Recombinant cells

Abstract

The present disclosure relates generally to the field of bio-production of low-molecular weight hyaluronic acids. The present disclosure also relates to compositions comprising said hyaluronic acid, and the various uses thereof.

Description

[0001] RECOMBINANT CELLS

[0002] TECHNICAL FIELD

[0003] The present disclosure relates generally to the field of bio-production of low-molecular weight hyaluronic acids. The present disclosure also relates to compositions comprising said hyaluronic acid, and the various uses thereof.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format, compliant with WIPO Standard ST.26, and is hereby incorporated by reference in its entirety. Said XML copy, created on October 15, 2025, is named 31544.xml and is 291,015 bytes in size.

[0006] BACKGROUND

[0007] Hyaluronic acid, also known as hyaluronan or HA, is a naturally occurring high molecular weight polysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine, linked via alternating P-(l— >4) and P~(l— >3) glycosidic bonds and having the chemical formula (Ci4H2iNOn)n. Hyaluronic acid can be 25,000 disaccharide repeats in length. Polymers of hyaluronic acid can range in size from 5,000 to 20,000,000 Da in vivo. The primary structure of hyaluronic acid is reproduced below:

[0008] Hyaluronic acid is found ubiquitously throughout the body, and either directly or indirectly involved in every physiological function of the body. Hyaluronic acid has a wide molecular weight spectrum which can reach 15,000 kDa and above, depending on the method for its production.

[0009] HA has many applications in the medical and cosmetic fields including scaffolding for tissue engineering, dermatological fillers, and viscosupplementation for osteoarthritis treatment. In particular, a reduction in HA mass or molecular weight via degradation or slowing of synthesis affects physical and chemical properties such as tissue volume, viscosity, and elasticity. There is therefore a constant need for HA production.

[0010] Today’s main known sources of HA are human umbilical cords, rooster combs and fermentation of certain microorganisms.

[0011] Fermentation methods for preparing HA from microorganisms are particularly of interest since they facilitate the production of a large quantity of HA through possible scale-up, at a reduced cost. Contrary to isolating HA from animal sources which provide hyaluronic acid of very high molecular weights, microbial fermentation allows to control, to a certain extent, the size of the starting molecular weight. This avoids the need for further fractionation steps by mechanical, physical or chemical means.

[0012] In this aim, bacterial cultures such as group A and C hemolytic streptococci have been shown to represent good sources of HA (US5316926, US4801539, JP2009011315). However, such bacteria, and in particular Streptococcus zooepidemicus which is mainly used in the art, is not generally recognized as safe. Recombinant Bacillus host cells have also shown the ability to produce HA in the range of 20 to 800 KDa (US2008038780). It has also been demonstrated in US2006168690 that transforming plant cells to include a DNA encoding hyaluronic acid synthase successfully allowed for the production of HA. Finally, production of HA has also been demonstrated in yeasts such as in a recombinant Pichia pastor is in CN 104263666 and Saccharomyces cerevisiae in WO2022207786A1.

[0013] Hyaluronic acid can have different properties and applications depending on its molecular weight. Although there are multiple methods available in the art for the production of relatively high- molecular weight HAs, there is still a need for further hyaluronic acid production methods allowing its highly efficient synthesis and secretion, and in particular, to provide production methods to obtain large amounts of hyaluronic acid of a particular and controlled size in the lower molecular weight range, e.g. below 5 kDa.

[0014] The inventors have surprisingly found that such production method can be achieved using recombinant host cells with the genetic compositions as described hereinafter. Moreover, the inventors have surprisingly found that the HA produced by the said recombinant cells, in particular when comprised in a skin care composition, show significant anti-ageing and anti-wrinkle properties when applied on skin. SUMMARY OF THE INVENTION

[0015] In accordance with a first aspect of the present disclosure there is provided a recombinant cell producing hyaluronic acid.

[0016] In accordance with a second aspect of the present disclosure there is provided a method of producing hyaluronic acid using the recombinant cell of the first aspect.

[0017] In accordance with a third aspect of the present disclosure there is provided hyaluronic acid obtained or obtainable from the recombinant cell according to the first aspect or from the method according the second aspect.

[0018] In accordance with a fourth aspect of the present disclosure there is provided a cultivation medium comprising the hyaluronic acid of the third aspect.

[0019] In accordance with a fifth aspect of the present disclosure there is provided a composition (e.g. a cosmetic composition and in particular a skin care composition) comprising the hyaluronic acid according to the third aspect, optionally comprising a carrier.

[0020] In accordance with a sixth aspect of the present disclosure there is provided the use of the composition according to the fifth aspect, or of the hyaluronic acid according to the third aspect, for anti-ageing treatment and / or anti-wrinkle treatment.

[0021] In accordance with a seventh aspect of the present disclosure there is provided a cosmetic method of skin treatment, comprising the step of applying to the skin an effective amount of the composition according to the fifth aspect, or of the hyaluronic acid according to the third aspect, wherein the skin treatment is preferably for anti-ageing treatment and / or anti-wrinkle treatment, and more preferably for treatment of sleep wrinkles.

[0022] These and other aspects of the invention will be better understood in view of the following detailed description of particular embodiments of the invention.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows analysis of molecular weight of a sample from a hyaluronic acid of the present invention (Micronhyal) by size exclusion chromatography.

[0025] Figure 2 shows profile of skin penetration by tracking Micronhyal Raman spectroscopic signal.

[0026] Figure 3 shows impact of Micronhyal on DMNT3B expression on human keratinocytes prematurely aged by senescence replicative. Keratinocytes were treated with Micronhyal at 5 and 10 pg / ml for 24h. Nuclear protein was extracted and DMNT3B was quantified. Data are expression ng / mg of proteins. Statistical analysis was done by Mann-Whitney test with * p<0.05. Figure 4 shows impact of Micronhyal on truncated transcripts number by PRO-seq method on human skin keratinocyte prematurely aged by replicative senescence. After 3 days of treatment with Micronhyal at 10 pg / ml, total RNA were extracted and truncated transcripts were isolated by PRO-seq methodology. Date is shown in number of abnormal isoforms reflecting aberrant transcript. Statistical analysis was done with # p<0.1.

[0027] Figure 5 shows impact of Micronhyal on Sirtuin-1 expression and activity. Human normal keratinocytes were treated for 24 h and then nuclear proteins were extracted and Sirtuin-1 expression and activity were analysed by ELISA. Data is shown in ng / mg protein for expression analysis and in ng / min / mg protein for activity. Statistical analysis was done by Mann-Whitney with # p<0.1 and * p<0.05.

[0028] Figure 6 shows impact of Micronhyal on PARP1 expression and activity. Human normal keratinocytes were treated for 24 h and then nuclear proteins were extracted and PARP1 expression and activity were analysed by ELISA. Data is shown in ng / mg protein for expression analysis and in U / mg protein for activity. Statistical analysis was done by Mann Whitney with # p<0.1 and * p<0.05.

[0029] Figure 7 shows telomerase expression and activity analysis in presence of Micronhyal. Telomerase expression (left panel): human primary keratinocytes were treated either with positive reference (FK228) or Micronhyal at 10 pg / mL for 24 h. After the incubation period, cell lysates were collected and telomerase expression was quantified in the nuclear extract. Statistical analysis was done by Mann-Whitney test with # p<0.1 and * p<0.05. Telomerase activity (right panel): human primary keratinocytes were prematurely aged (replicative senescence) then treated with Micronhyal at 10 pg / mL for 24 h. RNA were extracted and qPCR was performed on cDNA targeting telomere gene. Statistical analysis was done by student’s t-test with # p<0.1 and *** p<0.001.

[0030] Figure 8 shows in vitro collagen I synthesis by human fibroblasts under premature ageing condition in presence of Micronhyal. NHDFs were prematurely aged with H2O2 for 3 h or not. Then they were treated with Micronhyal at 0.1 mg / mL for 72 h. Immunostaining of pro-collagen I was performed on fixed cells. Statistical analysis was done by Mann-Whitney test with * p<0.05 and *** pO.OOL

[0031] Figure 9 shows comparison of effect of different grades of HA on DMNT3B expression on human keratinocytes prematurely aged by senescence replicative. Keratinocytes were treated with Micronhyal 10 pg / ml or P50Life at 5 mg / ml or P300 at 5 mg / ml to PUF at 1 mg / ml for 24 h. Nuclear protein was extracted and DMNT3B was quantified. Data is shown in ng / mg of proteins relative to mg of HA. Statistical analysis was done by Man-Whitney test with * p<0.05.

[0032] Figure 10 shows the data in the count wrinkles on all the face (left and right) after twice daily applications during 28 and 56 days with Micronhyal at 0.5% in comparison with the placebo formula.

[0033] Figure 11 shows the data in the fines wrinkles on the periorbital area on eyes after twice daily applications during 28 and 56 days with Micronhyal at 0.5% in comparison with the placebo formula.

[0034] Figure 12 shows the data on the count wrinkles on the above the eyes area after twice daily applications during 28 and 56 days with Micronhyal at 0.5% in comparison with the placebo formula.

[0035] Figure 13 shows the data on the count wrinkles on the above the eyes area after twice daily applications during 28 and 56 days with Micronhyal at 0.5% in comparison with the placebo formula.

[0036] Figure 14 shows the data on the count wrinkles on the 3 areas after twice daily applications during 28 and 56 days with Micronhyal at 0.5% in comparison with the placebo formula.

