Recombinant cells producing heparosan

Abstract

The present invention relates to the field of heparosan bioproduction. There is a need in the art for a heparosan production method that allows for highly efficient synthesis and secretion. The solution proposed in the present invention is the use of genetically modified cells that contain multiple modifications as described herein. The present invention further proposes a method that allows for the bioproduction of heparosan with controlled molecular weight using the genetically modified cells of the present invention.

Description

[Technical field]

[0001] The present invention relates to the field of the biological production of heparosan. [Background technology]

[0002] Heparosan, also known as N-acetylheparosan, is the naturally occurring eukaryotic biosynthetic precursor of heparin and heparan sulfate found in animals from Hydra to vertebrates. More specifically, it is composed of the repeating disaccharide units [→4) β-D-glucuronic acid (GlcA) (1→4) N-acetyl-α-D-glucosamine (GlcNAc) (l→]. n. In nature, Escherichia coli K5 and Pasteurella multocida type D strains produce heparosan as a capsular polysaccharide (Lindahl U. et al., (1998) J. Biol. Chem., 273(39):24979-24982).

[0003] Heparosan-derived derivatives bind to a variety of vital polypeptides, including hemostatic factors (e.g., antithrombin III, thrombin), growth factors (e.g., EGF, VEGF), and chemokines (e.g., IL-8, platelet factor 4), as well as adhesive proteins of viral pathogens (e.g., herpes, dengue). Among these derivatives, heparin, one of the most widely used anticoagulants or antithrombin drugs, is useful, for example, in the treatment of thromboembolism and disseminated intravascular coagulation (DIC), in extracorporeal circulation, and in the prevention of blood clotting during artificial dialysis. In addition, recent studies suggest that heparin may have significant antineoplastic activity separate and distinct from its anticoagulant activity, while other studies indicate a role for heparin in treating inflammation, infertility, and infectious diseases.

[0004] The main currently known sources of heparin are extractions from animal tissues, such as porcine intestinal mucosa, but these carry the attendant risks of impurities and contamination.

[0005] Fermentation methods for preparing heparosan from microorganisms are of particular interest because they facilitate the production of large amounts of heparosan at low cost, potentially through scale-up. Moreover, in contrast to the isolation of heparosan and its derivatives from animal sources, which yields heparosan of very high molecular weight, microbial fermentation allows to control, to a certain extent, the size of the starting molecular weight. This avoids the need for further fractionation steps by mechanical, physical or chemical means.

[0006] To this end, recombinant Bacillus megaterium host cells have shown the ability to produce heparosan in the range of 10-300 KDa (A. Williams et al., Microb. Cell. Fact. 2009 Aug 12; 18(1):132). Similarly, the E. coli K5 strain has shown the ability to produce heparosan (Wang Z. et al., (2010) Biotechnol. Bioeng., 107(6):964-973), however, many challenges remain to be overcome, such as improving the substrate consumption rate and increasing the oxygen supply in the fermenter.

[0007] In contrast to other microorganisms commonly used for the production of biological molecules, yeasts are generally recognized as safe. They can grow rapidly, can be cultured at high densities compared to bacteria, and do not require a sterile environment. Furthermore, yeast cells can be separated from the culture medium more easily than bacteria, greatly simplifying the process of product extraction and purification. Finally, yeasts present the advantage of being more resistant to changes in the pH of the culture medium, and therefore represent a more robust fermentation system.

[0008] However, among yeasts, distinguishing between species characteristics can also present certain challenges. This is mainly due to metabolic differences between species. For example, although genomic integration in P. pastoris is stable, one transformation often results in highly variable clones, showing different productive characteristics or changes in their physiology. This requires a time-consuming screening process to find clones with optimal properties for the desired application. This clonal variability is a significant drawback for the further development of P. pastoris as a platform for producing value-added chemicals. Another drawback of using P. pastoris is that it is a methanotroph.

[0009] In contrast, Saccharomyces cerevisiae has a long and safe history of use by humans (e.g., in wine, beer, or bread) and is therefore a well-established model whose genetic information is well known in the art, making it particularly useful as a tool for the production of molecules of interest. S. cerevisiae further presents the advantage of being generally recognized as safe for humans and animals. Furthermore, the acidification of the medium that occurs when culturing this yeast reduces the possibility of contamination of the biofermenter, thus eliminating the need to add antibiotics to the medium. Finally, numerous genetic tools have been developed that allow stable modification of its genome (integration into the chromosome). [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Lindahl U. et al. (1998) J. Biol. Chem., 273(39):24979-24982 [Non-Patent Document 2] A. Williams et al. Microb. Cell. Fact. 2009 Aug 12; 18(1):132 [Non-licensed document 3] Wang Z.ら, (2010) Biotechnol. Bioeng., 107(6):964-973

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[0011] Thus, there remains a need in the art for further heparosan production methods that allow for highly efficient synthesis and secretion. In particular, there remains a need to provide production methods that provide heparosan that is cost-effective and safe for human application.

[0012] There remains a need in the art for heparosan production methods that in certain cases make it possible to obtain large amounts of heparosan of specific and controlled sizes. [Means for solving the problem]

[0013] The present invention therefore relates to the following items:

[0014] Item 1: A recombinant yeast cell producing heparosan, (a) one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase (HSS) activity; (b) one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity; A recombinant yeast cell comprising:

[0015] As shown in the examples, the recombinant yeast of the invention allows the production of heparosan in yeast cells that are not naturally capable of producing heparosan.

[0016] The advantageous properties can be further increased by engineering the yeast with additional modifications as described hereinafter.

[0017] Item 2: The recombinant cell according to item 1, comprising a nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity, wherein the polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity comprises a secretion signal and optionally an anchoring signal.

[0018] Item 3: A recombinant host cell producing heparosan, (a) one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase (HSS) activity; (b) one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity; and (c) one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity; Including, The polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity comprises a secretion signal and optionally an anchoring signal so that heparosan, particularly one of the desired molecular weight, is produced by the host cell. Recombinant host cells.

[0019] Item 4: The recombinant cell according to item 2 or 3, wherein the nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity is obtained or derived from Pedobacter heparinus.

[0020] Item 5: The recombinant cell according to any one of items 2 to 4, wherein the nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity is under the control of a promoter selected from the group consisting of pTEF1, pCCW12, pCCW12.sba, pCCW12.Sar, pPDC1, pTEF3, pTDH3, pNUP57, and pCCW10.ago.

[0021] Item 6: The recombinant cell according to any one of Items 2 to 5, wherein the molecular weight of the heparosan is within the range of less than 50 kDa, preferably within the range of about 20 kDa to about 50 kDa.

[0022] Item 7: The recombinant cell according to any one of items 2 to 5, wherein the molecular weight of the heparosan is in the range of more than 50 kDa, preferably in the range of about 50 kDa to about 250 kDa.

[0023] Item 8: The recombinant cell according to any one of items 2 to 5, wherein the molecular weight of the heparosan is in the range of more than 100 kDa, preferably in the range of about 100 kDa to about 1500 kDa.

[0024] Item 9: The recombinant cell according to any one of items 1 to 8, wherein the nucleic acid encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity is obtained or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, in particular obtained or derived from Arabidopsis thaliana or Chlorella virus PBCV1.

[0025] Item 10: A recombinant cell according to any one of items 1 to 9, wherein the nucleic acid encoding a polypeptide having heparosan synthase (HSS) activity is obtained or derived from Pasteurella multocida.

[0026] Item 11: (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity 11. The recombinant cell according to any one of items 1 to 10, further comprising at least one recombinant nucleic acid encoding one or more of:

[0027] Item 12: (i) a polypeptide having phosphoglucomutase-1 (PGM1) activity, and / or (ii) a polypeptide having UTP-glucose-1-phosphate uridylyltransferase (UGP1) activity, and / or (iii) a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity, and / or (iv) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity 12. The recombinant cell according to any one of items 1 to 11, further comprising at least one recombinant nucleic acid encoding one or more of:

[0028] Item 13: - a nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or - a nucleic acid encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity, and / or - a nucleic acid encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity, and / or - a nucleic acid encoding a polypeptide having UTP-glucose-1-phosphate uridylyltransferase (UGP1) activity, and / or - a nucleic acid encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity, and / or - a nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase (PCM1) is obtained or derived from Saccharomyces cerevisiae, 13. The recombinant cell according to item 11 or 12.

[0029] Item 14: The recombinant host cell according to any one of items 3 to 13, wherein the recombinant host cell is a yeast.

[0030] Item 15: The recombinant cell according to any one of items 1 to 14, which belongs to the genus Saccharomyces, or Candida, or Kluyveromyces, or Ogataea, or Yarrowia, or Debaryomyces, or Ashbya, in particular the genus Saccharomyces.

[0031] Item 16: Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces castelli, Candida albicans, Candida glabrata, Candida tropicalis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus 16. The recombinant cell according to item 15, wherein the recombinant cell is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces thermotolerens, Ogataea polymorpha, Yarrowia lypolytica, Debaryomyces hansenii, and Ashbya gossypii, and is preferably Saccharomyces cerevisiae.

[0032] Item 17: A method for producing heparosan, comprising: (a) culturing the recombinant cell according to any one of items 1 to 16 in a culture medium for a time sufficient to produce heparosan; (b) optionally isolating or recovering the heparosan from the recombinant cells and / or the culture medium. The method includes:

[0033] Item 18: The method according to Item 17, wherein the heparosan has a molecular weight of about 20 kDa to about 50 kDa.

[0034] Item 19: The method according to Item 17, wherein the heparosan has a molecular weight of about 50 kDa to about 150 kDa.

[0035] Item 20: The method according to Item 17, wherein the heparosan has a molecular weight of about 150 kDa to about 1500 kDa.

[0036] Item 21: The method according to any one of items 17 to 20, wherein the recombinant cell is a yeast belonging to the genus Saccharomyces.

[0037] Item 22: The method according to any one of Items 17 to 21, wherein the time sufficient to produce heparosan is a period of about 35 to about 50 hours, preferably about 40 to about 50 hours, preferably about 48 hours.

[0038] Item 23: The method according to any one of items 17 to 22, wherein the molecular weight of the produced heparosan is controlled by adjusting the pH of the culture medium in step (a).

[0039] Item 24: The method according to any one of items 17 to 23, wherein the method is carried out on an industrial scale, preferably the culture medium is at least about 100 L, more preferably in the range of about 1000 L to about 3000 L, even more preferably about 10,000 L, or even more preferably about 100,000 L, or even about 250,000 L.

[0040] Item 25: A heparosan obtainable from a recombinant cell according to any one of items 1 to 15 or from the method according to any one of items 17 to 24.

[0041] Item 26: A culture medium comprising the heparosan according to item 25.

[0042] Item 27: A composition comprising the heparosan according to item 25.

[0043] Item 28: An industrial product, a consumer product, or a consumable product comprising (i) the heparosan according to item 25 having a molecular weight as defined in any one of items 6 to 8, (ii) the culture medium according to item 26, or (iii) the composition according to item 27.

[0044] Item 29: The industrial or consumer product or consumable according to item 28, which is a cosmetic product, a flavour product, a fragrance product, a foodstuff, a food, a beverage, a texturant, a pharmaceutical composition, a dietary supplement, a nutraceutical, a cleaning product, a dental and / or oral hygiene composition.

[0045] Item 30: Use of a recombinant cell according to any one of items 1 to 116 for producing heparosan having a molecular weight in the range of about 20 kDa to about 50 kDa or about 50 kDa to about 1000 kDa.

[0046] Certain embodiments of the present invention provide the following advantages: A process for producing heparosan of controlled molecular weight, A process for producing heparosan of controlled molecular weight in Saccharomyces yeast cells, as well as A process for producing heparosan of controlled molecular weight by varying genetic parameters (e.g., regulatory sequences) and / or process parameters (pH or fermentation time). may provide one or more of: [Brief description of the drawings]

[0047] [Figure 1] FIG. 1 shows a schematic pathway for the production of heparosan. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Overview of Arrays SEQ ID NO:1 is the recoded nucleic acid sequence of heparosan synthase originating from Pasteurella multocida (HSS1) SEQ ID NO:2 is the recoded nucleic acid sequence of heparosan synthase originating from Pasteurella multocida (HSS2) SEQ ID NO:3 is the amino acid sequence of heparosan synthase originating from Pasteurella multocida (HSS1) SEQ ID NO: 4 is the amino acid sequence of heparosan synthase originating from Pasteurella multocida (HSS2) SEQ ID NO:5 is the nucleic acid sequence of UDP-glucose dehydrogenase (HASB) originating from Arabidopsis thaliana SEQ ID NO:6 is the recoded nucleic acid sequence of UDP-glucose dehydrogenase (HASB) originating from the Chlorella virus PBCV-1. SEQ ID NO:7 is the recoded nucleic acid sequence of UDP-glucose dehydrogenase (HASB-A) originating from Chlorella virus PBCV-1 SEQ ID NO:8 is the amino acid sequence of UDP-glucose dehydrogenase (HASB) from Arabidopsis thaliana SEQ ID NO:9 is the amino acid sequence of UDP-glucose dehydrogenase (HASB) originating from Chlorella virus PBCV1 SEQ ID NO:10 is a recoded nucleic acid sequence of glucuronosyl-disulfoglucosamine glucuronidase originating from Pedobacter heparinus with an N-terminal secretion signal SEQ ID NO:11 is a recoded nucleic acid sequence of glucuronosyl-disulfoglucosamine glucuronidase originating from Pedobacter heparinus, with an N-terminal secretion signal and a C-terminal anchoring signal. SEQ ID NO:12 is a recoded nucleic acid sequence of glucuronosyl-disulfoglucosamine glucuronidase originating from Pedobacter heparinus with an N-terminal secretion signal SEQ ID NO:13 is a recoded nucleic acid sequence of glucuronosyl-disulfoglucosamine glucuronidase originating from Pedobacter heparinus, with an N-terminal secretion signal and a C-terminal anchoring signal. SEQ ID NO:14 is the amino acid sequence of glucuronosyl-disulfoglucosamine glucuronidase from Pedobacter heparinus with an N-terminal secretion signal SEQ ID NO:15 is the amino acid sequence of glucuronosyl-disulfoglucosamine glucuronidase originating from Pedobacter heparinus, with an N-terminal secretion signal and a C-terminal anchoring signal SEQ ID NO:16 is the nucleic acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Saccharomyces cerevisiae SEQ ID NO:17 is the recoded nucleic acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Chlorella virus 1 (PBCV-1) SEQ ID NO:18 is the recoded nucleic acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Chlorella virus 1 (PBCV-1) SEQ ID NO:19 is the amino acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Saccharomyces cerevisiae SEQ ID NO:20 is the amino acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Chlorella virus 1 (PBCV-1) SEQ ID NO:21 is the nucleic acid sequence of UDP-N-acetylglucosamine pyrophosphorylase (QRI1) originating from Saccharomyces cerevisiae SEQ ID NO:22 is the amino acid sequence of UDP-N-acetylglucosamine pyrophosphorylase (QRI1) originating from Saccharomyces cerevisiae SEQ ID NO:23 is the nucleic acid sequence of phosphoglucomutase-1 (PGM1) originating from Saccharomyces cerevisiae. SEQ ID NO:24 is the amino acid sequence of phosphoglucomutase-1 (PGM1) originating from Saccharomyces cerevisiae SEQ ID NO:25 is the nucleic acid sequence of UTP-glucose 1-phosphate uridylyltransferase (UGP1) originating from Saccharomyces cerevisiae SEQ ID NO:26 is the amino acid sequence of UTP-glucose 1-phosphate uridylyltransferase (UGP1) originating from Saccharomyces cerevisiae SEQ ID NO:27 is the nucleic acid sequence of glucosamine 6-phosphate N-acetyltransferase (GNA1) originating from Saccharomyces cerevisiae SEQ ID NO:28 is the amino acid sequence of glucosamine 6-phosphate N-acetyltransferase (GNA1) originating from Saccharomyces cerevisiae SEQ ID NO:29 is the nucleic acid sequence of phosphoacetylglucosamine mutase (PCM1) originating from Saccharomyces cerevisiae SEQ ID NO:30 is the amino acid sequence of phosphoacetylglucosamine mutase (PCM1) originating from Saccharomyces cerevisiae SEQ ID NO: 31 is the nucleic acid sequence of promoter pTDH3 SEQ ID NO: 32 is the nucleic acid sequence of promoter pTDH3.Sk SEQ ID NO: 33 is the nucleic acid sequence of the promoter pTDH3-1.sba SEQ ID NO: 34 is the nucleic acid sequence of the promoter pTDH3.Sar SEQ ID NO: 35 is the nucleic acid sequence of promoter pENO2 SEQ ID NO: 36 is the nucleic acid sequence of promoter pTEF3 SEQ ID NO: 37 is the nucleic acid sequence of the promoter pTEF1 SEQ ID NO: 38 is the nucleic acid sequence of the promoter pTEF1.ago SEQ ID NO: 39 is the nucleic acid sequence of the promoter pTEF1.Sba SEQ ID NO: 40 is the nucleic acid sequence of promoter pPDC1 SEQ ID NO: 41 is the nucleic acid sequence of promoter pCCW12 SEQ ID NO: 42 is the nucleic acid sequence of the promoter pCCW12.Sm SEQ ID NO: 43 is the nucleic acid sequence of promoter pCCW12.Sk SEQ ID NO: 44 is the nucleic acid sequence of the promoter pCCW12.Sba SEQ ID NO: 45 is the nucleic acid sequence of the promoter pCCW12.Sar SEQ ID NO: 46 is the nucleic acid sequence of the promoter pNUP57 SEQ ID NO: 47 is the nucleic acid sequence of the promoter pCCW10.ago SEQ ID NO: 48 is the nucleic acid sequence of promoter pCWP2 SEQ ID NO: 49 is the nucleic acid sequence of the promoter pCCW120.Sca SEQ ID NO: 50 is the nucleic acid sequence of promoter pRPLA1 SEQ ID NO: 51 is the nucleic acid sequence of the promoter pCUP1 SEQ ID NO: 52 is the nucleic acid sequence of the promoter pMET6 SEQ ID NO: 53 is the nucleic acid sequence of promoter pMET25 SEQ ID NO: 54 is the nucleic acid sequence of the promoter pSAM1 SEQ ID NO: 55 is the nucleic acid sequence of the terminator tTPI1 SEQ ID NO: 56 is the nucleic acid sequence of terminator tMET25 SEQ ID NO: 57 is the nucleic acid sequence of the terminator tDIT1 SEQ ID NO: 58 is the nucleic acid sequence of terminator tRPL3 SEQ ID NO: 59 is the nucleic acid sequence of terminator tRPL3.sm SEQ ID NO: 60 is the nucleic acid sequence of the terminator tRPL3.sba SEQ ID NO: 61 is the nucleic acid sequence of the terminator tRPL41B SEQ ID NO: 62 is the nucleic acid sequence of the terminator tRPL15A SEQ ID NO: 63 is the nucleic acid sequence of terminator tRPL15A.sba SEQ ID NO: 64 is the nucleic acid sequence of terminator tIDP1 SEQ ID NO: 65 is the nucleic acid sequence of the terminator tTEF1.sba SEQ ID NO:66 is the nucleic acid sequence of the secretory sequence added to the 5' end SEQ ID NO:67 is the amino acid sequence of the secretory sequence added to the N-terminus SEQ ID NO:68 is the nucleic acid sequence of the anchoring sequence added to the 3' SEQ ID NO:69 is the amino acid sequence of the anchoring sequence added to the C-terminus

[0049] Detailed Description of the Invention The inventors have envisaged genetically modified cells, in particular genetically modified yeast, that have the ability to produce heparosan compared to a parent cell that is not naturally capable of doing so, in particular compared to a parent yeast.

[0050] These genetically modified cells are described throughout the specification.

[0051] definition The term "heparosan" refers to the heparin derivatives consisting of the repeats [-4-N-acetylglucosamine-α1,4-glucuronic acid-β1-] [→4)β-D-glucuronic acid (GlcA) (1→4)N-acetyl-α-D-glucosamine (GlcNAc) (l→] n ) is a polysaccharide polymer composed of

[0052] Heparosan can be produced in recombinant cells.

[0053] As used herein, the term "recombinant" when used in reference to a cell indicates that the cell has been modified by the introduction of endogenous and / or heterologous nucleic acids or proteins into the cell or the alteration of a native cell, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes or nucleic acids that are not found in the native (non-recombinant) form of the cell, or expresses native (e.g., endogenous) genes at levels that differ from their native levels, or expresses additional or supplemental copies of native (e.g., endogenous) ones at levels that differ from their native levels.

[0054] As used herein, the term "recombinant" when used in reference to a nucleic acid or vector is a sequence formed / obtained by genetic engineering techniques well known to those skilled in the art. The US National Institute for Health (NIH) guidelines therefore state: "Recombinant […] nucleic acids are i. (a) a molecule constructed by joining nucleic acid molecules, and (b) a molecule capable of replicating in a living cell, i.e., a recombinant nucleic acid It is defined as which reflects the conventional use of the word "recombinant" attached to a nucleic acid sequence to mean recombined following insertion or joining of another nucleic acid.

[0055] A protein produced by expression of recombinant DNA or a recombinant vector in a living cell is also called a recombinant protein.

[0056] The term "recombinant" is therefore synonymous with the term "genetically modified." The term "gene" is synonymous with the term "nucleic acid" or "nucleotide sequence."

