Recombinant cells producing chondroitin

Abstract

The present invention relates to the field of biological production of chondroitin. There is a need in the art for a method of producing chondroitin that allows for highly efficient synthesis and secretion. The solution proposed in the present invention is the use of recombinant cells, in particular recombinant yeast, that contain a number of modifications described herein. The present invention further proposes a method that allows for biological production of chondroitin using recombinant cells of the present invention, in particular recombinant yeast.

Description

[Technical field]

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

[0002] Chondroitin is a high molecular weight polysaccharide that occurs naturally in the connective tissues of humans and animals. It belongs to a family of heteropolysaccharides called glycosaminoglycans.

[0003] Glycosaminoglycans (GAGs) or mucopolysaccharides are unbranched, negatively charged polysaccharide chains composed of repeating disaccharide units containing acidic and amino sugars (N-acetylglucosamine or N-acetylgalactosamine). Due to their immobile nature and high negative charge, GAGs are used in the body as lubricants or shock absorbers. For example, the cartilage matrix covering the knee joint, which is rich in GAGs, can support pressures of several hundred atmospheres by this mechanism.

[0004] Chondroitin, formed from D-glucuronic acid and N-acetyl-D-galactosamine, may be sulfated. Chondroitin sulfate is important in maintaining the strength and elasticity of cartilage and is sold as a dietary supplement to reduce joint pain and promote healthy cartilage and joint function. Clinical trials support the use of chondroitin and chondroitin sulfate for the treatment of osteoarthritis.

[0005] Chondroitin sulfate or chondroitin occurs naturally in the extracellular matrix of the connective tissue of the skin and is also used in cosmetic applications, particularly to moisturize, heal and soothe the skin, and as an ingredient in hair conditioners.

[0006] Currently, chondroitin is primarily produced by extraction from animal cartilage using chemical and enzymatic procedures to dissociate the polysaccharides from the proteins to produce polysaccharide products of various qualities, however, these methods can be laborious and expensive.

[0007] Therefore, microbial production of chondroitin has been proposed. Indeed, certain bacteria produce chondroitin and chondroitin-like polysaccharide polymers as components of their capsules. However, these known bacteria, e.g., Pasteurella multocida or Escherichia coli, are pathogens for many mammals and produce a low number of polysaccharides.

[0008] As a result, research has shifted to recombinant microorganisms, in particular recombinant bacteria, for the production of chondroitin. For example, reference may be made to International Publication No. WO2011109438, European Publication No. EP2142643, or US Publication No. US20090263867.

[0009] In contrast to other microorganisms commonly used for the production of biological molecules, yeasts are generally recognized as safe. Compared to bacteria, they can grow rapidly, can be cultured at high densities, 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.

[0010] However, among yeasts, distinguishing characteristics between species can also present certain challenges, mainly due to metabolic differences between species and their culture conditions.

[0011] However, the inventors have found that Saccharomyces cerevisiae is particularly useful as a tool for the production of molecules of interest, since it 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. S. cerevisiae further offers 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 reducing the need to add antibiotics to the medium. Finally, a number of genetic tools have been developed that allow for stable modification of the genome (integration into the chromosome).

[0012] Thus, there remains a need in the art for additional chondroitin production methods that allow for highly efficient synthesis and secretion. In particular, there remains a need to provide production methods that provide chondroitin that is cost-effective and safe for human application.

[0013] There remains a need in the art for methods of producing chondroitin that, in certain cases, allow obtaining large amounts of chondroitin of specific and controlled sizes. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. WO2011109438 [Patent Document 2] European Patent No. EP2142643 [Patent Document 3] US Publication No. US20090263867 [Non-patent literature]

[0015] [Non-Patent Document 1] Astら(2013) Cell 152: 1134-1145

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[0016] The present invention therefore relates to the following items:

[0017] Item 1: A recombinant yeast cell that produces chondroitin, (a) one or more recombinant nucleic acids encoding a polypeptide having chondroitin synthase (HCOS) 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 UDP-glucose-4-epimerase (kfoA or GNE1) activity; A recombinant yeast cell comprising:

[0018] As shown in the examples, the recombinant yeast of the invention allows the production of chondroitin in yeast cells that are not naturally capable of producing this glycosaminoglycan. It is further shown in the examples that the size of the chondroitin produced by the recombinant yeast can be controlled.

[0019] The advantageous properties can be further increased by engineering the yeast with additional modifications as described hereinafter.

[0020] Item 2: The recombinant cell of item 1, wherein the recombinant cell comprises one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity, wherein the polypeptide having chondroitinase activity comprises a secretion signal and optionally an anchoring signal.

[0021] Item 3: A recombinant host cell that produces chondroitin, (a) one or more recombinant nucleic acids encoding a polypeptide having chondroitin synthase (HCOS) 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 UDP-glucose-4-epimerase (kfoA or GNE1) activity; (d) one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity, the polypeptide having chondroitinase activity comprising a secretion signal or a secretion signal and optionally an anchoring signal such that chondroitin, particularly of a desired molecular weight, is produced by the host cell; A recombinant host cell comprising:

[0022] Item 4: The recombinant host cell according to item 2 or 3, wherein the recombinant nucleic acid encoding a polypeptide having chondroitinase activity is obtained or derived from at least one of Cupiennius salei, Tityus serrulatus, Bos taurus, Vespa magnifica, Macaca mulata, or Apis mellifera, preferably Tityus serrulatus.

[0023] Item 5: The recombinant cell according to any one of Items 2 to 4, wherein the molecular weight of the chondroitin is within the range of less than 50 kDa, preferably within the range of about 20 kDa to about 50 kDa.

[0024] Item 6: The recombinant cell according to any one of items 1 to 4, wherein the molecular weight of the chondroitin is in the range of more than 50 kDa, preferably in the range of about 50 kDa to about 250 kDa.

[0025] Item 7: The recombinant cell according to any one of items 1 to 4, wherein the molecular weight of the chondroitin is in the range of more than 100 kDa, preferably in the range of about 100 kDa to about 1500 kDa.

[0026] Item 8: The recombinant cell according to any one of items 1 to 7, 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 at least one of Arabidopsis thaliana or Chlorella virus PBCV1.

[0027] Item 9: A nucleic acid encoding a polypeptide having chondroitin synthase (HCOS) activity, (i) a nucleic acid encoding a chondroitin synthase, or (ii) a nucleic acid encoding a chimeric polypeptide having chondroitin synthase activity 9. The recombinant cell according to any one of items 1 to 8,

[0028] Item 10: The recombinant cell according to item 9, wherein the nucleic acid is obtained or derived from at least one of Pasteurella multocida, Chlorella virus PBCV1, Mycobacterium tuberculosis, Homo sapiens, Escherichia coli, Saccharomyces cerevisiae, or Penicillium oxalicum.

[0029] Item 11: The recombinant cell according to any one of items 1 to 10, wherein the nucleic acid encoding a polypeptide having UDP-glucose-4-epimerase (kfoA or GNE1) activity is obtained or derived from a bacterium, in particular a bacterium selected from the group consisting of Pseudomonas aeruginosa, Pasteurella multocida, and Escherichia coli.

[0030] Item 12: (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) 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:

[0031] Item 13: (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 13. The recombinant cell according to any one of items 1 to 12, further comprising at least one recombinant nucleic acid encoding one or more of:

[0032] Item 14: - a nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, - a nucleic acid encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity, - a nucleic acid encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity, - a nucleic acid encoding a polypeptide having UTP-glucose-1-phosphate uridylyltransferase (UGP1) activity, - 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 from or derived from Saccharomyces cerevisiae, 14. The recombinant cell according to item 12 or 13.

[0033] Item 15: The recombinant host cell according to any one of items 3 to 14, wherein the recombinant host cell is a yeast.

[0034] Item 16: The recombinant cell according to any one of items 1 to 15, which belongs to the genus Saccharomyces, or Candida, or Kluyveromyces, or Ogataea, or Yarrowia, or Debaryomyces, or Ashbya, in particular the genus Saccharomyces.

[0035] Item 17: Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces castelli, Candida albicans, Candida glabrata, Candida tropicalis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus 17. The recombinant cell according to item 16, 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.

[0036] Item 18: A method for producing chondroitin of a desired molecular weight, comprising: (a) culturing the recombinant cell according to any one of items 1 to 17 in a culture medium for a time sufficient to produce chondroitin of the desired molecular weight; (b) optionally isolating or recovering the chondroitin from the recombinant cells and / or the culture medium. The method includes:

[0037] Item 19: The method according to Item 18, wherein the chondroitin has a molecular weight of about 20 kDa to about 50 kDa.

[0038] Item 20: The method according to Item 18, wherein the chondroitin has a molecular weight of about 50 kDa to about 150 kDa.

[0039] Item 21: The method according to Item 18, wherein the chondroitin has a molecular weight of about 150 kDa to about 1500 kDa.

[0040] Item 22: The method according to any one of items 18 to 21, wherein the recombinant cell is a yeast belonging to the genus Saccharomyces, in particular Saccharomyces cerevisiae.

[0041] Item 23: The method according to any one of Items 18 to 22, wherein the time sufficient to produce chondroitin of the desired molecular weight is a period of about 35 hours to about 50 hours, preferably about 40 hours to about 50 hours, preferably about 48 hours.

[0042] Item 24: The method according to any one of items 18 to 23, wherein the molecular weight of the produced chondroitin is controlled by adjusting the pH of the culture medium in step (a).

[0043] Item 25: The method according to any one of items 18 to 24, wherein the method is carried out on an industrial scale, and preferably the culture medium is at least about 100 L, more preferably in the range of about 1,000 L to about 3,000 L, even more preferably about 10,000 L, or even more preferably about 100,000 L, or even about 250,000 L.

[0044] Item 26: Chondroitin obtainable from a recombinant cell according to any one of items 1 to 17 or from a method according to any one of items 18 to 25.

[0045] Item 27: A culture medium containing the chondroitin according to Item 26.

[0046] Item 28: A composition comprising the chondroitin according to Item 26.

[0047] Item 29: (i) a chondroitin having a molecular weight as defined in any one of Items 5 to 7, (ii) a culture medium according to Item 27, or (iii) an industrial product, a consumer product, or a consumable product comprising the composition according to Item 28.

[0048] Item 30: The industrial or consumer product or consumable according to item 29, 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.

[0049] Item 31: Use of a recombinant cell according to any one of items 2 to 17 for producing chondroitin having a molecular weight in the range of about 20 kDa to about 50 kDa or about 50 kDa to about 1000 kDa.

[0050] Item 32: A method for producing chondroitin, comprising: (a) culturing the recombinant yeast according to any one of items 1 to 17 in a culture medium; (b) recovering chondroitin from the culture medium; Including, The chondroitin recovered in step (b) is - the nature and origin of the nucleic acid encoding a polypeptide having recombinant yeast chondroitinase activity, - the nature and origin of the promoter controlling the expression of the recombinant nucleic acid encoding the polypeptide having chondroitinase activity of the recombinant yeast, - the presence of an anchoring and / or secretion signal associated with the recombinant yeast polypeptide having chondroitinase activity, - the pH of the culture medium during the cultivation process of the recombinant yeast, and / or - Duration of recombinant yeast cultivation having a molecular weight controlled through the selection of method.

[0051] Certain embodiments of the present invention provide the following advantages: A process for producing chondroitin of controlled molecular weight, A process for producing chondroitin of controlled molecular weight in Saccharomyces yeast cells, and A process for producing chondroitin 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:

[0052] The details, examples, and preferences provided in relation to any particular one or more of the mentioned aspects of the invention will be explained further herein and apply equally to all aspects of the invention. Any combination of the embodiments, examples, and preferences described herein in all possible variations thereof is encompassed by the present invention, unless otherwise indicated herein or clearly excluded by context. [Brief description of the drawings]