[0037] Figure 15 shows the data on the fine wrinkles on the 3 areas after twice daily applications during 28 and 56 days with Micronhyal at 0.5% in comparison with the placebo formula.

[0038] DETAILED DESCRIPTION

[0039] The inventors have conceived genetically modified cells, and especially genetically modified yeasts, having the ability to selectively produce hyaluronic acid with low molecular weight (e.g. an average molecular weight of less than 5 kDa), as compared to the parent cells, and especially as compared to the parent yeasts which are not naturally capable of doing so. These genetically modified cells are described throughout the present specification.

[0040] Furthermore, the inventors have discovered, surprisingly, that skin care compositions comprising low molecular weight hyaluronic acid, in particular the hyaluronic acid produced by the above- mentioned recombinant cells, exert very significant anti-aging and anti-wrinkle effects on human skin, even in comparison with formulations containing hyaluronic acids of higher molecular weights (e.g. higher than 20 kDa, or higher than 100 kDa). Skin-aging is a term that refers to the changes experienced by the skin with age, whether that is through chronological aging or through exposure to the sun (photo-aging) or through other environmental agents such as tobacco smoke, extreme climatic conditions of cold, heat, or wind, chemical contaminants or pollutants, and includes all the external visible and / or perceptible changes through touch, such as but not restricted to, the development of discontinuities on the skin such as wrinkles, fine lines, furrows, irregularities or roughness, increase in the size of pores, loss of elasticity, loss of firmness, loss of smoothness, loss of the capacity to recover from deformation, sagging of the skin such as sagging cheeks, the appearance of bags under the eyes or the appearance of a double chin, among others, changes to the colour of the skin such as marks, reddening, or the appearance of hyper-pigmented areas such as age spots or freckles among others, anomalous differentiation, hyper-keratinization, elastosis, keratosis, loss of collagen structure and other histological changes of the stratum corneum, of the dermis, or epidermis.

[0041] It is known that a decreased level of DMNT3B is among the key factors in skin-aging. DMNT3B has been identified as a key enzyme involved in DNA ageing which is related to longevity. It plays an active role in DNA methylation and controlling the quality of transcription. Indeed, DMNT3B expression and activity is significantly decreased during ageing, contributing to accumulated truncated transcripts and subsequently the increase of non-functional proteins.

[0042] Surprisingly, the inventors have found that the low molecular weight hyaluronic acids of the invention, and skin care compositions comprising thereof (e.g. as described in the Claims) can increase expression of DMNT3B in the skin. It is believed, although the applicant does not wish to be bound by theory, that said increase is among the mechanisms which contribute to the antiageing effect of the hyaluronic acid and the skin care compositions comprising thereof as disclosed herein. Other potential mechanisms, e.g. those involved in DNA damage, epigenetics, collagen production and telomerase activity, are also described in details in Examples.

[0043] Throughout this disclosure, the terms “hyaluronic acid”, “hyaluronan”, and “hyaluronate” are used interchangeably, if not otherwise noted. Hyaluronic acid has a wide molecular weight spectrum which can reach 15,000 kDa and above, depending on the method for its production.

[0044] The finding that low molecular weight hyaluronic acid, when applied to the skin, can elicit a biological effect that is observable as a reduction in the visible signs of skin aging, such as a reduction in skin-wrinkles enables the skilled person in the art to provide cosmetic preparations, e.g. skin care compositions, for application to the skin of a human subject. Cosmetic preparations, and in particular skin care compositions, of the present invention contain a cosmetically acceptable amount of low molecular weight hyaluronic acid as disclosed herein.

[0045] A cosmetically effective amount of low molecular weight hyaluronic acid is understood to be a non-toxic but sufficient quantity thereof to provide the desired effect. With regard to the total weight of a cosmetic preparation, in particular the skin care composition of the present invention, the low molecular weight hyaluronic acid may be present in amounts of about 0.005% w / v to about 5% w / v, and more particularly about 0.05% to about 3% w / v, even more particularly about 0.1% to about 1% w / v, preferably about 0.2% to 0.7% w / v, e.g. about 0.5% w / v.

[0046] Cosmetic preparations, and in particular skin care compositions, of the present invention may contain one or more cosmetically acceptable excipients. Any excipients commonly used in the preparation of cosmetic preparations for use on the human skin may be employed in the present invention. Suitable excipients include, but are not limited to ingredients that can influence organoleptic properties, penetration of the skin, and the bioavailability of the hyaluronic acid. More specifically, they include liquids, such as water, oils or surfactants, including those of petroleum, animal, plant or synthetic origin, such as and not restricted to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glycosides, maltosides, fatty alcohols, nonoxynols, poloxamers, polyoxyethylenes, polyethylene glycols, dextrose, glycerol, digitonin, and the like.

[0047] The cosmetic preparation, and in particular the skin care composition, may be in the form of a liposome composition, mixed liposomes, oleosomes, niosomes, ethosomes, milliparticles, microparticles, nanoparticles and solid-lipid nanoparticles, vesicles, micelles, mixed micelles of surfactants, surfactant-phospholipid mixed micelles, millispheres, microspheres and nanospheres, lipospheres, millicapsules, microcapsules and nanocapsules, as well as microemulsions and nanoemulsions, which can be added to achieve a greater penetration of the acetylated hyaluronic acid or its sodium salt.

[0048] The cosmetic preparation, and in particular the skin care composition, may be produced in any solid, liquid, or semi-solid form useful for application to the skin topically or by transdermal application. Thus, these preparations of topical or transdermal application include, but are not restricted to, creams, multiple emulsions, such as and not restricted to, oil and / or silicone in water emulsions, water-in-oil and / or silicone emulsions, water / oil / water or water / silicone / water type emulsions, and oil / water / oil or silicone / water / silicone type emulsions, micro-emulsions, emulsions and / or solutions, liquid crystals, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, aqueous or oily lotions, aqueous or oily gels, cream, hydro-alcoholic solutions, hydro-glycolic solutions, hydrogels, liniments, sera, soaps, face masks, serums, polysaccharide films, ointments, mousses, pomades, pastes, powders, bars, pencils and sprays or aerosols (sprays), including leave-on and rinse-off formulations.

[0049] The finding that low molecular weight hyaluronic acid as disclosed herein elicits a biological effect was particularly surprising considering that it was not possible to reproduce the effect using hyaluronic acid of higher molecular weights, confirming that the activity is related to the particular size of hyaluronic acid molecules.

[0050] Particularly preferred forms of hyaluronic acid for use in the present invention (also referred to as “Micronhyal” in the Examples and Figures) are characterized in that they have a relatively low molecular weight. In particular, said molecules have an average molecular weight of less than about 5 kDa, and preferably higher than 800 Da. In preferred embodiments, said hyaluronic acid has a median molecular weight of less than about 5 kDa, more particularly of less than about 3 kDa, e.g. about 2.8 kDa.

[0051] In a first aspect, the present disclosure relates to a recombinant cell, e.g. a recombinant yeast cell, producing hyaluronic acid (HA) wherein the recombinant cell comprises:

[0052] (a) one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity;

[0053] (b) one or more recombinant nucleic acids encoding a polypeptide having UDP- Glucose dehydrogenase (UDP-GlcDH or HASB) activity;

[0054] (c) one or more recombinant nucleic acids, under the control of a pCCW12.Spr promoter, encoding a polypeptide having hyaluronidase activity wherein the polypeptide having hyaluronidase activity comprises a secretion signal so that hyaluronic acid, in particular of a desired molecular weight (HAMW) is produced by the recombinant yeast cell, and

[0055] (d) (i) one or more recombinant nucleic acids encoding a polypeptide having a glutamine synthetase (GLN1) activity; and / or

[0056] (ii) one or more disrupted endogeneous nucleic acids encoding a glutamate synthase (GLT1); wherein said recombinant yeast cell belongs to the Saccharomyces genus, or to the Candida genus, or to the Kluyveromyces genus, or to the Ogataea genus, or to the Yarrowia genus, or to the Debaryomyces genus, or to the Ashbya genus.

[0057] In an embodiment, the recombinant cell is a yeast cell.

[0058] In an embodiment, the polypeptide having hyaluronidase activity further comprises an anchoring signal.

[0059] In an embodiment, the hyaluronic acid has an average molecular weight of less than about 5 kDa. In an embodiment, the hyaluronic acid has an average molecular weight of more than about 800 Da.

[0060] In an embodiment, the hyaluronic acid has a median molecular weight of less than about 5 kDa, more particularly of less than about 3 kDa, e.g. about 2.8 kDa.

[0061] In an embodiment, the nucleic acid encoding a polypeptide having a glutamine synthetase activity is obtained or derived from Saccharomyces cerevisiae.

[0062] In an embodiment, the nucleic acid encoding a polypeptide having hyaluronidase activity is obtained or derived from at least one of Cupiennius salei. Loxosceles intermedia, Hirudo nipponia, Bothrops atrox or Tityus serrulatus.

[0063] In an embodiment, the nucleic acid encoding a polypeptide having hyaluronan synthase activity is obtained or derived from Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviKl, Chlorella virus IL-5-2sl, Chlorella virus CZ-2, Chlorella virus CVG-1, Xenopus laevis or Pasteurella mullocida, and is in particular obtained or derived from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1 , Chlorella virus CviKl, Chlorella virus IL-5-2sl, Chlorella virus CZ-2, Chlorella virus CVG-1 or Xenopus laevis.

