Human milk oligosaccharide-producing microorganisms

Abstract

To provide a genetically modified microorganism that produces human milk oligosaccharide (HMO) and a method for producing human milk oligosaccharide using same.SOLUTION: A genetically modified microorganism for producing human milk oligosaccharide (HMO) which can be a bacterium, preferably E. coli, is provided. The microorganism of the present invention includes one or more modifications to increase production including an inducible lytic system that can readily extract intracellular and extracellular HMO, and a chaperone that can increase intracellular lactose levels and increase intracellular availability of a lactose permease enzyme variant or key enzymes used in the production of HMO.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a genetically modified microorganism that produces human milk oligosaccharides (HMOs), and a method for producing HMOs using the microorganism. The present invention further relates to a medium in which the microorganism of the present invention is cultured, purified HMOs produced by the production method of the present invention, the medium, and uses of the HMOs. [Background technology]

[0002] Human milk oligosaccharides (HMOs) are structurally diverse unconjugated glycans found in human breast milk. To date, over 200 HMOs have been identified, all of which are composed exclusively of five monosaccharide components: D-galactose (Gal), D-glucose (Glc), N-acetyl-D-glucosamine (GlcNAc), L-fucose (Fuc), and the sialic acid derivative, N-acetyl-neuraminic acid (NPL 1). The reducing end can be lactose or lactose extended with several disaccharide units in two structures: lacto-N-biose (Gal-β1,3-GlcNAc, type I) or lactosamine (Gal-β1,4-GlcNAc, type II, LacNAc). The structures and classification of HMOs are described, for example, in Figure 1 and Table 1 of NPL 1.

[0003] Among HMOs, 2'-fucosyllactose (2-FL) is the most abundant, accounting for approximately 30% of all HMOs. A further prominent HMO is 3'-fucosyllactose (3-FL). Other HMOs include lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-fucopentaose. Human milk also contains neutral HMOs, as well as acidic HMOs such as 3'-sialyllactose, 6'-sialyllactose, and 3-fucosyl-3'-sialyllactose and disialyl-lacto-N-tetraose. HMOs are known to protect against pathogens such as Campylobacter jejuni, enteropathogenic Escherichia coli (E. coli), and Entamoeba histolytica (Non-Patent Document 2). 2-FL is thought to function in the prevention of infectious diseases by preventing the adhesion of toxins and pathogens at the epithelial level. It stimulates the proliferation of certain bifidobacteria and receptor analogs, providing protection against the most common toxic and pathogenic challenges encountered in infants. Based on these findings, HMOs, particularly 2-FL and 3-FL, are of particular interest as functional ingredients in products such as infant nutrition, medical nutrition, functional foods, and animal feed.

[0004] HMOs can be extracted from human breast milk or cow's milk, but in either case they are difficult to obtain in large quantities and / or in sufficient purity.

[0005] HMOs may be chemically synthesized, but the technology for doing so is not stable and the cost of chemically synthesizing HMOs is expensive.

[0006] On the other hand, HMO production in genetically modified microorganisms promises to provide a stable, safe, and low-cost means of production. Patent document 1 describes a method for producing oligosaccharides from the precursor lactose by genetically modified Escherichia coli cells, including inactivation of the lacZ gene. Patent documents 2 to 5 further describe different fucosyltransferases that produce 2-FL or 3-FL in genetically modified microorganisms. Non-patent document 3 describes the biosynthesis of 2-FL or 3-FL in genetically modified Escherichia coli and a multiple modular optimization strategy therefor (Non-patent document 3). Patent document 4 describes Escherichia coli expressing a sugar exporter that enhances the release of 2-FL into the medium.

[0007] 2-FL consists of lactose and fucose (Non-Patent Document 4). The biosynthetic pathway of 2-(3-)-FL in Escherichia coli is shown schematically in Figure 1. E. coli can take up the precursors glucose and lactose into the cell via specific transporters. Glucose is absorbed via the phosphotransferase system and converted to glucose 6-phosphate. The latter functions as a carbon and energy source to promote growth and as a precursor to GDP-L-fucose, an intermediate in colanic acid biosynthesis. The de novo biosynthetic pathway of GDP-L-fucose is derived from the glycolytic intermediate fructose 6-phosphate and is based on the enzymes ManA (mannose 6-phosphate isomerase), ManB (phosphomannomutase), GTP-dependent ManC (mannose 1-phosphate guanylyltransferase), Gmd (GDP-D-mannose 4,6-dehydratase), and NADPH-dependent WcaG (GDP-L-fucose synthase), all of which are derived from Escherichia coli (Figure 2). The second precursor, lactose, is synthesized from lactose / H +It is absorbed into cells via the symporter lactose permease LacY. The key enzyme for 2-FL production is α-1,2-fucosyltransferase (FutC) from Helicobacter pylori, which transfers the fucose moiety of the donor GDP-L-fucose to the acceptor lactose to produce 2-FL (Figure 2). By replacing the futC gene with the futA gene from H. pylori, which encodes α-1,3-fucosyltransferase, this biosynthetic pathway can be utilized to produce 3'-fucosyllactose.

[0008] Despite recent advances, there remains a need for methods to improve the biotechnological production of HMOs, particularly 2-FL and / or 3-FL, in genetically modified microorganisms. The present invention significantly improves the synthesis of HMOs and their release into the medium by the known 2-FL biosynthetic pathway in E. coli. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 7,521,212 [Patent Document 2] European Patent No. 2675899 [Patent Document 3] European Patent No. 2877574 [Patent Document 4] European Patent No. 2440661 [Patent Document 5] European Patent No. 3191499 [Non-patent literature]

[0010] [Non-Patent Document 1] Petschacher and Nidetzky, J Biotechnol. 2016, 235:61-83 [Non-patent document 2] Bode, Early Hum Dev. 2015; 91(11):619-22 [Non-patent document 3] Huang et al., Metab Eng. 2017;(41):23-38 [Non-patent document 4] Pereira & McDonald, Sci Rep. 2012; 84(1):637-655 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention provides a microorganism that improves HMO production and a method for producing HMO using the same. [Means for solving the problem]

[0012] In particular, the present invention provides microorganisms containing an inducible lytic system that can facilitate the extraction of intracellular and extracellular HMOs without the need for further processing steps to obtain the intracellular fraction. Preferred examples of inducible lytic systems include those containing Mg 2+ There is an autoinducing bacteriolytic system regulated by Mg 2+ Deficiency conditions, i.e., extracellular Mg 2+ When the concentration of HMOs falls below a certain threshold, the cells are lysed. When the cells are lysed, the intracellular HMOs are released into the medium, increasing the HMO concentration in the medium. This can advantageously increase the yield of HMOs and save time and money.

[0013] The present invention also provides a microorganism comprising an exogenous gene encoding a lactose permease, preferably a LacY mutant that is less susceptible to inhibition by intracellular lactose compared to wild-type LacY, which can advantageously improve HMO yields.

[0014] The present invention further provides a method for producing HMO using the above-described microorganism of the present invention.

[0015] The present invention also provides a medium in which the microorganism of the present invention is cultured in the method for producing HMO, and purified HMO obtained by the method for producing HMO of the present invention.