[0053] [Figure 1] FIG. 1 shows a schematic pathway for the production of chondroitin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Overview of Arrays SEQ ID NO:1 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS1-1) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from a nucleic acid encoding a chondroitin synthase originating from Pasteurella multocida. SEQ ID NO:2 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS1-2) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida. SEQ ID NO:3 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS1-3) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida. SEQ ID NO: 4 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS.Sc) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida, a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida, and a fragment derived from a nucleic acid encoding chitin synthase 2 from Saccharomyces cerevisiae. SEQ ID NO:5 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HHASA.Sc) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from a nucleic acid encoding chitin synthase 2 from Saccharomyces cerevisiae. SEQ ID NO:6 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS1-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from E. coli. SEQ ID NO: 7 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS2-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase originating from E. coli. SEQ ID NO:8 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS3-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a galactofuranosyltransferase originating from Mycobacterium tuberculosis. SEQ ID NO:9 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS4-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from E. coli. SEQ ID NO:10 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS5-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from Penicillium oxalicum. SEQ ID NO:11 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS6-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida. SEQ ID NO:12 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS7-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida. SEQ ID NO:13 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS8-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida. SEQ ID NO:14 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS9-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin sulfate synthase originating from Homo sapiens. SEQ ID NO:15 is a recoded nucleic acid sequence for a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS10-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin sulfate synthase originating from Homo sapiens. SEQ ID NO:16 is a recoded nucleic acid sequence for a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS11-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin sulfate synthase originating from Homo sapiens. SEQ ID NO:17 is a recoded nucleic acid sequence for a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS12-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin sulfate synthase originating from Homo sapiens. SEQ ID NO: 18 is the amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS1-1) comprising a fragment derived from the amino acid sequence encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from the amino acid sequence encoding a chondroitin synthase from Pasteurella multocida. SEQ ID NO: 19 is the amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS1-2) comprising a fragment derived from the amino acid sequence encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from the amino acid sequence encoding a chondroitin synthase from Pasteurella multocida. SEQ ID NO: 20 is the amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS1-3) comprising a fragment derived from the amino acid sequence encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from the amino acid sequence encoding a chondroitin synthase from Pasteurella multocida. SEQ ID NO: 21 is the amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS.Sc) comprising a fragment derived from the amino acid sequence encoding hyaluronan synthase originating from Pasteurella multocida, a fragment derived from the amino acid sequence encoding chondroitin synthase from Pasteurella multocida, and a fragment derived from the amino acid sequence encoding chitin synthase 2 from Saccharomyces cerevisiae. SEQ ID NO:22 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HHASA.Sc) comprising a fragment derived from the amino acid sequence encoding hyaluronan synthase originating from Pasteurella multocida and a fragment derived from the amino acid sequence encoding chitin synthase 2 from Saccharomyces cerevisiae. SEQ ID NO: 23 is the amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS1-Vir) comprising a fragment derived from the amino acid sequence encoding the hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from the amino acid sequence encoding the chondroitin synthase from E. coli. SEQ ID NO:24 is a recoded amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS2-Vir) comprising a fragment derived from the amino acid sequence encoding the hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from the amino acid sequence encoding the chondroitin synthase from E. coli. SEQ ID NO:25 is a recoded amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS3-Vir) comprising a fragment derived from the amino acid sequence encoding the hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from the amino acid sequence encoding the chondroitin synthase from E. coli. SEQ ID NO:26 is a recoded amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS4-Vir) comprising a fragment derived from the amino acid sequence encoding the hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from the amino acid sequence encoding the chondroitin synthase from E. coli. SEQ ID NO: 27 is the amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS5-Vir) comprising a fragment derived from the amino acid sequence encoding the hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from the amino acid sequence encoding the chondroitin synthase from Penicillium oxalicum. SEQ ID NO: 28 is the amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS6-Vir) comprising a fragment derived from the amino acid sequence encoding the hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from the amino acid sequence encoding the chondroitin synthase from Pasteurella multocida. SEQ ID NO:29 is a recoded amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS7-Vir) containing a fragment derived from the amino acid sequence encoding the hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from the amino acid sequence encoding the chondroitin synthase from Pasteurella multocida. SEQ ID NO:30 is a recoded amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS8-Vir) comprising a fragment derived from the amino acid sequence encoding the hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from the amino acid sequence encoding the chondroitin synthase from Pasteurella multocida. SEQ ID NO:31 is the amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS9-Vir) comprising a fragment derived from an amino acid sequence encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from an amino acid sequence encoding a chondroitin sulfate synthase originating from Homo sapiens. SEQ ID NO:32 is a recoded amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS10-Vir) containing a fragment derived from an amino acid sequence encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from an amino acid sequence encoding a chondroitin sulfate synthase originating from Homo sapiens. SEQ ID NO:33 is a recoded amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS11-Vir) containing a fragment derived from an amino acid sequence encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from an amino acid sequence encoding a chondroitin sulfate synthase originating from Homo sapiens. SEQ ID NO:34 is a recoded amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS12-Vir) containing a fragment derived from an amino acid sequence encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from an amino acid sequence encoding a chondroitin sulfate synthase originating from Homo sapiens. SEQ ID NO:35 is the nucleic acid sequence of UDP-glucose dehydrogenase (HASB) originating from Arabidopsis thaliana SEQ ID NO:36 is the recoded nucleic acid sequence of UDP-glucose dehydrogenase (HASB) originating from Chlorella virus PBCV-1 SEQ ID NO:37 is the recoded nucleic acid sequence of UDP-glucose dehydrogenase (HASB-A) originating from Chlorella virus PBCV-1 SEQ ID NO:38 is the nucleic acid sequence of UDP-glucose dehydrogenase (HASB) originating from Streptococcus zooepidemicus SEQ ID NO:39 is the amino acid sequence of UDP-glucose dehydrogenase (HASB) from Arabidopsis thaliana SEQ ID NO: 40 is the amino acid sequence of UDP-glucose dehydrogenase (HASB) originating from the Chlorella virus PBCV-1 SEQ ID NO: 41 is the amino acid sequence of UDP-glucose dehydrogenase (HASB) originating from Streptococcus zooepidemicus SEQ ID NO:42 is the nucleic acid sequence of UDP-glucose-4-epimerase (GNE1) originating from Pseudomonas aeruginosa SEQ ID NO: 43 is the nucleic acid sequence of UDP-glucose-4-epimerase (KFOA) originating from Pasteurella multocida SEQ ID NO:44 is the nucleic acid sequence of UDP-glucose-4-epimerase (KFOA) originating from E. coli SEQ ID NO:45 is the amino acid sequence of UDP-glucose-4-epimerase (GNE1) originating from Pseudomonas aeruginosa SEQ ID NO: 46 is the amino acid sequence of UDP-glucose-4-epimerase (KFOA) originating from Pasteurella multocida SEQ ID NO: 47 is the amino acid sequence of UDP-glucose-4-epimerase (KFOA) originating from Escherichia coli SEQ ID NO:48 is a recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Kupienius salei with an N-terminal secretion signal SEQ ID NO:49 is a recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Titius serulatus with an N-terminal secretion signal SEQ ID NO:50 is a recoded nucleic acid sequence of hyaluronidase (HYAL) of B. taurus origin with an N-terminal secretion signal SEQ ID NO:51 is a recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Apis mellifera with an N-terminal secretion signal SEQ ID NO:52 is a recoded nucleic acid sequence of hyaluronidase (HYAL) from Macaca mulatta with an N-terminal secretory signal. SEQ ID NO:53 is a recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Vespa magnifica with an N-terminal secretion signal SEQ ID NO:54 is a recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Kupienius salei with an N-terminal secretion signal SEQ ID NO:55 is a recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Titius serulatus with an N-terminal secretion signal and a C-terminal anchoring signal. SEQ ID NO:56 is a recoded nucleic acid sequence of hyaluronidase (HYAL) of B. taurus origin with an N-terminal secretion signal and a C-terminal anchoring signal. SEQ ID NO:57 is a recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Apis mellifera with an N-terminal secretion signal and a C-terminal anchoring signal. SEQ ID NO:58 is a recoded nucleic acid sequence of hyaluronidase (HYAL) from Macaca mulatta with an N-terminal secretion signal and a C-terminal anchoring signal. SEQ ID NO:59 is a recoded nucleic acid sequence of hyaluronidase (HYAL) from Vespa magnifica with an N-terminal secretion signal and a C-terminal anchoring signal. SEQ ID NO:60 is the amino acid sequence of hyaluronidase (HYAL) from Kupienius salei with an N-terminal secretion signal SEQ ID NO:61 is the amino acid sequence of hyaluronidase (HYAL) from Titius serulatus with an N-terminal secretion signal SEQ ID NO:62 is the amino acid sequence of hyaluronidase (HYAL) of B. taurus origin with an N-terminal secretion signal SEQ ID NO:63 is the amino acid sequence of hyaluronidase (HYAL) from Apis mellifera with an N-terminal secretion signal SEQ ID NO:64 is the amino acid sequence of hyaluronidase (HYAL) from Macaca mulatta with an N-terminal secretory signal SEQ ID NO:65 is the amino acid sequence of hyaluronidase (HYAL) from Vespa magnifica with an N-terminal secretion signal SEQ ID NO:66 is the amino acid sequence of hyaluronidase (HYAL) from Kupienius salei with an N-terminal secretion signal and a C-terminal anchoring signal SEQ ID NO:67 is the amino acid sequence of hyaluronidase (HYAL) from Titius serulatus with an N-terminal secretion signal and a C-terminal anchoring signal SEQ ID NO:68 is the amino acid sequence of hyaluronidase (HYAL) of B. taurus origin with an N-terminal secretion signal and a C-terminal anchoring signal SEQ ID NO:69 is the amino acid sequence of hyaluronidase (HYAL) from Apis mellifera with an N-terminal secretion signal and a C-terminal anchoring signal SEQ ID NO:70 is the amino acid sequence of hyaluronidase (HYAL) from Macaca mulatta with an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO:71 is the amino acid sequence of hyaluronidase (HYAL) from Vespa magnifica with an N-terminal secretion signal and a C-terminal anchoring signal SEQ ID NO:72 is the nucleic acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Saccharomyces cerevisiae SEQ ID NO:73 is the recoded nucleic acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Chlorella virus 1 (PBCV-1) SEQ ID NO:74 is the recoded nucleic acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Chlorella virus 1 (PBCV-1) SEQ ID NO:75 is the amino acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Saccharomyces cerevisiae SEQ ID NO: 76 is the amino acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Chlorella virus 1 (PBCV-1) SEQ ID NO:77 is the nucleic acid sequence of UDP-N-acetylglucosamine pyrophosphorylase (QRI1) originating from Saccharomyces cerevisiae SEQ ID NO:78 is the amino acid sequence of UDP-N-acetylglucosamine pyrophosphorylase (QRI1) originating from Saccharomyces cerevisiae SEQ ID NO:79 is the nucleic acid sequence of phosphoglucomutase-1 (PGM1) originating from Saccharomyces cerevisiae. SEQ ID NO:80 is the amino acid sequence of phosphoglucomutase-1 (PGM1) originating from Saccharomyces cerevisiae SEQ ID NO:81 is the nucleic acid sequence of UTP-glucose 1-phosphate uridylyltransferase (UGP1) originating from Saccharomyces cerevisiae SEQ ID NO:82 is the amino acid sequence of UTP-glucose 1-phosphate uridylyltransferase (UGP1) originating from Saccharomyces cerevisiae SEQ ID NO:83 is the nucleic acid sequence of glucosamine 6-phosphate N-acetyltransferase (GNA1) originating from Saccharomyces cerevisiae SEQ ID NO:84 is the amino acid sequence of glucosamine 6-phosphate N-acetyltransferase (GNA1) originating from Saccharomyces cerevisiae SEQ ID NO:85 is the nucleic acid sequence of phosphoacetylglucosamine mutase (PCM1) originating from Saccharomyces cerevisiae SEQ ID NO:86 is the amino acid sequence of phosphoacetylglucosamine mutase (PCM1) originating from Saccharomyces cerevisiae SEQ ID NO: 87 is the nucleic acid sequence of promoter pTDH3 SEQ ID NO: 88 is the nucleic acid sequence of promoter pTDH3.Sk SEQ ID NO: 89 is the nucleic acid sequence of promoter pTDH3-1.sba SEQ ID NO: 90 is the nucleic acid sequence of the promoter pTDH3-1.Sar SEQ ID NO: 91 is the nucleic acid sequence of promoter pENO2 SEQ ID NO: 92 is the nucleic acid sequence of promoter pTEF3 SEQ ID NO: 93 is the nucleic acid sequence of promoter pTEF1 SEQ ID NO: 94 is the nucleic acid sequence of the promoter pTEF1.ago SEQ ID NO: 95 is the nucleic acid sequence of the promoter pTEF1.Sba SEQ ID NO: 96 is the nucleic acid sequence of promoter pPDC1 SEQ ID NO: 97 is the nucleic acid sequence of promoter pCCW12 SEQ ID NO: 98 is the nucleic acid sequence of the promoter pCCW12.Sm SEQ ID NO: 99 is the nucleic acid sequence of promoter pCCW12.Sk SEQ ID NO: 100 is the nucleic acid sequence of the promoter pCCW12.Sba SEQ ID NO: 101 is the nucleic acid sequence of the promoter pCCW12.Sar SEQ ID NO: 102 is the nucleic acid sequence of the promoter pNUP57 SEQ ID NO: 103 is the nucleic acid sequence of the promoter pCCW10.ago SEQ ID NO: 104 is the nucleic acid sequence of promoter pCWP2 SEQ ID NO: 105 is the nucleic acid sequence of promoter pFBA1 SEQ ID NO: 106 is the nucleic acid sequence of the promoter pCCW120.Sm SEQ ID NO: 107 is the nucleic acid sequence of the promoter pCUP1 SEQ ID NO: 108 is the nucleic acid sequence of promoter pMET6 SEQ ID NO: 109 is the nucleic acid sequence of promoter pMET25 SEQ ID NO: 110 is the nucleic acid sequence of the promoter pSAM1 SEQ ID NO: 111 is the nucleic acid sequence of the terminator tTPI1 SEQ ID NO: 112 is the nucleic acid sequence of terminator tMET25 SEQ ID NO: 113 is the nucleic acid sequence of the terminator tDIT1 SEQ ID NO: 114 is the nucleic acid sequence of terminator tRPL3 SEQ ID NO: 115 is the nucleic acid sequence of terminator tRPL3.sm SEQ ID NO: 116 is the nucleic acid sequence of terminator tRPL3.sba SEQ ID NO: 117 is the nucleic acid sequence of terminator tRPL41B SEQ ID NO: 118 is the nucleic acid sequence of terminator tRPL41B.Sba SEQ ID NO: 1119 is the nucleic acid sequence of terminator tRPL15A SEQ ID NO: 120 is the nucleic acid sequence of the terminator tRPL15A.Sm SEQ ID NO: 121 is the nucleic acid sequence of terminator tRPL15A.sba SEQ ID NO: 122 is the nucleic acid sequence of terminator tIDP1 SEQ ID NO: 123 is the nucleic acid sequence of the terminator tIDP1.Sba SEQ ID NO: 124 is the nucleic acid sequence of terminator tTEF1.sba SEQ ID NO:125 is the nucleic acid sequence of the secretory sequence added to the 5' end SEQ ID NO: 126 is the amino acid sequence of the secretory sequence added to the N-terminus SEQ ID NO: 127 is the nucleic acid sequence of the anchoring sequence added to the 3' SEQ ID NO: 128 is the amino acid sequence of the anchoring sequence added to the C-terminus

[0055] Detailed Description of the Invention The inventors have contemplated genetically modified cells, particularly genetically modified yeast, that have the ability to produce chondroitin compared to parent cells that are not naturally capable of doing so, particularly compared to parent yeast.

[0056] These genetically modified cells are described throughout the specification.

[0057] definition The term "chondroitin" refers to a compound of the chemical formula (C 14 H 21 NO 11 )n (a polymer of alternating β-glucuronic acid-(1→3)-N-acetyl-β-galactosamine-4-sulfate-(1→4)).

[0058] The term "chondroitin," as used herein, is intended to encompass chondroitin and its derivatives, including, but not limited to, chondroitin sulfate, 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.

[0059] Chondroitin sulfate includes, but is not limited to, chondroitin-4-sulfate, chondroitin-6-sulfate, chondroitin-2,6-sulfate, chondroitin-4,6-sulfate, and / or mixtures thereof.

[0060] The site of sulfation in chondroitin-4-sulfate is at the 4-carbon position of the N-acetylgalactosamine (GalNAc) sugar.

[0061] The site of sulfation in chondroitin-6-sulfate is the 6-carbon of the GalNAc sugar.

[0062] The sites of sulfation in chondroitin-2,6-sulfate are the 2-carbon of the glucuronic acid and the 6-carbon of the GalNAc sugar.

[0063] The sites of sulfation in chondroitin-4,6-sulfate are the 4- and 6-carbon positions of the GalNAc sugar.

[0064] Chondroitin derivatives, such as chondroitin sulfate, may be useful in alleviating the risk of, reducing, preventing, and / or treating osteoarthritis, cartilage deterioration, and osteoarthritis-related joint pain, tenderness, and / or swelling.

[0065] In particular, chondroitin derivatives, such as chondroitin sulfate, may be useful in alleviating the risk of, reducing, preventing, and / or treating osteoarthritis, osteoarthritis-related joint pain, joint tenderness, and / or joint swelling, and / or cartilage deterioration.

[0066] Chondroitin, particularly chondroitin sulfate, may be used alone or together with glucosamine as a dietary supplement for the treatment of osteoarthritis, osteoarthritis-related joint pain, joint tenderness, and joint swelling, joint degeneration, and / or cartilage deterioration.

[0067] Chondroitin, particularly chondroitin sulfate, can be prepared as a composition.

[0068] Chondroitin derivatives can be prepared from chondroitin through suitable chemical or enzymatic processes.

[0069] Chondroitin can be produced in recombinant cells.

[0070] 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.

[0071] 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. Guidelines from the US National Institute for Health (NIH) 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.

[0072] A protein produced by expression of recombinant DNA or a recombinant vector in a living cell is also called a recombinant protein.

[0073] The term "recombinant" is therefore synonymous with the term "genetically modified." The term "gene" is synonymous with the term "nucleic acid" or "nucleotide sequence."

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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 an "exogenous or heterologous gene" or "heterologous nucleotide sequence" or "heterologous nucleic acid" as defined hereinafter).

[0083] "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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] The nucleic acid expression construct may further comprise 5' and / or 3' recognition sequences and / or selectable markers.

[0090] 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.

[0091] 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.

[0092] As used herein, the term "anchoring signal" when used in connection with a protein or polypeptide, e.g., an enzyme (e.g., hyaluronidase, for example), 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., hyaluronidase, for example, to form a fusion protein), that enables the cellular transport machinery of a cell, particularly an S. cerevisiae cell, to properly anchor and / or position the second protein operably linked to the first protein in the cell membrane.

[0093] As used herein, the term "secretion signal", when used in connection with a protein or polypeptide, e.g., an enzyme (e.g., hyaluronidase, for example), 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., hyaluronidase, for example, to form a fusion protein), that enables the cellular transport machinery of a cell, particularly 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 is cleaved from the second protein.

[0094] As used herein, "secretion signal" and "anchoring signal", when used in conjunction with a protein or polypeptide, e.g., an enzyme (e.g., hyaluronidase, for example), 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., hyaluronidase, for example), that enables the cellular transport mechanisms of a cell, particularly an S. cerevisiae cell, to position at least the second protein at 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 an "anchoring signal". In some cases, a secretion-anchoring signal can provide dual secretion signal and anchoring signal functions.

[0095] The sequences of secretion and anchoring signals, methods for expression, anchoring, and / or secretion of heterologous proteins, such as enzymes (e.g., hyaluronidase, for example) on the surface of cells (e.g., yeast cells, for example) are well known in the art (see, for example, 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). 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 accomplished, for example, by altering 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 elements, or by increasing the strength of the promoter or operator to which the nucleotide sequence is operably linked.

[0096] 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.

[0097] 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 the 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. The terms "encode" or "encode" refer to the process by which a polynucleotide produces an amino acid sequence through the mechanisms of transcription and translation.

[0098] The genes encoding the enzymes contemplated in the present invention may be exogenous or endogenous.

[0099] 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.

[0100] 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).

[0101] 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.

[0102] 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).

[0103] 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.

[0104] 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.

[0105] 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 cell.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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. Comparison of sequences between two sequences and determination of the percentage of sequence identity may be accomplished using a mathematical algorithm. Those skilled in the art will be aware of 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).

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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 / .

[0115] 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.

[0116] 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.

[0117] In certain embodiments, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0118] "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.

[0119] 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.

[0120] The term "medium" refers to a culture medium, or a cultivation medium, or a fermentation medium.

[0121] For maximum production of chondroitin, recombinant cells used as production hosts preferably have a high carbohydrate utilization rate. These characteristics may be imparted 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 sources such as cheese whey permeate, corn steep liquor, sugar beet molasses, and malt. Other carbon substrates may include glycerol, acetate, and / or ethanol.

[0122] Thus, it is contemplated that the carbon source utilized in the present invention may encompass a wide range of carbon-containing substrates, limited only by the choice of cell, particularly yeast.

[0123] 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.

[0124] The preferred carbon substrate is glucose or sucrose.

[0125] 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.

[0126] Furthermore, additional genetic modifications suitable for the growth of recombinant cells according to the invention may be considered.

[0127] 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.

[0128] "Microaerobic conditions" refers to a culture medium in which the oxygen concentration is lower than that found in air, i.e., up to 6% O2.

[0129] "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.