[0057] Recombinant nucleic acid sequences for use in recombinant cells, particularly recombinant yeasts, of the present invention may be provided in the form of a nucleic acid construct. The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that is isolated or derived from a native (e.g., endogenous) naturally occurring gene, or is a heterologous nucleic acid, or has been modified to contain segments of nucleic acid that are combined and juxtaposed in a manner that would not occur in nature. The term "nucleic acid construct" is synonymous with the term "expression cassette" or "heterologous nucleic acid expression cassette" when the nucleic acid construct contains one or more regulatory elements required for the expression of a coding sequence, said control sequences being operably linked to said coding sequence. Non-limiting examples of regulatory elements include promoters, enhancers, silencers, terminators, and poly-A signals.

[0058] Recombinant nucleic acid sequences for use in the recombinant cells of the invention may be provided in the form of an expression vector, in which the polynucleotide sequence is operably linked to at least one control sequence for expression of the polynucleotide sequence in the recombinant cell.

[0059] The terms "obtained from" or "originate from" or "originating from" a microorganism or animal generally mean that a substance (e.g., a nucleic acid molecule or polypeptide) of microbial or animal origin is native to that microorganism or animal.

[0060] The term "derived from" a microorganism or animal means that a substance (e.g., a nucleic acid molecule or polypeptide) derived from a microorganism or animal is the result of modifications made to a substance native (i.e. present as is) in that microorganism or animal. For example, with respect to a nucleic acid sequence derived from a microorganism or animal, said nucleic acid sequence may represent a recoded and / or shortened version of the native nucleic acid sequence derived from this microorganism or animal. Other modifications known to those skilled in the art may be made to native substances of a microorganism or animal, resulting in the substance used being "derived from" said microorganism or animal.

[0061] As used herein, the term "polypeptide" refers to a molecule that contains amino acid residues linked by peptide bonds and contains more than five amino acid residues. Amino acids are identified by either single-letter or three-letter notations. The term "protein" as used herein is synonymous with the term "polypeptide" and may further refer to two or more polypeptides. Thus, the terms "protein", "peptide", and "polypeptide" may be used interchangeably. Polypeptides may optionally be modified to add functionality (e.g., glycosylation, phosphorylation, acylation, farnesylation, prenylation, sulfonation, etc.). Polypeptides that exhibit activity may be referred to as enzymes. It will be understood that as a result of the degeneracy of the genetic code, multiple nucleotide sequences encoding a given polypeptide may be produced.

[0062] Polypeptides encoded by recombinant nucleic acids for use in recombinant cells of the invention, particularly recombinant yeast, may include a signal peptide and / or a propeptide sequence. When a polypeptide expressed by a recombinant cell of the invention, particularly recombinant yeast, includes a signal peptide and / or a propeptide, sequence identity may be calculated over the mature polypeptide sequence.

[0063] The term "operably linked" as used herein refers to two or more nucleic acid sequence elements that are physically linked and in a functional relationship with each other. For example, a promoter is operably linked to a coding sequence if it is capable of initiating or regulating the transcription or expression of the coding sequence, in which case the coding sequence shall be understood to be "under the control" of the promoter. Generally, when two nucleic acid sequences are operably linked, they are in the same orientation and usually also in the same reading frame. They are usually essentially contiguous, although this is not required.

[0064] The terms "native" or "endogenous" as used herein with reference to molecules, particularly enzymes and nucleic acids, refer to molecules that are their origin or expressed in the organism in which they are found in nature.

[0065] The term "endogenous gene" means that the gene was present in the cell in a wild-type strain prior to any genetic modification. Endogenous genes may be overexpressed by introducing heterologous sequences in addition to or to replace endogenous regulatory elements, or by introducing one or more additional or supplementary copies of the gene into a chromosome or plasmid (said additional or supplementary copies are designated "exogenous or heterologous genes" or "heterologous nucleotide sequences" or "heterologous nucleic acids" as defined herein). Endogenous genes may also be modified to modulate their expression and / or activity. For example, mutations may be introduced into the coding sequence to modify the gene product, or heterologous sequences may be introduced in addition to or to replace endogenous regulatory elements. Modulation of endogenous genes may result in upregulation and / or enhancement of the activity of the gene product, or alternatively, downregulation and / or attenuation of the endogenous gene product. Another means of enhancing expression of an endogenous gene is to introduce one or more additional or auxiliary copies of the gene into a chromosome or plasmid (said auxiliary copies being referred to as an "exogenous or heterologous gene" or "heterologous nucleotide sequence" or "heterologous nucleic acid" as defined herein).

[0066] "One or more additional or supplementary copies of a gene" according to the present invention is understood, for example, in the present invention as 1 to 50 copies, in particular 1 to 30 copies, more particularly 1 to 20 copies, preferably 1 to 10 copies. Said copies may be inserted at the same locus or at different loci of the recombinant cell of the present invention.

[0067] The term "exogenous gene" means that a gene has been introduced into a cell by means well known to those skilled in the art, which gene is naturally present or not present in a wild type cell. A cell can express exogenous genes if these genes are introduced into the cell with all the elements that allow their expression in the cell. Transforming exogenous DNA into a cell is a routine task for those skilled in the art. Exogenous genes can be integrated into the host chromosome or expressed extrachromosomally from a plasmid or vector. A variety of plasmids that differ in terms of origin of replication and copy number in the cell are all known in the art. The sequence of the exogenous gene can be adapted for its expression in the cell. In fact, those skilled in the art are aware of the concept of codon usage bias and how to adapt a nucleic acid sequence to a particular codon usage bias without modifying the predicted protein. In certain embodiments, the codon-optimized gene expresses a native enzyme.

[0068] The term "heterologous gene" or "heterologous nucleic acid sequence" refers to a gene or nucleic acid sequence that is not normally found naturally in a given cell. Thus, a heterologous nucleic acid sequence can be (a) foreign to its host cell (i.e., "exogenous" to the cell), (b) naturally found in the host cell (i.e., "endogenous") but present in a non-native amount in the cell (i.e., greater or less than that naturally found in the host cell), or (c) naturally found in the host cell but located outside its natural locus.

[0069] In this application, all genes are referred to by their common name, by their nucleotide and, where applicable, by their amino acid sequence. Using the accession number reference for known genes, one skilled in the art can determine the equivalent genes in other organisms, bacterial strains, yeast, fungi, mammals, plants, etc. This routine is advantageously carried out by using consensus sequences, which can be determined by performing sequence alignments with genes from other cells, to design degenerate probes for cloning the corresponding genes in another organism.

[0070] Those skilled in the art are aware of different means for modulating, in particular up-regulating or down-regulating, the expression of an endogenous gene. For example, a means for enhancing or overexpressing the expression of an endogenous gene is to introduce one or more additional or supplementary copies of the gene into a chromosome or a plasmid.

[0071] Another approach is to replace the endogenous promoter of the gene with a stronger promoter. These promoters can be homologous or heterologous. Promoters of particular interest in the present invention are described in more detail elsewhere herein.

[0072] The nucleic acid expression construct may further comprise 5' and / or 3' recognition sequences and / or selectable markers.

[0073] The term "inducible promoter" refers to a - a promoter whose activity is induced, i.e. increased, in the presence of one or more specific metabolites (the higher the concentration of the metabolite in the medium, the stronger the promoter activity); or - promoters whose activity is induced, i.e., increased, in the presence or absence of low concentrations of one or more metabolites (these metabolites are different from those whose increased presence induces the activity of the promoter. The lower the concentration of the metabolite in the medium, the stronger the promoter activity). is used to specify.

[0074] The term "repressible promoter" refers to a - a promoter whose activity is repressed, i.e. reduced, in the presence of one or more specific metabolites (the higher the concentration of the metabolite in the medium, the weaker the promoter activity), or - promoters whose activity is repressed, i.e. reduced, in the presence or absence of low concentrations of one or more metabolites (these metabolites are different from those whose increased presence represses the activity of the promoter; the lower the concentration of the metabolite in the medium, the weaker the promoter activity). is used to specify.

[0075] As used herein, the term "anchoring signal", when used in conjunction with a protein or polypeptide, e.g., an enzyme (e.g., a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity), means, e.g., a first nucleic acid encoding a protein operably linked to a second nucleic acid encoding a protein or polypeptide, or a first protein or polypeptide operably linked to a second protein or polypeptide, e.g., an enzyme (e.g., a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity, such as, for example, to form a fusion protein), that enables the cellular transport machinery of a cell, in particular an S. cerevisiae cell, to correctly anchor and / or position the second protein operably linked to the first protein in the cell membrane.

[0076] As used herein, the term "secretion signal", when used in connection with a protein or polypeptide, e.g., an enzyme (e.g., for example, a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity), means, e.g., a first nucleic acid encoding a peptide or protein operably linked to a second nucleic acid encoding a protein, or a first protein linked to a second protein, e.g., an enzyme (e.g., for example, a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity to form a fusion protein), that enables the cellular transport machinery of a cell, in particular an S. cerevisiae cell, to position at least the second protein in the cell membrane and secrete the second protein outside the cell, e.g., after the first protein has been cleaved from the second protein.

[0077] As used herein, the terms "secretion signal" and "anchoring signal", when used in conjunction with a protein or polypeptide, e.g., an enzyme (e.g., a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity), refer to, e.g., a first nucleic acid encoding a peptide or protein operably linked to a second nucleic acid encoding a protein, or a first protein operably linked to a second protein, e.g., an enzyme (e.g., a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity), that allows the cellular transport mechanisms of a cell, particularly an S. cerevisiae cell, to position at least the second protein in the cell membrane, where the second protein is not secreted and remains attached to the cell membrane when the second protein is operably linked to the "anchoring signal". In some cases, a secretion-anchoring signal can provide dual secretion signal and anchoring signal functions.

[0078] Secretion and anchoring signal sequences, methods for expression, anchoring, and / or secretion of heterologous proteins, such as enzymes (e.g., for example, glucuronosyl-disulfoglucosamine glucuronidase) on the surface of cells (e.g., for example, yeast cells), are well known in the art (see, e.g., Ast et al. (2013) Cell 152: 1134-1145; Ast and Schuldiner (2013) Crit Rev Biochem Mol Biol 48(3) 273-288; Van der Vaart et al. (1997) Applied Environmental Microbiology 63(2) 615-620, the entire contents of each of which are incorporated herein by reference).

[0079] The "activity" of an enzyme is used interchangeably with the term "function" and refers, in the context of the present invention, to the ability of the enzyme to catalyze a desired reaction. The amount of an enzyme in a host cell can be altered by modifying the transcription of the gene encoding the enzyme. This can be achieved, for example, by modifying the copy number of the nucleotide sequence encoding the enzyme (e.g., by using a higher or lower copy number expression vector containing the nucleotide sequence, or by introducing additional copies of the nucleotide sequence into the genome of the host cell, or by deleting or disrupting the nucleotide sequence in the genome of the host cell), by changing the order of the coding sequences in the polycistronic mRNA of the operon, or by splitting the operon into individual genes, each with its own control element, or by increasing the strength of the promoter or operator to which the nucleotide sequence is operably linked.

[0080] Alternatively, or additionally, the copy number of an enzyme in a host cell can be altered by modifying the level of translation of the mRNA encoding the enzyme. This can be achieved, for example, by modifying the stability of the mRNA, modifying the sequence of the ribosome binding site, modifying the distance or sequence between the ribosome binding site and the start codon of the enzyme coding sequence, modifying the entire intercistronic region located "upstream" or immediately 5' to the start codon of the enzyme coding region, stabilizing the 3' end of the mRNA transcript using hairpins and specialized sequences, modifying the codon usage of the enzyme, altering the expression of tRNAs for rare codons used in the biosynthesis of the enzyme, and / or increasing the stability of the enzyme, for example, by mutation of its coding sequence.

[0081] The activity of an enzyme in a host cell can be altered in a number of ways, including, but not limited to, expressing an altered form of the enzyme that exhibits increased or decreased solubility in the host cell, expressing an altered form of the enzyme that lacks a domain through which activity of the enzyme is inhibited, expressing an altered form of the enzyme that has a higher or lower Kcat or a lower or higher Km for a substrate, or expressing an altered form of the enzyme that is more or less affected by feedback or feedforward regulation by another molecule in the pathway.

[0082] The terms "encode" or "encoding" refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an amino acid sequence.

[0083] The genes encoding the enzymes contemplated in the present invention may be exogenous or endogenous.

[0084] The method implemented in the present invention preferably requires the use of one or more chromosomal integration constructs for the stable introduction of heterologous nucleotide sequences into specific locations on chromosomes or for the functional disruption of one or more target genes in genetically modified cells.In some embodiments, the disruption of target genes prevents the expression of the relevant functional protein.In some embodiments, the disruption of target genes results in the expression of non-functional proteins from the disrupted genes.

[0085] Parameters of the chromosomal integration construct that may be varied in the practice of the present invention include, but are not limited to, the length of the homologous sequence, the nucleotide sequence of the homologous sequence, the length of the integration sequence, the nucleotide sequence of the integration sequence, and the nucleotide sequence of the target locus. In some embodiments, the effective range of the length of each homologous sequence is 20 to 5,000 base pairs, preferably 50 to 100 base pairs. In certain embodiments, the length of each homologous sequence is about 50 base pairs. For more information regarding the length of homology required for gene targeting, see D. Burke et al., Methods in yeast Genetics - A cold spring harbor laboratory course Manual (2000).

[0086] In some embodiments, the disrupted gene into which the above-mentioned DNA construct is intended to be inserted may advantageously contain one or more selectable markers useful for the selection of transformed cells. Preferably, said selectable markers are included in the DNA construct of the present invention.

[0087] In some embodiments, the selectable marker is an antibiotic resistance marker. Illustrative examples of antibiotic resistance markers include NAT1, AUR1-C, HPH, DSDA, KAN <r>These include, but are not limited to, the NAT gene product from S. noursei, which confers resistance to nourseothricin, the AURl-C gene product from Saccharomyces cerevisiae, which confers resistance to aureobasidin A (AbA), the HPH gene product from Klebsiella pneumoniae, which confers resistance to hygromycin B, the DSDA gene product from E. coli, which allows cells to grow on plates with D-serine as the sole nitrogen source, and the KAN of the Tn903 transposon. <r>The gene confers resistance to G418, and the SHBLE gene product from Streptoalloteichus hindustanus confers resistance to zeocin (bleomycin).

[0088] In some embodiments, the antibiotic resistance marker is removed after the genetically modified cells of the invention are isolated. One of skill in the art can select an appropriate marker in a particular genetic context.

[0089] In a particular embodiment, the recombinant cells according to the invention do not carry any antibiotic resistance markers, which advantageously obviates the need to add antibiotics to the selection medium.

[0090] In some embodiments, the selectable marker rescues a requirement (e.g., an auxotrophy) in the genetically modified cell. In such embodiments, the parent cell, particularly the parent yeast, contains a functional disruption in one or more gene products that function in an amino acid or nucleotide biosynthetic pathway, such as the HIS3, LEU2, LYS1, LYS2, MET15, TRP1, ADE2, and URA3 gene products in yeast, which renders the parent cell unable to grow in medium without supplementation of one or more nutrients (auxotrophic phenotype). The auxotrophic phenotype can then be rescued by transforming the parent cell with a chromosomal integration encoding a functional copy of the disrupted gene product (in some embodiments, the functional copy of the gene can originate from an adjacent species, e.g., Kluyveromyces, Candida, etc.), and the resulting genetically modified cells can be selected based on the loss of the auxotrophy phenotype of the parent microbial cell.

[0091] For each nucleic acid sequence, including a promoter sequence, a coding sequence (e.g., an enzyme coding sequence), or a terminator sequence, a reference sequence is described herein. The description herein also encompasses nucleic acid sequences that have a particular percentage of nucleic acid identity with the reference nucleic acid sequence.

[0092] For each amino acid sequence of interest, a reference sequence is described herein. The description herein also encompasses amino acid sequences (e.g., enzyme amino acid sequences) that have a certain percentage of amino acid identity with the reference amino acid sequence.

[0093] For obvious reasons, throughout this specification, a particular nucleic acid sequence or a particular amino acid sequence that matches the nucleotide or amino acid identity considered, respectively, should further lead to obtaining a protein (or enzyme) that exhibits the desired biological activity. As used herein, the "percentage of identity" between two nucleic acid sequences or two amino acid sequences is determined by comparing both optimally aligned sequences over a comparison window.

[0094] The portion of the nucleotide or amino acid sequence within the comparison window may therefore contain additions or deletions (e.g., "gaps") compared to the reference sequence (the reference sequence does not contain these additions or these deletions), such that optimal alignment is obtained between both sequences.

[0095] The terms "sequence homology" or "sequence identity" or "homology" or "identity" are used interchangeably herein. For the purposes of the present invention, in order to determine the percentage of sequence homology or sequence identity of two amino acid sequences or two nucleic acid sequences, it is defined herein that the sequences are aligned for optimal comparison purposes. To optimize the alignment between the two sequences, gaps may be introduced into either of the two sequences being compared. Such alignment may be performed over the entire length of the sequences being compared. Alternatively, alignment may be performed over a shorter length, for example, over about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Sequence identity is the percentage of identical matches between the two sequences over the reported alignment region.

[0096] The comparison of sequences between two sequences and the determination of the percentage of sequence identity can be accomplished using a mathematical algorithm. Those skilled in the art will recognize the fact that several different computer programs are available for aligning two sequences and determining the identity between two sequences (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pages 1-44, Addison Wesley).

[0097] The percent sequence identity between two amino acid sequences or two nucleotide sequences can be determined using the Needleman and Wunsch algorithm for alignment of two sequences. (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). Both amino acid and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE.

[0098] For the purposes of the present invention, the NEEDLE program of the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. Optional parameters used are a gap opening penalty of 10 and a gap extension penalty of 0.5. No end gap penalty is added. In the Output section, Yes is indicated in response to the question "Brief identity and similarity" and "SRS pairwise" indicates the Output alignment format.

[0099] After alignment with the above-mentioned program NEEDLE, the percentage of sequence identity between the query sequence and the sequence of the invention is calculated as follows: the number of corresponding positions in the alignment that show identical amino acids or identical nucleotides in both sequences is divided by the total length of the alignment after subtracting the total number of gaps in the alignment. Identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled as "longest-identity" on the program output.

[0100] Nucleotide and amino acid sequence similarity, i.e., percentage sequence identity, can be determined using several other art-known algorithms, preferably using the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), using hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or using the CLUSTAL algorithm available at https: / / www.ebi.ac.uk / Tools / msa / clustalo / (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), or the GAP program (mathematical algorithm of University of Iowa), or the mathematical algorithm of Myers and Miller (1989 - Cabios 4: 11-17), or the Clone Manager program. 9 can be used to determine the sequence alignment. The preferred parameters used are the default parameters set out at https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0101] Sequence identity grades (percentage of sequence identity) can be calculated using BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed with the BLASTN program, score=100, wordlength=12, to obtain polynucleotide sequences that are homologous to nucleic acids encoding related proteins.

[0102] To obtain amino acid sequences homologous to the SHC polypeptide, BLAST protein searches are performed using the BLASTP program, score=50, word length=3. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence match analysis can be assisted by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1: 154-162) or Markov Random Fields. When percentages of sequence identity are referred to in this application, these percentages are calculated with respect to the full length of the longer sequence, unless otherwise specifically indicated.

[0103] In certain embodiments, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0104] "Fermentation" or "cultivation" is generally carried out in a fermenter using an appropriate culture medium containing at least one simple carbon source adapted to the cells being cultured and, if necessary, supplementary substrates.

[0105] The term "fermentation composition" refers to a composition comprising genetically modified host cells and products or metabolites produced by the genetically modified host cells. An example of a fermentation composition is a whole cell culture, which can be the entire contents of a vessel (e.g., flask, plate, or fermenter) containing the cells, the aqueous phase, and the compounds produced from the genetically modified host cells.

[0106] The term "medium" refers to a culture medium, or a cultivation medium, or a fermentation medium.

[0107] For maximum production of heparosan, the recombinant cells used as production hosts preferably have a high carbohydrate utilization rate. These characteristics may be conferred by mutagenesis and selection, genetic engineering, or may be natural. The fermentation medium or "culture medium" or "cultivation medium" of the cells may contain at least about 10 g / L of glucose and / or sucrose. Additional carbon substrates may include, but are not limited to, monosaccharides such as fructose, mannose, xylose, and arabinose, oligosaccharides such as lactose, maltose, galactose, or sucrose, polysaccharides such as starch or cellulose, or mixtures thereof, as well as unrefined mixtures from renewable raw materials such as cheese whey permeate, corn steep liquor, sugar beet molasses, and malt. Other carbon substrates may include glycerol, acetate, and / or ethanol.

[0108] Thus, it is contemplated that the carbon source utilized in the present invention can encompass a wide range of carbon-containing substrates, limited only by the choice of cell, particularly yeast.

[0109] Although all of the above carbon substrates and mixtures thereof are contemplated to be suitable in the present invention, for cells, particularly yeast, engineered to use C5 sugars, more particularly glucose, the preferred carbon substrates are glucose, fructose, and sucrose, or mixtures thereof with C5 sugars, such as xylose and / or arabinose.

[0110] The preferred carbon substrate is glucose or sucrose.

[0111] In addition to a suitable carbon source, the fermentation medium may contain suitable minerals, salts, cofactors, buffers, and other components known to those of skill in the art suitable for growth of the culture and promoting the enzymatic pathways necessary for production of the desired product.

[0112] Furthermore, additional genetic modifications suitable for the growth of recombinant cells according to the invention may be considered.

