Recombinant cells that produce hyaluronic acid

Abstract

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

Description

[Technical Field]

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

[0002] Hyaluronic acid, also known as hyaluronan or HA, is composed of D-glucuronic acid and N-acetyl-D-glucosamine linked by alternating β-(1→4) and β-(1→3) glycosidic bonds and has the chemical formula (C 14 H 21 NO 11 Hyaluronic acid is a naturally occurring high molecular weight polysaccharide with a molecular weight of 1.25 (Hyaluronic acid). Hyaluronic acid can be 25,000 disaccharide repeats long. Polymers of hyaluronic acid can range in size from 5,000 to 20,000,000 Da in vivo.

[0003] HA has numerous applications in the medical and cosmetic fields, including scaffolding for tissue engineering, dermal fillers, and viscosupplements for osteoarthritis treatment. In particular, reduction of HA mass or molecular weight due to degradation or delayed synthesis affects physical and chemical properties, such as tissue volume, viscosity, and elasticity. Therefore, there is a constant need for HA production.

[0004] Currently, the main known sources of HA are human umbilical cord, rooster comb, and the fermentation of certain microorganisms.

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

[0006] For this purpose, bacterial cultures, such as group A and C hemolytic streptococci, have been shown to represent a good source of HA (U.S. Patent Nos. 5,316,926, 4,801,539, and JP 2009-11315). However, such bacteria, particularly Streptococcus zooepidemicus, which is primarily used in the art, are generally not recognized as safe. Recombinant Bacillus host cells have also demonstrated the ability to produce HA in the 20-800 kDa range (U.S. Publication No. 2008038780). Furthermore, successful transformation of plant cells to contain DNA encoding hyaluronan synthase has been shown to enable HA production (U.S. Publication No. 2006168690). Finally, in China Publication No. 104263666, the production of small molecules of HA has also been demonstrated in yeast, e.g., recombinant Pichia pastoris.

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

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

[0009] In contrast, Saccharomyces cerevisiae has a long history of safe use by humans (e.g., in wine, beer, or bread) and is therefore a well-established model whose genetic information is well known in the art, making it particularly useful as a tool for the production of molecules of interest. S. cerevisiae also 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 potential for contamination of biofermenters, thus eliminating the need for antibiotics to be added to the medium. Finally, numerous genetic tools have been developed that allow for stable modification of the genome (chromosomal integration). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 5,316,926 [Patent Document 2] U.S. Patent No. 4,801,539 [Patent Document 3] Patent Publication No. 2009-011315 [Patent Document 4] U.S. Publication No. 2008038780

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

[0013] There remains a need in the art for hyaluronic acid methods that allow for obtaining large amounts of hyaluronic acid of specific and controlled sizes in certain cases. [Means for solving the problem]

[0014] The present invention therefore relates to the following items:

[0015] Item 1: A recombinant yeast cell that produces hyaluronic acid (HA), (a) one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase 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 hyaluronidase activity, wherein the polypeptide having hyaluronidase activity comprises a secretion signal such that hyaluronic acid, particularly of a desired molecular weight (HAMW), is produced by the recombinant yeast cell; and (d)(i) one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase (GLN1) activity, and / or (ii) one or more disrupted endogenous nucleic acids encoding glutamate synthase (GLT1); Including, The recombinant yeast cell belongs to the genus Saccharomyces, or Candida, or Kluyveromyces, or Ogataea, or Yarrowia, or Debaryomyces, or Ashbya. Recombinant yeast cells.

[0016] As shown in the examples, the recombinant yeast of the present invention allows the production of hyaluronic acid in yeast cells that are not naturally capable of producing hyaluronic acid. The examples further demonstrate that the size of the hyaluronic acid produced by the recombinant yeast can be controlled.

[0017] The advantageous properties can be further increased by engineering the yeast with additional modifications as described hereinafter.

[0018] Item 2: A recombinant host cell that produces hyaluronic acid (HA), (a) one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase 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 hyaluronidase activity, wherein the polypeptide having hyaluronidase activity comprises a secretion signal and an anchoring signal such that hyaluronic acid, particularly of a desired molecular weight (HAMW), is produced by the host cell; and (d)(i) one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase (GLN1) activity, and / or (ii) one or more disrupted endogenous nucleic acids encoding glutamate synthase (GLT1); A recombinant host cell comprising:

[0019] Item 3: The recombinant cell according to Item 1 or 2, wherein the molecular weight of the HA is within the range of less than 50 kDa, preferably within the range of about 20 kDa to about 50 kDa.

[0020] Item 4: The recombinant cell according to Item 1 or 2, wherein the molecular weight of the HA is in the range of more than 50 kDa, preferably in the range of about 50 kDa to about 250 kDa.

[0021] Item 5: The recombinant cell according to Item 1 or 2, wherein the molecular weight of the HA is in the range of more than 100 kDa, preferably in the range of about 100 kDa to about 1500 kDa.

[0022] Item 6: The recombinant cell according to any one of items 1 to 5, wherein the nucleic acid encoding a polypeptide having glutamine synthetase activity is obtained or derived from Saccharomyces cerevisiae.

[0023] Item 7: The recombinant cell according to any one of Items 1 to 6, wherein the nucleic acid encoding a polypeptide having hyaluronidase activity is obtained from or derived from Cupiennius salei, Loxosceles intermedia, Hirudo nipponia, Bothrops atrox, or Tityus serrulatus.

[0024] Item 8: The recombinant cell according to any one of Items 1 to 7, wherein the nucleic acid encoding a polypeptide having hyaluronan synthase activity is obtained or derived from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviK1, Chlorella virus IL-5-2s1, Chlorella virus CZ-2, Chlorella virus CVG-1, Xenopus laevis, or Pasteurella multocida, in particular, obtained or derived from Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviK1, Chlorella virus IL-5-2s1, Chlorella virus CZ-2, Chlorella virus CVG-1, or Xenopus laevis.

[0025] Item 9: The recombinant cell according to any one of items 1 to 8, wherein the nucleic acid encoding a polypeptide having UDP-glucose dehydrogenase activity is obtained or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, in particular Arabidopsis thaliana or Chlorella virus PBCV1.

[0026] Item 10: (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity 10. The recombinant cell of any one of items 1 to 9, comprising a recombinant nucleic acid encoding one or more of:

[0027] Item 11: (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 11. The recombinant cell of any one of items 1 to 10, comprising at least one recombinant nucleic acid encoding one or more of:

[0028] Item 12: The recombinant yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces castelli, Candida albicans, Candida glabrata, Candida tropicalis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus, and the like. 12. The recombinant yeast cell according to any one of items 1 and 3 to 11, wherein the recombinant yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces polysporus, Kluyveromyces thermotolerens, Ogataea polymorpha, Yarrowia lypolytica, Debaryomyces hansenii, and Ashbya gossypii, and is preferably Saccharomyces cerevisiae.

[0029] Item 13: The recombinant host cell according to any one of Items 2 to 11, wherein the recombinant host cell belongs to the order Saccharomycetales, in particular the family Saccharomycetaceae, and is in particular 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.

[0030] Item 14: A method for producing hyaluronic acid (HA) of a desired molecular weight (HAMW), comprising: (a) culturing the recombinant cell according to any one of items 1 to 13 in a culture medium for a time sufficient to produce hyaluronic acid (HA) of a desired molecular weight; (b) optionally isolating or recovering hyaluronic acid (HA) from the recombinant cells and / or culture medium; A method comprising:

[0031] Item 15: The method according to Item 14, wherein the HA has a molecular weight of about 20 kDa to about 50 kDa, preferably about 20 kDa to about 30 kDa.

[0032] Item 16: The method according to Item 15, wherein the HA has a molecular weight of about 30 kDa to about 50 kDa.

[0033] Item 17: The method according to Item 14, wherein the molecular weight of the HA is about 50 kDa to about 150 kDa.

[0034] Item 18: The method according to Item 14, wherein the molecular weight of the HA is about 150 kDa to about 1500 kDa.

[0035] Item 19: The recombinant cell (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity 19. The method of any one of items 14 to 18, comprising at least one recombinant nucleic acid encoding one or more of:

[0036] Item 20: The recombinant cell (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 20. The method of any one of items 14 to 19, comprising at least one recombinant nucleic acid encoding one or more of:

[0037] Item 21: The method according to any one of items 14 to 20, wherein the recombinant cell is a member of the genus Saccharomyces, in particular Saccharomyces cerevisiae.

[0038] Item 22: The method according to any one of Items 14 to 21, wherein the time sufficient to produce hyaluronic acid (HA) 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.

[0039] Item 23: The method according to any one of items 14 to 22, wherein the molecular weight of the hyaluronic acid is controlled by adjusting the pH of the culture medium.

[0040] Item 24: The method according to any one of items 14 to 23, 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 1000 L to about 3000 L, even more preferably about 10,000 L, even more preferably about 100,000 L, or even about 250,000 L.

[0041] Item 25: Hyaluronic acid (HA) obtainable from a recombinant cell according to any one of items 1 to 10 or from a method according to any one of items 14 to 24.

[0042] Item 26: A culture medium containing the HA according to Item 25.

[0043] Item 27: A composition comprising hyaluronic acid (HA) according to Item 24.

[0044] Item 28: An industrial product, consumer product, or consumable product comprising (i) the HA according to Item 25, (ii) the culture medium according to Item 26, or (iii) the composition according to Item 27.

[0045] Item 29: The industrial or consumer product or consumable according to item 27, which is a cosmetic product, a flavor product, a fragrance product, a foodstuff, a food, a beverage, a texturant, a pharmaceutical composition, a dietary supplement, a nutraceutical, a cleaning product, and / or a dental and / or oral hygiene composition.

[0046] Item 30: Use of the recombinant cell according to any one of items 1 to 13 for producing hyaluronic acid (HA) having a molecular weight in the range of about 20 kDa to about 50 kDa or about 50 kDa to about 1000 kDa.

[0047] Item 31: A method for producing hyaluronic acid, comprising: (a) culturing the recombinant yeast according to any one of items 1 and 3 to 12 in a culture medium; (b) recovering hyaluronic acid from the culture medium; Including, The hyaluronic acid recovered in step (b) - the nature and origin of the nucleic acid encoding the recombinant yeast hyaluronidase; - the nature and origin of the promoter controlling the expression of the nucleic acid encoding the recombinant yeast hyaluronidase; - the presence of an anchoring and / or secretion signal associated with the recombinant yeast encoded hyaluronidase; - 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.

[0048] Certain embodiments of the present invention provide the following advantages: A process for producing hyaluronic acid of controlled molecular weight, A process for producing hyaluronic acid of controlled molecular weight in Saccharomyces yeast cells, and A process for producing hyaluronic acid 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:

[0049] The details, examples, and preferences provided in connection with any particular one or more of the mentioned aspects of the invention will be further explained 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 explanation of the drawings]

[0050] [Figure 1] FIG. 1 shows a schematic pathway for the production of hyaluronic acid. [Figure 2]Figure 2A shows agarose gels used to determine the molecular weight of hyaluronic acid produced by strains according to the invention, for example, strain YA5235-1 (Figure 2A). The agarose gels were obtained after running aliquots of the supernatant of the strains considered and staining them with "stains all" (CAS Number 7423-31-6). HA molecular weight standards are present on each gel for reference. Figure 2B shows agarose gels used to determine the molecular weight of hyaluronic acid produced by strains according to the invention, for example, strain YA5359-10 (Figure 2B). The agarose gels were obtained after running aliquots of the supernatant of the strains considered and staining them with "stains all" (CAS Number 7423-31-6). HA molecular weight standards are present on each gel for reference. DETAILED DESCRIPTION OF THE INVENTION

[0051] Array Overview SEQ ID NO: 1 is the recoded nucleic acid sequence of hyaluronan synthase (HASA) originating from the Chlorella virus PBCV-1 SEQ ID NO:2 is the recoded nucleic acid sequence of hyaluronan synthase (HASA) originating from the Chlorella virus PBCV-1 SEQ ID NO: 3 is the recoded nucleic acid sequence of hyaluronan synthase (HASA) originating from Pasteurella multocida SEQ ID NO: 4 is the recoded nucleic acid sequence of hyaluronan synthase (HASA) originating from Pasteurella multocida SEQ ID NO: 5 is the recoded nucleic acid sequence of hyaluronan synthase (HASA) originating from Pasteurella multocida SEQ ID NO: 6 is the recoded nucleic acid sequence of hyaluronan synthase (HASA) originating from Xenopus laevis SEQ ID NO: 7 is the recoded nucleic acid sequence of hyaluronan synthase (HASA) originating from Streptococcus zooepidemicus SEQ ID NO: 8 is the amino acid sequence of hyaluronan synthase (HASA) originating from the Chlorella virus PBCV-1 SEQ ID NO: 9 is the amino acid sequence of hyaluronan synthase (HASA) originating from Pasteurella multocida SEQ ID NO: 10 is the amino acid sequence of hyaluronan synthase (HASA) from Xenopus laevis SEQ ID NO: 11 is the amino acid sequence of hyaluronan synthase (HASA) originating from Streptococcus zooepidemicus SEQ ID NO: 12 is the nucleic acid sequence of UDP-glucose dehydrogenase (HASB) originating from Arabidopsis thaliana SEQ ID NO: 13 is the recoded nucleic acid sequence of UDP-glucose dehydrogenase (HASB) originating from the Chlorella virus PBCV-1 SEQ ID NO: 14 is the recoded nucleic acid sequence of UDP-glucose dehydrogenase (HASB) originating from the Chlorella virus PBCV-1 SEQ ID NO: 15 is the recoded nucleic acid sequence of UDP-glucose dehydrogenase (HASB) originating from Streptococcus zooepidemicus SEQ ID NO: 16 is the amino acid sequence of UDP-glucose dehydrogenase (HASB) from Arabidopsis thaliana SEQ ID NO: 17 is the amino acid sequence of UDP-glucose dehydrogenase (HASB) originating from the Chlorella virus PBCV1 SEQ ID NO: 18 is the amino acid sequence of UDP-glucose dehydrogenase (HASB) originating from Streptococcus zooepidemicus SEQ ID NO: 19 is the recoded nucleic acid sequence of hyaluronidase (HYAL) from Bothropus atrox with an N-terminal secretory signal SEQ ID NO: 20 is the recoded nucleic acid sequence of hyaluronidase (HYAL) from Bothropus atrox, with an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO: 21 is the recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Cupienius salei with an N-terminal secretory signal SEQ ID NO: 22 is the recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Cupienius salei, with an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO: 23 is the recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Hirudo Nipponia with an N-terminal secretory signal SEQ ID NO: 24 is the recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Hirudo Nipponia having an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO: 25 is the recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Loxosceles intermedia with an N-terminal secretory signal SEQ ID NO: 26 is the recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Loxosceles intermedia, with an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO: 27 is the recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Titius serulatus with an N-terminal secretion signal SEQ ID NO: 28 is the recoded nucleic acid sequence of hyaluronidase (HYAL) originating from Titius serulatus, with an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO: 29 is the recoded nucleic acid sequence of hyaluronidase (HYAL) from Vespa magnifica with an N-terminal secretory signal SEQ ID NO: 30 is the recoded nucleic acid sequence of hyaluronidase (HYAL) from Vespa magnifica, with an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO: 31 is the amino acid sequence of hyaluronidase (HYAL) from Bothropus atrox with an N-terminal secretory signal SEQ ID NO: 32 is the amino acid sequence of hyaluronidase (HYAL) from Bothropus atrox, with an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO: 33 is the amino acid sequence of hyaluronidase (HYAL) from Cupienius salei with an N-terminal secretory signal SEQ ID NO: 34 is the amino acid sequence of hyaluronidase (HYAL) from Cupienius salei, with an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO: 35 is the amino acid sequence of hyaluronidase (HYAL) from Hirudo Nipponia with an N-terminal secretory signal SEQ ID NO: 36 is the amino acid sequence of hyaluronidase (HYAL) from Hirudo Nipponia having an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO: 37 is the amino acid sequence of hyaluronidase (HYAL) from Loxosceles intermedia with an N-terminal secretory signal SEQ ID NO: 38 is the amino acid sequence of hyaluronidase (HYAL) originating from Loxosceles intermedia, with an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO: 39 is the amino acid sequence of hyaluronidase (HYAL) from Titius serrata with an N-terminal secretory signal SEQ ID NO: 40 is the amino acid sequence of hyaluronidase (HYAL) from Titius serrata, with an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO: 41 is the amino acid sequence of hyaluronidase (HYAL) from Vespa magnifica with an N-terminal secretory signal SEQ ID NO: 42 is the amino acid sequence of hyaluronidase (HYAL) from Vespa magnifica, with an N-terminal secretory signal and a C-terminal anchoring signal. SEQ ID NO: 43 is the nucleic acid sequence of the secretory sequence added to the 5' end SEQ ID NO: 44 is the amino acid sequence of the secretory sequence added to the N-terminus SEQ ID NO: 45 is the nucleic acid sequence of the anchoring sequence added to the 3' end SEQ ID NO: 46 is the amino acid sequence of the anchoring sequence added to the C-terminus SEQ ID NO: 47 is the nucleic acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Saccharomyces cerevisiae SEQ ID NO: 48 is the recoded nucleic acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Chlorella virus 1 (PBCV-1) SEQ ID NO: 49 is the recoded nucleic acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Chlorella virus 1 (PBCV-1) SEQ ID NO: 50 is the amino acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Saccharomyces cerevisiae SEQ ID NO: 51 is the amino acid sequence of glutamine-fructose-6-phosphate amidotransferase (GFA1) originating from Chlorella virus 1 (PBCV-1) SEQ ID NO: 52 is the nucleic acid sequence of UDP-N-acetylglucosamine pyrophosphorylase (QRI1) originating from Saccharomyces cerevisiae SEQ ID NO: 53 is the amino acid sequence of UDP-N-acetylglucosamine pyrophosphorylase (QRI1) originating from Saccharomyces cerevisiae SEQ ID NO: 54 is the nucleic acid sequence of phosphoglucomutase-1 (PGM1) originating from Saccharomyces cerevisiae. SEQ ID NO: 55 is the amino acid sequence of phosphoglucomutase-1 (PGM1) originating from Saccharomyces cerevisiae SEQ ID NO: 56 is the nucleic acid sequence of UTP-glucose 1-phosphate uridylyltransferase (UGP1) originating from Saccharomyces cerevisiae SEQ ID NO: 57 is the amino acid sequence of UTP-glucose 1-phosphate uridylyltransferase (UGP1) originating from Saccharomyces cerevisiae SEQ ID NO: 58 is the nucleic acid sequence of glucosamine 6-phosphate N-acetyltransferase (GNA1) originating from Saccharomyces cerevisiae SEQ ID NO: 59 is the amino acid sequence of glucosamine 6-phosphate N-acetyltransferase (GNA1) originating from Saccharomyces cerevisiae SEQ ID NO: 60 is the nucleic acid sequence of phosphoacetylglucosamine mutase (PCM1) originating from Saccharomyces cerevisiae SEQ ID NO: 61 is the amino acid sequence of phosphoacetylglucosamine mutase (PCM1) originating from Saccharomyces cerevisiae SEQ ID NO: 62 is the nucleic acid sequence of promoter pTDH3 SEQ ID NO: 63 is the nucleic acid sequence of promoter pTDH3.Sk SEQ ID NO: 64 is the nucleic acid sequence of promoter pTDH3-1.sba SEQ ID NO: 65 is the nucleic acid sequence of the promoter pTDH3.Sar SEQ ID NO: 66 is the nucleic acid sequence of promoter pENO2 SEQ ID NO: 67 is the nucleic acid sequence of promoter pTEF3 SEQ ID NO: 68 is the nucleic acid sequence of promoter pTEF1 SEQ ID NO: 69 is the nucleic acid sequence of the promoter pTEF1.ago SEQ ID NO: 70 is the nucleic acid sequence of the promoter pTEF1.Sba SEQ ID NO: 71 is the nucleic acid sequence of promoter pPDC1 SEQ ID NO: 72 is the nucleic acid sequence of promoter pCCW12 SEQ ID NO: 73 is the nucleic acid sequence of promoter pCCW12.Sm SEQ ID NO: 74 is the nucleic acid sequence of promoter pCCW12.Sk SEQ ID NO: 75 is the nucleic acid sequence of promoter pCCW12.Sba SEQ ID NO: 76 is the nucleic acid sequence of the promoter pCCW12.Sar SEQ ID NO: 77 is the nucleic acid sequence of promoter pNUP57 SEQ ID NO: 78 is the nucleic acid sequence of the promoter pCCW10.ago SEQ ID NO: 79 is the nucleic acid sequence of promoter pCWP2 SEQ ID NO: 80 is the nucleic acid sequence of promoter pRPLA1 SEQ ID NO: 81 is the nucleic acid sequence of the promoter pCUP1 SEQ ID NO: 82 is the nucleic acid sequence of promoter pMET6 SEQ ID NO: 83 is the nucleic acid sequence of promoter pMET25 SEQ ID NO: 84 is the nucleic acid sequence of the promoter pSAM1 SEQ ID NO: 85 is the nucleic acid sequence of the terminator tTPI1 SEQ ID NO: 86 is the nucleic acid sequence of the terminator tMET25 SEQ ID NO: 87 is the nucleic acid sequence of the terminator tDIT1 SEQ ID NO: 88 is the nucleic acid sequence of the terminator tRPL3 SEQ ID NO: 89 is the nucleic acid sequence of the terminator tRPL3.sm SEQ ID NO: 90 is the nucleic acid sequence of the terminator tRPL3.sba SEQ ID NO: 91 is the nucleic acid sequence of the terminator tRPL41B SEQ ID NO: 92 is the nucleic acid sequence of terminator tRPL15A SEQ ID NO: 93 is the nucleic acid sequence of terminator tRPL15A.sba SEQ ID NO: 94 is the nucleic acid sequence of the terminator tIDP1 SEQ ID NO: 95 is the nucleic acid sequence of the terminator tTEF1.sba SEQ ID NO: 96 is the nucleic acid sequence of glutamine synthetase (GLN1) originating from Saccharomyces cerevisiae SEQ ID NO: 97 is the amino acid sequence of glutamine synthetase (GLN1) originating from Saccharomyces cerevisiae SEQ ID NO: 98 is the nucleic acid sequence of glutamate synthase (GLT1) originating from Saccharomyces cerevisiae SEQ ID NO: 99 is the amino acid sequence of glutamate synthase (GLT1) originating from Saccharomyces cerevisiae SEQ ID NO: 100 is the nucleic acid sequence of the terminator tTDH3 SEQ ID NO: 101 is the recoded nucleic acid sequence of hyaluronan synthase (HASA) originating from the Chlorella virus CviKl SEQ ID NO: 102 is the recoded nucleic acid sequence of hyaluronan synthase (HASA) originating from Chlorella virus IL-5-2s1 SEQ ID NO: 103 is the recoded nucleic acid sequence of hyaluronan synthase (HASA) originating from Chlorella virus CZ-2 SEQ ID NO: 104 is the recoded nucleic acid sequence of hyaluronan synthase (HASA) originating from the Chlorella virus CVG-1 SEQ ID NO: 105 is the amino acid sequence of hyaluronan synthase (HASA) from the Chlorella virus CviKl SEQ ID NO: 106 is the amino acid sequence of hyaluronan synthase (HASA) from Chlorella virus IL-5-2s1 SEQ ID NO: 107 is the amino acid sequence of hyaluronan synthase (HASA) from Chlorella virus CZ-2 SEQ ID NO: 108 is the amino acid sequence of hyaluronan synthase (HASA) from the Chlorella virus CVG-1

[0052] Detailed Description of the Invention The inventors have envisioned genetically modified cells, particularly genetically modified yeast, that have the ability to produce hyaluronic acid compared to parental cells, particularly parental yeast, that are not naturally capable of doing so.