[0130] Particularly suitable culture media for producing the recombinant cells of the invention, in particular the recombinant yeasts of the invention, are described further below. The "controlled" molecular weight of the chondroitin of the invention means that at least 80%, in particular at least 85%, of the chondroitin produced by the method of the invention is controlled by at least one parameter of the method and / or recombinant cell of the invention, such as, for example: - the nature and origin of the nucleic acid encoding the polypeptide having chondroitinase activity of the recombinant cell; - the nature and origin of the promoter controlling the expression of the nucleic acid encoding the polypeptide having chondroitinase activity in the recombinant cell; - the presence of an anchoring and / or secretion signal associated with the polypeptide having chondroitinase 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 predetermined range of molecular weights.

[0131] 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.

[0132] 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: Prom1-ORF1-term1-ORF2-gene2-term2- … / … -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 indicate 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.

[0133] 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).

[0134] - 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.

[0135] - 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.

[0136] - 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.

[0137] Recombinant cells according to the present invention The present inventors have envisaged recombinant cells, in particular recombinant yeast, that have the ability to produce chondroitin.

[0138] The present invention relates to recombinant cells, in particular recombinant yeast, having the ability to produce chondroitin, where this ability to produce chondroitin is obtained through multiple alterations that have been introduced into its genome by genetic engineering methods.

[0139] The present invention relates to a recombinant yeast cell that produces chondroitin, comprising: (a) one or more recombinant nucleic acids encoding a polypeptide having chondroitin synthase (HCOS) 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 UDP-glucose-4-epimerase (kfoA or GNE1) activity; The present invention relates to a recombinant yeast cell comprising:

[0140] The present invention further provides a recombinant host cell that produces chondroitin, comprising: (a) one or more recombinant nucleic acids encoding a polypeptide having chondroitin synthase (HCOS) 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 UDP-glucose-4-epimerase (kfoA or GNE1) activity; (d) one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity, the polypeptide having chondroitinase activity comprising a secretion signal and optionally an anchoring signal such that chondroitin, particularly of a desired molecular weight, is produced by the host cell; The present invention relates to a recombinant host cell comprising the

[0141] The present inventors have found that the ability of cells, particularly yeast cells, to produce chondroitin can be achieved by introducing multiple genetic alterations into the genome of these cells.

[0142] The production of chondroitin by the cells of the invention, particularly the yeast cells of the invention, is achieved by optimizing the endogenous metabolism of UDP-glucose and UDP-N-acetyl-glucosamine and then directing the artificially engineered metabolic pathway primarily towards chondroitin, while maintaining optimal viability of the resulting genetically engineered cells.

[0143] It has been determined that production of chondroitin 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-glucose, UDP-glucuronate, and chondroitin, or (ii) fructose-6-phosphate, glucosamine-6-phosphate, N-acetyl-glucosamine-6-phosphate, N-acetyl-glucosamine-1-phosphate, UDP-N-acetyl-glucosamine, UDP-N-acetyl-galactosamine, and chondroitin, while maintaining a metabolic balance that allows for good viability of the resulting recombinant cells.

[0144] 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.

[0145] Unexpected technical challenges were encountered in establishing suitable conditions for the preparation of recombinant cells capable of producing chondroitin, particularly chondroitin having a controlled molecular weight.

[0146] Indeed, after extensive research and experimental testing, the inventors have determined that the following parameters: - selection of the nature and origin of the nucleic acid sequence encoding a polypeptide having chondroitinase 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 chondroitinase 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 chondroitinase activity in the recombinant cell according to the invention, in particular in the recombinant yeast, and / or the pH of the culture medium during the process of culturing the recombinant cell according to the invention, in particular the recombinant yeast, and / or - the duration of the cultivation of the recombinant cell according to the invention, in particular the recombinant yeast; We have discovered that it was possible to cultivate recombinant cells, in particular recombinant yeast cells, more specifically recombinant Saccharomyces cerevisiae yeast cells, that are capable of producing chondroitin with a controlled molecular weight using a method that uses recombinant yeast cells, such as recombinant yeast cells, and more specifically recombinant Saccharomyces cerevisiae yeast cells, that are capable of producing chondroitin with a controlled molecular weight.

[0147] To the inventors' knowledge, this has never been achieved before.

[0148] The nucleic acid encoding a polypeptide having chondroitinase activity in the recombinant cell of the present invention can be obtained or derived from at least one of Cupienius salei, Titius serulatus, Bos taurus, Vespa magnifica, Macaca mulatta, or Apis mellifera, preferably Titius serulatus.

[0149] The molecular weight of the chondroitin can be in the range of less than 50 kDa, preferably in the range of about 20 kDa to about 50 kDa.

[0150] Alternatively, the molecular weight of the chondroitin may be in the range above 50 kDa, preferably in the range of about 50 kDa to about 250 kDa.

[0151] In another alternative, the molecular weight of the chondroitin may be in the range above 100 kDa, preferably in the range of about 100 kDa to about 1500 kDa.

[0152] 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, obtained or derived from Arabidopsis thaliana or Chlorella virus PBCV1.

[0153] The nucleic acid encoding a polypeptide having chondroitin synthase (HCOS) activity is (i) a nucleic acid encoding a chondroitin synthase, or (ii) a nucleic acid encoding a chimeric polypeptide having chondroitin synthase activity It could be.

[0154] These nucleic acids can be obtained or derived from at least one of Pasteurella multocida, Chlorella virus PBCV1, Mycobacterium tuberculosis, Homo sapiens, Escherichia coli, Saccharomyces cerevisiae, or Penicillium oxalicum.

[0155] The nucleic acid encoding a polypeptide having UDP-glucose-4-epimerase (kfoA or GNE1) activity can be obtained or derived from a bacterium, in particular a bacterium selected from the group consisting of Pseudomonas aeruginosa, Pasteurella multocida, and Escherichia coli.

[0156] The recombinant cell is (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:

[0157] The recombinant cell is (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:

[0158] In certain embodiments of the recombinant cell, - a nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, - a nucleic acid encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity, - a nucleic acid encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity, - a nucleic acid encoding a polypeptide having UTP-glucose-1-phosphate uridylyltransferase (UGP1) activity, - 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,

[0159] In certain embodiments, the recombinant host cell is yeast.

[0160] In particular, the recombinant cell belongs to the genus Saccharomyces, or Candida, or Kluyveromyces, or Ogataea, or Yarrowia, or Debaryomyces, or Ashbya, and in particular to the genus Saccharomyces.

[0161] Specifically, the recombinant cell 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, and is preferably Saccharomyces cerevisiae.

[0162] Another object of the present invention is a method for producing chondroitin of a desired molecular weight, comprising the steps of: (a) culturing a recombinant cell as defined herein in a culture medium for a period of time sufficient to produce chondroitin of a desired molecular weight; (b) optionally isolating or recovering the chondroitin from the recombinant cells and / or the culture medium. The present invention relates to a method comprising the steps of:

[0163] In this method, the chondroitin can have a molecular weight of about 20 kDa to about 50 kDa.

[0164] In another embodiment, the chondroitin may have a molecular weight of about 50 kDa to about 150 kDa.

[0165] In another embodiment, the chondroitin may have a molecular weight of about 150 kDa to about 1500 kDa.

[0166] In the method of the present invention, the recombinant cell may be a yeast belonging to the genus Saccharomyces, and in particular may be Saccharomyces cerevisiae.

[0167] The time sufficient to produce chondroitin 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, and preferably about 48 hours.

[0168] In certain embodiments, the molecular weight of the chondroitin produced can be controlled by adjusting the pH of the culture medium in step (a).

[0169] In certain embodiments, 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 1,000 L to about 3,000 L, even more preferably about 10,000 L, or even more preferably about 100,000 L, or even about 250,000 L.

[0170] Another object of the invention relates to a chondroitin obtainable from a recombinant cell according to the invention or from a method according to the invention.

[0171] The present invention also relates to a culture medium comprising the chondroitin of the present invention.

[0172] Another object of the invention is a composition comprising a chondroitin according to the invention.

[0173] The present invention further relates to an industrial or consumer product or consumable product comprising (i) a chondroitin having a molecular weight according to the present invention, (ii) a culture medium according to the present invention, or (iii) a composition according to the present invention.

[0174] In particular, the industrial or consumer product or consumable product may be 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.

[0175] The present invention also relates to the use of the recombinant cell of the present invention for producing chondroitin having a molecular weight within the range of about 20 kDa to about 50 kDa or about 50 kDa to about 1000 kDa.

[0176] Another object of the present invention is a method for producing chondroitin, comprising the steps of: (a) culturing a recombinant yeast as defined herein in a culture medium; (b) recovering chondroitin from the culture medium; Including, The chondroitin recovered in step (b) is - the nature and origin of the recombinant nucleic acid encoding a polypeptide having recombinant yeast chondroitinase activity; - the nature and origin of the promoter controlling the expression of the recombinant nucleic acid encoding the polypeptide having chondroitinase activity of the recombinant yeast, - the presence of an anchoring and / or secretion signal associated with the recombinant yeast polypeptide having chondroitinase activity, - the pH of the culture medium during the cultivation process of the recombinant yeast, and / or - Duration of recombinant yeast cultivation having a molecular weight controlled through the selection of It concerns the method.

[0177] The present invention further relates to a recombinant yeast that produces chondroitin, - (A) one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity; - (B) one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity; - (C) one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity; - (D) one or more recombinant nucleic acids encoding a polypeptide having chondroitin synthase (HCOS) activity; - (E) one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase (kfoA or GNE1) activity; Including, The recombinant yeast is Saccharomyces cerevisiae. Concerning recombinant yeast.

[0178] In certain embodiments, a recombinant cell according to the invention, in particular a recombinant yeast according to the invention, further comprises (F) a nucleic acid encoding a polypeptide having chondroitinase activity, wherein the nucleic acid encoding the polypeptide having chondroitinase activity comprises an anchoring signal and / or a secretion signal such that chondroitin, in particular of a desired molecular weight, is produced by the host cell.

[0179] In a particular embodiment, a recombinant cell according to the invention, in particular a recombinant yeast, comprises only one recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity.

[0180] In a particular embodiment, the recombinant cell according to the invention, in particular the recombinant yeast, comprises 1 to 10, preferably 2 to 8, recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity.

[0181] In a particular embodiment, a recombinant cell according to the invention, in particular a recombinant yeast, comprises from 1 to 5 recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity.

[0182] In another embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) are obtained or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, in particular, obtained or derived from Chlorella virus PBCV1 or Arabidopsis thaliana.

[0183] In another embodiment, the recombinant cell according to the invention, in particular the recombinant yeast, comprises from 1 to 5 recombinant nucleic acids encoding a polypeptide having chondroitin synthase (HCOS) activity.

[0184] In certain embodiments, the one or more nucleic acids encoding a polypeptide having chondroitin synthase (HCOS) activity are obtained or derived from at least one of Pasteurella multocida, Chlorella virus PBCV1, Mycobacterium tuberculosis, Homo sapiens, Escherichia coli, Saccharomyces cerevisiae, or Penicillium oxalicum.

[0185] In another embodiment, a recombinant cell according to the invention, in particular a recombinant yeast, comprises from 1 to 5 recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase (kfoA or GNE1) activity.

[0186] In certain embodiments, the one or more nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase (kfoA or GNE1) activity are obtained or derived from at least one of Pseudomonas aeruginosa, Pasteurella multocida, and Escherichia coli.

[0187] In a particular embodiment, a recombinant cell according to the invention, in particular a recombinant yeast, comprises only one recombinant nucleic acid encoding a polypeptide having chondroitinase activity.

[0188] In another embodiment, the one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity are obtained or derived from at least one of Cupienius salei, Titius serlatus, Bos taurus, Apis mellifera, Macaca mulatta, or Vespa magnifica.

[0189] In another embodiment, the recombinant cell according to the invention, in particular a recombinant yeast cell, (A) a polypeptide having phosphoglucomutase-1 (PGM1) activity, and / or (B) a polypeptide having UTP-glucose-1-phosphate uridylyltransferase (UGP1) activity, and / or (C) a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity, and / or (D) A polypeptide having phosphoacetylglucosamine mutase (PCM1) activity The recombinant nucleic acid may comprise at least one recombinant nucleic acid encoding one or more of:

[0190] In particular, a recombinant cell according to the invention, in particular a recombinant yeast cell according to the invention, comprises at least two, in particular at least three, more particularly all of the modifications set out above.

[0191] In certain embodiments, the recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity, the recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase (UGP1) activity, the recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, the recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity, the recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity, and the recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity are recombinant nucleic acids obtained or derived from a yeast, preferably Saccharomyces cerevisiae.

[0192] In a particular embodiment, the recombinant nucleic acid or nucleic acids encoding a polypeptide as defined above contained in a recombinant cell according to the invention, in particular a recombinant yeast according to the invention, are under the control of a promoter selected from the group consisting of pPDC1, pTDH3, pTDH3.Sk, pTDH3-1.Sba, pCCW12, pCCW120.Sm, pTEF1, pENO2 pTEF1.Ago, and pTEF1.Sba.

[0193] In certain embodiments, the inducible or repressible promoter referred to herein is selected from the group consisting of copper-inducible or repressible promoters or methionine-inducible or repressible promoters, in particular pCUP1.

[0194] In a specific embodiment, the recombinant nucleic acid is under the control of the promoter pCWP2.

[0195] Another object of the present invention is a method for producing chondroitin, comprising the steps of: (a) culturing a recombinant cell according to the invention, in particular a recombinant yeast as defined herein, in a culture medium; (b) recovering chondroitin from the culture medium; The present invention relates to a method comprising the steps of:

[0196] In certain embodiments, the chondroitin recovered in step (b) is - the nature and origin of recombinant nucleic acids encoding polypeptides having chondroitinase activity of recombinant cells, in particular recombinant yeasts; - the nature and origin of the promoter controlling the expression of the recombinant nucleic acid encoding the polypeptide having chondroitinase activity in the recombinant cell, in particular in the recombinant yeast; - the presence of an anchoring or secretion signal associated with the recombinant nucleic acid encoding a polypeptide having chondroitinase activity in a recombinant cell, in particular in a recombinant yeast, the pH of the culture medium during the process of culturing recombinant cells, in particular recombinant yeasts, and / or - the period during which the recombinant cells, in particular the recombinant yeast, are cultivated; The molecular weight is controlled through the selection of

[0197] In a particular embodiment, the method of the present invention is for producing chondroitin having a molecular weight strictly less than 50 kDa, in particular strictly less than 50 kDa and greater than or equal to 20 kDa, and (c) the step of recovering chondroitin from the culture medium is carried out 48 hours after the initiation of the culture of the recombinant cell, in particular the recombinant yeast.

[0198] In another embodiment, the method of the present invention is for producing chondroitin having a molecular weight of 50 kDa or more and less than 1000 kDa, and (c) the step of recovering chondroitin from the culture medium is performed 48 hours after the initiation of culture of the recombinant yeast.

[0199] In another embodiment, the method of the invention is for producing chondroitin having a molecular weight strictly greater than 1000 kDa, in particular having a molecular weight strictly greater than 1000 kDa up to 1,500,000 kDa, and (c) the step of recovering chondroitin from the culture medium is carried out 48 hours after the initiation of the culture of the recombinant cell, in particular the recombinant yeast.

[0200] In particular, the culture medium comprises at least a carbon source, preferably a carbon source selected from the group consisting of glucose and sucrose.

[0201] Another object of the invention relates to the use of a recombinant cell, in particular a recombinant yeast, according to the invention as defined herein for the production of chondroitin, in particular chondroitin of controlled molecular weight.

[0202] Recombinant nucleic acids encoding polypeptides having chondroitin synthase activity (HCOS) 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 chondroitin synthase activity.

[0203] A polypeptide having chondroitin synthase activity according to the present invention means a polypeptide that converts the intermediate metabolic products UDP-glucuronate and UDP-N-acetyl-galactosamine into chondroitin ((β-D-1,3-GalNAc-β-D-1,4-GlcA)n).

[0204] In some embodiments, the nucleic acid encoding a polypeptide having chondroitin synthase activity can be under the control of an inducible or repressible promoter.

[0205] One or more of the recombinant nucleic acids encoding a polypeptide having chondroitin synthase activity can be under the control of the pTDH3-1.Sba or pTDH3.Sar promoter.

[0206] The one or more recombinant nucleic acids encoding a polypeptide having chondroitin synthase (HCOS) activity include (i) a nucleic acid encoding a chondroitin synthase, or (ii) a nucleic acid encoding a chimeric polypeptide having chondroitin synthase activity It could be.

[0207] The nucleic acid encoding a polypeptide having chondroitin synthase activity can be obtained or derived from at least one of Pasteurella multocida, Chlorella virus PBCV1, Mycobacterium tuberculosis, Homo sapiens, Escherichia coli, Saccharomyces cerevisiae, or Penicillium oxalicum.