[0016] Preferred embodiments are as follows: 1. Genetically modified microorganisms that produce oligosaccharides found in human milk. 2. The genetically modified microorganism described in 1., wherein the microorganism is a bacterium. 3. A genetically modified microorganism according to 1. or 2., wherein the microorganism is Escherichia coli. 4. A genetically modified microorganism according to any one of 1. to 3., characterized in that the genetically modified microorganism comprises an inducible lysis system. 5. The genetically modified microorganism described in 4., wherein the inducible lytic system is autoinducible. 6. The induction lysis system is Mg 2+ 4. or 5. A genetically modified microorganism according to 4. or 5., wherein the gene is regulated by 7. The induced lysis system is free from exogenous free Mg. 2+ The genetically modified microorganism according to any one of 4. to 6., wherein the concentration is adjusted. 8. The microorganism is Mg 2+ 8. The genetically modified microorganism according to any one of 4. to 7., which comprises a regulatable promoter. 9. Said Mg 2+ 9. The genetically modified microorganism according to 8., wherein the regulatable promoter is the Pmgt promoter derived from the mgtB gene of Salmonella typhimurium or the PmgtA promoter of Escherichia coli. 10. The microorganism further comprises additional Mg 2+ 10. The genetically modified microorganism according to 8. or 9, comprising a regulatory element, preferably the 5'UTR of the mgtA gene of Escherichia coli. 11. The genetically modified microorganism described in any one of 4. to 10., wherein the microorganism contains a lytic gene. 12. The genetically modified microorganism according to 11, wherein the lysis gene is a lysis gene derived from a bacteriophage. 13. The genetically modified microorganism according to 12, wherein the bacteriophage is a lambda bacteriophage. 14. The genetically modified microorganism according to 13, wherein the lytic gene is the lambda bacteriophage lytic gene SRz or the lambda bacteriophage mutant Sam7 lytic gene. 15. The lytic gene is a cytoplasmic ... 2+ 15. The genetically modified microorganism according to any one of 11 to 14, which is inducibly expressed under starvation conditions. 16. The microorganism is Mg 2+ a regulated promoter and a lytic gene, wherein expression of the lytic gene is regulated by Mg 2+ A regulated promoter and, optionally, additional Mg 2+ 16. The genetically modified microorganism according to any one of 4. to 15, wherein the genetically modified microorganism is controlled by a regulatory element. 17. The genetically modified microorganism described in any one of 1. to 16., wherein the genetically modified microorganism is unable to decompose lactose into glucose and galactose. 18. A genetically modified microorganism of any of 1. to 17. in which the lac operon is inactivated. 19. A genetically modified microorganism described in any one of 1. to 18., wherein the microorganism contains an exogenous gene encoding a lactose permease. 20. The genetically modified microorganism described in 19., wherein the lactose permease is Escherichia coli LacY protein. 21. A genetically modified microorganism according to 19 or 20, which has the A198V and / or S209I mutations in the lactose permease. 22. A genetically modified microorganism described in any one of 1. to 20., wherein the human milk oligosaccharide is 2'-fucosyllactose and / or 3'-fucosyllactose. 23. A genetically modified microorganism described in any one of 1. to 22., which contains an exogenous gene encoding a fucosyltransferase. 24. The genetically modified microorganism according to 23., wherein the fucosyltransferase is α-1,2-fucosyltransferase and / or α-1,3-fucosyltransferase. 25. A genetically modified microorganism according to item 23 or 24, wherein the gene encoding the fucosyltransferase is a heterologous gene and / or is driven by a heterologous promoter. 26. A genetically modified microorganism described in any one of 23. to 25., wherein the codons of the gene encoding the fucosyltransferase have been optimized for expression in the microorganism. 27. The genetically modified microorganism according to any one of 23 to 26, wherein the gene encoding the fucosyltransferase is futC derived from Helicobacter pylori or futA derived from Helicobacter pylori. 28. A genetically modified microorganism described in any one of 1. to 27., which contains a heterologous gene encoding a chaperone. 29. The genetically modified microorganism according to 28, wherein the chaperone is human Hsp70. 30. The genetically modified microorganism according to any one of 1. to 29., wherein the microorganism overexpresses a transcriptional regulatory protein that enhances expression of a gene involved in the de novo synthesis of GDP-L-fucose. 31. A genetically modified microorganism according to any one of 1. to 30., in which the rcsA gene is overexpressed. 32. A genetically modified microorganism according to 30. or 31., wherein the rcsA gene is derived from Escherichia coli. 33. A genetically modified microorganism according to any one of 1. to 32., in which the gene encoding the Lon protease family protein is inactivated, preferably by deletion. 34. The genetically modified microorganism according to 33, wherein the Lon protease family protein is Escherichia coli Lon protease. 35. A genetically modified microorganism according to any one of 1. to 34., in which the wcaJ gene is inactivated, preferably inactivated by deletion. 36. A genetically modified microorganism described in any one of 1. to 35., in which the zwf gene and the two pntAB genes are overexpressed. 37. The genetically modified microorganism according to 36, wherein the zwf gene and the pntAB gene are derived from Escherichia coli. 38. A genetically modified microorganism described in any one of 1. to 37., in which the gsk gene is overexpressed. 39. A genetically modified microorganism according to 38., wherein the gsk gene is derived from Escherichia coli. 40. A genetically modified microorganism described in any one of 1. to 39., in which the trxA gene is overexpressed. 41. The genetically modified microorganism according to 39., wherein the trxA gene is derived from Escherichia coli. 42. A genetically modified microorganism according to any one of 1. to 41., in which the trxB gene is inactivated, preferably by deletion. 43. A method for producing human milk oligosaccharides, comprising the steps of: (a) A method for culturing a genetically modified microorganism according to any one of 1. to 42. in a medium. A method comprising: 44. A method for producing human milk oligosaccharides according to 43., wherein the medium further contains lactose. 45. The medium contains free Mg 2+ 45. The method for producing human milk oligosaccharides according to 43. or 44., further comprising: 46. ​​Free Mg in the medium before the start of the culture 2+ 45. The method for producing human milk oligosaccharides according to 45., wherein the concentration is 5 mM or more. 47. Free Mg in the medium 2+ 45. The method for producing human milk oligosaccharides according to 46. or 47., wherein the concentration is 10 μM or less at the end of the culture. 48. A method for producing human milk oligosaccharides described in any one of 43. to 47., wherein the culture is a batch culture, or optionally a fed-batch culture. 49. A method for producing human milk oligosaccharides according to any one of items 43 to 48, wherein the volume of the culture medium is 10 L or more, preferably 100 L or more. 50. A method for producing human milk oligosaccharides described in any one of 43. to 49., wherein the concentration of the human milk oligosaccharides in the culture medium is at least 15 g / L, preferably at least 20 g / L, at the end of the culture. 51. A method for producing human milk oligosaccharides described in any one of 43 to 50, wherein the human milk oligosaccharides are 2'-fucosyllactose and / or 3'-fucosyllactose. 52. Further comprising the steps of: (b) purifying the human milk oligosaccharides from the culture medium and / or the microorganism itself; 52. The method for producing human milk oligosaccharides according to any one of 43. to 51., comprising: 53. A method for producing human milk oligosaccharides according to 52., wherein the human milk oligosaccharides are purified from the culture medium after inducing lysis via an inducible lysis system. 54. Free Mg in the medium 2+ 52. The method for producing human milk oligosaccharides according to 53., wherein the human milk oligosaccharides are purified from the culture medium when the concentration of is 10 μM or less and / or when the lysis can be directly visualized. 55. The lysis occurs when the OD of the culture 600 54. The method for producing human milk oligosaccharides described in 54., wherein the decrease in the value can be directly visualized. 56. A culture medium obtained by the method described in any one of 43. to 51. 57. Human milk oligosaccharides obtainable by the method described in any one of 52. to 55. 58. Use of the culture medium of 56. or the human milk oligosaccharides of 57. in preparing animal feed. [Brief explanation of the drawings]