[0053] These genetically modified cells are described throughout this specification.

[0054] definition The term "hyaluronic acid," also known as hyaluronan or HA, refers to a molecule composed of D-glucuronic acid and N-acetyl-D-glucosamine linked by alternating β-(1→4) and β-(1→3) glycosidic bonds, with the chemical formula (C 14 H 21 NO 11 )n.

[0055] Hyaluronic acid can be produced in recombinant cells.

[0056] 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 supplementary copies of native (e.g., endogenous) genes at levels that differ from their native levels.

[0057] As used herein, the term "recombinant" when used in reference to a nucleic acid or vector is a sequence formed / obtained by genetic engineering techniques well known to those skilled in the art. The US National Institutes for Health (NIH) guidelines therefore state: “Recombinant […] nucleic acids are i. (a) a molecule constructed by joining nucleic acid molecules, and (b) a molecule that can replicate in living cells, i.e., 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 after insertion of or joining together with another nucleic acid.

[0058] Proteins produced by expression of recombinant DNA or recombinant vectors in living cells are also called recombinant proteins.

[0059] The term "recombinant" is therefore synonymous with the term "genetically modified." The term "gene" is synonymous with the term "nucleic acid" or "nucleotide sequence."

[0060] Recombinant nucleic acid sequences for use in the recombinant cells, particularly recombinant yeast, of the present invention can 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 from or derived from a native (e.g., endogenous), naturally occurring gene, or is a heterologous nucleic acid, or that has been modified to contain segments of nucleic acid 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 expression of a coding sequence, the control sequence being operably linked to the coding sequence. Non-limiting examples of regulatory elements include promoters, enhancers, silencers, terminators, and poly-A signals.

[0061] Recombinant nucleic acid sequences for use in the recombinant cells of the present invention may be provided in the form of an expression vector, wherein the polynucleotide sequence is operably linked to at least one control sequence for expression of the polynucleotide sequence in the recombinant cell.

[0062] 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.

[0063] 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 to (i.e., present as is) that microorganism or animal. For example, with respect to a nucleic acid sequence derived from a microorganism or animal, the nucleic acid sequence may represent a recoded and / or shortened version of the native nucleic acid sequence derived from that microorganism or animal. Other modifications known to those skilled in the art can be made to a substance native to a microorganism or animal such that the substance used is "derived from" that microorganism or animal.

[0064] As used herein, the term "polypeptide" refers to a molecule comprising amino acid residues linked by peptide bonds and comprising more than five amino acid residues. Amino acids are identified by either single-letter or three-letter codes. As used herein, the term "protein" is synonymous with the term "polypeptide" and may further refer to two or more polypeptides. Thus, the terms "protein," "peptide," and "polypeptide" can be used interchangeably. Polypeptides can optionally be modified to add functionality (e.g., glycosylation, phosphorylation, acylation, farnesylation, prenylation, sulfonation, etc.). Polypeptides that exhibit activity can 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 can be produced.

[0065] Polypeptides encoded by recombinant nucleic acids for use in recombinant cells of the invention, particularly recombinant yeast, may include signal peptide and / or propeptide sequences. When a polypeptide expressed by a recombinant cell of the invention, particularly recombinant yeast, includes a signal peptide and / or propeptide, sequence identity can be calculated over the mature polypeptide sequence.

[0066] As used herein, the term "operably linked" refers to two or more nucleic acid sequence elements that are physically linked and in a functional relationship to 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, and the coding sequence is then 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.

[0067] The terms "native" or "endogenous" as used herein with reference to molecules, particularly enzymes and nucleic acids, refer to molecules that are expressed in the organism in which they originate or in which they are found in nature.

[0068] 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 can be overexpressed by introducing heterologous sequences in addition to or to replace endogenous regulatory elements, or by introducing one or more additional or supplemental copies of the gene into a chromosome or plasmid (the additional or supplemental copies are referred to as "exogenous or heterologous genes" or "heterologous nucleotide sequences" or "heterologous nucleic acids" as defined herein). Endogenous genes can also be modified to modulate their expression and / or activity. For example, mutations can be introduced into the coding sequence to alter the gene product, or heterologous sequences can be introduced in addition to or to replace endogenous regulatory elements. Modulation of an endogenous gene can 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 (such auxiliary copies are referred to as "exogenous or heterologous genes" or "heterologous nucleotide sequences" or "heterologous nucleic acids" as defined herein).

[0069] "One or more additional or supplementary copies of a gene" according to the present invention is understood to mean, for example, 1 to 50 copies, particularly 1 to 30 copies, more particularly 1 to 20 copies, and preferably 1 to 10 copies. The copies may be inserted into the same or different loci of the recombinant cell of the present invention.

[0070] The term "exogenous gene" means that a gene has been introduced into a cell by means well known to those skilled in the art, but the gene may or may not naturally exist in a wild-type cell. A cell can express exogenous genes if these genes are introduced into the cell along 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 a host chromosome or expressed extrachromosomally from a plasmid or vector. A variety of plasmids, differing 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. Indeed, those skilled in the art are familiar with the concept of codon usage bias and how to adapt a nucleic acid sequence to a particular codon usage bias without altering the predicted protein. In certain embodiments, the codon-optimized gene expresses a native enzyme.

[0071] 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 the cell in a non-native amount (i.e., greater or lesser than that naturally found in the host cell), or (c) naturally found in the host cell but located outside its natural locus.

[0072] In this application, all genes are referred to by their common name, by their nucleotide and, where occurring, by their amino acid sequence. Using the accession number reference for known genes, one skilled in the art can determine the equivalent gene 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 gene in another organism.

[0073] Those skilled in the art are aware of different means for modulating, in particular up-regulating or down-regulating, the expression of endogenous genes. 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.

[0074] Another approach is to replace the gene's endogenous promoter 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.

[0075] The nucleic acid expression construct may further comprise 5' and / or 3' recognition sequences and / or selectable markers.

[0076] 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 - A promoter 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 promoter's activity; the lower the concentration of the metabolite in the medium, the stronger the promoter activity). is used to define

[0077] 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 metabolite in the medium, the weaker the promoter activity), or - A promoter 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 define

[0078] 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 the 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.

[0079] 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), refers to, for example, 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 extracellularly, e.g., after the first protein is cleaved from the second protein.

[0080] As used herein, the terms "secretion signal" and "anchoring signal," when used in connection with a protein or polypeptide, e.g., an enzyme (e.g., hyaluronidase), refer to, for example, a first nucleic acid encoding a peptide or protein operably linked to a second nucleic acid encoding the protein, or a first protein operably linked to a second protein, e.g., an enzyme (e.g., hyaluronidase), that enables the cellular transport machinery of a cell, particularly an S. cerevisiae cell, to locate at least the second protein in the cell membrane, where the second protein is not secreted but remains attached to the cell membrane when operably linked to the "anchoring signal." In some cases, a secretion-anchoring signal can provide dual secretion signal and anchoring signal functions.

[0081] Secretion and anchoring signal sequences, methods for expressing, anchoring, and / or secreting heterologous proteins, such as enzymes (e.g., hyaluronidase) on the surface of cells (e.g., yeast cells), are well known in the art (see, e.g., Ast et al. (2013) Cell 152: 1134-1145; Ast and Schuldiner (2013) Crit Rev Biochem Mol Biol 48(3) 273-288; Van der Vaart et al. (1997) Applied Environmental Microbiology 63(2) 615-620, the entire contents of each of which are incorporated herein by reference).

[0082] The "activity" of an enzyme is used interchangeably with the term "function" and, in the context of the present invention, refers to the ability of the enzyme to catalyze a desired reaction. The amount of an enzyme in a host cell can be altered by modifying the transcription of the gene encoding the enzyme. This can be achieved, for example, by modifying the copy number of the nucleotide sequence encoding the enzyme (e.g., by using a higher or lower copy number expression vector containing the nucleotide sequence, or by introducing additional copies of the nucleotide sequence into the genome of the host cell, or by deleting or disrupting the nucleotide sequence in the genome of the host cell), by changing the order of the coding sequences in the polycistronic mRNA of the operon, or by splitting the operon into individual genes each with its own control elements, or by increasing the strength of the promoter or operator to which the nucleotide sequence is operably linked.

[0083] 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.

[0084] The activity of an enzyme in a host cell can be altered in several ways, including, but not limited to, expressing a modified form of the enzyme that exhibits increased or decreased solubility in the host cell, expressing a modified form of the enzyme that lacks a domain through which the activity of the enzyme is inhibited, expressing a modified form of the enzyme that has a higher or lower Kcat or a lower or higher Km for the substrate, or expressing a modified form of the enzyme that is more or less affected by feedback or feedforward regulation by another molecule in the pathway.

[0085] The terms "encode" or "encoding" refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an amino acid sequence.

[0086] The genes encoding the enzymes contemplated in this invention may be exogenous or endogenous.

[0087] The methods implemented in the present invention preferably require the use of one or more chromosomal integration constructs for the stable introduction of heterologous nucleotide sequences into specific locations on a chromosome or for the functional disruption of one or more target genes in genetically modified cells. In some embodiments, disruption of the target gene prevents expression of the associated functional protein. In some embodiments, disruption of the target gene results in the expression of a non-functional protein from the disrupted gene.

[0088] Thus, a "disrupted endogenous nucleic acid" in the present invention relates to an endogenous nucleic acid or gene that is unable to encode the functional or fully functional protein or polypeptide that it encoded before being disrupted. A nucleic acid can be disrupted, for example, by introducing an integration construct into the nucleic acid, as illustrated in the Examples. The integration can, for example, prevent expression of the relevant functional protein or polypeptide or result in the expression of a non-functional or incompletely functional protein or polypeptide from the disrupted gene.

[0089] Parameters of the chromosomal integration construct that can 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 useful range for 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 approximately 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).

[0090] 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 selecting transformed cells. Preferably, said selectable markers are included in the DNA construct of the present invention.

[0091] 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 Escherichia 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).

[0092] In some embodiments, the antibiotic resistance marker is removed after the genetically modified cells of the present invention are isolated. Those skilled in the art will be able to select appropriate markers for a particular genetic context.

[0093] In certain embodiments, 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.

[0094] 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, e.g., the HIS3, LEU2, LYS1, LYS2, MET15, TRP1, ADE2, and URA3 gene products in yeast, resulting in the parent cell being 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 integrant encoding a functional copy of the disrupted gene product (in some embodiments, the functional copy of the gene can originate from an adjacent species, e.g., Kluyveromyces, Candida, etc.), and the resulting genetically modified cells can be selected based on the loss of the auxotrophy phenotype of the parent microbial cell.

[0095] 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 set forth herein. The description herein also encompasses nucleic acid sequences having a specified percentage of nucleic acid identity with the reference nucleic acid sequence.

[0096] 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 specified percentage of amino acid identity with the reference amino acid sequence.

[0097] For obvious reasons, throughout this specification, a particular nucleic acid sequence or a particular amino acid sequence matching the considered nucleotide or amino acid identity, respectively, should also lead to obtaining a protein (or enzyme) exhibiting the desired biological activity. As used herein, the "percentage identity" between two nucleic acid sequences or two amino acid sequences is determined by comparing both optimally aligned sequences over a comparison window.

[0098] 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 deletions) to ensure optimal alignment between both sequences.

[0099] The terms "sequence homology" or "sequence identity" or "homology" or "identity" are used interchangeably herein. For purposes of the present invention, to determine the percentage of sequence homology or sequence identity between 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.

[0100] The comparison of sequences and determination of the percentage sequence identity between two sequences can be accomplished using a mathematical algorithm. Those skilled in the art will recognize 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, pp. 1-44, Addison Wesley).

[0101] The percent sequence identity between two amino acid sequences or two nucleotide sequences can be determined using the Needleman and Wunsch algorithm for aligning two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE.

[0102] For the purposes of this invention, the NEEDLE program from 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 as 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.

[0103] After alignment with the above-mentioned program NEEDLE, the percentage sequence identity between the query sequence and a sequence of the invention is calculated as follows: the number of corresponding positions in the alignment that show the same amino acid or the same nucleotide 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.

[0104] Nucleotide and amino acid sequence similarity, i.e., percentage sequence identity, can be determined using several other algorithms known in the art, 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 the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), available at https: / / www.ebi.ac.uk / Tools / msa / clustalo / , or the GAP program (a mathematical algorithm from the University of Iowa), or the mathematical algorithm of Myers and Miller (1989 - Cabios 4: 11-17), or Clone Manager. 9. The preferred parameters used are the default parameters set out at https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0105] The grade of sequence identity (percent 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 using the BLASTN program, score=100, word length=12, to obtain polynucleotide sequences homologous to nucleic acids encoding related proteins.

[0106] 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 identity analysis can be aided 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 over the entire length of the longer sequence unless otherwise specifically indicated.

[0107] In certain embodiments, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0108] "Fermentation" or "cultivation" is generally carried out in a fermenter using an appropriate culture medium adapted to the cells being cultured, containing at least one simple carbon source and, if necessary, supplementary substrates.

[0109] 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 broth, which can be the entire contents of a vessel (e.g., a flask, plate, or fermenter) containing the cells, the aqueous phase, and the compounds produced from the genetically modified host cells.

[0110] The term "medium" refers to a culture medium, or a cultivation medium, or a fermentation medium.

[0111] For maximum production of hyaluronic acid, recombinant cells used as production hosts preferably have a high carbohydrate utilization rate. These characteristics may be conferred by mutagenesis and selection, genetic engineering, or may be natural. The fermentation medium or "culture medium" or "cultivation medium" of the cells may contain at least about 10 g / L of glucose and / or sucrose. Additional carbon substrates may include, but are not limited to, monosaccharides such as fructose, mannose, xylose, and arabinose; oligosaccharides such as lactose, maltose, galactose, or sucrose; polysaccharides such as starch or cellulose, or mixtures thereof; and crude mixtures derived 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.

[0112] Thus, it is contemplated that the carbon source utilized in the present invention can encompass a wide range of carbon-containing substrates, limited only by the choice of cell, particularly yeast.

[0113] While all of the above carbon substrates and mixtures thereof are contemplated as 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 of these with C5 sugars, such as xylose and / or arabinose.

[0114] The preferred carbon substrate is glucose or sucrose.

[0115] 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 promotion of the enzymatic pathways necessary for production of the desired product.

[0116] Furthermore, additional genetic modifications suitable for the growth of recombinant cells according to the present invention may be considered.

[0117] 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.

[0118] "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.

[0119] A "suitable culture medium" refers to a culture medium (e.g., a sterile, liquid medium) containing 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 supply source" according to the present invention refers to any carbon source that can be used by those skilled in the art 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.

[0120] Particularly suitable culture media for producing the recombinant cells of the invention, and in particular the recombinant yeasts of the invention, are described in more detail below.

[0121] 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.

[0122] General characteristics of the genetic modifications introduced by the present invention - All genome modifications are inserted into recombinant cells, in particular recombinant yeast, according to known genetic engineering techniques: - the contiguous nucleic acid sequence contained in the genetic construct to be introduced into the genome of the 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 in newly synthesized mRNA by providing a signal that triggers the process of releasing the mRNA from the transcription complex; - "1", "2", ... / ... "n" may or may not refer to the same ORF (open reading frame), promoter, or terminator. The order of the nucleic acid sequences does not matter. "n" is usually an integer ranging from 5 to 20. These constructs are inserted into one of the recombinant cell chromosomes at a controlled location. In some embodiments, the insertion site is not important for the functionality of the inserted construct or the viability of the resulting genetically modified cell.

[0123] 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 utilized in a species are referred to as optimal codons, while those utilized 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 "controlling for 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 a 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. The 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).

[0124] - When the recombinant cell is a yeast cell, in particular a Saccharomyces cerevisiae yeast cell, nucleic acid sequences originating from other organisms than Saccharomyces cerevisiae that are introduced into the yeast genome are generally "transcoded" (generally "codon optimized"), meaning that these nucleic acid sequences are synthesized with a codon usage that is optimal 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 endogenous copies of the gene.

[0125] - 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 gene constructs described above, or alternatively, the gene targeted for deletion is replaced by a short stretch of nucleotides.

[0126] - 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, including induction or repression by abiotic factors, such as compounds not naturally found in the cells of interest, particularly yeast, light, oxygen levels, heat, or low temperature. Lists and sequences of inducible or repressible promoters are provided elsewhere herein.

[0127] Recombinant cells according to the present invention The present inventors have envisioned recombinant cells, particularly recombinant yeast, that have the ability to produce hyaluronic acid.

[0128] The present invention relates to recombinant cells, in particular recombinant yeast, that have the ability to produce hyaluronic acid, where this ability to produce hyaluronic acid is obtained through multiple alterations that have been introduced into their genome by genetic engineering methods.

[0129] The present invention provides a recombinant yeast cell that produces hyaluronic acid (HA), comprising: (a) one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase 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 hyaluronidase activity, wherein the polypeptide having hyaluronidase activity comprises a secretion signal such that hyaluronic acid, particularly of a desired molecular weight (HAMW), is produced by the recombinant yeast cell; and (d)(i) one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase (GLN1) activity, and / or (ii) one or more disrupted endogenous nucleic acids encoding glutamate synthase (GLT1); Including, The recombinant yeast cell belongs to the genus Saccharomyces, or Candida, or Kluyveromyces, or Ogataea, or Yarrowia, or Debaryomyces, or Ashbya, and in particular to Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces castellii, or Candida albica. lance, Candida glabrata, Candida tropicalis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus, Kluyveromyces thermotolerans, Ogataea polymorpha, Yarrowia lipolytica, Debaryomyces hansenii, and Ashbya gossypii, preferably Saccharomyces cerevisiae; Recombinant yeast cells.

[0130] The present invention further provides a recombinant host cell that produces hyaluronic acid (HA), comprising: (a) one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase 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 hyaluronidase activity, wherein the polypeptide having hyaluronidase activity comprises a secretion signal and an anchoring signal such that hyaluronic acid, particularly of a desired molecular weight (HAMW), is produced by the host cell; and (d)(i) one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase (GLN1) activity, and / or (ii) one or more disrupted endogenous nucleic acids encoding glutamate synthase (GLT1); The present invention relates to a recombinant host cell comprising the

[0131] The present inventors have discovered that the ability of cells, particularly yeast cells, to produce hyaluronic acid can be achieved by introducing multiple genetic alterations into the genome of these cells.

[0132] The production of hyaluronic acid by the cells of the present invention, particularly the yeast cells of the present invention, is achieved by optimizing the endogenous metabolism of UDP-glucose, and optionally UDP-N-acetyl-glucosamine, and directing the subsequent artificially engineered metabolic pathway primarily towards hyaluronic acid, while maintaining optimal viability of the resulting genetically engineered cells.

[0133] It has been determined that the production of hyaluronic acid by recombinant cells according to the present invention can be increased by increasing the conversion of glucose-6-phosphate to the continuous intermediate metabolic products (i) glucose-1-phosphate, UDP-glucose, UDP-glucuronate, and hyaluronic acid, and (ii) fructose-6-phosphate, glucosamine-6-phosphate, N-acetyl-glucosamine-6-phosphate, N-acetyl-glucosamine-1-phosphate, UDP-N-acetyl-glucosamine, and hyaluronic acid, while maintaining a metabolic balance that allows for good viability of the resulting recombinant cells.

[0134] Indeed, in order to obtain viable recombinant cells of the present invention, many different constructs were tested in order to obtain viable and efficient recombinant cells, in particular viable recombinant yeast, which have been difficult to obtain because the transient accumulation of some intermediates appeared to be toxic to the yeast.

[0135] Unexpected technical challenges were encountered in establishing suitable conditions for the preparation of recombinant cells capable of producing hyaluronic acid, particularly hyaluronic acid with a controlled molecular weight.

[0136] A "controlled" molecular weight of the hyaluronic acid of the present invention means that at least 80%, in particular at least 85%, of the hyaluronic acid produced by the method of the present invention is controlled by at least one parameter of the method and / or recombinant cell of the present invention, such as, for example: - the nature and origin of the nucleic acid encoding the hyaluronidase of the recombinant cell; - the nature and origin of the promoter controlling expression of the nucleic acid encoding the hyaluronidase in the recombinant cell; - the presence of an anchoring and / or secretion signal associated with the encoded hyaluronidase of the recombinant cell; - pH of the culture medium during the recombinant cell culture process, and / or - Duration of recombinant cell culture By adjusting for is intended to mean having a molecular weight that falls within a certain range of molecular weights.

[0137] Indeed, after extensive research and experimental testing, the present inventors have cultured recombinant cells, in particular recombinant yeast cells, more particularly recombinant Saccharomyces cerevisiae yeast cells, to obtain the following parameters: - selection of the nature and source of the nucleic acid sequence encoding the polypeptide having hyaluronidase activity in the recombinant cell of the invention, in particular in the recombinant yeast; and / or - the nature and origin of the promoter controlling the expression of the nucleic acid sequence encoding the polypeptide having hyaluronidase activity in the recombinant cell of the invention, in particular in the recombinant yeast, and / or the optional presence of an anchoring signal, in addition to the secretion signal, associated with the encoded polypeptide having hyaluronidase activity in a recombinant cell, in particular a recombinant yeast, according to the invention; and / or the pH of the culture medium during the process of culturing the recombinant cells according to the invention, in particular the recombinant yeast cells, and / or - the duration of the cultivation of the recombinant cells, in particular recombinant yeast cells, according to the invention; It has been discovered that it is possible to produce hyaluronic acid with a controlled molecular weight using

[0138] To the inventors' knowledge, this has never been achieved before.