[0064] In an embodiment, 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, and in particular from Arabidopsis thaliana or Chlorella virus PBCV1.

[0065] In an embodiment, the recombinant cell comprises at least one recombinant nucleic acid encoding one or more of:

[0066] (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity; and / or

[0067] (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity. In an embodiment, the recombinant cell comprises at least one recombinant nucleic acid encoding one or more of:

[0068] (i) a polypeptide having Phosphoglucomutase- 1 (PGM1) activity; and / or

[0069] (ii) a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase (UGP1) activity; and / or

[0070] (iii) a polypeptide having Glucosamine-6-phosphate N-acetyltransf erase (GNA1) activity; and / or

[0071] (iv) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity.

[0072] In an embodiment, the recombinant cell belongs to the Saccharomycetales order, and is in particular 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, Kluyveromyces thermotolerens, Ogataea polymorpha, Yarrowia lypolytica, Debaryomyces hansenii, and Ashbya gossypii, and is preferably Saccharomyces cerevisiae.

[0073] In a second aspect, the present disclosure relates to a hyaluronic acid having i) a molecular weight of more than about 800 Da and less than about 5 kDa; and / or ii) a median molecular weight of less than about 5 kDa, more particularly of less than about 3 kDa, e.g. about 2.8 kDa, comprising:

[0074] (a) cultivating a recombinant cell according to the first aspect in a cultivation medium for a time sufficient to produce said hyaluronic acid; and

[0075] (b) optionally isolating or recovering the hyaluronic acid from the recombinant cell and / or from the cultivation medium.

[0076] In an embodiment, the recombinant cell comprises at least one recombinant nucleic acid encoding one or more of:

[0077] (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity; and / or

[0078] (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity. In an embodiment, the recombinant cell comprises at least one recombinant nucleic acid encoding one or more of:

[0079] (i) a polypeptide having Phosphoglucomutase- 1 (PGM1) activity; and / or (ii) a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase (UGP1) activity; and / or

[0080] (iii) a polypeptide having Glucosamine-6-phosphate N-acetyltransf erase (GNA1) activity; and / or

[0081] (iv) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity.

[0082] In an embodiment, the recombinant cell is a member of the genus Saccharomyces. and in particular is Saccharomyces cerevisiae.

[0083] In an embodiment, the time sufficient to produce the hyaluronic acid is a period of from about 35 hours to about 60 hours, preferably from about 40 hours to about 55 hours, in particular about 52 hours.

[0084] In an embodiment, the molecular weight of the hyaluronic acid is controlled by regulating the pH of the cultivation medium.

[0085] In a third aspect, the present disclosure relates to hyaluronic acid obtained or obtainable from a recombinant cell according to the first aspect, or from the method according to the second aspect. In a fourth aspect, the present disclosure relates to a cultivation medium comprising the hyaluronic acid according to the third aspect.

[0086] In a fifth aspect, the present disclosure relates to a composition comprising the hyaluronic acid according to the third aspect, and optionally a carrier. In an embodiment, said composition is a cosmetic preparation, and in particular a skin care composition.

[0087] In an embodiment, the hyaluronic acid in said composition is present in an amount of 0.1 to 1.0% (w / v), more preferably 0.2 to 0.7% (w / v), e.g. about 0.5% (w / v), of the composition.

[0088] In an embodiment, the skin care composition comprises the hyaluronic acid as a first active cosmetic ingredient, and a carrier.

[0089] In accordance with a sixth aspect of the present disclosure there is provided the use of the composition according to the fifth aspect, or of the hyaluronic acid according to the third aspect, for anti-ageing treatment and / or anti-wrinkle treatment, more preferably for treatment of sleep wrinkles. In an embodiment, the use is a cosmetic use.

[0090] In accordance with a seventh aspect of the present disclosure there is provided a cosmetic method of skin treatment, comprising the step of applying to the skin an effective amount of the composition according to the fifth aspect, or of the hyaluronic acid according to the third aspect, wherein the skin treatment is preferably for anti-ageing treatment and / or anti-wrinkle treatment, and more preferably for treatment of sleep wrinkles.

[0091] In an embodiment of the sixth or seventh aspect, the treatment leads to one or more of i) an increase in expression of DMNT3B; ii) a reduction in aberrant transcripts; iii) an increase in expression and activity of sirtuin-1; iv) an increase in expression and activity of PARP-1; v) an increase in expression and activity of telomerase; vi) an increase in production of type-1 collagen by fibroblasts. In an embodiment, the increase in expression of DMNT3B is at least 10 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, or at least 500 times higher than a similar treatment using PrimalHyal Ultrafiler™ (as described e.g. in WO2018162672A1), Primalhyal™ 50, or Primalhyal™ 300. Primalhyal™ 50 comprises HA in a molecular weight range of 20-50 kDa; Primalhyal™ 300 comprises HA in a molecular weight range of 100-300 kDa.

[0092] Definitions

[0093] As used herein, the term "recombinant", when used in reference to a cell, indicates that the cell has been modified by the introduction of an endogenous and / or heterologous nucleic acid or protein into the cell or the alteration of a native cell or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes or nucleic acid that are not found within the native (non-recombinant) form of the cell or express native (e.g. endogenous) genes at a different level than their native level or express additional or supplementary copies of native (e.g. endogenous) at a different level than their native level.

[0094] As used herein, the term “recombinant”, when used in reference to a nucleic acid or vector, are sequences formed / obtained by technics of genetic engineering well known to the man skilled in the art. The guidelines of the US National Institute for Health (NIH) accordingly indicate that: “recombinant [...] nucleic acids are defined as: i. molecules that (a) are constructed by joining nucleic acid molecules and (b) that can replicate in a living cell, i.e., recombinant nucleic acids;", which reflects the conventional use of the word “recombinant” attached to nucleic acid sequences to mean recombined following the insertion of, or joining together with, another nucleic acid.

[0095] Proteins that result from the expression of recombinant DNA or recombinant vector within living cells are also termed recombinant proteins. The term "recombinant" is accordingly synonymous with the term "genetically modified". The term “gene” is synonymous with the term “nucleic acid” or “nucleotide sequence”.

[0096] A recombinant nucleic acid sequence for use in a recombinant cell, and in particular a recombinant yeast, of the invention may be provided in the form of a nucleic acid construct. The term "nucleic acid construct" refers to as a nucleic acid molecule, either single-or double-stranded, which is isolated or derived from a native (e.g. endogenous) naturally occurring gene or is a heterologous nucleic acid or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist 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, wherein said control sequences are operably linked to said coding sequence. Non-limiting examples of regulatory elements include promoters, enhancers, silencers, terminators, and poly-A signals.

[0097] A recombinant nucleic acid sequence for use in a recombinant cell of the 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 the expression of the polynucleotide sequence in a recombinant cell.

[0098] The terms “obtained from” or “originate from” or “originating from” a microorganism or animal generally means that a substance (e.g., a nucleic acid molecule or polypeptide) originating from the microorganism or animal is native to that microorganism or animal.

[0099] The terms "derived from" a microorganism or animal generally means that a substance (e.g., a nucleic acid molecule or polypeptide) derived from the microorganism or animal is the result of modifications brought to the substance native to (i.e. present as such) in that microorganism or animal. For example, regarding a nucleic acid sequence derived from a microorganism or animal, said nucleic acid sequence can correspond to a re-encoded and / or truncated version of the native nucleic acid sequence from this microorganism or animal. Other modifications well known to the man skilled in the art can be brought to the native substance of a microorganism or animal leading to the substance used being “derived from” said microorganism or animal.

[0100] As used herein, the term "polypeptide" refers to a molecule comprising amino acid residues linked by peptide bonds and containing more than five amino acid residues. The amino acids are identified by either the single-letter or three-letter designations. The term "protein" as used herein is synonymous with the term "polypeptide" and may also refer to two or more polypeptides. Thus, the terms "protein", "peptide" and "polypeptide" can be used interchangeably. Polypeptides may optionally be modified (e.g., glycosylated, phosphorylated, acylated, famesylated, prenylated, sulfonated, and the like) to add functionality. Polypeptides exhibiting activity may be referred to as enzymes. It will be understood that, as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding a given polypeptide may be produced.

[0101] A polypeptide encoded by a recombinant nucleic acid for use in a recombinant cell, and in particular in a recombinant yeast, of the invention may comprise a signal peptide and / or a propeptide sequence. In the event that a polypeptide expressed by a recombinant cell, and in particular a recombinant yeast, of the invention comprises a signal peptide and / or a pro-peptide, sequence identity may be calculated over the mature polypeptide sequence.

[0102] The term "operably linked" as used herein refers to two or more nucleic acid sequence elements that are physically linked and are in a functional relationship with each other. For instance, a promoter is operably linked to a coding sequence if the promoter is able to initiate or regulate the transcription or expression of the coding sequence, in which case the coding sequence should be understood as being "under the control of' the promoter. Generally, when two nucleic acid sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They usually will be essentially contiguous, although this may not be required.

[0103] The term “native” or “endogenous” as used herein with reference to molecules, and in particular enzymes and nucleic acids, indicates molecules that are expressed in the organism in which they originated or are found in nature.

