionally, or alternatively, MFS transporters may also facilitate the efflux of molecules that are not considered HMOs or oligosaccharides according to the invention, such as lactose, glucose, cellular metabolites or toxins.

[0033] In the context of two or more nucleic acid or amino acid sequences, the term "% sequence identity" means that when the maximum correspondence comparison and alignment is performed on a comparison window or a specified nucleic acid or amino acid sequence, the two or more sequences have common nucleotides or amino acid residues in a given percentage (i.e., the sequences have at least 90% (%) identity). The percent identity of nucleic acid or amino acid sequences can be measured using the BLAST 2.0 sequence comparison algorithm with default parameters, or by manual alignment and visual inspection (see, e.g., http: / / www.ncbi.nlm.nih.gov / BLAST / ). This definition also applies to the complementary sequence of the test sequence and sequences with deletions and / or additions, as well as sequences with substitutions. An example of an algorithm suitable for determining percent identity, sequence similarity, and alignment is the BLAST 2.2.20+ algorithm, which is described in Nucl. Acids Res. 25, 3389 (1997) by Altschul et al. BLAST 2.2.20+ is used to determine the sequence identity percentage of nucleic acid of the present invention and protein.Software for BLAST analysis can be publicly obtained through National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).The example of sequence alignment algorithm is CLUSTALO mega (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ), EMBOSS Needle (http: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ), MAFFT (http: / / mafft.cbrc.jp / alignment / server / ) or MUSCLE (http: / / www.ebi.ac.uk / Tools / msa / muscle / ).

[0034] In the context of the present invention, the term "oligosaccharide" means a sugar polymer containing multiple monosaccharide units. In some embodiments, preferred oligosaccharides are sugar polymers composed of three or four monosaccharide units, ie trisaccharides or tetrasaccharides. Preferred oligosaccharides of the present invention are human milk oligosaccharides (HMOs).

[0035] The term "human milk oligosaccharide" or "HMO" herein means a complex carbohydrate found in human milk (see Urashima et al.: Milk Oligosaccharides., Nova Biomedical Books, 2011; Chen, Progress in Carbohydrate Chemistry and Biochemistry, 72, 113 (2015)). HMO has a core structure comprising a lactose unit at the reducing end, which can be extended by one or more β-N-acetyl-lactosamine units and / or one or more β-lacto-N-biotinyl units, and the core structure can be substituted by α-L-ribofuranosyl and / or α-N-acetylneuraminoyl (sialyl) moieties. In this regard, non-acidic (or neutral) HMOs do not contain sialic acid residues, while acidic HMOs have at least one sialic acid residue in their structure. Non-acidic (or neutral) HMOs can be fucosylated or non-fucosylated. Examples of such neutral non-fucosylated HMOs include lacto-N-triose 2 (LNT-2), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexose (LNnH), p-lacto-N-neohexose (pLNnH), p-lacto-N-hexaose (pLNH), and lacto-N-hexaose (LNH). Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2'-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-I II), 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-neohexose II (F-pLNnHII) and fucosyl-lacto-N-neohexose (FLNnH).Examples of acidic HMOs include 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl-3'-sialyllactose (FSL), 3'-O-sialyllacto-N-tetraose a (LST a), fucosyl-LST a (FLST a), 6'-O-sialyllacto-N-tetraose b (LST b), fucosyl-LST b (FLST b), 6'-O-sialyllacto-N-neotetraose (LST c), fucosyl-LST c (FLST c), 3'-O-sialyllacto-N-neotetraose (LST d), and fucosyl-LST d (FLST d). d), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I (SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II) and disialyl-lacto-N-tetraose (DSLNT). In the context of the present invention, lactose is not considered to be an HMO species.

[0036] In some embodiments of the invention, tri-HMOs and tetra-HMOs may be preferred, such as the trisaccharides 2'-FL, 3-FL, LNT-2, 3'-SL, 6'-SL and the tetrasaccharides DFL, LNT, LNnT, FSL.

[0037] In order to be able to synthesize one or more HMOs, the recombinant cells of the present invention comprise at least one recombinant nucleic acid encoding a functional enzyme with glycosyltransferase activity. The galactosyltransferase gene may be integrated into the genome of the host cell (via chromosomal integration), or alternatively, it may be contained in plasmid DNA and expressed as plasmid-borne. If two or more glycosyltransferases are required for the production of HMOs, such as LNT or LNnT, two or more recombinant nucleic acids encoding different enzymes with glycosyltransferase activity may be integrated into the genome and / or expressed from a plasmid, such as a β-1,3-N-acetylglucosaminyltransferase (a first recombinant nucleic acid encoding a first glycosyltransferase) combined with a β-1,3-galactosyltransferase (a second recombinant nucleic acid encoding a second glycosyltransferase) for the production of LNT, wherein the first and second recombinant nucleic acids may be chromosomally integrated or integrated on a plasmid independently of each other. In a preferred embodiment, both the first and second recombinant nucleic acids are stably integrated into the chromosome of the production cell; in another embodiment, at least one of the first and second glycosyltransferases is plasmid-borne. The protein / enzyme having glycosyltransferase activity (glycosyltransferase) can be selected in different embodiments from enzymes having the activity of: α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3 / 4-fucosyltransferase, α-1,4-fucosyltransferase, α-2,3-sialyltransferase, α-2,6-sialyltransferase, β-1,3-N-acetylglucosaminetransferase, β-1,6-N-acetylglucosaminetransferase, β-1,3-galactosyltransferase and β-1,4-galactosyltransferase. For example, the production of 2'-FL requires that the modified cell express an active α-1,2-fucosyltransferase; to produce 3-FL, the modified cell needs to express an active α-1,3-fucosyltransferase; to produce LNT, the modified cell needs to express at least two glycosyltransferases, a β-1,3-N-acetylglucosaminyltransferase and a β-1,3-galactosyltransferase; to produce 6'-SL, the modified cell must express an active α-2,6-sialyltransferase and a CMP-sialic acid synthesis pathway; to produce 3'-SL, the modified cell must express an active α-2,3-sialyltransferase and a CMP-sialic acid synthesis pathway. Some non-limiting embodiments of proteins with glycosyltransferase activity that can be encoded by a recombinant gene contained in a production cell can be selected from the non-limiting examples in Table 1.

[0038] Table 1

[0039]

[0040]

[0041]

[0042] One aspect of the present invention is to provide a nucleic acid construct comprising a heterologous nucleic acid sequence (one or more) encoding a protein capable of sugar transport, the protein being a major facilitator superfamily (MFS) protein as shown in SEQ ID NO:1, or a functional homolog thereof, whose amino acid sequence has at least 80% identity with SEQ ID NO:1, wherein the nucleic acid sequence encoding the MFS protein has at least 70% sequence identity with SEQ ID NO:2.

[0043] The term "heterologous nucleic acid sequence", "recombinant gene / nucleic acid / DNA coding" or "coding nucleic acid sequence" means an artificial nucleic acid sequence (i.e., a nucleic acid sequence prepared in vitro using standard laboratory methods) that contains a set of consecutive, non-overlapping triplets (codons) that are transcribed into mRNA and translated into a polypeptide when placed under the control of an appropriate control sequence (i.e., a promoter). The boundaries of the coding sequence are generally determined by a ribosome binding site, a transcription start codon (AUG, GUG or UUG), and a translation stop codon (UAA, UGA or UAG) located upstream of the 5' end of the open reading frame of the mRNA. Coding sequences may include, but are not limited to, genomic DNA, cDNA, synthetic and recombinant nucleic acid sequences. The term "nucleic acid" includes RNA, DNA and cDNA molecules. It should be understood that due to the degeneracy of the genetic code, a large number of nucleotide sequences encoding a given protein can be generated. The term nucleic acid can be used interchangeably with the term "polynucleotide". An "oligonucleotide" is a short-chain nucleic acid molecule. A "primer" is an oligonucleotide, whether naturally occurring, such as in a purified restriction digest, or synthetically produced, which is capable of serving as a starting point for synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand (i.e., in the presence of nucleotides and an inducing agent, such as a DNA polymerase, and at a suitable temperature and pH). For maximum efficiency in amplification, the primer is preferably single-stranded, but may also be double-stranded. If the primer is double-stranded, it is first treated to separate its strands before being used to prepare an extension product. Preferably, the primer is a deoxyribonucleic acid. The primer must be long enough to initiate the synthesis of an extension product in the presence of an inducing agent. The exact length of the primer depends on many factors, including temperature, primer source, and method of use.