[0139] The molecular weight of hyaluronic acid (HA) can be in the range of less than 50 kDa, preferably in the range of about 20 kDa to about 50 kDa.

[0140] Alternatively, the molecular weight of the hyaluronic acid (HA) can be in the range above 50 kDa, preferably in the range of about 50 kDa to about 250 kDa.

[0141] In another alternative, the molecular weight of the HA may be in the range above 100 kDa, preferably in the range of about 100 kDa to about 1500 kDa.

[0142] Nucleic acid encoding a polypeptide having glutamine synthetase activity can be obtained or derived from Saccharomyces cerevisiae.

[0143] The nucleic acid encoding a polypeptide having hyaluronidase activity in the recombinant cells of the present invention can be obtained or derived from at least one of Cupienius salei, Loxosceles intermedia, Hirudo nipponia, Bothropus atrox, or Titius serulatus.

[0144] The nucleic acid encoding a polypeptide having hyaluronan synthase activity in the recombinant cell of the invention can be obtained or derived from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviK1, Chlorella virus IL-5-2s1, Chlorella virus CZ-2, Chlorella virus CVG-1, Xenopus laevis, or Pasteurella multocida, and in particular, can be obtained or derived from Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviK1, Chlorella virus IL-5-2s1, Chlorella virus CZ-2, Chlorella virus CVG-1, or Xenopus laevis.

[0145] The nucleic acid encoding a polypeptide having UDP-glucose dehydrogenase activity in the recombinant cell of the present invention can be obtained or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, and in particular can be obtained or derived from Arabidopsis thaliana or Chlorella virus PBCV1.

[0146] The recombinant cell according to the present invention comprises: (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity The recombinant nucleic acid may include a recombinant nucleic acid encoding one or more of:

[0147] The recombinant cell according to the present invention comprises: (i) a polypeptide having phosphoglucomutase-1 (PGM1) activity, and / or (ii) a polypeptide having UTP-glucose-1-phosphate uridylyltransferase (UGP1) activity, and / or (iii) a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity, and / or (iv) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity The recombinant nucleic acid may comprise at least one recombinant nucleic acid encoding one or more of:

[0148] The recombinant yeast cell according to 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.

[0149] The recombinant host cell according to the invention may belong to the order Saccharomycetales, and in particular 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.

[0150] Another object of the present invention is a method for producing hyaluronic acid (HA) of a desired molecular weight (HAMW), comprising: (a) culturing a recombinant cell as defined above, i.e., a recombinant yeast cell or a recombinant host cell of the present invention, in a culture medium for a time sufficient to produce hyaluronic acid (HA) of the desired molecular weight; (b) optionally isolating or recovering hyaluronic acid (HA) from the recombinant cells and / or culture medium; The present invention relates to a method comprising:

[0151] In the methods of the present invention, the HA may have a molecular weight of about 20 kDa to about 50 kDa, and preferably 20 kDa to 30 kDa.

[0152] In the methods of the present invention, the HA may alternatively have a molecular weight of between 30 kDa and 50 kDa.

[0153] In the methods of the present invention, the HA may alternatively have a molecular weight of about 50 kDa to about 150 kDa.

[0154] In the methods of the present invention, the HA may alternatively have a molecular weight of about 150 kDa to about 1500 kDa.

[0155] In the methods of the present invention, a nucleic acid encoding a polypeptide having hyaluronidase activity can be obtained or derived from Cupienius salei, Loxosceles intermedia, Hirudo nipponia, Bothropus atrox, or Titius serulatus.

[0156] In the methods of the present invention, the nucleic acid encoding a polypeptide having hyaluronan synthase activity can be obtained or derived from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviK1, Chlorella virus IL-5-2s1, Chlorella virus CZ-2, Chlorella virus CVG-1, Xenopus laevis, or Pasteurella multocida, and in particular from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviK1, Chlorella virus IL-5-2s1, Chlorella virus CZ-2, Chlorella virus CVG-1, and Xenopus laevis.

[0157] In the methods of the present invention, the nucleic acid encoding a polypeptide having UDP-glucose dehydrogenase activity can be obtained or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, and in particular can be obtained or derived from Arabidopsis thaliana or Chlorella virus PBCV1.

[0158] In the method of the present invention, the recombinant cell (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity The recombinant nucleic acid may comprise at least one recombinant nucleic acid encoding one or more of:

[0159] In the method of the present invention, the recombinant cell (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 The recombinant nucleic acid may comprise at least one recombinant nucleic acid encoding one or more of:

[0160] In the methods of the present invention, the recombinant cell may be a member of the genus Saccharomyces, and in particular Saccharomyces cerevisiae.

[0161] In the method of the present invention, the molecular weight of hyaluronic acid can be controlled by the fermentation time.

[0162] In the methods of the present invention, the time sufficient to produce hyaluronic acid (HA) 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.

[0163] In the method of the present invention, the molecular weight of hyaluronic acid can be controlled by the pH of the culture medium.

[0164] In the method of the present invention, the molecular weight of hyaluronic acid can be controlled by adjusting the pH of the culture medium during the culture step (a) of the method of the present invention.

[0165] In the methods of the present invention, the molecular weight of the hyaluronic acid can be controlled by removing biomass from the culture medium.

[0166] The method of the present invention is carried out on an industrial scale, preferably the culture medium is at least about 100 L, more preferably in the range of about 1000 L to about 3000 L, even more preferably about 10,000 L, even more preferably about 100,000 L, or even about 250,000 L.

[0167] Another object of the present invention relates to hyaluronic acid (HA) obtained or obtainable from the recombinant cell of the invention or the method of the invention.

[0168] A further object of the present invention relates to a culture medium comprising hyaluronic acid (HA) according to the invention.

[0169] The present invention further relates to a composition comprising hyaluronic acid (HA) according to the present invention.

[0170] The present invention further relates to (i) HA having a molecular weight in the range above 100 kDa, preferably in the range of about 100 kDa to about 1500 kDa, (ii) a culture medium of the present invention, or (iii) an industrial or consumer product or consumable product comprising the composition of the present invention.

[0171] The present invention also relates to the use of the recombinant cells according to the present invention for producing hyaluronic acid (HA) having a molecular weight in the range of about 20 kDa to about 50 kDa or about 50 kDa to about 1000 kDa.

[0172] Another object of the present invention is a method for producing hyaluronic acid, comprising: (a) culturing the recombinant yeast according to the present invention in a culture medium; (b) recovering hyaluronic acid from the culture medium; Including, The hyaluronic acid recovered in step (b) - the nature and origin of the nucleic acid encoding the recombinant yeast hyaluronidase; - the nature and origin of the promoter controlling the expression of the nucleic acid encoding the recombinant yeast hyaluronidase; - the presence of an anchoring and / or secretion signal associated with the recombinant yeast encoded hyaluronidase; - 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 Regarding the method.

[0173] More specifically, the present invention relates to a recombinant yeast that produces hyaluronic acid, comprising: (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; and (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 hyaluronan synthase (HASA) activity; (E) one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity, wherein the polypeptide having hyaluronidase activity comprises a secretion signal such that hyaluronic acid, particularly of a desired molecular weight (HAMW), is produced by the recombinant yeast cell; and Including, The recombinant yeast is Saccharomyces cerevisiae. Concerning recombinant yeast.

[0174] In a particular embodiment, a recombinant cell according to the invention, in particular a recombinant yeast according to the invention, comprises one recombinant nucleic acid encoding a polypeptide having glutamine synthetase (GLN1) activity.

[0175] In a particular embodiment, the recombinant cell according to the invention, in particular the recombinant yeast according to the invention, is such that at least one, in particular all, of its endogenous nucleic acids encoding glutamate synthase (GLT1) are disrupted.

[0176] In a particular embodiment, the recombinant cell according to the invention, in particular the recombinant yeast according to the invention, - one recombinant nucleic acid encoding a polypeptide having glutamine synthetase (GLN1) activity, in particular a polypeptide having glutamine synthetase (GLN1) activity obtained or derived from Saccharomyces cerevisiae, - such that at least one, and in particular all, of its endogenous nucleic acids encoding glutamate synthase (GLT1) are disrupted.

[0177] In a particular embodiment, a recombinant cell according to the invention, in particular a recombinant yeast according to the invention, comprises only one recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity.

[0178] In certain embodiments, a recombinant cell, particularly a recombinant yeast, according to the invention comprises 5 to 10 recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity.

[0179] In certain embodiments, a recombinant cell, particularly a recombinant yeast, according to the invention comprises 3 to 7 recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity.

[0180] In another embodiment, the one or more nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity are obtained or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, and in particular, are obtained or derived from Arabidopsis thaliana or Chlorella virus PBCV1.

[0181] In certain embodiments, recombinant cells, particularly recombinant yeast, according to the invention comprise 4 to 8 recombinant nucleic acids encoding polypeptides having hyaluronan synthase (HASA) activity.

[0182] In another embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase (HASA) activity are obtained or derived from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviK1, Chlorella virus IL-5-2s1, Chlorella virus CZ-2, Chlorella virus CVG-1, Xenopus laevis, or Pasteurella multocida, in particular obtained or derived from Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviK1, Chlorella virus IL-5-2s1, Chlorella virus CZ-2, Chlorella virus CVG-1, or Xenopus laevis.

[0183] In certain embodiments, recombinant cells according to the invention, particularly recombinant yeast cells according to the invention, contain only one recombinant nucleic acid encoding a polypeptide having hyaluronidase activity.

[0184] In another embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity are obtained or derived from Cupienius salei, Loxosceles intermedia, Hirudo nipponia, Bothropus atrox, or Titius serulatus.

[0185] In another embodiment, the recombinant cell according to the invention, in particular the 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) Polypeptide having phosphoacetylglucosamine mutase (PCM1) activity The recombinant nucleic acid may comprise at least one recombinant nucleic acid encoding one or more of:

[0186] 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 in particular all of the modifications set out above.

[0187] In certain embodiments, the nucleic acids encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity, the nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase (UGP1) activity, the nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, the nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity, the nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity, and the nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity are nucleic acids originating from or derived from yeast, preferably Saccharomyces cerevisiae.

[0188] In a particular embodiment, one or more recombinant nucleic acids encoding a polypeptide as defined above contained in a recombinant cell according to the invention, in particular contained in a recombinant yeast of the invention, are under the control of a promoter selected from the group consisting of pPDC1, pTDH3, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, pCCW12.sar, pTEF1, pENO2, pRPLA1, pNUP57, and pTEF3.

[0189] 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, and in particular selected from the group consisting of pMET6, pMET25, and pSAM1.

[0190] The present invention further provides a method for producing the aforementioned hyaluronic acid, comprising: (a) culturing a recombinant cell according to the invention, in particular a recombinant yeast as defined herein, in a culture medium; (b) recovering hyaluronic acid from the culture medium; Including, The hyaluronic acid recovered in step (b) - the nature and origin of the nucleic acid encoding the hyaluronidase of the recombinant cell according to the invention, in particular of the recombinant yeast according to the invention; and / or - the nature and origin of the promoter controlling the expression of the nucleic acid encoding the hyaluronidase in the recombinant cell according to the invention, in particular in the recombinant yeast, and / or - the presence of an anchoring and / or secretion signal associated with the encoded hyaluronidase in a recombinant cell, particularly a recombinant yeast, according to the invention; 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 period for which the recombinant cell, in particular the recombinant yeast, according to the invention is cultivated; having a molecular weight controlled through the selection of Regarding the method.

[0191] Recombinant nucleic acids encoding polypeptides having hyaluronan synthase activity Recombinant cells according to the invention, particularly recombinant yeasts according to the invention, comprise one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity.

[0192] A polypeptide having hyaluronan synthase activity according to the present invention refers to a polypeptide that converts the intermediate metabolites UDP-glucuronate and UDP-N-acetylglucosamine (UDP-GlcNAc) into hyaluronic acid ((β-D-1,3-GlcNAc-β-D-1,4-GlcA)n).

[0193] In one embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having hyaluronan synthase 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 pCUP1 or pMET25.

[0194] One or more of the recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity can be under the control of a promoter selected from the group consisting of pCCW12, pCCW12.Sm, pTDH3-1.Sba, and pTDH3.Sar.

[0195] The one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may originate from or be derived from at least one of Streptococcus zooepidemicus (sz), Chlorella virus PBCV1 (Vir), Chlorella virus CviKl (Vir), Chlorella virus IL-5-2s1 (Vir), Chlorella virus CZ-2 (Vir), Chlorella virus CVG-1 (Vir), Xenopus laevis (xl), or Pasteurella multocida (pm), and in particular, as shown in the Examples herein, may originate from or be derived from Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviKl, Chlorella virus IL-5-2s1, Chlorella virus CZ-2, Chlorella virus CVG-1, or Xenopus laevis.

[0196] Recombinant cells according to the present invention, particularly recombinant yeast according to the present invention, may comprise 2 to 8, specifically 4 to 8, recombinant nucleic acids encoding polypeptides having hyaluronan synthase (HASA) activity. Recombinant cells according to the present invention, particularly recombinant yeast according to the present invention, may comprise, for example, 2 or 6, specifically 6, recombinant nucleic acids encoding polypeptides having hyaluronan synthase (HASA) activity.

[0197] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity can be inserted into the JLP1 gene, and / or SAM3 gene, and / or TRP1 gene, and / or LYP1 gene of a recombinant cell, particularly a recombinant yeast, as shown in the Examples herein.

[0198] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast, - 2 to 8, particularly 4 to 8, recombinant nucleic acids encoding a polypeptide having hyaluronan synthase (HASA) activity; - one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity originating from or derived from at least one of Streptococcus zooepidemicus (sz), Chlorella virus PBCV1 (Vir), Xenopus laevis (xl), or Pasteurella multocida (pm), in particular originating from or derived from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1, or Xenopus laevis; and - one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity, which is functional in a recombinant cell of the invention, such as, for example, under the control of the inducible or repressible promoter pCUP or pMET25, and / or under the control of a promoter selected from the group consisting of pCCW12, pCCW12.Sm, pTDH3-1.Sba, and pTDH3.Sar. Includes:

[0199] Recombinant nucleic acids encoding polypeptides having UDP-glucose dehydrogenase activity Recombinant cells according to the invention, in particular recombinant yeasts according to the invention, comprise one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity.

[0200] A polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity according to the present invention means a polypeptide that converts the intermediate metabolite uridine-diphosphate-glucose (UDP-glucose) to UDP-glucuronate.

[0201] In one embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase (UDP-GlcDH or HASB) activity is under the control of an inducible or repressible promoter that is functional in a recombinant cell of the invention, such as, for example, the inducible or repressible promoter pMET25 or pMET6.

[0202] One or more of the recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity may be under the control of pCCW12, in particular a promoter selected from the group consisting of the pCCW12.sk and pCCW12.sba promoters, pTEF1.Sba, and pTDH3.Sk.

[0203] The one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase dehydrogenase activity may originate from or be derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, and in particular may originate from or be derived from Arabidopsis thaliana or Chlorella virus PBCV1.

[0204] The recombinant cell according to the present invention, particularly the recombinant yeast according to the present invention, may comprise 2 to 7, specifically 3 to 7, recombinant nucleic acids encoding polypeptides having UDP-glucose dehydrogenase activity. The recombinant cell according to the present invention, particularly the recombinant yeast according to the present invention, may comprise, for example, 2 or 5 recombinant nucleic acids encoding polypeptides having UDP-glucose dehydrogenase activity.

[0205] 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 TRP1 gene, and / or the LYP1 gene of a recombinant cell, particularly a recombinant yeast, as shown in the Examples herein.

[0206] In one embodiment of the invention, the recombinant cell of the invention, in particular the recombinant yeast, - 2 to 7, in particular 3 to 7, recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity, - one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity originating from or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, in particular originating from or derived from Arabidopsis thaliana or Chlorella virus PBCV1; and - one or more recombinant nucleic acids 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, for example under the control of the inducible or repressible promoter pMET25 or pMET6, and / or under the control of a promoter selected from the group consisting of the pCCW12.sk and pCCW12.sba promoters, pTEF1.Sba, and pTDH3.Sk. Includes:

[0207] Recombinant nucleic acids encoding polypeptides having hyaluronidase activity Recombinant cells according to the invention, particularly recombinant yeast according to the invention, comprise one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity.

[0208] A polypeptide having hyaluronidase activity according to the present invention refers to a polypeptide that degrades hyaluronic acid, i.e., converts hyaluronic acid of a given molecular weight into hyaluronic acid of a lower molecular weight.

[0209] As described above, the polypeptides having hyaluronidase activity of the present invention contain a secretion signal.

[0210] In some embodiments, the polypeptide having hyaluronidase activity comprises both a secretory signal and an anchoring signal.

[0211] In one embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity are under the control of an inducible or repressible promoter that is functional in the recombinant cell of the invention.

[0212] One or more of the recombinant nucleic acids encoding a polypeptide having hyaluronidase activity can be under the control of a promoter selected from the group consisting of pTEF1, pCCW12, pCCW12.sba, pCCW12.Sar, pPDC1, pTEF3, pTDH3, pNUP57, pCWP2, and pCCW10.ago.

[0213] The one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity can originate from or be derived from at least one of Cupienius salei (Csa), Loxosceles intermedia (Li), Hirudo nipponia (Hn), Bothropus atrox (Ba), or Titius serulatus (Ts), as shown in the Examples herein.

[0214] Recombinant cells according to the invention, particularly recombinant yeast according to the invention, can contain only one recombinant nucleic acid encoding a polypeptide having hyaluronidase activity.

[0215] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity can be inserted into the JLP1 gene and / or the LYP1 gene, as shown in the Examples herein.

[0216] 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 hyaluronidase activity, - a recombinant nucleic acid encoding a polypeptide having hyaluronidase activity, the recombinant nucleic acid originating from or derived from Cupienius salei (Csa), Loxosceles intermedia (Li), Hirudo nipponia (Hn), Bothropus atrox (Ba), or Titius serrata (Ts), - a recombinant nucleic acid encoding a polypeptide having hyaluronidase activity, wherein the recombinant nucleic acid (i) comprises a secretory signal and no anchoring signal, or (ii) comprises a secretory signal and an anchoring signal; and - the recombinant nucleic acid encoding a polypeptide having hyaluronidase activity is under the control of a promoter selected from the group consisting of pTEF1, pCCW12, pCCW12.sba, pCCW12.Sar, pPDC1, pTEF3, pTDH3, pNUP57, pCWP2, and pCCW10.ago Includes:

[0217] Recombinant nucleic acids encoding polypeptides having glutamine synthetase activity Recombinant cells according to the invention, in particular recombinant yeast cells according to the invention, can comprise one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity.

[0218] The polypeptides having glutamine synthetase activity according to the present invention convert glutamate to glutamine, and at the same time, one ATP and one NH + "Consuming" refers to a polypeptide.

[0219] In certain embodiments, one or more of the recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity is under the control of an inducible or repressible promoter that is functional in a recombinant cell of the invention.

[0220] One or more of the recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity can be under the control of a promoter selected from the group consisting of pTEF1 and pTEF1.Ago.

[0221] The one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity can originate from or be derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0222] 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 synthetase activity.

[0223] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity can be inserted into the LYP1 or GLT1 gene of a recombinant cell, particularly a recombinant yeast cell, as shown in the Examples herein.

[0224] In one embodiment of the invention, the recombinant cell of the invention, particularly the recombinant yeast cell, - only one recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity, - a recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity originating from or derived from Saccharomyces cerevisiae; and - the recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity, under the control of a promoter selected from the group consisting of pTEF1 and pTEF1.Ago Includes:

[0225] In a further 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 glutamine synthase activity, - a recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity, said recombinant nucleic acid originating from or derived from Saccharomyces cerevisiae; - said recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity under the control of a promoter selected from the group consisting of pTEF1 and pTEF1.Ago; and - a recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity, which is inserted into the LYP1 or GLT1 gene of a recombinant cell, in particular a recombinant yeast cell; Includes:

[0226] Recombinant nucleic acids encoding polypeptides having glutamine-fructose-6-phosphate amidotransferase activity Recombinant cells according to the invention, in particular recombinant yeast cells according to the invention, can comprise one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity.

[0227] 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.

[0228] 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.

[0229] One or more of the recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity can be under the control of a promoter selected from the group consisting of pTEF1 and pTEF1.Ago.

[0230] The one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may originate from or be derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0231] 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.

[0232] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity can be inserted into the SAM3 gene of a recombinant cell, particularly a recombinant yeast cell, as shown in the Examples herein.

[0233] In one embodiment of the invention, the recombinant cell of the invention, particularly the recombinant yeast cell, - only one 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 originating from or derived from Saccharomyces cerevisiae; and - the recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity, under the control of a promoter selected from the group consisting of pTEF1 and pTEF1.Ago Includes:

[0234] Recombinant nucleic acids encoding polypeptides having UDP-N-acetylglucosamine pyrophosphorylase activity Recombinant cells according to the invention, particularly recombinant yeast cells according to the invention, can comprise one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity.

[0235] A polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity according to the present invention means a polypeptide that can convert N-acetylglucosamine to UDP-N-acetylglucosamine.

[0236] 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.

[0237] 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.

[0238] The one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity can originate from or be derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0239] A recombinant cell according to the present invention, particularly a recombinant yeast cell according to the present invention, may contain 5 to 10 recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity. A recombinant cell according to the present invention, particularly a recombinant yeast cell according to the present invention, may contain, for example, 5, 7, or 8 recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity.

[0240] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity can be inserted into the HIS3 gene, and / or the JLP1 gene, and / or the SAM3 gene of a recombinant cell, particularly a recombinant yeast, as shown in the Examples herein.

[0241] In one embodiment of the invention, the recombinant cell of the invention, particularly the recombinant yeast cell, - 5 to 10 recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity; - one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity, said nucleic acid or nucleic acids originating from or derived from Saccharomyces cerevisiae; - one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity, under the control of an inducible or repressible promoter that is functional in the recombinant cell of the invention, such as, for example, the inducible or repressible promoter pMET6 or pCUP1 and / or the promoter pTDH3. Includes:

[0242] Recombinant nucleic acids encoding polypeptides having phosphoglucomutase-1 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 phosphoglucomutase-1 (PGM1) activity.