[0208] A recombinant cell according to the invention, in particular a recombinant yeast according to the invention, may comprise one recombinant nucleic acid encoding a polypeptide having chondroitin synthase activity.

[0209] Illustratively, the chondroitin synthase gene can be inserted into the JLP1 gene and / or into the SAM3 gene, as shown in the Examples herein.

[0210] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast, - a recombinant nucleic acid encoding a polypeptide having chondroitin synthase activity, - a recombinant nucleic acid encoding a polypeptide having chondroitin synthase activity obtained or derived from at least one of Pasteurella multocida, Chlorella virus PBCV1, Mycobacterium tuberculosis, Homo sapiens, Escherichia coli, Saccharomyces cerevisiae, or Penicillium oxalicum; - one recombinant nucleic acid encoding a polypeptide having chondroitin synthase activity under the control of an inducible or repressible promoter and / or under the control of the pTDH3-1.Sba or pTDH3.Sar promoter; Includes.

[0211] Recombinant nucleic acids encoding polypeptides having UDP-glucose dehydrogenase activity (HASB) 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.

[0212] 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.

[0213] The one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity can be under the control of a promoter selected from the group consisting of pTEF1.sba, pCCW12, and pTDH3.Sk.

[0214] The one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase dehydrogenase activity can be obtained or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, in particular at least one of Arabidopsis thaliana or Chlorella virus PBCV1.

[0215] The recombinant cell according to the present invention, in particular the recombinant yeast according to the present invention, may contain 1 to 5 recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity.

[0216] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity can be inserted into the JLP1 gene and / or the SAM3 gene of a recombinant cell, in particular a recombinant yeast, as shown in the Examples herein.

[0217] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast, - 1 to 5 recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity, - said recombinant nucleic acid encoding a polypeptide having UDP-glucose dehydrogenase activity, which is obtained or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1 or Streptococcus zooepidemicus, in particular from at least one of Arabidopsis thaliana or Chlorella virus PBCV1, - said recombinant nucleic acid encoding a polypeptide having UDP-glucose dehydrogenase 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 pTEF1.Sba, pCCW12, and pTDH3.Sk Includes.

[0218] Recombinant nucleic acid encoding a polypeptide having UDP-glucose-4-epimerase activity (kfoA or GNE1) 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 UDP-glucose-4-epimerase activity.

[0219] A polypeptide having UDP-glucose-4-epimerase activity according to the present invention means a polypeptide which converts the intermediate metabolic product UDP-N-acetyl-glucosamine into UDP-N-acetyl-galactosamine.

[0220] In certain embodiments, one or more of the recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase activity can be under the control of a promoter selected from the group consisting of pCCW12 or pCCW120.Sm.

[0221] The one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase activity can be obtained or derived from a bacterium, in particular a bacterium selected from the group consisting of Pseudomonas aeruginosa, Pasteurella multocida, and Escherichia coli.

[0222] The recombinant cell according to the present invention, in particular the recombinant yeast according to the present invention, may contain 1 to 5 recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase activity.

[0223] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase activity can be inserted into the JLP1 gene and / or the SAM3 gene of a recombinant cell, in particular a recombinant yeast, as shown in the Examples herein.

[0224] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast cell, - 1 to 5 recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase activity, - said recombinant nucleic acid encoding a polypeptide having UDP-glucose-4-epimerase activity, said recombinant nucleic acid being derived from or obtained from the group consisting of Pseudomonas aeruginosa, Pasteurella multocida and Escherichia coli, - said recombinant nucleic acid encoding a polypeptide having UDP-glucose-4-epimerase 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 or pCCW120.Sm Includes.

[0225] Recombinant Nucleic Acids Encoding Polypeptides Having Chondroitinase Activity 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 chondroitinase activity.

[0226] A polypeptide having chondroitinase activity according to the present invention means a polypeptide which degrades chondroitin, i.e. converts chondroitin of a given molecular weight into chondroitin of a lower molecular weight.

[0227] A polypeptide having chondroitinase activity according to the present invention can include a hyaluronoglucosaminidase, e.g., a chondroitinase or a hyaluronidase.

[0228] As mentioned above, the polypeptide having chondroitinase activity of the present invention comprises a secretion signal.

[0229] In certain embodiments, a polypeptide having chondroitinase activity includes both a secretion signal and an anchoring signal.

[0230] In certain embodiments, the one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity are under the control of an inducible or repressible promoter that is functional in the recombinant cell of the invention.

[0231] One or more of the recombinant nucleic acids encoding a polypeptide having chondroitinase activity may 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, in particular the pCCW12.Sba promoter.

[0232] The one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity can be obtained or derived from at least one of Cupienius salei (Csa), Titius serlatus (Ts), Bos taurus (Bt), Apis mellifera (Am), Macaca mulatta (Mm), or Vespa magnifica (Vm), in particular Titius serlatus (Ts).

[0233] 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 chondroitinase activity.

[0234] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity can be inserted within the JLP1 gene, and / or within the LYP1 gene, and / or within the LEU2 gene, in particular within the LEU2 gene.

[0235] In a preferred embodiment, the recombinant nucleic acid encoding a polypeptide having chondroitinase activity is a recombinant nucleic acid encoding a hyaluronidase. In particular, the polypeptide having chondroitinase activity is a hyaluronidase.

[0236] All of the features described above regarding recombinant nucleic acids encoding polypeptides having chondroitinase activity apply to recombinant nucleic acids encoding hyaluronidase.

[0237] 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 chondroitinase activity, - said recombinant nucleic acid encoding a polypeptide having chondroitinase activity derived from or obtained from at least one of Cupienius salei (Csa), Titius serlatus (Ts), Bos taurus (Bt), Apis mellifera (Am), Macaca mulatta (Mm) or Vespa magnifica (Vm), in particular Titius serlatus (Ts), - said recombinant nucleic acid encoding a polypeptide having chondroitinase activity, which (i) comprises a secretion signal and no anchoring signal, or (ii) comprises a secretion signal and an anchoring signal, - the polypeptide having a chondroitinase that is a hyaluronidase, said recombinant nucleic acid encoding a polypeptide having chondroitinase 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 pTEF1, pCCW12, pCCW12.sba, pCCW12.Sar, pPDC1, pTEF3, pTDH3, pNUP57 and pCCW10.ago, in particular the pCCW12.Sba promoter; Includes.

[0238] Recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity (GFA1) A recombinant cell according to the invention, in particular a recombinant yeast cell according to the invention, may contain only one recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity.

[0239] 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.

[0240] 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.

[0241] One or more of the recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity can be under the control of the pTEF1.Ago promoter.

[0242] The one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may be obtained or derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0243] A recombinant cell according to the invention, in particular a recombinant yeast cell according to the invention, may contain only one recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity.

[0244] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity can be inserted into the JLP1 gene of a recombinant cell, in particular a recombinant yeast cell, as shown in the Examples herein.

[0245] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast cell, - a single recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity, - a recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity, said recombinant nucleic acid having origin or originating from Saccharomyces cerevisiae, and - the recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity, which is under the control of the pTEF1.Ago promoter Includes.

[0246] Recombinant nucleic acid encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity (QRI1) 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.

[0247] A polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity according to the present invention means a polypeptide that converts N-acetylglucosamine into UDP-N-acetylglucosamine.

[0248] 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.

[0249] One or more of the recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity can be under the control of promoter pTDH3-1.Sba or pTDH3.

[0250] The one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity can be obtained or derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0251] The recombinant cell according to the present invention, in particular the recombinant yeast according to the present invention, may contain 1 to 10 recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity, in particular may contain 2 to 8 recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity, for example, 2, 6, or 7 recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity.

[0252] 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 MET14 gene, and / or the JLP1 gene of a recombinant cell, in particular a recombinant yeast, as shown in the Examples herein.

[0253] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast cell, - 2 to 8 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 recombinant 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 functional in a recombinant cell of the invention, such as, for example, under the control of the inducible or repressible promoter pMET6 or pCUP1 and / or under the control of the promoter pTDH3-1.Sba or pTDH3. Includes.

[0254] Recombinant Nucleic Acids Encoding Polypeptides Having Phosphoglucomutase-1 Activity (PGM1) A recombinant cell according to the invention, in particular a recombinant yeast cell according to the invention, can comprise one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity.

[0255] 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.

[0256] 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 the recombinant cell of the invention.

[0257] One or more of the recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity can be under the control of promoter pTDH3.

[0258] One or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity can be obtained or derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0259] A recombinant cell according to the invention, in particular a recombinant yeast cell according to the invention, may contain only one recombinant nucleic acid encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity.

[0260] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity can be inserted into the MET14 gene of a recombinant cell, in particular a recombinant yeast.

[0261] 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 originating from or derived from Saccharomyces cerevisiae, - said recombinant nucleic acid encoding a polypeptide having phosphoglucomutase-1 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 the promoter pTDH3; Includes.

[0262] Recombinant nucleic acid encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity (UGP1) Recombinant cells according to the invention, in particular recombinant yeast cells according to the invention, comprise one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase (UGP1) activity.

[0263] 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.

[0264] 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.

[0265] One or more of the recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity can be under the control of the pFBA1, pENO2, or pPDC1 promoter.

[0266] One or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity can be obtained or derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0267] A recombinant cell according to the present invention, in particular a recombinant yeast cell according to the present invention, may comprise 1 to 10 recombinant nucleic acids encoding a polypeptide having UTP-glucose-1-phosphate uridylyltransferase activity, in particular 2 to 8 recombinant nucleic acids encoding a polypeptide having UTP-glucose-1-phosphate uridylyltransferase activity, for example 2, 6, or 8 recombinant nucleic acids encoding a polypeptide having UTP-glucose-1-phosphate uridylyltransferase activity.

[0268] 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 MET14 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.

[0269] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast cell, - 2 to 8 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 under an inducible or repressible promoter that 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 pFBA1, pENO2, and pPDC1. Includes.

[0270] Recombinant Nucleic Acids Encoding Polypeptides Having Glucosamine-6-Phosphate N-Acetyltransferase (GNA1) Activity A recombinant cell according to the invention, in particular a recombinant yeast cell according to the invention, may contain only one recombinant nucleic acid encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] The one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity can be obtained or derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0275] A recombinant cell according to the invention, in particular a recombinant yeast cell according to the invention, may contain only one recombinant nucleic acid encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity.

[0276] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity can be inserted into the MET14 gene and / or the SAM3 gene of a recombinant cell, in particular a recombinant yeast, as shown in the Examples herein.

[0277] 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 an inducible or repressible promoter that is functional in a recombinant cell of the invention and / or under the control of the promoter pCWP2; Includes.

[0278] Recombinant nucleic acids encoding polypeptides having phosphoacetylglucosamine mutase (PCM1) 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.

[0279] A polypeptide having phosphoacetylglucosamine mutase activity according to the present invention means a polypeptide which converts N-acetylglucosamine-6-phosphate to N-acetylglucosamine-1-phosphate.

[0280] 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.

[0281] One or more of the recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity can be under the control of the pTEF1 promoter.

[0282] The one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may be obtained or derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0283] 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.

[0284] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity can be inserted into the MET14 gene and / or the SAM3 gene of a recombinant cell, in particular a recombinant yeast, as shown in the Examples herein.

[0285] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast cell, - 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, - said recombinant nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase 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 the pTEF1 promoter; Includes.

[0286] Chondroitin synthase (HCOS) The enzyme chondroitin synthase is a protein described in the art for catalyzing the conversion of UDP-glucuronate and UDP-N-acetyl-galactosamine to chondroitin.

[0287] In certain embodiments, the nucleic acid encoding a polypeptide having chondroitin synthase (HCOS) activity is (i) a nucleic acid encoding a chondroitin synthase, or (ii) a nucleic acid encoding a chimeric polypeptide having chondroitin synthase activity It is.

[0288] Chimeric polypeptides, also known as fusion polypeptides, are polypeptides that consist of at least two domains or fragments, preferably from two different polypeptides, encoded by separate nucleic acids that are linked to be transcribed and translated as a single unit to produce a single polypeptide. Chimeric polypeptides can be produced in vivo or in vitro using recombinant DNA techniques. Such methods known in the art notably include DNA solid-phase synthesis, but also conventional molecular techniques involving the use of recombinant PCR, restriction enzymes, ligases, recombinases, techniques such as Gibson assembly (Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009). Nature Methods. 6 (5): 343-345), or the use of linear DNA recombination in vivo, preferably in yeast.

[0289] A chimeric polypeptide having chondroitin synthase activity suitable according to the present invention may be a hybrid protein, as known in the art, encoded by a nucleic acid comprising a nucleic acid encoding a polypeptide having hyaluronan synthase activity and a nucleic acid encoding a polypeptide having chondroitin synthase activity (the concept of chimeric chondroitin synthase is described in Tracy et al. (2006) Journal of biological chemistry, 282, 337-344 and Jing and DeAngelis (2003) Glycobiology 13, 661-71). The resulting polypeptide is a chimeric polypeptide of hyaluronan synthase and chondroitin synthase.

[0290] Another chimeric polypeptide with chondroitin synthase activity that is preferred according to the invention is encoded by a nucleic acid comprising a nucleic acid encoding a polypeptide with hyaluronan synthase activity and a nucleic acid encoding a polypeptide with galactofuranosyltransferase activity, the resulting polypeptide being a fusion protein of hyaluronan synthase and galactofuranosyltransferase (galactofuranosyltransferase GlfT2 Mycobacterium tuberculosis UNIPROT O53585 EC 2.1.4.288).

[0291] Another chimeric polypeptide with chondroitin synthase activity that is preferred according to the invention is encoded by a nucleic acid comprising a nucleic acid encoding a polypeptide with hyaluronan synthase activity and a nucleic acid encoding a polypeptide with chitin synthase activity, the resulting polypeptide being a fusion protein of hyaluronan synthase and chitin synthase (chitin synthase CHS2 Saccharomyces cerevisiae UNIPROT P14180 EC 2.1.4.16).

[0292] Examples of chimeric polypeptides having chondroitin synthase activity that are suitable according to the invention are - a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida (e.g. HCOS1-1, HCOS1-2 or HCOS1-3), - a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida, a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida, and a fragment derived from a nucleic acid encoding chitin synthase 2 from Saccharomyces cerevisiae (e.g. HCOS.Sc), - a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from a nucleic acid encoding chitin synthase 2 from Saccharomyces cerevisiae (e.g. HHASA.Sc), - a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from E. coli (e.g. HCOS1-Vir, HCOS2-Vir or HCOS4-Vir), - a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a galactofuranosyltransferase from Mycobacterium tuberculosis (e.g. HCOS3-Vir), - a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from Penicillium oxalicum (e.g. HCOS5-Vir, HCOS6-Vir, HCOS7-Vir or HCOS8-Vir), - a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin sulfate synthase originating from Homo sapiens (e.g., HCOS9-Vir, HCOS10-Vir, HCOS11-Vir, or HCOS12-Vir); It can be encoded by a nucleic acid sequence comprising:

[0293] Methods implemented for measuring the activity level of a polypeptide having chondroitin synthase activity belong to the general knowledge of the person skilled in the art.

[0294] In this regard, the skilled artisan can advantageously refer to a method in which the ability of the enzyme to produce chondroitin is evaluated by the presence of chondroitin in the supernatant assayed using the carbazole method (Bitter and Muir (1962) analytical biochemistry 4, 330-334).

[0295] Preferred polypeptides having chondroitin synthase activity herein are enzymes having the two activities EC 2.4.1.175 and 2.4.1.226.

[0296] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having chondroitin synthase activity can be obtained or 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 chondroitin synthase activity can be obtained or derived from an archaea. In some preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having chondroitin synthase activity can be obtained or derived from the group consisting of Pasteurella multocida (Pm), Chlorella virus PBCV1 (Vir), Mycobacterium tuberculosis (Mt), Homo sapiens (Hs), Escherichia coli (Ec), Saccharomyces cerevisiae (Sc), or Penicillium oxalicum (Po).