[0104] The term "endogenous gene" means that the gene was present in the cell before any genetic modification, in the wild-type strain. Endogenous genes may be overexpressed by introducing heterologous sequences in addition to, or to replace endogenous regulatory elements, or by introducing one or more additional or supplementary copies of the gene into the chromosome or a plasmid (said additional or supplementary copies being designated “exogenous or heterologous genes” or “heterologous nucleotide sequences” or “heterologous nucleic acids” as defined herein). Endogenous genes may also be modified to modulate their expression and / or activity. For example, mutations may be introduced into the coding sequence to modify the gene product or heterologous sequences may be introduced in addition to or to replace endogenous regulatory elements. Modulation of an endogenous gene may result in the up-regulation and / or enhancement of the activity of the gene product, or alternatively, in the down-regulation and / or attenuation of the activity of the endogenous gene product. Another way to enhance expression of endogenous genes is to introduce one or more additional or supplementary copies of the gene onto the chromosome or a plasmid (said supplementary copies being designated “exogenous or heterologous genes” or “heterologous nucleotide sequences” or heterologous nucleic acids” as defined herein).

[0105] By “one or more additional or supplementary copies of a gene” according to the invention, it is for example understood in the present invention from 1 to 50 copies, in particular from 1 to 30 copies, more particularly from 1 to 20 copies, and preferably from 1 to 10 copies. Said copies may be inserted into the same locus or into different loci of a recombinant cell of the invention.

[0106] The term "exogenous gene" means that the gene was introduced into a cell, by means well known to the man skilled in the art, whereas this gene is or is not naturally occurring in the wild-type cell. Cells can express exogenous genes if these genes are introduced into the cell with all the elements allowing their expression in the cell. Transforming cells with exogenous DNA is a routine task for the man skilled in the art. Exogenous genes may be integrated into the host chromosome, or be expressed extra-chromosomally from plasmids or vectors. A variety of plasmids, which differ with respect to their origin of replication and their copy number in the cell, are all known in the art. The sequence of exogenous genes may be adapted for its expression in the cell. Indeed, the man skilled in the art knows the notion of codon usage bias and how to adapt nucleic sequences for a particular codon usage bias without modifying the deduced protein. In particular embodiments, codon optimized genes express native enzymes.

[0107] The term “heterologous gene” or heterologous nucleic acid sequence” refers to a gene or nucleic acid sequence not normally found in a given cell in nature. As such, a heterologous nucleic acid sequence may be: (a) foreign to its host cell (i.e. is “exogenous” to the cell); (b) naturally found in the host cell (i.e. “endogenous”) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); or (c) be naturally found in the host cell but positioned outside of its natural locus.

[0108] In the present application, all genes are referenced with their common names and with references to their nucleotide sequences and, the case arising, to their amino acid sequences. Using the references given in accession number for known genes, those skilled in the art are able to determine the equivalent genes in other organisms, bacterial strains, yeast, fungi, mammals, plants, etc. This routine work is advantageously done using consensus sequences that can be determined by carrying out sequence alignments with genes derived from other cells and designing degenerated probes to clone the corresponding gene in another organism.

[0109] The man skilled in the art knows different means to modulate, and in particular up-regulate or down-regulate, the expression of endogenous genes. For example, a way to enhance expression of, or over express, endogenous genes is to introduce one or more additional or supplementary copies of the gene onto the chromosome or a plasmid.

[0110] Another way is to replace the endogenous promoter of a gene with a stronger promoter. These promoters may be homologous or heterologous. Promoters particularly interesting in the present invention are described in more detail elsewhere in the present specification.

[0111] The nucleic acid expression construct may further comprise 5' and / or 3' recognition sequences and / or selection markers.

[0112] The term “inducible promoter” is used to qualify a promoter whose activity is induced, i.e. increased:

[0113] - in the presence of one or more particular metabolite(s). The higher the metabolite concentration in the medium, the stronger the promoter activity; or

[0114] - in the presence of a low concentration, or in the absence, of one or more metabolite(s). These metabolites are different from those whose increasing presence induces the activity of the promoter. The lower the metabolite concentration in the medium, the stronger the promoter activity.

[0115] The term “repressible promoter” is used to qualify a promoter whose activity is repressed, i.e. reduced:

[0116] - in the presence of one or more particular metabolite(s). The higher the metabolite concentration in the medium, the weaker the promoter activity; or

[0117] - in the presence of a low concentration, or in the absence, of one or more metabolite(s). These metabolites are different from those whose increasing presence represses the activity of the promoter. The lower the metabolite concentration in the medium, the weaker the promoter activity.

[0118] As used herein, the term “anchoring signal” when used in conjunction with a protein or polypeptide such as an enzyme (such as, for example hyaluronidase) means for example a first nucleic acid encoding a protein that is operably linked to a second nucleic acid encoding a protein or a polypeptide, or a first protein or polypeptide that is operably linked to a second protein or polypeptide, such as an enzyme (such as, for example hyaluronidase to form, for example a fusion protein), and that enables the cellular transport machinery of a cell, in particular of a S. cerevisiae cell, to correctly anchor and / or position in the membrane of the cell the second protein operably linked to the first protein.

[0119] As used herein, the term “secretion signal” when used in conjunction with a protein or polypeptide such as an enzyme (such as, for example hyaluronidase) means for example a first nucleic acid encoding a peptide or protein that is operably linked to a second nucleic acid encoding a protein, or a first protein that is linked to a second protein, such as an enzyme (such as, for example hyaluronidase to form, for example a fusion protein), and that enables the cellular transport machinery of a cell, in particular of a S. cerevisiae cell, to locate at least the second protein to the membrane of the cell and to secrete the second protein outside of the cell, after the first protein has, for example, been cleaved from the second protein.

[0120] As used herein the terms “secretion signal” and “anchoring signal” when used in conjunction with a protein or polypeptide such as an enzyme (such as, for example, hyaluronidase), mean for example a first nucleic acid encoding a peptide or protein that is operably linked to a second nucleic acid encoding a protein, or a first protein that is operably linked to a second protein, such as an enzyme (such as, for example hyaluronidase), and that enables the cellular transport machinery of a cell, in particular of a S. cerevisiae cell, to locate at least the second protein to the membrane of the cell wherein if the second protein is also operably linked to an “anchoring signal” the second protein is not secreted but remains attached to the membrane of the cell. In some cases, a secretion-anchoring signal may provide a dual secretion signal and anchoring signal function.

[0121] Sequences of secretion and anchoring signals, methods for the expression, anchoring and / or secretion of heterologous proteins, such as enzymes (such as, for example, hyaluronidase) on the surface of a cell (such, as for example, a yeast cell) are well known in the art (see for example, Ast et al (2013) Cell 152: 1134-1145, Ast and Schuldiner (2013) Crit Rev Biochem Mol Biol 48(3) 273-288, Van der Vaart et al (1997) Applied Environmental Microbiology 63(2) 615-620 and the entire contents of each of these publications are incorporated herein by reference).

[0122] The “activity” of an enzyme is used interchangeably with the term “function” and designates, in the context of the invention, the capacity of an enzyme to catalyze a desired reaction. The amount of an enzyme in a host cell may be altered by modifying the transcription of the gene that encodes 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 comprising 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 coding sequences on a polycistronic mRNA of an operon or breaking up an 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.

[0123] Alternatively, or in addition, the copy number of an enzyme in a host cell may be altered by modifying the level of translation of an mRNA that encodes 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 of or adjacent to the 5’ side of 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 enzyme, altering expression of rare codon tRNAs used in the biosynthesis of the enzyme, and / or increasing the stability of the enzyme, as, for example, via mutation of its coding sequence.

[0124] The activity of an enzyme in a host cell can be altered in a number of ways, including, but not limited to, expressing a modified form of the enzyme that exhibits increased or decreased solubility in the host cell, expressing an altered form of the enzyme that lacks a domain through which the activity of the enzyme is inhibited, expressing a modified form of the enzyme that has a higher or lower KCat or a lower or higher Kmfor the substrate, or expressing an altered form of the enzyme that is more or less affected by feedback or feed-forward regulation by another molecule in the pathway.

[0125] The terms "encoding" or "coding for" refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an amino-acid sequence.

[0126] The gene(s) encoding the enzyme(s) considered in the present invention can be exogenous or endogenous.

[0127] The methods implemented in the present invention preferably require the use of one or more chromosomal integration constructs for the stable introduction of a heterologous nucleotide sequence into a specific location on a chromosome or for the functional disruption of one or more target genes in a genetically modified cell. In some embodiments, disruption of a target gene prevents the expression of the related functional protein. In some embodiments, disruption of a target gene results in the expression of a non-functional protein from the disrupted gene.

[0128] Accordingly, a “disrupted endogenous nucleic acid” in the present invention relates to an endogenous nucleic acid, or gene, unable to encode the functional, or fully functional, protein or polypeptide it codes for before it was disrupted. The nucleic acid can for example be disrupted by the introduction of an integration construct into the nucleic acid, as illustrated in the examples. Said integration can for example prevent the expression of the related functional protein or polypeptide or result in the expression of a non-functional, or non-fully functional protein or polypeptide from the disrupted gene.

[0129] Parameters of chromosomal integration constructs that may be varied in the practice of the present invention include, but are not limited to, the lengths of the homologous sequences; the nucleotide sequence of the homologous sequences; the length of the integrating sequence; the nucleotide sequence of the integrating sequence; and the nucleotide sequence of the target locus. In some embodiments, an effective range for the length of each homologous sequence is 20 to 5,000 base pairs, preferentially 50 to 100 base pairs. In particular embodiments, the length of each homologous sequence is about 50 base pairs. For more information on 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).