[0113] The term "aerobic conditions" refers to a concentration of oxygen in the culture medium that is sufficient for aerobic or facultative anaerobic cells, particularly yeast, to use dioxygen as a terminal electron donor.

[0114] "Microaerophilic conditions" are conditions in which the oxygen concentration is lower than that in air, i.e., up to 6% O 2 This refers to a culture medium having an oxygen concentration of 0.1%.

[0115] "Suitable culture medium" refers to a medium (e.g., sterile, liquid medium) that contains nutrients essential or beneficial for the maintenance and / or growth of cells, such as a carbon source or carbon substrate, a nitrogen source, such as peptone, yeast extract, meat extract, malt extract, urea, ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium phosphate, a phosphorus source, such as monopotassium phosphate or dipotassium phosphate, trace elements (e.g., metal salts), such as magnesium salts, cobalt salts, and / or manganese salts, and growth factors, such as amino acids, vitamins, growth promoters, etc. The term "carbon source" or "carbon substrate" or "carbon source" according to the present invention refers to any carbon source that can be used by the skilled artisan to support the normal growth of cells, including hexoses (e.g., glucose, galactose, or lactose), pentoses, monosaccharides, oligosaccharides, disaccharides (e.g., sucrose, cellobiose, or maltose), molasses, starch or derivatives thereof, cellulose, hemicellulose, and combinations thereof.

[0116] Particularly suitable culture media for producing the recombinant cells of the invention, and in particular the recombinant yeasts of the invention, are described in more detail below.

[0117] A "controlled" molecular weight of the heparosan of the present invention means that at least 80%, in particular at least 85%, of the heparosan produced by the method of the present invention is controlled by at least one parameter of the method and / or recombinant cell of the present invention, such as, for example: - the nature and origin of the nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity of a recombinant cell; - the nature and origin of the promoter controlling the expression of the nucleic acid encoding the polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity in the recombinant cell; - the presence of an anchoring and / or secretion signal associated with the polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity of the recombinant cell; - the pH of the culture medium during the recombinant cell culture process, and / or - Duration of recombinant cell culture By adjustment, it is intended to mean having a molecular weight that falls within a given molecular weight range.

[0118] As used herein, the term "about" refers to a reasonable range for a value as determined by one of ordinary skill in the art. In certain embodiments, the term about refers to ±1, 2, or 3 standard deviations. In certain embodiments, the term about refers to ±5%, 10%, 20%, or 25%. In certain embodiments, the term about refers to ±0.1, 0.2, or 0.3 logarithmic units, e.g., pH units.

[0119] General characteristics of the genetic modifications introduced according to the present invention - all genome modifications are inserted into recombinant cells, in particular recombinant yeasts, according to known genetic engineering techniques: the contiguous nucleic acid sequence contained in the genetic construct to be introduced into the genome of a recombinant cell according to the invention is of the following structure: Prom 1 -ORF 1 -term 1 -ORF 2 -gene 2 -term 2 - … / … -Prom n -ORF n -term n , where: - Prom1 is a sequence regulating the expression of the coding sequence ORF1, - ORF1 is a nucleic acid sequence encoding the desired protein PROT1, in particular the desired enzyme PROT1, - Term1 is a transcription terminator sequence that mediates transcription termination by providing a signal in newly synthesized mRNA that triggers the process of releasing the mRNA from the transcription complex; - "1", "2", ... / ..."n" may or may not refer to the same ORF (open reading frame), promoter, or terminator. The order of the nucleic acid sequences does not matter. "n" is usually an integer ranging from 5 to 20. These constructs are inserted into one of the recombinant cell chromosomes at a controlled location. In some embodiments, the insertion site is not important for the functionality of the inserted construct or for the viability of the resulting genetically modified cell.

[0120] As will be appreciated by those skilled in the art, it may be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant, with 64 possible codons, but most organisms typically use a subset of these codons. The codons most frequently used in a species are referred to as optimal codons, and those used less frequently are classified as rare or low-usage codons. Codons may be substituted to reflect the host's preferred codon usage in a process sometimes referred to as "codon optimization" or "control of species codon bias." Codon optimization for other host cells can be readily determined using codon usage tables or can be performed using commercially available software, such as CodonOp from Integrated DNA Technologies (www.idtdna.com / CodonOptfrom). Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (Murray et al., 1989, Nucl Acids Res. 17: 477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts with desirable properties, such as longer half-life, compared to transcripts produced from non-optimized sequences. Translation stop codons can also be modified to reflect host preferences. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. A typical stop codon for monocotyledonous plants is UGA, while insects and E. coli generally use UAA as the stop codon (Dalphin et al., 1996, Nucl Acids Res. 24: 216-8).

[0121] - When the recombinant cell is a yeast cell, in particular a Saccharomyces cerevisiae yeast cell, the nucleic acid sequences originating from other organisms that are not Saccharomyces cerevisiae and that are introduced into the yeast genome are generally "transcoded" (generally "codon optimized"), meaning that these nucleic acid sequences are synthesized with optimal codon usage for expression in S. cerevisiae. The nucleotide sequence (but not the protein sequence) of some nucleic acid sequences derived from S. cerevisiae has also been modified ("transcoded") to minimize recombination with the endogenous copy of said gene.

[0122] - Genes may be deleted through standard procedures used in cell genetic engineering. In some embodiments, the gene targeted for deletion may be disrupted by insertion of one of the above mentioned genetic constructs, or alternatively, the gene targeted for deletion is replaced by a short stretch of nucleotides.

[0123] - A nucleic acid sequence can be made "inducible or repressible" by deleting the endogenous copy of the nucleic acid sequence (if necessary) and placing the new copy of the ORF under the control of an inducible or repressible promoter. An inducible or repressible promoter is a promoter whose activity is modulated or controlled, i.e. increased or decreased, by a change in environmental conditions or external stimuli. Induction or repression may also be artificially controlled, which includes induction or repression by abiotic factors, such as compounds not naturally found in the cells of interest, in particular yeast, light, oxygen levels, heat, or low temperature. A list and sequences of inducible or repressible promoters are provided elsewhere in this specification.

[0124] Recombinant cells according to the present invention The present inventors have envisaged recombinant cells, in particular recombinant yeast, that have the ability to produce heparosan.

[0125] The present invention relates to recombinant cells, in particular recombinant yeasts, capable of producing heparosan, in which this ability to produce heparosan is obtained through multiple alterations that have been introduced into its genome by genetic engineering methods.

[0126] The present invention relates to a recombinant yeast cell producing heparosan, comprising: (a) one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase (HSS) activity; (b) one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity; The present invention relates to a recombinant yeast cell comprising:

[0127] The present invention further provides a recombinant host cell producing heparosan, comprising: (a) one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase (HSS) activity; (b) one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity; and (c) one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity; Including, The polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity comprises a secretion signal and optionally an anchoring signal so that heparosan, particularly one of the desired molecular weight, is produced by the host cell. Concerning recombinant host cells.

[0128] The present inventors have found that the ability of cells, in particular yeast cells, to produce heparosan can be achieved by introducing multiple genetic alterations into the genome of these cells.

[0129] The production of heparosan by the cells of the invention, in particular the yeast cells of the invention, is achieved by optimizing the endogenous metabolism of UDP-glucose and, optionally, UDP-N-acetyl-glucosamine, and directing the subsequent artificially engineered metabolic pathways primarily towards heparosan, while at the same time maintaining optimal viability of the resulting genetically engineered cells.

[0130] It has been determined that the production of heparosan by recombinant cells according to the invention can be increased by increasing the conversion of glucose-6-phosphate to the ongoing intermediate metabolic products (i) glucose-1-phosphate, UDP-glucuronate, and heparosan, and (ii) fructose-6-phosphate, glucosamine-6-phosphate, N-acetyl-glucosamine-6-phosphate, N-acetyl-glucosamine-1-phosphate, UDP-N-acetyl-glucosamine, and heparosan, while maintaining a balance that allows for good viability of the resulting recombinant cells.

[0131] Indeed, in order to obtain a viable recombinant cell of the invention, many different constructs were tested in order to obtain a viable and efficient recombinant cell, in particular a viable recombinant yeast, which was difficult to obtain because the transient accumulation of some intermediates appeared to be toxic to the yeast.

[0132] In establishing suitable conditions for the preparation of recombinant cells capable of producing heparosan, in particular heparosan having a controlled molecular weight, unexpected technical challenges were encountered.

[0133] A "controlled" molecular weight of the heparosan of the present invention means that at least 80%, in particular at least 85%, of the heparosan produced by the method of the present invention is controlled by at least one parameter of the method and / or recombinant cell of the present invention, such as, for example: - the nature and origin of the nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity of a recombinant cell; - the nature and origin of the promoter controlling the expression of the nucleic acid encoding the polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity in the recombinant cell; - the presence of an anchoring and / or secretion signal associated with the polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity of the recombinant cell; - the pH of the culture medium during the recombinant cell culture process, and / or - Duration of recombinant cell culture By adjustment, it is intended to mean having a molecular weight that falls within a certain range of molecular weights.

[0134] Indeed, after extensive research and experimental testing, the present inventors have cultured recombinant cells, in particular recombinant yeast cells, more particularly recombinant Saccharomyces cerevisiae yeast cells, to obtain a microbial cell culture medium that exhibits the following characteristics: - selection of the nature and origin of the nucleic acid sequence encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity of the recombinant cell of the invention, in particular of a recombinant yeast, and / or the nature and origin of the promoter controlling the expression of the nucleic acid sequence encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity in the recombinant cell of the invention, in particular in the recombinant yeast, and / or the optional presence, in addition to the secretion signal, of an anchoring signal associated with the encoded polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity of the recombinant cell according to the invention, in particular of the recombinant yeast, and / or the pH of the culture medium during the process of culturing the recombinant cells according to the invention, in particular the recombinant yeast cells, and / or - the duration of the cultivation of the recombinant cell according to the invention, in particular the recombinant yeast cell; It has been found that it was possible to produce heparosan with a controlled molecular weight using a method that is controlled by the use of a glycerol derivative.

[0135] To the inventors' knowledge, this has never been achieved before.

[0136] A nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity can be obtained or derived from Pedobacter heparinus.

[0137] The nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity can be under the control of a promoter selected from the group consisting of pTEF1, pCCW12, pCCW12.sba, pCCW12.Sar, pPDC1, pTEF3, pTDH3, pNUP57, and pCCW10.ago.

[0138] The molecular weight of the heparosan may be in the range of less than 50 kDa, preferably in the range of about 20 kDa to about 50 kDa.

[0139] Alternatively, the molecular weight of the heparosan may be in the range above 50 kDa, preferably in the range of about 50 kDa to about 250 kDa.

[0140] In another alternative, the molecular weight of the heparosan may be in the range above 100 kDa, preferably in the range of about 100 kDa to about 1500 kDa.

[0141] The nucleic acid encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity can be obtained or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, in particular, can be obtained or derived from Arabidopsis thaliana or Chlorella virus PBCV1.

[0142] A nucleic acid encoding a polypeptide having heparosan synthase (HSS) activity can be obtained or derived from Pasteurella multocida.

[0143] The recombinant cell according to the present invention comprises: (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity The method may further comprise at least one recombinant nucleic acid encoding one or more of:

[0144] The recombinant cell according to the present invention comprises: (i) a polypeptide having phosphoglucomutase-1 (PGM1) activity, and / or (ii) a polypeptide having UTP-glucose-1-phosphate uridylyltransferase (UGP1) activity, and / or (iii) a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity, and / or (iv) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity The method may further comprise at least one recombinant nucleic acid encoding one or more of:

[0145] The recombinant cell of the invention further comprises: - a nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or - a nucleic acid encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity, and / or - a nucleic acid encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity, and / or - a nucleic acid encoding a polypeptide having UTP-glucose-1-phosphate uridylyltransferase (UGP1) activity, and / or - a nucleic acid encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity, and / or - a nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase (PCM1) may be such that it is obtained or derived from Saccharomyces cerevisiae.

[0146] The recombinant host cell of the present invention may in particular be a yeast.

[0147] The recombinant cell of the present invention may be a yeast cell belonging to the genus Saccharomyces, or Candida, or Kluyveromyces, or Ogataea, or Yarrowia, or Debaryomyces, or Ashbya, in particular to the genus Saccharomyces, more specifically Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces cerevisia ... castellii, Candida albicans, Candida glabrata, Candida tropicalis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus, Kluyveromyces thermotolerans, Ogataea polymorpha, Yarrowia lipolytica, Debaryomyces hansenii, and Ashbya gossypii, and preferably Saccharomyces cerevisiae.

[0148] Another object of the present invention is a method for producing heparosan of a desired molecular weight, comprising the steps of: (a) culturing a recombinant cell of the invention, i.e. a recombinant yeast cell or a recombinant host cell of the invention, in a culture medium for a time sufficient to produce heparosan of the desired molecular weight; (b) optionally isolating or recovering the heparosan from the recombinant cells and / or the culture medium. The present invention relates to a method comprising the steps of:

[0149] In the methods of the present invention, the heparosan may have a molecular weight of about 20 kDa to about 50 kDa, and preferably 20 kDa to 30 kDa.

[0150] In the methods of the invention, the heparosan may alternatively have a molecular weight of between 30 kDa and 50 kDa.

[0151] In the methods of the present invention, the heparosan may alternatively have a molecular weight of from about 50 kDa to about 150 kDa.

[0152] In the methods of the present invention, the heparosan may alternatively have a molecular weight of from about 150 kDa to about 1500 kDa.

[0153] In the methods of the present invention, the recombinant cell may be a yeast, in particular a member of the genus Saccharomyces, and in particular Saccharomyces cerevisiae.

[0154] In the method of the present invention, in particular, the time sufficient to produce heparosan of the desired molecular weight can be a period of about 35 hours to about 50 hours, preferably about 40 hours to about 50 hours, preferably about 48 hours.

[0155] In the method of the present invention, the molecular weight of the produced heparosan can be controlled by the pH of the culture medium.

[0156] In the method of the present invention, the molecular weight of the heparosan can be controlled by adjusting the pH of the culture medium during the culture step (a) of the method of the present invention.

[0157] In the method of the invention, the molecular weight of the heparosan can be controlled by removing biomass from the culture medium.

[0158] The method of the present invention is carried out on an industrial scale, preferably the culture medium is at least about 100 L, more preferably in the range of about 1000 L to about 3000 L, even more preferably about 10,000 L, even more preferably about 100,000 L, or even about 250,000 L.

[0159] Another object of the invention relates to a heparosan obtained or obtainable from a recombinant cell of the invention or from a method of the invention.

[0160] The present invention further relates to a culture medium comprising a heparosan according to the invention.

[0161] The present invention further relates to a composition comprising a heparosan according to the invention.

[0162] The present invention further relates to (i) a heparosan having a molecular weight of less than 50 kDa, preferably in the range of about 20 kDa to about 50 kDa, or greater than 50 kDa, preferably in the range of about 50 kDa to about 250 kDa, or greater than 100 kDa, preferably in the range of about 100 kDa to about 1500 kDa, (ii) a culture medium of the present invention, or (iii) an industrial product, or a consumer product, or a consumable product comprising the composition of the present invention.

[0163] The industrial or consumer product or consumable product of the present invention may be a cosmetic product, a flavor product, a fragrance product, a foodstuff, a food, a beverage, a texturant, a pharmaceutical composition, a dietary supplement, a nutraceutical, a cleaning product, a dental and / or oral hygiene composition.

[0164] Another object of the present invention relates to the use of the recombinant cell of the present invention for the production of heparosan having a molecular weight within the range of about 20 kDa to about 50 kDa or about 50 kDa to about 1000 kDa.

[0165] Recombinant Nucleic Acids Encoding Polypeptides Having Heparosan Synthase Activity - Patent application The recombinant cells according to the invention, in particular the recombinant yeasts according to the invention, comprise one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase activity.

[0166] The polypeptide having heparosan synthase activity according to the present invention converts the intermediate metabolic products UDP-glucuronate and UDP-N-acetylglucosamine (UDP-GlcNAc) into heparosan [→4) β-D-glucuronic acid (GlcA) (1→4) N-acetyl-α-D-glucosamine (GlcNAc) (1→] n This means a polypeptide which converts

[0167] In certain embodiments, one or more of the recombinant nucleic acids encoding a polypeptide having heparosan synthase activity is under the control of an inducible or repressible promoter that is functional in the recombinant cell of the invention.

[0168] One or more of the recombinant nucleic acids encoding a polypeptide having heparosan synthase activity can be under the control of a promoter selected from the group consisting of pCCW12.Sar, pCCW120.sca, pTDH3-1.Sba, and pTDH3.Sar.

[0169] The one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase activity may originate from or be derived from Streptococcus zooepidemicus (sz), Chlorella virus PBCV1 (Vir), Xenopus laevis (xl), or Pasteurella multocida (pm), in particular Pasteurella multocida, as shown in the examples herein.

[0170] The recombinant cell according to the invention, in particular the recombinant yeast according to the invention, may contain 1 to 5, specifically 1 to 3, recombinant nucleic acids encoding a polypeptide having heparosan synthase activity. The recombinant cell according to the invention, in particular the recombinant yeast according to the invention, may contain, for example, 1, 2 or 3 recombinant nucleic acids encoding a polypeptide having heparosan synthase activity.

[0171] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase activity may be inserted into the JLP1 gene, and / or the LEU2 gene, and / or the SAM3 gene of a recombinant cell, in particular a recombinant yeast, as shown in the examples herein.

[0172] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast, - 1 to 5, in particular 1 to 3, recombinant nucleic acids encoding a polypeptide having heparosan synthase activity, - one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase activity originating from or derived from Streptococcus zooepidemicus (sz), Chlorella virus PBCV1 (Vir), Xenopus laevis (xl) or Pasteurella multocida (pm), in particular originating from or derived from Pasteurella multocida, - one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase activity under the control of an inducible or repressible promoter that is functional in a recombinant cell of the invention and / or under the control of a promoter selected from the group consisting of pCCW12.Sar, pCCW120.sca, pTDH3-1.Sba, and pTDH3.Sar. Includes.

[0173] Recombinant Nucleic Acids Encoding Polypeptides Having UDP-Glucose Dehydrogenase Activity A recombinant cell according to the invention, in particular a recombinant yeast according to the invention, comprises one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity.

[0174] A polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity according to the present invention means a polypeptide which converts the intermediate metabolic product uridine-diphosphate-glucose (UDP-glucose) to UDP-glucuronate.

[0175] In certain embodiments, one or more of the recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity is under the control of an inducible or repressible promoter that is functional in a recombinant cell of the invention.

[0176] One or more of the recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity may be under the control of a pCCW12 promoter, in particular under the control of a promoter selected from the group consisting of pCCW12.sk and pCCW12.sba.

[0177] The one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase dehydrogenase activity may originate from or be derived from Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, in particular originate from or are derived from Arabidopsis thaliana or Chlorella virus PBCV1.

[0178] The recombinant cell according to the present invention, particularly the recombinant yeast according to the present invention, may contain 1 to 7, specifically 2 to 7, recombinant nucleic acids encoding polypeptides having UDP-glucose dehydrogenase activity. The recombinant cell according to the present invention, particularly the recombinant yeast according to the present invention, may contain, for example, 2 or 5 recombinant nucleic acids encoding polypeptides having UDP-glucose dehydrogenase activity.

[0179] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity can be inserted into the JLP1 gene, and / or into the LEU2 gene, and / or into the SAM3 gene of a recombinant cell, in particular a recombinant yeast, as shown in the examples herein.

[0180] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast, - 1 to 7, in particular 2 to 7, recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity, - one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity originating from or derived from Arabidopsis thaliana, Chlorella virus PBCV1 or Streptococcus zooepidemicus, in particular originating from or derived from Arabidopsis thaliana or Chlorella virus PBCV1, and - said one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity, which may be under the control of an inducible or repressible promoter that is functional in the recombinant cell of the invention and / or under the control of a pCCW12 promoter, in particular a promoter selected from the group consisting of pCCW12.sk and pCCW12.sba; Includes.

[0181] Recombinant Nucleic Acids Encoding Polypeptides Having Glucuronosyl-Disulfoglucosamine Glucuronidase Activity Recombinant cells according to the invention, in particular recombinant yeasts according to the invention, comprise one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity.

[0182] A polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity according to the present invention means a polypeptide which degrades heparosan, ie converts heparosan of a given molecular weight into heparosan of a lower molecular weight.

[0183] As mentioned above, the polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity of the present invention comprises a secretion signal.

[0184] In certain embodiments, the polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity contains a secretion signal but no anchoring signal.

[0185] In certain embodiments, a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity includes both a secretion signal and an anchoring signal.

[0186] In one embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity are under the control of an inducible or repressible promoter that is functional in the recombinant cell of the invention.

[0187] One or more of the recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity can be under the control of a promoter selected from the group consisting of pTEF1, pCCW12, pCCW12.sba, pCCW12.Sar, pPDC1, pTEF3, pTDH3, pNUP57, and pCCW10.ago.

[0188] In certain embodiments, the nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity is under the control of a promoter selected from the group consisting of pTEF1, pCCW12, pCCW12.sba, pCCW12.Sar, pPDC1, pTEF3, pTDH3, pNUP57, and pCCW10.ago.