[0044] The recombinant nucleic acid sequence of the present invention can be a coding DNA sequence, such as a gene, or a non-coding DNA sequence, such as a regulatory DNA, such as a promoter sequence. One aspect of the present invention relates to providing a recombinant cell comprising a recombinant DNA sequence encoding an enzyme required for producing one or more HMOs and a DNA sequence encoding a Nec transporter. Therefore, in one embodiment, the present invention 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 glycosyltransferase gene or a nec gene), and a non-coding DNA sequence, such as a promoter DNA sequence, such as a recombinant promoter sequence derived from a promoter of a lac operon or a 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 connected. The term "operably connected" refers to a functional relationship between two or more nucleic acids (e.g., DNA). Typically, it refers to a functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, if the promoter sequence stimulates or regulates the transcription of the coding sequence in a suitable host cell or other expression system, the promoter sequence is operably connected to the coding sequence. Promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically adjacent to the transcribed sequence, ie, they are cis-acting.

[0045] In one embodiment, the nucleic acid construct of the present invention may be part of a vector DNA, in another embodiment, the construct is an expression cassette / cassette integrated into the host cell genome. By the term "nucleic acid structure" is meant an artificially constructed nucleic acid fragment, particularly a DNA fragment, which is intended to be "transplanted" into a target cell, such as a bacterial cell, to modify the expression of a genomic gene or to express a gene / coding DNA sequence that may be included in the construct. In the context of the present invention, a nucleic acid construct comprises a recombinant DNA sequence containing two or more recombinant DNA sequences: essentially, a non-coding DNA sequence comprises a promoter DNA sequence and a coding DNA sequence encoding a target gene, such as a Nec protein, a glycosyltransferase, or another gene for producing HMOs in a host cell. Preferably, the construct further comprises a non-coding DNA sequence that regulates the transcription or translation of the construct encoding DNA, such as a DNA sequence that promotes ribosome binding to the transcript, a leader DNA sequence that stabilizes the transcript.

[0046] Integration of the desired recombinant nucleic acid contained in the construct (expression cassette) into the bacterial genome can be achieved by conventional methods, for example by using a linear cassette containing flanking sequences homologous to specific sites on the chromosome, as described for the attTn7-site (Waddell CS and Craig NL, Genes Dev. (1988) February; 2(2): 137-49.); methods for genomic integration of nucleic acid sequences, wherein recombination is mediated by the red recombinase function of bacteriophage λ or the RecE / RecT recombinase function of the Rac prophage (Murphy, Journal of Bacteriology (J Bacteriol.) 1998); 180(8): 2063-7; Zhang et al., Nature Genetics (Nature Genetics) (1998) s (1998) 20: 123-128 Muyrers et al., EMBO Reports (EMBO Rep.) (2000) 1(3):239-243); Red / ET recombination-based methods (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, for example, by loss or gain of marker gene or gene function.

[0047] According to the present invention, a single copy of the expression cassette comprising the gene of interest is sufficient to ensure the production of the desired HMO and achieve the desired effect. Thus, in some preferred embodiments, the present invention relates to recombinant HMO producing cells comprising one, two or three copies of the gene of interest integrated into the genomic DNA of the cell. In some embodiments, a single copy of the gene is preferred.

[0048] In a preferred embodiment, the recombinant encoding nucleic acid sequence of the nucleic acid construct of the present invention is heterologous with respect to the promoter, which means that it is transcribed under the control of another promoter sequence (i.e., not the promoter sequence of the construct) in an equivalent natural coding sequence in the genome of the species of origin. However, with respect to the host cell, the encoding DNA may be heterologous (i.e., derived from another biological species or genus), such as a DNA sequence encoding a Nec protein expressed in an E. coli host cell, or homologous (i.e., derived from the host cell), such as genes of the colic acid operon, the wca gene.

[0049] The term, "regulatory element" or "promoter" or "promoter region" or "promoter element" is a nucleic acid sequence that is identified and bound by a DNA-dependent RNA polymerase during the initiation of transcription. Promoters and other transcription and translation regulatory nucleic acid sequences (also referred to as "control sequences") are necessary for the expression of a given gene or genome (operon). In general, transcription and translation regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancer or activator sequences. "Transcription start site" means the first transcribed nucleotide, designated as +1. The nucleotides downstream of the start site are numbered +2, +3, +4, etc., while the nucleotides on the 5' opposite (upstream) are numbered -1, -2, -3, etc. The promoter DNA sequence of the construct can be derived from the promoter region of any gene in the genome of the selected species, preferably the promoter region of the Escherichia coli genomic DNA. Therefore, any promoter DNA sequence that can bind to RNA polymerase and initiate transcription is suitable for implementing the present invention. In principle, any promoter DNA sequence can be used to control the transcription of the target recombinant gene of the construct, and different or identical promoter sequences can be used to drive the transcription of different target genes integrated in the host cell genome or expression vector DNA. In order to optimally express the recombinant gene included in the construct, the construct may include further regulatory sequences, such as a leading DNA sequence, such as a DNA sequence derived from the 5'-untranslated region (5'UTR) of the Escherichia coli glp gene, a sequence for ribosome binding. Examples of the latter sequences are described in WO2019123324 (incorporated herein by reference), and are illustrated in the non-limiting working examples herein.

[0050] In some preferred embodiments, the regulatory element used to regulate the expression of the recombinant gene included in the construct of the present invention is the glpFKX operator promoter, PglpF, and in other preferred embodiments, the promoter is the lac operator promoter Plac.

[0051] On the other hand, the regulatory element used to regulate the expression of the recombinant gene included in the construct of the present invention is mgIBAC; the galactose / methylgalactoside transporter promoter PmglB or a variant thereof, such as but not limited to PmglB_70UTR of SEQ ID NO:15 or PmglB_70UTR_SD4 of SEQ ID NO:16.

[0052] In another aspect, the regulatory element for regulating the expression of the recombinant gene included in the construct of the present invention is gatYZABCD; tagatose-1,6-bisP aldolase promoter PgatY or a variant thereof, such as but not limited to PgatY_U70UTR of SEQ ID NO: 17.

[0053] Preferred regulatory elements present in the genetically modified cells or nucleic acid constructs of the present invention are selected from: PgatY_70UTR, PglpF, PglpF_SD1, PglpF_SD10, PglpF_SD2, PglpF_SD3, PglpF_SD4, PglpF_SD5, PglpF_SD6, PglpF_SD7, PglpF_SD8, PglpF_SD9, Plac_16UTR, Plac, PmglB_70UTR and PmglB_70UTR_SD4.

[0054] Particularly preferred regulatory elements present in the genetically modified cell or nucleic acid construct of the invention are selected from the group consisting of PglpF and Plac.

[0055] However, any promoter capable of transcribing and / or regulating the transcription level of one or more recombinant nucleic acids encoding one or more proteins (or one or more regulatory nucleic acids) that are necessary or beneficial for achieving optimal levels of biosynthetic production of one or more HMOs in a host cell, such as proteins involved in transmembrane transport of HMOs or HMO precursors, degradation of HMO production byproducts, gene expression regulatory proteins, etc., and that allows the desired effects according to the present invention to be achieved, is suitable for use in the practice of the present invention.

[0056] Preferably, the constructs of the present invention comprise genes associated with HMO biosynthetic products, promoter DNA sequences and other regulatory sequences expressed in host cells, such as ribosomal binding site sequences (e.g., Shine-Dalgarno sequences), enabling production of HMOs in 1 liter of fermentation medium containing a suspension of host cells at a level of at least 0.03 g / OD (optical density), such as levels of about 0.05 g / l / OD to about 0.1 g / l / OD. For purposes of the present invention, the latter level of HMO production is considered "sufficient," and host cells capable of producing this level of the desired HMOs are considered "suitable host cells," i.e., the cells can be further modified to express an HMO transporter, such as Nec, to achieve at least one effect described herein that favors HMO production.

[0057] The genetically modified cells or nucleic acid constructs of the present invention comprise a nucleic acid sequence, such as a heterologous gene encoding a putative MFS (major facilitator superfamily) transporter.

[0058] A MFS transporter of particular interest in the present invention is the Nec protein.Thus, the nucleic acid construct of the present invention comprises a nucleic acid sequence having at least 70% sequence identity to the gene nec, SEQ ID NO:2.

[0059] The nucleic acid sequence contained in the genetically modified cell or nucleic acid construct encodes the protein of SEQ ID NO: 1 or a functional homologue thereof, and its amino acid sequence has at least 80% identity with SEQ ID NO: 1.