[0017] [Figure 1]FIG. 1 shows the biosynthesis of 2'-(3')-fucosyllactose in E. coli. Glc-6-P: glucose 6-phosphate; Fru-6-P: fructose 6-phosphate; Man-6-P: mannose 6-phosphate; 6-P-Gluc: 6-phosphate-gluconate; Ribu-5-P: ribulose 5-phosphate; Ribo-5-P: ribose 5-phosphate; PPP: phosphoribosyl pyrophosphate; gal: galactose; glc: glucose; XMP: xanthosine 5'-monophosphate; IMP: inosine 5-monophosphate; Pgi: phosphoglucose isomerase; ManA: mannose 6-phosphate isomerase; ManB: phosphomannomutase; ManC: α-D-mannose 1-phosphate guanylate transferase; Gmd: GDP-mannose 6-dehydrogenase; WcaG: GDP-L-fucose synthase; WcaJ: UDP-glucose carrier transferase; Wca AF, IM: colanic acid biosynthetic genes; LacA: β-galactoside transacetylase; LacZ: β-galactosidase; LacY: lactose permease; Lon: protease; ClpYQ (HslUV): protease; RscA: positive transcriptional regulator; guaA: GMP synthetase; GuaB: IMP-dehydrogenase; GuaC: GMP-reductase; DeoD: purine nucleotide phosphorylase; Gpt: guanine phosphoribosyltransferase; Gnd: 6-phosphogluconate dehydrogenase; Ndk: nucleotide diphosphate kinase. [Figure 2] FIG. 1 shows the E. coli expression vector pETDuet-1. The vector contains a dual T7 promoter (PT7), a T7 terminator (TT7), a ColE1 ori, an ampicillin resistance gene (ampR), and a lac repressor (lacI). [Figure 3]FIG. 1 shows pET-manCB-gmd-wcaG, in which the E. coli expression vector pETDuet-1 comprises a dual T7 promoter (PT7), a T7 terminator (TT7), a pBR322 on, an ampicillin resistance gene (ampR), and a lac repressor (lacI), and also comprises manB: phosphomannomutase, manC: α-D-mannose 1-phosphate guanylyltransferase, gmd: GDP-mannose 6-dehydrogenase, and wcaG: GDP-L-fucose synthase. [Figure 4] FIG. 1 shows the E. coli expression vector pCDFDuet-1E, which contains a dual T7 promoter (PT7), a T7 terminator (TT7), a CloDF13 ori, a streptomycin resistance gene (SmR), and a lac repressor (lacI). [Figure 5] FIG. 1 shows the E. coli cloning vector pUC19, which contains the ampicillin resistance gene (AmpR), the lacZ alpha peptide (lacZ'), and the on. [Figure 6] FIG. 1 shows the E. coli expression vector pCDF-futC-lacY*-hHSP70-trxA-SRz. The E. coli expression vector pCDFDuet-1 contains a dual T7 promoter (PT7), a T7 terminator (TT7), a CloDF13 ori, a streptomycin resistance gene (SmR), and a lac repressor (lacI), and contains the following genes: futC: α-1,2-fucosyltransferase; lacY*: S209I-containing lactose permease; hHSP70: human heat shock protein 70; trxA: thioredoxin; SRRz: 5'-UTR; and a lysis gene from bacteriophage lambda under the control of the mtgA promoter with the 3'LuxICDABEG terminator. [Figure 7] FIG. 1 shows the E. coli expression vector pCOLADuet-1, which contains a dual T7 promoter (PT7), a T7 terminator (TT7), a ColA ori, a kanamycin resistance gene (KmR), and a lac repressor (lacI). [Figure 8]FIG. 1 shows the pCOLA-pntAB-rcsA-zwf-gsk vector. The E. coli expression vector pCOLADuet-1 contains a dual T7 promoter (PT7), a T7 terminator (TT7), a ColA ori, a kanamycin resistance gene (KmR), and a lac repressor (lacI), and also contains pntAB: membrane-bound transhydrogenase, rcsA: positive transcriptional regulator of colanic acid biosynthesis, zwf: glucose-6-phosphate dehydrogenase, and gsk: guanosine inosine kinase. DETAILED DESCRIPTION OF THE INVENTION

[0018] [Definitions and general methods] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms "comprise" and "comprising" may each be used interchangeably with the terms "consists of" and "consisting of," where appropriate. The terms "include" and "including" may each be used interchangeably with the term "consists of" and "consisting of" where appropriate.

[0019] The term "genetically modified" refers to any alteration introduced into the genetic material of a cell, including the introduction of new genetic material, deletion of genetic material, or mutation of genetic material. The term "inducible lysis system" means that cell lysis may be induced by an external stimulus. The term "autoinducible" means that the external stimulus does not necessarily have to be actively applied by the skilled artisan practicing the invention. The term "external stimulus" refers to a chemical or physical stimulus applied to a cell from outside the cell.

[0020] The term “Mg 2+ "Adjustment" refers to the free magnesium (Mg 2+ The term "exogenous free Mg" refers to the regulation of a substance by the presence or absence of the ion. 2+ "Mg concentration" refers to the amount of free Mg acting on cells from the outside, such as the medium surrounding the cells. 2+ The term "Mg" refers to the concentration of ions. 2+ "Deficient conditions" refers to the absence of free Mg in the medium. 2+ Low concentration or free Mg in the medium 2+ means that there is no such thing as

[0021] A "lytic gene" is a gene that encodes a factor, for example, a protein, which, when expressed intracellularly, causes cell lysis. The term "inducibly expressed" refers to a gene that is not constitutively expressed; instead, expression may be induced by an external stimulus.

[0022] The term "lytic genes" refers to Mg 2+ "Controlled by a regulated promoter" refers to the free Mg 2+ Depending on the presence or absence of an ion, the gene is expressed or not expressed when the promoter is active or inactive, respectively. "Free Mg 2+ "Ions" refers to those dissolved in, for example, the culture medium, that are not complexed with a chelating agent such as EDTA. That is, for example, by adding a chelating agent to the free Mg in the medium. 2+ In this specification, unless otherwise specified, the term "Mg 2+ " is "Free Mg 2+ "Ion".

[0023] The term "exogenous gene" refers to a gene that is introduced into a cell from outside the cell, for example, by gene transfer. However, the term "exogenous gene" does not dictate the origin of the gene; for example, an exogenous gene in E. coli may be derived from E. coli or from another organism. An exogenous gene may be episomal, present in an (expression) vector, such as a plasmid, or may be integrated into the chromosome of the cell.

[0024] The term "heterologous gene" refers to a gene that is introduced into a cell from another organism than the cell into which it is introduced; for example, a heterologous gene in E. coli does not originate from E. coli. A heterologous gene may be episomal, for example present in an (expression) vector, such as a plasmid, or may be integrated into the chromosome of the cell.

[0025] A "heterologous promoter" refers to a promoter that, when operably linked to a gene, is not the promoter naturally associated with that gene.

[0026] Exogenous, or heterologous, genetic material can be introduced into microorganisms by techniques commonly known in the art. For example, genetic material can be introduced into bacteria by gene transfer. Gene transfer can be performed, for example, by heat shock of chemically competent bacteria or by electroporation of electrocompetent bacteria (see, e.g., Hanahan et al., Methods Enzymol. 1991;204:63-113; Miller and Nickoloff, Methods Mol Biol. 1995;47:105-13).

[0027] Exogenous, or heterologous, genetic material can also be integrated into the chromosomes of a cell by techniques commonly known in the art, such as lambda Red-mediated integration or CRISPR / Cas9 (see, e.g., Juhas and Ajioka, Microb Cell Fact. 2016;15(1):172; Reisch and Prather, Sci Rep. 2015;5:15096).

[0028] A gene "derived from" a particular organism, such as E. coli, may be a respective gene (obtained directly from) said organism (i.e., without modifying the sequence by mutation and / or codon optimization), or may include mutants obtained by introducing (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3) mutations into the original sequence from the organism, and / or codon-optimized variants are also included.

[0029] The term "codon optimized for expression in a microorganism" means that the nucleic acid sequence of the open reading frame (ORF) of a gene is optimized for translation into a protein in the organism into which the gene is introduced.

[0030] When a microorganism according to the present invention is identified as containing a gene encoding a particular protein, it is intended that the microorganism also expresses the gene to produce the encoded protein.

[0031] The term "overexpressed" or "overexpression" in the context of a gene refers to a higher expression level of the gene in a microorganism in which the gene is overexpressed than the expression level of the same gene in the same microorganism in which the gene is not overexpressed. Genes can be overexpressed by techniques commonly known in the art. For example, additional copies of an exogenous gene can be introduced into the cell, the cell can be treated with an exogenous transcriptional activator, or an exogenous gene encoding a transcriptional activator can be introduced into the cell. In the present invention, introduction of additional copies of the gene is preferred.

[0032] "Inactivation" of a gene refers to the failure of the gene to express any functional protein. This includes near-total or complete absence of expression of a dysfunctional protein. Near-total or complete absence of expression can be achieved, for example, by deletion of a regulatory sequence such as a promoter, deletion of (part of) the open reading frame (ORF) of the gene, or deletion of the entire gene including the promoter and ORF. Thus, gene knockout is also encompassed by the term. Expression of a dysfunctional protein can be achieved, for example, by partial deletion, point mutation, and / or insertion in / to the ORF, resulting in expression of, for example, a truncated and / or catalytically inactive protein. Methods for introducing deletions, point mutations, and / or insertions are well known in the art. See, for example, Reisch and Prather, 2015 (supra), Zerbini et al. (Microb Cell Fact. 2017; 16(1): 68), or Jensen et al. (Sci Rep. 2015; 5: 17874).

[0033] Specific nucleotide sequences, such as sequences underlying a particular gene of interest or promoter sequences, can be amplified by polymerase chain reaction from the genomic sequence of an organism such as E. coli, or can be chemically synthesized by methods generally known in the art.

[0034] GenBank Accession number refers to a unique identifier used in GenBank, which may be accessed, for example, at https: / / www.ncbi.nlm.nih.gov / genbank / . Gene identifier (GI) refers to a unique number used to identify a particular gene, which may be queried, for example, at https: / / www.ncbi.nlm.nih.gov / gene / .

[0035] [Embodiment] <Genetically Recombinant Microorganisms for Producing HMO> The present invention relates to the provision of genetically recombinant microorganisms for producing HMO.