[0297] According to yet a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having chondroitin synthase activity have (i) a sequence identity at least 65%, advantageously at least, with the nucleic acid sequence as set forth in the sequence SEQ ID NO:1 (HCOS1-1), SEQ ID NO:2 (HCOS1-2), SEQ ID NO:3 (HCOS-1-3), SEQ ID NO:4 (HCOS.Sc), SEQ ID NO:5 (HHASA.Sc), SEQ ID NO:6 (HCOS1-Vir), SEQ ID NO:7 (HCOS2-Vir), SEQ ID NO:8 (HCOS3-Vir), SEQ ID NO:9 (HCOS4-Vir), SEQ ID NO:10 (HCOS5-Vir), SEQ ID NO:11 (HCOS6-Vir), SEQ ID NO:12 (HCOS7-Vir), SEQ ID NO:13 (HCOS8-Vir), SEQ ID NO:14 (HCOS9-Vir), SEQ ID NO:15 (HCOS10-Vir), SEQ ID NO:16 (HCOS11-Vir) or SEQ ID NO:17 (HCOS12-Vir). and (ii) having the same qualitative biological activity as a nucleic acid sequence having the nucleic acid sequence set forth as the sequence of SEQ ID NO:1 (HCOS1-1), SEQ ID NO:2 (HCOS1-2), SEQ ID NO:3 (HCOS-1-3), SEQ ID NO:4 (HCOS.Sc), SEQ ID NO:5 (HHASA.Sc), SEQ ID NO:6 (HCOS1-Vir), SEQ ID NO:7 (HCOS2-Vir), SEQ ID NO:8 (HCOS3-Vir), SEQ ID NO:9 (HCOS4-Vir), SEQ ID NO:10 (HCOS5-Vir), SEQ ID NO:11 (HCOS6-Vir), SEQ ID NO:12 (HCOS7-Vir), SEQ ID NO:13 (HCOS8-Vir), SEQ ID NO:14 (HCOS9-Vir), SEQ ID NO:15 (HCOS10-Vir), SEQ ID NO:16 (HCOS11-Vir), or SEQ ID NO:17 (HCOS12-Vir), respectively.

[0298] A similar biological activity for this sequence is the ability to encode a polypeptide that converts UDP-glucuronate and UDP-N-acetyl-galactosamine to chondroitin, as previously described.

[0299] 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.

[0300] 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.

[0301] 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.

[0302] For the amino acid sequence of a chimeric polypeptide having chondroitin synthase activity, which contains amino acids including a fragment derived from an amino acid encoding hyaluronan synthase originating from Pasteurella multocida, and a fragment derived from an amino acid encoding chondroitin synthase derived from Pasteurella multocida, a person skilled in the art can refer to the sequences described in the UniProt database accession numbers Q7BLV3 and Q9CMP0, respectively, or SEQ ID NO: 18 (HCOS1-1), SEQ ID NO: 19 (HCOS1-2), and SEQ ID NO: 20 (HCOS-1-3) described herein.

[0303] For the amino acid sequences of chimeric polypeptides having chondroitin synthase activity, including a fragment derived from the amino acid encoding hyaluronan synthase originating from Pasteurella multocida, a fragment derived from the amino acid encoding chondroitin synthase derived from Pasteurella multocida, and a fragment derived from the amino acid encoding chitin synthase 2 derived from Saccharomyces cerevisiae, those skilled in the art can refer to the sequences set forth in the accession numbers Q7BLV3, Q9CMP0, and P14180 of the UniProt database, respectively, or SEQ ID NO: 21 (HCOS.Sc) described herein.

[0304] For the amino acid sequence of a chimeric polypeptide having chondroitin synthase activity, which comprises a fragment derived from the amino acid sequence encoding hyaluronan synthase originating from Pasteurella multocida and a fragment derived from the amino acid sequence encoding chitin synthase 2 derived from Saccharomyces cerevisiae, a person skilled in the art can refer to the sequence set forth in the UniProt database accession numbers Q7BLV3 and P14180, respectively, or SEQ ID NO: 22 (HHASA.Sc) described herein.

[0305] For the amino acid sequence of a chimeric polypeptide having chondroitin synthase activity, including a fragment derived from the amino acid encoding hyaluronan synthase originating from Chlorella virus PBCV-1 and a fragment derived from the amino acid encoding chondroitin synthase derived from Escherichia coli, a person skilled in the art can refer to the sequences described in the UniProt database accession numbers Q84419 and Q8L0V4, respectively, or SEQ ID NO: 23 (HCOS1-Vir), SEQ ID NO: 24 (HCOS2-Vir), or SEQ ID NO: 26 (HCOS4-Vir) described herein.

[0306] For the amino acid sequence of a chimeric polypeptide having chondroitin synthase activity, which comprises a fragment derived from the amino acid encoding hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from the amino acid encoding galactofuranosyltransferase derived from Mycobacterium tuberculosis, a person skilled in the art can refer to the sequence set forth in the UniProt database accession numbers Q84419 and O53585, respectively, or SEQ ID NO: 25 (HCOS3-Vir) described herein.

[0307] For the amino acid sequence of a chimeric polypeptide having chondroitin synthase activity, which comprises a fragment derived from the amino acid encoding hyaluronan synthase originating from Chlorella virus PBCV-1 and a fragment derived from the amino acid encoding chondroitin synthase derived from Penicillium oxalicum, a person skilled in the art can refer to the sequence set forth in the UniProt database accession numbers Q84419 and S7Z8F8, respectively, or SEQ ID NO: 27 (HCOS5-Vir) described herein.

[0308] For the amino acid sequence of a chimeric polypeptide having chondroitin synthase activity, which comprises a fragment derived from the amino acid encoding hyaluronan synthase originating from Chlorella virus PBCV-1 and a fragment derived from the amino acid encoding chondroitin synthase derived from Pasteurella multocida, a person skilled in the art can refer to the sequences described in the UniProt database accession numbers Q84419 and Q9CMP0, respectively, or SEQ ID NO: 28 (HCOS6-Vir), SEQ ID NO: 29 (HCOS7-Vir), or SEQ ID NO: 30 (HCOS8-Vir) described herein.

[0309] For the amino acid sequence of a chimeric polypeptide having chondroitin synthase activity, comprising a fragment derived from the amino acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from the amino acid encoding a chondroitin sulfate synthase derived from Homo sapiens, a person skilled in the art can refer to the sequences set forth in the UniProt database accession numbers Q84419 and Q86X52, respectively, or SEQ ID NO: 31 (HCOS9-Vir), SEQ ID NO: 32 (HCOS10-Vir), SEQ ID NO: 33 (HCOS11-Vir), or SEQ ID NO: 34 (HCOS12-Vir) described herein.

[0310] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having chondroitin synthase activity are selected from the group consisting of SEQ ID NO:18 (HCOS1-1), SEQ ID NO:19 (HCOS1-2), SEQ ID NO:20 (HCOS-1-3), SEQ ID NO:21 (HCOS.Sc), SEQ ID NO:22 (HHASA.Sc), SEQ ID NO:23 (HCOS1-Vir), SEQ ID NO:24 (HCOS2-Vir), SEQ ID NO:25 (HCOS3-Vir), SEQ ID NO:26 (HCOS4-Vir), SEQ ID NO:27 (HCOS5-Vir), SEQ ID NO:28 (HCOS6-Vir), SEQ ID NO:29 (HCOS7-Vir), SEQ ID NO:30 (HCOS8-Vir), SEQ ID NO:31 (HCOS9-Vir), SEQ ID NO:32 (HCOS10-Vir), SEQ ID NO:33 (HCOS11-Vir), SEQ ID NO:34 (HCOS12-Vir), SEQ ID NO:35 (HCOS13-Vir), SEQ ID NO:36 (HCOS14-Vir), SEQ ID NO:37 (HCOS15-Vir), SEQ ID NO:38 (HCOS16-Vir), SEQ ID NO:39 (HCOS17-Vir), SEQ ID NO:40 (HCOS18-Vir), SEQ ID NO:41 (HCOS19-Vir), SEQ ID NO:42 (HCOS20-Vir), SEQ ID NO:43 (HCOS21-Vir), SEQ ID NO:44 (HCOS22-Vir), SEQ ID NO:45 (HCOS23-Vir), SEQ ID NO:46 (HCOS24-Vir), SEQ ID NO:47 (HCOS25-Vir), SEQ ID NO:48 (HCOS26-Vir), The nucleic acid 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: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 (HCOS8-Vir), SEQ ID NO: 31 (HCOS9-Vir), SEQ ID NO: 32 (HCOS10-Vir), SEQ ID NO: 33 (HCOS11-Vir) and SEQ ID NO: 34 (HCOS12-Vir) and also having the same qualitative biological activity.

[0311] The same qualitative biological activity for this sequence is as previously described, namely the ability to catalyze the conversion of UDP-glucuronate and UDP-N-acetyl-galactosamine to UDP-N-acetyl-galactosamine.

[0312] 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.

[0313] 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.

[0314] 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.

[0315] As described above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having chondroitin 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 chondroitin synthase activity, respectively.

[0316] 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.

[0317] 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.

[0318] In this respect, the skilled person may advantageously refer to the method described by Oka and Jigami (FEBS Journal 273, 2645-2657, 2006).

[0319] A preferred polypeptide having UDP-glucose dehydrogenase activity herein is the enzyme having the EC number 1.1.1.22.

[0320] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity can be obtained or 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 can be obtained or derived from an archaea. In some preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity can be obtained or derived from a yeast, in particular Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, preferably Arabidopsis thaliana or Chlorella virus PBCV1.

[0321] According to yet a preferred embodiment, 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 sequences set forth as sequences SEQ ID NO: 35 (At), SEQ ID NO: 36 (Vir), SEQ ID NO: 37 (Vir) and SEQ ID NO: 38 (Sz), and (ii) biological activity of the same nature as those set forth as sequences SEQ ID NO: 35 (At), SEQ ID NO: 36 (Vir), SEQ ID NO: 37 (Vir) and SEQ ID NO: 38 (Sz). The nucleic acid sequences set forth as SEQ ID NO:35 (At), SEQ ID NO:36 (Vir), SEQ ID NO:37 (Vir), and SEQ ID NO:38 (Sz) respectively encode polypeptides having UDP-glucose dehydrogenase activity obtained or derived from Arabidopsis thaliana (At), Chlorella virus PBCV1 (Vir), or Streptococcus zooepidemicus (Sz), which may collectively be referred to herein as HASB.

[0322] A similar biological activity for this sequence is the ability to encode a polypeptide that converts UDP-glucose to UDP-glucuronate, as previously explained.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] 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 SEQ ID NO: 39 (At), SEQ ID NO: 40 (Vir), and SEQ ID NO: 41 (Sz) described herein.

[0327] According to another particular embodiment, the nucleic acid encoding a UDP-glucose dehydrogenase may be a nucleic acid encoding 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: 39 (At), SEQ ID NO: 40 (Cv) and SEQ ID NO: 41 (Sz) and also having the same qualitative biological activity and the same qualitative biological activity as the amino acid sequences of SEQ ID NO: 39 (At), SEQ ID NO: 40 (Vir) and SEQ ID NO: 41 (Sz).

[0328] 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.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] As mentioned above, the expression level of a polypeptide having UDP-glucose dehydrogenase activity according to the invention is regulated by at least one promoter and at least one terminator, e.g. as defined in more detail hereinafter, which are present at the 5' and 3' positions, respectively, of the nucleic acid encoding said polypeptide having UDP-glucose dehydrogenase activity.

[0333] UDP-glucose-4-epimerase (kfoA or GNE1) UDP-glucose-4-epimerase is a protein known in the art to catalyze the conversion of UDP-N-acetyl-glucosamine to UDP-N-acetyl-galactosamine. UDP-glucose-4-epimerase obtained or derived from Pseudomonas aeruginosa, Pasteurella multocida, or Escherichia coli may be referred to as kfoA or GNE1.

[0334] Methods implemented for measuring the activity level of a polypeptide having UDP-glucose-4-epimerase activity belong to the general knowledge of a person skilled in the art.

[0335] In this respect, the skilled person may advantageously refer to the method described in Creuzenet et al. (2000, Journal of Biological Chemistry 275, 19060-67).

[0336] A preferred polypeptide having UDP-glucose-4-epimerase activity herein is the enzyme having the EC number 5.1.3.7.

[0337] According to preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase activity can be obtained or 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-glucose-4-epimerase activity can be obtained or derived from an archaebacteria. In some preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase activity can be obtained or derived from Pseudomonas aeruginosa, Pasteurella multocida (Pm), or Escherichia coli (Ec).

[0338] According to yet a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase 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 sequences set forth in the sequences SEQ ID NO: 42 (Pa), SEQ ID NO: 43 (Pm) and SEQ ID NO: 44 (Ec), and (ii) the same qualitative biological activity as those set forth in the sequences SEQ ID NO: 42 (Pa), SEQ ID NO: 43 (Pm) and SEQ ID NO: 44 (Ec).

[0339] A similar biological activity for this sequence is the ability to encode a polypeptide which converts UDP-N-acetyl-glucosamine to UDP-N-acetyl-galactosamine, as previously explained.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] For the amino acid sequences of polypeptides having UDP-glucose-4-epimerase activity derived from Pseudomonas aeruginosa, Pasteurella multocida, and Escherichia coli, those skilled in the art can refer to the sequences set forth as accession numbers Q8KN66, AAK02370, and Q8L0V2 in the UniProt database, respectively, or SEQ ID NO: 45 (Pa), SEQ ID NO: 46 (Pm), and SEQ ID NO: 47 (Ec) described herein.

[0344] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase 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 sequence set forth as SEQ ID NO: 45 (Pa), SEQ ID NO: 46 (Pm) or SEQ ID NO: 47 (Ec) and also having the same qualitative biological activity as the amino acid sequence of SEQ ID NO: 45 (Pa), SEQ ID NO: 46 (Pm) or SEQ ID NO: 47 (Ec).

[0345] The same qualitative biological activity for this sequence is as previously described, namely the ability to catalyze the conversion of UDP-N-acetyl-glucosamine to UDP-N-acetyl-galactosamine.

[0346] 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.

[0347] 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.

[0348] 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.

[0349] As mentioned above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase 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-4-epimerase activity, respectively.

[0350] Chondroitinase (HYAL) Chondroitinase enzymes are proteins described in the art for catalyzing the breakdown of chondroitin molecules into smaller chondroitin molecules. As described above, a polypeptide having chondroitinase activity can be a hyaluronoglucosaminidase, e.g., chondroitinase or hyaluronidase.

[0351] In certain embodiments, the polypeptide having chondroitinase activity is a hyaluronidase.

[0352] A polypeptide having chondroitinase activity may be encoded by the genome of Cupienius salei, Titius serlatus, Bos taurus, Apis mellifera, Macaca mulatta, or Vespa magnifica and may be referred to as HYAL.

[0353] A polypeptide having chondroitinase activity 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. When an encoded polypeptide having chondroitinase activity has both a secretion signal and an anchoring signal, it may be referred to as HYAL-31.

[0354] When the encoded polypeptide having chondroitinase activity has a secretion signal but no anchoring signal, it may be referred to as HYAL-3.

[0355] Methods implemented for measuring the activity level of a polypeptide having chondroitinase activity belong to the general knowledge of the person skilled in the art.

[0356] In this regard, the skilled person can advantageously monitor the molecular weight of the resulting chondroitin on an agarose gel.

[0357] A preferred polypeptide having chondroitinase activity herein is an enzyme having the EC number n° EC 3.2.1.35.

[0358] According to preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity can be obtained or 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 chondroitinase activity can be obtained or derived from an Archaea. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity can be obtained or derived from an organism, preferably selected from yeast. In some other preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity can be obtained or derived from Cupienius salei (Csa), Titius serlatus (Ts), Bos taurus (Bt), Apis mellifera (Am), Macaca mulatta (Mm), or Vespa magnifica (Vm), in particular Titius serlatus (Ts).

[0359] According to yet a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity (i) have at least 65%, advantageously at least 70%, preferably at least 80% nucleic acid identity with a nucleic acid of SEQ ID NO: 48 (Csa), SEQ ID NO: 49 (Ts), SEQ ID NO: 50 (Bt), SEQ ID NO: 51 (Am), SEQ ID NO: 52 (Mm) or SEQ ID NO: 53 (Vm), (ii) encode a polypeptide having chondroitinase activity and a secretion signal but not including an anchoring signal, and (iii) obtained or derived from Cupienius salei, Titius serlatus, Bos taurus, Apis mellifera, Macaca mulatta, or Vespa magnifica, respectively; and (iv) having the same qualitative biological activity as the sequence of SEQ ID NO: 48 (Csa), SEQ ID NO: 49 (Ts), SEQ ID NO: 50 (Bt), SEQ ID NO: 51 (Am), SEQ ID NO: 52 (Mm), or SEQ ID NO: 53 (Vm).

[0360] According to another preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having chondroitinase 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: 54 (Csa), SEQ ID NO: 55 (Ts), SEQ ID NO: 56 (Bt), SEQ ID NO: 57 (Am), SEQ ID NO: 58 (Mm) or SEQ ID NO: 59 (Vm); (ii) encoding a polypeptide having chondroitinase activity and comprising a secretion signal and an anchoring signal, and being obtained or derived from Cupienius salei, Titius serlatus, Bos taurus, Apis mellifera, Macaca mulatta or Vespa magnifica, respectively; and (iii) having the same qualitative biological activity as the sequence of SEQ ID NO: 54 (Csa), SEQ ID NO: 55 (Ts), SEQ ID NO: 56 (Bt), SEQ ID NO: 57 (Am), SEQ ID NO: 58 (Mm) or SEQ ID NO: 59 (Vm).