[0060] Functional homologues of the protein of SEQ ID NO: 1 can be obtained by mutagenesis. Compared to the functionality of the amino acid sequence of SEQ ID NO: 1, the functional homologue should have a residual functionality of at least 50%, such as 60%, 70%, 80%, 90% or 100%. Compared to the functionality of the amino acid sequence of SEQ ID NO: 1, the functional homologue may have a higher functionality. The functional homologue of SEQ ID NO: 1 should be able to increase the HMO production of the genetically modified cells according to the invention.

[0061] The genetically modified cells (host cells or recombinant cells) may be, for example, bacteria or yeast cells. In a preferred embodiment, bacterial cells are genetically modified. With regard to bacterial host cells, there are in principle no restrictions; they may be true bacteria (Gram-positive or Gram-negative) or archaea, as long as they allow genetic manipulation for the insertion of the gene of interest and can be cultured on a manufacturing scale. Preferably, the host cells have properties that allow cultivation to high cell densities. Non-limiting examples of bacterial host cells suitable for the recombinant industrial production of HMO(s) according to the invention may be Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum or Xanthomonas campestris. Bacillus bacteria may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans.Similarly, bacteria of the genera Lactobacillus and Lactococcus can be modified using the methods of the invention, including, but not limited to, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophilus and Proprionibacterium freudenreichii are also suitable bacterial species for the invention described herein. As part of the present invention, bacterial strains from Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophilus), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium), Sporolactobacillus, Micromonospora, Micrococcus, Rhodococcus and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa) are also included. Bacteria comprising the characteristics described herein are cultivated in the presence of lactose, and oligosaccharides such as HMO produced by the cells are extracted from the bacteria themselves or the bacterial culture supernatant. In a preferred embodiment, the genetically modified cells of the present invention are Escherichia coli cells.

[0062] In another preferred embodiment, the host cell is a yeast cell, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Kluveromyces lactis, Kluveromyces marxianus, and the like.

[0063] The HMO produced by the recombinant cells of the present invention can be purified using appropriate procedures available in the art (for example, as described in WO2015188834, WO2017182965 or WO2017152918).

[0064] The genetically modified cells of the present invention can be provided using standard methods in the art, such as the methods described in the manuals by Sambrook et al., Wilson & Walker, Maniatise et al., and Ausubel et al.

[0065] A host 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 gene (e.g., GenBank Accession No. V00296 (GI:41901)). For the purposes of the present invention, the host cell for producing HMO is genetically manipulated to contain any β-galactosidase gene or to contain an inactivated gene. The gene may be inactivated by completely or partially deleting the corresponding nucleic acid sequence from the bacterial genome, or the gene sequence may be mutated in the manner in which it is transcribed, or if transcribed, the transcript is not translated, or if translated into a protein (i.e., β-galactosidase), the protein does not have the corresponding enzyme activity. In this way, the HMO producer accumulates a larger intracellular lactose pool, which is beneficial to the production of HMO.

[0066] In some embodiments, the engineered cell (e.g., bacteria) comprises a defective sialic acid decomposition pathway. "Sialic acid decomposition pathway" means a series of reactions that are often controlled and catalyzed by enzymes and that cause sialic acid degradation. The exemplary sialic acid decomposition pathway described herein is an Escherichia coli pathway. In this pathway, sialic acid (Neu5Ac; N-acetylneuraminic acid) is degraded by NanA (N-acetylneuraminic acid lyase) and NanK (N-acetylmannosamine kinase) and NanE (N-acetylmannosamine-6-phosphate epimerase), which are all encoded by the nanATEK-yhcH operon and inhibited by NanR (http: / / ecocyc.org / ECOLI). By introducing mutations in endogenous nanA (N-acetylneuraminic acid lyase) (e.g., GenBank Accession No. D00067.1 (GL216588)) and / or nanK (N-acetylmannosamine kinase) genes (e.g., GenBank Accession No. (amino acid) BAE77265.1 (GL85676015)) and / or nanE (N-acetylmannosamine-6-phosphate differential enzyme, GI: 947745, incorporated herein by reference), a defective sialic acid decomposition pathway is presented in an E. coli host. Optionally, the nanT (N-acetylneuraminic acid transporter) gene is also inactivated or mutated. Other intermediates of sialic acid metabolism include: (ManNAc-6-P) N-acetylmannosamine-6-phosphate; (GlcNAc-6-P) N-acetylglucosamine-6-phosphate; (GlcN-6-P) glucosamine-6-phosphate and (Fruc-6-P) fructose-6-phosphate. In some preferred embodiments, nanA is mutated. In other preferred embodiments, nanA and nanK are mutated, while nanE remains functional. In another preferred embodiment, nanA and nanE are mutated, while nanK is not mutated, inactivated or deleted. Mutations are one or more changes in the nucleic acid sequence encoding the gene product of nanA, nanK, nanE and / or nanT. For example, the mutations in the nucleic acid sequence can be 1, 2, up to 5, up to 10, up to 25, up to 50 or up to 100 changes. For example, the nanA, nanK, nanE and / or nanT genes are mutated by null mutations. Null mutations described herein include amino acid substitutions, additions, deletions or insertions that either result in loss of enzyme function (i.e., reduced or inactive activity) or loss of enzyme (i.e., no gene product). "Deletion" means complete or partial deletion of a coding region, so that no (functional) gene product is produced. Inactivation means that the coding sequence has been altered so that the resulting gene product is functionally inactive, or the activity of the encoded gene product is less than 100%, such as 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the natural, naturally occurring, endogenous gene product.A "non-mutated" gene or protein does not differ from the native, naturally occurring or endogenous coding sequence by 1, 2, at most 5, at most 10, at most 20, at most 50, at most 100, at most 200, or at most 500 or more codons, or from the corresponding encoded amino acid sequence.

[0067] In addition, the bacterium (eg, E. coli) also comprises sialic acid synthesis capability. For example, the bacterium has sialic acid synthesis capability by providing exogenous UDP-GIcNAc 2-differentiase (e.g., neuC of Campylobacter jejuni (GenBank AAK91727.1) or equivalent (e.g., GenBank CAR04561.1), Neu5Ac synthase (e.g., neuB of Campylobacter jejuni (GenBank AAK91726.1) or equivalent (e.g., Flavobacterium limnosediminis sialic acid synthase, GenBank WP_023580510.1), and / or CMP-Neu5Ac synthetase (e.g., neuA of Campylobacter jejuni (GenBank AAK91728.1) or equivalent (e.g., Vibrio brasiliensis CMP-sialic acid synthase, GenBank WP_006881452.1).

[0068] The production of neutral N-acetylglucosamine-containing HMOs in engineered bacteria is also known in the art (see, for example, Gebus C et al. (2012) Carbohydrate Research 363 83-90).

[0069] For the production of HMOs containing N-acetylglucosamine, such as lacto-N-triose (LNT-2), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-fucopentaose I (LNFP-I), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose I (LDFH-I), lacto-N-difucohexaose II (LDFH-II) and lacto-N-neodifucohexaose II (LNDFH-III), as described above, the bacteria contain a functional lacY and a dysfunctional lacZ gene, and are engineered to contain an exogenous UDP-GlcNAc:Galα / β-Rβ-3-N-acetylglucosamine transferase gene or a functional variant or fragment thereof. Such exogenous UDP-GlcNAc:Galα / β-Rβ-3-N-acetylglucosamine transferase genes can be obtained from any of a variety of sources, such as the IgtA gene described in Neisseria meningitidis (N. meningitides) (Genbank protein accession number AAF42258.1) or Neisseria gonorrhoeae (N. gonorrhoeae) (Genbank protein accession number ACF31229.1). Optionally, additional exogenous glycosyltransferase genes can be co-expressed in bacteria containing exogenous UDP-GlcNAc:Galα / β-Rβ-3-N-acetylglucosamine transferase. For example, a β-1,4-galactosyltransferase gene is co-expressed with a UDP-GlcNAc:Galα / β-Rβ-3-N-acetylglucosamine transferase gene. Such an exogenous β-1,4-galactosyltransferase gene can be obtained from any of a variety of sources, such as the IgtB gene described in Neisseria meningitidis (Genbank Protein Accession No. AAF42257.1) or the HP0826 / galT gene described in Helicobacter pylori (H. pylori) (Genbank Protein Accession No. NP_207619.1). Optionally, the additional exogenous glycosyltransferase gene co-expressed in the bacteria comprising the exogenous UDP-GlcNAc:Galα / β-Rβ-3-N-acetylglucosamine transferase gene is a PI,3-galactosyltransferase gene, such as the wbgO gene described from Escherichia coli 055:H7 (Genbank protein accession number WP_000582563.1) or the jhp0563 gene described from Helicobacter pylori (Genbank protein accession number AEZ55696.1), or the cpslBJ gene described from Streptococcus agalactiae type lbO / 2 (Genbank protein accession number AB050723).Functional variants and fragments of any of the above enzymes are also encompassed by the present invention.