[0189] The one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity may originate from or be derived from Pedobacter heparinus (Ph), Cupiennius salei (Csa), Loxosceles intermedia (Li), Hirudo nipponia (Hn), Bothrops atrox (Ba), or Tityus serrulatus (Ts), as shown in the Examples herein. In a particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity originate from or are derived from Pedobacter heparinus (Ph).

[0190] The recombinant cell according to the invention, in particular the recombinant yeast according to the invention, may contain only one recombinant nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity. The recombinant cell according to the invention, in particular the recombinant yeast according to the invention, may alternatively contain 1 to 8, in particular 1 to 5, recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity.

[0191] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity can be inserted into the JLP1 gene, and / or the LYP1 gene, and / or the SAM3 gene, and / or the HIS3 gene, as shown in the Examples herein.

[0192] Recombinant Nucleic Acids Encoding Polypeptides Having Glutamine-Fructose-6-Phosphate Amidotransferase Activity Recombinant cells according to the invention, in particular recombinant yeast cells according to the invention, can comprise one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity.

[0193] A polypeptide having glutamine-fructose-6-phosphate amidotransferase activity according to the present invention means a polypeptide that converts fructose-6-phosphate into glucosamine-6-phosphate.

[0194] In one embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity is under the control of an inducible or repressible promoter that is functional in the recombinant cell of the invention.

[0195] One or more of the recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase synthase activity may be under the control of the pTEF1 promoter, in particular pTEF1.Ago.

[0196] The one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may originate from or be derived from the group consisting of Saccharomyces cerevisiae and Chlorella virus PBCV1.

[0197] The recombinant cell according to the present invention, in particular the recombinant yeast according to the present invention, may contain 1 to 5, specifically 1 to 3, recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity.

[0198] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity can be inserted into the SAM3 gene and / or the JLP1 gene of a recombinant cell, in particular a recombinant yeast, as shown in the Examples herein.

[0199] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast, - 1 to 5, in particular 1 to 3, recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity, - said recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity, said nucleic acid originating from or derived from the group consisting of Saccharomyces cerevisiae and Chlorella virus PBCV1, - said recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity, under the control of the pTEF1 promoter, in particular pTEF1.Ago Includes.

[0200] Recombinant Nucleic Acids Encoding Polypeptides Having UDP-N-Acetylglucosamine Pyrophosphorylase Activity Recombinant cells according to the invention, in particular recombinant yeast cells according to the invention, can contain one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity.

[0201] A polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity according to the present invention means a polypeptide that converts N-acetylglucosamine into UDP-N-acetylglucosamine.

[0202] In one embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity is under the control of an inducible or repressible promoter that is functional in a recombinant cell of the invention, such as, for example, the inducible or repressible promoter pMET6 or pCUP1.

[0203] One or more of the recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity can be under the control of promoter pTDH3.

[0204] The one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may originate from or be derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0205] The recombinant cell according to the present invention, particularly the recombinant yeast according to the present invention, may contain 5 to 15, specifically 5 to 12, recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity. The recombinant cell according to the present invention, particularly the recombinant yeast according to the present invention, may contain, for example, 8 or 10 recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity.

[0206] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity can be inserted into the HIS3 gene, and / or the JLP1 gene, and / or the SAM3 gene of a recombinant cell, in particular a recombinant yeast, as shown in the Examples herein.

[0207] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast, - 5 to 15, in particular 5 to 12, recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity, - one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity, said nucleic acid or nucleic acids originating from or derived from Saccharomyces cerevisiae; - one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity, which is / are under the control of an inducible or repressible promoter that is functional in a recombinant cell of the invention, such as, for example, the inducible or repressible promoter pMET6 or pCUP1 and / or the promoter pTDH3. Includes.

[0208] Recombinant Nucleic Acids Encoding Polypeptides Having Phosphoglucomutase-1 Activity A recombinant cell according to the invention, in particular a recombinant yeast according to the invention, may comprise one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity.

[0209] A polypeptide having phosphoglucomutase-1 (PGM1) activity according to the present invention means a polypeptide which converts glucose-6-phosphate into the intermediate metabolite glucose-1-phosphate.

[0210] In one embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity is under the control of an inducible or repressible promoter that is functional in a recombinant cell of the invention, such as, for example, an inducible or repressible promoter.

[0211] One or more of the recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity can be under the control of promoter pPDC1.

[0212] The one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity can originate from or be derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0213] A recombinant cell according to the invention, in particular a recombinant yeast according to the invention, may contain only one recombinant nucleic acid encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity.

[0214] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity can be inserted into the SAM3 gene of a recombinant cell, in particular a recombinant yeast, as shown in the Examples herein.

[0215] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast, - a single recombinant nucleic acid encoding a polypeptide having phosphoglucomutase-1 activity, - a recombinant nucleic acid encoding a polypeptide having phosphoglucomutase-1 activity, said recombinant nucleic acid originating from or derived from Saccharomyces cerevisiae, and - said recombinant nucleic acid encoding a polypeptide having phosphoglucomutase-1 activity, said nucleic acid being under the control of the promoter pPDC1 Includes.

[0216] Recombinant Nucleic Acids Encoding Polypeptides Having UTP-Glucose 1-Phosphate Uridylyltransferase Activity A recombinant cell according to the invention, in particular a recombinant yeast according to the invention, can comprise one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase (UGP1) activity.

[0217] A polypeptide having UTP-glucose 1-phosphate uridylyltransferase (UGP1) activity according to the present invention means a polypeptide which converts the intermediate metabolite glucose-1-phosphate into the intermediate metabolite UDP-glucose.

[0218] In one embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity is under the control of an inducible or repressible promoter that is functional in a recombinant cell of the invention, such as, for example, the inducible or repressible promoter pSAM1 or pCUP1.

[0219] One or more of the recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity can be under the control of a promoter selected from the group consisting of pPDC1 and pENO2.

[0220] The one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity can originate from or be derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0221] The recombinant cell according to the present invention, particularly the recombinant yeast according to the present invention, may contain 5 to 15, specifically 5 to 12, recombinant nucleic acids encoding a polypeptide having UTP-glucose-1-phosphate uridylyltransferase activity. The recombinant cell according to the present invention, particularly the recombinant yeast according to the present invention, may contain, for example, 8 or 11 recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity.

[0222] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having a UTP-glucose 1-phosphate uridylyltransferase activity gene may be inserted into the HIS3 gene, and / or the JLP1 gene, and / or the SAM3 gene of a recombinant cell, in particular a recombinant yeast, as shown in the Examples herein.

[0223] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast, - 5 to 15, in particular 5 to 12, recombinant nucleic acids encoding a polypeptide having UTP-glucose-1-phosphate uridylyltransferase activity, - one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity originating from or derived from Saccharomyces cerevisiae, and - one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose-1-phosphate uridylyltransferase activity, which is functional in a recombinant cell of the invention, for example under the control of the inducible or repressible promoters pSAM1 or pCUP1 and / or under the control of a promoter selected from the group consisting of pPDC1 and pENO2 Includes.

[0224] Recombinant Nucleic Acids Encoding Polypeptides Having Glucosamine-6-Phosphate N-Acetyltransferase Activity Recombinant cells according to the invention, in particular recombinant yeast cells according to the invention, can contain one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity.

[0225] A polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity according to the present invention means a polypeptide that converts glucosamine-6-phosphate to N-acetyl-glucosamine-6-phosphate.

[0226] In one embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity is under the control of an inducible or repressible promoter that is functional in the recombinant cell of the invention.

[0227] One or more of the recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity can be under the control of promoter pCWP2.

[0228] The one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity may originate from or be derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0229] A recombinant cell according to the invention, in particular a recombinant yeast according to the invention, may contain only one recombinant nucleic acid encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity.

[0230] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity can be inserted into the SAM3 gene of a recombinant cell, in particular a recombinant yeast, as shown in the Examples herein.

[0231] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast, - a single recombinant nucleic acid encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity, - a recombinant nucleic acid encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity, said recombinant nucleic acid having origin or originating from Saccharomyces cerevisiae, - said recombinant nucleic acid encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity, under the control of the promoter pCWP2; Includes.

[0232] Recombinant Nucleic Acids Encoding Polypeptides Having Phosphoacetylglucosamine Mutase Activity Recombinant cells according to the invention, in particular recombinant yeast cells according to the invention, can comprise one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity.

[0233] A polypeptide having phosphoacetylglucosamine mutase (PCM1) activity according to the present invention means a polypeptide that converts N-acetylglucosamine-6-phosphate to N-acetylglucosamine-1-phosphate.

[0234] In certain embodiments, one or more of the recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity is under the control of an inducible or repressible promoter that is functional in the recombinant cell of the invention.

[0235] One or more of the recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity can be under the control of the pTEF1 promoter.

[0236] The one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may originate from or be derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0237] A recombinant cell according to the invention, in particular a recombinant yeast according to the invention, may contain only one recombinant nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase activity.

[0238] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity can be inserted into the SAM3 gene of a recombinant cell, in particular a recombinant yeast, as shown in the Examples herein.

[0239] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast, - only one recombinant nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase activity, - a recombinant nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase activity originating from or derived from Saccharomyces cerevisiae, - the recombinant nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase activity, which is under the control of the pTEF1 promoter; Includes.

[0240] Heparosan synthase (HSS) Heparosan synthase enzymes are proteins that have been described in the art to catalyze the conversion of UDP-glucuronate or UDP-N-acetyl-glucose to heparosan. Heparosan synthases originating from Streptococcus zooepidemicus, Chlorella virus PBCV1, Xenopus laevis, or Pasteurella multocida may be referred to as HSS.

[0241] Methods implemented to measure the activity level of a polypeptide having heparosan synthase activity belong to the general knowledge of the person skilled in the art.

[0242] In this regard, the skilled person may advantageously refer to the method of colorimetric determination after treatment with concentrated sulfuric acid and carbazole described by Bitter and Muir (Analytical Biochemistry, 4, 330-334, 1962).

[0243] A preferred polypeptide having heparosan synthase activity herein is an enzyme having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 (HSS1) and SEQ ID NO: 2 (HSS2).

[0244] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase activity may originate from or be derived from an organism, preferably selected from the group consisting of prokaryotes and eukaryotes. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase activity may originate from or be derived from archaea. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase activity may originate from or be derived from bacteria, in particular Streptococcus zooepidemicus (Sz), Chlorella virus PBCV1 (Vir), Xenopus laevis (Xl), or Pasteurella multocida (Pm), in particular Pasteurella multocida.

[0245] According to yet a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase activity may be selected from the group consisting of nucleic acid sequences having (i) at least 65%, advantageously at least 70%, preferably at least 80% nucleic acid identity with the nucleic acid sequence set forth as sequence SEQ ID NO:1 (HSS1-Pm) or SEQ ID NO:2 (HSS2-Pm), and (ii) the same qualitative biological activity as a nucleic acid sequence having the nucleic acid sequence set forth as sequence SEQ ID NO:1 (HSS1-Pm) or SEQ ID NO:2 (HSS2-Pm), respectively.

[0246] A similar biological activity for this sequence is the ability to encode an enzyme that converts UDP-glucuronate or UDP-N-acetyl-glucose into heparosan, as previously explained.

[0247] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0248] As described herein, a nucleic acid sequence having at least 70% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0249] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0250] With regard to the amino acid sequence of a polypeptide having heparosan synthase activity originating from Pasteurella multocida, a person skilled in the art may refer to the UniProt database accession numbers Q8RP78 and Q5SGE1, respectively, or to SEQ ID NO: 3 (HSS1-Pm) or SEQ ID NO: 4 (HSS2-Pm) described in this specification.

[0251] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase activity may be a nucleic acid encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 50%, advantageously at least 65%, preferably at least 80% amino acid identity with the amino acid sequence set forth as SEQ ID NO: 3 (Pm HSS1) or SEQ ID NO: 4 (Pm HSS2) and also having the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO: 3 (Pm HSS1) or SEQ ID NO: 4 (Pm HSS2).

[0252] A biological activity of the same nature for this sequence is as previously described, namely the ability to catalyse the conversion of UDP-glucuronate or UDP-N-acetyl-glucose to heparosan.

[0253] As described herein, an amino acid sequence having at least 50% amino acid identity to a reference amino acid sequence is at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109, 109, 102, 103, 104, 105, 106, 107, 108, 109 ... The present invention encompasses amino acid sequences that have 3%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also have the same qualitative biological activity as the reference amino acid sequence.

[0254] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence, and also having the same qualitative biological activity as the reference amino acid sequence.

[0255] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also having the same qualitative biological activity as the reference amino acid sequence.

[0256] As mentioned above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase activity in the present invention is regulated by at least one promoter and at least one terminator, such as those defined in more detail hereinafter, present at the 5' and 3' positions of one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase activity, respectively.

[0257] UDP-glucose dehydrogenase (UDP-GlcDH or HASB) UDP-glucose dehydrogenase is a protein known in the art to catalyze the conversion of UDP-glucose to UDP-glucuronate. UDP-glucose dehydrogenase originating from the genomes of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus may be referred to as HASB.

[0258] Methods implemented for measuring the activity level of a polypeptide having UDP-glucose dehydrogenase activity belong to the general knowledge of the person skilled in the art.

[0259] In this respect, the skilled person may advantageously refer to the method described by Oka and Jigami (FEBS Journal 273, 2645-2657, 2006).

[0260] A preferred polypeptide having UDP-glucose dehydrogenase activity herein is the enzyme having the EC number 1.1.1.22.

[0261] According to preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity may originate from or be derived from an organism, preferably selected in the group including prokaryotes and eukaryotes. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity may originate from or be derived from an archaea. In some preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity may originate from or be derived from a yeast, in particular Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, more specifically Arabidopsis thaliana or Chlorella virus PBCV1.

[0262] According to yet preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity may be selected from the group consisting of nucleic acid sequences having (i) at least 65%, advantageously at least 70%, preferably at least 80% nucleic acid identity with the nucleic acid set forth as sequence SEQ ID NO:5(At), SEQ ID NO:6(Vir) or SEQ ID NO:7(Vir), and (ii) the same qualitative biological activity as the nucleic acid set forth as sequence SEQ ID NO:5(At), SEQ ID NO:6(Vir) or SEQ ID NO:7(Vir). The nucleic acids set forth as sequence SEQ ID NO:5(At), SEQ ID NO:6(Vir) and SEQ ID NO:7(Vir) respectively encode a polypeptide having UDP-glucose dehydrogenase activity, which may be collectively referred to herein as HASB, originating from Arabidopsis thaliana (At) or Chlorella virus PBCV1 (Vir).

[0263] A similar biological activity for this sequence is the ability to encode a polypeptide that converts UDP-glucose to UDP-glucuronate, as previously explained.

[0264] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0265] As described herein, a nucleic acid sequence having at least 70% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0266] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0267] With regard to the amino acid sequence of a polypeptide having UDP-glucose dehydrogenase activity derived from Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, a person skilled in the art may refer to the sequences set forth in the UniProt database under accession numbers NP_173979.1, NP_048965, or KIS19289, respectively, or in SEQ ID NO: 8 (At) and SEQ ID NO: 9 (Vir) described herein.

[0268] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity may be a nucleic acid encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 55%, advantageously at least 65%, preferably at least 80% amino acid identity with the amino acid sequences of SEQ ID NO:8 (At) and SEQ ID NO:9 (Vir) and also having the same qualitative biological activity as the amino acid sequences of SEQ ID NO:8 (At) and SEQ ID NO:9 (Vir).

[0269] The same qualitative biological activity for this sequence is as previously described, namely the ability to catalyze the conversion of UDP-glucose to UDP-glucuronate.

[0270] As described herein, an amino acid sequence having at least 55% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with said reference amino acid sequence, and also having the same qualitative biological activity as said reference amino acid sequence.

[0271] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence, and also having the same qualitative biological activity as the reference amino acid sequence.

[0272] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also having the same qualitative biological activity as the reference amino acid sequence.

[0273] As mentioned above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity in the present invention is regulated by at least one promoter and at least one terminator, such as those defined in more detail hereinafter, present at the 5' and 3' positions of one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity, respectively.

[0274] Glucuronosyl-disulfoglucosamine glucuronidase The glucuronosyl-disulfoglucosamine glucuronidase enzyme is a protein that has been described in the art to catalyze the breakdown of heparosan molecules into smaller heparosan molecules. The glucuronosyl-disulfoglucosamine glucuronidase encoded by the genome of Pedobacter heparinus may be referred to as HEPC.

[0275] A polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity of the present invention may have both a secretion signal and an anchoring signal, or may have a secretion signal but no anchoring signal, or may have a secretion-anchor signal with dual secretion and anchoring functions.

[0276] Methods implemented for measuring the activity level of a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity belong to the general knowledge of a person skilled in the art.

[0277] In this regard, the skilled person can advantageously monitor the molecular weight of the obtained heparosan on an agarose gel.

[0278] A preferred polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity herein is the enzyme having the EC number 3.2.1.56.

[0279] According to preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity may originate from or be derived from an organism, preferably selected in the group including prokaryotes and eukaryotes. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity may originate from or be derived from an archaea. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity may originate from or be derived from an organism, preferably selected from yeast. In some other preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity may originate from or be derived from Pedobacter heparinus.

[0280] According to yet preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity may be selected from the group consisting of nucleic acid sequences having at least 65%, advantageously at least 70%, preferably at least 80% nucleic acid identity with the nucleic acid of SEQ ID NO: 10(Ph) or SEQ ID NO: 12(Ph) and also having the same qualitative biological activity as the nucleic acid of SEQ ID NO: 10(Ph) or SEQ ID NO: 12(Ph). The nucleic acids of SEQ ID NO: 10(Ph) and SEQ ID NO: 12(Ph) encode a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity, comprising a secretion signal and not comprising an anchoring signal, originating or derived from Pedobacter heparinus.

[0281] According to another preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity may be selected from the group consisting of nucleic acid sequences having at least 65%, advantageously at least 70%, preferably at least 80% nucleic acid identity with the nucleic acid of SEQ ID NO: 11 (Ph) or SEQ ID NO: 13 (Ph) and also having the same qualitative biological activity as the nucleic acid of SEQ ID NO: 11 (Ph) or SEQ ID NO: 13 (Ph). The nucleic acids of SEQ ID NO: 11 (Ph) and SEQ ID NO: 13 (Ph) encode a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity, contain a secretion signal and an anchoring signal, and originate or are derived from Pedobacter heparinus.

[0282] A biological activity of the same nature for this sequence is the ability to encode a polypeptide which catalyzes the degradation of heparosan molecules into smaller heparosan molecules, as previously explained.

[0283] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0284] As described herein, a nucleic acid sequence having at least 70% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0285] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0286] For the amino acid sequence of one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity derived from Pedobacter heparinus, a person skilled in the art can refer to UniProt database accession number Q59289, which corresponds to both SEQ ID NO: 10(Ph) and SEQ ID NO: 12(Ph) described in this specification.

[0287] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity may be nucleic acids encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences that include a secretion signal and do not include an anchoring signal, have at least 50%, advantageously at least 65%, preferably at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 14(Ph) and also have the same qualitative biological activity as the amino acid sequence of SEQ ID NO: 14(Ph).

[0288] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity may be nucleic acids encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 55%, advantageously at least 65%, preferably at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 15(Ph), including a secretion signal and an anchoring signal, and also having the same qualitative biological activity as the amino acid sequence of SEQ ID NO: 15(Ph).

[0289] The biological activity of the same nature for this sequence is as previously described, namely the ability to catalyse the degradation of heparosan molecules into smaller heparosan molecules.

[0290] As described herein, an amino acid sequence having at least 55% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with said reference amino acid sequence, and also having the same qualitative biological activity as said reference amino acid sequence.

[0291] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence, and also having the same qualitative biological activity as the reference amino acid sequence.

[0292] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also having the same qualitative biological activity as the reference amino acid sequence.

[0293] As described above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity in the present invention is regulated by at least one promoter and at least one terminator, such as those defined in more detail hereinafter, present at the 5' and 3' positions of one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity, respectively.

[0294] Glutamine-fructose-6-phosphate amidotransferase (GFA1) The glutamine-fructose-6-phosphate amidotransferase enzyme is a protein described in the art for catalyzing the conversion of fructose-6-phosphate to glucosamine-6-phosphate. The glutamine-fructose-6-phosphate amidotransferase originating from Saccharomyces cerevisiae and Chlorella virus PBCV1 may be referred to as GFA1.

[0295] Methods implemented for measuring the activity level of a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity belong to the general knowledge of a person skilled in the art.

[0296] In this regard, the skilled person may advantageously refer to the method described by Shiga Shibatan and Hiroaki Kitazawa (Plant Biotechnology 26, 149-152, 2009).

[0297] A preferred polypeptide having glutamine-fructose-6-phosphate amidotransferase activity according to the invention is the enzyme having the EC number n° EC 2.6.1.16.

[0298] According to preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may originate from or be derived from an organism, preferably selected in the group including prokaryotes and eukaryotes. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may originate from or be derived from an archaea. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may originate from or be derived from an organism, preferably selected from Bacillus subtilis and yeast. In some other preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may originate from or be derived from a yeast, in particular Saccharomyces cerevisiae.