[0036] The HMO produced in the present invention is based on the disaccharide lactose or N-acetyl lactosamine. HMO based on lactose is preferred.

[0037] Preferred oligosaccharides in human milk related to the present invention include, but are not limited to, 2'-fucosyllactose (2-Fucoyllactose (FL)), 3'-fucosyllactose (3-FL), 3'-sialyllactose (3-SL), 6'-sialyllactose (6-SL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-hexaose (LNH), iso-lacto-N-octaose, iso-lacto-N-neooctaose, para-lacto-N-octaose, lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), LS-tetraose a (LST a), LS-tetraose b (LST b), LS-tetraose c (LST c), and disialyllacto-N-tetraose (DSLNT).

[0038] [[ID=A]]

[0039] The trisaccharides of 2-FL and / or 3-FL are preferably produced in the present invention, and 2-FL is particularly preferred. The structures of 2-FL and 3-FL are shown in Non-Patent Document 4.The present invention is described in more detail with respect to the genetic engineering and pathway for the production of 2-FL and / or 3-FL in the enterobacterial bacterium Escherichia coli (E. coli) K-12. However, the present invention is equally applicable to the production of other HMOs in E. coli or microorganisms other than E. coli. Microbial production of HMOs other than 2-FL and / or 3-FL has been previously reported. For example, Priem et al. showed that expression of the β1,3-N-acetylglucosaminyltransferase LgtA (e.g., GI:904226) from Neisseria meningitides could be used to glycosylate lactose to yield GlcNAc-β1,3-Galβ1,4-Glc. This can be further elongated to lacto-N-neotetraose (LNnT) by the β1,4-galactosyltransferase LgtB (e.g., GI:904227), also from Neisseria meningitidis (Priem et al., Glycobiology. 2002;12(4):235-40). Furthermore, intracellular fucosylation of LNnT catalyzed by the β1,3-fucosyltransferases FutA and FutB of Helicobacter pylori can produce a mixture of HMOs, with lacto-N-neodifucohexaose II (LNnDFHII) as the main product (Dumon et al., Biotechnol Prog. 2004;20(2):412-9). Han et al. further discuss different production methods (Han et al., 2012, Biotechnol Adv. 2012;30(6):1268-78).

[0040] The microorganisms used in the present invention are not particularly limited. Preferably, they are microorganisms that have the inherent ability to produce HMOs, or that can produce precursor molecules that produce HMOs intracellularly, or that can introduce the precursors from the extracellular space. Examples of microorganisms include bacteria and yeast. Examples of bacteria include Escherichia coli, Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus can 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 Similarly, bacteria of the genera Lactobacillus and Lactococcus may be modified using the methods of the present invention, including Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Examples of suitable bacterial species include, but are not limited to, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophilus and Propionibacterium freudenreichii are also suitable bacterial species for the present invention.Also included as part of the present invention are bacteria of the genus Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), the genus Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), bacteria of the genus Spororolactobacillus, bacteria of the genus Micromospora, bacteria of the genus Micrococcus, bacteria of the genus Rhodococcus, and bacteria of the genus Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa). Examples of yeast include strains modified from Saccharomyces cerevisiae, Kluyveromyces lactis, and Pichia pastoris. The microorganism is preferably a bacterium, with Escherichia coli being particularly preferred. There is no particular limitation on the Escherichia coli strain used.

[0041] The release of HMOs from bacterial cells is an essential process for industrial production. Approximately 50% of the 2-FL yield remains intracellularly, and the remainder is released into the medium (Non-Patent Document 3). To obtain the entire amount of 2-FL, heat-induced lysis at 100°C is required. In the microorganism of the present invention, an autolytic system is established to improve the recovery process. That is, the microorganism of the present invention may contain an inducible lysis system. The type of the lysis system is not particularly limited as long as lysis can be induced by an external stimulus. For example, the lysis genes of bacteriophage lambda (S, R, and Rz) are capable of activating Mg 2+ -It can be controlled by the repressible PmgtA-UTR promoter (Tamekou Lacmata et al., Bioengineered. 2017;26:1-6). Free Mg2+ When Mg is consumed (for example, to 10 μM or less), the synthesis of lytic genes is induced, and the degradation of the cell wall component peptidoglycan promotes the release of 2-FL into the medium. 2+ Regulatory promoters include the nickel-inducible system described by Liu and Curtiss (Proc Natl Acad Sci USA. 2009;106(51):21550-4), the thermosensitive lambda repressor (Juhas et al., PLoS One. 2016;11(10):e0165778), the nisin-inducible promoter (Visweswaran et al., Appl Microbiol Biotechnol. 2017 Feb;101(3):1099-1110), the arabinose-inducible promoter (Masuda et al., FEMS Microbiol Lett. 2016;363(6)), the arabinose-inducible promoter in a MgSO4-opposed system (Li et al., Curr Microbiol. 2016;72(4):390-6), or lytic induction by solvent addition (Hajnal et al., Appl Microbiol There are several alternatives available for establishing autolytic systems based on the ELISA kit (Biotechnol. 2016;100(21):9103-9110). These systems are also summarized in Table 1.

[0042] Table 1 Induced autolytic system

[0043] [Table 1] Mg 2+ Regulatory systems include, for example, ethylenediaminetetraacetic acid (EDTA), (Ca 2+ and Mg 2+ The addition of a chelating agent that sequesters metal ions (such as divalent metal ions) can be advantageous in promoting cell lysis. 2+is required for α-1,2- and α-1,3-fucosyltransferase activity (Zhang et al., Glycobiology. 2010;20(9):1077-88; Zhao et al., Chem Commun (Camb). 2016;52(20):3899-902). 2+ In the presence of Mg, 2'-fucosyllactose synthesis can be improved due to the promotion of enzyme activity. 2+ In the absence of Mg, cell lysis is induced through cell wall disruption, facilitating product recovery. 2+ The regulatory system is preferably used in the present invention, and preferably Mg 2+ The regulatory system is Mg 2+ It is inhibitory.

[0044] In the induced lysis system, Mg 2+ Regulated promoters can be used. 2+ The regulated promoter preferably requires exogenous free Mg 2+ The concentration is adjusted. Preferably, Mg 2+ Active under Mg deficiency conditions. 2+ The depletion condition is when the growth medium contains free Mg 2+ Preferably, the condition includes 500 μM or less, 100 μM or less, 50 μM or less, 10 μM or less, 1 μM or less, or 0 μM of Mg. 2+ Deficiency conditions include free Mg 2+ However, it is less than 10 μM. 2+The regulatable promoter is preferably the PmgtB promoter derived from the mgtB gene of Salmonella typhimurium (e.g., GI: 1250998), the PpagC promoter derived from the pagC gene of Salmonella typhimurium (e.g., 1248241), or the PmgtA promoter derived from the mgtA gene of Escherichia coli (e.g., GI: 948778). The PmgtB promoter derived from the mgtB gene of Salmonella typhimurium is, for example, as described in Zhang et al. (2009, supra), and can be obtained, for example, by polymerase chain reaction (PCR) using the Salmonella typhimurium genome as a template and the following primers: 5'-GTATACTCCGGAGCAAAACGCCTGAACTCC-3' (SEQ ID NO: 1) and 5'-TCTAGAGAAGAAAGAAAGAAAGTACGTGCTATTTTAG-3' (SEQ ID NO: 2). The PmgtA promoter derived from Escherichia coli is described, for example, in Tamekou Lacmata et al., 2017 (ibid.), and can be obtained, for example, by polymerase chain reaction (PCR) using the Escherichia coli genome as a template and the following primers: 5'-CGAGCTCCTTTTTTTTATTCATCATCCGCG-3' (SEQ ID NO: 3) and 5'-CGAGCTCGCGCGATAATATACCTCTGGGC-3' (SEQ ID NO: 4).

[0045] Mg 2+ Regulated promoters include additional Mg promoters, such as the 5'UTR of the mgtA gene of E. coli. 2+ The 5' UTR of the mgtA gene from Escherichia coli is as described in, for example, Tamekou Lacmata et al., 2017 (supra), and the PmgtA promoter, together with the mgtA 5' UTR, can be obtained by polymerase chain reaction (PCR) using, for example, the E. coli genome as a template and primers 5'-CGAGCTCTTTTTTTTATTCATCAGCCCG-3' (SEQ ID NO: 3) and 5'-CGAGCTCAAGGTCCTCCCGCACTGT-3' (SEQ ID NO: 5).