[0070] N-acetylglucosamine transferase gene and / or galactosyltransferase gene are also operably linked to Pglp and expressed from the corresponding genomic integration box. In one embodiment, the genomic integration gene is a gene encoding galactosyltransferase, such as the HP0826 gene encoding the GaIT enzyme from Helicobacter pylori (Genbank protein accession number NP_207619.1); in another embodiment, the genomic integration gene is a gene encoding β-1,3-N-acetylglucosamine transferase, such as the IgtA gene from Neisseria meningitidis (Genbank protein accession number AAF42258.1). In these embodiments, the second gene, i.e., the gene encoding β-1,3-N-acetylglucosamine transferase or galactosyltransferase, can be expressed from a genomic integration box or a plasmid vector box, respectively. The second gene is optionally expressed under the control of the glp promoter or under the control of any other promoter (e.g., Plac) suitable for the expression system.

[0071] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Singleton et al. (1994) Dictionary of Microbiology and Molecular Biology, Second Edition, John Wiley & Sons (New York) provides a general dictionary of many of the terms used in the present invention for those skilled in the art. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described. Most of the nomenclature and general laboratory procedures required in this application can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (2012); Wilson K. and Walker J., Principles and Techniques of Biochemistry and Molecular Biology (2010), Cambridge University Press; or Manatise et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (2012); or in Ausubel et al., Current protocols in molecular biology, John Wiley and Sohns (2010). These manuals are hereinafter referred to as "Sambrook et al.," "Wilson & Walker," "Maniatise et al.," "Ausubel et al.," respectively.

[0072] A second aspect of the present invention relates to a method for producing one or more HMOs, the method comprising the steps of:

[0073] (i) providing a genetically modified cell capable of producing HMO, wherein the cell comprises a recombinant nucleic acid encoding a protein of SEQ ID NO: 1 or a functional homologue thereof, the amino acid sequence of the functional homologue having at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity to SEQ ID NO: 1;

[0074] (ii) culturing the cell according to (i) in a suitable cell culture medium to allow HMO production and expression of the DNA sequence, thereby producing a protein having the amino acid sequence of SEQ ID NO: 1, or a functional homologue thereof, whose amino acid sequence has at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity to SEQ ID NO: 1;

[0075] (iii) harvesting the one or more HMOs produced in step (ii).

[0076] According to the present invention, the term "culturing" (or "cultivating" or "cultivation", also called "fermentation") relates to the propagation of bacterial expression cells in a controlled bioreactor according to methods known in the art.

[0077] To produce one or more HMOs, the HMO-producing bacteria described herein are cultured in the presence of a suitable carbon source (e.g., glucose, glycerol, lactose, etc.) according to procedures known in the art, and the produced HMOs are harvested from the culture medium and the microbial biomass formed during the culture process. Thereafter, the HMOs are purified according to procedures known in the art (e.g., as described in WO2015188834, WO2017182965, or WO2017152918), and the purified HMOs are used as nutraceuticals, medicines, or for any other purpose, such as for research. The manufacture of HMOs is typically accomplished by mass cultivation. The terms "manufacturing" and "manufacturing scale" in the meaning of the present invention define fermentation with a minimum volume of 5 L of culture broth. Often, a "manufacturing scale" process is defined as being able to process a large amount of a preparation containing the desired HMO or HMOs, and to produce a certain amount of the target protein, such as in the case of a therapeutic compound or composition, to meet the needs of clinical trials and market supply. In addition to the large volumes, manufacturing-scale processes, in contrast to simple laboratory-scale processes such as shake flask cultures, are characterized by the use of technological systems of bioreactors (fermenters) equipped with means for stirring, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.) To a large extent, the behavior of the expression system in laboratory-scale processes, such as shake flasks, bench-top bioreactors or the deep well format described in the examples of the present disclosure, does allow predicting the behavior of the system in the complex environment of a bioreactor.

[0078] With regard to suitable cell culture media used in the fermentation process, there are no restrictions. The culture medium can be semi-defined, i.e. containing complex culture media compounds (e.g. yeast extract, soy peptone, tyrosine amino acids, etc.), or chemically defined, not containing any complex compounds.

[0079] The term "one or more HMOs" means that the HMO producing cell can produce a single HMO structure (a first HMO) or multiple HMO structures (a second, a third HMO, etc.). In some embodiments, a host cell that produces a single HMO may be preferred, and in other preferred embodiments, a host cell that produces multiple HMO structures may be preferred. Non-limiting examples of host cells that produce a single HMO structure are 2'-FL, 3-FL, 3'-SL, 6'-SL or LNT-2 production cells. Non-limiting examples of host cells that can produce multiple HMO structures may be DFL, FSL, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFP IV, LNFP V, pLNnH, pLNH2 production cells.

[0080] The term "harvesting" in the context of the present invention relates to the collection of the produced HMO(s) after the fermentation is finished. In different embodiments, it may include the collection of the HMO(s) included in the biomass (i.e., host cells) and the culture medium, i.e., before / without separation of the fermentation broth from the biomass. In other embodiments, the produced HMO(s) may be collected separately from the biomass and the fermentation broth, i.e., after separation of the biomass from the culture medium (i.e., the fermentation broth). The cells may be separated from the culture medium using any method well known to those skilled in the art, such as any suitable type of centrifugation or filtration. Separation of cells from the culture medium may be performed immediately after harvesting the fermentation broth, or at a later stage after storage of the fermentation broth under appropriate conditions. Recovering the produced HMO(s) from the remaining biomass (or total fermentation) includes extracting the HMO(s) from the biomass (producing cells). It may be accomplished by any suitable method in the art, such as by sonication, boiling, homogenization, enzymatic lysis using lysozyme, or freezing and grinding.

[0081] After fermentation recovery, the HMO(s) may be used for further processing and purification.

[0082] Fermentation-produced HMOs may be purified using appropriate procedures described in WO2016095924, WO2015188834, WO2017152918, WO2017182965, US20190119314 (all incorporated by reference).

[0083] In some embodiments of the invention, a host cell may produce multiple HMOs, one of which is a "product" HMO, and some / all of the other HMOs are "byproduct" HMOs. Typically, a byproduct HMO is a product of a major HMO precursor or further modification of a major HMO. In some embodiments, it may be desirable to produce a large amount of a product HMO and a small amount of a byproduct HMO. The cells and methods for HMO production described herein allow for the controlled production of an HMO product with a defined HMO distribution, for example, in one embodiment, a mixture of HMOs is produced in which the product HMO is the main HMO compared to the other HMOs of the mixture (i.e., the byproduct HMOs), i.e., the yield of the product HMO is higher than that of the other byproduct HMOs; in other embodiments, cells producing the same mixture of HMOs may be regulated to produce more of one or more byproduct HMOs than the product HMO. For example, during the production of 2'-FL, a product HMO is produced, typically a large amount of DFL, i.e., a byproduct HMO. Using the genetically modified cells of the invention, the level of DFL in the 2'-FL product can be significantly reduced.

[0084] Advantageously, the present invention provides a reduced ratio of byproducts to products and an increased overall yield of products (and / or total amount of HMO). This reduced byproduct formation relative to product formation promotes increased product yields and improves the efficiency of the production and product recovery processes, providing a high quality HMO manufacturing process.

[0085] In different preferred embodiments, different host cells that produce one or both of 2'-FL, 3-FL, 3'-SL, 6'-SL, LNT-2, DFL, FSL, LNT, DFL, FSL, LNT, LNnT, LNFP I, LNFP II, LNFP III, LNFP IV, LNFP V, pLNnH, pLNH2 as product or by-product HMOs can be selected. In a preferred embodiment, the product is 3-FL and the by-product is DFL. In another preferred embodiment, the product is 2'-FL and the by-product is DFL. In another preferred embodiment, the product is LNT-2 and the by-products are LNT and LNFP I.