[0299] According to yet a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may be selected from the group consisting of nucleic acid sequences having (i) at least 65%, advantageously at least 70%, preferably at least 80% nucleic acid identity with the nucleic acid sequence set forth as sequence SEQ ID NO: 47(Sc), and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as sequence SEQ ID NO: 47(Sc). The nucleic acid set forth as sequence SEQ ID NO: 47 encodes a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity originating from Saccharomyces cerevisiae or Chlorella virus PBCV1, which may also be referred to as GFA1.

[0300] According to yet a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may be selected from the group consisting of nucleic acid sequences having (i) at least 65%, advantageously at least 70%, preferably at least 80% nucleic acid identity with the nucleic acid sequence set forth as sequence SEQ ID NO: 16(Sc) or EQ, and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as sequence SEQ ID NO: 16(Sc). The nucleic acid set forth as sequence SEQ ID NO: 16 encodes a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity originating from Saccharomyces cerevisiae, which may also be referred to as GFA1.

[0301] According to yet another embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may be selected from the group consisting of a nucleic acid sequence having (i) at least 65%, advantageously at least 70%, preferably at least 80% nucleic acid identity with the nucleic acid sequence set forth as sequence SEQ ID NO: 15 or SEQ ID NO: 16, and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as sequence SEQ ID NO: 17 or SEQ ID NO: 18. The nucleic acid sequence set forth as sequence SEQ ID NO: 17 or SEQ ID NO: 18 encodes a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity originating from the Chlorella virus PBCV1.

[0302] A similar biological activity for this sequence is the ability to encode a polypeptide which converts fructose-6-phosphate to glucosamine-6-phosphate, as previously explained.

[0303] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0304] As described herein, a nucleic acid sequence having at least 70% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0305] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0306] For the amino acid sequence of a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity originating from Saccharomyces cerevisiae, those skilled in the art can refer to the UniProt database accession number NP8012818 or the sequence of SEQ ID NO: 19 described in this specification.

[0307] For the amino acid sequence of a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity originating from the Chlorella virus PBCV1, those skilled in the art can also refer to the UniProt database accession number NP_048448 or the sequence of SEQ ID NO: 20 described in this specification.

[0308] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may be a nucleic acid encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 35%, advantageously at least 65%, preferably at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20 and also having the same qualitative biological activity as the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20.

[0309] The biological activity of the same nature for this sequence is as previously described, namely the ability to catalyze the conversion of fructose-6-phosphate to glucosamine-6-phosphate.

[0310] As described herein, an amino acid sequence having at least 35% amino acid identity with a reference amino acid sequence is at least 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65% identity with the reference amino acid sequence. , 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also have the same qualitative biological activity as the reference amino acid sequence.

[0311] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence, and also having the same qualitative biological activity as the reference amino acid sequence.

[0312] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also having the same qualitative biological activity as the reference amino acid sequence.

[0313] As mentioned above, the expression level of a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity in the present invention is regulated by at least one promoter and at least one terminator, such as those defined in more detail hereinafter, present at the 5' and 3' positions, respectively, of one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity.

[0314] UDP-N-acetylglucosamine pyrophosphorylase (QRI1) The UDP-N-acetylglucosamine pyrophosphorylase enzyme is a protein that has been described in the art for catalyzing the conversion of N-acetylglucosamine-6-phosphate to UDP-N-acetylglucose. The UDP-N-acetylglucosamine pyrophosphorylase originating from Saccharomyces cerevisiae may be referred to as QRI1.

[0315] Methods implemented for measuring the activity level of a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity belong to the general knowledge of a person skilled in the art.

[0316] In this regard, the skilled artisan may refer to the method described by Mio et al. (The Journal of Biological Chemistry, Col. 273, No 23, 5 June 1998, 14392-14397), except that UDP-N-acetyl-glucosamine is detected by LC MS / MS using a Synergi RP Fusion column.

[0317] A preferred polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity according to the invention is the enzyme having the EC number n° EC 2.7.7.23.

[0318] According to preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may originate from or be derived from an organism, preferably selected in the group consisting of prokaryotes and eukaryotes. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may originate from or be derived from an archaea. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may originate from or be derived from an organism, preferably selected from Bacillus subtilis and yeast. In some other preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may originate from or be derived from a yeast, in particular Saccharomyces cerevisiae.

[0319] According to yet a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may be selected from the group consisting of nucleic acid sequences having (i) at least 65%, advantageously at least 70%, preferably at least 80% nucleic acid identity with the nucleic acid sequence set forth as SEQ ID NO: 21, and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as SEQ ID NO: 21. The nucleic acid sequence set forth as SEQ ID NO: 21 encodes a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity originating from Saccharomyces cerevisiae, which may also be referred to as QRI1.

[0320] A similar biological activity for this sequence is the ability to encode a polypeptide that converts N-acetylglucosamine-6-phosphate to UDP-N-acetylglucose, as previously described.

[0321] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0322] As described herein, a nucleic acid sequence having at least 70% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0323] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0324] For the amino acid sequence of a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity originating from Saccharomyces cerevisiae, those skilled in the art can refer to the UniProt database accession number NP_010180 or SEQ ID NO: 22 described in this specification.

[0325] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may be a nucleic acid encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 35%, advantageously at least 45%, preferably at least 80% amino acid identity with the amino acid sequence set forth as sequence SEQ ID NO:22 and also having the same qualitative biological activity as the amino acid sequence set forth as sequence SEQ ID NO:22.

[0326] The biological activity of the same nature for this sequence is as previously described, namely the ability to catalyze the conversion of N-acetylglucosamine-6-phosphate to UDP-N-acetyl-glucose.

[0327] As described herein, an amino acid sequence having at least 35% amino acid identity with a reference amino acid sequence is at least 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65% identity with the reference amino acid sequence. , 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also have the same qualitative biological activity as the reference amino acid sequence.

[0328] As described herein, an amino acid sequence having at least 45% amino acid identity with a reference amino acid sequence is at least 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ... , 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also have the same qualitative biological activity as the reference amino acid sequence.

[0329] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also having the same qualitative biological activity as the reference amino acid sequence.

[0330] As described above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity in the present invention is regulated by at least one promoter and at least one terminator, such as those defined in more detail hereinafter, located at the 5' and 3' positions of one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity, respectively.

[0331] Phosphoglucomutase-1 (PGM1) The phosphoglucomutase-1 enzyme is a protein described in the art for catalyzing the conversion of glucose-6-phosphate to glucose-1-phosphate. Phosphoglucomutase-1 originating from Saccharomyces cerevisiae can be referred to as PGM1.

[0332] Methods implemented for measuring the activity level of a polypeptide exhibiting phosphoglucomutase-1 activity belong to the general knowledge of the person skilled in the art.

[0333] In this regard, the skilled person may advantageously refer to the method described by Tiwari and Bhat (Biochemical and Biophysical Research Communications 366, 340-345, 2008).

[0334] A preferred polypeptide having phosphoglucomutase-1 activity according to the invention is the enzyme having the EC number n° 5.4.2.2.

[0335] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity may originate from an organism, preferably selected in the group consisting of prokaryotes and eukaryotes. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity may originate from or be derived from Archaea. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity may originate from or be derived from an organism, preferably selected from Bacteria. In a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity may originate from or be derived from Saccharomyces cerevisiae.

[0336] According to certain embodiments, the one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity may be selected from the group consisting of: (i) a nucleic acid sequence originating from Saccharomyces cerevisiae having at least 80% nucleic acid identity with the nucleic acid sequence set forth as sequence SEQ ID NO:23; and (ii) a nucleic acid sequence having the same qualitative biological activity as the nucleic acid sequence set forth as sequence SEQ ID NO:23.

[0337] A similar biological activity for this sequence is the ability to encode a polypeptide which converts glucose-6-phosphate to glucose-1-phosphate, as previously explained.

[0338] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0339] For the amino acid sequence of a peptide having phosphoglucomutase-1 activity derived from Saccharomyces cerevisiae, those skilled in the art can refer to the UniProt database accession number NP33401 or SEQ ID NO: 24 described herein.

[0340] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity may be a nucleic acid encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 80% amino acid identity with the amino acid sequence set forth as sequence SEQ ID NO:24 and also having the same qualitative biological activity as the amino acid sequence set forth as sequence SEQ ID NO:24.

[0341] The biological activity of the same nature for this sequence is as previously described, namely the ability to catalyze the conversion of glucose-6-phosphate to glucose-1-phosphate.

[0342] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also having the same qualitative biological activity as the reference amino acid sequence.

[0343] As mentioned above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity in the present invention is regulated by at least one promoter and at least one terminator, such as those defined in more detail hereinafter, present at the 5' and 3' positions of one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity, respectively.

[0344] UTP-glucose 1-phosphate uridylyltransferase (UGP1) The UTP-glucose 1-phosphate uridylyltransferase enzyme is a protein that has been described in the art for catalyzing the conversion of glucose-1-phosphate to UDP-glucose. The UTP-glucose 1-phosphate uridylyltransferase originating from Saccharomyces cerevisiae may be referred to as UGP1.

[0345] Methods implemented for measuring the activity level of a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity belong to the general knowledge of the person skilled in the art.

[0346] In this respect, the skilled person can advantageously refer to the method described by Roeben (J. Mol. Biol 364, 551-560, 2006).

[0347] A preferred polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity according to the invention is the enzyme having the EC number n° 2.7.7.9.

[0348] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity may originate from or be derived from an organism, preferably selected in the group consisting of prokaryotes and eukaryotes. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity may originate from or be derived from an archaea. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity may originate from or be derived from an organism, preferably selected from bacteria. In a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity may originate from or be derived from Saccharomyces cerevisiae.

[0349] According to certain embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity may be selected from the group consisting of nucleic acid sequences having (i) at least 80% nucleic acid identity with the nucleic acid set forth as sequence SEQ ID NO:25 originating from Saccharomyces cerevisiae, and (ii) the same qualitative biological activity as the nucleic acid set forth as sequence SEQ ID NO:25.

[0350] A similar biological activity for this sequence is the ability to encode a polypeptide which converts glucose-1-phosphate to UDP-glucose, as previously explained.

[0351] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0352] For the amino acid sequence of a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity originating from Saccharomyces cerevisiae, a person skilled in the art can refer to the sequence set forth under accession number NP_32861 in the UniProt database or as sequence number 26 described herein.

[0353] According to another specific embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity may be a nucleic acid encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 80% amino acid identity with the amino acid sequence set forth as SEQ ID NO:26 and also having the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO:26.

[0354] The same qualitative biological activity for this sequence is as previously described, namely the ability to catalyze the conversion of glucose-1-phosphate to UDP-glucose.

[0355] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also having the same qualitative biological activity as the reference amino acid sequence.

[0356] As mentioned above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity in the present invention is regulated by at least one promoter and at least one terminator, such as those defined in more detail hereinafter, present at the 5' and 3' positions of one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity, respectively.

[0357] Glucosamine-6-phosphate N-acetyltransferase (GNA1) Glucosamine-6-phosphate N-acetyltransferase enzymes are proteins that have been described in the art to catalyze the conversion of glucosamine-6-phosphate to N-acetyl-glucosamine-6-phosphate. The glucosamine-6-phosphate N-acetyltransferase originating from Saccharomyces cerevisiae can be referred to as GNA1.

[0358] Methods implemented to measure the activity level of a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity belong to the general knowledge of a person skilled in the art.

[0359] In this regard, the skilled person may advantageously refer to the method described by Li et al. (Anal. Biochem. 370, 142-146, 2007).

[0360] A preferred polypeptide having glucosamine-6-phosphate N-acetyltransferase activity according to the invention is the enzyme having the EC number n° 2.3.1.4.

[0361] According to preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity may originate from or be derived from an organism, preferably selected from the group consisting of prokaryotes and eukaryotes. In some preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity may originate from or be derived from a yeast, in particular Saccharomyces cerevisiae.

[0362] According to certain embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity may be selected from the group consisting of nucleic acid sequences having (i) at least 80% nucleic acid identity with the nucleic acid sequence set forth as sequence SEQ ID NO: 27, and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as sequence SEQ ID NO: 27. The nucleic acid sequence set forth as sequence SEQ ID NO: 27 encodes a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity originating from Saccharomyces cerevisiae, which may also be referred to as GNA1.

[0363] A similar biological activity for this sequence is the ability to encode a polypeptide that converts glucosamine-6-phosphate to N-acetyl-glucosamine-6-phosphate, as previously described.

[0364] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0365] For the amino acid sequence of a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity originating from Saccharomyces cerevisiae, those skilled in the art can refer to the UniProt database accession number NP_116637 or SEQ ID NO: 28 described in this specification.

[0366] According to another specific embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity may be a nucleic acid encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 80% amino acid identity with the amino acid sequence set forth as SEQ ID NO:28 and also having the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO:28.

[0367] The biological activity of the same nature for this sequence is as previously described, namely the ability to catalyze the conversion of glucosamine-6-phosphate to N-acetyl-glucosamine-6-phosphate.

[0368] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also having the same qualitative biological activity as the reference amino acid sequence.

[0369] As mentioned above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity in the present invention is regulated by at least one promoter and at least one terminator, such as those defined in more detail hereinafter, located at the 5' and 3' positions of the one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity, respectively.

[0370] Phosphoacetylglucosamine mutase (PCM1) The phosphoacetylglucosamine mutase enzyme is a protein that has been described in the art to catalyze the conversion of N-acetylglucosamine-6-phosphate to N-acetylglucosamine-1-phosphate. The phosphoacetylglucosamine mutase originating from Saccharomyces cerevisiae may be referred to as PCM1.

[0371] Methods implemented for measuring the activity level of a polypeptide having phosphoacetylglucosamine mutase activity belong to the general knowledge of a person skilled in the art.

[0372] In this regard, the skilled person can advantageously refer to the method described by Bandini et al. (Molecular Microbiology 85(3), 513-534, 2012).

[0373] A preferred polypeptide having phosphoacetylglucosamine mutase activity according to the invention is the enzyme having the EC number n° 5.4.2.3.

[0374] According to preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may originate from or be derived from an organism, preferably selected in the group consisting of prokaryotes and eukaryotes. In some preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may originate from or be derived from a yeast, in particular Saccharomyces cerevisiae.

[0375] According to certain embodiments, the one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may be selected from the group consisting of: (i) at least 80% nucleic acid identity with the nucleic acid sequence set forth as SEQ ID NO: 29, and (ii) a nucleic acid sequence having the same qualitative biological activity as the nucleic acid sequence set forth as SEQ ID NO: 29. The nucleic acid set forth as SEQ ID NO: 29 encodes a polypeptide having phosphoacetylglucosamine mutase activity originating from Saccharomyces, which may also be referred to as PCM1.

[0376] A similar biological activity for this sequence is the ability to encode a polypeptide that converts N-acetyl-glucosamine-6-phosphate to N-acetyl-glucosamine-1-phosphate, as previously described.

[0377] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence includes nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with said reference nucleic acid sequence and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0378] For the amino acid sequence of a polypeptide having phosphoacetylglucosamine mutase activity originating from Saccharomyces cerevisiae, those skilled in the art may refer to the UniProt database accession number NP_010856 or SEQ ID NO: 30 described herein.

[0379] According to another specific embodiment, the one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may be a nucleic acid encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 80% amino acid identity with the amino acid sequence set forth as SEQ ID NO:30 and also having the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO:30.

[0380] The biological activity of the same nature for this sequence is as previously described, namely the ability to catalyze the conversion of N-acetyl-glucosamine-6-phosphate to N-acetyl-glucosamine-1-phosphate.

[0381] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence and also having the same qualitative biological activity as the reference amino acid sequence.

[0382] As described above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity in the present invention is regulated by at least one promoter and at least one terminator, such as those defined in more detail hereinafter, located at the 5' and 3' positions of one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity, respectively.

[0383] promoter As disclosed herein, the expression of a gene of interest that has been genetically engineered to obtain a recombinant cell according to the present invention comprises appropriate regulatory sequences that are functional in the recombinant cell of the present invention, in particular in a recombinant yeast cell of the present invention, including specifically Saccharomyces cerevisiae.

[0384] A variety of promoters may be used to direct the desired expression of the coding sequence of interest.

[0385] The promoter according to the present invention is the following promoter: pTDH3 (SEQ ID NO: 31), pTDH3.sk (SEQ ID NO: 32), pTDH3-1.Sba (SEQ ID NO: 33), pTDH3.Sar (SEQ ID NO: 34), pENO2 (SEQ ID NO: 35), pTEF3 (SEQ ID NO: 36), pTEF1 (SEQ ID NO: 37), pTEF1.ago (SEQ ID NO: 38), pTEF1.sba (SEQ ID NO: 39), pPDC1 (SEQ ID NO: 40), pCCW12 (SEQ ID NO: 41), pCCW12.Sm (SEQ ID NO: 42), pCCW12.sk (SEQ ID NO: 43), pCCW12.sba (SEQ ID NO: 44), pCCW12.sar (SEQ ID NO: 45), pNUP57 (SEQ ID NO: 46), pCCW10.ago (SEQ ID NO: 37), pCWP2 (SEQ ID NO: 48), pCCW120.Sca (SEQ ID NO: 49), and pRPLA1 (SEQ ID NO: 50) may be selected from the group consisting of:

[0386] Promoters of more particular interest in the present invention are pTDH3 (SEQ ID NO: 31), pTDH3.sk (SEQ ID NO: 32), pTDH3-1.Sba (SEQ ID NO: 33), pTDH3.Sar (SEQ ID NO: 34), pENO2 (SEQ ID NO: 35), pTEF3 (SEQ ID NO: 36), pTEF1 (SEQ ID NO: 37), pTEF1.ago (SEQ ID NO: 38), pTEF1.sba (SEQ ID NO: 39), pPDC1 (SEQ ID NO: 40), pCCW12 (SEQ ID NO: 41), pCCW12.Sm (SEQ ID NO: 42), pCCW12.sk (SEQ ID NO: 43), pCCW12.sba (SEQ ID NO: 44), and pCCW12.sar (SEQ ID NO:45) may be selected from the group consisting of:

[0387] The promoter may in particular be selected from the group consisting of pTDH3, pTDH3-1.Sba, pTDH3.Sar, pENO2, pTEF3, pTEF1, pPDC1, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, and pCCW12.sar.

[0388] Alternatively, the promoter of interest in the present invention is pNUP57 (SEQ ID NO: 46), and pCCW10.ago (SEQ ID NO: 47) may be selected from the group consisting of:

[0389] Other promoters of interest in the present invention are pCWP2 (SEQ ID NO: 48), pCCW120.Sca (SEQ ID NO: 49), and pRPLA1 (SEQ ID NO: 50) It could be.

[0390] As mentioned above, an inducible or repressible promoter is a promoter whose activity is controlled by the presence or absence of a biotic or abiotic factor, and also by the amount of said factor. Thus, for some promoters, their activity is induced, and therefore increased, in particular when the amount of a given factor is increased or increased, and therefore the activity of these same promoters can be repressed, and therefore reduced, when the amount of said factor is decreased or reduced. The amount of said factor in the culture medium of the recombinant yeast cell of the present invention containing an inducible or repressible promoter can be determined by those skilled in the art and controlled accordingly.

[0391] For example, increasing the amount of copper in the culture medium of a recombinant yeast cell according to the invention containing the pCUP-1 promoter will induce and thus increase the transcription of genes under the control of this promoter. In contrast, decreasing the amount of copper in said culture medium will cause repression and thus a decrease in the transcription of genes under the control of this promoter.

[0392] In another example, increasing the amount of methionine in the culture medium of a recombinant yeast cell according to the invention containing the pMET6 promoter will repress and thus decrease the transcription of genes under the control of this promoter, whereas decreasing the amount of methionine in said culture medium will cause induction and thus increase the transcription of genes under the control of this promoter.

[0393] For this reason, the following promoters are referred to herein as "inducible or repressible promoters."

[0394] According to a first embodiment, the inducible or repressible promoter according to the invention may be selected from the group comprising copper-inducible or repressible promoters, methionine-inducible or repressible promoters and threonine-inducible or repressible promoters, in particular pCUP1-copper-inducible or repressible promoters.

[0395] According to this embodiment, the inducible or repressible promoter according to the invention is in particular pCUP1 (SEQ ID NO: 51).

[0396] The activity of this promoter is induced by increasing the presence of methionine, copper, or threonine, as shown above, and their activity is decreased, i.e., repressed, when the amounts of methionine, copper, or threonine are reduced.

[0397] According to a second embodiment, the inducible or repressible promoter according to the invention may be selected from the group comprising copper-inducible or repressible promoters, lysine-inducible or repressible promoters and methionine-inducible or repressible promoters, in particular pMET6-methionine-inducible or repressible (SEQ ID NO: 52), pMET25-methionine inducible or repressible (SEQ ID NO: 53), and pSAM1-methionine-inducible or -repressible (SEQ ID NO:54) may be selected from the group consisting of:

[0398] According to this particular embodiment, the inducible or repressible promoter according to the invention may be selected from the group consisting of pMET6, pMET25, and pSAM1.

[0399] The activity of these promoters is therefore repressed by increasing the presence of methionine, copper, lysine, or glucose, as shown above, and their activity is increased, i.e., induced, when the amounts of methionine, copper, lysine, or glucose are reduced.

[0400] In certain embodiments, the inducible or repressible promoter according to the present invention may be selected from the group comprising copper-inducible or repressible promoters, glucose-inducible or repressible promoters, lysine-inducible or repressible promoters, methionine-inducible or repressible promoters, and threonine-inducible or repressible promoters.