[0046] A microorganism containing an inducible lysis system may further contain one or more lysis genes. The lysis gene is not particularly limited, as long as it is capable of lysing the cell when expressed intracellularly. For example, the lysis gene may be derived from a bacteriophage such as lambda bacteriophage, MS2 bacteriophage, phiX174 bacteriophage, or RLT bacteriophage. Examples of lysis genes include ydfD from Escherichia coli, sicG from Streptococcus dysgalactiase subsp. equisimilis, vanX from Escherichia coli, SRRz from Salmonella phage P22, yncE from Salmonella enterica serovar Paratyphi A, and lysis genes from Staphylococcus aureus phage P68, as described by Masuda et al., 2016 (supra).

[0047] Preferred examples of lytic genes include the SRRz gene from lambda bacteriophage (e.g., as found in the sequence of GenBank Accession No. NC_001416), its Sam7 mutant in which G at position 161 of the S gene is mutated to A, the MS2 phage lytic gene encoding the E protein, or the phiX174 bacteriophage lytic gene encoding the L protein. The SRRz gene is, for example, as described in Zhang et al. (2009; supra), and can be obtained by PCR using the lambda bacteriophage genome as a template and primers 5'-CCGCATATGAAAAAAACATGACCTGATGAG-3' (SEQ ID NO: 6) and 5'-TTAGTCGACGCAACTCTTCTTGCTC-3' (SEQ ID NO: 7). The MS2 bacteriophage lytic gene or the phiX174 bacteriophage lytic gene is as described in Juhas et al. (2016; supra).

[0048] The microorganism may also contain two or more lysis genes, either the same or different. In some cases, a combination of two or more different lysis genes can be used, as different mechanisms of action may lead to more efficient lysis. For example, the microorganism may contain lysis genes from lambda and MS2 bacteriophages, lambda and phi X174 bacteriophages, MS2 and phi X174 bacteriophages, or lambda, MS2, and phi X174 bacteriophages. However, the present invention is not limited to these combinations, and the other lysis genes described may be used alone or in combination. Preferably, the two or more lysis genes are arranged in tandem, polycistronic, under the control of the same promoter and the same, and optionally additional, regulatory elements.

[0049] One or more lytic genes can be 2+ It can be inducibly expressed under conditions of Mg deficiency, preferably with or without additional regulatory elements. 2+ When the 5'UTR of the mgtA gene is used as an additional regulatory element, the expression cassette of the lysis gene is preferably 2+ It contains, in that order, a regulatable promoter, the 5'UTR of the mgtA gene, and the ORF of a lysis gene.

[0050] The expression cassettes that can be used for inducible lysis as described above can be present in the microorganism as an episomal vector, such as an expression vector, for example a plasmid, or can be integrated into the host chromosome.

[0051] Furthermore, since lactose is a precursor of HMOs, a high intracellular level of lactose is desirable for intracellular production of HMOs. Therefore, the microorganism of the present invention may not be capable of cleaving lactose into glucose and galactose. This can be achieved by inactivating the lacZ gene encoding the β-galactosidase LacZ, which is involved in lactose cleavage, or by inactivating the entire lac operon encoding LacZ, the lactose repressor LacI, the transacetylase LacA, and the lactose permease LacY. Inactivation can be achieved by expression inhibitors, by introducing inactivating mutations, or by deletion. Inactivating the entire lac operon, including the lacI, lacZ, lacY, and lacA genes, can increase the availability of lactose in the cells by i) preventing the breakdown of lactose into glucose and galactose and ii) preventing the production of allolactose or acetyllactose. The latter can further simplify the purification process by removing the two sugar molecules from the mixture.

[0052] Lactose permease is responsible for the uptake of lactose into cells. Many bacteria possess the inherent ability to transport lactose from the medium into cells using transport proteins that are either homologs of the E. coli lactose permease (e.g., as found in Bacillus licheniformis) or members of the PTS sugar transport family (e.g., as found in Lactobacillus casei and Lactobacillus rhamnosus). Therefore, introduction of an exogenous lactose permease gene, such as E. coli LacY, can increase intracellular lactose levels. For bacteria lacking the inherent ability to transport extracellular lactose into cells (including bacteria in which the entire lac operon has been inactivated), this function can be imparted or restored by introducing an exogenous lactose permease gene, such as E. coli lacY. Therefore, the microorganisms of the present invention may contain an exogenous gene encoding a lactose permease. Preferably, the lactose permease is E. coli LacY (e.g., GI:949083). It is particularly preferred that E. coli LacY contains the A198V and / or S209I mutations (Wilson et al., Biochim Biophys Acta. 1990;1029(1):113-6). Both mutations are predicted to disrupt the interaction between LacY and its inhibitor protein, Enzyme-IIaGlc (Hogema et al., Mol Microbiol. 1999;31(6):1825-33). As a result, the autoregulatory mechanism may be suppressed when intracellular lactose concentrations are high. Thus, HMO yields may be increased by increasing lactose uptake. Furthermore, because lactose is a natural inducer of the T7 promoter expression system (described below), the expression of enzymes required for 2-FL synthesis, for example, may be further improved by increasing the availability of intracellular lactose.

[0053] Preferably, the microorganism of the present invention is for producing 2-FL and / or 3-FL.

[0054] Exemplary genetic modifications that enhance 2-FL production in E. coli are shown in Table 2. Table 2 Genetic modifications that enhance 2'-fucosyllactose production in E. coli

[0055] [Table 2] 2-FL or 3-FL can be produced by coupling the activated sugar GDP-L-fucose to lactose. As mentioned above, E. coli expresses the lactose permease LacY (lactose / H + E. coli can import the precursor molecule lactose into the cell via a phosphotransporter (cotransporter). Furthermore, E. coli can import glucose into the cell via a phosphotransferase system. Glucose can function as both an energy source to promote growth and a precursor for the production of GDP-L-fucose, a natural intermediate in colanic acid biosynthesis, via a de novo pathway (see Figure 1). The de novo pathway for biosynthesis of the activated sugar GDP-L-fucose branches off from the glycolytic intermediate fructose-6-phosphate and is based on the E. coli enzymes ManA and ManB, the GTP-dependent ManC, the NADPH-dependent Gmd, and WcaG. Alternatively, GDP-L-fucose can also be produced from imported fucose via a salvage pathway.

[0056] The fucose moiety of GDP-L-fucose can be transferred to intracellular acceptor lactose via fucosyltransferase (EC 2.4.1.x), thereby producing 2-FL or 3-FL (see Figure 1). The enzyme important for 2-FL production is α-1,2-fucosyltransferase, such as FutC from Helicobacter pylori. For 3-FL production, α-1,3-fucosyltransferase, such as FutA from Helicobacter pylori, can be used. Therefore, the microorganism of the present invention also contains an exogenous gene encoding a fucosyltransferase, preferably an α-1,2-fucosyltransferase and / or an α-1,3-fucosyltransferase. The gene encoding the fucosyltransferase is preferably a heterologous gene and / or is driven by a heterologous promoter. Several fucosyltransferases have been identified in bacteria, such as Bacteroides fragilis, Bacteroides vulgatus, Campylobacter jejuni, Escherichia coli, Helicobacter bilis, H. hepaticus, H. mustelae, Helicobacter pylori, and Thermosynechococcus elongatus. Because codon usage differs among many organisms, codons with limited availability of cognate tRNA anticodons can lead to ribosome stalling and reduced expression of heterologous genes. To ensure high protein expression in the microorganisms of the present invention, the codon usage of the α-1,2- and α-1,3-fucosyltransferase-encoding genes (e.g., futC and futA) can be optimized for the microorganisms of the present invention. Thus, genes encoding fucosyltransferases can be codon-optimized for expression in the microorganisms. Codon optimization may increase intracellular fucosyltransferase protein levels and improve the yield of 2-FL and / or 3-FL.

[0057] Examples of α-1,2-fucosyltransferases that are preferred for use in the present invention include FutC from Helicobacter pylori (e.g., NCBI accession number KY499613), FutL from H. mustelae (e.g., NCBI accession number WP.013023529.1), FutF from H. bilis (e.g., NCBI accession number WP.020995676.1), and FutF from Bacteroides WcfB from Bacillus fragilis NCTC 9343 (e.g., NCBI accession number WP.005817145.1), WbsJ from E. coli O128 (e.g., NCBI accession number AAO3768.1), WbwK from E. coli O86 (e.g., NCBI accession number AA37719.1), WbgL from E. coli O126 (e.g., NCBI accession number ADN43847.1), WbiQ from E. coli O127 (e.g., NCBI accession number CAS09719.1), FutG from Campylobacter jejuni (e.g., NCBI accession number WP_002861859.1), and Bacteroides vulgatus ATCC Examples include FutN from Bacteroides fragilis NCTC9343 (e.g., NCBI accession number ALK85429.1), and WcfB and WcfW from Bacteroides fragilis NCTC9343 (e.g., NCBI accession numbers WP_005817145.1 and WP_005813010.1).