[0086] The present invention also relates to the use of the genetically modified cells or nucleic acid constructs according to the invention in the production of one or more oligosaccharides, preferably one or more human milk oligosaccharides (one or more). In one embodiment, the genetically modified cells or nucleic acid constructs according to the invention are used to produce a specific HMO selected from 2'-FL, 3-FL, DLF, LNT, LNT-II, LNnT, LNFP-1, pLNnH and pLNH-II. In a preferred embodiment, the genetically modified cells or nucleic acid constructs according to the invention are used to produce a specific HMO selected from 2'-FL, 3-FL, DLF, LNT, LNT-II, LNnT, pLNnH and pLNH-II.

[0087] In a particularly preferred embodiment, the genetically modified cells or nucleic acid constructs according to the invention are used for the production of a specific HMO selected from the group consisting of 2'-FL, 3-FL, LNT, LNT-II, LNnT and pLNH-II.

[0088] The present invention is further illustrated by the following non-limiting examples and embodiments.

[0089] Example

[0090] Materials and methods

[0091] Unless otherwise indicated, standard techniques, vectors, control sequence elements and other expression system elements known in the field of molecular biology are used for nucleic acid manipulation, transformation and expression. Such standard techniques, vectors and elements can be found in the following: Ausubel et al. (eds.), Current Protocols in Molecular Biology (1995) (John Wiley & Sons); Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning (Molecular Cloning) (1989) (Cold Spring Harbor Laboratory Press, New York); Berger and 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, New York); Miller, JH Experiments in Molecular Genetics (Experiments in molecular genetics) (1972) (Cold Spring Harbor Laboratory Press, New York).

[0092] strains

[0093] The bacterial strain MDO used was constructed from Escherichia coli K12 DH1. The genotype of Escherichia coli K12 DH1 is: F - , λ-, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. Table 2 describes the strains used in this example.

[0094] Table 2

[0095]

[0096] Culture medium

[0097] Luria broth (LB) medium was prepared using LB broth powder, Millers (Fisher Scientific), and LB agar plates were prepared using LB agar powder, Millers (Fisher Scientific). Appropriate amounts of ampicillin (100 μg / mL) or any appropriate antibiotic) and / or chloramphenicol (20 μg / mL) were added.

[0098] The composition of the minimal medium is 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), trace mineral solution (5 mL / L). The trace mineral stock solution contains: ZnSO4*7H2O 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 is adjusted to 7.0 with 5N NaOH and autoclaved. Prior to inoculation, the minimal medium was provided with 1 mM MgSO4, 4 μg / mL thiamine, 0.5% of a given carbon source (glycerol (Carbosynth)), and isopropyl-β-D-thiogalactoside (IPTG) (0.2 mM) was added when appropriate. Thiamine, antibiotics, and IPTG were sterilized by filtration. All percentage concentrations of glycerol are expressed as v / v, and all percentage concentrations of glucose are expressed as w / v.

[0099] The composition of M9 plates containing 2-deoxy-galactose was as follows: 15 g / L agar (Fisher Scientific), 2.26 g / L 5×M9 minimal salts (Sigma-Aldrich), 2 mM MgSO 4 , 4 μg / mL thiamine, 0.2% glycerol, and 0.2% 2-deoxy-D-galactose (Carbosynth).

[0100] The composition of the MacConkey indicator plate is as follows: 40 g / L MacConkey agar base (BD-Difco TM ) and a carbon source at a final concentration of 1%.

[0101] nourish

[0102] Unless otherwise stated, E. coli strains were propagated with agitation in Luria Bertani (LB) medium containing 0.2% glucose at 37° C. Agar plates were incubated overnight at 37° C.

[0103] Chemically competent cells and transformation

[0104] E. coli was inoculated from LB plates into 5 mL LB containing 0.2% glucose at 37°C and shaken until OD600-0.4. 2 mL of culture was obtained by centrifugation at 13.000 g for 25 seconds. The supernatant was removed and the cell pellet was resuspended in 600 μL cold TB solution (10 mM pipe, 15 mM CaCl2, 250 mM KCl). The cells were cultured on ice for 20 minutes and then precipitated at 13.000 g for 15 seconds. The supernatant was removed and the cell pellet was resuspended in 100 μL cold TB solution. Plasmid transformation was performed using 100 μL competent cells and 1 to 10 ng plasmid DNA. The cells and DNA were cultured on ice for 20 minutes and then heat-shocked at 42°C for 45 seconds. After incubation on ice for 2 minutes, 400 μL SOC (20 g / L tryptone, 5 g / L yeast extract, 0.5 g / L NaCl, 0.186 g / L KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose) was added, and the cell culture was shaken at 37°C for 1 hour and then plated on selective plates.

[0105] Plasmids were transformed into TOP10 chemically competent cells under conditions recommended by the supplier (ThermoFisher Scientific).

[0106] DNA Technology

[0107] Plasmid DNA was isolated from E. coli using the QIAprep Spin-Miniprep Kit (Qiagen). Chromosomal DNA was isolated from E. coli using the QIAmp DNA Mini Kit (Qiagen). PCR products were purified using the QIAquick PCR Purification Kit (Qiagen). DreamTaq PCR Master Mix (Thermofisher), Phusion U Hot Start PCR Master Mix (Thermofisher), USEREnzyme (New England Biolabs) were used according to the supplier's recommendations. Primers were provided by Eurofins Genomics, Germany. PCR fragments and plasmids were sequenced by Eurofins Genomics. Colony PCR was performed using DreamTaq PCR Master Mix on a T100 TM The experiments were performed in a thermal cycler (Bio-Rad).

[0108] Table 3: Oligos used to amplify plasmid backbone, promoter elements and nec

[0109]

[0110] Table 4: Heterologous proteins expressed in HMO production cells

[0111]

[0112] Table 5: Synthetic DNA elements for expression of nec

[0113]

[0114]

[0115] Plasmid construction

[0116] A plasmid backbone containing two I-Scel endonuclease sites was synthesized, separated by two DNA fragments suitable for homologous recombination into the E. coli genome and a T1 transcription terminator sequence. For example, the galactose operon (required for homologous recombination in galK) and the T1 transcription terminator sequence (pUC57::gal) were synthesized in one plasmid backbone (GeneScript). The DNA sequence used for homologous recombination in the gal operon includes base pairs 3.628.621-3.628.720 and 3.627.572-3.627.671 in the sequence E. coli K-12MG155 complete genome GenBank: ID: CP014225.1. Insertion by homologous recombination will result in a 949 base pair deletion of the galK and galK- phenotypes. In a similar manner, a backbone based on pUC57 (GeneScript) or any other suitable vector containing two I-Scel endonuclease sites separated by two DNA fragments suitable for homologous recombination into the E. coli genome and a T1 transcription terminator sequence can be synthesized. Standard techniques well known in the field of molecular biology are used to design primers and amplify specific DNA sequences of E. coli K-12 DH1 chromosomal DNA. For example, such standard techniques, vectors and elements can be found in: Ausubel et al. (eds.), Current Protocols in Molecular Biology (1995) (John Wiley & Sons); Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, New York); Berger and Kimmel, Methods in Enzymology, 152: A Guide to Molecular Cloning Techniques (1987) (Academic Press); Bukhari et al. (eds.).

[0117] Chromosomal DNA obtained from E. coli DH1 was used to amplify a 300 bp DNA fragment containing the promoter PglpF using oligomers O261 and O262, and a 195 bp DNA fragment containing Plac using oligomers O68 and IO113 (Table 3).

[0118] A 1.185 bp DNA fragment containing a codon-optimized version of the nec gene from Rosenbergiella nectarea was synthesized using GeneScript (Table 5). The nec gene was amplified by PCR using oligonucleotides O741 and O742.

[0119] All PCR fragments (plasmid backbone, promoter-containing elements and nec gene) are purified, and the plasmid backbone, promoter element (PglpF or Plac) and DNA fragment containing nec are assembled. The plasmid is cloned by standard user cloning method. Any standard DNA cloning technique can be used to clone in any appropriate plasmid. The plasmid is transformed into TOP10 cells and selected on LB plates containing 100 μg / mL ampicillin (or any suitable antibiotic) and 0.2% glucose. The constructed plasmid is purified and the promoter sequence and the 5' end of the nec gene are verified by DNA sequencing (MWG Eurofins Genomics). In this way, a gene cassette containing any promoter associated with the nec gene is constructed.

[0120] Table 6: Examples of helper and donor plasmids used for strain construction

[0121] Plasmids Related genotypes Marker genes PcbS Para-I-Scel-λRed,p15A ori,cam* cam pUC57 pMB1,bla bla pUC57::gal pUC57::galTK' / T1-galKM' bla

[0122] Construction of strains

[0123] The bacterial strain MDO used was constructed from Escherichia coli K-12DH1. The genotype of Escherichia coli K-12DH1 is: F - , λ-, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. In addition to the E. coli K-12DH1 genotype, MDO also had the following modifications: lacZ: 1.5 kbp deletion; lacA: 0.5 kbp deletion; nanKETA: 3.3 kbp deletion; melA: 0.9 kbp deletion; wcaJ: 0.5 kbp deletion; mdoH: 0.5 kbp deletion, and the insertion of the Plac promoter upstream of the gmd gene.