[0130] In some embodiments, (a) disrupted gene(s) in which the above-mentioned DNA construct(s) is / are intended to be inserted may advantageously comprises one or more selectable markers useful for the selection of transformed cells. Preferably, said selectable marker(s) are comprised in the DNA construct(s) according to the present invention.

[0131] In some embodiments, the selectable marker is an antibiotic resistance marker. Illustrative examples of antibiotic resistance markers include, but are not limited to the, NAT1, AUR1-C, HPH, DSD A, KAN<R>, and SH BLE gene products. The NAT 1 gene product from S. noursei confers resistance to nourseothricin; the AUR1-C gene product from Saccharomyces cerevisiae confers resistance to Auerobasidin A (AbA); the HPH gene product of Klebsiella pneumoniae confers resistance to Hygromycin B; the DSDA gene product of E. coli allows cells to grow on plates with D-serine as the sole nitrogen source; the KAN<R> gene of the Tn903 transposon confers resistance to G418; and the SH BLE gene product from Streptoalloteichus hindustanus confers resistance to Zeocin (bleomycin).

[0132] In some embodiments, the antibiotic resistance marker is deleted after the genetically modified cell of the invention is isolated. The man skilled in the art is able to choose suitable marker in specific genetic context.

[0133] In a particular embodiment, a recombinant cell according to the invention is devoid of any antibiotic resistance marker. This advantageously prevents the necessity to add antibiotics in the selection medium.

[0134] In some embodiments, the selectable marker rescues an auxotrophy (e.g., a nutritional auxotrophy) in the genetically modified cell. In such embodiments, a parent cell, and in particular a parent yeast, comprises a functional disruption in one or more gene products that function in an amino acid or nucleotide biosynthetic pathway, such as, for example, the HIS3, LEU2, LYS1, LYS2, MET 15, TRP1, ADE2, and URA3 gene products in yeast, which renders the parent cell incapable of growing in media without supplementation with one or more nutrients (auxotrophic phenotype). The auxotrophic phenotype can then be rescued by transforming the parent cell with a chromosomal integration encoding a functional copy of the disrupted gene product (in some embodiments the functional copy of the gene may originate from close species, such as Kluveromyces, Candida etc.), and the genetically modified cell generated can be selected based on the loss of the auxotrophic phenotype of the parent microbial cell.

[0135] For each of the nucleic acid sequences comprising a promoter sequence, a coding sequence (e.g. an enzyme coding sequence), or a terminator sequence, reference sequences are described herein. The present description also encompasses nucleic acid sequences having specific percentages of nucleic acid identity with a reference nucleic acid sequence.

[0136] For each or the amino acid sequences of interest, reference sequences are described herein. The present description also encompasses amino acid sequences (e.g. enzyme amino acid sequences), having specific percentages of amino acid identity with a reference amino acid sequence.

[0137] For obvious reasons, in all the present description, a specific nucleic acid sequence or a specific amino acid sequence which complies with, respectively, the considered nucleotide or amino acid identity, should further lead to obtaining a protein (or enzyme) which displays the desired biological activity. As used herein, the "percentage of identity" between two nucleic acid sequences or between two amino acid sequences is determined by comparing both optimally aligned sequences through a comparison window.

[0138] The portion of the nucleotide or amino-acid sequence in the comparison window may thus include additions or deletions (for example "gaps") as compared to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment between both sequences.

[0139] The terms "sequence homology" or "sequence identity" or "homology" or "identity" are used interchangeably herein. For the purpose of the invention, it is defined here that in order to determine the percentage of sequence homology or sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids / based or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.

[0140] A comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley).

[0141] The percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman- Wunsch algorithm has been implemented in the computer program NEEDLE.

[0142] For the purpose of the 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 for the substitution matrix. For nucleotide sequence, EDNAFULL is used. The 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” has been indicated in response to the question “Brief identity and similarity”, and “SRS pairwise” indicated as Output alignment format.

[0143] After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as "longest-identity".

[0144] The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments using several other art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http: / / hmmer.wustl.edu / ) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. on https: / / www.ebi.ac.uk / Tools / msa / clustalo / or the GAP program (mathematical algorithm of the University of Iowa) or the mathematical algorithm of Myers and Miller (1989 - Cabios 4: 11-17) or Clone Manager 9. Preferred parameters used are the default parameters as they are set on https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0145] The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed with the BLASTN program, score = 100, word length = 12, to obtain polynucleotide sequences that are homologous to those nucleic acids which encode the relevant protein.

[0146] BLAST protein searches are performed with the BLASTP program, score = 50, word length = 3, to obtain amino acid sequences homologous to the SHC polypeptide. To obtain gapped alignments for comparative 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 matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1 : 154-162) or Markov random fields. When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.

[0147] In particular embodiments, % identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0148] The "fermentation" or "culture" is generally conducted in fermenters with an appropriate culture medium adapted to the cell being cultivated, containing at least one simple carbon source, and if necessary co-substrates.

[0149] The term “fermentation composition” refers to a composition which comprises 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 broth, which can be the entire contents of a vessel (e.g., a flask, plate, or fermentor), including cells, aqueous phase, and compounds produced from the genetically modified host cells.

[0150] The term “medium” refers to a culture medium or cultivation medium or fermentation medium.

[0151] For maximal production of hyaluronic acid, the recombinant cells used as production hosts preferably have a high rate of carbohydrate utilization. These characteristics may be conferred by mutagenesis and selection, genetic engineering, or may be natural. Fermentation media, or “culture medium” or “cultivation medium”, for the present 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 unpurified mixtures from renewable feedstocks such as cheese whey permeate cornsteep liquor, sugar beet molasses, and barley malt. Other carbon substrates may include glycerol, acetate and / or ethanol.

[0152] Hence, it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of cell, and in particular of yeast.

[0153] Although it is contemplated that all of the above-mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and / or arabinose for cells, and in particular yeasts, modified to use C5 sugars, and more particularly glucose.

[0154] Preferred carbon substrates are glucose or sucrose. In addition to an appropriate carbon source, fermentation media may contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for the production of the desired product.

[0155] Besides, additional genetic modifications suitable for the growth of recombinant cells according to the invention may be considered.

[0156] The terms "Aerobic conditions" refers to concentrations of oxygen in the culture medium that are sufficient for an aerobic or facultative anaerobic cell, and in particular yeast, to use di-oxygene as a terminal electron acceptor.

[0157] “Microaerobic condition” refers to a culture medium in which the concentration of oxygen is less than that in air, i.e. oxygen concentration up to 6% O2.

[0158] An "appropriate culture medium" designates a medium (e.g. a sterile, liquid medium) comprising nutrients essential or beneficial to the maintenance and / or growth of the cell such as carbon sources or carbon substrate, nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorus sources, for example, monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), for example magnesium salts, cobalt salts and / or manganese salts; as well as growth factors such as amino acids, vitamins, growth promoters, and the like. The term "carbon source" or "carbon substrate" or "source of carbon" according to the present invention denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a cell, including hexoses (such as glucose, galactose or lactose), pentoses, monosaccharides, oligosaccharides, disaccharides (such as sucrose, cellobiose or maltose), molasses, starch or its derivatives, cellulose, hemicelluloses and combinations thereof. Culture mediums that are particularly appropriate to produce recombinant cells of the invention, and in particular recombinant yeasts of the invention, are described in greater details further below in the text.

[0159] As used herein, the term “about” refers to a reasonable range about a value as determined by the practitioner of skill. In certain embodiments, the term about refers to ± one, two, or three 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. General features of genetic modifications introduced according to the invention

[0160] - All the genome modifications are inserted in recombinant cells, and in particular recombinant yeasts, according to known genetic engineering techniques:

[0161] - The successive nucleic acid sequences included in a gene construct that is introduced in the recombinant cell genome according to the invention are of the following structure:

[0162] Prom i -ORF i -term wherein:

[0163] - Proml is a sequence regulating the expression of the coding sequence ORF1,

[0164] - ORF1 is a nucleic acid sequence encoding a desired protein PROT1, and especially a desired enzyme PROT1,

[0165] - Terml is a transcription terminator sequence that mediates transcriptional termination by providing signals in the newly synthesized mRNA that trigger processes which release the mRNA from the transcriptional complex, and

[0166] > “n” may or may not describe the same ORF (Open Reading Frame), promoter or terminator. The order of the nucleic acid sequences does not matter, “n” is an integer usually ranging from 5 and 20. These constructs are inserted in one of the recombinant cell chromosome at a controlled location. In some embodiments, the insertion site is neither essential for the functionality of the inserted construct, nor for the viability of the resulting genetically modified cell.

[0167] As will be understood by those of skill in the art, it can 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 that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. Codons can be substituted to reflect the preferred codon usage of the host, in a process sometimes called “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 (www.idtdna.com / CodonOptfrom) from Integrated DNA Technologies. 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 having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al, 1996, Nucl Acids Res. 24: 216-8).

[0168] - When the recombinant cell is a yeast cell, and in particular is Saccharomyces cerevisiae yeast cell, nucleic acid sequences introduced in the yeast genome and originating from other organisms than Saccharomyces cerevisiae are generally “transcoded” (generally “codon- optimized”), meaning that these nucleic acid sequences are synthesized with an optimal codon usage for expression in S. cerevisiae. The nucleotide sequence (and not the protein sequence) of some nucleic acid sequences from S. cerevisiae has also been modified (“transcoded”) to minimize recombination with an endogenous copy of the said gene.