[0361] A similar biological activity for this sequence is the ability to encode a polypeptide that catalyzes the breakdown of chondroitin molecules into smaller chondroitin molecules, as previously explained.

[0362] 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.

[0363] 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.

[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 hyaluronidase from Cupienius salei, Titius serlatus, Bos taurus, Apis mellifera, Macaca mulatta, or Vespa magnifica, a person skilled in the art can refer to the UniProt database accession numbers A0A0S4JYH2, P85841, Q7YS45, Q08169, G7ML68, or P86875, respectively, or SEQ ID NO: 60 (Csa), SEQ ID NO: 61 (Ts), SEQ ID NO: 62 (Bt), SEQ ID NO: 63 (Am), SEQ ID NO: 64 (Mm), or SEQ ID NO: 65 (Vm) described herein.

[0366] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having chondroitinase 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 sequence of SEQ ID NO: 60 (Csa), SEQ ID NO: 61 (Ts), SEQ ID NO: 62 (Bt), SEQ ID NO: 63 (Am), SEQ ID NO: 64 (Mm) or SEQ ID NO: 65 (Vm), including a secretion signal and not including an anchoring signal, and also having the same qualitative biological activity as the sequence of SEQ ID NO: 60 (Csa), SEQ ID NO: 61 (Ts), SEQ ID NO: 62 (Bt), SEQ ID NO: 63 (Am), SEQ ID NO: 64 (Mm) or SEQ ID NO: 65 (Vm).

[0367] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having chondroitinase 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 sequence of SEQ ID NO: 66 (Csa), SEQ ID NO: 67 (Ts), SEQ ID NO: 68 (Bt), SEQ ID NO: 69 (Am), SEQ ID NO: 70 (Mm) or SEQ ID NO: 71 (Vm), 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: 66 (Csa), SEQ ID NO: 67 (Ts), SEQ ID NO: 68 (Bt), SEQ ID NO: 69 (Am), SEQ ID NO: 70 (Mm) or SEQ ID NO: 71 (Vm).

[0368] The biological activity of the same nature for this sequence is as previously described, namely the ability to catalyze the breakdown of chondroitin molecules into smaller chondroitin molecules.

[0369] 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.

[0370] 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.

[0371] 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.

[0372] As described above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having chondroitinase 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 chondroitinase activity, respectively.

[0373] 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 may be referred to as GFA1.

[0374] 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.

[0375] 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).

[0376] 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.

[0377] 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.

[0378] 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: 72(Sc), and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as sequence SEQ ID NO: 72. The nucleic acid set forth as sequence SEQ ID NO: 72 encodes a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity obtained or derived from Saccharomyces cerevisiae, which may also be referred to as GFA1.

[0379] 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: 73 or SEQ ID NO: 74, and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as sequence SEQ ID NO: 73 or SEQ ID NO: 74. The nucleic acid sequence set forth as sequence SEQ ID NO: 73 or SEQ ID NO: 74 encodes a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity originating from the Chlorella virus PBCV1.

[0380] 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.

[0381] 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.

[0382] 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.

[0383] 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.

[0384] 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: 75 described in this specification.

[0385] 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: 76 described in this specification.

[0386] 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: 75 or SEQ ID NO: 76 and also having the same qualitative biological activity as the amino acid sequence of SEQ ID NO: 75 or SEQ ID NO: 76.

[0387] 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.

[0388] 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%, 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%, 102%, 109%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ... These include amino acid sequences that have 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 and also have the same qualitative biological activity as the reference amino acid sequence.

[0389] 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.

[0390] 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.

[0391] 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.

[0392] 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.

[0393] 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.

[0394] 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.

[0395] 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.

[0396] 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.

[0397] 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: 77, and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as SEQ ID NO: 77. The nucleic acid sequence set forth as SEQ ID NO: 77 encodes a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity originating from Saccharomyces cerevisiae, which may also be referred to as QRI1.

[0398] 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.

[0399] 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.

[0400] 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.

[0401] 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.

[0402] 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: 78 described in this specification.

[0403] 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: 78 and also having the same qualitative biological activity as the amino acid sequence set forth as sequence SEQ ID NO: 78.

[0404] 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-acetylglucose.

[0405] 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%, 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%, 102%, 109%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ... These include amino acid sequences that have 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 and also have the same qualitative biological activity as the reference amino acid sequence.

[0406] 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.

[0407] 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.

[0408] 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.

[0409] 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.

[0410] Methods implemented for measuring the activity level of a polypeptide having phosphoglucomutase-1 activity belong to the general knowledge of the person skilled in the art.

[0411] 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).

[0412] A preferred polypeptide having phosphoglucomutase-1 activity according to the invention is the enzyme having the EC number n° 5.4.2.2.

[0413] 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.

[0414] 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:79; and (ii) a nucleic acid sequence having the same qualitative biological activity as the nucleic acid sequence set forth as sequence SEQ ID NO:79.

[0415] 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.

[0416] 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.

[0417] 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: 80 described herein.

[0418] 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: 80 and also having the same qualitative biological activity as the amino acid sequence set forth as sequence SEQ ID NO: 80.

[0419] 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.

[0420] 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.

[0421] 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.

[0422] 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.

[0423] 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.

[0424] In this respect, the skilled person can advantageously refer to the method described by Roeben (J. Mol. Biol 364, 551-560, 2006).

[0425] 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.

[0426] 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.

[0427] 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 in SEQ ID NO:81 originating from Saccharomyces cerevisiae, and (ii) the same qualitative biological activity as the nucleic acid set forth in SEQ ID NO:81.

[0428] 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.

[0429] 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.

[0430] 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 82 described herein.

[0431] 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:82 and also having the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO:82.

[0432] 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.

[0433] 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.

[0434] 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.

[0435] 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.

[0436] 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.

[0437] In this regard, the skilled person may advantageously refer to the method described by Li et al. (Anal. Biochem. 370, 142-146, 2007).

[0438] 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.

[0439] 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.

[0440] 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: 83, and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as sequence SEQ ID NO: 83. The nucleic acid sequence set forth as sequence SEQ ID NO: 83 encodes a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity originating from Saccharomyces cerevisiae, which may also be referred to as GNA1.

[0441] 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.

[0442] 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.

[0443] For the amino acid sequence of a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity obtained or derived from Saccharomyces cerevisiae, the skilled person can refer to the UniProt database accession number NP_116637 or to SEQ ID NO: 84 described herein.

[0444] 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:84 and also having the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO:84.

[0445] 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.

[0446] 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.

[0447] 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.

[0448] 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.

[0449] 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.

[0450] In this regard, the skilled person can advantageously refer to the method described by Bandini et al. (Molecular Microbiology 85(3), 513-534, 2012).

[0451] A preferred polypeptide having phosphoacetylglucosamine mutase activity according to the invention is the enzyme having the EC number n° 5.4.2.3.

[0452] 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.

[0453] 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) a nucleic acid sequence having at least 80% nucleic acid identity with the nucleic acid sequence set forth as SEQ ID NO: 85, and (ii) a nucleic acid sequence having the same qualitative biological activity as the nucleic acid sequence set forth as SEQ ID NO: 85. The nucleic acid set forth as SEQ ID NO: 85 encodes a polypeptide having phosphoacetylglucosamine mutase activity originating from Saccharomyces, which may also be referred to as PCM1.

[0454] 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.

[0455] 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.

[0456] For the amino acid sequence of a polypeptide having phosphoacetylglucosamine mutase activity originating from Saccharomyces cerevisiae, those skilled in the art can refer to the UniProt database accession number NP_010856 or SEQ ID NO: 86 described in this specification.

[0457] 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:86 and also having the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO:86.

[0458] 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.

[0459] 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.

[0460] 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.

[0461] 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.

[0462] A variety of promoters may be used to direct the desired expression of the coding sequence of interest.

[0463] The promoter according to the present invention is the following promoter: pTDH3 (SEQ ID NO: 87), pTDH3.sk (SEQ ID NO: 88), pTDH3-1.Sba (SEQ ID NO: 89), pTDH3.Sar (SEQ ID NO: 90), pENO2 (SEQ ID NO: 91), pTEF3 (SEQ ID NO: 92), pTEF1 (SEQ ID NO: 93), pTEF1.ago (SEQ ID NO: 94), pTEF1.sba (SEQ ID NO: 95), pPDC1 (SEQ ID NO: 96), pCCW12 (SEQ ID NO: 97), pCCW12.Sm (SEQ ID NO: 98), pCCW12.sk (SEQ ID NO: 99), pCCW12.sba (SEQ ID NO: 100), pCCW12.sar (SEQ ID NO: 101), pNUP57 (SEQ ID NO: 102), pCCW10.ago (SEQ ID NO: 103), pCWP2 (SEQ ID NO: 104), pFBA1 (SEQ ID NO: 105), pCCW120.Sm (SEQ ID NO: 106) may be selected from the group consisting of:

[0464] Promoters of more particular interest in the present invention are pTDH3 (SEQ ID NO: 87), pTDH3.sk (SEQ ID NO: 88), pTDH3-1.Sba (SEQ ID NO: 89), pTDH3.Sar (SEQ ID NO: 90), pENO2 (SEQ ID NO: 91), pTEF3 (SEQ ID NO: 92), pTEF1 (SEQ ID NO: 93), pTEF1.ago (SEQ ID NO: 94), pTEF1.sba (SEQ ID NO: 95), pPDC1 (SEQ ID NO: 96), pCCW12 (SEQ ID NO: 97), pCCW12.sba (SEQ ID NO: 100), pCCW120.Sm (SEQ ID NO: 106), pNUP57 (SEQ ID NO: 102), pCCW10.ago (SEQ ID NO: 103), pCWP2 (SEQ ID NO: 104), and pFBA1 (SEQ ID NO: 105) may be selected from the group consisting of:

[0465] The promoter may in particular be selected from the group consisting of pTDH3, pTDH3.Sk, pTDH3-1.Sba, pTEF1, pTEF1.Ago, pTEF1.sba, pCCW12, pCCW120.Sm, pCWP2, and pFBA1.

[0466] Alternatively, the promoter of interest in the present invention is pNUP57 (SEQ ID NO: 102), and pCCW10.ago (SEQ ID NO: 103) may be selected from the group consisting of:

[0467] Another promoter of interest in the present invention is pCWP2 (SEQ ID NO: 104) It could be.

[0468] 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.

[0469] For example, increasing the amount of copper in the culture medium of a recombinant yeast cell according to the invention containing the pCUP1 promoter will induce and thus increase the transcription of the genes under the control of this promoter. In contrast, decreasing the amount of copper in the culture medium will cause the repression and thus the reduction of the transcription of the genes under the control of this promoter.

[0470] 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.

[0471] For this reason, the following promoters are referred to herein as inducible or repressible promoters.

[0472] 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 copper-inducible or repressible CUP-1.

[0473] According to this embodiment, the inducible or repressible promoter according to the invention is in particular pCUP1 (SEQ ID NO: 107).

[0474] 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.

[0475] 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: 108), pMET25-methionine inducible or repressible (SEQ ID NO: 109), and pSAM1-methionine-inducible or repressible (SEQ ID NO: 110) may be selected from the group consisting of:

[0476] According to this particular embodiment, the inducible or repressible promoter according to the invention may be selected from the group consisting of pMET6 and pSAM1.

[0477] 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.

[0478] 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.

[0479] Synthetic promoters as described in Blazeck & Alper (2013) Biotechnol. J. 8 46-58 can also be used.

[0480] 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:

[0481] 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), Saccharomyces arboricola (Sar), and Ashbya gossypii (Ago).

[0482] 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.

[0483] The same or different transcription terminators can be found in the publication Yamanishi et al. (2013) ACS synthetic biology 2, 337-347.

[0484] Terminators of more particular interest in the present invention are tTPI1 (SEQ ID NO: 111), derived from the gene encoding triosephosphate isomerase, tMET25 (SEQ ID NO: 112), derived from the gene encoding O-acetylhomoserine-O-acetylserine sulfhydrylase, tDIT1 (SEQ ID NO: 113), tRPL3 (SEQ ID NO: 114), tRPL3.sm (SEQ ID NO: 115), tRPL3.sba (SEQ ID NO: 116), tRPL41B (SEQ ID NO: 117), tRPL41B.Sba (SEQ ID NO: 118), tRPL15A (SEQ ID NO: 119), tRPL15A.Sm (SEQ ID NO: 120), tRPL15A.sba (SEQ ID NO: 121), tIDP1 (SEQ ID NO: 122), tIDP1.Sba (SEQ ID NO: 123), and tTEF1.sba (SEQ ID NO: 124) may be selected from the group comprising:

[0485] Terminators of more particular interest in the present invention are tTPI1 (SEQ ID NO: 111), derived from the gene encoding triosephosphate isomerase, tMET25 (SEQ ID NO: 112), derived from the gene encoding O-acetylhomoserine-O-acetylserine sulfhydrylase, tDIT1 (SEQ ID NO: 113) tRPL3 (SEQ ID NO: 114) tRPL3.Sm (SEQ ID NO: 115) tRPL3.sba (SEQ ID NO: 116) tRPL41B (SEQ ID NO: 117) tRPL41B.Sba (SEQ ID NO: 118) tRPL15A (SEQ ID NO: 119) tRPL15A.Sm (SEQ ID NO: 120) tIDP1 (SEQ ID NO: 122) tIDP1.Sba (SEQ ID NO: 123), and tTEF1.sba (SEQ ID NO: 124) may be selected from the group consisting of:

[0486] In particular, the terminator may be selected from the group consisting of tTPI1, tDIT11, tRPL3, tRPL3.sm, tRPL41B, tRPL41B.Sba, tRPL15A, tRPL15A.Sm, tIDP1, tIDP1.Sba, and tTEF1.sba.

[0487] The terminator of the present invention may originate from any organism from the class Hemiascomycota, in particular from an organism selected from the group consisting of Saccharomyces cerevisiae (Sc), Saccharomyces mikatae (Sm) and Saccharomyces bayanus (sba).

[0488] Recombinant cells The recombinant cell of the present invention may be selected from the group consisting of yeast and bacteria.

[0489] A recombinant cell of the invention, eg, a recombinant host cell of the invention, is preferably a recombinant yeast cell.

[0490] 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.

[0491] A recombinant cell of the invention, in particular a recombinant yeast cell of the invention, is preferably a Saccharomycetales cell.

[0492] 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.

[0493] 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.

[0494] 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.

[0495] 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.

[0496] 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.

[0497] 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.

[0498] 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).

[0499] 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.

[0500] 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.

[0501] 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.

[0502] 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.

[0503] The recombinant cell of the present invention may more preferably be a Saccharomyces cerevisiae cell.

[0504] As described above, the recombinant cell according to the present invention has the ability to produce chondroitin by inserting one or more recombinant nucleic acids according to the present invention. In a particular embodiment, the recombinant yeast according to the present invention has the ability to produce chondroitin with a controlled size (controlled molecular weight) by inserting one or more recombinant nucleic acids according to the present invention.

[0505] 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.

[0506] However, unexpected technical problems were encountered because the outcome of inserting a DNA construct into the genome of a cell, particularly a yeast genome, such as the genome of Saccharomyces cerevisiae, is unpredictable. In particular, the viability of cells, particularly yeast, and their ability to grow and produce the desired chondroitin are also unpredictable.

[0507] In order to obtain the recombinant cells, in particular recombinant yeast, of the present invention, a large number of different constructs have been tested by the inventors in order to obtain viable and efficient recombinant cells, in particular yeast.

[0508] Culture conditions The present invention also relates to the use of the recombinant cells of the invention for the production of chondroitin, in particular chondroitin of controlled molecular weight.

[0509] The present invention further provides a method for producing chondroitin of a desired molecular weight, comprising the steps of: (a) culturing a recombinant cell as defined herein in a culture medium for a period of time sufficient to produce chondroitin of a desired molecular weight; (b) optionally isolating or recovering the chondroitin from the recombinant cells and / or the culture medium. Typically, the cells of the invention, in particular the yeast 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 to 34°C.

[0510] 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.

[0511] 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.

[0512] 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.

[0513] 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.

[0514] 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.

[0515] 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.

[0516] The culture medium of the present invention may further contain rare elements, such as CuSO4·5H2O, KI, FeCl3, ZnSO4·7H2O, MnSO4·H2O, or H2SO4, MgCl2, CaCl2, NaCl, K2HPO4, KH2PO4, ZnCl, H3BO3, MnSO4, Na2MoO4.

[0517] The term "suitable culture medium" is defined above.