[0124] Insertion of an expression cassette containing a promoter linked to the nec gene and the T1 transcription termination sequence is essentially accomplished by gene engulfment as described by Herring et al. (Herring, CD, Glasner, JD and Blattner, FR (2003). Gene (311). 153-163). Briefly, the donor plasmid and the helper plasmid were co-transformed into MDO and selected on LB plates containing 0.2% glucose, ampicillin (100 μg / mL) or kanamycin (50 mg / mL) and chloramphenicol (20 μg / mL). A single colony was inoculated in 1 mL of LB containing chloramphenicol (20 μg / mL) and 10 μL of 20% L-arabinose and cultured with shaking at 37°C for 7 to 8 hours. To integrate the galK site of the E. coli cells, they were then inoculated on M9-DOG plates and incubated at 37°C for 48 hours. Individual colonies formed on MM-DOG plates were re-streaked on LB plates containing 0.2% glucose and cultured at 37°C for 24 hours. White colonies appeared on MacConkey galactose agar plates and were sensitive to both ampicillin and chloramphenicol, and it was expected that these colonies had lost the donor and helper plasmids and contained insertions in the galK site. Insertions in the galK site were identified by colony PCR using primers O48 (SEQ ID NO: 13) and O49 (SEQ ID NO: 14), and the inserted DNA was verified by sequencing (Eurofins Genomics, Germany).

[0125] The genetic cassettes are inserted into other sites of the E. coli chromosomal DNA in a similar manner using different selectable marker genes.

[0126] Deep hole measurement

[0127] Individual bacterial colonies from LB flat plates were pre-cultured in 10 mL 24-well plates (Axygen) in 1 mL basic minimum medium containing 5 g / L glucose, 1 M MgSO4 and 4 mg / L thiamine. Before cultivation using a hydrophobic gas permeable adhesive seal (Axygen), the culture plates were sealed and cultured 24 hours at 34 ° C in an orbital shaker (Edmund Buhler GmbH) with a rotating speed vibration of 700 rpm. The cell density of the culture was monitored at 600 nm using an S-20 spectrophotometer (Germany Boeco). 40 μl overnight cultures were inoculated in 2 mL basic minimum medium containing 0.1 g / L glucose, 5 g / L lactose, 20 g / L sucrose, 1 M MgSO4, 4 mg / L thiamine and 0.25 mg / L SUH (Sigma). If appropriate, IPTG was added. The deep well plates were covered with sealing foil and incubated at 28°C for 48 or 72 hours with orbital shaking at 700 rpm. After incubation, the OD600 was measured and the plates were heated (Saveen Werner) covered with sealing tape and incubated at 100°C in a thermomixer with shaking at 400 rpm for 1 hour.

[0128] For analysis of total samples, cell lysates prepared by boiling were pelleted by centrifugation at 4.700 rpm for 10 minutes. The HMO concentration in the supernatant was determined by HPLC or HPAC methods.

[0129] After the initial centrifugation of the deep well plate at 4.700 rpm for 10 minutes, the supernatant sample was retained for analytical measurement. In order to analyze the cell precipitation, the remaining supernatant was discarded and the cells were washed with 2 mL of cold PBS. After this washing step and re-centrifugation, the precipitation was resuspended in 500 mL of MQ water, and the entire deep well plate was shaken and boiled at 400 rpm in a thermal mixer for 1 hour. The cell lysate prepared by boiling was precipitated by centrifugation at 4.700 rpm for 10 minutes. The HMO concentration in the supernatant was determined by HPLC or HPAC method.

[0130] result

[0131] Example 1. Engineering of E. coli to produce 2'-FL expressing the nec gene

[0132] E. coli K-12 (DH1) MDO strains can be manipulated to express heterologous genes of interest. For example, strain MPA1 is a 2'-FL production strain that overexpresses the α-1,2-fucosyltransferase gene, futC, and the colic acid gene (gmd-fcl-gmm-wcal-cpsB-cpsG). Insertion of an expression cassette containing a promoter element (PglpF) linked to the nec gene into MPA2 chromosomal DNA resulted in i) relatively high titers of 2'-FL ( Figure 1A ), ii) lower amounts of 2'-FL in cellular components and higher amounts of 2'-FL in the culture medium ( Figure 1B ), iii) a relatively low ratio of DFL to 2'-FL ( Figure 1C ), and iv) a relatively low optical density when measuring cell density at 600 nm ( Figure 1D ).

[0133] Example 2. Engineering of E. coli to produce 3-FL expressing the nec gene

[0134] E. coli K-12 (DH1) MDO strains can be manipulated to express heterologous genes of interest. For example, strain MPA3 is a 3-FL production strain that overexpresses the α-1,3-fucosyltransferase gene futA and the colic acid genes (gmd-fcl-gmm-wcal-cpsB-cpsG). An expression cassette containing a promoter element (Plac) linked to the nec gene was inserted in a single copy into the MPA3 background strain (see MPA4 strains), resulting in i) relatively higher amounts of 3-FL in the media components, and ii) relatively lower amounts of 3-FL within the cells.

[0135] Example 3. Engineering of E. coli to produce LNT2 expressing the nec gene

[0136] E. coli K-12 (DH1) MDO strains can be manipulated to express heterologous genes of interest. For example, strain MP4002 is the best LNT2 production strain that overexpresses the β-1,3-N-acetylglucosamine transferase gene IgtA (Table 4).

[0137] Total sample analysis ( Figure 3 ) showed that when an expression cassette containing a promoter element (PglpF) linked to the nec gene was integrated into the chromosomal DNA of strain MP4002 to generate strain MP4039, LNT2 titers could be significantly increased (Table 2). Interestingly, the extracellular fraction of LNT2 from strain MP4039 was also much larger than that from strain MP4002, a fact also reflected in the fact that the levels of trisaccharides detected in the pellet fraction of strain MP4039 expressing nec were lower than those in the non-transporter expressing strain MP4002 ( Figure 3 ).

[0138] Example 4. Engineering of E. coli for LNT production using the nec gene

[0139] E. coli K-12 (DH1) MDO strains can be manipulated to express heterologous genes of interest. For example, strain MP4473 is an LNT production strain that overexpresses the β-1,3-N-acetylglucosamine transferase gene IgtA and the β-1,3-galactosyltransferase gene galTK (Table 4).

[0140] Insertion of an expression cassette containing a promoter element (PglpF) linked to the nec gene into the chromosomal DNA of strain MP4473 to generate strain MP4537 (Table 2) resulted in i) a more than 2-fold increase in total LNT titer, ii) a modest increase in total LNT2 concentration, and iii) a more than 2-fold increase in total pLNH2 formation ( Figure 4 ).

[0141] Compared with the concentration measured in the culture medium of MP4473, the concentration of LNT in the culture supernatant of strain MP4537 increased by 2-fold ( Figure 4 ). Although limited Nec-mediated LNT2 export was observed, the fraction of extracellular LNT2 in strain MP4537 was only slightly higher than that in strain MP4473. LNT2 transport events may occur more slowly than Nec-mediated LNT export events. Interestingly, despite the large increase in total pLNH2 formation in transporter-expressing cells, pLNH2 was only found in the cell pellet of strain MP4537 ( Figure 4 ).