[0243] A polypeptide having phosphoglucomutase-1 (PGM1) activity according to the present invention means a polypeptide that converts glucose-6-phosphate into the intermediate metabolite glucose-1-phosphate.

[0244] In one embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity is under the control of an inducible or repressible promoter that is functional in a recombinant cell of the invention, such as, for example, an inducible or repressible promoter.

[0245] One or more of the recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity can be under the control of promoter pPDC1.

[0246] The one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 (PGM1) activity can originate from or be derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0247] 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.

[0248] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity can be inserted into the SAM3 gene of a recombinant cell, particularly a recombinant yeast, as shown in the Examples herein.

[0249] 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 phosphoglucomutase-1 activity, - a recombinant nucleic acid encoding a polypeptide having phosphoglucomutase-1 activity, said recombinant nucleic acid originating from or derived from Saccharomyces cerevisiae; and - said recombinant nucleic acid encoding a polypeptide having phosphoglucomutase-1 activity, under the control of the promoter pPDC1 Includes:

[0250] Recombinant nucleic acids encoding polypeptides having UTP-glucose 1-phosphate uridylyltransferase activity 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.

[0251] A polypeptide having UTP-glucose 1-phosphate uridylyltransferase (UGP1) activity according to the present invention means a polypeptide that converts the intermediate metabolite glucose-1-phosphate into the intermediate metabolite UDP-glucose.

[0252] 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.

[0253] One or more of the recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity can be under the control of a promoter selected from the group consisting of pPDC1 and pENO2.

[0254] The one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity can originate from or be derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0255] A recombinant cell according to the present invention, particularly a recombinant yeast cell according to the present invention, may comprise 5 to 10 recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity. A recombinant cell according to the present invention, particularly a recombinant yeast cell according to the present invention, may comprise, for example, 5, 7, or 8 recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity.

[0256] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having a UTP-glucose 1-phosphate uridylyltransferase activity gene can be inserted into the HIS3 gene, and / or the JLP1 gene, and / or the SAM3 gene of a recombinant cell, particularly a recombinant yeast, as shown in the Examples herein.

[0257] In one embodiment of the invention, the recombinant cell of the invention, particularly the recombinant yeast cell, - 5 to 10 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, said polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity originating from or derived from Saccharomyces cerevisiae; and - said 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 promoter pSAM1 or pCUP1, and / or under the control of a promoter selected from the group consisting of pPDC1 and pENO2; Includes:

[0258] Recombinant nucleic acids encoding polypeptides having glucosamine-6-phosphate N-acetyltransferase activity Recombinant cells according to the invention, in particular recombinant yeast cells according to the invention, can comprise one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase (GNA1) activity.

[0259] 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.

[0260] 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 a recombinant cell of the invention.

[0261] 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.

[0262] The one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity can originate from or be derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0263] 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.

[0264] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity can be inserted into the SAM3 gene of a recombinant cell, particularly a recombinant yeast, as shown in the Examples herein.

[0265] 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 glucosamine-6-phosphate N-acetyltransferase activity, - a recombinant nucleic acid encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity, said recombinant nucleic acid originating from or derived from Saccharomyces cerevisiae; and - the recombinant nucleic acid encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity, under the control of the promoter pCWP2 Includes:

[0266] Recombinant nucleic acids encoding polypeptides having phosphoacetylglucosamine mutase activity Recombinant cells according to the invention, in particular recombinant yeast cells according to the invention, can comprise one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity.

[0267] A polypeptide having phosphoacetylglucosamine mutase (PCM1) activity according to the present invention refers to a polypeptide that converts N-acetyl-glucosamine-6-phosphate to N-acetyl-glucosamine-1-phosphate.

[0268] 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.

[0269] One or more of the recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity can be under the control of a promoter selected from the group consisting of pTEF3 and pTEF1.

[0270] The one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity can originate from or be derived from Saccharomyces cerevisiae, as shown in the Examples herein.

[0271] 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 phosphoacetylglucosamine mutase activity.

[0272] Illustratively, one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity can be inserted into the SAM3 gene of a recombinant cell, particularly a recombinant yeast, as shown in the Examples herein.

[0273] In one embodiment of the invention, the recombinant cell of the invention, particularly 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; and - the recombinant nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase activity, under the control of a promoter selected from the group consisting of promoters pTEF3 and pTEF1 Includes:

[0274] Hyaluronan synthase (HASA) Hyaluronan synthase enzymes are proteins described in the art for catalyzing the conversion of UDP-glucuronate or UDP-N-acetyl-glucose to hyaluronic acid. Hyaluronan synthases originating from Streptococcus zooepidemicus, Chlorella virus PBCV1, Xenopus laevis, or Pasteurella multocida may be referred to as HASA.

[0275] Methods implemented to measure the activity level of a polypeptide having hyaluronan synthase activity belong to the general knowledge of a person skilled in the art.

[0276] In this regard, the skilled person can advantageously refer to the method of colorimetric determination after treatment with concentrated sulfuric acid and carbazole described by Bitter and Muir (Analytical Biochemistry, 4, 330-334, 1962).

[0277] A preferred polypeptide having hyaluronan synthase activity according to the present invention is the enzyme having EC number no. 2.4.1.212.

[0278] According to preferred embodiments, one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may originate from or be derived from an organism, preferably selected from the group consisting of prokaryotes and eukaryotes. In some embodiments, one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may originate from or be derived from archaea. In some embodiments, one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may originate from or be derived from bacteria, in particular Streptococcus zooepidemicus (Sz), or Pasteurella multocida (Pm), Chlorella virus PBCV1 (Vir), Chlorella virus CviKl (Vir), Chlorella virus IL-5-2s1 (Vir), Chlorella virus CZ-2 (Vir), Chlorella virus CVG-1 (Vir), or Xenopus laevis (Xl).

[0279] According to still preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity have an identity of at least 65%, advantageously at least, with the nucleic acid sequence set forth as the sequence of SEQ ID NO:1 (Vir), SEQ ID NO:2 (Vir), SEQ ID NO:3 (Pm), SEQ ID NO:4 (Pm), SEQ ID NO:5 (Pm), SEQ ID NO:6 (Xl), SEQ ID NO:7 (sz), SEQ ID NO:101 (Vir), SEQ ID NO:102 (Vir), SEQ ID NO:103 (Vir), or SEQ ID NO:104 (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 (Vir), SEQ ID NO:2 (Vir), SEQ ID NO:3 (Pm), SEQ ID NO:4 (Pm), SEQ ID NO:5 (Pm), SEQ ID NO:6 (Xl), SEQ ID NO:7 (sz), SEQ ID NO:101 (Vir), SEQ ID NO:102 (Vir), SEQ ID NO:103 (Vir), or SEQ ID NO:104 (Vir), respectively.

[0280] A similar biological activity for this sequence is the ability to encode a polypeptide that converts UDP-glucuronate or UDP-N-acetyl-glucose into hyaluronic acid, as previously described.

[0281] As described herein, a nucleic acid sequence having at least 65% nucleotide identity to 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 to said reference nucleic acid sequence, and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0282] As described herein, a nucleic acid sequence having at least 70% nucleotide identity to 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 to the reference nucleic acid sequence, and also having the same qualitative biological activity as the reference nucleic acid sequence.

[0283] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to 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.

[0284] With respect to the amino acid sequences of polypeptides having hyaluronan synthase activity originating from Streptococcus zooepidemicus, Chlorella virus PBCV1, Xenopus laevis, or Pasteurella multocida, those skilled in the art will recognize the amino acid sequences of the polypeptides having hyaluronan synthase activity, which are respectively identified in the UniProt database under accession numbers B4U0D4, Q84419, P13563, Q7BLV3, M1GZS8, M1H5V3, M1H2Q1, and M1HN86, or SEQ ID NO:8 (Vir), SEQ ID NO:9 (Pm), SEQ ID NO:10 (Vir), SEQ ID NO:11 (Pm), SEQ ID NO:12 (Pm), SEQ ID NO:13 (Pm), SEQ ID NO:14 (Pm), SEQ ID NO:15 (Pm), SEQ ID NO:16 (Pm), SEQ ID NO:17 (Pm), SEQ ID NO:18 (Pm), SEQ ID NO:19 (Pm), SEQ ID NO:20 (Pm), SEQ ID NO:21 (Pm), SEQ ID NO:22 (Pm), SEQ ID NO:23 (Pm), SEQ ID NO:24 (Pm), SEQ ID NO:25 (Pm), SEQ ID NO:26 (Pm), SEQ ID NO:27 (Pm), SEQ ID NO:28 (Pm), SEQ ID NO:29 (Pm), SEQ ID NO:30 (Pm), SEQ ID NO:31 (Pm), SEQ ID NO:32 (Pm), SEQ ID NO:33 (Pm), SEQ ID NO:34 (Pm), SEQ ID NO:35 (Pm), SEQ ID NO:36 (Pm), SEQ ID NO:37 (Pm), SEQ ID NO:38 (Pm), SEQ ID NO:40 (Pm), S Reference may be made to SEQ ID NO: 10(Xl), SEQ ID NO: 11(Sz), SEQ ID NO: 105(Vir), SEQ ID NO: 106(Vir), SEQ ID NO: 107(Vir), or SEQ ID NO: 108(Vir), particularly SEQ ID NO: 8(Vir), SEQ ID NO: 9(Pm), SEQ ID NO: 10(Xl), SEQ ID NO: 11(Sz), SEQ ID NO: 105(Vir), SEQ ID NO: 106(Vir), or SEQ ID NO: 108(Vir), more particularly SEQ ID NO: 8(Vir), SEQ ID NO: 9(Pm), SEQ ID NO: 10(Xl), SEQ ID NO: 11(Sz).

[0285] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may be a nucleic acid encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 50%, advantageously at least 65%, preferably at least 80% amino acid identity with the amino acid sequence set forth as SEQ ID NO:8 (Vir), SEQ ID NO:9 (Pm), SEQ ID NO:10 (X1), SEQ ID NO:11 (Sz), SEQ ID NO:105 (Vir), SEQ ID NO:106 (Vir), SEQ ID NO:107 (Vir), or SEQ ID NO:108 (Vir), and which also have the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO:8 (Vir), SEQ ID NO:9 (Pm), SEQ ID NO:10 (X1), SEQ ID NO:11 (Sz), SEQ ID NO:105 (Vir), SEQ ID NO:106 (Vir), SEQ ID NO:107 (Vir), or SEQ ID NO:108 (Vir).

[0286] The biological activity of the same nature for this sequence is as previously described, namely the ability to catalyze the conversion of UDP-glucuronate or UDP-N-acetyl-glucose to hyaluronic acid.

[0287] As described herein, an amino acid sequence having at least 50% amino acid identity to a reference amino acid sequence is at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 15 These include amino acid sequences that have 3%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity and also have the same qualitative biological activity as the reference amino acid sequence.

[0288] 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.

[0289] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences that have 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 have the same qualitative biological activity as the reference amino acid sequence.

[0290] As noted above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having hyaluronan 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, located 5' and 3' to the one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity, respectively.

[0291] 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 dehydrogenases originating from the genomes of Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus are sometimes referred to as HASB.

[0292] Methods implemented to measure the activity level of a polypeptide having UDP-glucose dehydrogenase activity belong to the general knowledge of a person skilled in the art.

[0293] In this respect, the skilled person may advantageously refer to the method described by Oka and Jigami (FEBS Journal 273, 2645-2657, 2006).

[0294] A preferred polypeptide having UDP-glucose dehydrogenase activity herein is the enzyme having the EC number 1.1.1.22.

[0295] According to preferred embodiments, one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity may originate from or be derived from an organism, preferably selected from the group consisting of prokaryotes and eukaryotes. In some embodiments, one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity may originate from or be derived from archaea. In some preferred embodiments, one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity may originate from or be derived from yeast, in particular Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus or Halomyces.

[0296] According to a still 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 set forth as sequence SEQ ID NO: 12 (At), SEQ ID NO: 13 (Vir), SEQ ID NO: 14 (Vir) or SEQ ID NO: 15 (Sz), and (ii) the same qualitative biological activity as the nucleic acid set forth as sequence SEQ ID NO: 12 (At), SEQ ID NO: 13 (Vir), SEQ ID NO: 14 (Vir) or SEQ ID NO: 15 (Sz). The nucleic acids set forth as sequences SEQ ID NO:12 (At), SEQ ID NO:13 (Vir), SEQ ID NO:14 (Vir), and SEQ ID NO:15 (Sz) encode polypeptides having UDP-glucose dehydrogenase activity, which may collectively be referred to herein as HASB, originating from Arabidopsis thaliana (At), Chlorella virus PBCV1 (Vir), or Streptococcus zooepidemicus (Sz), respectively.

[0297] A similar biological activity for this sequence is the ability to encode a polypeptide that converts UDP-glucose to UDP-glucuronate, as previously explained.

[0298] As described herein, a nucleic acid sequence having at least 65% nucleotide identity to 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 to said reference nucleic acid sequence, and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0299] As described herein, a nucleic acid sequence having at least 70% nucleotide identity to 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 to the reference nucleic acid sequence, and also having the same qualitative biological activity as the reference nucleic acid sequence.

[0300] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to 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.

[0301] For the amino acid sequences of polypeptides having UDP-glucose dehydrogenase activity derived from Arabidopsis thaliana, Chlorella virus PBCV1, or Streptococcus zooepidemicus, those skilled in the art can 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: 16 (At), SEQ ID NO: 17 (Vir), and SEQ ID NO: 18 (Sz) described herein.

[0302] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity may be nucleic acids encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 55%, advantageously at least 65%, preferably at least 80% amino acid identity with the amino acid sequences of SEQ ID NO: 16 (At), SEQ ID NO: 17 (Vir) and SEQ ID NO: 18 (Sz) and also having the same qualitative biological activity as the amino acid sequences of SEQ ID NO: 16 (At), SEQ ID NO: 17 (Vir) and SEQ ID NO: 18 (Sz).

[0303] The biological activity of the same nature for this sequence is as previously described, namely the ability to catalyze the conversion of UDP-glucose to UDP-glucuronate.

[0304] As described herein, an amino acid sequence having at least 55% amino acid identity to 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 to the reference amino acid sequence, and also having the same qualitative biological activity as the reference amino acid sequence.

[0305] 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.

[0306] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences that have 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 have the same qualitative biological activity as the reference amino acid sequence.

[0307] As mentioned above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity in the present invention is regulated by at least one promoter and at least one terminator, such as those defined in more detail hereinafter, located at the 5' and 3' positions of one or more recombinant nucleic acids encoding a polypeptide having UDP-glucose dehydrogenase activity, respectively.

[0308] Hyaluronidase (HYAL) Hyaluronidase enzymes are proteins described in the art for catalyzing the breakdown of hyaluronic acid molecules into smaller hyaluronic acid molecules. Hyaluronidases originating from Cupienius salei, Loxosceles intermedia, Hirudo nipponia, Bothropus atrox, Titius serulatus, or Vespa magnifica may be referred to as HYAL.

[0309] Polypeptides with hyaluronidase activity of the present invention may have both a secretory signal and an anchoring signal, or may have a secretory signal but no anchoring signal, or may have a secretory-anchor signal with dual secretory and anchoring functions. When the encoded polypeptide with hyaluronidase activity has both a secretory signal and an anchoring signal, it may be referred to as HYAL-31, as shown in the Examples. When the encoded polypeptide with hyaluronidase activity has a secretory signal but no anchoring signal, it may be referred to as HYAL-3, as shown in the Examples.

[0310] Methods implemented to measure the activity level of a polypeptide having hyaluronidase activity are within the general knowledge of a person skilled in the art.

[0311] In this regard, the skilled person can advantageously monitor the molecular weight of the resulting hyaluronic acid on an agarose gel.

[0312] A preferred polypeptide having hyaluronidase activity herein is an enzyme having the EC number n° EC 3.2.1.35.

[0313] According to preferred embodiments, one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may originate from or be derived from an organism, preferably selected from the group including prokaryotes and eukaryotes. In some embodiments, one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may originate from or be derived from an archaea. In some embodiments, one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may originate from or be derived from an organism, preferably selected from yeast. In some other preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may originate from or be derived from Cupienius salei (Csa), Loxosceles intermedia (Li), Hirudo Nipponia (Hn), Bothropus atrox (Ba), Titius serlatus (Ts), or Vespa magnifica (Vm), in particular Cupienius salei (Csa), Loxosceles intermedia (Li), Hirudo Nipponia (Hn), Bothropus atrox (Ba), and Titius serlatus (Ts).

[0314] According to still preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be selected from the group consisting of nucleic acid sequences having at least 65%, advantageously at least 70%, and preferably at least 80% nucleic acid identity with the nucleic acid of SEQ ID NO: 19 (Ba), SEQ ID NO: 25 (Li), SEQ ID NO: 21 (Csa), SEQ ID NO: 27 (Ts), SEQ ID NO: 23 (Hn), or SEQ ID NO: 29 (Vm), and also having the same qualitative biological activity as the nucleic acid of SEQ ID NO: 19 (Ba), SEQ ID NO: 25 (Li), SEQ ID NO: 21 (Csa), SEQ ID NO: 27 (Ts), SEQ ID NO: 23 (Hn), or SEQ ID NO: 29 (Vm). The nucleic acids of SEQ ID NO:19 (Ba), SEQ ID NO:25 (Li), SEQ ID NO:21 (Csa), SEQ ID NO:27 (Ts), SEQ ID NO:23 (Hn), or SEQ ID NO:29 (Vm) encode a polypeptide having hyaluronidase activity, containing a secretory signal but not containing an anchoring signal, and originating from or derived from Bothropus atrox, Loxosceles intermedia, Cupienius salei, Titius serulatus, Hirudo nipponia, or Vespa magnifica, respectively.

[0315] According to another preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be selected from the group consisting of nucleic acid sequences having at least 65%, advantageously at least 70%, and preferably at least 80% nucleic acid identity with the nucleic acid of SEQ ID NO: 22 (Csa), SEQ ID NO: 26 (Li), SEQ ID NO: 24 (Hn), SEQ ID NO: 20 (Ba), SEQ ID NO: 28 (Ts), or SEQ ID NO: 30 (Vm), and also having the same qualitative biological activity as the nucleic acid of SEQ ID NO: 22 (Csa), SEQ ID NO: 26 (Li), SEQ ID NO: 24 (Hn), SEQ ID NO: 20 (Ba), SEQ ID NO: 28 (Ts), or SEQ ID NO: 30 (Vm). The nucleic acids of SEQ ID NO:22 (Csa), SEQ ID NO:26 (Li), SEQ ID NO:24 (Hn), SEQ ID NO:20 (Ba), SEQ ID NO:28 (Ts), or SEQ ID NO:30 (Vm) encode a polypeptide having hyaluronidase activity and contain a secretory signal and an anchoring signal, and originate from or are derived from Cupienius salei, Loxosceles intermedia, Hirudo nipponia, Bothropus atrox, Titius serulatus, or Vespa magnifica, respectively.

[0316] In certain embodiments, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be selected from the group consisting of nucleic acid sequences that (i) have at least 65%, advantageously at least 70%, and preferably at least 80% nucleic acid identity with the nucleic acid of SEQ ID NO: 19 (Ba), SEQ ID NO: 25 (Li), SEQ ID NO: 21 (Csa), SEQ ID NO: 27 (Ts), or SEQ ID NO: 23 (Hn); and (ii) have the same qualitative biological activity as the nucleic acid of SEQ ID NO: 19 (Ba), SEQ ID NO: 25 (Li), SEQ ID NO: 21 (Csa), SEQ ID NO: 27 (Ts), or SEQ ID NO: 23 (Hn).

[0317] In certain embodiments, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be selected from the group consisting of nucleic acid sequences that (i) have at least 65%, advantageously at least 70%, and preferably at least 80% nucleic acid identity with the nucleic acid of SEQ ID NO:22 (Csa), SEQ ID NO:26 (Li), SEQ ID NO:24 (Hn), SEQ ID NO:20 (Ba), or SEQ ID NO:28 (Ts), and (ii) have the same qualitative biological activity as the nucleic acid of SEQ ID NO:22 (Csa), SEQ ID NO:26 (Li), SEQ ID NO:24 (Hn), SEQ ID NO:20 (Ba), or SEQ ID NO:28 (Ts).

[0318] A biological activity of the same nature for this sequence is the ability to encode a polypeptide that catalyzes the degradation of hyaluronic acid molecules into smaller hyaluronic acid molecules, as previously explained.

[0319] As described herein, a nucleic acid sequence having at least 65% nucleotide identity to 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 to said reference nucleic acid sequence, and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0320] As described herein, a nucleic acid sequence having at least 70% nucleotide identity to 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 to the reference nucleic acid sequence, and also having the same qualitative biological activity as the reference nucleic acid sequence.

[0321] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to 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.

[0322] For the amino acid sequences of one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity from Cupienius salei, Loxosceles intermedia, Hirudo nipponia, Bothropus atrox, or Titius serratus, those skilled in the art can refer to UniProt database accession numbers A0A0S4JYH2, R4J7Z9, X4Y2L4, A0A2H4Z8F4, or P85841, respectively, or SEQ ID NO: 33 (Csa), SEQ ID NO: 37 (Li), SEQ ID NO: 35 (Hn), SEQ ID NO: 31 (Ba), SEQ ID NO: 39 (Ts), or SEQ ID NO: 41 (Vm) described herein.

[0323] According to another specific embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be a nucleic acid encoding a polypeptide having an amino acid sequence selected from the group consisting of: a sequence comprising a secretory signal and no anchoring signal, having at least 50%, advantageously at least 65%, and preferably at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 33 (Csa), SEQ ID NO: 37 (Li), SEQ ID NO: 35 (Hn), SEQ ID NO: 31 (Ba), SEQ ID NO: 39 (Ts), or SEQ ID NO: 41 (Vm), and also having the same qualitative biological activity as the amino acid sequence of SEQ ID NO: 33 (Csa), SEQ ID NO: 37 (Li), SEQ ID NO: 35 (Hn), SEQ ID NO: 31 (Ba), SEQ ID NO: 39 (Ts), or SEQ ID NO: 41 (Vm).