[0401] According to more specific embodiments, the inducible or repressible promoter according to the invention may be selected from the group consisting of pCUP1, pMET6, pSAM1, and pMET25.

[0402] In a particular embodiment of the invention, the promoter of interest in the present invention is pTDH3 (SEQ ID NO: 31), pTEF1 (SEQ ID NO: 37), pTEF1.ago (SEQ ID NO: 38), pPDC1 (SEQ ID NO: 40), pCCW12 (SEQ ID NO: 41), pCCW12.sk (SEQ ID NO: 43), pCCW12.sba (SEQ ID NO: 44), pCCW12.sar (SEQ ID NO: 45), pCWP2 (SEQ ID NO: 48), pCCW120.Sca (SEQ ID NO: 49), pCUP1 (SEQ ID NO: 51), pMET6 (SEQ ID NO: 52), and pSAM1 (SEQ ID NO:54) may be selected from the group consisting of:

[0403] Synthetic promoters as described in Blazeck & Alper (2013) Biotechnol. J. 8 46-58 can also be used.

[0404] The promoter of the present invention may originate from any organism of the Saccharomycetes class, in particular Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces castellii, Saccharomyces bayanus, Saccharomyces arboricola, Saccharomyces kudriavzevii, Saccharomyces mikatae, Ashbya gossypii, Kluveromyces lactis, Pichia pastoris, Candida glabrata, Candida tropicalis, Debaryomyces castelii, Yarrowia lipolitica, and Cyberlindnera jadinii. The organism may originate from an organism selected from the group consisting of at least one of the following:

[0405] The promoter of the present invention may preferably originate from an organism selected from the group consisting of Saccharomyces cerevisiae (sc), Saccharomyces mikatae (Sm), Saccharomyces kudriabzevii (sk), Saccharomyces bayanus (sba), and Saccharomyces arboricola (Sar).

[0406] Terminator As disclosed herein, the expression of a gene of interest that has been genetically engineered to obtain a recombinant cell according to the invention, in particular a recombinant yeast cell according to the invention, comprises a suitable transcription terminator sequence that is functional in the recombinant cell of the invention, in particular a recombinant yeast cell of the invention, in particular Saccharomyces cerevisiae.

[0407] The same or different transcription terminators can be found in the publication Yamanishi et al. (2013) ACS synthetic biology 2, 337-347.

[0408] Terminators of more particular interest in the present invention are tTPI1 (SEQ ID NO: 55), derived from the gene encoding triosephosphate isomerase, tMET25 (SEQ ID NO: 56), derived from the gene encoding O-acetylhomoserine-O-acetylserine sulfhydrylase, tDIT1 (SEQ ID NO: 57), tRPL3 (SEQ ID NO: 58), tRPL3.sm (SEQ ID NO:59), tRPL3.sba (SEQ ID NO: 60), tRPL41B (SEQ ID NO: 61), tRPL15A (SEQ ID NO: 62), tRPL15A.sba (SEQ ID NO: 63), tIDP1 (SEQ ID NO: 64), and tTEF1.sba (SEQ ID NO:65) may be selected from the group comprising:

[0409] In particular, the terminator may be selected from the group consisting of tTPI1, tMET25, tDIT1, tRPL3, tRPL3.sm, tRPL3.sba, tRPL41B, tRPL15A, tRPL15A.sba, tIDP1, and tTEF1.sba.

[0410] The terminator of the invention may originate from any organism from the class Hemiascomycota, in particular from an organism selected from the group consisting of Saccharomyces cerevisiae and Saccharomyces bayanus.

[0411] Recombinant yeast The recombinant cell of the present invention may be selected from the group consisting of yeast and bacteria.

[0412] A recombinant cell of the invention, eg, a recombinant host cell of the invention, is preferably a recombinant yeast cell.

[0413] In general, yeast can grow rapidly, can be cultured at high densities compared to bacteria, and does not require a sterile environment in an industrial environment. Furthermore, yeast cells can be easily separated from the culture medium compared to bacterial cells, greatly simplifying the process of product extraction and purification.

[0414] A recombinant cell of the invention, in particular a recombinant yeast cell of the invention, is preferably a Saccharomycetales cell.

[0415] The recombinant cell of the invention, in particular the recombinant yeast of the invention, may in particular belong to the genus Saccharomyces, or Candida, or Kluyveromyces, or Ogataea, or Yarrowia, or Debaryomyces, or Ashbya.

[0416] The recombinant cell of the present invention belonging to the genus Saccharomyces may be selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces castellii, Saccharomyces cariocanus, Saccharomyces kudriabzevii, Saccharomyces arboricolus, Saccharomyces pastorianus, Saccharomyces uvarum, and Saccharomyces delbrueckii.

[0417] The recombinant cell of the present invention belonging to the genus Candida may be selected from the group consisting of Candida albicans, Candida glabrata, Candida tropicalis, Candida dubliniensis, Candida parapsilosis, Candida lusitaniae, and Candida guilliermondii.

[0418] The recombinant cell of the present invention belonging to the genus Kluyveromyces may be selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus, Kluyveromyces thermotolerans, Kluyveromyces dobzhanskii, and Kluyveromyces wickerhamii.

[0419] The recombinant cell of the present invention belonging to the genus Ogataea may be selected from the group consisting of Ogataea polymorpha, Ogataea histrianica, Ogataea deakii, Ogataea kolombanensis, Ogataea philodendra, Ogataea siamensis, Ogataea angusta, Ogataea parapolymorpha, Ogataea minuta, Ogataea nonfermentans, and Ogataea kodamae.

[0420] The recombinant cell of the present invention belonging to the genus Yarrowia can be selected from the group consisting of Yarrowia lipolytica, Yarrowia parophonii, Yarrowia galli, Yarrowia oslonensis, Yarrowia alimentaria, Yarrowia hollandica, and Yarrowia yakushimensis.

[0421] The recombinant cell of the present invention belonging to the genus Debaryomyces includes Debaryomyces hansenii, Debaryomyces carsonii, Debaryomyces castellii, Debaryomyces marama, Debaryomyces occidentalis, Debaryomyces oviformis, Debaryomyces nepalensis, Debaryomyces coudertii, Debaryomyces udenii, Debaryomyces psychrosporus, and Debaryomyces yamadae. yamadae).

[0422] The recombinant cell of the present invention belonging to the genus Ashbya may be selected from the group consisting of Ashbya gossypii and Ashbya aceri.

[0423] The recombinant cell of the invention, in particular the recombinant yeast cell of the invention, may in particular be selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces castellii, Candida albicans, Candida glabrata, Candida tropicalis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus, Kluyveromyces thermotolerans, Ogataea polymorpha, Yarrowia lipolytica, Debaryomyces hansenii and Ashbya gossypii, and is preferably Saccharomyces cerevisiae.

[0424] In certain embodiments, the recombinant yeast cell according to the invention is of the genus Saccharomyces, Kluyveromyces, or Eremothecium, more specifically of a species selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces marxianus, Ogataea polymorpha, and Ashbya gossypii.

[0425] In one embodiment, the recombinant host cell of the invention is a yeast selected from the order Saccharomycetales, in particular from the family Saccharomycetaceae, in particular selected from the group consisting of Yarrowia lipolytica, Kluyveromyces marxianus, Ogataea polymorpha, Ashbya gossypii, and Saccharomyces cerevisiae.

[0426] The recombinant cell of the present invention may more preferably be a Saccharomyces cerevisiae cell.

[0427] As mentioned above, the recombinant cell according to the invention has the ability to produce heparosan by inserting one or more recombinant nucleic acids according to the invention. In a particular embodiment, the recombinant yeast according to the invention has the ability to produce heparosan with a controlled size by inserting one or more recombinant nucleic acids according to the invention.

[0428] The methods implemented to insert specific DNA constructs into genes are within the general knowledge of the person skilled in the art. The relevant methods are described in more detail in the Examples below in this specification.

[0429] However, unexpected technical problems were encountered because the outcome of inserting a DNA construct into the genome of a cell, in particular the genome of a yeast, for example the genome of Saccharomyces cerevisiae, is unpredictable. In particular, the viability of the cells, in particular the yeast, and their ability to grow and produce the desired heparosan are also unpredictable.

[0430] To obtain the recombinant cells of the invention, in particular recombinant cells, a large number of different constructs have been tested by the inventors in order to obtain viable and efficient recombinant cells, in particular yeast.

[0431] Culture conditions The present invention also relates to the use of the recombinant cells of the invention for the production of heparosan, in particular heparosan of controlled molecular weight.

[0432] The present invention further provides a method for producing heparosan, in particular heparosan of a desired molecular weight, comprising the steps of: (a) culturing a recombinant cell according to the invention in a culture medium for a time sufficient to produce heparosan; (b) optionally isolating or recovering the heparosan from the recombinant cells and / or the culture medium. The present invention relates to a method comprising the steps of:

[0433] Typically, the cells of the invention, in particular the yeasts of the invention, are grown in a suitable culture medium at a temperature of about 20°C to about 37°C, preferably at a temperature of 27-34°C.

[0434] Suitable growth media for the cells of the invention, particularly the yeasts of the invention, are generally commercially prepared media such as media containing a yeast nitrogen source, ammonium sulfate, and dextrose as a carbon / energy source, or YPD medium, which is a blend of peptone, yeast extract, and dextrose in optimal ratios for best growth. Other defined or synthetic growth media may also be used, and suitable media for the growth of particular cells, particularly yeasts, will be known to those skilled in the art of microbiology or fermentation science.

[0435] A particular medium that is preferred herein is SY medium, which contains the following elements: KH2PO4:100mM, MgSO4 7H2O:2,8mM, K2SO4:11,5mM, Na2SO4:1,1mM, NaCl:2,6mM, CaCl2 2H2O:0,7mM, CuSO4 5H2O:15μM, KI:6μM, FeCl3:30μM, ZnSO4 7H2O:61μM, MnSO4 H2O: 25 μM, H2SO4: 110 μM, pantothenic acid hemicalcium salt: 42 μM, thiamine hydrochloride: 59 μM, pyridoxine hydrochloride: 49 μM, myo-inositol (C6H12O6): 555 μM, nicotinic acid (C6H5NO2): 29 μM, D-biotin: 0.82 μM, tribasic ammonium citrate: 33 mM, and glucose or sucrose 2-30%. Includes.

[0436] Carbon sources that can be used in the culture medium include fructose, mannose, xylose, and arabinose, oligosaccharides such as lactose, maltose, galactose, or sucrose, polysaccharides such as starch or cellulose, or mixtures thereof, as well as unrefined mixtures from renewable raw materials such as cheese whey permeate, corn steep liquor, sugar beet molasses, and malt.

[0437] Nitrogen sources that may be included in the culture medium include peptone, yeast extract, meat extract, malt extract, urea, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium citrate, and combinations thereof.

[0438] The culture medium may further comprise trace elements (eg, metal salts), such as magnesium salts, cobalt salts, and / or manganese salts, as well as growth factors, such as amino acids, vitamins, growth promoting factors, and the like.

[0439] Examples of vitamins that may be included are hemicalcium pantothenate, thiamine hydrochloride, pyridoxine hydrochloride, myo-inositol, nicotinic acid, D-biotin, folic acid, p-aminobenzoic acid, and riboflavin.

[0440] The culture medium of the present invention may contain rare elements, such as CuSO 4 5H 2 O, KI, FeCl 3 , ZnSO 4 7H 2 O, MnSO 4 H 2 O or H 2 SO 4 , MgCl2, CaCl2, NaCl, K2HPO4, KH2PO4, ZnCl, H3BO3, MnSO4, Na2MoO4.

[0441] The term "suitable culture medium" is defined above.

[0442] Examples of known culture media for recombinant cells according to the present invention are known to those skilled in the art and are provided in the following publications: D. Burke et al., Methods in yeast Genetics - A cold spring harbor laboratory course Manual (2000).

[0443] The suitable pH range for fermentation is pH 3.0 to pH 7.5, with pH 4 to pH 6 being preferred as initial conditions.

[0444] As described elsewhere herein, the pH value of the culture medium can be adjusted during the culture step of the method of the invention in order to modulate the activity of the polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity, which will affect the molecular weight of the heparosan molecule produced by the recombinant cell of the invention, in particular the recombinant yeast of the invention.

[0445] In particular, the pH of the culture medium may be modified depending on the heparosan intended to be produced by the recombinant yeast. For example, the pH of the culture medium may remain at a pH of 4, 5.5, or 6 during the culture period.

[0446] In certain embodiments, the pH of the culture medium may change or be allowed to vary during the length of time of culturing a recombinant cell of the invention, particularly a recombinant yeast of the invention. As previously mentioned, Saccharomyces cerevisiae oxidizes the medium in which it is cultured, reducing the pH of the culture medium. For example, the pH of the culture medium may start at 6, drop to 4, and then return to 6. In another example, the pH of the culture medium may start at 6, remain at or be maintained at 6, and then be lowered to 4.

[0447] In certain embodiments, the pH of the culture medium may be modulated during the culturing step (a) of the method of the invention, such that at the end of the culturing step of the method of the invention, the pH of the culture medium is the same as the pH at the start of said culturing step (a).

[0448] In another embodiment, the pH of the culture medium may remain the same throughout the length of time of the culture of the recombinant cells of the invention, particularly the recombinant yeast cells of the invention.

[0449] Said time length of cultivation of the recombinant cell according to the invention, in particular the recombinant yeast cell of the invention, can vary depending on the molecular weight of the heparosan of interest: the longer said time length, the lower the molecular weight of the heparosan in a given culture medium of the recombinant cell of the invention, in particular the recombinant yeast cell of the invention.

[0450] The length of time for culturing the recombinant cells of the present invention, particularly the recombinant yeast of the present invention, can be from about 35 hours to about 50 hours, preferably from about 40 hours to about 50 hours, and is particularly about 48 hours.

[0451] Fermentation can be carried out under aerobic or microaerobic conditions.

[0452] The amount of heparosan product in the fermentation medium can be determined using several methods known in the art, such as high performance liquid chromatography (HPLC) or gas chromatography (GC).

[0453] The process may utilize a batch fermentation method. Classical batch fermentation is a closed system in which the composition of the medium is set at the beginning of the fermentation and is not subject to artificial changes during the fermentation. Thus, at the beginning of the fermentation, the medium is inoculated with the desired organisms and the fermentation is allowed to occur without adding anything to the system. Typically, however, a "batch" fermentation method or system is batch with respect to the addition of the carbon source, and control of factors such as temperature, pH, and oxygen concentration is often attempted. In a batch system, the composition of the metabolic products and biomass of the system changes constantly until the time when the fermentation is stopped. Within a batch culture, cells progress from a stationary lag phase to a high growth logarithmic phase and finally to a stationary phase where the growth rate is reduced or stopped. If not treated, the cells in the stationary phase will eventually die. Generally, the cells in the logarithmic phase are responsible for the majority of the production of the end product or intermediate.

[0454] Fed-batch systems may also be used in the present invention. A fed-batch system is similar to a typical batch system, except that the carbon source substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression (e.g., glucose repression) tends to inhibit the metabolism of the cells and when it is desirable to limit the amount of substrate in the medium. Measurement of the actual substrate concentration in a fed-batch system is difficult, and therefore, pH, dissolved oxygen, and CO2 are important parameters to be considered. 2 The estimated values ​​are based on changes in measurable factors such as the partial pressure of exhaust gases, etc.

[0455] Batch and fed-batch culture methods are common and well known in the art, and examples can be found in Biotechnology: A Textbook of Industrial Microbiology, Crueger, Crueger, and Brock, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA, or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36, 227, (1992). Although the present invention is carried out in batch mode, it is contemplated that the method could be adapted for continuous fermentation.

[0456] Continuous fermentation is an open system in which a defined fermentation medium is continuously added to a bioreactor and an equal amount of conditioned medium is simultaneously removed for processing. Continuous fermentation generally maintains the culture at a constant high density where the cells are primarily in log phase growth.

[0457] Continuous fermentation allows for the modulation of one or any number of factors that affect cell growth or end product concentration. For example, one method would maintain the limiting nutrient, e.g., carbon source or nitrogen level, at a fixed rate and allow all other parameters to vary. In other systems, some factors that affect growth can be continuously altered while the cell concentration, measured by medium turbidity, is kept constant. Continuous systems attempt to maintain steady-state growth conditions, and therefore cell loss due to medium runoff must be balanced against the cell growth rate during fermentation. Methods for modulating nutrients and growth factors in continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the field of industrial microbiology.

[0458] It is contemplated that the present invention may be practiced using either batch, fed-batch, or continuous processes, and that any known fermentation mode would be suitable. In addition, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for production.

[0459] To further improve heparosan production, a particular embodiment may consist in culturing the recombinant cell of the invention, in particular the recombinant yeast cell of the invention, in a suitable culture medium, such as those mentioned above, wherein said culture medium comprises an optimal amount of a carbon source, in particular glucose or sucrose.

[0460] In a preferred embodiment, the carbon source contained in the optimal culture medium consists of glucose and / or sucrose. In a preferred embodiment, the optimal culture medium comprises 1% w / w or more of glucose and / or sucrose, in particular 5% w / w or more of glucose and / or sucrose, in particular 10% w / w or more of glucose and / or sucrose, in particular 15% w / w or more of glucose and / or sucrose. In a preferred embodiment, the optimal culture medium comprises at most 40% w / w of glucose, including at most 35% w / w of glucose.

[0461] In a preferred embodiment, the process of the invention is carried out on an industrial scale.

[0462] More specifically, the culture medium of the method according to the present invention can be at least about 100L, more preferably in the range of about 1000L to about 3000L, even more preferably about 10,000L, even more preferably 100,000L, or even about 250,000L.

[0463] The present invention further relates to a method for producing the aforementioned heparosan, comprising the steps of: (a) culturing a recombinant cell of the present invention in a culture medium; (b) recovering heparosan from the culture medium; Including, The heparosan recovered in step (b) - the nature and origin of the recombinant cell of the invention, in particular the recombinant nucleic acid(s) encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity of the recombinant cell of the invention, - the nature and origin of the promoter controlling the expression of the recombinant nucleic acid or nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity of the recombinant cell of the invention, in particular of the recombinant yeast of the invention; - the presence or absence of an anchoring signal associated with one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity of a recombinant cell of the invention, in particular a recombinant yeast of the invention, the pH of the culture medium during the process of culturing the recombinant cell of the invention, in particular the recombinant yeast of the invention, and / or - the period during which the recombinant cell of the invention, in particular the recombinant yeast of the invention, is cultured; having a molecular weight controlled through the selection of It concerns the method.

[0464] The molecular weight of heparosan is a particular molecular weight, or more preferably, a particular range of molecular weight, such as, for example, less than 50 kDa, in the range of about 20 kDa to about 50 kDa, 50 kDa or more, in the range of about 50 kDa to about 150 kDa, in the range of about 50 kDa to about 250 kDa, 100 kDa or more, in the range of about 100 kDa to about 1500 kDa, in the range of about 150 kDa to about 1500 kDa, greater than 1000 kDa, or greater than 1500 kDa.

[0465] The present invention also relates to the use of a recombinant cell according to the present invention, in particular a recombinant yeast cell according to the present invention, for the production of heparosan having a molecular weight in the range of about 20 kDa to about 50 kDa or about 50 kDa to about 1000 kDa.

[0466] The secretion signal of the present invention is, for example, - the nucleic acid sequence set forth as SEQ ID NO: 66, and / or - the amino acid sequence set forth as SEQ ID NO: 67 may have:

[0467] The anchoring signal of the present invention is, for example, - the nucleic acid sequence set forth as SEQ ID NO: 68, and / or - the amino acid sequence of SEQ ID NO: 69 may have:

[0468] A secretion signal can be fused to a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity by creating a chimeric nucleic acid beginning with a nucleic acid sequence encoding a signal peptide and followed by a recombinant nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity as defined above.

[0469] Secretion signals and anchoring signals can be fused to a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity by creating a chimeric nucleic acid beginning with a nucleic acid sequence encoding a signal peptide, followed by a recombinant nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity as defined above, followed by a nucleic acid sequence encoding the anchoring signal.

[0470] Such chimeric nucleic acid sequences can be obtained by techniques known to those skilled in the art, such as, for example, chemical synthesis of nucleic acids or by any recombinant technique, such as, for example, cloning or PCR.

[0471] Purification of heparosan According to a particular embodiment of the present invention, the fermentative production of heparosan preferably includes a step of isolation of the produced heparosan from the culture medium. The recovery of heparosan from the culture medium is a routine operation for those skilled in the art. It can be achieved by several techniques well known in the art, including but not limited to pervaporation, selective precipitation, filtration, centrifugation, spray drying, freeze drying or liquid extraction. Those skilled in the art know how to adapt the parameters of each technique depending on the characteristics of the material to be separated.

[0472] Yeast as a model cell in the present invention is preferred in that the synthesized heparosan is transported entirely outside the cell, thus simplifying the purification process.

[0473] Gas stripping is accomplished using a stripping gas selected from helium, argon, carbon dioxide, hydrogen, nitrogen, or mixtures thereof.

[0474] Liquid extraction is accomplished using an organic solvent such as pentane, hexane, heptane, or dodecane as the hydrophobic phase. A regenerating solvent may also be used.