[0142] Example 5. Engineering of E. coli for LNFP-I production using the nec gene

[0143] E. coli K-12 (DH1) MDO strains can be manipulated to express heterologous genes of interest. For example, strain MP2789 is a LNFP-I production strain that overexpresses the β-1,3-N-acetylglucosamine transferase gene IgtA, the β-1,3-galactosyltransferase gene galTK, the α-1,2-fucosyltransferase gene futC, and the native colic acid genes (gmd-fcl-gmm-wcal-cpsB-cpsG) (Table 4). An expression cassette containing a promoter element (PglpF) linked to the nec gene was inserted into the chromosomal DNA of strain MP2789 to generate strain MP4597 (Table 2), resulting in i) similar LNFP-I titers, ii) a more than 2-fold increase in total 2'-FL titers, and iii) significantly higher total HMO sums (2'-FL, LNFP-I, and LNT) ( Figure 5The data presented here indicate that 2'-FL, but not LNFP-I and LNT, is efficiently transported out of cells expressing the nec transporter and that in this way the formation and subsequent export of 2'-FL is favored in LNFP-I producing cells expressing Nec. Sequence Listing <110> Gleikam AG <120> HMO Production <130> P2937WO00 <150> DK202000833 <151> 2020-07-13 <150> DK202000085 <151> 2020-01-23 <160> 17 <170> BiSSAP 1.3.6 <210> 1 <211> 394 <212> PRT <213> Rosenbergiella nectarea <220> <223> Nec_WP_092672081.1\Optimized translation <400> 1 Met Gln Ser Phe Thr Pro Pro Ala Pro Lys Gly Gly Asn Pro Val Phe 1 5 10 15 Met Met Phe Met Leu Val Thr Phe Phe Val Ser Ile Ala Gly Ala Leu 20 25 30 Gln Ala Pro Thr Leu Ser Leu Tyr Leu Ser Gln Glu Leu Ala Ala Lys 35 40 45 Pro Phe Met Val Gly Leu Phe Phe Thr Ile Asn Ala Val Thr Gly Ile 50 55 60 Ile Ile Ser Phe Ile Leu Ala Lys Arg Ser Asp Arg Lys Gly Asp Arg 65 70 75 80 Arg Arg Leu Leu Met Phe Cys Cys Ala Met Ala Ile Ala Asn Ala Leu 85 90 95 Met Phe Ala Phe Val Arg Gln Tyr Val Val Leu Ile Thr Leu Gly Leu 100 105 110 Ile Leu Ser Ala Leu Thr Ser Val Val Met Pro Gln Leu Phe Ala Leu 115 120 125 Ala Arg Glu Tyr Ala Asp Arg Thr Gly Arg Glu Val Val Met Phe Ser 130 135 140 Ser Val Met Arg Thr Gln Met Ser Leu Ala Trp Val Ile Gly Pro Pro 145 150 155 160 Ile Ser Phe Ala Leu Ala Leu Asn Tyr Gly Phe Ile Thr Leu Tyr Leu 165 170 175 Val Ala Ala Ala Leu Phe Leu Leu Ser Leu Ile Leu Ile Lys Thr Thr 180 185 190 Leu Pro Ser Val Pro Arg Leu Tyr Pro Ala Glu Asp Leu Ala Lys Ser 195 200 205 Ala Ala Ser Gly Trp Lys Arg Thr Asp Val Arg Phe Leu Phe Ala Ala 210 215 220 Ser Val Leu Met Trp Val Cys Asn Leu Met Tyr Ile Ile Asp Met Pro 225 230 235 240 Leu Tyr Ile Ser Lys Ser Leu Gly Met Pro Glu Ser Phe Ala Gly Val 245 250 255 Leu Met Gly Thr Ala Ala Gly Leu Glu Ile Pro Val Met Leu Leu Ala 260 265 270 Gly Tyr Leu Ala Lys Arg Val Gly Lys Arg Pro Leu Val Ile Val Ala 275 280 285 Ala Val Cys Gly Leu Ala Phe Tyr Pro Ala Met Leu Val Phe His Gln 290 295 300 Gln Thr Gly Leu Leu Ile Ile Gln Leu Leu Asn Ala Val Phe Ile Gly 305 310 315 320 Ile Val Ala Gly Leu Val Met Leu Trp Phe Gln Asp Leu Met Pro Gly 325 330 335 Lys Ala Gly Ala Ala Thr Thr Leu Phe Thr Asn Ser Val Ser Thr Gly 340 345 350 Met Ile Phe Ala Gly Leu Cys Gln Gly Leu Leu Ser Asp Leu Leu Gly 355 360 365 His Gln Ala Ile Tyr Val Leu Ala Thr Val Leu Met Val Ile Ala Leu 370 375 380 Leu Leu Leu Leu Arg Val Lys Glu Gln Ala 385 390 <210> 2 <211> 1185 <212> DNA <213> Rosenbergiella nectarea <220> <223> nec nucleotide sequence <400> 2 atgcagagct tcaccccgcc ggcgccgaag ggtggcaacc cggtgttcat gatgtttatg 60 ctggtgacct tctttgtgag cattgcgggt gcgctgcagg cgccgaccct gagcctgtac 120 ctgagccaag agctggcggc gaaaccgttc atggtgggcc tgttctttac cattaacgcg 180 gttaccggta tcattatcag ctttatcctg gcgaagcgta gcgaccgtaa aggtgaccgt 240 cgtcgtctgc tgatgttctg ctgcgcgatg gcgatcgcga acgcgctgat gttcgcgttt 300 gttcgtcagt acgtggttct gattaccctg ggcctgatcc tgagcgcgct gaccagcgtg 360 gttatgccgc aactgttcgc gctggcgcgt gagtatgcgg accgtaccgg tcgtgaagtg 420 gttatgttta gcagcgtgat gcgtacccaa atgagcctgg cgtgggttat tggcccgccg 480 atcagcttcg cgctggcgct gaactacggt tttattaccc tgtatctggt ggctgcggcg 540 ctgtttctgc tgagcctgat tctgatcaag accaccctgc cgagcgttcc gcgtctgtat 600 ccggcggaag acctggcgaa gagcgcggcg agcggttgga aacgtaccga tgtgcgtttc 660 ctgtttgcgg cgagcgtgct gatgtgggtt tgcaacctga tgtacattat cgatatgccg 720 ctgtatatca gcaaaagcct gggtatgccg gagagcttcg cgggtgttct gatgggcacc 780 gcggcgggtc tggaaattcc ggtgatgctg ctggcgggct acctggcgaa gcgtgttggt 840 aaacgtccgc tggtgattgt tgcggcggtg tgcggcctgg cgttctatcc ggcgatgctg 900 gtttttcacc agcaaaccgg tctgctgatt atccagctgc tgaacgcggt gttcattggc 960 atcgtggcgg gtctggttat gctgtggttt caagacctga tgccgggtaa agcgggtgcg 1020 gcgaccaccc tgttcaccaa cagcgttagc accggcatga tctttgcggg cctgtgccag 1080 ggtctgctga gcgatctgct gggtcaccaa gcgatttacg tgctggcgac cgtgctgatg 1140 gttatcgcgc tgctgctgct gctgcgtgtt aaagagcagg cgtaa 1185 <210> 3 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> PglpF expression element <300> <310> WO2019123324 <311> 19.12.2018 <312> 27.06.2019 <400> 3 gcggcacgcc ttgcagatta cggtttgcca cacttttcat ccttctcctg gtgacataat 60 ccacatcaat cgaaaatgtt aataaatttg ttgcgcgaat gatctaacaa acatgcatca 120 tgtacaatca gatggaataa atggcgcgat aacgctcatt ttatgacgag gcacacacat 180 tttaagttcg atatttctcg tttttgctcg ttaacgataa gtttacagca tgcctacaag 240 catcgtggag gtccgtgact ttcacgcata caacaaacat taaccaagga ggaaacagct 300 <210> 4 <211> 195 <212> DNA <213> Artificial sequence <220> <223> Plac expression element <300> <310> WO2019123324 <311> 19.12.2018 <312> 27.06.2019 <400> 4 atgcgcaaat tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 60 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 120 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctgggtacct 180 aaggaggaaa cagct 195 <210> 5 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primer O40 backbone.for <220> <221> misc_RNA <222> 9 <400> 5 attaacccuc caggcatcaa ataaaacgaa aggc 34 <210> 6 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Plac.for <400> 6 atgcgcaaau tgtgagttag ctcactcatt ag 32 <210> 7 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primer O79 backbone.rev <220> <221> misc_RNA <222> 10 <400> 7 atttgcgcau caccaatcaa attcacgcgg cc 32 <210> 8 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Plac.rev <400> 8 agctgttucc tccttaggta cccagctttt gttccc 36 <210> 9 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Primers O261, PglpF.for <220> <221> misc_RNA <222> 10 <400> 9 atgcgcaaau gcggcacgcc ttgcagatta cg 32 <210> 10 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Primers O262, PglpF.rev <220> <221> misc_RNA <222> 8 <400> 10 agctgttucc tccttggtta atgtttgttg tatgcg 36 <210> 11 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Primer O741, nec.for <220> <221> misc_RNA <222> 8 <400> 11 aaacagcuat gcagagcttc accccgcc 28 <210> 12 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primer O742, nec.rev <220> <221> misc_RNA <222> 9 <400> 12 agggttaaut tacgcctgct ctttaacacg cagc 34 <210> 13 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primer O48, galK.for <400> 13 cccagcgaga cctgaccgca gaac 24 <210> 14 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Primer O49, glaK.rev <400> 14 ccccagtcca tcagcgtgac tacc 24 <210> 15 <211> 203 <212> DNA <213> Artificial sequence <220> <223> Promoter sequence Pmg1B_70UTR <400> 15 tgcgtcgcca ttctgtcgca acacgccaga atgcggcggc gatcactaac tcaacaaatc 60 aggcgatgta accgctttca atctgtgagt gatttcacag tatcttaaca atgtgatagc 120 tatgattgca ccgtgcctac aagcatcgtg gaggtccgtg actttcacgc atacaacaaa 180 cattaaccaa ggaggaaaca gct 203 <210> 16 <211> 203 <212> DNA <213> Artificial sequence <220> <223> Promoter sequence PmglB_70UTR_SD4 <400> 16 tgcgtcgcca ttctgtcgca acacgccaga atgcggcggc gatcactaac tcaacaaatc 60 aggcgatgta accgctttca atctgtgagt gatttcacag tatcttaaca atgtgatagc 120 tatgattgca ccgtgcctac aagcatcgtg gaggtccgtg actttcacgc atacaacaaa 180 cattaaccaa ctaggaaaca gct 203 <210> 17 <211> 291 <212> DNA <213> Artificial sequence <220> <223> Promoter sequence PgatY_70UTR <400> 17 cggcaaccta tgcctgatgc gacgctgaag cgtcttatca tgcctacata gcactgccac 60 gtatgtttac accgcatccg gcataaaaac acgcgcactt tgctacggct tccctatcgg 120 gaggccgttt ttttgccttt cactcctcga ataattttca tattgtcgtt tttgtgatcg 180 ttatctcgat atttaaaaac aaataatttc attatatttt gtgcctacaa gcatcgtgga 240 ggtccgtgac tttcacgcat acaacaaaca ttaaccaagg aggaaacagc t 291