[0169] - Genes may be deleted through standard procedures used in cell genetic engineering. In some embodiments, the genes targeted for deletion may be interrupted by insertion of one of the above-described gene constructs, or alternatively the genes targeted for deletion are replaced by a short stretch of nucleotide.

[0170] - A nucleic acid sequences may be rendered “inducible or repressible” by deleting an endogenous copy of the nucleic acid sequences (if necessary) and placing a new copy of the ORF under the control of an inducible or repressible promoter. An inducible or repressible promoter is a promoter which activity is modulated or controlled, i.e. either increased or decreased, upon a change in the environmental conditions or external stimuli. Induction or repression may be artificially controlled, which encompasses induction or repression by abiotic factors such as chemical compounds not found naturally in the cell, and in particular yeast, of interest, light, oxygen levels, heat or cold. A list and sequences of inducible or repressible promoters are described elsewhere in the present specification.

[0171] Recombinant cells according to the invention

[0172] The inventors have conceived recombinant cells, in particular recombinant yeasts, having an ability of producing low molecular weight hyaluronic acid (e.g. the hyaluronic acid as described under the second aspect of the invention). This ability is obtained through a plurality of alterations that have been introduced in the genome of said cells, by genetic engineering methods.

[0173] The production of low molecular weight hyaluronic acid by cells of the invention, and in particular by yeast cells of the invention, has been achieved by optimizing the endogenous metabolism of UDP-Glucose, and optionally UDP-N-Acetyl-glucosamine, and directing the subsequent artificially modified metabolic pathway mainly towards hyaluronic acid production while in the same time maintaining an optimal viability of the resulting genetically modified cells.

[0174] It has been determined that a hyaluronic acid production by recombinant cells according to the invention may be increased by increasing the conversion of glucose-6-phosphate into the successive intermediate metabolites (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 allowing a good viability of the resulting recombinant cells.

[0175] Indeed, in order to obtain a viable recombinant cell of the invention, many different constructs were tested in order to obtain a viable and efficient recombinant cell, and in particular a viable recombinant yeast. In particular, such recombinant yeast were difficult to obtain because the temporary accumulation of some intermediates appeared to be toxic for yeasts.

[0176] Unexpected technical difficulties were encountered in establishing the conditions suitable for the preparation of a recombinant cell able to produce hyaluronic acid, and in particular to produce hyaluronic acid with a controlled molecular weight, i.e. the low molecular weight hyaluronic acid as described in the second aspect of the present invention.

[0177] By “controlled” molecular weight of a hyaluronic acid of the invention is intended to mean that at least 80%, in particular at least 85%, of the hyaluronic acid produced by a method of the invention has a molecular weight comprised within a certain molecular weight’ s range through the regulation of at least one parameter of the method and / or of the recombinant cell of the invention, such as for example:

[0178] - the nature and origin of the nucleic acid encoding the hyaluronidase of the recombinant cell, and / or

[0179] - the nature and origin of the promoter controlling the expression of the nucleic acid encoding the hyaluronidase(s) of the recombinant cell, and / or

[0180] - the presence of an anchoring and / or of a secretion signal associated to the encoded hyaluronidase(s) of the recombinant cell, and / or

[0181] - the pH of the culture medium during the step of culturing the recombinant cell, and / or

[0182] - the duration of the culturing of the recombinant cell. Indeed, after much research and experimental trial, the inventors discovered that it was possible to cultivate a recombinant cell, and in particular a recombinant yeast cell, more particularly a recombinant Saccharomyces cerevisiae yeast cell, able to produce hyaluronic acid having a controlled molecular weight (i.e. the low molecular weight hyaluronic acid as described in the second aspect of the present invention) controlled using the following parameters:

[0183] - the selection of the nature and origin of the nucleic acid sequences encoding the polypeptide having hyaluronidase activity of the recombinant cell, in particular the recombinant yeast, of the invention; and / or

[0184] - the nature and origin of the promoter controlling the expression of the nucleic acid sequences encoding the polypeptide having hyaluronidase activity of the recombinant cell, in particular the recombinant yeast, of the invention; and / or

[0185] - the optional presence of an anchoring signal, in addition to a secretion signal, associated to the encoded polypeptide having hyaluronidase activity of the recombinant cell, in particular the recombinant yeast, according to the invention; and / or

[0186] - the pH of the culture medium during the step of culturing the recombinant cell, in particular the recombinant yeast cell, according to the invention; and / or

[0187] - the duration of the culturing of the recombinant cell, in particular the recombinant yeast cell, according to the invention.

[0188] Among all the different factors, the inventors have surprisingly found that the promoter controlling the expression of the nucleic acid sequences encoding the polypeptide having hyaluronidase activity of the recombinant cell plays a critical role in determining the molecular weight of the produced HA. Although promoters of different strength leading to the production of various amount of protein are known in the art, the precise amount of the protein produced and its effect in the complex gene expression circuitry of a eukaryotic cell such as yeast are unpredictable and should be empirically determined. The inventors have surprisingly found that expressing the exogenous hyaluronidase under the promoter pCCW12.spr leads to production of HA of the desired molecular weight, i.e. the low-molecular- weight HA of the invention. In such embodiments, one or both of the exogenous hyaluronidase is expressed under the promoter pCCW12.spr, the hyaluronidase is preferably anchored at the membrane, and preferably the hyaluronidase expressed is from Cupiennius salei (herein referred to as HYAL.Csa).

[0189] To the inventors’ knowledge, this has never been achieved before. The molecular weight of the hyaluronic acid (HA) produced by the recombinant cells of the invention will be in the range of less than 5 kDa, preferably in the range of about 800 Da to about 5 kDa, and particularly with a median molecular weight of about 2.8 kDa.

[0190] The nucleic acid encoding a polypeptide having a glutamine synthetase activity may be obtained or derived from Saccharomyces cerevisiae.

[0191] The nucleic acid encoding a polypeptide having hyaluronidase activity in a recombinant cell of the invention may be obtained or derived from at least one of Cupiennius salei. Loxosceles intermedia, Hirudo nipponia, Bothrops atrox or Tityus serrulatus.

[0192] The nucleic acid encoding a polypeptide having hyaluronan synthase activity in a recombinant cell of the invention may be obtained or derived from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviKl, Chlorella virus IL-5-2sl, Chlorella virus CZ-2, Chlorella virus CVG-1, Xenopus laevis or Pasteur ella mullocida, and is in particular obtained or derived from Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviKl, Chlorella virus IL-5-2sl, Chlorella virus C7.-2, Chlorella virus CVG-1 o Xenopus laevis.

[0193] The nucleic acid encoding a polypeptide having UDP-Glucose dehydrogenase activity in a recombinant cell of the invention may be obtained or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1 or Streptococcus zooepidemicus, and in particular from Arabidopsis thaliana or Chlorella virus PBCV1.

[0194] The recombinant cell according to the invention can comprise at least one recombinant nucleic acid encoding one or more of:

[0195] (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity; and / or

[0196] (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRH) activity.

[0197] The recombinant cell according to the invention can comprise at least one recombinant nucleic acid encoding one or more of:

[0198] (i) a polypeptide having Phosphoglucomutase- 1 (PGM1) activity; and / or

[0199] (ii) a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase (UGP1) activity; and / or

[0200] (iii) a polypeptide having Glucosamine-6-phosphate N-acetyltransf erase (GNA1) activity; and / or

[0201] (iv) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity. The recombinant yeast cell according to the invention can be in particular 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, Kluyveromyces thermotolerens, Ogataea polymorpha, Yarrowia lypolytica, Debaryomyces hansenii, and Ashbya gossypii, and is preferably Saccharomyces cerevisiae.

[0202] The recombinant host cell according to the invention can belong to the Saccharomycetales order and is in particular 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, Kluyveromyces thermotolerens, Ogataea polymorpha, Yarrowia lypolytica, Debaryomyces hansenii, and Ashbya gossypii, and is preferably Saccharomyces cerevisiae.

[0203] In a method of the invention, the nucleic acid encoding a polypeptide having hyaluronidase activity may be obtained or derived from Cupiennius salei, Loxosceles intermedia, Hirudo nipponia, Bothrops atrox or Tityus serrulatus.

[0204] In a method of the invention, the nucleic acid encoding a polypeptide having hyaluronan synthase activity may be obtained or derived from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviKl, Chlorella virus IL-5-2sl, Chlorella virus CZ-2, Chlorella virus CVG-1, Xenopus laevis or Pasteurella multocida, and is in particular obtained or derived from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviKl, Chlorella virus IL-5-2sl, Chlorella virus CZ-2, Chlorella virus CVG-1, o Xenopus laevis.

[0205] In a method of the invention, the nucleic acid encoding a polypeptide having UDP-Glucose dehydrogenase activity may be obtained or derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1 or Streptococcus zooepidemicus, and in particular obtained or derived from Arabidopsis thaliana or Chlorella virus PBCV1.

[0206] In a method of the invention, the recombinant cell may comprise at least one recombinant nucleic acid encoding one or more of:

[0207] (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity; and / or (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity.