[0518] 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).

[0519] 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.

[0520] As described elsewhere herein, the pH value of the culture medium can be adjusted during the culture step of the method of the present invention to modulate the activity of the polypeptide having chondroitin activity, which will affect the molecular weight of the chondroitin molecule produced by the recombinant cell of the present invention, in particular the recombinant yeast of the present invention.

[0521] Specifically, the pH of the culture medium may be modified depending on the chondroitin 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.

[0522] 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.

[0523] 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).

[0524] 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.

[0525] Said time period for culturing 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 chondroitin of interest: the longer said time period, the lower the molecular weight of the chondroitin in a given culture medium of the recombinant cell of the invention, in particular the recombinant yeast cell of the invention.

[0526] The length of time for culturing the recombinant cells of the present invention, particularly the recombinant yeast cells 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.

[0527] Fermentation can be carried out under aerobic or microaerobic conditions.

[0528] The amount of chondroitin 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).

[0529] 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.

[0530] Fed-batch systems can also be used in the present invention. Fed-batch systems are similar to typical batch systems, except that the carbon source substrate is added incrementally as the fermentation progresses. Fed-batch systems are useful when catabolite repression (e.g., glucose repression) tends to inhibit the metabolism of cells and when it is desirable to limit the amount of substrate in the medium. The actual substrate concentration in a fed-batch system is difficult to measure and is therefore estimated based on changes in measurable factors such as pH, dissolved oxygen, and partial pressure of waste gases such as CO2.

[0531] 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.

[0532] 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.

[0533] 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.

[0534] 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.

[0535] To further improve chondroitin production, a particular embodiment may consist in culturing the recombinant cells of the invention, in particular the recombinant yeast cells of the invention, in a suitable culture medium, such as those described above, wherein said culture medium contains an optimal amount of a carbon source, in particular glucose or sucrose.

[0536] 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.

[0537] In a preferred embodiment, the process of the invention is carried out on an industrial scale.

[0538] More specifically, the culture medium of the method according to the present invention can be at least about 100 L, more preferably in the range of about 1,000 L to about 3,000 L, even more preferably about 10,000 L, even more preferably 100,000 L, or even about 250,000 L.

[0539] The present invention further provides a method for producing the aforementioned chondroitin, comprising: (a) culturing a recombinant cell of the present invention in a culture medium; (b) recovering chondroitin from the culture medium; Including, The chondroitin recovered in step (b) is - the nature and origin of the recombinant nucleic acid(s) encoding a polypeptide having chondroitinase activity of the recombinant cell of the invention, in particular of the recombinant yeast 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 chondroitin 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 chondroitin 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.

[0540] The molecular weight of chondroitin is a specific molecular weight, or more preferably, a specific 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.

[0541] The present invention also relates to the use of the recombinant cell according to the present invention, in particular the recombinant yeast of the present invention, for producing chondroitin having a molecular weight in the range of about 20 kDa to about 50 kDa or about 50 kDa to about 1000 kDa.

[0542] The one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity of the recombinant cell of the present invention may be selected from, for example, those obtained or derived from at least one of Cupienius salei, Titius serlatus, Bos taurus, Apis mellifera, Macaca mulatta, or Vespa magnifica, in particular, when it is secreted (i.e., in the presence of a secretion signal and in the absence of an anchoring signal), from the sequence set forth as SEQ ID NO: 48 (Csa), SEQ ID NO: 49 (Ts), SEQ ID NO: 50 (Bt), SEQ ID NO: 51 (Am), SEQ ID NO: 52 (Mm), or SEQ ID NO: 53 (Vm).

[0543] The one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity of the recombinant cell of the present invention may be selected from, for example, those obtained or derived from at least one of Cupienius salei, Titius serlatus, Bos taurus, Apis mellifera, Macaca mulatta, or Vespa magnifica, particularly those of the sequence set forth as SEQ ID NO: 54 (Csa), SEQ ID NO: 55 (Ts), SEQ ID NO: 56 (Bt), SEQ ID NO: 57 (Am), SEQ ID NO: 58 (Mm), or SEQ ID NO: 59 (Vm), when it is anchored (i.e., in the presence of both a secretion signal and an anchoring signal).

[0544] The one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity of the recombinant cell of the present invention may be under the control of a promoter selected from the group consisting of pTDH3, pTDH3.Sk, pTDH3-1.Sba, pTEF1, pTEF1.Ago, pTEF1.sba, pCCW12, pCCW12.Sba, pCCW120.Sm, pCWP2, pCW10.Ago, pNUP57, and pFBA1, in particular, the group consisting of pCCW12.Sba, pCW10.Ago, and pNUP57.

[0545] Specifically, the promoter of one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity of a recombinant cell of the invention, particularly a recombinant yeast of the invention, can be obtained or derived from Saccharomyces bayanus, Saccharomyces kudriabzevii, Saccharomyces mikatae, Saccharomyces arboricola, or other Saccharomycetales, or Abishia gossypii.

[0546] The secretion signal of the present invention is, for example, - the nucleic acid sequence set forth as SEQ ID NO: 125, and / or - the amino acid sequence set forth as SEQ ID NO: 126 may have:

[0547] The anchoring signal of the present invention is, for example, - the nucleic acid sequence set forth as SEQ ID NO: 127, and / or - the amino acid sequence of SEQ ID NO: 128 as set forth may have:

[0548] A secretion signal can be fused to a polypeptide having chondroitinase 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 chondroitinase activity as defined above.

[0549] Secretion signals and anchoring signals can be fused to a polypeptide having chondroitinase 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 chondroitinase activity as defined above, followed by a nucleic acid sequence encoding the anchoring signal.

[0550] 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.

[0551] The present invention further relates to a method as described above for producing chondroitin having a molecular weight of less than about 10 kDa, comprising: (a) the pH of the medium is greater than 4, and in particular in the range of about 5 to about 7; (b) one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity to be integrated into the genome of a recombinant cell of the invention originate from or are derived from Titius serrulatus, have a secretion signal but lack an anchoring signal, and are under the control of the pCCW12.sba promoter; (c) the polypeptide having chondroitinase activity is hyaluronidase; (d) a step of recovering chondroitin from the culture medium is carried out about 48 hours after the initiation of the culture of the recombinant cell of the invention, in particular the recombinant yeast cell of the invention; The recombinant cell is in particular a recombinant yeast cell, more particularly a Saccharomyces cerevisiae cell, It concerns the method.

[0552] The present invention further relates to the aforementioned method for producing chondroitin having a molecule of about 10 kDa to about 50 kDa, (a) the pH of the medium is greater than 4, and in particular in the range of about 5 to about 7; (b) one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity to be integrated into the genome of a recombinant cell of the invention originate from or are derived from Titius serrulatus, have a secretion signal but lack an anchoring signal, and are under the control of the pCCW10.Ago promoter; (c) the polypeptide having chondroitinase activity is hyaluronidase; (d) a step of recovering chondroitin from the culture medium is carried out about 48 hours after the initiation of the culture of the recombinant cell of the invention, in particular the recombinant yeast cell of the invention; The recombinant cell is in particular a recombinant yeast cell, more particularly a Saccharomyces cerevisiae cell, It concerns the method.

[0553] The present invention further relates to the aforementioned method for producing chondroitin having a molecule of about 50 kDa to about 500 kDa, (a) the pH of the medium is greater than 4, and in particular in the range of about 5 to about 7; (b) one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity to be integrated into the genome of the recombinant cell of the invention originate from or are derived from Titius serrulatus, have a secretion signal but lack an anchoring signal, and are under the control of the pNUP57 promoter; (c) the polypeptide having chondroitinase activity is hyaluronidase; (d) a step of recovering chondroitin from the culture medium is carried out about 48 hours after the initiation of the culture of the recombinant cell of the invention, in particular the recombinant yeast cell of the invention; The recombinant cell is in particular a recombinant yeast cell, more particularly a Saccharomyces cerevisiae cell, It concerns the method.

[0554] Another aspect of the invention relates to chondroitin obtained or obtainable from a recombinant cell of the invention or a method according to the invention.

[0555] A further aspect of the invention is a culture medium comprising the chondroitin of the invention.

[0556] The present invention further relates to a composition comprising a chondroitin according to the invention.

[0557] The present invention also relates to an industrial product, a consumer product, or a consumable product comprising (i) a chondroitin of the present invention, (ii) a culture medium comprising a chondroitin of the present invention, or (iii) a composition comprising a chondroitin of the present invention.

[0558] In particular, said industrial or consumer product or consumable product according to the invention may be 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, and / or a dental and / or oral hygiene composition.

[0559] Purification of chondroitin According to a particular embodiment of the present invention, the fermentation production of chondroitin preferably includes a step of isolating the produced chondroitin from the culture medium. The recovery of chondroitin 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.

[0560] Yeast as a model cell in the present invention is preferred in that the synthesized chondroitin is transported entirely outside the cell, thus simplifying the purification process.

[0561] Gas stripping is accomplished using a stripping gas selected from helium, argon, carbon dioxide, hydrogen, nitrogen, or mixtures thereof.

[0562] 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.

[0563] Formulations and Products The compositions of the invention may be incorporated into formulations / products, for example nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulations / products.

[0564] 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.

[0565] 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.

[0566] "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.

[0567] "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.

[0568] "Mixture" means a mixture or solution composed of two or more substances.

[0569] The present invention also provides the use of the compositions of the invention in nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulations / products.

[0570] Such formulations or products are hereinafter referred to as "formulations or products of the invention".

[0571] 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.

[0572] "Cosmetic" or "cosmetic active ingredient" refers to 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, blends, compositions, formulations (including, but not limited to, skin moisturizers, creams, balms, serums, oils, eye, facial cosmetics, rinse-off hair products, leave-in hair products, hair dyes (including, but not limited to, natural hair dyes), and / or combinations thereof), either individually or as part of a mixture of compounds, ingredients, intermediates, molecules, substances, raw materials, or products, final products and related technology 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 - means a method of making that is useful / used in / intended to provide a cosmetic and / or dermatological function and / or benefit together with a biological activity benefit (but without affecting the structure or function of the body). 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.

[0573] "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.

[0574] "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.

[0575] Alternatively, the nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulation / product may consist or consist essentially of the composition of the present invention.

[0576] The cosmetic formulation / product may be an anti-ageing formulation.

[0577] 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.

[0578] "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.

[0579] The nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulations / products may be in liquid or solid form.

[0580] Liquid dosage formulations / products for oral administration include solutions, emulsions, aqueous or oily suspensions, syrups, and elixirs.

[0581] 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.

[0582] 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.

[0583] 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.

[0584] 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.

[0585] 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.

[0586] 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.

[0587] 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.

[0588] 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.

[0589] 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.

[0590] Typically, such carriers are dermatologically acceptable.

[0591] 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.

[0592] 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).

[0593] 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.

[0594] 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.

[0595] 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.

[0596] 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.

[0597] 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.

[0598] 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.

[0599] 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.

[0600] 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.

[0601] 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.

[0602] 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.

[0603] 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.

[0604] 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.

[0605] 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."

[0606] 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.

[0607] 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).

[0608] 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.

[0609] The following examples and figures are offered for illustrative purposes and are not meant to imply limitations of the present invention. EXAMPLES

[0610] 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).

[0611] 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.

[0612] In addition, with the aim of better understanding the following genotypes: - jlp1, his3, leu2, sam3, and met14 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.

[0613] 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.

[0614] 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.

[0615] Example 2 Comparative Example for the Production of Chondroitin First, three recombinant strains are obtained: YA5809, YA5810, and YA5571.

[0616] Thus, these three strains are: YA5809: MAT-α, his3::[tRPL3-UGP1-pSAM1, pMET6-QRI1-tIDP1, HIS3]x5, jlp1::[LEU2.Sba-loxP, pTEF1.Sba-HASB.Vir-tRPL3.Sm, pTDH3.Sk-HASB-A.Vir-tTEF1.Sba, pTDH3-1.Sba-HCOS1-3.Pm-tRPL15A.Sm, pCCW12-KFOA.Pm-tRPL41B.Sba, pCCW120.Sm-KFOA.Pm-tRPL15A.Sba, pTEF1.Ago-GFA1.Vir-tRPL15A], leu2, met14::[TRP1.Sba-RS, pTDH3-PGM1-tIDP1, pCWP2-GNA1-tTPI1, pTEF1-PCM1-tRPL41B, pFBA1-UGP1-tRPL3, pTDH3-1.Sba-QRI1-tIDP1.Sba, pTEF1.Ago-GFA1-tRPL15A], trp1, bottom3 YA5810: MAT-α, his3::[tRPL3-UGP1-pSAM1, pMET6-QRI1-tIDP1, HIS3]x5, jlp1::[LEU2.Sba-loxP, pTEF1.Sba-HASB.Vir-tRPL3.Sm, pTDH3.Sk-HASB-A.Vir-tTEF1.Sba, pTDH3-1.Sba-HCOS1-3.Pm-tRPL15A.Sm, pCCW12-KFOA.Pm-tRPL41B.Sba, pCCW120.Sm-KFOA.Pm-tRPL15A.Sba, pTEF1.Ago-GFA1.Vir-tRPL15A], leu2, met14::[TRP1.Sba-RS, pTDH3-PGM1-tIDP1, pCWP2-GNA1-tTPI1, pTEF1-PCM1-tRPL41B, pFBA1-UGP1-tRPL3, pTDH3-1.Sba-QRI1-tIDP1.Sba], trp1, ura3 YA5571: MAT-α, his3::[tRPL3-UGP1-pSAM1, pMET6-QRI1-tIDP1, HIS3]x5, jlp1::[LEU2.Sba-loxP, pTEF1.Sba-HASB.Vir-tRPL3.Sm, pTDH3.Sk-HASB-A.Vir-tTEF1.Sba, leu2, trp1, ura3

[0617] HASB and HASB-A represent nucleic acid sequences encoding a polypeptide having UDP-glucose 6-dehydrogenase activity. They differ from each other in that they are different recoded versions of a nucleic acid sequence encoding an enzyme. HASB has the sequence of SEQ ID NO: 36 and HASB-A has the sequence of SEQ ID NO: 37. HCOS1-3.Pm represents a nucleic acid sequence encoding a polypeptide having chondroitin synthase activity, which is a nucleic acid comprising a fragment of a nucleic acid encoding a polypeptide having hyaluronan synthase activity and a nucleic acid encoding a polypeptide having chondroitin synthase activity. HCOS1-3.Pm has the sequence of SEQ ID NO: 3.

[0618] These strains were incubated in Erlenmeyer flasks in 25 ml of SY medium supplemented with 2% glucose at 28° C. for 48 hours.

[0619] 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, tribasic ammonium citrate: 33 mM, and glucose or sucrose 2-30%.

[0620] Growth medium was collected at 48 hours and assayed for chondroitin content.

[0621] An aliquot of the supernatant was loaded and run on a 0.5% agarose gel, which was subsequently stained with bromophenol blue.

[0622] The amount of chondroitin 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).

[0623] The amounts obtained for these different strains were, respectively: - YA5809: 200mg.L -1 - YA5810: 200mg.L -1 - YA5571: 200mg.L -1 It is.

[0624] In comparison, the native strain from which the three other strains are derived (Cherest et al. (2000) J. Biol. Chem. 275: 14056-14063) does not produce chondroitin.

[0625] This experiment showed that recombinant strains containing modifications according to the present invention produced greater amounts of chondroitin when cultured under the same conditions as other strains that did not contain all of the genetic modifications according to the present invention.

[0626] Example 3 Example for the production of chondroitin with controlled molecular weight 1. Production of chondroitin with a molecular weight of less than 10 kDa Two recombinant strains are then obtained: YA5887 and 5902.

[0627] Thus, these two strains are: YA5887: MAT-a, his3, jlp1::[LEU2.Kl, pCUP1-UGP1-tRPL3, pCUP1-QRI1-tIDP1, pPDC1-UGP1-tTPI1, pTDH3-QRI1-tMET25, pCCW12-HASB.At-tRPL15A], leu2::[TRP1.Sba, pCCW12.Sba-HYAL-3.Ts-tRPL15A], sam3::[HASB.Vir-tTEF1.Sba, pTDH3-1.Sba-HCOS1-3.Pm-tRPL3.Sba, pTDH3.Sar-HCOS1-3.Pm-tRPL15A.Sm, pCCW12-KFOA.Pm-tRPL41B.Sba, pCCW120.Sm-KFOA.Pm-tRPL15A.Sba, HIS3.Sba], trp1 YA5902: MAT-α, his3::[ pSAM1-UGP1-tRPL3, pMET6-QRI1-tIDP1, HIS3]x5, jlp1::[LEU2.Kl, pCUP1-UGP1-tRPL3, pCUP1-QRI1-tIDP1, pPDC1-UGP1-tTPI1, pTDH3-QRI1-tMET25, pCCW12-HASB.At-tRPL15A], leu2::[TRP1.Sba, pCCW12.Sba-HYAL-3.Ts-tRPL15A], sam3::[LEU2.Kl, pENO2-UGP1-tRPL3, pCWP2-GNA1-tTPI1, pTEF1-PCM1-tRPL41B, trp1

[0628] HYAL-3.Ts represents a nucleic acid sequence encoding a polypeptide with chondroitinase activity associated with a secretion signal but without an anchoring signal. HYAL-3.Ts has the amino acid sequence of SEQ ID NO:23.