Claims

1. A genetically modified cell capable of producing one or more human milk oligosaccharides (HMOs), wherein the cell comprises a recombinant nucleic acid encoding the protein of SEQ ID NO: 1, and the HMO is selected from 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), lacto-N-triose-2 (LNT-2), lacto-N-tetraose (LNT) and lacto-N-fucopentaose 1 (LNFP 1); and When the HMO is 2'-fucosyllactose (2'-FL), the genetically modified cell further comprises a recombinant nucleic acid encoding an α-1,2-fucosyltransferase; When the HMO is 3-fucosyllactose (3-FL), the genetically modified cell further comprises a recombinant nucleic acid encoding α-1,3-fucosyltransferase; When the HMO is 3'-sialyllactose (3'-SL), the genetically modified cell further comprises a recombinant nucleic acid encoding an α-2,3-sialyltransferase; When the HMO is lacto-N-triose-2 (LNT-2), the genetically modified cell further comprises a recombinant nucleic acid encoding β-1,3-N-acetylglucosamine transferase; When the HMO is lacto-N-tetraose (LNT), the genetically modified cell further comprises recombinant nucleic acids encoding β-1,3-N-acetylglucosamine transferase and β-1,3-galactosyl transferase; When the HMO is lacto-N-fucopentaose I (LNFP I), the genetically modified cell further comprises recombinant nucleic acids encoding β-1,3-N-acetylglucosamine transferase, β-1,3-galactosyltransferase and α-1,2-fucosyltransferase.

2. The genetically modified cell according to claim 1, wherein the HMO is selected from 2'-fucosyllactose (2'-FL), 3'-sialyllactose (3'-SL) and lacto-N-tetraose (LNT).

3. The genetically modified cell of claim 1, wherein the HMO is 2'-fucosyllactose (2'-FL).

4. The genetically modified cell of any one of claims 1-3, wherein the genetically modified cell is Escherichia coli.

5. The genetically modified cell according to claim 1 or 2, wherein the cell further comprises a recombinant DNA sequence comprising a regulatory element for regulating the expression of the recombinant nucleic acid.

6. The genetically modified cell according to claim 5, wherein the regulatory element for regulating the expression of the recombinant nucleic acid is a lac promoter Plac, or a glp promoter PglpF.

7. Use of a nucleic acid construct for producing one or more human milk oligosaccharides (HMOs), the nucleic acid construct comprising a nucleic acid sequence encoding the protein of SEQ ID NO: 1, wherein the nucleic acid sequence encoding the protein of SEQ ID NO: 1 has at least 70% sequence identity with SEQ ID NO: 2, and the HMO is selected from 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), lacto-N-triose-2 (LNT-2), lacto-N-tetraose (LNT) and lacto-N-fucopentaose I (LNFP I); and When the HMO is 2'-fucosyllactose (2'-FL), the genetically modified cell further comprises a recombinant nucleic acid encoding an α-1,2-fucosyltransferase; When the HMO is 3-fucosyllactose (3-FL), the genetically modified cell further comprises a recombinant nucleic acid encoding α-1,3-fucosyltransferase; When the HMO is 3'-sialyllactose (3'-SL), the genetically modified cell further comprises a recombinant nucleic acid encoding an α-2,3-sialyltransferase; When the HMO is lacto-N-triose-2 (LNT-2), the genetically modified cell further comprises a recombinant nucleic acid encoding β-1,3-N-acetylglucosamine transferase; When the HMO is lacto-N-tetraose (LNT), the genetically modified cell further comprises recombinant nucleic acids encoding β-1,3-N-acetylglucosamine transferase and β-1,3-galactosyl transferase; When the HMO is lacto-N-fucopentaose I (LNFP I), the genetically modified cell further comprises recombinant nucleic acids encoding β-1,3-N-acetylglucosamine transferase, β-1,3-galactosyltransferase and α-1,2-fucosyltransferase.

8. The use according to claim 7, wherein the construct further comprises a nucleic acid sequence containing a regulatory element.

9. The use according to claim 8, wherein the regulatory element regulates the expression of the nucleic acid sequence having at least 70% sequence identity with SEQ ID NO:

2.

10. The use according to claim 8 or 9, wherein the regulatory element for regulating the expression of the recombinant nucleic acid is a lac promoter Plac, or a glp promoter PglpF.

11. A method for producing one or more human milk oligosaccharides (HMOs), the method comprising the steps of: (i) providing a genetically modified cell capable of producing an HMO, wherein the cell comprises a recombinant nucleic acid encoding the protein of SEQ ID NO: 1; (ii) culturing the cell according to (i) in a suitable cell culture medium to express the recombinant nucleic acid; (iii) harvesting the one or more HMOs produced in step (ii), wherein the HMO is selected from 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyl lactose (3'-SL), lacto-N-triose-2 (LNT-2), lacto-N-tetraose (LNT) and lacto-N-fucopentaose I (LNFP I); and When the HMO is 2'-fucosyllactose (2'-FL), the genetically modified cell further comprises a recombinant nucleic acid encoding an α-1,2-fucosyltransferase; When the HMO is 3-fucosyllactose (3-FL), the genetically modified cell further comprises a recombinant nucleic acid encoding α-1,3-fucosyltransferase; When the HMO is 3'-sialyllactose (3'-SL), the genetically modified cell further comprises a recombinant nucleic acid encoding an α-2,3-sialyltransferase; When the HMO is lacto-N-triose-2 (LNT-2), the genetically modified cell further comprises a recombinant nucleic acid encoding β-1,3-N-acetylglucosamine transferase; When the HMO is lacto-N-tetraose (LNT), the genetically modified cell further comprises recombinant nucleic acids encoding β-1,3-N-acetylglucosamine transferase and β-1,3-galactosyl transferase; When the HMO is lacto-N-fucopentaose I (LNFP I), the genetically modified cell further comprises recombinant nucleic acids encoding β-1,3-N-acetylglucosamine transferase, β-1,3-galactosyltransferase and α-1,2-fucosyltransferase.

12. The method of claim 11, wherein the HMO is selected from 2'-fucosyllactose (2'-FL), 3'-sialyllactose (3'-SL) and lacto-N-tetraose (LNT).

13. The method of claim 11, wherein the HMO is 2'-fucosyllactose (2'-FL).

14. Use of the genetically modified cell according to any one of claims 1 to 5 in the production of one or more human milk oligosaccharides (HMOs), wherein the HMO is selected from 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), 3'-sialyllactose (3'-SL), lacto-N-triose-2 (LNT-2), lacto-N-tetraose (LNT) and lacto-N-fucopentaose 1 (LNFP 1).

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