[0208] In the method of the invention, the recombinant cell may comprise at least one recombinant nucleic acid encoding one or more of:

[0209] (i) a polypeptide having Phosphoglucomutase- 1 (PGM1) activity; and / or

[0210] (ii) a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase (UGP1) activity; and / or

[0211] (iii) a polypeptide having Glucosamine-6-phosphate N-acetyltransf erase (GNA1) activity; and / or

[0212] (iv) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity.

[0213] In a method of the invention, the recombinant cell may be a member of the genus Saccharomyces. and in particular is Saccharomyces cerevisiae.

[0214] In a method of the invention, the molecular weight of the hyaluronic acid may be controlled by the fermentation time.

[0215] In a method of the invention, the time sufficient to produce hyaluronic acid (HA) of a desired molecular weight (e.g. the low molecular weight hyaluronic acid as described in the second aspect of the present invention) may be a period of from about 35 hours to about 60 hours, preferably from about 40 hours to about 55 hours, in particular about 52 hours.

[0216] In a method of the invention, the molecular weight of the hyaluronic acid may be controlled by the pH of the cultivation medium.

[0217] In a method of the invention, the molecular weight of the hyaluronic acid may be controlled by regulating the pH of the cultivation medium during the cultivation step (a) of a method of the invention.

[0218] In a method of the invention, the molecular weight of the hyaluronic acid may be controlled by removing the biomass from the cultivation medium.

[0219] In an embodiment, the method of the third aspect of the invention can be carried out on an industrial scale, preferably where the cultivation medium is at least about 100 L, more preferably in the range of about 1000 L to about 3000 L, even more preferably about 10,000 L or even more preferably about 100,000 L, or even about 250,000 L.

[0220] Another object of the invention relates to hyaluronic acid (HA) obtained or obtainable from a recombinant cell of the invention or from the method of the third aspect of the invention. In a particular embodiment, the recombinant cell according to the invention, and in particular the recombinant yeast according to the invention, comprises one recombinant nucleic acid encoding a polypeptide having glutamine synthetase (GLN1) activity.

[0221] In a particular embodiment, the recombinant cell according to the invention, and in particular the recombinant yeast according to the invention, is such that at least one, and in particular all, its endogeneous nucleic acids encoding a glutamate synthase (GLT1) are disrupted.

[0222] In a particular embodiment, the recombinant cell according to the invention, and in particular the recombinant yeast according to the invention:

[0223] - comprises one recombinant nucleic acid encoding a polypeptide having glutamine synthetase (GLN1) activity, and in particular a polypeptide having glutamine synthetase (GLN1) activity obtained or derived from Saccharomyces cerevisiae: and

[0224] - is such that at least one, and in particular all, its endogeneous nucleic acids encoding a glutamate synthase (GLT1) are disrupted.

[0225] In a particular embodiment, the recombinant cell according to the invention, and in particular the recombinant yeast according to the invention comprises only one recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity. In a particular embodiment, the recombinant cell according to the invention, and in particular the recombinant yeast comprises between 5 and 10 recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity.

[0226] In a particular embodiment, the recombinant cell according to the invention, and in particular the recombinant yeast comprises between 3 and 7 recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase (UDP-GlcDH or HASB) activity.

[0227] In another embodiment, the one or more nucleic acids encoding a polypeptide having a UDP- Glucose dehydrogenase (UDP-GlcDH or HASB) activity is obtained or derived from at least one of Arabidopsis thaliana. Chlor ella virus PBCV1 or Streptococcus zooepidemicus. and in particular obtained or derived from Arabidopsis thaliana or Chlor ella virus PBCV1.

[0228] In a particular embodiment, the recombinant cell according to the invention, and in particular the recombinant yeast comprises between 4 and 8 recombinant nucleic acids encoding a polypeptide having hyaluronan synthase (HAS A) activity.

[0229] In another embodiment, the one or more recombinant nucleic acids encoding a polypeptide having a hyaluronan synthase (HASA) activity is obtained or derived from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1 , Chlorella virus CviKl, Chlor ella virus IL-5-2sl , Chlor ella virus CV-2, Chlorella virus CVG-1, Xenopus laevis or Pasteurella mullocida. and is in particular obtained or derived from Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviKl, Chlorella virus IL-5-2sl, Chlorella virus C7.-2, Chlorella virus CVG-1 or Xenopus laevis. In a particular embodiment, the recombinant cell according to the invention, and in particular the recombinant yeast of the invention only comprises one recombinant nucleic acid encoding a polypeptide having hyaluronidase activity.

[0230] In another embodiment, the one or more recombinant nucleic acids encoding a polypeptide having a hyaluronidase activity is obtained or derived from Cupiennius salei, Loxosceles intermedia, Hirudo nipponia, Bothrops atrox or Tityus serrulatus.

[0231] In another embodiment, the recombinant cell according to the invention, and in particular the recombinant yeast cell may comprise at least one recombinant nucleic acid encoding one or more of:

[0232] (A) a polypeptide having Phosphoglucomutase- 1 (PGM1) activity; and / or

[0233] (B) a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase (UGP1) activity; and / or

[0234] (C) a polypeptide having Glucosamine-6-phosphate N-acetyltransferase (GNA1) activity; and / or

[0235] (D) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity.

[0236] In particular, the recombinant cell according to the invention, and in particular the recombinant yeast cell of the invention comprises at least two, in particular at least three, and more particularly all of the modifications indicated above.

[0237] In a particular embodiment, the nucleic acid encoding a polypeptide having Phosphoglucomutase- 1 (PGM1) activity, the nucleic acid encoding a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase (UGP1) activity, the nucleic acid encoding a polypeptide having glutamine- fructose-6-phosphate amidotransferase (GFA1) activity, the nucleic acid encoding a polypeptide having Glucosamine-6-phosphate N-acetyltransferase (GNA1) activity, the nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity and the nucleic acid encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity are nucleic acids originating or derivating from a yeast, preferably from Saccharomyces cerevisiae. In a particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide defined above and comprised in a recombinant cell according to the invention, and in particular comprised in the recombinant yeast of the invention are under the control of a promoter selected from the group consisting of pPDCl, pTDH3, pCCW12, pCCW12.Spr, pCCW12.Sm, pCCW12.sk, pCCW12.sba, pCCW12.sar, pTEFl, pENO2, pRPLAl, pNUP57 and pTEF3.

[0238] In a particular embodiment, the inducible or repressible promoters mentioned in the present specification are selected from the group consisting of promoters inducible or repressible with copper or promoters inducible or repressible with methionine, in particular selected from the group consisting of pMET6, pMET25 and pSAMl.

[0239] Recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity

[0240] A recombinant cell according to the invention, and in particular a recombinant yeast of the invention, comprises one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity.

[0241] A polypeptide having hyaluronan synthase activity according to the invention means a polypeptide that converts the intermediate metabolites UDP-Glucoronate and UDP-N-acetylglucosamine (UDP-GlcNAc) into hyaluronic acid ((P-D-l,3-GlcNAc-P-D-l,4-GlcA)n).

[0242] In an embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity are under the control of an inducible or repressible promoter that is functional in the recombinant cells of the invention, such as for example the inducible or repressible promoters pCUPl or pMET25.

[0243] One or more of the recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may be under the control of a promoter selected from the group consisting of pCCW12, pCCW12.Sm, pTDH3-l.Sba and pTDH3.Sar.

[0244] The said one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may originate or be derived from at least one of Streptococcus zooepidemicus (sz), Chlorella virus PBCV1 (Vir), Chlorella virus CviKl (Vir), Chlorella virus IL-5-2sl (Vir), Chlorella virus C7.-2 (Vir), Chlorella virus CVG-1 (Vir), Xenopus laevis (xl) or Pasteurella multocida (pm), and may originate or derive in particular from Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviKl, Chlorella virus IL-5-2sl, Chlorella virus CV-2, Chlorella virus CVG-1 or Xenopus laevis, as shown in the examples herein. A recombinant cell according to the invention, and in particular a recombinant yeast according to the invention, may comprise between 2 and 8, in particular between 4 and 8 recombinant nucleic acids encoding a polypeptide having hyaluronan synthase (HASA) activity. A recombinant cell according to the invention, and in particular a recombinant yeast according to the invention, may for example comprise 2 or 6, and in particular 6, recombinant nucleic acids encoding a polypeptide having hyaluronan synthase (HASA) activity.

[0245] Illustratively, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may be inserted within the JLP1 gene and / or within the SAM3 gene and / or within the TRP1 gene and / or within the LYP1 gene of the recombinant cell, and in particular of the recombinant yeast, as it is shown in the examples herein.

[0246] In an embodiment of the invention, a recombinant cell, and in particular a recombinant yeast, of the invention comprises:

[0247] - between 2 and 8, in particular between 4 and 8 recombinant nucleic acids encoding a polypeptide having hyaluronan synthase (HASA) activity;

[0248] - said one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity originating or being derived from at least one of Streptococcus zooepidemicus (sz), Chlorella virus PBCV1 (Vir), Xenopus laevis (xl) or Pasteurella multocida (pm), and originating or being derived in particular from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1 or Xenopus laevis; and

[0249] - said one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity being under the control of an inducible or repressible promoter that is functional in the recombinant cells of the invention, such as for example the inducible or repressible promoters pCUP or pMET25 and / or under the control of a promoter selected from the group consisting of pCCW12, pCCW12.Sm, pTDH3-l.Sba and pTDH3.Sar.