[0324] According to another specific embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be nucleic acids encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 55%, advantageously at least 65%, and preferably at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 34 (Csa), SEQ ID NO: 38 (Li), SEQ ID NO: 36 (Hn), SEQ ID NO: 32 (Ba), SEQ ID NO: 40 (Ts), or SEQ ID NO: 42 (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: 34 (Csa), SEQ ID NO: 38 (Li), SEQ ID NO: 36 (Hn), SEQ ID NO: 32 (Ba), SEQ ID NO: 40 (Ts), or SEQ ID NO: 42 (Vm).

[0325] In certain embodiments, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be nucleic acids encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 55%, advantageously at least 65%, and preferably at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 33 (Csa), SEQ ID NO: 37 (Li), SEQ ID NO: 35 (Hn), SEQ ID NO: 31 (Ba), or SEQ ID NO: 39 (Ts), and also having the same qualitative biological activity as the amino acid sequence of SEQ ID NO: 33 (Csa), SEQ ID NO: 37 (Li), SEQ ID NO: 35 (Hn), SEQ ID NO: 31 (Ba), or SEQ ID NO: 39 (Ts).

[0326] In another specific embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase 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%, and preferably at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 34 (Csa), SEQ ID NO: 38 (Li), SEQ ID NO: 36 (Hn), SEQ ID NO: 32 (Ba), or SEQ ID NO: 40 (Ts), and also having the same qualitative biological activity as the amino acid sequence of SEQ ID NO: 34 (Csa), SEQ ID NO: 38 (Li), SEQ ID NO: 36 (Hn), SEQ ID NO: 32 (Ba), or SEQ ID NO: 40 (Ts).

[0327] The biological activity of the same nature for this sequence is as previously described, namely the ability to catalyze the breakdown of hyaluronic acid molecules into smaller hyaluronic acid molecules.

[0328] As described herein, an amino acid sequence having at least 55% amino acid identity to 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 to the reference amino acid sequence, and also having the same qualitative biological activity as the reference amino acid sequence.

[0329] 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.

[0330] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences that have 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 have the same qualitative biological activity as the reference amino acid sequence.

[0331] As discussed above, the expression level of one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase 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 5' and 3' to the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity, respectively.

[0332] Glutamine synthetase (GLN1) The glutamine synthetase enzyme is a protein described in the art for catalyzing the conversion of glutamate to glutamine. The glutamine synthetase originating from Saccharomyces cerevisiae can be referred to as GLN1.

[0333] Methods implemented to measure the activity level of a polypeptide having glutamine synthetase activity belong to the general knowledge of a person skilled in the art.

[0334] In this respect, the skilled person may advantageously refer to the method described by Legrain et al. (1982) European Journal of Biochemistry 123, 611-616.

[0335] A preferred polypeptide having glutamine synthetase activity according to the present invention is the enzyme having the EC number n° EC 6.3.1.2.

[0336] According to preferred embodiments, one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity may originate from or be derived from an organism, preferably selected from the group including prokaryotes and eukaryotes. In some embodiments, one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity may originate from or be derived from archaea. In some embodiments, one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity may originate from or be derived from yeast, in particular Saccharomyces cerevisiae.

[0337] According to still preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity may be selected from the group consisting of nucleic acid sequences having (i) at least 65%, advantageously at least 70%, and preferably at least 80% nucleic acid identity with the nucleic acid sequence set forth as sequence SEQ ID NO: 96(Sc), and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as sequence SEQ ID NO: 96(Sc). The nucleic acid set forth as sequence SEQ ID NO: 96 encodes a polypeptide having glutamine synthetase activity originating from Saccharomyces cerevisiae, which may also be referred to as GLN1.

[0338] The biological activity of the same nature for this sequence is as previously explained, in particular one ATP and one NH4 + The ability to encode a polypeptide that converts glutamate to glutamine through consumption of glutamate.

[0339] As described herein, a nucleic acid sequence having at least 65% nucleotide identity to 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 to said reference nucleic acid sequence, and also having the same qualitative biological activity as said reference nucleic acid sequence.

[0340] As described herein, a nucleic acid sequence having at least 70% nucleotide identity to 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 to the reference nucleic acid sequence, and also having the same qualitative biological activity as the reference nucleic acid sequence.

[0341] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to 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.

[0342] For the amino acid sequence of a polypeptide having glutamine synthetase activity originating from Saccharomyces cerevisiae, those skilled in the art can refer to the UniProt database accession number P32288 or the sequence of SEQ ID NO: 97 described herein.

[0343] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase 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:97 and also having the same qualitative biological activity as the amino acid sequence of SEQ ID NO:97.

[0344] The biological activity of the same nature for this sequence is as previously described, namely the ability to catalyze the conversion of glutamate to glutamine.

[0345] 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 14 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.

[0346] 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.

[0347] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences that have 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 have the same qualitative biological activity as the reference amino acid sequence.

[0348] As noted above, the expression level of a polypeptide having glutamine synthetase 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 5' and 3', respectively, of one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity.

[0349] Glutamate synthase (GLT1) The glutamate synthase enzyme is a protein described in the art for catalyzing the conversion of glutamine to glutamate. The glutamate synthase originating from Saccharomyces cerevisiae can be referred to as GLT1.

[0350] Methods implemented to measure the activity level of a polypeptide having glutamate synthase activity belong to the general knowledge of a person skilled in the art.

[0351] In this respect, the skilled person may advantageously refer to the method described by Roon et al. (1974) Journal of bacteriology 118, 89-95.

[0352] A preferred polypeptide having glutamate synthase activity according to the present invention is the enzyme having the EC number n° EC 1.4.1.14 (SEQ ID NO: 98).

[0353] For the amino acid sequence of a polypeptide having glutamate synthase activity derived from Saccharomyces cerevisiae, those skilled in the art can refer to the UniProt database accession number Q12680 or the sequence of SEQ ID NO: 99 described herein.

[0354] As mentioned above, the expression level of a polypeptide having glutamate synthase activity may be reduced in a recombinant cell, in particular a recombinant yeast, according to the invention compared to said cell, in particular a yeast, in its non-recombinant form, i.e. at least one endogenous gene of the recombinant cell, in particular the recombinant yeast, is disrupted.

[0355] 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 from Saccharomyces cerevisiae can be referred to as GFA1.

[0356] Methods implemented to measure 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.

[0357] In this respect, the skilled person can advantageously refer to the method described by Shiga Shibatan and Hiroaki Kitazawa (Plant Biotechnology 26, 149-152, 2009).

[0358] A preferred polypeptide having glutamine-fructose-6-phosphate amidotransferase activity according to the present invention is an enzyme having the EC number n° EC 2.6.1.16.

[0359] According to preferred embodiments, 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 the group consisting of prokaryotes and eukaryotes. In some embodiments, one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may originate from or be derived from archaea. In some embodiments, 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, one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may originate from or be derived from yeast, particularly Saccharomyces cerevisiae.

[0360] According to a still 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%, and preferably at least 80% nucleic acid identity with the nucleic acid sequence set forth as SEQ ID NO: 47(Sc), and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as SEQ ID NO: 47(Sc). The nucleic acid set forth as SEQ ID NO: 47 encodes a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity originating from Saccharomyces cerevisiae, which may also be referred to as GFA1.

[0361] 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 nucleic acid sequences having (i) at least 65%, advantageously at least 70%, and preferably at least 80% nucleic acid identity with the nucleic acid sequence set forth as SEQ ID NO: 48 or SEQ ID NO: 49, and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as SEQ ID NO: 48 or SEQ ID NO: 49. The nucleic acid sequence set forth as SEQ ID NO: 48 or SEQ ID NO: 49 encodes a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity originating from the Chlorella virus PBCV1.

[0362] A similar biological activity for this sequence is the ability to encode a polypeptide that converts fructose-6-phosphate to glucosamine-6-phosphate, as previously described.

[0363] As described herein, a nucleic acid sequence having at least 65% nucleotide identity to 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 to 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 70% nucleotide identity to 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 to the reference nucleic acid sequence, and also having the same qualitative biological activity as the reference nucleic acid sequence.

[0365] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to 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.

[0366] 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: 50 described herein.

[0367] 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: 51 described herein.

[0368] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may be nucleic acids 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: 50 or SEQ ID NO: 51, and also having the same qualitative biological activity as the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 51.

[0369] 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.

[0370] 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 14 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.

[0371] 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.

[0372] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences that have 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 have the same qualitative biological activity as the reference amino acid sequence.

[0373] As described 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, located at the 5' and 3' positions, respectively, of one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity.

[0374] UDP-N-acetylglucosamine pyrophosphorylase (QRI1) The UDP-N-acetylglucosamine pyrophosphorylase enzyme is a protein described in the art for catalyzing the conversion of N-acetylglucosamine-6-phosphate to UDP-N-acetylglucose. The UDP-N-acetylglucosamine pyrophosphorylase from Saccharomyces cerevisiae can be designated QRI1.

[0375] Methods implemented to measure the activity level of a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity belong to the general knowledge of a person skilled in the art.

[0376] In this regard, those skilled in the art can refer to the method described by Mio et al. (The Journal of Biological Chemistry, Col. 273, No. 23, June 5, 1998, pp. 14392-14397), except that UDP-N-acetyl-glucosamine is detected by LC MS / MS using a Synergi RP Fusion column.

[0377] A preferred polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity according to the present invention is an enzyme having the EC number n° EC 2.7.7.23.

[0378] According to preferred embodiments, 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 the group consisting of prokaryotes and eukaryotes. In some embodiments, one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may originate from or be derived from archaea. In some embodiments, 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, one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may originate from or be derived from yeast, particularly Saccharomyces cerevisiae.

[0379] According to still preferred embodiments, 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%, and preferably at least 80% nucleic acid identity with the nucleic acid sequence set forth as SEQ ID NO: 52, and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as SEQ ID NO: 52. The nucleic acid sequence set forth as SEQ ID NO: 52 encodes a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity originating from Saccharomyces cerevisiae, which may also be referred to as QRI1.

[0380] A similar biological activity for this sequence is the ability to encode a polypeptide that converts N-acetyl-glucosamine-6-phosphate to UDP-N-acetyl-glucose, as previously explained.

[0381] As described herein, a nucleic acid sequence having at least 65% nucleotide identity to 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 to 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 to 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 to the reference nucleic acid sequence, and also having the same qualitative biological activity as the reference nucleic acid sequence.

[0383] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to 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 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: 53 described herein.

[0385] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may be nucleic acids 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 SEQ ID NO: 53, and also having the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO: 53.

[0386] 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 UDP-N-acetyl-glucose.

[0387] 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 14 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.

[0388] As described herein, an amino acid sequence having at least 45% amino acid identity to 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 15 , 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.

[0389] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences that have 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 have the same qualitative biological activity as the reference amino acid sequence.

[0390] 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.

[0391] Phosphoglucomutase-1 (PGM1) The enzyme phosphoglucomutase-1 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.

[0392] Methods implemented to measure the activity level of a polypeptide exhibiting phosphoglucomutase-1 activity belong to the general knowledge of a person skilled in the art.

[0393] In this respect, the skilled person may advantageously refer to the method described by Tiwari and Bhat (Biochemical and Biophysical Research Communications 366, 340-345, 2008).

[0394] A preferred polypeptide having phosphoglucomutase-1 activity according to the invention is the enzyme having EC number n° 5.4.2.2.

[0395] According to preferred embodiments, one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity may originate from an organism, preferably selected from the group consisting of prokaryotes and eukaryotes. In some embodiments, one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity may originate from or be derived from archaea. In some embodiments, 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, one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity may originate from or be derived from Saccharomyces cerevisiae.

[0396] 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 SEQ ID NO: 54; and (ii) a nucleic acid sequence having the same qualitative biological activity as the nucleic acid sequence set forth as SEQ ID NO: 54.

[0397] A similar biological activity for this sequence is the ability to encode a polypeptide that converts glucose-6-phosphate to glucose-1-phosphate, as previously explained.

[0398] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to 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.

[0399] 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: 55 described herein.

[0400] 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 SEQ ID NO: 55 and also having the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO: 55.

[0401] 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.

[0402] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences that have 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 have the same qualitative biological activity as the reference amino acid sequence.

[0403] As described 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, located at the 5' and 3' positions of one or more recombinant nucleic acids encoding a polypeptide having phosphoglucomutase-1 activity, respectively.

[0404] UTP-glucose 1-phosphate uridylyltransferase (UGP1) The enzyme UTP-glucose 1-phosphate uridylyltransferase is a protein described in the art for catalyzing the conversion of glucose-1-phosphate to UDP-glucose. UTP-glucose 1-phosphate uridylyltransferase from Saccharomyces cerevisiae can be referred to as UGP1.

[0405] Methods implemented to measure the activity level of a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity belong to the general knowledge of a person skilled in the art.

[0406] In this respect, the skilled person can advantageously refer to the method described by Roeben (J. Mol. Biol 364, 551-560, 2006).

[0407] A preferred polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity according to the present invention is an enzyme having EC number no. 2.7.7.9.

[0408] According to preferred embodiments, 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 the group consisting of prokaryotes and eukaryotes. In some embodiments, one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity may originate from or be derived from archaea. In some embodiments, 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, 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.

[0409] 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: (i) a nucleic acid sequence originating from Saccharomyces cerevisiae having at least 80% nucleic acid identity with the nucleic acid set forth as sequence SEQ ID NO: 56; and (ii) a nucleic acid sequence having the same qualitative biological activity as the nucleic acid set forth as sequence SEQ ID NO: 56.

[0410] A similar biological activity for this sequence is the ability to encode a polypeptide that converts glucose-1-phosphate to UDP-glucose, as previously explained.

[0411] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to 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.

[0412] For the amino acid sequence of a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity originating from Saccharomyces cerevisiae, those skilled in the art can refer to the sequence set forth in the UniProt database under accession number NP_32861 or as sequence number 57 described herein.

[0413] 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:57 and also having the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO:57.

[0414] The biological activity of the same nature for this sequence is as previously described, namely the ability to catalyze the conversion of glucose-1-phosphate to UDP-glucose.

[0415] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences that have 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 have the same qualitative biological activity as the reference amino acid sequence.

[0416] As described 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, located at the 5' and 3' positions of one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose 1-phosphate uridylyltransferase activity, respectively.

[0417] Glucosamine-6-phosphate N-acetyltransferase (GNA1) Glucosamine-6-phosphate N-acetyltransferase enzymes are proteins described in the art for catalyzing the conversion of glucosamine-6-phosphate to N-acetyl-glucosamine-6-phosphate. Glucosamine-6-phosphate N-acetyltransferases originating from Saccharomyces cerevisiae can be referred to as GNA1.

[0418] 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.

[0419] In this respect, the skilled person can advantageously refer to the method described by Li et al. (Anal. Biochem. 370, 142-146, 2007).

[0420] A preferred polypeptide having glucosamine-6-phosphate N-acetyltransferase activity according to the present invention is an enzyme having EC number n° 2.3.1.4.

[0421] According to preferred embodiments, 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, one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity may originate from or be derived from yeast, in particular Saccharomyces cerevisiae.

[0422] 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 SEQ ID NO: 58, and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as SEQ ID NO: 58. The nucleic acid sequence set forth as SEQ ID NO: 58 encodes a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity originating from Saccharomyces cerevisiae, which may also be referred to as GNA1.

[0423] 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.

[0424] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to 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.

[0425] For the amino acid sequence of a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity originating from Saccharomyces cerevisiae, those skilled in the art can refer to the UniProt database accession number NP_116637 or SEQ ID NO: 59 described herein.

[0426] According to another specific embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity may be nucleic acids 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:59 and also having the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO:59.

[0427] 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.

[0428] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences that have 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 have the same qualitative biological activity as the reference amino acid sequence.

[0429] As described 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 one or more recombinant nucleic acids encoding a polypeptide having glucosamine-6-phosphate N-acetyltransferase activity, respectively.

[0430] Phosphoacetylglucosamine mutase (PCM1) The phosphoacetylglucosamine mutase enzyme is a protein described in the art for catalyzing the conversion of N-acetyl-glucosamine-6-phosphate to N-acetyl-glucosamine-1-phosphate. The phosphoacetylglucosamine mutase from Saccharomyces cerevisiae can be referred to as PCM1.

[0431] Methods implemented to measure the activity level of a polypeptide having phosphoacetylglucosamine mutase activity belong to the general knowledge of a person skilled in the art.

[0432] In this respect, the skilled person can advantageously refer to the method described by Bandini et al. (Molecular Microbiology 85(3), 513-534, 2012).

[0433] A preferred polypeptide having phosphoacetylglucosamine mutase activity according to the present invention is an enzyme having EC number no. 5.4.2.3.

[0434] According to preferred embodiments, one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may originate from or be derived from an organism, preferably selected from the group consisting of prokaryotes and eukaryotes. In some preferred embodiments, one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may originate from or be derived from yeast, particularly Saccharomyces cerevisiae.

[0435] 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 nucleic acid sequences having (i) at least 80% nucleic acid identity with the nucleic acid sequence set forth as SEQ ID NO: 60, and (ii) the same qualitative biological activity as the nucleic acid sequence set forth as SEQ ID NO: 60. The nucleic acid set forth as SEQ ID NO: 60 encodes a polypeptide having phosphoacetylglucosamine mutase activity originating from Saccharomyces, which may also be referred to as PCM1.

[0436] 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.

[0437] As described herein, a nucleic acid sequence having at least 80% nucleotide identity to 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.

[0438] 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: 61 described herein.

[0439] According to another specific embodiment, the one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may be nucleic acids 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:61 and also having the same qualitative biological activity as the amino acid sequence set forth as SEQ ID NO:61.

[0440] 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.

[0441] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences that have 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 have the same qualitative biological activity as the reference amino acid sequence.

[0442] 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.

[0443] 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, particularly the recombinant yeast cell of the present invention, including specifically Saccharomyces cerevisiae.

[0444] A variety of promoters may be used to direct the desired expression of the coding sequence of interest.

[0445] The promoter according to the present invention is the following promoter: pTDH3 (SEQ ID NO: 62), pTDH3.sk (SEQ ID NO: 63), pTDH3-1.Sba (SEQ ID NO: 64), pTDH3.Sar (SEQ ID NO: 65), pENO2 (SEQ ID NO: 66), pTEF3 (SEQ ID NO: 67), pTEF1 (SEQ ID NO: 68), pTEF1.ago (SEQ ID NO: 69), pTEF1.sba (SEQ ID NO: 70), pPDC1 (SEQ ID NO: 71), pCCW12 (SEQ ID NO: 72), pCCW12.Sm (SEQ ID NO: 73), pCCW12.sk (SEQ ID NO: 74), pCCW12.sba (SEQ ID NO: 75), pCCW12.sar (SEQ ID NO: 76), pNUP57 (SEQ ID NO: 77), pCCW10.ago (SEQ ID NO: 78), pCWP2 (SEQ ID NO: 79), and pRPLA1 (SEQ ID NO: 80) may be selected from the group consisting of:

[0446] Promoters of more particular interest in the present invention are: pTDH3 (SEQ ID NO: 62), pTDH3.sk (SEQ ID NO: 63), pTDH3-1.Sba (SEQ ID NO: 64), pTDH3.Sar (SEQ ID NO: 65), pENO2 (SEQ ID NO: 66), pTEF3 (SEQ ID NO: 67), pTEF1 (SEQ ID NO: 68), pTEF1.ago (SEQ ID NO: 69), pTEF1.sba (SEQ ID NO: 70), pPDC1 (SEQ ID NO: 71), pCCW12 (SEQ ID NO: 72), pCCW12.Sm (SEQ ID NO: 73), pCCW12.sk (SEQ ID NO: 74), pCCW12.sba (SEQ ID NO: 75), and pCCW12.sar (SEQ ID NO: 76) may be selected from the group consisting of:

[0447] The promoter may in particular be selected from the group consisting of pTDH3, pTDH3-1.Sba, pTDH3.Sar, pENO2, pTEF3, pTEF1, pPDC1, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, and pCCW12.sar.

[0448] Alternatively, promoters of interest in the present invention are pNUP57 (SEQ ID NO: 77), and pCCW10.ago (SEQ ID NO: 78) may be selected from the group consisting of:

[0449] Other promoters of interest in the present invention include: pCWP2 (SEQ ID NO: 79), and pRPLA1 (SEQ ID NO: 80) It could be.

[0450] 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, particularly when the amount of a given factor increases or is increased; therefore, the activity of these same promoters can be repressed, and therefore decreased, when the amount of said factor decreases or is reduced. The amount of said factor in the culture medium of a recombinant yeast cell of the present invention containing an inducible or repressible promoter can be determined and controlled accordingly by those skilled in the art.

[0451] For example, increasing the amount of copper in the culture medium of a recombinant yeast cell according to the invention containing the pCUP-1 promoter will induce and thus increase the transcription of genes under the control of this promoter, whereas decreasing the amount of copper in the culture medium will cause repression and thus a decrease in the transcription of genes under the control of this promoter.

[0452] 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 therefore decrease, the transcription of genes under the control of this promoter. In contrast, decreasing the amount of methionine in the culture medium will induce, and therefore increase, the transcription of genes under the control of this promoter.

[0453] For this reason, the following promoters are referred to herein as inducible or repressible promoters.

[0454] 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 CUP1 (SEQ ID NO: 81).

[0455] According to this embodiment, the inducible or repressible promoter according to the invention is, in particular, pCUP1.

[0456] The activity of this promoter is induced by increasing the presence of methionine, copper, or threonine, as shown above, and its activity is decreased, i.e., repressed, when the amount of methionine, copper, or threonine is reduced.

[0457] 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: 82), pMET25-methionine inducible or repressible (SEQ ID NO: 83), and pSAM1 - methionine-inducible or repressible (SEQ ID NO: 84) may be selected from the group consisting of:

[0458] According to this particular embodiment, the inducible or repressible promoter according to the invention may be selected from the group consisting of pMET6, pMET25, and pSAM1.

[0459] 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 amount of methionine, copper, lysine, or glucose is reduced.

[0460] 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.

[0461] According to more specific embodiments, the inducible or repressible promoter according to the present invention may be selected from the group consisting of pCUP1, pMET6, pSAM1, and pMET25.

[0462] Synthetic promoters as described in Blazeck & Alper (2013) Biotechnol. J. 8 46-58 can also be used.

[0463] The promoter of the present invention may originate from any organism of the class Saccharomycetes, 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:

[0464] The promoter of the present invention may preferably originate from an organism selected from the group consisting of Saccharomyces cerevisiae (sc), Saccharomyces mikatae (Sm), Saccharomyces kudriabzevii (sk), Saccharomyces bayanus (sba), and Saccharomyces arboricola (Sar).