[0475] derivative The term "heparosan", as used herein, is also intended to encompass derivatives of heparosan, such as heparin (sulfated heparosan), that are useful for applications in cosmetic products, flavor products, fragrance products, foodstuffs, foods, beverages, texturants, pharmaceutical compositions, dietary supplements, nutraceuticals, cleaning products, and / or dental and / or oral hygiene compositions, or combinations thereof.

[0476] As used herein, a "derivative of heparosan" is a heparosan compound that has been modified by chemical or enzymatic methods and therefore contains a similar structure to heparosan. These derivatives may exhibit the same biological activity as heparosan, or they may exhibit different biological activity.

[0477] Suitable heparosan derivatives include, but are not limited to, heparin, enoxaparin, dalteparin, and tinzaparin.

[0478] Heparin is useful as an anticoagulant (blood thinner) and in mitigating the risk of, reducing, preventing, and / or treating heart attacks and unstable angina.

[0479] In particular, heparin is generally Acute coronary syndromes, e.g. NSTEMI, Atrial fibrillation, Deep vein thrombosis and pulmonary embolism, Cardiopulmonary bypass for cardiac surgery, -ECMO circuits for extracorporeal life support, Hemofiltration and / or Indwelling central or peripheral venous catheters and for anticoagulant purposes to mitigate the risk of, reduce, prevent, and / or treat conditions such as, but not limited to,

[0480] Heparin and its low molecular weight derivatives (eg, enoxaparin, dalteparin, tinzaparin) are effective in preventing deep vein thrombosis and pulmonary embolism in at-risk individuals.

[0481] Heparin is given by injection into a vein or under the skin.

[0482] Other uses include applications in test tubes and kidney dialysis machines.

[0483] Heparosan, and in particular heparin, can be prepared as a composition.

[0484] Formulations and Products The compositions of the invention may be incorporated into formulations / products, for example nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulations / products.

[0485] Thus, the present invention provides a formulation comprising the composition of the present invention. For example, the present invention may provide a cosmetic formulation comprising the composition of the present invention.

[0486] The present invention also provides a product comprising the composition of the present invention. For example, the present invention may provide a cosmetic product comprising the composition of the present invention.

[0487] "Cosmetic product" is intended to mean any substance or mixture intended to be placed in contact with the exterior of the human body (the epidermis, hair system, nails, lips, and external genitalia) or with the teeth and mucous membranes of the oral cavity, exclusively or primarily with a view to cleaning them, perfume them, modifying their appearance, protecting them, keeping them in good condition, correcting body odor, and / or combinations thereof.

[0488] "Substance" is intended to mean chemical elements and their compounds in the natural state or obtained by any manufacturing process, including any additives necessary to preserve their stability, and any impurities resulting from the process used, but excluding any solvent that can be separated without affecting the stability of the substance or changing its composition.

[0489] "Mixture" means a mixture or solution composed of two or more substances.

[0490] The present invention also provides the use of the compositions of the invention in nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulations / products.

[0491] Such formulations or products are hereinafter referred to as "formulations or products of the invention".

[0492] The nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulations / products may optionally further comprise pharmaceutical / veterinary / cosmetic (e.g., cosmetically active) ingredients, such as excipients, carriers, and mixtures thereof, as appropriate.

[0493] "Cosmetic" or "cosmetic active ingredient" means any and all natural, naturally occurring, nature-identical, synthetic, synthetically produced, biosynthetically produced, sustainable, renewable, and / or biodegradable compounds, ingredients, intermediates, molecules, substances, raw materials, or products, either individually or as part of a mixture of compounds, ingredients, intermediates, molecules, substances, raw materials, or products; Blends, compositions, formulations (including, but not limited to, skin moisturizers, creams, balms, serums, oils, eye, facial makeup, rinse-off hair products, leave-in hair products, hair dyes (including, but not limited to, natural hair dyes), and / or combinations thereof), final products and related technologies such as, for example, ingredients incorporated into cosmetic formulations (e.g., natural colorants, preservatives, emulsifiers, antioxidants, etc., which have no activity on, for example, the skin, hair, scalp, etc., but play a role in the formulation of the final product), delivery systems, marketing aids (e.g., colored unispheres applied to clear formulations), and - direct application onto the human or animal body by rubbing, pouring, sprinkling, spraying or other methods and / or by contact with various external and / or surface parts of the human or animal body (including but not limited to the skin, hair, hair, hair system, scalp, nails, lips, external genitalia, teeth, oral and / or nasal mucosa, etc.); and / or indirect application to the human or animal body, such as, for example, application as part of a textile or application to a textile as part of a delivery device (e.g., capsule) or delivery system (e.g., blend or formulation) that is applied to the textile; and / or - primarily for cleansing or perfume the skin, oral mucosa, scalp, or hair, or for the purpose of cleansing, caring for, cooling, beautifying, conditioning, treating, soothing, texturizing, enhancing, protecting, maintaining, improving, enhancing, altering, and / or changing the exterior and / or surfaces of the human or animal body (e.g., but not limited to, the scalp) or the aesthetic appearance of the human or animal body, and / or by providing soothing, healing, repair, or regeneration, hydration of the skin, or to relieve, smooth, moisturize, color-regulate, heal, sterilize, relieve, correct, and / or improve conditions of dryness, irritation, injury, or fatigue; with a view to protecting or maintaining the good condition of the skin, or neutralizing its body odor, or changing its appearance, or correcting or repairing an imbalance, and / or correcting pigmentation disorders, or providing non-pharmaceutical prevention and / or treatment of dandruff, acne, irritation and / or inflammation, etc., and / or restoring balance to the bacterial flora (e.g., for example, the microflora) on the surface of the skin (e.g., for example, by promoting levels of beneficial bacterial flora on the skin surface), and / or for the purpose of keeping the human or animal body in good condition for health and / or well-being purposes, and / or for the purpose of improving the appearance of the human or animal body, for example by improving the appearance of a product applied to the human or animal body, and / or - Providing cosmetic and / or dermatological functions and / or benefits together with the benefit of biological activity (but without affecting the structure or function of the body) means a method of making that is useful / used in / intended for, or related to. For the avoidance of doubt, a cosmetic or cosmetically active ingredient, or part thereof, may also qualify as a functional ingredient and / or a nutraceutical.

[0494] "Functional ingredient" means a food ingredient or portion of a food that provides a medicinal or health benefit and includes any of the following: carotenoids, dietary fiber, fatty acids, saponins, antioxidants, flavonoids, isothiocyanates, phenols, polyphenols (e.g., resveratrol), plant sterols or stanols (phytosterols and phytostanols), polyols, prebiotics, phytoestrogens, soy proteins, sulfides / thiols, vitamins, glucosamine, preservatives, humectants, edible gelling ingredients, edible gel mixtures and gel compositions, long chain primary aliphatic saturated alcohols, colorants, texture modifiers, emulsifiers, and combinations thereof.

[0495] "Nutraceutical" means any and all natural, naturally occurring, sustainable, synthetically produced, and biosynthetically produced compounds, mixtures of compounds, functional ingredients, molecules, compositions, raw materials, and intermediates, including ingredients and their associated delivery devices (e.g., capsules), delivery systems thereof (e.g., blends or formulations), and methods of making the foregoing, associated with improving or maintaining the health and / or cosmetic benefits and appearance of the human body. For the avoidance of doubt, nutraceuticals include compounds that can be used as a supplement to food or beverages, whether in solid formulations, capsules, tablets, liquid formulations, solutions, or suspensions.

[0496] Alternatively, the nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulation / product may consist or consist essentially of the composition of the present invention.

[0497] The cosmetic formulation / product may be an anti-ageing formulation.

[0498] As used herein, references to a medicament, veterinary, or cosmetically acceptable excipient may refer to medicament, veterinary, or cosmetically acceptable adjuvants, diluents, and / or carriers known to those skilled in the art.

[0499] "Pharmaceutically / veterinarily / cosmetically acceptable" means that the additional components of the composition are generally safe, non-toxic, and not biologically or otherwise undesirable. For example, the additional components may generally be sterile and pyrogen-free. Such components must be "acceptable" in the sense of being compatible with the composition of the present invention and not harmful to the recipient thereof. Thus, a "pharmaceutically acceptable excipient" includes any compound used to form part of a formulation that is intended to act merely as an excipient, i.e., not intended to have biological activity itself.

[0500] The nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulations / products may be in liquid or solid form.

[0501] Liquid dosage formulations / products for oral administration include solutions, emulsions, aqueous or oily suspensions, syrups, and elixirs.

[0502] The formulations and products described herein (e.g., pharmaceutical, veterinary, or cosmetic formulations / products), e.g., those intended for oral administration, can be prepared according to methods known to those skilled in the art, e.g., by mixing the ingredients of the formulation / product together.

[0503] A formulation or product (e.g., a pharmaceutical, veterinary, or cosmetic formulation / product) may include one or more additional ingredients, e.g., medicinal ingredients and excipients, e.g., sweeteners, flavoring agents, coloring agents, and preservatives.

[0504] The formulations or products (e.g., pharmaceutical, veterinary, or cosmetic formulations / products) may also include one or more additional active ingredients, e.g., cosmetic or pharmaceutical active ingredients, such as hyaluronic acid, centella asiatica extracts, peptides, e.g., Matrixyl® and Argireline®, and mixtures thereof.

[0505] The formulations or products of the present invention may contain the active ingredient in admixture with non-toxic pharma- ceutically acceptable excipients (or ingredients), which may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, such as corn starch, maltodextrin, or alginic acid; binders, such as starch, gelatin, or acacia; or lubricants, such as magnesium stearate, stearic acid, talc, and mixtures thereof.

[0506] A liquid formulation or product (e.g., a pharmaceutical, veterinary, or cosmetic formulation / product) may be contained within a capsule, which may be uncoated or coated as defined above.

[0507] Suitable pharmaceutical or veterinary carriers include inert solid diluents or fillers, sterile aqueous solutions, and various organic solvents. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, and water.

[0508] Additionally, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

[0509] Suitable pharmaceutical carriers include sterile aqueous solutions and various organic solvents.Examples of liquid carriers are syrup, vegetable oils, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.Furthermore, carriers or diluents can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with wax.

[0510] Suitable cosmetic carriers are typically those that are suitable for topical administration to external surfaces of the human body, for example the skin and / or hair and / or scalp.

[0511] Typically, such carriers are dermatologically acceptable.

[0512] The phrase "dermatologically acceptable carrier" means that the carrier is suitable for topical application to keratinous tissue, has good aesthetic properties, is compatible with the active substances in the composition, and does not pose any unreasonable safety or toxicity concerns.

[0513] The carrier may be in a wide variety of forms. In some cases, the solubility or dispersibility of the ingredients (e.g., extracts, sunscreen actives, additional ingredients) may determine the form and characteristics of the carrier. Non-limiting examples include simple solutions (e.g., aqueous or anhydrous solutions), dispersions, emulsions, and solid forms (e.g., gel sticks, flowable solids, or amorphous materials).

[0514] The dermatologically acceptable carrier may be in the form of an emulsion. Emulsions may generally be classified as having a continuous aqueous phase (e.g., oil-in-water and water-in-oil-in-water) or a continuous oily phase (e.g., oil-in-water or oil-in-water). The oily phase of the present invention may include silicone oils, non-silicone oils, such as hydrocarbon oils, esters, ethers, and the like, and mixtures thereof. The aqueous phase typically includes water and water-soluble components (e.g., water-soluble humectants, conditioning agents, antimicrobial agents, moisturizers, and / or other skin care actives). However, in some cases, the aqueous phase may include components other than water, including, but not limited to, water-soluble humectants, conditioning agents, antimicrobial agents, moisturizers, and / or other water-soluble skin care actives. In some cases, the non-water components of the composition include moisturizers, such as glycerin and / or other polyols. The emulsion may also include an emulsifier. The emulsifier may be nonionic, anionic, or cationic.

[0515] The carrier may include one or more dermatologically acceptable hydrophilic diluents. As used herein, "diluent" includes materials in which the compositions of the present invention can be dispersed, dissolved, or otherwise incorporated. Hydrophilic diluents include water, organic hydrophilic diluents such as lower monohydric alcohols (e.g., C1-C4), and low molecular weight glycols and polyols such as propylene glycol, polyethylene glycol, polypropylene glycol, glycerol, butylene glycol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol, ethanol, isopropanol, sorbitol esters, butanediol, ether propanol, ethoxylated ethers, propoxylated ethers, and combinations thereof.

[0516] The cosmetic formulation / product may optionally contain one or more additional ingredients commonly used in cosmetic compositions (e.g., colorants, skin color adjusting agents, skin anti-aging agents, anti-inflammatory agents, sunscreens, combinations thereof, etc.), provided that the additional ingredients do not unnecessarily alter the anti-glycation benefits provided by the composition.

[0517] In some cases, it may be desirable to select skin color adjusting agents that function through different biological pathways so that the actives do not interfere with each other, which may reduce the effectiveness of both agents. The additional ingredients, when incorporated into the composition, should be suitable for use in contact with human skin tissue without undue toxicity, incompatibility, instability, allergic reaction, and the like.

[0518] The term "carrier" as used herein may also refer to a natural product or a product of natural origin that has been transformed or modified so as to differ from the original natural product, e.g., maltodextrin.

[0519] The amount of the composition of the present invention present in a nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulation or product will vary depending on the application.

[0520] Typically, the amount of a composition of the invention that may be present in a nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulation or product will be from about 0.001 to about 50% by weight of the nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulation or product, such as from about 0.01% to about 30% or from about 1% to about 20%, for example from about 0.01 to about 20% by weight, or from about 0.1 to 10% by weight, or from about 1 to about 5% by weight of the formulation or product.

[0521] Those skilled in the art will recognize that due to the degenerate nature of the genetic code, various DNA molecules differing in nucleotide sequence may be used to code for a given enzyme of the present disclosure. The native DNA sequences encoding the biosynthetic enzymes described above are referred to herein merely to exemplify embodiments of the present disclosure, and the present disclosure includes DNA molecules of any sequence that encode the amino acid sequences of the enzyme polypeptides and proteins utilized in the methods of the present disclosure. In a similar manner, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of the desired activity. The present disclosure includes such polypeptides having amino acid sequences that differ from the specific proteins described herein, so long as the modified or variant polypeptide has the enzymatic anabolic or catabolic activity of the reference polypeptide. Moreover, the amino acid sequences encoded by the DNA sequences shown herein are merely exemplifying embodiments of the present disclosure.

[0522] Specific genes and proteins useful in the methods, compositions, and organisms of the present disclosure are described herein, however, it will be recognized that absolute identity to such genes is not required. For example, alterations in a particular gene or polynucleotide containing a sequence encoding a polypeptide or enzyme may be made and screened for activity. Typically, such alterations include conservative mutations and silent mutations. Such altered or mutated polynucleotides and polypeptides may be screened for expression of a functional enzyme using methods known in the art.

[0523] Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionally equivalent polypeptide can also be used to clone and express the polynucleotides encoding such enzymes.

[0524] Techniques known to those skilled in the art may be suitable for identifying further homologous genes and homologous enzymes. In general, similar genes and / or similar enzymes may be identified by functional analysis and will have functional similarities.

[0525] Techniques known to those skilled in the art may be suitable for identifying similar genes and similar enzymes, or any biosynthetic pathway genes, proteins, or enzymes, including, but not limited to, cloning genes by PCR using primers based on the published sequence of the gene / enzyme of interest, or by degenerate PCR using degenerate primers designed to amplify conserved regions within the gene of interest. Furthermore, those skilled in the art may use techniques to identify homologous or similar genes, proteins, or enzymes with functional homology or similarity. Techniques include testing cells or cell cultures for catalytic activity of the enzyme through in vitro enzyme assays for said activity (e.g., as described herein or in Kiritani, K., Branched-Chain Amino Acids Methods Enzymology, 1970), followed by isolating the enzyme with said activity by purification, determining the protein sequence of the enzyme by techniques such as Edman denaturation, designing PCR primers to potential nucleic acid sequences, amplifying said DNA sequences by PCR, and cloning said nucleic acid sequences. To identify homologous or similar genes and / or homologous or similar enzymes, similar genes and / or similar enzymes or proteins, techniques also include comparing data on candidate genes or enzymes to databases such as BRENDA, KEGG, or MetaCYC. Candidate genes or enzymes can be identified in the aforementioned databases following the teachings herein.

[0526] The terms "to" and "range of" are to be understood to include the limits unless otherwise specified. Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises," "comprising," and "possesses" are to be understood to imply the inclusion of a stated integer or step or group of integers or steps, and not the exclusion of any other integer or step or group of integers or steps. The term "comprising" also means "including" as well as "consisting," e.g., a composition "comprising" X may consist exclusively of X or may include some addition, e.g., X+Y. It should also be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. By way of example, a reference to "a gene" or "an enzyme" is a reference to "one or more genes" or "one or more enzymes."

[0527] It is to be understood that the disclosure is not limited to the specific methods, protocols, and reagents described herein, as they may vary. It is also to be understood that the terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In accordance with the present disclosure, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of one of ordinary skill in the art may be employed.

[0528] The present disclosure is not limited in its application to the details of construction and arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or carried out in various ways. Furthermore, the phraseology and terminology used herein are for the purpose of description and are not to be regarded as limiting. Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Kolbl, H. (eds.) (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0529] Several documents are cited throughout this specification. Each of the documents cited herein (including all patents, patent applications, chemical publications, manufacturer's specifications, instructions, GenBank Accession Number sequence entries, etc.), whether supra or infra, is hereby incorporated by reference in its entirety.

[0530] The following examples and figures are offered for illustrative purposes and are not meant to imply limitations of the present invention. EXAMPLES

[0531] Example 1 Protocol for generating recombinant Saccharomyces cerevisiae strains according to the present invention All recombinant Saccharomyces cerevisiae strains implemented herein below were constructed from standard strains using standard yeast molecular genetic procedures (Methods in yeast Genetics - A cold spring harbor laboratory course Manual (2000) by D. Burke, D. Dawson, T. Stearns CSHL Press).

[0532] The cluster of genes mentioned below was integrated one at a time into recombinant yeast using the ability of yeast to efficiently recombine free DNA ends that have sequence homology.

[0533] In addition, with the aim of better understanding the following genotypes: - jlp1, lyp1, sam3, his3, leu2, trp1, and ura3 are insertion sites. - Lower case letters mean that the gene under consideration is inactive, upper case letters represent active genes. - "::" after a gene name means that the gene is disrupted by the one that follows it (if more than one gene is inserted, they are indicated in brackets []). The disruption of the gene occurs concomitantly with the total deletion of the coding sequence, but the promoter is preserved. As a result, the gene followed by "::" is inactive and is indicated in lower case. If not specified, the transcription of the inserted gene is controlled by the promoter of the disrupted gene. - "Gene.Kl" means that the gene originates from Kluyveromyces lactis. If nothing is indicated after the gene in this example, this means that the gene originates from Saccharomyces cerevisiae.

[0534] More specifically, the coding sequences to be cloned were artificially synthesized. For heterologous sequences (non-yeast), the nucleic acid sequences were modified to obtain synonymous coding sequences using yeast codon usage. Using restriction enzymes and classical cloning techniques, each synthetic sequence was cloned between a transcription promoter and a transcription terminator. Each promoter sequence is preceded by a sequence of 50-200 nucleotides that is homologous to the sequence of the terminator of the upstream gene. Similarly, the terminator of each gene (gene containing promoter-coding sequence-terminator) is followed by a sequence that is homologous to the gene immediately following it. Thus, each unit to be integrated has an overlap of 50-200 nucleotides with both the upstream unit and the downstream unit. For the first unit, the promoter is preceded by 50-200 nucleotides that are homologous to the yeast chromosomal nucleotides of the locus to be integrated. Similarly, for the last unit, the terminator is followed by 50-200 nucleotides that are homologous to the yeast chromosomal nucleotides of the locus to be integrated.

[0535] Each unit is then PCR amplified from the plasmid construct to obtain linear DNA X units with overlapping sequences. At least one of the genes is a requirement marker to select for recombination events. All linear fragments are transformed into yeast at once, and recombinant yeast cells are selected for the requirement associated with the marker used. The integrity of the sequence is then verified by PCR and sequencing.

[0536] Example 2 Comparative Example for the Production of Heparosan Seven recombinant strains were derived from the wild type strain CC788-2D (Cherest et al. (2000) J. Biol. Chem. 275: 14056-14063), but they do not produce any heparosan: YA5398-3, YA5402-1, YA5400-1, YA5401-1, YA5437-18, YA5435-2, and DA2531-12.