[0629] HASB, HASB-A, and HCOS-1.Pm are as defined above.

[0630] These strains were incubated in Erlenmeyer flasks in 25 ml of SY medium supplemented with 2% glucose at 28° C. for 48 hours.

[0631] Growth medium was collected at 48 hours and assayed for chondroitin content.

[0632] An aliquot of the supernatant was loaded and run on a 0.5% agarose gel, which was subsequently stained with bromophenol blue.

[0633] The amount of chondroitin 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).

[0634] The amounts obtained for these different strains were, respectively: - YA5887: 600mg.L -1 - YA5902: 600mg.L -1 It is.

[0635] In comparison, the native strain from which the two other strains are derived (Cherest et al. (2000) J. Biol. Chem. 275: 14056-14063) does not produce chondroitin.

[0636] This experiment showed that recombinant strains containing modifications according to the present invention produced greater amounts of chondroitin when cultured under the same conditions as other strains that did not contain all of the genetic modifications according to the present invention.

[0637] Furthermore, these two strains resulted in the production of chondroitin with a molecular weight of less than 10 kDa after 48 hours.

[0638] 2. Production of chondroitin with a molecular weight of about 10 kDa to about 50 kDa Two other recombinant strains were then obtained: YA5888 and 5903.

[0639] Thus, these two strains are: YA5888: MAT-a, his3, jlp1::[LEU2.Kl, pCUP1-UGP1-tRPL3, pCUP1-QRI1-tIDP1, pPDC1-UGP1-tTPI1, pTDH3-QRI1-tMET25, pCCW12-HASB.At-tRPL15A], leu2::[TRP1.Sba, pCCW10.Ago-HYAL-3.Ts-tRPL15A], sam3::[HASB.Vir-tTEF1.Sba, pTDH3-1.Sba-HCOS1-3.Pm-tRPL3.Sba, pTDH3.Sar-HCOS1-3.Pm-tRPL15A.Sm, pCCW12-KFOA.Pm-tRPL41B.Sba, pCCW120.Sm-KFOA.Pm-tRPL15A.Sba, HIS3.Sba], trp1 YA5903: MAT-α, his3::[ pSAM1-UGP1-tRPL3, pMET6-QRI1-tIDP1, HIS3]x5, jlp1::[LEU2.Kl, pCUP1-UGP1-tRPL3, pCUP1-QRI1-tIDP1, pPDC1-UGP1-tTPI1, pTDH3-QRI1-tMET25, pCCW12-HASB.At-tRPL15A], leu2::[TRP1.Sba, pCCW10.Ago-HYAL-3.Ts-tRPL15A], sam3::[LEU2.Kl, pENO2-UGP1-tRPL3, pCWP2-GNA1-tTPI1, pTEF1-PCM1-tRPL41B, trp1

[0640] HYAL-3.Ts, HASB, HASB-A, and HCOS-1.Pm are as defined above.

[0641] These strains were incubated in Erlenmeyer flasks in 25 ml of SY medium supplemented with 2% glucose at 28° C. for 48 hours.

[0642] Growth medium was collected at 48 hours and assayed for chondroitin content.

[0643] An aliquot of the supernatant was loaded and run on a 0.5% agarose gel, which was subsequently stained with bromophenol blue.

[0644] The amount of chondroitin 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).

[0645] The amounts obtained for these different strains were, respectively: - YA5888: 600mg.L -1 - YA5903: 600mg.L -1 It is.

[0646] In comparison, the native strain from which the two other strains are derived (Cherest et al. (2000) J. Biol. Chem. 275: 14056-14063) does not produce chondroitin.

[0647] This experiment showed that recombinant strains containing modifications according to the present invention produced greater amounts of chondroitin when cultured under the same conditions as other strains that did not contain all of the genetic modifications according to the present invention.

[0648] Furthermore, these two strains resulted in the production of chondroitin having a molecular weight in the range of about 10 kDa to about 50 kDa after 48 hours.

[0649] 3. Production of chondroitin with a molecular weight of about 50 kDa to about 500 kDa Subsequently, two other recombinant strains are obtained: YA5889 and 5904.

[0650] Thus, these two strains are: YA5889: MAT-a, his3, jlp1::[LEU2.Kl, pCUP1-UGP1-tRPL3, pCUP1-QRI1-tIDP1, pPDC1-UGP1-tTPI1, pTDH3-QRI1-tMET25, pCCW12-HASB.At-tRPL15A], leu2::[TRP1.Sba, pNUP57-HYAL-3.Ts-tRPL15A], sam3::[HASB.Vir-tTEF1.Sba, pTDH3-1.Sba-HCOS1-3.Pm-tRPL3.Sba, pTDH3.Sar-HCOS1-3.Pm-tRPL15A.Sm, pCCW12-KFOA.Pm-tRPL41B.Sba, pCCW120.Sm-KFOA.Pm-tRPL15A.Sba, HIS3.Sba], trp1 YA5904: MAT-α, his3::[ pSAM1-UGP1-tRPL3, pMET6-QRI1-tIDP1, HIS3]x5, jlp1::[LEU2.Kl, pCUP1-UGP1-tRPL3, pCUP1-QRI1-tIDP1, pPDC1-UGP1-tTPI1, pTDH3-QRI1-tMET25, pCCW12-HASB.At-tRPL15A], leu2::[TRP1.Sba, pNUP57-HYAL-3.Ts-tRPL15A], sam3::[LEU2.Kl, pENO2-UGP1-tRPL3, pCWP2-GNA1-tTPI1, pTEF1-PCM1-tRPL41B, pTEF1.Sba-HASB.vir-tRPL3.Sm, pTDH3.Sk-HASB-A.Vir-tTEF1.Sba, pTDH3-1.Sba-HCOS1-3.Pm-tRPL15A.Sm, pCCW12-KFOA.Pm-tRPL15A.Sba, HIS3.Sba-loxP], trp1

[0651] HYAL-3.Ts, HASB, HASB-A, and HCOS-1.Pm are as defined above.

[0652] These strains were incubated in Erlenmeyer flasks in 25 ml of SY medium supplemented with 2% glucose at 28° C. for 48 hours.

[0653] Growth medium was collected at 48 hours and assayed for chondroitin content.

[0654] An aliquot of the supernatant was loaded and run on a 0.5% agarose gel, which was subsequently stained with bromophenol blue.

[0655] The amount of chondroitin 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).

[0656] The amounts obtained for these different strains were, respectively: - YA5889: 600mg.L -1 - YA5904: 600mg.L -1 It is.

[0657] In comparison, the native strain from which the two other strains are derived (Cherest et al. (2000) J. Biol. Chem. 275: 14056-14063) does not produce chondroitin.

[0658] This experiment showed that recombinant strains containing modifications according to the present invention produced greater amounts of chondroitin when cultured under the same conditions as other strains that did not contain all of the genetic modifications according to the present invention.

[0659] Furthermore, these two strains resulted in the production of chondroitin having a molecular weight in the range of about 10 kDa to about 50 kDa after 48 hours.

[0660] array SEQ ID NO:1 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS1-1) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from a nucleic acid encoding a chondroitin synthase originating from Pasteurella multocida.

[0661] [ka]

[0662] [ka]

[0663] SEQ ID NO:2 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS1-2) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida.

[0664] [ka]

[0665] [ka]

[0666] SEQ ID NO:3 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS1-3) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida.

[0667] [ka]

[0668] [ka]

[0669] SEQ ID NO: 4 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS.Sc) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida, a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida, and a fragment derived from a nucleic acid encoding chitin synthase 2 from Saccharomyces cerevisiae.

[0670] [ka]

[0671] [ka]

[0672] SEQ ID NO:5 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HHASA.Sc) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from a nucleic acid encoding chitin synthase 2 from Saccharomyces cerevisiae.

[0673] [ka]

[0674] [ka]

[0675] SEQ ID NO:6 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS1-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from E. coli.

[0676] [ka]

[0677] SEQ ID NO: 7 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS2-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase originating from E. coli.

[0678] [ka]

[0679] SEQ ID NO:8 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS3-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a galactofuranosyltransferase originating from Mycobacterium tuberculosis.

[0680] [ka]

[0681] SEQ ID NO:9 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS4-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from E. coli.

[0682] [ka]

[0683] SEQ ID NO:10 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS5-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from Penicillium oxalicum.

[0684] [ka]

[0685] SEQ ID NO:11 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS6-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida.

[0686] [ka]

[0687] [ka]

[0688] SEQ ID NO:12 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS7-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida.

[0689] [ka]

[0690] SEQ ID NO:13 is a recoded nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS8-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin synthase from Pasteurella multocida.

[0691] [ka]

[0692] SEQ ID NO:14 is the nucleic acid sequence of a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS9-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin sulfate synthase originating from Homo sapiens.

[0693] [ka]

[0694] [ka]

[0695] SEQ ID NO:15 is a recoded nucleic acid sequence for a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS10-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin sulfate synthase originating from Homo sapiens.

[0696] [ka]

[0697] [ka]

[0698] SEQ ID NO:16 is a recoded nucleic acid sequence for a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS11-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin sulfate synthase originating from Homo sapiens.

[0699] [ka]

[0700] SEQ ID NO:17 is a recoded nucleic acid sequence for a polypeptide having chondroitin synthase activity: a chimeric nucleic acid (HCOS12-Vir) comprising a fragment derived from a nucleic acid encoding a hyaluronan synthase originating from the Chlorella virus PBCV-1 and a fragment derived from a nucleic acid encoding a chondroitin sulfate synthase originating from Homo sapiens.

[0701] [ka]

[0702] SEQ ID NO: 18 is the amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS1-1) comprising a fragment derived from the amino acid sequence encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from the amino acid sequence encoding a chondroitin synthase from Pasteurella multocida.

[0703] [ka]

[0704] SEQ ID NO: 19 is the amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS1-2) comprising a fragment derived from the amino acid sequence encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from the amino acid sequence encoding a chondroitin synthase from Pasteurella multocida.

[0705] [ka]

[0706] SEQ ID NO: 20 is the amino acid sequence of a polypeptide having chondroitin synthase activity: a chimeric amino acid sequence (HCOS1-3) comprising a fragment derived from the amino acid sequence encoding a hyaluronan synthase originating from Pasteurella multocida and a fragment derived from the ...

Claims

1. A recombinant yeast cell that produces chondroitin, comprising: (a) one or more recombinant nucleic acids encoding a polypeptide having chondroitin synthase (HCOS) activity; (b) one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity; (c) one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose-4-epimerase (kfoA or GNE1) activity; A recombinant yeast cell comprising:

2. The recombinant cell of claim 1, comprising one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity, the polypeptide having chondroitinase activity comprising a secretion signal and optionally an anchoring signal.

3. A recombinant host cell that produces chondroitin, comprising: (a) one or more recombinant nucleic acids encoding a polypeptide having chondroitin synthase (HCOS) 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 UDP-glucose-4-epimerase (kfoA or GNE1) activity; (d) one or more recombinant nucleic acids encoding a polypeptide having chondroitinase activity, the polypeptide having chondroitinase activity comprising a secretion signal and optionally an anchoring signal such that chondroitin, particularly of a desired molecular weight, is produced by the host cell; A recombinant host cell comprising:

4. 4. The recombinant host cell of claim 2 or 3, wherein the recombinant nucleic acid encoding a polypeptide having chondroitinase activity is obtained or derived from at least one of Cupienius salei, Titius serlatus, Bos taurus, Vespa magnifica, Macaca mulatta, or Apis mellifera, preferably Titius serlatus.

5. 5. The recombinant cell according to any one of claims 2 to 4, wherein the molecular weight of the chondroitin is in the range of less than 50 kDa, preferably in the range of about 20 kDa to about 50 kDa.

6. 5. The recombinant cell according to any one of claims 1 to 4, wherein the molecular weight of the chondroitin is in the range above 50 kDa, preferably in the range of about 50 kDa to about 250 kDa.

7. 5. The recombinant cell according to any one of claims 1 to 4, wherein the molecular weight of the chondroitin is in the range above 100 kDa, preferably in the range of about 100 kDa to about 1500 kDa.

8. 8. The recombinant cell according to claim 1, 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 at least one of Arabidopsis thaliana or Chlorella virus PBCV1.

9. A nucleic acid encoding a polypeptide having chondroitin synthase (HCOS) activity, (i) a nucleic acid encoding a chondroitin synthase, or (ii) a nucleic acid encoding a chimeric polypeptide having chondroitin synthase activity 9. The recombinant cell according to any one of claims 1 to 8,

10. 10. The recombinant cell of claim 9, wherein the nucleic acid is obtained or derived from at least one of Pasteurella multocida, Chlorella virus PBCV1, Mycobacterium tuberculosis, Homo sapiens, Escherichia coli, Saccharomyces cerevisiae, or Penicillium oxalicum.

11. 11. The recombinant cell according to any one of claims 1 to 10, wherein the nucleic acid encoding a polypeptide having UDP-glucose-4-epimerase (kfoA or GNE1) activity is obtained or derived from a bacterium, in particular a bacterium selected from the group consisting of Pseudomonas aeruginosa, Pasteurella multocida, and Escherichia coli.

12. (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity 12. The recombinant cell of claim 1 , further comprising at least one recombinant nucleic acid encoding one or more of the following:

13. (i) a polypeptide having phosphoglucomutase-1 (PGM1) activity; (ii) a polypeptide having UTP-glucose-1-phosphate uridylyltransferase (UGP1) activity; (iii) a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity, and / or (iv) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity 13. The recombinant cell of claim 1 , further comprising at least one recombinant nucleic acid encoding one or more of:

14. - a nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, - a nucleic acid encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity, - a nucleic acid encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity, - a nucleic acid encoding a polypeptide having UTP-glucose-1-phosphate uridylyltransferase (UGP1) activity, - 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, A recombinant cell according to claim 12 or 13.

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

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

17. 17. The recombinant cell of claim 16, 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.

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

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

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

21. 19. The method of claim 18, wherein the chondroitin has a molecular weight of about 150 kDa to about 1500 kDa.

22. 22. The method according to any one of claims 18 to 21, wherein the recombinant cell is a yeast belonging to the genus Saccharomyces, in particular Saccharomyces cerevisiae.

23. 23. The method according to any one of claims 18 to 22, wherein the time sufficient to produce chondroitin of the desired molecular weight is a period of about 35 hours to about 50 hours, preferably about 40 hours to about 50 hours, preferably about 48 hours.

24. 24. The method according to any one of claims 18 to 23, wherein the molecular weight of the produced chondroitin is controlled by adjusting the pH of the culture medium in step (a).

25. 25. The method according to any one of claims 18 to 24, 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 1,000 L to about 3,000 L, even more preferably about 10,000 L, or even more preferably about 100,000 L, or even about 250,000 L.

26. A chondroitin obtainable from a recombinant cell according to any one of claims 1 to 17 or from a method according to any one of claims 18 to 25.

27. A culture medium comprising the chondroitin of claim 26.

28. 27. A composition comprising the chondroitin of claim 26.

29. 29. An industrial product, a consumer product, or a consumable product comprising: (i) a chondroitin having a molecular weight as defined in any one of claims 5 to 7; (ii) a culture medium as defined in claim 27; or (iii) a composition as defined in claim 28.

30. 30. The industrial or consumer product or consumable of claim 29 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.

31. 18. Use of a recombinant cell as defined in any one of claims 2 to 17 for the production of chondroitin having a molecular weight in the range of about 20 kDa to about 50 kDa or about 50 kDa to about 1000 kDa.

32. 1. A method for producing chondroitin, comprising: (a) culturing a recombinant yeast as defined in any one of claims 1 to 17 in a culture medium; (b) recovering chondroitin from the culture medium; Including, The chondroitin recovered in step (b) is - the nature and origin of the recombinant nucleic acid encoding a polypeptide having recombinant yeast chondroitinase activity; - the nature and origin of the promoter controlling the expression of the recombinant nucleic acid encoding the polypeptide having chondroitinase activity of the recombinant yeast, - the presence of an anchoring and / or secretion signal associated with the recombinant yeast polypeptide having chondroitinase activity, - the pH of the culture medium during the cultivation process of the recombinant yeast, and / or - Duration of recombinant yeast cultivation having a molecular weight controlled through the selection of method.

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