[0465] 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, particularly a recombinant yeast cell according to the invention, comprises a suitable transcription terminator sequence that is functional in the recombinant cell of the invention, particularly a recombinant yeast cell of the invention, particularly a Saccharomyces cerevisiae cell.

[0466] The same or different transcription terminators can be found in Yamanishi et al. (2013) ACS synthetic biology 2, 337-347.

[0467] Terminators of more particular interest in the present invention are: tTPI1 (SEQ ID NO: 85), derived from the gene encoding triosephosphate isomerase, tMET25 (SEQ ID NO: 86), derived from the gene encoding O-acetylhomoserine-O-acetylserine sulfhydrylase, tDIT1 (SEQ ID NO: 87), tRPL3 (SEQ ID NO: 88), tRPL3.sm (SEQ ID NO: 89), tRPL3.sba (SEQ ID NO: 90), tRPL41B (SEQ ID NO: 91), tRPL15A (SEQ ID NO: 92), tRPL15A.sba (SEQ ID NO: 93), tIDP1 (SEQ ID NO: 94), tTEF1.sba (SEQ ID NO: 95), and tTDH3 (SEQ ID NO: 100) may be selected from the group comprising:

[0468] In particular, the terminator may be selected from the group consisting of tTPI1, tMET25, tDIT1, tRPL3, tRPL3.sm, tRPL3.sba, tRPL41B, tRPL15A, tRPL15A.sba, tIDP1, tTEF1.sba, and tTDH3.

[0469] The terminator of the present invention may originate from any organism from the class Hemiascomycetes, and in particular from an organism selected from the group consisting of Saccharomyces cerevisiae and Saccharomyces bayanus.

[0470] Recombinant cells The recombinant cell of the present invention may be selected from the group consisting of yeast and bacteria.

[0471] A recombinant cell of the invention, eg, a recombinant host cell of the invention, is preferably a recombinant yeast cell.

[0472] 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 setting. Furthermore, yeast cells can be easily separated from the culture medium compared to bacterial cells, which can greatly simplify the process of product extraction and purification.

[0473] Recombinant cells of the invention, particularly recombinant yeast cells of the invention, are preferably Saccharomycetales cells.

[0474] 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.

[0475] 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.

[0476] 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.

[0477] 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.

[0478] 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.

[0479] 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.

[0480] The recombinant cells of the present invention belonging to the genus Debaryomyces include Debaryomyces hansenii, Debaryomyces carsonii, Debaryomyces castellii, Debaryomyces marama, Debaryomyces occidentalis, Debaryomyces oviformis, Debaryomyces nepalensis, Debaryomyces coudertii, Debaryomyces udenii, Debaryomyces psychrosporus, and Debaryomyces yamadae. yamadae).

[0481] 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.

[0482] The recombinant cell of the present invention, in particular the recombinant yeast cell of the present 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.

[0483] In certain embodiments, the recombinant yeast cell according to the invention is of the genus Saccharomyces, Kluyveromyces, or Eremothecium, more particularly of a species selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces marxianus, Ogataea polymorpha, and Ashbya gossypii.

[0484] In one embodiment, the recombinant host cell of the invention is a yeast selected from the order Saccharomycetales, particularly the family Saccharomycetaceae, and in particular selected from the group consisting of Yarrowia lipolytica, Kluyveromyces marxianus, Ogataea polymorpha, Ashbya gossypii, and Saccharomyces cerevisiae.

[0485] The recombinant cell of the present invention may more preferably be a Saccharomyces cerevisiae cell.

[0486] As described above, the recombinant cells of the present invention have the ability to produce hyaluronic acid by inserting one or more recombinant nucleic acids of the present invention. In certain embodiments, the recombinant yeast of the present invention has the ability to produce hyaluronic acid with a controlled size (controlled molecular weight) by inserting one or more recombinant nucleic acids of the present invention.

[0487] The methods implemented to insert specific DNA constructs into genes are within the general knowledge of those skilled in the art, and the relevant methods are described in more detail in the Examples below.

[0488] However, unexpected technical problems were encountered because the outcome of inserting a DNA construct into a cell's genome, particularly a yeast genome, such as the genome of Saccharomyces cerevisiae, is unpredictable. Specifically, the viability of cells, particularly yeast, and their ability to grow and produce the desired hyaluronan are also unpredictable.

[0489] In order to obtain the recombinant cells, particularly recombinant yeast, of the present invention, a number of different constructs were tested by the inventors in order to obtain viable and efficient recombinant cells, particularly yeast.

[0490] Culture conditions The present invention also relates to the use of the recombinant cells of the present invention for the production of hyaluronic acid, particularly hyaluronic acid of controlled molecular weight.

[0491] The present invention further provides a method for producing hyaluronic acid (HA) of a desired molecular weight (HAMW), comprising: (a) culturing a recombinant cell of the present invention in a culture medium for a time sufficient to produce hyaluronic acid (HA) of a desired molecular weight; (b) optionally isolating or recovering hyaluronic acid (HA) from the recombinant cells and / or culture medium; The present invention relates to a method comprising:

[0492] Typically, cells of the invention, particularly yeasts of the invention, are grown in a suitable culture medium at a temperature of about 20°C to about 37°C, preferably 27-34°C.

[0493] 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 appropriate media for the growth of particular cells, particularly yeasts, will be known to those skilled in the art of microbiology or fermentation science.

[0494] A particular medium that is suitable 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:

[0495] 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, and crude mixtures derived from renewable raw materials such as cheese whey permeate, corn steep liquor, sugar beet molasses, and malt.

[0496] Nitrogen sources that can 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.

[0497] 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.

[0498] 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.

[0499] 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.

[0500] The term "suitable culture medium" is defined above.

[0501] 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 publication: D. Burke et al., Methods in Yeast Genetics - A Cold Spring Harbor Laboratory Course Manual (2000).

[0502] The pH range suitable for fermentation is pH 3.0 to pH 7.5, with pH 4 to pH 6 being preferred as initial conditions.

[0503] As described elsewhere herein, the pH value of the culture medium can be adjusted during the culturing step of the methods of the present invention to modulate the activity of the polypeptide exhibiting hyaluronidase activity, which will affect the molecular weight of the hyaluronic acid molecule produced by the recombinant cells of the present invention, particularly the recombinant yeast of the present invention.

[0504] Specifically, the pH of the culture medium can be modified depending on the hyaluronic acid intended to be produced by the recombinant yeast. For example, the pH of the culture medium can be maintained at 4, 5, 5, or 6 throughout the culture period.

[0505] In certain embodiments, the pH of the culture medium may be changed or varied over 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 6, or be maintained at 6, and then be lowered to 4.

[0506] In certain embodiments, the pH of the culture medium may be modulated during the culturing step (a) of the method of the invention so 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).

[0507] In another embodiment, the pH of the culture medium may remain the same throughout the length of time that the recombinant cells of the invention, particularly recombinant yeast cells of the invention, are cultivated.

[0508] The time period for culturing the recombinant cells of the present invention, in particular the recombinant yeast cells of the present invention, can vary depending on the molecular weight of the hyaluronic acid of interest: the longer the time period, the lower the molecular weight of the hyaluronic acid in a given culture medium of the recombinant cells of the present invention, in particular the recombinant yeast cells of the present invention.

[0509] The length of time for culturing the recombinant cells of the present invention, particularly the recombinant yeast cells of the present invention, can be about 35 hours to about 50 hours, preferably about 40 hours to about 50 hours, and particularly about 48 hours.

[0510] Fermentation can be carried out under aerobic or microaerobic conditions.

[0511] The amount of hyaluronic acid 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).

[0512] 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 organism, and fermentation occurs without adding anything to the system. Typically, however, a "batch" fermentation method or system is batch with respect to the addition of a carbon source, and control of factors such as temperature, pH, and oxygen concentration is often attempted. In a batch system, the metabolic and biomass composition of the system changes constantly until the time the fermentation is stopped. Within a batch culture, cells progress from a static lag phase to a high-growth logarithmic phase and eventually to a stationary phase where growth rate decreases or stops. If untreated, the stationary phase cells will eventually die. Generally, the logarithmic phase cells are responsible for the majority of the production of the end product or intermediate.

[0513] Fed-batch systems can also be used in the present invention. A fed-batch system is similar to a typical batch system, except that the carbon source substrate is added incrementally as the fermentation progresses. Fed-batch systems are useful when catabolite repression (e.g., glucose repression) tends to inhibit cellular metabolism and when it is desirable to limit the amount of substrate in the medium. Measuring the actual substrate concentration in a fed-batch system is difficult and therefore must be estimated based on changes in measurable factors such as pH, dissolved oxygen, and the partial pressure of waste gases such as CO2.

[0514] 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). While the present invention is conducted in batch mode, it is contemplated that the method would be adaptable to continuous fermentation.

[0515] 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.

[0516] Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end-product concentration. For example, one method would maintain a limiting nutrient, such as the carbon source or nitrogen level, at a fixed rate and allow all other parameters to vary. In other systems, some factors that affect growth may be continuously altered, while the cell concentration, as measured by medium turbidity, is held constant. Continuous systems attempt to maintain steady-state growth conditions; 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.

[0517] It is contemplated that the present invention may be practiced using either a batch, fed-batch, or continuous process, 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.

[0518] To further improve hyaluronic acid production, a particular embodiment may consist of 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.

[0519] 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 contains 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 contains at most 40% w / w of glucose, including at most 35% w / w of glucose.

[0520] In a preferred embodiment, the method of the present invention is carried out on an industrial scale.

[0521] 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 1000 L to about 3000 L, even more preferably about 10,000 L, even more preferably 100,000 L, or even about 250,000 L.

[0522] The present invention further provides a method for producing the aforementioned hyaluronic acid, comprising: (a) culturing a recombinant cell of the present invention in a culture medium; (b) recovering hyaluronic acid from the culture medium; Including, The hyaluronic acid recovered in step (b) - the nature and origin of the recombinant nucleic acid(s) encoding a polypeptide having hyaluronidase activity of the recombinant cell of the invention, particularly the recombinant yeast of the invention; - the nature and origin of the promoter controlling the expression of one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity in the recombinant cell of the invention, in particular in the recombinant yeast of the invention; - the presence or absence of anchoring signals associated with one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity in a recombinant cell of the invention, particularly 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 for culturing the recombinant cell of the present invention, in particular the recombinant yeast of the present invention having a molecular weight controlled through the selection of Regarding the method.

[0523] The molecular weight of hyaluronic acid is a specific molecular weight, or more preferably, a molecular weight within a specific range, 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.

[0524] The present invention also relates to the use of recombinant cells according to the present invention, in particular recombinant yeast cells according to the present invention, for the production of hyaluronic acid (HA) having a molecular weight in the range of about 20 kDa to about 50 kDa or about 50 kDa to about 1000 kDa.

[0525] The one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity of the recombinant cells of the present invention can be selected from, for example, those originating from or derived from at least one of Cupienius salei (Csa), Loxosceles intermedia (Li), Hirudonipponia (Hn), Bothropus atrox (Ba), Titius serrata (Ts), or Vespa magnifica (Vm), particularly those originating from or derived from at least one of Cupienius salei (Csa), Loxosceles intermedia (Li), Hirudonipponia (Hn), Bothropus atrox (Ba), or Titius serrata (Ts), more particularly those having the sequences set forth as SEQ ID NO: 19 (Ba), SEQ ID NO: 25 (Li), SEQ ID NO: 21 (Csa), SEQ ID NO: 27 (Ts), or SEQ ID NO: 23 (Hn) (in the presence of a secretion signal and in the absence of an anchoring signal).

[0526] The one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity of the recombinant cells of the present invention can be selected from, for example, those originating from or derived from at least one of Cupienius salei (Csa), Loxosceles intermedia (Li), Hirudonipponia (Hn), Bothropus atrox (Ba), Titius serrata (Ts), or Vespa magnifica (Vm), particularly Cupienius salei (Csa), Loxosceles intermedia (Li), Hirudonipponia (Hn), Bothropus atrox (Ba), or Titius serrata (Ts), particularly those having a sequence set forth as SEQ ID NO: 22 (Csa), SEQ ID NO: 26 (Li), SEQ ID NO: 24 (Hn), SEQ ID NO: 20 (Ba), or SEQ ID NO: 28 (Ts) (in the presence of both a secretion signal and an anchoring signal).

[0527] The one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity of the recombinant cells of the present invention can be under the control of a promoter selected from the group consisting of, for example, pTEF1, pCCW12, pCCW12.sba, pCCW12.Sar, pPDC1, pTEF3, pTDH3, pNUP57, and pCCW10.ago.

[0528] Specifically, the promoter of one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity of a recombinant cell of the invention, particularly a recombinant yeast of the invention, can originate from or be derived from Saccharomyces bayanus, Saccharomyces kudriabzevii, Saccharomyces mikatae, Saccharomyces arboricola, or other Saccharomycetales species, or Abishia gossypii.

[0529] The secretory signal of the present invention may be, for example: - the nucleic acid sequence set forth as SEQ ID NO: 43, and / or - the amino acid sequence set forth as SEQ ID NO: 44 may have:

[0530] The anchoring signal of the present invention can be, for example: - the nucleic acid sequence set forth as SEQ ID NO: 45, and / or - the amino acid sequence of SEQ ID NO: 46 may have:

[0531] A secretion signal can be fused to a polypeptide having hyaluronidase activity by creating a chimeric nucleic acid that begins with a nucleic acid sequence encoding the signal peptide and is followed by a recombinant nucleic acid encoding a polypeptide having hyaluronidase activity as defined above.

[0532] Secretion signals and anchoring signals can be fused to a polypeptide with hyaluronidase activity by creating a chimeric nucleic acid that begins with a nucleic acid sequence encoding a signal peptide, followed by a recombinant nucleic acid encoding a polypeptide with hyaluronidase activity as defined above, followed by a nucleic acid sequence encoding the anchoring signal.

[0533] Such chimeric nucleic acid sequences can be obtained by techniques known to those skilled in the art, such as chemical synthesis of nucleic acids or by any recombinant technique, such as cloning or PCR.

[0534] The present invention further relates to the aforementioned method for producing hyaluronic acid having a molecular weight of about 50 kDa or less, particularly less than 50 kDa and greater than about 20 kDa, comprising: (a) the pH of the medium is greater than 4, particularly in the range of about 5 to about 7; (b) one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity that are integrated into the genome of a recombinant cell of the invention, (i) originating from or derived from Titius serrulatus, having a secretion signal but lacking an anchoring signal, and under the control of a promoter selected from the group consisting of pTDH3, pENO2, pTEF3, pTEF1, pPDC1, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, pCCW12.sar, pTDH3-1.Sba, and pTDH3.Sar; (ii) originating from or derived from Cupienius salei, having a secretion signal but lacking an anchoring signal, and under the control of a promoter selected from the group consisting of pNUP57 and pCCW10.ago; (iii) originating from or derived from Cupienius salei, having a secretion signal and an anchoring signal, and under the control of promoter pCWP2; (iv) originating from or derived from Loxosceles intermedia, having a secretion signal but lacking an anchoring signal, and under the control of a promoter selected from the group consisting of pTDH3, pENO2, pTEF3, pTEF1, pPDC1, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, pCCW12.sar, pTDH3-1.Sba, and pTDH3.Sar; or (v) originating from or derived from Hirudonipponia, having a secretion signal but lacking an anchoring signal, and being under the control of a promoter selected from the group consisting of pNUP57 and pCCW10.ago; or (vi) a vector originating from or derived from Hirudonipponia, having both a secretion signal and an anchoring signal, and being under the control of a promoter selected from the group consisting of pTDH3, pENO2, pTEF3, pTEF1, pPDC1, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, pCCW12.sar, pTDH3-1.Sba, and pTDH3.Sar; (c) the step of recovering hyaluronic acid from the culture medium is carried out about 48 hours after the start of culturing 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, Regarding the method.

[0535] The present invention further provides the aforementioned method for producing hyaluronic acid having a molecular weight of about 50 kDa or more and about 1000 kDa or less, comprising: (a) the pH of the medium is between about 4 and about 6, and optionally varies from about 6 to about 4 during the process of culturing the recombinant cells, particularly recombinant yeast cells, of the invention; (b) one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity that are integrated into the genome of a recombinant cell of the invention, (i) originating from or derived from Titius serrulatus, having a secretion signal but lacking an anchoring signal, and under the control of a promoter selected from the group consisting of pTDH3, pENO2, pTEF3, pTEF1, pPDC1, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, pCCW12.sar, pTDH3-1.Sba, and pTDH3.Sar; (ii) originating from or derived from Titius serrulatus, having both a secretion signal and an anchoring signal, and under the control of a promoter selected from the group consisting of pTDH3, pENO2, pTEF3, pTEF1, pPDC1, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, pCCW12.sar, pTDH3-1.Sba, and pTDH3.Sar; (iii) originating from or derived from Loxosceles intermedia, having both a secretion signal and an anchoring signal, and under the control of a promoter selected from the group consisting of pNUP57 and pCCW10.ago; (iv) a vector originating from or derived from Loxosceles intermedia, having a secretion signal but lacking an anchoring signal, and under the control of a promoter selected from the group consisting of pTDH3, pENO2, pTEF3, pTEF1, pPDC1, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, pCCW12.sar, pTDH3-1.Sba, and pTDH3.Sar; (v) originating from or derived from Cupienius salei, having both a secretion signal and an anchoring signal, and under the control of a promoter selected from the group consisting of pNUP57 and pCCW10.ago; (vi) originating from or derived from Cupienius salei, having a secretion signal but lacking an anchoring signal, and under the control of a promoter selected from the group consisting of pNUP57 and pCCW10.ago; or (vii) a vector originating from or derived from Hirudonipponia, having both a secretion signal and an anchoring signal, and being under the control of a promoter selected from the group consisting of pTDH3, pENO2, pTEF3, pTEF1, pPDC1, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, pCCW12.sar, pTDH3-1.Sba, and pTDH3.Sar; (c) the step of recovering hyaluronic acid from the culture medium is carried out about 48 hours after the start of culturing 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, Regarding the method.

[0536] The present invention further relates to the aforementioned method for producing hyaluronic acid having a molecular weight of more than about 1000 kDa, particularly having a molecular weight of about 1000 kDa to about 1,500,000 Da, comprising: (a) the pH of the medium is between about 4 and about 6, and optionally varies from about 6 to about 4 during the process of culturing the recombinant cell, particularly the recombinant yeast, of the invention; (b) one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity that are integrated into the genome of a recombinant cell of the invention, (i) originating from or derived from Titius serrulatus, having a secretion signal but lacking an anchoring signal, and under the control of a promoter selected from the group consisting of pNUP57 and pCCW10.ago; (ii) originating from or derived from Loxosceles intermedia, having a secretion signal but lacking an anchoring signal, and under the control of a promoter selected from the group consisting of pNUP57 and pCCW10.ago; (iii) originating from or derived from Bothropus atrox, having a secretion signal but lacking an anchoring signal, and being under the control of a promoter selected from the group consisting of pTDH3, pENO2, pTEF3, pTEF1, pPDC1, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, pCCW12.sar, pTDH3-1.Sba, and pTDH3.Sar; or (iv) a vector originating from or derived from Bothropus atrox, having both a secretion signal and an anchoring signal, and under the control of a promoter selected from the group consisting of pTDH3, pENO2, pTEF3, pTEF1, pPDC1, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, pCCW12.sar, pTDH3-1.Sba, and pTDH3.Sar; (c) the step of recovering hyaluronic acid from the culture medium is carried out about 48 hours after the start of culturing the recombinant cell of the invention, in particular the recombinant yeast cell of the invention, The recombinant cell is in particular a recombinant yeast, more particularly a Saccharomyces cerevisiae cell, Regarding the method.

[0537] Another aspect of the present invention relates to hyaluronic acid (HA) obtained or obtainable from the recombinant cell of the present invention or the method according to the present invention.

[0538] A further aspect of the present invention is a culture medium comprising the hyaluronic acid of the present invention.

[0539] The present invention further relates to a composition comprising hyaluronic acid according to the invention.

[0540] The present invention also relates to industrial or consumer products or consumables comprising (i) the hyaluronic acid of the present invention, (ii) a culture medium comprising the hyaluronic acid of the present invention, or (iii) a composition comprising the hyaluronic acid of the present invention.

[0541] In particular, said industrial or consumer product or consumable product according to the present invention may be a cosmetic product, a flavor product, a fragrance product, a foodstuff, a food, a beverage, a texturant, a pharmaceutical composition, a dietary supplement, a nutraceutical, a cleaning product, and / or a dental and / or oral hygiene composition.

[0542] Purification of hyaluronic acid According to a particular aspect of the present invention, the fermentation production of hyaluronic acid preferably includes the step of isolating the produced hyaluronic acid from the culture medium.Recovering hyaluronic acid from the culture medium is a routine procedure 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.

[0543] Yeast is preferred as a model cell in the present invention because the synthesized hyaluronic acid is transported entirely outside the cell, thus simplifying the purification process.

[0544] Gas stripping is accomplished using a stripping gas selected from helium, argon, carbon dioxide, hydrogen, nitrogen, or mixtures thereof.

[0545] 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.

[0546] Those skilled in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA molecules differing in nucleotide sequence can be used to encode a given enzyme of the present disclosure. The native DNA sequences encoding the biosynthetic enzymes described above are referenced herein solely 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 possesses the enzymatic anabolic or catabolic activity of the reference polypeptide. Furthermore, the amino acid sequences encoded by the DNA sequences set forth herein are solely illustrative of embodiments of the present disclosure.

[0547] 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 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.

[0548] 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 polynucleotides encoding such enzymes.

[0549] Techniques known to those skilled in the art may be suitable for identifying additional homologous genes and enzymes. Generally, similar genes and / or similar enzymes can be identified by functional analysis and will have functional similarities.

[0550] Techniques known to those skilled in the art may be suitable for identifying similar genes and similar enzymes, or any biosynthetic pathway gene, protein, or enzyme, 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 can use techniques to identify homologous or similar genes, proteins, or enzymes with functional homology or similarity. Techniques include testing cells or cell cultures for the catalytic activity of an 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 having said activity by purification, and determining the protein sequence of the enzyme by techniques such as Edman denaturation, designing PCR primers to potential nucleic acid sequences, amplifying the DNA sequence by PCR, and cloning the nucleic acid sequence. To identify homologous or similar genes and / or homologous or similar enzymes, analogous genes and / or analogous 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.

[0551] derivative The term "hyaluronic acid (HA)," as used herein, is also intended to encompass derivatives of hyaluronic acid, such as acetylated or sulfated hyaluronic acid, that are useful for applications in cosmetic products, flavored 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.