[0537] These seven strains are as follows: YA5398-3: MAT-α, can1-100, his3::[tRPL3-UGP1-pSAM1, pMET6-QRI1-tIDP1, HIS3]x8, leu2::[URA3.Sba-loxP, pCCW12.Sba-HASB.Vir-tRPL3.Sm, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW12.Sar-HSS2.Pm-tRPL3.Sba, pCCW120.Sca-HSS2.Pm-tRPL15A.Sm], leu2, trp1, ura3 YA5402-1: MAT-α, can1-100, his3::[tRPL3-UGP1-pSAM1, pMET6-QRI1-tIDP1, HIS3]x8, jlp1::[LEU2.Sba-loxP, pCCW12.Sba-HASB.Vir-tRPL3.Sm, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW12.Sar-HSS2.Pm-tRPL3.Sba, pCCW120.Sca-HSS2.Pm-tRPL15A.Sm], leu2, trp1, ura3 YA5400-1: MAT-α, can1-100, his3::[tRPL3-UGP1-pSAM1, pMET6-QRI1-tIDP1, HIS3]x8, jlp1::[LEU2.Sba-loxP, pCCW12.Sba-HASB.Vir-tRPL3.Sm, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW12.Sar-HSS2.Pm-tRPL3.Sba, pCCW120.Sca-HSS2.Pm-tRPL15A.Sm, pTEF1.Ago-GFA1-tRPL15A], leu2, trp1, ura3 <h2 style=";text-align:left;direction:ltr">YA5401-1 : MAT-α, can1-100, his3::[tRPL3-UGP1-pSAM1, pMET6-QRI1-tIDP1, HIS3]x8, jlp1::[LEU2.Sba-loxP, pCCW12.Sba-HASB.Vir-tRPL3.Sm, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW12.Sar-HSS2.Pm-tRPL3.Sba, pCCW120.Sca-HSS2.Pm-tRPL15A.Sm, pTEF1.Ago-GFA1.Vir-tRPL15A], leu2, trp1, ura3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YA5437-18: MAT-α, can1-100, his3::[tRPL3-UGP1-pSAM1, pMET6-QRI1-tIDP1, HIS3]x8, jlp1::[LEU2.Sba-loxP, pCCW12.Sba-HASB.Vir-tRPL3.Sm, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW12.Sar-HSS1.Pm-tRPL15A.Sm], leu2, trp1, ura3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> YA5435-2: MAT-α, can1-100, his3::[tRPL3-UGP1-pSAM1, pMET6-QRI1-tIDP1, HIS3]x8, jlp1::[LEU2.Sba-loxP, pCCW12.Sba-HASB.Vir-tRPL3.Sm, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW12.Sar-HSS1.Pm-tRPL15A.Sm, pTEF1.Ago-GFA1.Vir-tRPL15A], leu2, trp1, ura3<h2 style=";text-align:left;direction:ltr"> DA2531-12 : MAT-a / MAT-α, CAN1 / can1-100, his3 / his3::[tRPL3-UGP1-pSAM1, pMET6-QRI1-tIDP1, HIS3]x8, jlp1::[LEU2.Kl-RS, pCUP1-UGP1-tRPL3, pCUP1-QRI1-tIDP1, pPDC1-UGP1-tTPI1, pTDH3-QRI1-tMET25, pCCW12-HASB.At-tRPL15A] / jlp1::[LEU2.Sba-loxP, pCCW12.Sba-HASB.Vir-tRPL3.Sm, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW12.Sar-HSS1.Pm-tRPL15A.Sm, pTEF1.Ago-GFA1.Vir-tRPL15A], leu2 / leu2, SAM3 / sam3::[LEU2.Kl-RS, pPDC1-PGM1-tIDP1, pTEF1.Ago-GFA1-tRPL15A, pENO2-UGP1-tRPL3, pCWP2-GNA1-tTPI1, pTEF1-PCM1-tRPL41B, pCCW12.Sba-HASB.vir-tRPL3.Sm, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW12.Sar-HSS2.Pm-tRPL3.Sba, pCCW120.Sca-HSS2.Pm-tRPL15A.Sm, pTDH3-1.Sba-QRI1-tIDP1.Sba, HIS3.Sba-loxP], trp1 / trp1, ura3

[0538] HSS1.Pm represents the sequence encoding heparosan synthase A from Pasteurella multocida (UniProt reference number Q8RP78).HSS2.Pm represents the sequence encoding heparosan synthase B from Pasteurella multocida (UniProt reference number Q5SGE1).

[0539] All these strains were incubated in Erlenmeyer flasks in 25 ml of SY medium buffered with MES 0.1M ph 5.5 in baffled Erlenmeyer flasks at 28°C under vigorous agitation for 48 hours.

[0540] SY medium contains the following elements: KH2PO4:100mM, MgSO4 7H2O:2,8mM, K2SO4:11,5mM, Na2SO4:1,1mM, NaCl:2,6mM, CaCl2 2H2O:0,7mM, CuSO4 5H2O:15μM, KI:6μM, FeCl3:30μM, ZnSO4 7H2O:61μM, MnSO4 H2O: 25 μM, H2SO4: 110 μM, pantothenic acid hemicalcium salt: 42 μM, thiamine hydrochloride: 59 μM, pyridoxine hydrochloride: 49 μM, myo-inositol (C6H12O6): 555 μM, nicotinic acid (C6H5NO2): 29 μM, D-biotin: 0.82 μM, ammonium citrate tribasic: 33 mM, and glucose 2%.

[0541] Growth medium was harvested at 48 hours and assayed for heparosan content and quality. An aliquot of the medium was loaded and run on a 0.5% agarose gel, followed by staining with bromophenol blue. The amount of heparosan present in the medium was assessed by colorimetric determination after treatment with concentrated sulfuric acid and carbazole (Bitter and Muir (1962) analytical biochemistry 4, 330-334).

[0542] The amount of heparosan obtained from these different strains was, respectively: - YA5398-3: 170mg.L -1 - YA5402-1: 170mg.L -1 - YA5400-1: 110mg.L -1 - YA5401-1: 180mg.L -1 - YA5437-18: 200mg.L -1 - YA5435-2: 190mg.L -1 - DA2531-12: 235mg.L -1 It is.

[0543] In comparison, as mentioned above, the native strain does not produce heparosan.

[0544] This experiment showed that the recombinant strains containing the modifications according to the invention produced greater amounts of heparosan when cultured under the same conditions compared to other recombinant strains that did not contain all the genetic modifications according to the invention.

[0545] array SEQ ID NO:1 is the recoded nucleic acid sequence of heparosan synthase originating from Pasteurella multocida (HSS1)

[0546] [ka]

[0547] SEQ ID NO:2 is the recoded nucleic acid sequence of heparosan synthase originating from Pasteurella multocida (HSS2)

[0548] [ka]

[0549] SEQ ID NO:3 is the amino acid sequence of heparosan synthase originating from Pasteurella multocida (HSS1)

[0550] [ka]

[0551] SEQ ID NO: 4 is the amino acid sequence of heparosan synthase originating from Pasteurella multocida (HSS2)

[0552] [ka]

[0553] SEQ ID NO:5 is the nucleic acid sequence of UDP-glucose dehydrogenase (HASB) originating from Arabidopsis thaliana

[0554] [ka]

[0555] SEQ ID NO:6 is the recoded nucleic acid sequence of UDP-glucose dehydrogenase (HASB) originating from the Chlorella virus PBCV-1.

[0556] [ka]

[0557] SEQ ID NO:7 is the recoded nucleic acid sequence of UDP-glucose dehydrogenase (HASB-A) originating from Chlorella virus PBCV-1

[0558] [ka]

[0559] SEQ ID NO:8 is the amino acid sequence of UDP-glucose dehydrogenase (HASB) from Arabidopsis thaliana

[0560] [ka]

[0561] SEQ ID NO:9 is the amino acid sequence of UDP-glucose dehydrogenase (HASB) originating from Chlorella virus PBCV1

[0562] [ka]

[0563] SEQ ID NO:10 is a recoded nucleic acid sequence of glucuronosyl-disulfoglucosamine glucuronidase originating from Pedobacter heparinus with an N-terminal secretion signal

[0564] [ka]

[0565] SEQ ID NO:11 is a recoded nucleic acid sequence of glucuronosyl-disulfoglucosamine glucuronidase originating from Pedobacter heparinus, with an N-terminal secretion signal and a C-terminal anchoring signal.

[0566] [ka]

[0567] SEQ ID NO:12 is a recoded nucleic acid sequence of glucuronosyl-disulfoglucosamine glucuronidase originating from Pedobacter heparinus with an N-terminal secretion signal

[0568] [ka]

[0569] SEQ ID NO:13 is a recoded nucleic acid sequence of glucuronosyl-disulfoglucosamine glucuronidase originating from Pedobacter heparinus, with an N-terminal secretion signal and a C-terminal anchoring signal.

[0570] [ka]

[0571] SEQ ID NO:14 is the amino acid sequence of glucuronosyl-disulfoglucosamine glucuronidase from Pedobacter heparinus with an N-terminal secretion signal

[0572] [ka]

[0573] SEQ ID NO:15 is the amino acid sequence of glucuronosyl-disulfoglucosamine glucuronidase originating from Pedobacter heparinus, with an N-terminal secretion signal and a C-terminal anchoring signal

[0574] [ka]

[0575] SEQ ID NO:16 is the nucleic acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Saccharomyces cerevisiae

[0576] [ka]

[0577] SEQ ID NO:17 is the recoded nucleic acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Chlorella virus 1 (PBCV-1)

[0578] [ka]

[0579] SEQ ID NO:18 is the recoded nucleic acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Chlorella virus 1 (PBCV-1)

[0580] [ka]

[0581] SEQ ID NO:19 is the amino acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Saccharomyces cerevisiae

[0582] [ka]

[0583] SEQ ID NO:20 is the amino acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Chlorella virus 1 (PBCV-1)

[0584] [ka]

[0585] SEQ ID NO:21 is the nucleic acid sequence of UDP-N-acetylglucosamine pyrophosphorylase (QRI1) originating from Saccharomyces cerevisiae

[0586] [ka]

[0587] SEQ ID NO:22 is the amino acid sequence of UDP-N-acetylglucosamine pyrophosphorylase (QRI1) originating from Saccharomyces cerevisiae

[0588] [ka]

[0589] SEQ ID NO:23 is the nucleic acid sequence of phosphoglucomutase-1 (PGM1) originating from Saccharomyces cerevisiae

[0590] [ka]

[0591] SEQ ID NO:24 is the amino acid sequence of phosphoglucomutase-1 (PGM1) originating from Saccharomyces cerevisiae

[0592] [ka]

[0593] SEQ ID NO:25 is the nucleic acid sequence of UTP-glucose 1-phosphate uridylyltransferase (UGP1) originating from Saccharomyces cerevisiae

[0594] [ka]

[0595] SEQ ID NO:26 is the amino acid sequence of UTP-glucose 1-phosphate uridylyltransferase (UGP1) originating from Saccharomyces cerevisiae

[0596] [ka]

[0597] SEQ ID NO:27 is the nucleic acid sequence of glucosamine 6-phosphate N-acetyltransferase (GNA1) originating from Saccharomyces cerevisiae

[0598] [ka]

[0599] SEQ ID NO:28 is the amino acid sequence of glucosamine 6-phosphate N-acetyltransferase (GNA1) originating from Saccharomyces cerevisiae

[0600] [ka]

[0601] SEQ ID NO:29 is the nucleic acid sequence of phosphoacetylglucosamine mutase (PCM1) originating from Saccharomyces cerevisiae

[0602] [ka]

[0603] SEQ ID NO:30 is the amino acid sequence of phosphoacetylglucosamine mutase (PCM1) originating from Saccharomyces cerevisiae

[0604] [ka]

[0605] SEQ ID NO: 31 is the nucleic acid sequence of promoter pTDH3

[0606] [ka]

[0607] SEQ ID NO: 32 is the nucleic acid sequence of promoter pTDH3.Sk

[0608] [ka]

[0609] SEQ ID NO: 33 is the nucleic acid sequence of the promoter pTDH3-1.sba

[0610] [ka]

[0611] SEQ ID NO: 34 is the nucleic acid sequence of the promoter pTDH3.Sar

[0612] [ka]

[0613] SEQ ID NO: 35 is the nucleic acid sequence of promoter pENO2

[0614] [ka]

[0615] SEQ ID NO: 36 is the nucleic acid sequence of promoter pTEF3

[0616] [ka]

[0617] SEQ ID NO: 37 is the nucleic acid sequence of the promoter pTEF1

[0618] [ka]

[0619] SEQ ID NO: 38 is the nucleic acid sequence of the promoter pTEF1.ago

[0620] [ka]

[0621] SEQ ID NO: 39 is the nucleic acid sequence of the promoter pTEF1.Sba

[0622] [ka]

[0623] SEQ ID NO: 40 is the nucleic acid sequence of promoter pPDC1

[0624] [ka]

[0625] SEQ ID NO: 41 is the nucleic acid sequence of promoter pCCW12

[0626] [ka]

[0627] SEQ ID NO: 42 is the nucleic acid sequence of the promoter pCCW12.Sm

[0628] [ka]

[0629] SEQ ID NO: 43 is the nucleic acid sequence of promoter pCCW12.Sk

[0630] [ka]

[0631] SEQ ID NO: 44 is the nucleic acid sequence of the promoter pCCW12.Sba

[0632] [ka]

[0633] SEQ ID NO: 45 is the nucleic acid sequence of the promoter pCCW12.Sar

[0634] [ka]

[0635] SEQ ID NO: 46 is the nucleic acid sequence of the promoter pNUP57

[0636] [ka]

[0637] SEQ ID NO: 47 is the nucleic acid sequence of the promoter pCCW10.ago

[0638] [ka]

[0639] SEQ ID NO: 48 is the nucleic acid sequence of promoter pCWP2

[0640] [ka]

[0641] SEQ ID NO: 49 is the nucleic acid sequence of the promoter pCCW120.Sca

[0642] [ka]

[0643] SEQ ID NO: 50 is the nucleic acid sequence of promoter pRPLA1

[0644] [ka]

[0645] SEQ ID NO: 51 is the nucleic acid sequence of the promoter pCUP1

[0646] [ka]

[0647] SEQ ID NO: 52 is the nucleic acid sequence of the promoter pMET6

[0648] [ka]

[0649] SEQ ID NO: 53 is the nucleic acid sequence of promoter pMET25

[0650] [ka]

[0651] SEQ ID NO: 54 is the nucleic acid sequence of the promoter pSAM1

[0652] [ka]

[0653] SEQ ID NO: 55 is the nucleic acid sequence of the terminator tTPI1

[0654] [ka]

[0655] SEQ ID NO: 56 is the nucleic acid sequence of terminator tMET25

[0656] [ka]

[0657] SEQ ID NO: 57 is the nucleic acid sequence of the terminator tDIT1

[0658] [ka]

[0659] SEQ ID NO: 58 is the nucleic acid sequence of terminator tRPL3

[0660] [ka]

[0661] SEQ ID NO: 59 is the nucleic acid sequence of terminator tRPL3.sm

[0662] [ka]

[0663] SEQ ID NO: 60 is the nucleic acid sequence of the terminator tRPL3.sba

[0664] [ka]

[0665] SEQ ID NO: 61 is the nucleic acid sequence of the terminator tRPL41B

[0666] [ka]

[0667] SEQ ID NO: 62 is the nucleic acid sequence of the terminator tRPL15A

[0668] [ka]

[0669] SEQ ID NO: 63 is the nucleic acid sequence of terminator tRPL15A.sba

[0670] [ka]

[0671] SEQ ID NO: 64 is the nucleic acid sequence of terminator tIDP1

[0672] [ka]

[0673] SEQ ID NO: 65 is the nucleic acid sequence of the terminator tTEF1.sba

[0674] [ka]

[0675] SEQ ID NO:66 is the nucleic acid sequence of the secretory sequence added to the 5' end ATGCAATTTAGCACAGTCGCATCAGTAGCCTTCGTTGCCTTGGCCAACTTCGTGGCAGCA

[0676] SEQ ID NO:67 is the amino acid sequence of the secretory sequence added to the N-terminus MQFSTVASVAFVALANFVAA

[0677] SEQ ID NO:68 is the nucleic acid sequence of the anchoring sequence added to the 3'

[0678] [ka]

[0679] SEQ ID NO:69 is the amino acid sequence of the anchoring sequence added to the C-terminus AISQITDGQIQATTTATTEATTTAAPSSTVETVSPSSTETISQQTENGAAKAAVGMGAGALAAAAMLL< / r> < / r>

Claims

1. A recombinant yeast cell producing heparosan, comprising: (a) one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase (HSS) activity; (b) one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity; A recombinant yeast cell comprising:

2. The recombinant cell of claim 1, comprising a nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity, the polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity comprising a secretion signal and optionally an anchoring signal.

3. A recombinant host cell producing heparosan, comprising: (a) one or more recombinant nucleic acids encoding a polypeptide having heparosan synthase (HSS) activity; (b) one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity; and (c) one or more recombinant nucleic acids encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity; Including, The polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity comprises a secretion signal and optionally an anchoring signal so that heparosan, particularly one of the desired molecular weight, is produced by the host cell. Recombinant host cells.

4. 4. The recombinant cell of claim 2 or 3, wherein the nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity is obtained or derived from Pedobacter heparinus.

5. 5. A recombinant cell according to any one of claims 2 to 4, wherein the nucleic acid encoding a polypeptide having glucuronosyl-disulfoglucosamine glucuronidase activity is under the control of a promoter selected from the group consisting of pTEF1, pCCW12, pCCW12.sba, pCCW12.Sar, pPDC1, pTEF3, pTDH3, pNUP57, and pCCW10.ago.

6. The recombinant cell according to any one of claims 2 to 5, wherein the molecular weight of the heparosan is in the range of less than 50 kDa, preferably in the range of about 20 kDa to about 50 kDa.

7. A recombinant cell according to any one of claims 2 to 5, wherein the molecular weight of the heparosan is in the range above 50 kDa, preferably in the range of about 50 kDa to about 250 kDa.

8. A recombinant cell according to any one of claims 2 to 5, wherein the molecular weight of the heparosan is in the range above 100 kDa, preferably in the range of about 100 kDa to about 1500 kDa.

9. 9. The recombinant cell according to any one of claims 1 to 8, wherein the nucleic acid encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity is obtained or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, in particular from Arabidopsis thaliana or Chlorella virus PBCV1.

10. 10. A recombinant cell according to any one of claims 1 to 9, wherein the nucleic acid encoding a polypeptide having heparosan synthase (HSS) activity is obtained or derived from Pasteurella multocida.

11. (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity 11. The recombinant cell of claim 1, further comprising at least one recombinant nucleic acid encoding one or more of:

12. (i) a polypeptide having phosphoglucomutase-1 (PGM1) activity, and / or (ii) a polypeptide having UTP-glucose-1-phosphate uridylyltransferase (UGP1) activity, and / or (iii) a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity, and / or (iv) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity 12. The recombinant cell of claim 1 , further comprising at least one recombinant nucleic acid encoding one or more of the following:

13. - a nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or - a nucleic acid encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity, and / or - a nucleic acid encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity, and / or - a nucleic acid encoding a polypeptide having UTP-glucose-1-phosphate uridylyltransferase (UGP1) activity, and / or - a nucleic acid encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity, and / or - a nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase (PCM1) is obtained or derived from Saccharomyces cerevisiae, 13. A recombinant cell according to claim 11 or 12.

14. 14. The recombinant host cell according to any one of claims 3 to 13, wherein the recombinant host cell is a yeast.

15. 15. A recombinant cell according to any one of claims 1 to 14, belonging to the genus Saccharomyces, or Candida, or Kluyveromyces, or Ogataea, or Yarrowia, or Debaryomyces, or Ashbya, in particular belonging to the genus Saccharomyces.

16. 16. The recombinant cell of claim 15, which is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces castellii, Candida albicans, Candida glabrata, Candida tropicalis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus, Kluyveromyces thermotolerans, Ogataea polymorpha, Yarrowia lipolytica, Debaryomyces hansenii, and Ashbya gossypii, preferably Saccharomyces cerevisiae.

17. A method for producing heparosan, comprising the steps of: (a) culturing a recombinant cell according to any one of claims 1 to 16 in a culture medium for a time sufficient to produce heparosan; (b) optionally isolating or recovering the heparosan from the recombinant cells and / or the culture medium. The method includes:

18. 18. The method of claim 17, wherein the heparosan has a molecular weight of about 20 kDa to about 50 kDa.

19. 18. The method of claim 17, wherein the heparosan has a molecular weight of about 50 kDa to about 150 kDa.

20. 18. The method of claim 17, wherein the heparosan has a molecular weight of about 150 kDa to about 1500 kDa.

21. 21. The method according to any one of claims 17 to 20, wherein the recombinant cell is a yeast belonging to the genus Saccharomyces, in particular Saccharomyces cerevisiae.

22. 22. The method according to any one of claims 17 to 21, wherein the time sufficient to produce heparosan is for a period of about 35 to about 50 hours, preferably about 40 to about 50 hours, preferably about 48 hours.

23. 23. The method according to any one of claims 17 to 22, wherein the molecular weight of the heparosan produced is controlled by adjusting the pH of the culture medium during step (a).

24. 24. The method according to any one of claims 17 to 23, wherein the method is carried out on an industrial scale, preferably wherein the culture medium is at least about 100 L, more preferably in the range of about 1000 L to about 3000 L, even more preferably about 10,000 L, or even more preferably about 100,000 L, or even about 250,000 L.

25. Heparosan obtainable from a recombinant cell according to any one of claims 1 to 16 or from a method according to any one of claims 17 to 24.

26. 26. A culture medium comprising the heparosan of claim 25.

27. 26. A composition comprising the heparosan of claim 25.

28. 27. An industrial or consumer product or consumable comprising: (i) a heparosan as described in claim 25 having a molecular weight as defined in any one of claims 6 to 8; (ii) a culture medium as described in claim 26; or (iii) a composition as described in claim 27.

29. 29. The industrial or consumer product or consumable of claim 28 which is a cosmetic, a flavour product, a fragrance product, a foodstuff, a food, a beverage, a texturant, a pharmaceutical composition, a dietary supplement, a nutraceutical, a cleaning product, a dental and / or oral hygiene composition.

30. 17. Use of a recombinant cell according to any one of claims 1 to 16 for the production of heparosan having a molecular weight in the range of about 20 kDa to about 50 kDa or about 50 kDa to about 1000 kDa.

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