[0058] Examples of preferred α-1,3-fucosyltransferases for use in the present invention include FutA from H. pylori (e.g., NCBI accession number AAD07447.1), FutB from H. pylori 26695 (e.g., NCBI accession number AAD07710.1), FutD from H. trogontum (e.g., NCBI accession number WP_052089242.1), FutE from H. bilis (e.g., NCBI accession number WP_03 48028283.1), FutH from H. typhlonius (e.g., NCBI accession number WP_052082154.1), FutJ (Hh0072) and FutK (Hh1776) from H. hepaticus (e.g., NCBI accession numbers WP_011114915.1 and AAP78373), and FutM from B. fragilis (e.g., NCBI accession number CAH09151.1.1).

[0059] In a preferred embodiment, fucosyltransferase activity is determined by the presence of Mg 2+ The presence of

[0060] Fucosyltransferases can form insoluble intracellular inclusion bodies, which can reduce the availability of active fucosyltransferases within the cells. The presence of an exogenously introduced chaperone may favorably affect the lytic activity of fucosyltransferases, thereby improving the production of 2-FL and / or 3-FL. Therefore, the microorganisms of the present invention may further comprise an exogenous gene encoding a chaperone. In a preferred embodiment, the chaperone is human Hsp70 (e.g., GenBank accession number BC112963). In addition to its chaperone function, human Hsp70 can be utilized to establish an autoinducible expression system without adding IPTG to the culture medium (Briand et al., Sci Rep. 2016;6:33037). Because IPTG is also a substrate for lactose permease, the absence of IPTG can enhance lactose uptake and further improve the production of HMOs, particularly 2-FL and / or 3-FL.

[0061] In addition to the above-mentioned modifications, many other genes can be introduced or inactivated into the cells to further optimize the production of HMOs, particularly 2-FL and / or 3-FL. RcsA is a positive transcriptional regulator of colanic acid biosynthesis in E. coli, which uses GDP-L-fucose as an intermediate. Overexpression of rcsA (e.g., GenBank Accession No. M58003) enhances the transcription of genes involved in the biosynthesis of the GDP-L-fucose intermediate (see Figure 1), thereby enhancing the GDP-L-fucose pathway and improving the synthesis of 2-FL and / or 3-FL (Non-Patent Document 3). Therefore, the microorganism of the present invention may also preferably overexpress the rcsA gene derived from E. coli.

[0062] However, RcsA is subject to the control of Lon and ClpYQ proteases. Therefore, the lon and clpYQ genes can be inactivated to prevent downregulation of the desired GDP-L-fucose biosynthetic genes. Combining rcsA overexpression with lon / clpYQ inactivation can dramatically improve GDP-L-fucose, i.e., 2-FL, production. In other words, in the microorganism of the present invention, the Lon protease family genes, preferably the E. coli Lon protease (e.g., GenBank accession number L20572) and / or the clpYQ gene (NC_000913.3), can be inactivated.

[0063] Furthermore, the wcaJ gene (e.g., GenBank accession number (amino acid) BAA15900) encodes a UDP-glucose lipid carrier transferase predicted to initiate colanic acid biosynthesis. Inactivation of the wcaJ gene i) prevents the consumption of GDP-L-fucose by the competing colanic acid pathway, ii) prevents an increase in culture medium viscosity due to the release of the exopolysaccharide colanic acid, and iii) simplifies the 2-FL purification process by removing other contaminating sugar molecules (colanic acid). Therefore, in the microorganism of the present invention, the wcaJ gene, preferably the E. coli wcaJ gene, may be inactivated.

[0064] The cofactors NADPH and GTP also play important roles in GDP-L-fucose biosynthesis, and their availability can limit the synthesis of activated sugars (GDP-L-fucose) and thus the overall yield of HMOs such as 2-FL and / or 3-FL. Overexpression of the four genes in the microorganisms of the present invention can increase the availability of cofactors for GDP-L-fucose synthesis. Zwf is part of the pentose phosphate pathway, while PntA and PntB belong to the transhydrogenase system. Overexpression of zwf and pntAB can significantly contribute to NADPH regeneration by providing the cofactors required for the NADPH-dependent WcaG enzyme, which is involved in GDP-L-fucose synthesis (Figure 1). Therefore, the microorganism of the present invention can co-overexpress the zwf gene and both pntAB genes (KEGG Entry, E. coli K-12 MG1655: b1852, b1603 and b1602), but preferably they are derived from E. coli.

[0065] The second cofactor, GTP, can be synthesized via two alternative pathways: a de novo GTP biosynthetic pathway via the generation of phosphoribosyl pyrophosphate, or a salvage pathway derived from exogenously delivered guanosine. In either case, a sufficient supply of GMP increases the GTP concentration and improves the efficiency of GDP-L-fucose production. Overexpression of the gsk gene (guanosine inosine kinase, e.g., GI:946584), which is involved in both metabolic pathways, in the microorganisms of the present invention may increase the availability of GTP required for ManC (Figure 1). This may result in dramatic improvements in GDP-L-fucose synthesis and HMO production (Lee et al., Appl Microbiol Biotechnol. 2012;93(6):2327-34). Therefore, the microorganisms of the present invention may overexpress the gsk gene, preferably the gsk gene derived from Escherichia coli.

[0066] Primers for cloning the above exemplary genes can be found, for example, in Table S2 of Non-Patent Document 3 (supra).

[0067] Further optimization may involve manipulating thioredoxin activity. The trxA gene (KEGG Entry, E. coli K-12 MG1655:b3781) encodes the major physiological electron donor in E. coli. Overexpression of thioredoxin (TrxA) is known to promote disulfide bond formation in insoluble proteins, thereby improving heterologous protein expression. However, two other functions have also been associated with TrxA: a potential chaperone function and functioning as a processivity factor for T7-DNA polymerase (Weickert et al., Curr Opin Biotechnol. 1996;7(5):494-9). Therefore, overexpression of trxA may i) improve the solubility of enzymes required for HMO synthesis and ii) positively affect the expression of each gene. Therefore, the microorganism of the present invention may overexpress the thioredoxin trxA gene, preferably the E. coli-derived thioredoxin trxA gene.

[0068] Furthermore, the trxB gene (KEGG Entry, E. coli BL21(DE3):ECD_00892) encodes an NADPH-dependent thioredoxin reductase (TrxB), which regenerates TrxA from its oxidized form. Inactivation of trxB limits the reduction potential in the cytoplasm, allowing disulfide bonds to form (Weickert et al., 1996, supra). Inactivation of trxB i) improves the lytic and enzymatic activity of the protein, and ii) prevents the depletion of the cofactor NADPH required for WcaG (FIG. 1). Therefore, the trxB gene may be inactivated in the microorganism of the present invention.

[0069] [HMO manufacturing method] The present invention also provides a method for producing an HMO using the genetically modified microorganism of the present invention.

[0070] HMO can be produced by culturing the microorganism of the present invention in a medium.