[0552] The hyaluronic acid may be prepared as a composition.

[0553] Formulations and Products The compositions of the present invention may be incorporated into formulations / products, for example, nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulations / products.

[0554] Therefore, 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.

[0555] 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.

[0556] "Cosmetic product" is intended to mean any substance or mixture intended to be placed in contact with the exterior of the human body (epidermis, hair system, nails, lips, and external genitalia) or the teeth and mucous membranes of the oral cavity, exclusively or primarily with a view to cleansing them, perfume them, modifying their appearance, protecting them, keeping them in good condition, correcting body odor, and / or combinations thereof.

[0557] "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.

[0558] "Mixture" means a mixture or solution composed of two or more substances.

[0559] The present invention also provides the use of the compositions of the present invention in nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulations / products.

[0560] Such formulations or products are hereinafter referred to as "formulations or products of the invention."

[0561] 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.

[0562] "Cosmetic" or "cosmetic active ingredient" means any and all natural, naturally occurring, nature-identical, synthetic, synthetically produced, biosynthetically produced, sustainable, renewable, and / or biodegradable compounds, ingredients, intermediates, molecules, substances, raw materials, or products, either individually or as part of a mixture of compounds, ingredients, intermediates, molecules, substances, raw materials, or products; Blends, compositions, formulations (including, but not limited to, skin moisturizers, creams, balms, serums, oils, eye, facial makeup, rinse-off hair products, leave-in hair products, hair dyes (including, but not limited to, natural hair dyes), and / or combinations thereof), final products and related technology, such as, for example, ingredients incorporated into cosmetic formulations (e.g., natural colorants, preservatives, emulsifiers, antioxidants, etc., which, for example, have no activity on 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 by rubbing, pouring, sprinkling, spraying, or otherwise onto the human or animal body and / or by contact with various external and / or superficial parts of the human or animal body (including, but not limited to, the skin, hair, body 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) applied to the textile; and / or - cleansing, caring for, cooling, beautifying, conditioning, treating, soothing, texturizing, enhancing, protecting, maintaining, improving, enhancing, altering, and / or changing the exterior and / or surface 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 cleansing or perfumes the skin, oral mucosa, scalp, or hair, primarily by providing soothing, healing, repairing, or regenerating skin, hydration, or to relieve, smooth, moisturize, adjust color, 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 a human or animal body, 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, 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 products applied to the human or animal body, and / or - Providing cosmetic and / or dermatological functions and / or benefits along with biological activity benefits (but without affecting the structure or function of the body) "cosmetic" means a method of making a cosmetic or cosmetically active ingredient, or part thereof, that is useful in / used in / intended for, or related to. For the avoidance of doubt, a cosmetic or cosmetically active ingredient, or part thereof, may also qualify as a functional ingredient and / or a nutraceutical.

[0563] "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.

[0564] "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 associated with improving or maintaining health and / or cosmetic benefits and the appearance of the human body, including ingredients and their associated delivery devices (e.g., capsules), delivery systems thereof (e.g., blends or formulations), and methods of making the foregoing. For the avoidance of doubt, nutraceuticals include compounds that can be used as a supplement to food or beverages, whether in solid formulation, capsule, tablet, liquid formulation, solution, or suspension.

[0565] Alternatively, a nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulation / product may consist of or consist essentially of the composition of the present invention.

[0566] The cosmetic formulation / product may be an anti-aging formulation.

[0567] As used herein, references to pharmaceutically, veterinarily, or cosmetically acceptable excipients may refer to pharmaceutically, veterinarily, or cosmetically acceptable adjuvants, diluents, and / or carriers known to those skilled in the art.

[0568] "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 compositions 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 solely to act as an excipient, i.e., not intended to have biological activity itself.

[0569] Nutriceutical, pharmaceutical, veterinary, oenological or cosmetic formulations / products may be in liquid or solid form.

[0570] Liquid dosage formulations / products for oral administration include solutions, emulsions, aqueous or oily suspensions, syrups, and elixirs.

[0571] 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.

[0572] A formulation or product (e.g., a pharmaceutical, veterinary, or cosmetic formulation / product) may include one or more additional ingredients, e.g., pharmaceutical ingredients and excipients, such as sweeteners, flavoring agents, coloring agents, and preservatives.

[0573] The formulation or product (e.g., pharmaceutical, veterinary, or cosmetic formulation / product) may also include one or more additional active ingredients, for example, cosmetic or pharmaceutical active ingredients such as hyaluronic acid, centella asiatica extract, peptides such as Matrixyl® and Argireline®, and mixtures thereof.

[0574] The formulations or products of the present invention may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients (or ingredients), such as 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.

[0575] The 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.

[0576] 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.

[0577] 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.

[0578] Suitable pharmaceutical carriers include sterile aqueous solutions and various organic solvents.Examples of liquid carriers are syrup, vegetable oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.In addition, carriers or diluents can comprise any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with wax.

[0579] 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.

[0580] Typically, such carriers are dermatologically acceptable.

[0581] 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 problems.

[0582] The carrier can 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).

[0583] The dermatologically acceptable carrier may be in the form of an emulsion. Emulsions can 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., water-in-oil 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 ingredients (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 ingredients 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.

[0584] The carrier may comprise 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.

[0585] The cosmetic formulation / product may optionally contain one or more additional ingredients commonly used in cosmetic compositions (e.g., colorants, skin tone adjusting agents, skin anti-aging agents, anti-inflammatory agents, sunscreen agents, combinations thereof, etc.), provided that the additional ingredients do not unnecessarily alter the anti-glycation benefits provided by the composition.

[0586] 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, etc.

[0587] 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.

[0588] 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.

[0589] Typically, the amount of a composition of the present 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% by weight of the formulation or product, 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.

[0590] Those skilled in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA molecules differing in nucleotide sequence can be used to encode a given enzyme of the present disclosure. The native DNA sequences encoding the biosynthetic enzymes described above are referenced herein solely 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 possesses the enzymatic anabolic or catabolic activity of the reference polypeptide. Furthermore, the amino acid sequences encoded by the DNA sequences set forth herein are solely illustrative of embodiments of the present disclosure.

[0591] 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 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.

[0592] 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 polynucleotides encoding such enzymes.

[0593] Techniques known to those skilled in the art may be suitable for identifying additional homologous genes and enzymes. Generally, similar genes and / or similar enzymes can be identified by functional analysis and will have functional similarities.

[0594] Techniques known to those skilled in the art may be suitable for identifying similar genes and similar enzymes, or any biosynthetic pathway gene, protein, or enzyme, 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 can use techniques to identify homologous or similar genes, proteins, or enzymes with functional homology or similarity. Techniques include testing cells or cell cultures for the catalytic activity of an 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 having said activity by purification, and determining the protein sequence of the enzyme by techniques such as Edman denaturation, designing PCR primers to potential nucleic acid sequences, amplifying the DNA sequence by PCR, and cloning the nucleic acid sequence. To identify homologous or similar genes and / or homologous or similar enzymes, analogous genes and / or analogous 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.

[0595] 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," should 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 refers to "including" as well as "consisting of," 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 dictates 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."

[0596] It is understood that this disclosure is not limited to the particular methodologies, protocols, and reagents described herein, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is 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 one of ordinary skill 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.

[0597] 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, terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," Leuenberger, H.G.W., Nagel, B. and Kolbl, H. (eds.) (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0598] Several documents are cited throughout this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank accession number sequence entries, etc.), whether stated above or below, is hereby incorporated by reference in its entirety.

[0599] The following examples and figures are offered for illustrative purposes and are not intended to imply limitations of the present invention. [Example]

[0600] 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).

[0601] The clusters of genes mentioned below were integrated into recombinant yeast one at a time using the ability of yeast to efficiently recombine free DNA ends that have sequence homology.

[0602] In addition, with the aim of better understanding the following genotypes: - jlp1, lyp1, sam3, his3, leu2, trp1, and ura3 are insertion sites. - Lower case letters mean that the gene under consideration is inactive, upper case letters represent active genes. - "::" after a gene name means that the gene is disrupted by the one that follows it (if more than one gene is inserted, they are indicated in brackets []). The disruption of the gene occurs simultaneously with the complete deletion of the coding sequence, but the promoter is preserved. Consequently, 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.

[0603] More specifically, the coding sequences to be cloned were artificially synthesized. For heterologous (non-yeast) sequences, 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 50-200 nucleotide sequence homologous to the terminator sequence of the upstream gene. Similarly, the terminator of each gene (a gene comprising a promoter, coding sequence, and terminator) is followed by a sequence homologous to the immediately following gene. Thus, each unit to be integrated has a 50-200 nucleotide overlap with both the upstream and downstream units. For the first unit, the promoter is preceded by 50-200 nucleotides homologous to the yeast chromosomal nucleotides of the integration locus. Similarly, for the last unit, the terminator is followed by 50-200 nucleotides homologous to the yeast chromosomal nucleotides of the integration locus.

[0604] Each unit is then PCR amplified from the plasmid construct to obtain X units of linear DNA with overlapping sequences. At least one of the genes is a requirement marker for selecting 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.

[0605] Example 2 Comparative Example for the Production of Hyaluronic Acid 1. Production of hyaluronic acid less than 50 kDa A. First, three recombinant strains are obtained: YA5234-1, YA5235-1, and YA5302-2.

[0606] Thus, these three strains are: <h2 style=";text-align:left;direction:ltr">YA5234-1 : MAT-α、can1-100、his3::[pSAM1-UGP1-tRPL3-pMET6-QRI1-tIDP1- HIS3]x5,jlp1::[LEU2.Kl,pCUP1-HASA2.Sz-tRPL41B,pCUP1-UGP1-tTPI1,pTDH3-QRI1-tMET25,pCCW12-HASB.At-tRPL15A],leu2,lyp1::[pCCW12.sba-HYAL-3.Ts-tRPL15A,pCCW12.Sk-HASB-A.Vir-tTEF1.Sba,pCCW12-HASA-1.Vir-tDIT1,pCCW12.Sba-HASB.vir-tRPL3,p CCW12.Sm-HASA-A.Vir-tRPL15A.Sba]、sam3::[LEU2.Kl、pPDC1-PGM1-tIDP1、pTEF1-GFA1-tRPL15A、pENO2-UGP1-tRPL3、pCWP2-GNA1-tT PI1、pTEF3-PCM1-tIDP1、pCCW12-XHASA2. TRP1]x2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">YA5235-1:MAT-α、can1-100、his3::[pSAM1-UGP1-tRPL3-pMET6-QRI1-tIDP1- HIS3]x5,jlp1::[LEU2.Kl,pCUP1-HASA2.Sz-tRPL41B,pCUP1-UGP1-tTPI1,pTDH3-QRI1-tMET25,pCCW12-HASB.At-tRPL15A],leu2,lyp1::[pNUP57-HYAL-3.Csa-tRPL15A,pCCW12.Sk-HASB-A.Vir-tTEF1.Sba,pCCW12-HASA-1.Vir-tDIT1,pCCW12.Sba-HASB.vir-tRPL3,pC CW12.Sm-HASA-A.Vir-tRPL15A.Sba]、sam3::[LEU2.Kl、pPDC1-PGM1-tIDP1、pTEF1-GFA1-tRPL15A、pENO2-UGP1-tRPL3、pCWP2-GNA1-tT PI1、pTEF3-PCM1-tIDP1、pCCW12-XHASA2. TRP1]x2<h2 style=";text-align:left;direction:ltr"> YA5302-2: MAT-α, can1-100, his3::[pSAM1-UGP1- tRPL3-pMET6-QRI1-tIDP1- HIS3]x5, jlp1::[LEU2.Kl, pCUP1-HASA2.Sz-tRPL41B, pCUP1-UGP1-tRPL3, pCUP1-QRI1-tIDP1, pPDC1-UGP1-tTPI1, pTDH3-QRI1-tMET25, pCCW12- HASB.At-tRPL15A], leu2, lyp1::[pCCW12.Sba-HYAL-3.Li-tRPL15A, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW12-HASA-1.Vir-tDIT1, pCCW12.Sba -HASB.vir-tRPL3, pCCW12.Sm-HASA-A.Vir-tRPL15A.Sba], sam3::[LEU2.Kl, pPDC1-PGM1-tIDP1, pTEF1-GFA1-tRPL15A, pENO2-UGP1-tRPL3, pCWP 2-GNA1-tTPI1, pTEF3-PCM1-tIDP1, pCCW12-XHASA2.Xl-tRPL3, pTDH3-QRI1-tIDP1], trp1::[pMET6-HASB.Vir-tRPL3, pMET25-HASA-1.Vir-tIDP1- TRP1]x2

[0607] HYAL-3 refers to a nucleic acid sequence that encodes a polypeptide with hyaluronidase activity that has a secretion signal but no anchoring signal.

[0608] HASA, HASA-1, HASA-A, HASA2, and XHASA2 all represent nucleic acids encoding polypeptides having hyaluronan synthase activity. They differ from one another in that they are different recoded versions of a nucleic acid sequence encoding a hyaluronan synthase enzyme. HASA-1 has the sequence of SEQ ID NO:1, HASA-A has the sequence of SEQ ID NO:2, HASA2 has the sequence of SEQ ID NO:7, and XHASA2 has the sequence of SEQ ID NO:6.

[0609] HASB and HASB-A represent nucleic acid sequences encoding polypeptides having UDP-glucose 6-dehydrogenase activity. They differ from each other in that they are different recoded versions of the nucleic acid sequence encoding the enzyme. HASB.At has the sequence of SEQ ID NO: 12, HASB.Vir has the sequence of SEQ ID NO: 13, and HASB-A.Vir has the sequence of SEQ ID NO: 14.

[0610] All these strains were incubated in baffled Erlenmeyer flasks in 25 ml of SY medium buffered with MES 0.1 M pH 5.5 at 28° C. for 48 hours under vigorous agitation.

[0611] 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%.

[0612] Growth medium was collected at 48 hours and assayed for hyaluronic acid content and quality. Aliquots of medium were loaded and run on a 0.5% agarose gel, followed by staining with "stains all" (Sigma Aldrich CAS No. 7423-31-6).

[0613] The amount of hyaluronic acid 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).

[0614] For illustrative purposes, an agarose gel obtained after running an aliquot of the supernatant of strain YA5235-1 and staining it with "stains all" (CAS number 7423-31-6) is provided in Figure 2A. Classically, HA molecular weight (MW) standards (from left to right, 10 kDa, 20 kDa, 50 kDa, and 300 kDa MW) were run side by side on the gel.

[0615] The amounts obtained for these different strains were, respectively: - YA5234-1: 2.4gL -1 - YA5235-1: 4.5gL -1 - YA5302-2: 3.7gL -1 is.

[0616] In comparison, native yeast strains, such as strain CC788-2D (Cherest et al. (2000) J. Biol. Chem. 275: 14056-14063), do not produce hyaluronic acid.

[0617] This experiment showed that recombinant strains containing modifications according to the present invention produced greater amounts of hyaluronic acid when cultured under the same conditions as other recombinant strains that did not contain all of the genetic modifications according to the present invention.

[0618] Furthermore, these three strains resulted in the production of hyaluronic acid with molecular weights of 20 kDa to 50 kDa after 48 hours.

[0619] B. Three other recombinant strains were also obtained: YA5110-13 and YA5260-4.

[0620] These three strains are: <h2 style=";text-align:left;direction:ltr">YA5110-13: MAT-α, can1-100, his3::[pSAM1-UGP1-tRPL3, pMET6-QRI1-tIDP1-HIS3]x5, jlp1::[LEU2.Kl, pCUP1-HASA2.Sz-tRPL41B, pCUP1-UGP1-tTPI1, pTDH3-QRI1-tMET25, pCCW12-HASB.At-tRPL15A], leu2, lyp1::[pNUP57-HYAL-3.Hn-tRPL15A, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW12-HASA-1.Vir-tDIT1, p CCW12.Sba-HASB.vir-tRPL3、pCCW12.Sm-HASA-A.Vir-tRPL15A.Sba]、sam3::[LEU2.Kl、pPDC1-PGM1-tIDP1、pTEF1-GFA1-tRPL15A、pENO2-UGP1-tRPL3、 pCWP2-GNA1-tTPI1、pTEF3-PCM1-tIDP1、pCCW12-XHASA2. TRP1]x2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">YA5260-4:MAT-α、can1-100、his3::[pSAM1-UGP1-tRPL3-pMET6-QRI1-tIDP1- HIS3]x5,jlp1::[LEU2.Kl,pCUP1-HASA2.Sz-tRPL41B,pCUP1-UGP1-tRPL3,pCUP1-QRI1-tIDP1,pPDC1-UGP1-tTPI1,pTDH3-QRI1-tMET25,pCCW12-HASB.At-tRPL15A],leu2,lyp1::[pTEF3-HYAL-31.Hn-tRPL15A,pCCW12.Sk-HASB-A.Vir-tTEF1.Sba,pCCW12-HASA-1.Vir-tDIT1,pCCW12.Sba-H ASB.vir-tRPL3、pCCW12.Sm-HASA-A.Vir-tRPL15A.Sba]、sam3::[LEU2.Kl、pPDC1-PGM1-tIDP1、pTEF1-GFA1-tRPL15A、pENO2-UGP1-tRPL3、pCWP2 -GNA1-tTPI1、pTEF3-PCM1-tIDP1、pCCW12-XHASA2. TRP1]x2<h2 style=";text-align:left;direction:ltr"> YA5569 : MAT-α, his3::[pSAM1-UGP1- tRPL3, pMET6-QRI1-tIDP1, HIS3]x5, jlp1::[LEU2.Kl, pCUP1-HASA-1.Sz-tRPL41B, pCUP1-UGP1-tRPL3, pCUP1-QRI1-tIDP1, pPDC1-UGP1-tTPI1, pTDH3-QRI1 -tMET25, pCCW12-HASB.At-tRPL15A], leu2, lyp1::[pCWP2-HYAL-31.Csa-tRPL15A, pCCW12.Sba-HASB.vir-tRPL3, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW 12-HASA-1.Vir-tRPL41B, pCCW12.Sm-HASA-A.Vir-tRPL15A.Sba], sam3::[LEU2.Kl, pPDC1-PGM1-tIDP1, pTEF1.Ago-GFA1-tRPL15A, pENO2-UGP1-tRPL3, pCW P2-GNA1-tTPI1, pTEF1-PCM1-tIDP1, pCCW12-XHASA2.Xl-tRPL3, pTDH3-QRI1-tIDP1], trp1::[tRPL3-HASB.Vir-pMET6, pMET25-HASA-1.Vir-tIDP1, TRP1]x2

[0621] HYAL-31 refers to a nucleic acid sequence that encodes a polypeptide with hyaluronidase activity that has both a secretory signal and an anchoring signal.

[0622] HASA-1, HASA-A, HASA2, and XHASA2 all represent nucleic acid sequences encoding polypeptides having hyaluronan synthase activity. They differ from one another in that they are different recoded versions of a nucleic acid sequence encoding a hyaluronan synthase enzyme. HASA-1 has the sequence of SEQ ID NO: 1, HASA-A has the sequence of SEQ ID NO: 2, HASA2 has the sequence of SEQ ID NO: 7, and XHASA2 has the sequence of SEQ ID NO: 6.

[0623] HASB and HASB-A represent nucleic acid sequences encoding polypeptides having UDP-glucose 6-dehydrogenase activity. They differ from each other in that they are different recoded versions of the nucleic acid sequence encoding the enzyme. HASB.At has the sequence of SEQ ID NO: 12, HASB.Vir has the sequence of SEQ ID NO: 13, and HASB-A.Vir has the sequence of SEQ ID NO: 14.

[0624] The strains were inoculated into 2 liters of SY medium pH 6 as defined above in a bioreactor containing 2% sucrose and 500 μM CuSO. They were then grown in fed-batch with 500 μM CuSO added at 7, 24, and 31 hours and 130–175 g L of sucrose added for 10–48 hours.

[0625] For YA5110-13, the pH was maintained at 5.8-6.2 using 15% H2SO4 and 10% NH4OH from t=31 h.

[0626] For YA5260-4, the pH was maintained at 5.8-6.2 throughout the fermentation using 15% H2SO4 and 10% NH4OH.

[0627] For YA5569, the pH was maintained at 5.8-6.2 throughout the fermentation using 15% H2SO4 and 10% NH4OH.

[0628] Growth medium was collected at 48 hours and assayed for hyaluronic acid content and quality as described above.

[0629] The amounts of hyaluronic acid obtained from these two strains were, respectively: - YA5110-13: 23g.L -1 - YA5260-4: 16g.L -1 - YA5569: 32g.L -1 is.

[0630] In comparison, the native strain does not produce hyaluronic acid.

[0631] This experiment showed that recombinant strains containing modifications according to the present invention produced greater amounts of hyaluronic acid when cultured under the same conditions as other recombinant strains that did not contain all of the genetic modifications according to the present invention.

[0632] Furthermore, the hyaluronic acid produced by the YA5110-13 and YA5260 strains had a molecular weight of approximately 20 kDa, while the hyaluronic acid produced by YA5569 had a molecular weight of approximately 20 kDa to 50 kDa.

[0633] Using these three recombinant strains, we performed the exact same experiment, substituting glucose for sucrose. After 48 hours, the resulting hyaluronic acid had the same molecular weight of approximately 20 kDa or between approximately 20 kDa and approximately 50 kDa.

[0634] C. Another recombinant strain, 5672-29A, was obtained as follows. 5672-29A: MAT-α, his3::[pSAM1-UGP1-tRPL3, pMET6-QRI1-tIDP1, HIS3]x5, jlp1::[LEU2.Sba, pTEF1.Sba-HASB.V ir-tRPL3.Sm, pTDH3.Sk-HASB.Vir-tTEF1.Sba, pTDH3-1.Sba-HASA-1.Vir-tRPL3.Sba, pTDH3.Sar-HASA- 1.Vir-tRPL15A.Sba, pTEF1-HYAL-31.Hn-tRPL15A], leu2, sam3::[LEU2.Kl, pPDC1-PGM1-tIDP1, pTEF1.A go-GFA1-tRPL15A, pENO2-UGP1-tRPL3, pCWP2-GNA1-tTPI1, pTEF1-PCM1-tRPL41B, TRP1.Sba-loxP], trp1

[0635] HYAL-31 represents a hyaluronidase with an anchoring signal.

[0636] 1 HASA-1 represents a nucleic acid sequence encoding a polypeptide having hyaluronan synthase activity. HASA-1 has the sequence of SEQ ID NO:1.