[0071] The medium is not particularly limited as long as it supports the growth of the microorganism. Examples of media for culturing the microorganisms of the present invention include LB medium (10 g / L tryptone; 5 g / L yeast extract; 10 g / L NaCl, pH 7.5), modified M9 minimal medium containing glucose (or glycerol) as a carbon source (36 g / L glucose; 12.8 g / L NaHPO·7H2O, 3 g / L KH2PO4, 2 g / L NH4Cl, 0.5 g / L NaCl, 0.25 g / L MgSO4·7H2O, 14.7 mg / L CaCl2·2H2O, 10 mg / L thiamine, 2 g / L yeast extract, 0.1% (v / v) Triton-X 100, and 1 mL / L trace metal solution; stock metal solution: 25 g / L FeCl3·6H2O, 2 g / L CaCl2·2H2O, 2 g / L ZnCl2, 2 g / L Na2MoO4·2H2O, 1.9g / L CuSO4·5H2O, 0.5g / L H3BO3, pH 7.2 (non-patent document 3, see above), 2.68g / L (NH4)2SO4, 1g / L (NH4)2-H-citric acid, 26.42g / L glycerol, 14.6g / L K2HPO4, 0.241g / L MgSO4, 2g / L Na2SO4, 4g / L NaH2PO4·H2O, 0.5g / L NH4Cl, 10mg / L thiamine, and 3ml / L trace element solution (0.5g / L CaCl2·2H2O, 16.7g / L FeCl3·6H2O, 20.1g / L Na2-EDTA, 0.18g / L ZnSO4·7H2O, 0.1g / L The medium was either minimal medium (Baumgaertner et al., Microb Cell Fact. 2013;12:40) containing 20 g / L glycerol, 13.5 g / L KH2PO4, 4.0 g / L (NH4)2HPO4, 1.7 g / L citric acid, 1.4 g / L MgSO4·7H2O, 10 ml / L trace element solution (10 g / L iron(III) citrate, ZnSO4·7H2O, 1.0 g / L CuSO4·5H2O, 0.35 g / L MnSO4·H2O, 0.23 g / L Na2B4O7·10H2O, 0.11 g / L (NH4)6Mo7O 24, and minimal medium containing 2.0 g / L CaCl2·2H2O (Chin et al., J Biotechnol. 2016. pii:S0168-1656(16)31632-7).

[0072] The microorganism of the present invention may be cultured at any temperature suitable for growth, ie, a temperature between 16°C and 37°C, for example, between 16°C and 30°C, preferably between 16°C and 25°C.

[0073] In a preferred embodiment, the medium contains lactose. The lactose concentration in the medium before the start of culture is 20 g / L or more, 12 g / L or more, 10 g / L or more, 8 g / L or more, 8 to 20 g / L, 10 to 20 g / L, or 12 to 20 g / L. Preferably, the lactose concentration in the medium before the start of culture is 10 to 20 g / L.

[0074] In a further preferred embodiment, the medium contains free Mg 2+ Includes.

[0075] Free Mg in the medium before starting culture 2+ concentration (i.e., free Mg 2+ The concentration) may be 80 mM or more, 70 mM or more, 60 mM or more, 50 mM or more, 40 mM or more, 30 mM or more, 20 mM or more, 10 mM or more, 5 mM or more, 1 mM or more, 1 to 50 mM, 5 to 50 mM, 10 to 50 mM, 20 to 50 mM, 1 to 10 mM, 1 to 5 mM, or about 50 to 60 μM.

[0076] Free Mg at the end of culture 2+ The concentration can be 500 μM or less, 100 μM or less, 50 μM or less, 10 μM or less, 1 μM or less, or 0 μM. Preferably, the free Mg at the end of the culture 2+ The concentration is 10 μM or less.

[0077] Preferably, Mg 2+ is added to the medium as MgSO4.

[0078] At the end of the culture, a chelating agent such as EDTA or EGTA may be added in some cases, preferably to remove residual free Mg. 2+ It may be added in an amount sufficient to sequester ions and induce bacteriolysis.

[0079] The method for producing HMOs of the present invention is preferably carried out by batch culture or fed-batch culture. Batch culture is a closed system in which cells are grown in a fixed amount of nutrient medium under specific environmental conditions without the addition of additional nutrients. In fed-batch culture, nutrients are supplied to the bioreactor during the culture. In both cultures, the cells and product remain in the bioreactor until the end of the culture.

[0080] The volume of the culture medium in the method for producing an HMO of the present invention is preferably 10 L or more, 100 L or more, 1000 L or more, 10,000 L or more, 50,000 L or more, 10 L to 100 L, 100 L to 1,000 L, 1,000 L to 10,000 L, or 10,000 L or more. Preferably, the volume of the culture medium is 100 L to 1,000 L.

[0081] The HMO concentration in the medium at the end of the culture is preferably at least 15 g / L, more preferably at least 20 g / L, even more preferably at least 30 g / L, and most preferably at least 50 g / L.

[0082] In the present invention, a single HMO or two or more HMOs can be produced simultaneously, preferably in a single genetically modified microorganism. For example, expression of α-1,2-fucosyltransferase and α-1,3-fucosyltransferase in the same cell may produce difucosylated structures such as 2-FL, 3-FL, and / or lactodifucotetraose or lacto-N-difucohexaose.

[0083] The HMO produced in the present invention is preferably 2-FL and / or 3-FL, with 2-FL being particularly preferred.

[0084] The HMO can be recovered from the culture medium and / or the microorganism itself. To recover the HMO from the microorganism, the microorganism must be disrupted. Cell disruption by cell lysis can be achieved by chemical or physical means commonly known in the art, or lysis can be induced by an inducible lysis system such as those described above.

[0085] The method for producing an HMO of the present invention may further comprise purifying the HMO from the culture medium. Purification in this context means that the HMO is present in a pure or essentially pure form after purification. After purification, the HMO, e.g., 2-FL and / or 3FL, preferably comprises at least 90%, 95%, 98%, 99%, or 100% (w / w) of the purified product, based on dry weight.

[0086] Purification may be performed by techniques known to those skilled in the art. For example, HMOs may be purified from the medium by methods commonly known to those skilled in the art, such as column chromatography using an ethanol gradient or size exclusion, a charcoal step, and elution with 35-50% ethanol.

[0087] Purity can be assessed by any known method commonly known in the art, such as thin layer chromatography or other electrophoretic or chromatographic techniques.

[0088] Preferably, the HMOs are purified from the culture medium after cell lysis is induced via an inducible lysis system. When the cells are lysed, intracellular HMOs are released into the medium, increasing the HMO concentration in the medium. This improves HMO productivity and reduces processing steps, saving time and costs. Therefore, the HMOs can be purified by reducing the amount of free Mg in the medium. 2+It is preferred that the concentration of is below 10 μM and / or that lysis can be directly visualized, for example by visually removing the culture and / or by measuring the OD of the culture at 600 nm (optical density). 600 It can be visualized directly by measuring the absorbance value. 600 The OD value can be measured by methods commonly known to those skilled in the art, for example, using a spectrophotometer. 600 A decrease in the value indicates that the cells have been lysed. Therefore, in the present invention, the OD 600 The decrease in values ​​allows direct visualization of bacteriolysis.

[0089] The present invention further provides a medium obtainable by the method for producing HMO of the present invention. The medium contains HMO for culturing the microorganism of the present invention to produce HMO. The HMO concentration in the medium at the end of the culture is preferably at least 15 g / L, more preferably at least 20 g / L, even more preferably at least 30 g / L, and most preferably at least 50 g / L. The medium of the present invention can be further dehydrated, for example, by freeze-drying.

[0090] The present invention also provides purified HMOs obtainable by the methods for producing HMOs of the present invention.

[0091] The media and purified HMOs of the present invention may be used in the preparation of products for human consumption, such as infant nutrition and / or animals. The media and purified HMOs of the present invention may be used in the preparation of animal feed. The animal feed may be for companion animals (dogs, cats), livestock (cows, horses, sheep, goats, or pigs, and poultry), and / or fish. Preferably, the animal feed is for cattle. Preferably, the animal feed is a dairy replacer for calves.

[0092] The invention is illustrated by the following non-limiting examples. [Example]

[0093] Materials and Methods The strains, plasmids, and primers used are listed in Tables 3 and 4. E. coli BL21(DE3) was used as the host strain for HMO production, and E. coli TOP10 was used for plasmid construction and maintenance. E. coli was cultured in LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) with ampicillin (100 μg / ml), streptomycin (50 μg / ml), or kanamycin (50 μg / ml) added as needed. Gene deletions were performed using the lambda Red recombinase / flippase system described by Jensen et al. (2015) (Sci Rep 2015;5:17874). The vector pSIJ8 was transfected into competent E. coli BL21(DE3) cells. The lambda Red gene was induced in the presence of 15 mM L-arabinose. A kanamycin resistance cassette (kanR) is amplified from the vector pKD13 (Datsenko and Wanner, 2000), flanked by 50-bp-long homologous target sequences and FRT sites, and transfected into L-arabinose-inducible competent cells. After deletion, the antibiotic cassette is removed by induction of flippase recombinase in the presence of 50 mM L-rhamnose. Cells are grown at 42°C to contain a temperature-sensitive helper plasmid (pSIJ8). For gene expression, vectors pETDuet-1, pCDFDuet-1, and pCOLADuet-1 may be used (Table 3, Figures 2, 4, and 7). Plasmids are transfected into competent E. coli BL21(DE3) cells. For batch fermentation, E. coli is grown in 100 ml of LB containing 36 g / L glucose. OD 600 When the RI reaches 0.6-0.8, induction is performed by adding 0.1 mM IPTG or 0.2% lactose. After growth at 16-25°C for 2-10 hours, 5 g / L lactose is added to initiate HMO production.

[0094] Table 3. Strains and plasmids

[0095] [Table 3] Table 4 Primers used for gene overexpression and deletion

[0096] [Table 4] [Table 5] Underlined: restriction site (overexpression), vector binding site (knockout) [Construction of expression vector] pET-manCB-gmd-wcaG The manC-manB and gmd-wcaG genes were amplified from genomic DNA of E. coli K-12 strain MG1655 using the primers listed in Table 4. First, the gmd-wcaG PCR product was digested with NdeI / KpnI and inserted into the similarly cut vector pETDuet-1 (Figure 2) to generate pET-gmd-wcaG. Next, the manC-manB PCR fragment was digested with NcoI / HindII and inserted into the similarly cut vector pET-gmd-wcaG to generate vector pET-manCB-gmd-wcaG (Figure 3). Selected clones were grown in LB medium containing ampicillin (100 μg / ml) and verified by restriction enzyme mapping and DNA sequencing.

[0097] pCDF-futC-lacY * -hHSP70-trxA-SRRz A human HSP70 gene with E. coli-optimized codons was synthesized and provided in the pUC57 vector (GeneScript). The hHSP70 gene was isolated from the vector via NdeI / BglII restriction and inserted into the similarly cut vector pCDFDuet-1 (Figure 4) to obtain pCDF-hHSP70. The lacY gene was amplified from genomic DNA of E. coli K12-MG1655 using the primers listed in Table 4. For subcloning, the PCR product was inserted into the vector pUC19 (Figure 5) via BamHI / HindIII restriction. Site-directed mutagenesis was performed to introduce the amino acid substitution S209I (lacY*). The lacY* gene was then digested from the vector pUC19-lacY* via BamHI / HindIII and inserted into the similarly cut vector pCDF-hHSP70 to generate pCDF-lacY*-hHSP70. The H. pylori futC gene, with or without an N-terminal triple Asp-tag, was ligated into the pUC57 vector (GeneScript) and codon-optimized for E. coli. The futC fragment was isolated from pUC57 via NcoI / BamHI restriction and inserted into pCDF-lacY*-hHSP70 / NcoI / BamHI to generate pCDF-futC-lacY*-hHSP70. The trxA gene was amplified from E. coli K12-MG1655 strain and inserted into pCDF-futC-lacY*-hHSP70 via BglII / KpnI digestion to generate the plasmid pCDF-futC-lacY*-hHSP70-trxA. Finally, the SRRz gene from bacteriophage lambda was amplified from E. coli BL21(DE3), the bidirectional LuxICDABEG terminator from Vibrio fischeri was introduced at the 3' end, digested with SacI / PstI, and inserted into the similarly cut vector pUC19. The mgtA promoter was amplified with the 5' UTR region from E. coli BL21(DE3), digested with SacI, and inserted into pUC19-SRz-laxTerm / SacI to generate pUC-PmgtA-UTR-SRz-laxTerm.Finally, the gene cassette PmgtA-UTR-SRz-laxTerm was amplified and digested with Bsu36i, followed by insertion into pCDF-futC-lacY*-hHSP70-trxA / Bsu36i to generate pCDF-futC-lacY*-hHSP70-trxA-SRz (Figure 6). Selected clones were grown in LB medium containing streptomycin (50 μg / ml) and verified by restriction enzyme mapping and DNA sequencing.

[0098] pCOLA-pntAB-rcsA-zwf-gsk The rcsA gene was amplified from E. coli K12-MG1655 strain, digested with NdeI / BglII, and inserted into the similarly cut vector pCOLADuet-1 (Figure 7) to generate pCOL-rcsA. Next, the zwf gene was amplified from E. coli K12-MG1655 strain and inserted into pCOLA-rcsA / BglII / KpnI to generate pCOLA-rcsA-zwf. Subsequently, the gsk gene was amplified from E. coli K12-ATCC10798 strain, digested with KpnI / XhoI, and inserted into pCOLA-rcsA-zwf / KpnI / XhoI to generate pCOLA-rcsA-zwf-gsk. Finally, the pntAB genes were amplified from E. coli K12-MG1655, digested with NcoI / BamHI, and inserted into pCOLA-rcsA-zwf-gsk / NcoI / BamHI to generate the vector pCOLA-pntAB-rcsA-zwf-gsk (Figure 8). Selected clones were grown in LB containing kanamycin (50 μg / ml) and verified by restriction enzyme mapping and DNA sequencing. [Industrial Applicability]

[0099] The present invention provides an improved means for producing human milk oligosaccharides (HMOs). The HMOs produced by the microorganisms and production methods of the present invention can be widely applied industrially, for example, as functional ingredients in infant nutrition, medical nutrition, functional foods, and animal feed.

Claims

1. A composition comprising a genetically modified microorganism for producing 2'-fucosyllactose, said microorganism comprising: (a) an exogenous gene encoding an α-1,2-fucosyltransferase, wherein the gene encoding the α-1,2-fucosyltransferase is a heterologous gene encoding futC derived from Helicobacter pylori; (b) an inducible lytic system, the inducible lytic system comprising Mg ２＋ a regulated autoinducible lytic system; and (c) a bacteriophage-derived lysis gene and Mg ２＋ a regulated promoter, wherein expression of the lytic gene is regulated by Mg ２＋ controlled by a regulated promoter, The composition is a bacterium comprising:

2. The composition of claim 1 , wherein the bacterium is E. coli.

3. The induced lysis system is free from exogenous free Mg. ２＋ and / or the bacteria are further treated with additional Mg ２＋ The composition of claim 1 or 2, comprising a regulatory element.

4. The expression of the lytic gene may further comprise the addition of additional Mg. ２＋ The composition of claim 1 , which is controlled by a regulatory element.

5. 2. The composition of claim 1, wherein the bacterium is unable to degrade lactose into glucose and galactose and / or the lac operon is inactivated.

6. 10. The composition of claim 1, wherein the bacterium comprises an exogenous gene encoding a lactose permease.

7. The composition of claim 1, wherein the gene encoding the α-1,2-fucosyltransferase is driven by a heterologous promoter.

8. The composition of claim 1 , wherein the bacterium further comprises a heterologous gene encoding a chaperone.

9. 2. The composition of claim 1, wherein the rcsA gene is overexpressed, or a gene encoding a Lon protease family protein is inactivated, or the wcaJ gene is inactivated, or the zwf gene and two pntAB genes are overexpressed, or the gsk gene is overexpressed, or the trxA gene is overexpressed, or the trxB gene is inactivated.

10. A method for producing 2'-fucosyllactose, comprising: (a) culturing the genetically modified microorganism contained in the composition according to claim 1 in a medium, the medium containing lactose and / or free magnesium ２＋ A method comprising:

11. The method of claim 10, further comprising (b) purifying 2'-fucosyllactose from the culture medium and / or the microorganism itself.

12. Mg ２＋ The composition of claim 3, wherein the regulatory element is the 5'UTR of the mgtA gene of Escherichia coli.

13. 10. The composition of claim 5 or 9, which is inactivated by deletion.

14. 7. The composition of claim 6, wherein the lactose permease is Escherichia coli LacY protein.

15. The composition of claim 14, wherein the E. coli LacY protein comprises the mutations A198V and / or S209I.

16. The composition of claim 7, wherein the gene encoding the α-1,2-fucosyltransferase is codon-optimized and expressed in bacteria.

17. The composition of claim 8, wherein the chaperone is human Hsp70.

18. The composition according to claim 9, wherein the rcsA gene is derived from Escherichia coli, and / or the gene encoding a Lon protease family protein is a gene encoding Escherichia coli Lon protease, and / or the zwf gene and two pntAB genes are derived from Escherichia coli, and / or the gsk gene is derived from Escherichia coli, and / or the trxA gene is derived from Escherichia coli.

19. The method according to claim 10, wherein the concentration of 2'-fucosyllactose in the medium at the end of the culture is at least 30 g / L.

20. The method according to claim 10, wherein the concentration of 2'-fucosyllactose in the medium at the end of the culture is at least 50 g / L.

21. Purifying 2'-fucosyllactose from the medium after inducing lysis by the inducible lysis system, or ２＋ The method of claim 11, wherein 2'-fucosyllactose is purified from the culture medium when the concentration of is 10 μM or less or when dissolution can be directly visualized.

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