[0637] The strain was inoculated into 25 ml of SY medium as defined above, pH 5.5, buffered with MES 0.1 M, in a baffled Erlenmeyer flask at 28° C. under vigorous agitation.

[0638] Growth medium was collected at 48 hours and assayed for hyaluronic acid content and quality as described above.

[0639] The amount of hyaluronic acid obtained from this strain was 3.7 g L -1 It was.

[0640] 2. Production of hyaluronic acid with a molecular weight of 50 kDa to 1000 kDa A. First, three recombinant strains are obtained: YA5233-1, YA5359-10, and YA5381-1.

[0641] Thus, these three strains are: <h2 style=";text-align:left;direction:ltr">YA5233-1:MAT-α、can1-100、his3::[pSAM1-UGP1-tRPL3-pMET6-QRI1-tIDP1- HIS3]x5, jlp1::[LEU2.Kl, pCUP1-HASA2.Sz-tRPL41B, pCUP1-UGP1-tTPI1, pTDH3-QRI1-tMET25, pCCW12-HASB.At-tRPL15A], leu2, lyp1::[pTEF1-HYAL-3.Ts-tRPL15A, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW12-HASA-1.Vir-tDIT1, pCCW12.Sba-HASB.vir-tRPL3, pCC W12.Sm-HASA-A.Vir-tRPL15A.Sba]、sam3::[LEU2.Kl、pPDC1-PGM1-tIDP1、pTEF1-GFA1-tRPL15A、pENO2-UGP1-tRPL3、pCWP2-GNA1-tTP I1、pTEF3-PCM1-tIDP1、pCCW12-XHASA2. TRP1]x2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">YA5359-10: MAT-α、can1-100、his3::[pSAM1-UGP1- tRPL3-pMET6-QRI1-tIDP1- HIS3]x5、jlp1::[LEU2.Kl、pCUP1-HASA2.Sz-tRPL41B、pCUP1-UGP1- tTPI1、pTDH3-QRI1-tMET25、pCCW12-HASB.At-tRPL15A]、leu2、lyp1::[pCCW12.sba-HYAL-31.Ts-tRPL15A、pCCW12.Sk- HASB-A.Vir-tTEF1.Sba、pCCW12-HASA-1.Vir-tDIT1、pCCW12.Sba-HASB.vir-tRPL3、pCCW12.Sm-HASA-A.Vir-tRPL15A. Sba]、sam3::[LEU2.Kl、pPDC1-PGM1-tIDP1、pTEF1-GFA1-tRPL15A、pENO2-UGP1-tRPL3、pCWP2-GNA1-tTPI1、pTEF3-PCM1 -tIDP1、pCCW12-XHASA2. TRP1]x2<h2 style=";text-align:left;direction:ltr"> YA5381-1: MAT-α, can1-100, his3::[pSAM1-UGP1- tRPL3-pMET6-QRI1-tIDP1- HIS3]x5, jlp1::[LEU2.Kl, pCUP1-HASA2.Sz-tRPL41B, pCUP1-UGP1- tTPI1, pTDH3-QRI1-tMET25, pCCW12-HASB.At-tRPL15A], leu2, lyp1::[pCCW12.Sar-HYAL2-31.Ts-tRPL15A, pCCW12.Sk -HASB-A.Vir-tTEF1.Sba, pCCW12-HASA-1.Vir-tDIT1, pCCW12.Sba-HASB.vir-tRPL3, pCCW12.Sm-HASA-A.Vir-tRPL15A .Sba], sam3::[LEU2.Kl, pPDC1-PGM1-tIDP1, pTEF1-GFA1-tRPL15A, pENO2-UGP1-tRPL3, pCWP2-GNA1-tTPI1, pTEF3-PCM 1-tIDP1, pCCW12-XHASA2.Xl-tRPL3, pTDH3-QRI1-tIDP1], trp1::[pMET6-HASB.Vir-tRPL3, pMET25-HASA-1.Vir-tIDP1- TRP1]x2

[0642] HASA-1, HASA-A, HASA2, and XHASA2 have been previously defined.

[0643] HASB and HASB-A have been defined previously.

[0644] All these strains were incubated in baffled Erlenmeyer flasks in 25 ml of SY medium as defined above, buffered with MES 0.1 M pH 5.5, at 28° C. for 48 hours under vigorous agitation.

[0645] Growth medium was collected at 48 hours and assayed for hyaluronic acid content and quality as defined above.

[0646] For illustrative purposes, agarose gels obtained after running aliquots of supernatant from strain YA5359-10 and staining them with "stains all" (CAS number 7423-31-6) after 31 and 48 hours are provided in Figure 2B. Classically, HA molecular weight (MW) standards (from left to right, 20 kDa, 50 kDa, 60-120 kDa, 300 kDa, and 750 kDa MW) were run side by side on the gel.

[0647] The amounts obtained for these different strains were, respectively: - YA5233-1: 3.1gL -1 - YA5359-10: 3.4gL -1 - YA5381-1: 3.0gL -1 is.

[0648] In comparison, the native strain does not produce hyaluronic acid.

[0649] This experiment showed that recombinant strains containing modifications according to the present invention produced greater amounts of hyaluronic acid when cultured under the same conditions as other recombinant strains that did not contain all of the genetic modifications according to the present invention.

[0650] Furthermore, these three strains showed the following after 48 hours: - YA5233-1: 50kDa to 500kDa, - YA5359-10, 100kDa to 750kDa, and - YA5381-1: 50kDa to 250kDa This resulted in the production of hyaluronic acid with a molecular weight of 1.0.

[0651] B. Six other recombinant strains were also obtained: YA5262-16, YA5326-3, YA5300-15, YA5358-4, YA5264-1, and YA5331-6.

[0652] These six strains are: <h2 style=";text-align:left;direction:ltr">YA5262-16:MAT-α、can1-100、his3::[pSAM1-UGP1-tRPL3-pMET6-QRI1-tIDP1- HIS3]x5,jlp1::[LEU2.Kl,pCUP1-HASA2.Sz-tRPL41B,pCUP1-UGP1-tRPL3,pCUP1-QRI1-tIDP1,pPDC1-UGP1-tTPI1,pTDH3-QRI1-tMET25,pCCW12-HASB.At-tRPL15A],leu2,lyp1::[pCCW10.ago-HYAL-31.Hn-tRPL15A,pCCW12.Sk-HASB-A.Vir-tTEF1.Sba,pCCW12-HASA-1.Vir-tDIT1,pCCW12.Sb a-HASB.vir-tRPL3、pCCW12.Sm-HASA-A.Vir-tRPL15A.Sba]、sam3::[LEU2.Kl、pPDC1-PGM1-tIDP1、pTEF1-GFA1-tRPL15A、pENO2-UGP1-tRPL3、pCWP 2-GNA1-tTPI1、pTEF3-PCM1-tIDP1、pCCW12-XHASA2. TRP1]x2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">YA5326-3:MAT-α、can1-100、his3::[pSAM1-UGP1-tRPL3-pMET6-QRI1-tIDP1- HIS3]x5,jlp1::[LEU2.Kl,pCUP1-HASA2.Sz-tRPL41B,pCUP1-UGP1-tTPI1,pTDH3-QRI1-tMET25,pCCW12-HASB.At-tRPL15A],leu2,lyp1::[pCCW10.ago-HYAL-31.Csa-tRPL15A,pCCW12.Sk-HASB-A.Vir-tTEF1.Sba,pCCW12-HASA-1.Vir-tDIT1,pCCW12.Sba-HASB.vir-tRPL3, pCCW12.Sm-HASA-A.Vir-tRPL15A.Sba]、sam3::[LEU2.Kl、pPDC1-PGM1-tIDP1、pTEF1-GFA1-tRPL15A、pENO2-UGP1-tRPL3、pCWP2-GNA1-t TPI1、pTEF3-PCM1-tIDP1、pCCW12-XHASA2. TRP1]x2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">YA5300-15:MAT-α、can1-100、his3::[pSAM1-UGP1-tRPL3-pMET6-QRI1-tIDP1- HIS3]x5,jlp1::[LEU2.Kl,pCUP1-HASA2.Sz-tRPL41B,pCUP1-UGP1-tRPL3,pCUP1-QRI1-tIDP1,pPDC1-UGP1-tTPI1,pTDH3-QRI1-tMET25,pCCW12-HASB.At-tRPL15A],leu2,lyp1::[pTDH3-HYAL-3.Ts-tRPL15A,pCCW12.Sk-HASB-A.Vir-tTEF1.Sba,pCCW12-HASA-1.Vir-tDIT1,pCCW12.Sba-H ASB.vir-tRPL3、pCCW12.Sm-HASA-A.Vir-tRPL15A.Sba]、sam3::[LEU2.Kl、pPDC1-PGM1-tIDP1、pTEF1-GFA1-tRPL15A、pENO2-UGP1-tRPL3、pCWP2 -GNA1-tTPI1、pTEF3-PCM1-tIDP1、pCCW12-XHASA2. TRP1]x2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">YA5358-4:MAT-α、can1-100、his3::[pSAM1-UGP1-tRPL3-pMET6-QRI1-tIDP1- HIS3]x5,jlp1::[LEU2.Kl,pCUP1-HASA2.Sz-tRPL41B,pCUP1-UGP1-tRPL3,pCUP1-QRI1-tMET25,pCCW12-HASB.At-tRPL15A],leu2,lyp1::[pCCW12.Sar-HYAL-31.Li-tRPL15A,pCCW12.Sk-HASB-A.Vir-tTEF1.Sba,pCCW12-HASA-1.Vir-tDIT1,pCCW12.Sba-HASB.vir-tRPL3, pCCW12.Sm-HASA-A.Vir-tRPL15A.Sba]、sam3::[LEU2.Kl、pPDC1-PGM1-tIDP1、pTEF1-GFA1-tRPL15A、pENO2-UGP1-tRPL3、pCWP2-GNA1-t TPI1、pTEF3-PCM1-tIDP1、pCCW12-XHASA2. TRP1]x2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">YA5264-1:MAT-α、can1-100、his3::[pSAM1-UGP1-tRPL3-pMET6-QRI1-tIDP1- HIS3]x5,jlp1::[LEU2.Kl,pCUP1-HASA2.Sz-tRPL41B,pCUP1-UGP1-tTPI1,pTDH3-QRI1-tMET25,pCCW12-HASB.At-tRPL15A],leu2,lyp1::[pTDH3-HYAL-31.Ts-tRPL15A,pCCW12.Sk-HASB-A.Vir-tTEF1.Sba,pCCW12-HASA-1.Vir-tDIT1,pCCW12.Sba-HASB.vir-tRPL3,pCC W12.Sm-HASA-A.Vir-tRPL15A.Sba]、sam3::[LEU2.Kl、pPDC1-PGM1-tIDP1、pTEF1-GFA1-tRPL15A、pENO2-UGP1-tRPL3、pCWP2-GNA1-tTP I1、pTEF3-PCM1-tIDP1、pCCW12-XHASA2. TRP1]x2<h2 style=";text-align:left;direction:ltr"> YA5331-6: MAT-α, can1-100, his3::[pSAM1-UGP1- tRPL3-pMET6-QRI1-tIDP1- HIS3]x5, jlp1::[LEU2.Kl, pCUP1-HASA2.Sz-tRPL41B, pCUP1-UGP1-tRPL3, pCUP1-QRI1-tIDP1, pPDC1-UGP1-tTPI1, pTDH3-QRI1-tMET25, pCCW12 -HASB.At-tRPL15A], leu2, lyp1::[pTEF3-HYAL-31.Li-tRPL15A, pCCW12.Sk-HASB-A.Vir-tTEF1.Sba, pCCW12-HASA-1.Vir-tDIT1, pCCW12.Sba-H ASB.vir-tRPL3, pCCW12.Sm-HASA-A.Vir-tRPL15A.Sba], sam3::[LEU2.Kl, pPDC1-PGM1-tIDP1, pTEF1-GFA1-tRPL15A, pENO2-UGP1-tRPL3, pCWP2 -GNA1-tTPI1, pTEF3-PCM1-tIDP1, pCCW12-XHASA2.Xl-tRPL3, pTDH3-QRI1-tIDP1], trp1::[pMET6-HASB.Vir-tRPL3, pMET25-HASA-1.Vir-tIDP1- TRP1]x2

[0653] HASA-1, HASA-A, HASA2, and XHASA2 have been previously defined.

[0654] HASB and HASB-A have been defined previously.

[0655] YA5262-16 was inoculated into 2 liters of SY medium as defined above, pH 4, in 2% glucose in a bioreactor, and the pH was maintained at 4 throughout the fermentation using 15% H2SO4 and 10% NH4OH.

[0656] YA5326-3, - 2 liters of SY medium as defined above, pH 6, in 2% glucose was inoculated in a bioreactor, the pH being maintained at 6 throughout the fermentation using 15% H2SO4 and 10% NH4OH, or In a bioreactor, 2 liters of SY medium as defined above, pH 6, in 2% sucrose was inoculated and the pH was maintained at 6 with 15% H2SO4 and 10% NH4OH for up to 34 hours and at pH 4 from 34 to 48 hours.

[0657] YA5300-15 was inoculated into 2 liters of SY medium as defined above, pH 6, in 2% sucrose in a bioreactor, and the pH was maintained at 6 throughout the fermentation using 15% H2SO4 and 10% NH4OH.

[0658] YA5358-4 was inoculated into 2 liters of SY medium as defined above, pH 6, in 2% glucose in a bioreactor, and the pH was maintained at 6 throughout the fermentation using 15% H2SO4 and 10% NH4OH.

[0659] YA5264-1 was inoculated into 2 liters of SY medium as defined above, pH 6, in 2% sucrose in a bioreactor, and the pH was maintained at 6 throughout the fermentation using 15% H2SO4 and 10% NH4OH.

[0660] YA5331-6 was inoculated into 2 liters of SY medium as defined above, pH 6, in 2% glucose in a bioreactor, and the pH was maintained at 6 throughout the fermentation using 15% H2SO4 and 10% NH4OH.

[0661] Growth medium was collected at 48 hours and assayed for hyaluronic acid content and quality as described above. The amounts of hyaluronic acid obtained from these two strains were, respectively: - YA5262-16: 19g.L -1 , - YA5326-3: 13g.L each -1 and 15 g.L. -1 , - YA5300-15: 9g.L -1 , - YA5358-4: 14g.L -1 , - YA5264-1: 6g.L -1 ,and - YA5331-6: 9g.L -1 is.

[0662] In comparison, the native strain does not produce hyaluronic acid.

[0663] This experiment showed that recombinant strains containing modifications according to the present invention produced greater amounts of hyaluronic acid when cultured under the same conditions as other recombinant strains that did not contain all of the genetic modifications according to the present invention.

[0664] Furthermore, the hyaluronic acid produced by these strains after 48 hours was - YA5262-16: 50kDa, - YA5326-3: 50 kDa or 50 kDa to 250 kDa, respectively; - YA5300-15: 100kDa~1000kDa, - YA5358-4: 100kDa~1000kDa, - YA5264-1: 100kDa~1000kDa, and - YA5331-6: 100kDa~1000kDa The molecular weight of the compound was 1.0.

[0665] Furthermore, the recombinant strain YA5235-1 described above was inoculated in a bioreactor in 2% sucrose at pH 6, and the pH was maintained at 6 with 15% H2SO4 and 10% NH4OH for up to 34 hours and at 4 from 34 to 48 hours.

[0666] After 48 hours, the medium was diluted to 18 g.L -1 Contains 100 to 1000 kDa of hyaluronic acid.

[0667] The same experiment was performed using YA5235-1 but using glucose instead of sucrose. The resulting hyaluronic acid also has a molecular weight of 100-1000 kDa.

[0668] These results indicate, in particular, that pH regulation throughout the fermentation process and the production of hyaluronidase linked to a secretory signal or a secretory signal and an anchoring signal can affect or even control the size of the hyaluronic acid polymers produced.

[0669] 3. Production of hyaluronic acid with a molecular weight greater than 1000 kDa Four recombinant strains were obtained: YA5232-1, YA5303-3, YA5374-4, and YA5382-1.

[0670] Thus, these four strains are: <h2 style=";text-align:left;direction:ltr">YA5232-1:MAT-α、can1-100、his3::[pSAM1-UGP1-tRPL3-pMET6-QRI1-tIDP1- HIS3]x5,jlp1::[LEU2.Kl,pCUP1-HASA2.Sz-tRPL41B,pCUP1-UGP1-tTPI1,pTDH3-QRI1-tMET25,pCCW12-HASB.At-tRPL15A],leu2,lyp1::[pNUP57-HYAL-3.Ts-tRPL15A,pCCW12.Sk-HASB-A.Vir-tTEF1.Sba,pCCW12-HASA-1.Vir-tDIT1,pCCW12.Sba-HASB.vir-tRPL3,pCC W12.Sm-HASA-A.Vir-tRPL15A.Sba]、sam3::[LEU2.Kl、pPDC1-PGM1-tIDP1、pTEF1-GFA1-tRPL15A、pENO2-UGP1-tRPL3、pCWP2-GNA1-tTP I1、pTEF3-PCM1-tIDP1、pCCW12-XHASA2. TRP1]x2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">YA5303-2: MAT-α、can1-100、his3::[pSAM1-UGP1- tRPL3-pMET6-QRI1-tIDP1- HIS3]x5、jlp1::[LEU2.Kl、pCUP1-HASA2.Sz-tRPL41B、pCUP1-UGP1- tTPI1, pTDH3-QRI1-tMET25, pCCW12-...

Claims

1. A recombinant yeast cell that produces hyaluronic acid (HA), comprising: (a) one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase 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 hyaluronidase activity, wherein the polypeptide having hyaluronidase activity comprises a secretion signal such that hyaluronic acid is produced by the recombinant yeast cell; and (d)(i) one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase (GLN1) activity, and / or (ii) one or more disrupted endogenous nucleic acids encoding glutamate synthase (GLT1); Including, The recombinant yeast cell belongs to the genus Saccharomyces. Recombinant yeast cells.

2. A recombinant host cell that produces hyaluronic acid (HA), comprising: (a) one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase 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 hyaluronidase activity, wherein the polypeptide having hyaluronidase activity comprises a secretion signal and an anchoring signal such that hyaluronic acid is produced by the host cell; and (d)(i) one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase (GLN1) activity, and / or (ii) one or more disrupted endogenous nucleic acids encoding glutamate synthase (GLT1); wherein the recombinant host cell is a recombinant yeast cell of the genus Saccharomyces.

3. 3. The recombinant yeast cell or host cell of claim 1 or 2, wherein the molecular weight of the HA is in the range of less than 50 kDa.

4. 3. The recombinant yeast cell or host cell of claim 1 or 2, wherein the molecular weight of the HA is in the range above 50 kDa.

5. 3. The recombinant yeast cell or host cell of claim 1 or 2, wherein the molecular weight of the HA is in the range above 100 kDa.

6. 6. A recombinant yeast cell or host cell according to any one of claims 1 to 5, wherein the nucleic acid encoding a polypeptide having glutamine synthetase activity is obtained or derived from Saccharomyces cerevisiae.

7. 7. The recombinant yeast cell or host cell of any one of claims 1 to 6, wherein the nucleic acid encoding a polypeptide having hyaluronidase activity is obtained or derived from at least one of Cupienius salei, Loxosceles intermedia, Hirudo nipponia, Bothropus atrox, or Titius serrata.

8. 8. The recombinant yeast cell or host cell of any one of claims 1 to 7, wherein the nucleic acid encoding a polypeptide having hyaluronan synthase activity is obtained or derived from Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviK1, Chlorella virus IL-5-2s1, Chlorella virus CZ-2, Chlorella virus CVG-1, Xenopus laevis, or Pasteurella multocida.

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

10. (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity 10. The recombinant yeast cell or host cell of any one of claims 1 to 9, comprising a recombinant nucleic acid encoding one or more of:

11. (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 11. A recombinant yeast cell or host cell according to any one of claims 1 to 10, comprising at least one recombinant nucleic acid encoding one or more of:

12. 12. The recombinant yeast cell of any one of claims 1 and 3 to 11, wherein the recombinant yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces mikatae, and Saccharomyces castellii.

13. 1. A method for producing hyaluronic acid (HA) of a desired molecular weight (HAMW), comprising: (a) culturing the recombinant yeast cell or host cell of any one of claims 1 to 12 in a culture medium for a time sufficient to produce hyaluronic acid (HA) of the desired molecular weight; (b) optionally isolating or recovering hyaluronic acid (HA) from the recombinant cells and / or culture medium; wherein the recombinant yeast cell or host cell is a member of the genus Saccharomyces.

14. 14. The method of claim 13, wherein the HA has a molecular weight of 20 kDa to 50 kDa.

15. 15. The method of claim 14, wherein the HA has a molecular weight of 30 kDa to 50 kDa.

16. 14. The method of claim 13, wherein the molecular weight of the HA is between 50 kDa and 150 kDa.

17. 14. The method of claim 13, wherein the molecular weight of the HA is between 150 kDa and 1500 kDa.

18. The recombinant cell (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity, and / or (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity 18. The method of any one of claims 13 to 17, comprising at least one recombinant nucleic acid encoding one or more of:

19. The recombinant cell (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 19. The method of any one of claims 13 to 18, comprising at least one recombinant nucleic acid encoding one or more of:

20. 20. The method of any one of claims 13 to 19, wherein the time sufficient to produce hyaluronic acid (HA) of the desired molecular weight is a period of 35 to 50 hours.

21. 21. The method according to any one of claims 13 to 20, wherein the molecular weight of the hyaluronic acid is controlled by adjusting the pH of the culture medium.

22. 22. The method according to any one of claims 13 to 21, wherein the method is carried out on an industrial scale.

23. 13. Use of a recombinant yeast cell or host cell according to any one of claims 1 to 12 for the production of hyaluronic acid (HA) having a molecular weight in the range of 20 kDa to 50 kDa or 50 kDa to 1000 kDa.

24. 1. A method for producing hyaluronic acid, comprising: (a) culturing the recombinant yeast according to any one of claims 1 and 3 to 12 in a culture medium; (b) recovering hyaluronic acid from the culture medium; Including, The hyaluronic acid recovered in step (b) - the nature and origin of the nucleic acid encoding the recombinant yeast hyaluronidase; - the nature and origin of the promoter controlling the expression of the nucleic acid encoding the recombinant yeast hyaluronidase; - the presence of an anchoring and / or secretion signal associated with the recombinant yeast encoded hyaluronidase; - 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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