[0250] Recombinant nucleic acids encoding a polypeptide having UDP -Glucose dehydrogenase activity A recombinant cell according to the invention, and in particular a recombinant yeast according to the invention, comprises one or more recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase (UDP-GlcDH or HASB) activity.

[0251] A polypeptide having UDP-Glucose dehydrogenase (UDP-GlcDH or HASB) activity according to the invention means a polypeptide that converts the intermediate metabolite Uridine- Diphosphate-Glucose (UDP-Glucose) into UDP-Glucuronate. In an embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase (UDP-GlcDH or HASB) activity are under the control of an inducible or repressible promoter that is functional in recombinant cells of the invention, such as for example the inducible or repressible promoters pMET25 or pMET6.

[0252] One or more of the recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase activity may be under the control of a promoter selected from the group consisting of pCCW12, in particular the pCCW12.sk and the pCCW12.sba promoters; pTEFl.Sba and pTDH3.Sk.

[0253] The one or more recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase activity may originate or be derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1 or Streptococcus zooepidemicus. and in particular may originate or derive from Arabidopsis thaliana or Chlorella virus PBCV1.

[0254] A recombinant cell according to the invention, and in particular a recombinant yeast according to the invention, may comprise between 2 and 7, in particular between 3 and 7 recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase activity. A recombinant cell according to the invention, and in particular a recombinant yeast according to the invention, may for example comprise 2 or 5 recombinant nucleic acids encoding a polypeptide having UDP- Glucose dehydrogenase activity.

[0255] Illustratively, the one or more recombinant nucleic acids encoding a polypeptide having UDP- Glucose dehydrogenase activity may be inserted within the JLP1 gene and / or within the TRP1 gene, and / or within the LYP1 gene of the recombinant cell, and in particular of the recombinant yeast, as it is shown in the examples herein.

[0256] In an embodiment of the invention, a recombinant cell, and in particular a recombinant yeast, of the invention comprises:

[0257] - between 2 and 7, and in particular between 3 and 7 recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase activity;

[0258] - said one or more recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase activity originating or being derived from at least one of Arabidopsis thaliana, Chlorella virus PBCV1 or Streptococcus zooepidemicus, and originating or being derived in particular from Arabidopsis thaliana or Chlorella virus PBCVP, and - said one or more recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase activity being under the control of an inducible or repressible promoter that is functional in the recombinant cells of the invention, such as for example the inducible or repressible promoters pMET25 or pMET6 and / or under the control of a promoter selected from the group consisting of pCCW12, in particular the pCCW12.sk and the pCCW12.sba promoters; pTEFl.Sba and pTDH3.Sk.

[0259] Recombinant nucleic acids encoding a polypeptide having Hyaluronidase activity

[0260] A recombinant cell according to the invention, and in particular a recombinant yeast according to the invention, comprises one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity.

[0261] A polypeptide having hyaluronidase activity according to the invention means a polypeptide that degrades hyaluronic acid, i.e. that converts a hyaluronic acid of a given molecular weight into a hyaluronic acid of a lower molecular weight.

[0262] As previously indicated, polypeptides having hyaluronidase activity of the present invention comprise a secretion signal.

[0263] In an embodiment, polypeptide having hyaluronidase activity comprise both a secretion signal and an anchoring signal.

[0264] In an embodiment, the said 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 cells of the invention.

[0265] One or more of the recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be under the control of a promoter selected from the group consisting of pCCW12.Spr, pTEFl, pCCW12, pCCW12.sba, pCCW12.Sar, pPDCl, pTEF3, pTDH3, pNUP57, pCWP2 and pCCWlO.ago. In an embodiment, the recombinant cell has at least one nucleic acids encoding a polypeptide having hyaluronidase activity under the control of a pCCW12.Spr promoter.

[0266] The said one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may originate or be derived from at least one of Cupiennius salei (Csa), Loxosceles intermedia (Li), Hirudo nipponia (Hn), Bothrops atrox (Ba) or Tityus serrulatus (Ts) as shown in the examples herein. A recombinant cell according to the invention, and in particular a recombinant yeast according to the invention, may comprise only one recombinant nucleic acid encoding a polypeptide having hyaluronidase activity. In an embodiment, said nucleic acid is under the control of a pCCW12.Spr promoter. In a preferred embodiment, the recombinant cell, and in particular the recombinant yeast, comprises two recombinant nucleic acid encoding a polypeptide having hyaluronidase activity, both of which under the control of a pCCW 12.Spr promoter.

[0267] Illustratively, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be inserted within the JLP1 gene and / or within the LYP1 gene, as it is shown in the examples herein.

[0268] In an embodiment of the invention, a recombinant cell, and in particular a recombinant yeast, of the invention comprises:

[0269] - at least one, preferably two, recombinant nucleic acid encoding a polypeptide having hyaluronidase activity;

[0270] - said recombinant nucleic acid encoding a polypeptide having hyaluronidase activity originating or being derived from Cupiennius salei (Csa), Loxosceles intermedia (Li), Hirudo nipponia (Hn), Bothrops atrox (Ba) or Tityus serrulatus (Ts);

[0271] - said recombinant nucleic acid encoding a polypeptide having hyaluronidase activity comprising either (i) a secretion signal and no anchoring signal or (ii) a secretion signal and an anchoring signal; and

[0272] - said recombinant nucleic acid encoding a polypeptide having hyaluronidase activity being under the control of a promoter selected from the group consisting of pCCW 12.Spr, pTEFl, pCCW12, pCCW12.sba, pCCW12.Sar, pPDCl, pTEF3, pTDH3, pNUP57, pCWP2 and pCCWlO.ago.

[0273] Recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity

[0274] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, may comprise one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity.

[0275] A polypeptide having glutamine synthetase activity according to the invention means a polypeptide that converts glutamate into glutamine while consuming one ATP and one NH . In an embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity are under the control of an inducible or repressible promoter that is functional in the recombinant cells of the invention.

[0276] One or more of the recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity may be under the control of a promoter selected from the group consisting of pTEFl and pTEFl.Ago.

[0277] The said one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity can originate or be derived from Saccharomyces cerevisiae. as shown in the examples herein.

[0278] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, may comprise only one recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity.

[0279] Illustratively, the one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity may be inserted within the LYP1 or GLT1 gene of the recombinant cell, and in particular of the recombinant yeast cell, as it is shown in the examples herein.

[0280] In an embodiment of the invention, a recombinant cell, and in particular a recombinant yeast cell, of the invention comprises:

[0281] - only one recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity;

[0282] - said recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity originating or being derived from Saccharomyces cerevisiae,' and

[0283] - said recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity being under the control of a promoter selected from the group consisting of pTEFl and pTEFl.Ago.

[0284] In a further embodiment of the invention, a recombinant cell, and in particular a recombinant yeast cell, of the invention comprises:

[0285] - only one recombinant nucleic acid encoding a polypeptide having glutamine synthase activity;

[0286] - said recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity originating or being derived from Saccharomyces cerevisiae,' - said recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity being under the control of a promoter selected from the group consisting of pTEFl and pTEFl.Ago; and

[0287] - said recombinant nucleic acid encoding a polypeptide having glutamine synthetase activity being inserted within the LYP1 or GLT1 gene of the recombinant cell, and in particular of the recombinant yeast cell.

[0288] Recombinant nucleic acids encoding a polypeptide having Glutamine-fructose-6-phosphate amidotransferase activity

[0289] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, may comprise one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity.

[0290] A polypeptide having glutamine-fructose-6-phosphate amidotransferase activity according to the invention means a polypeptide that converts fructose-6-phosphate into glucosamine-6-phosphate. In an embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity are under the control of an inducible or repressible promoter that is functional in the recombinant cells of the invention.

[0291] One or more of the recombinant nucleic acids encoding a polypeptide having glutamine-fructose- 6-phosphate amidotransferase activity may be under the control of a promoter selected from the group consisting of pTEFl and pTEFl.Ago.

[0292] The said one or more recombinant nucleic acids encoding a polypeptide having glutamine- fructose-6-phosphate amidotransferase activity can originate or be derived from Saccharomyces cerevisiae. as shown in the examples herein.

[0293] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, may comprise only one recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity.

[0294] Illustratively, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may be inserted within the SAM3 gene of the recombinant cell, and in particular of the recombinant yeast cell, as it is shown in the examples herein.

[0295] In an embodiment of the invention, a recombinant cell, and in particular a recombinant yeast cell, of the invention comprises: - only one recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6- phosphate amidotransferase activity;

[0296] - said recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6- phosphate amidotransferase activity originating or being derived from Saccharomyces cerevisiae and

[0297] - said recombinant nucleic acid encoding a polypeptide having glutamine-fructose-6- phosphate amidotransferase activity being under the control of a promoter selected from the group consisting of pTEFl and pTEFl.Ago.

[0298] Recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity

[0299] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, can comprise one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity.

[0300] A polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity according to the invention means a polypeptide that can convert N-acetyl-glucosamine into UDP-N-acetyl- glucosamine.

[0301] In an embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity are under the control of an inducible or repressible promoter that is functional in the recombinant cells of the invention, such as for example the inducible or repressible promoters pMET6 or pCUPl.

[0302] One or more of the recombinant nucleic acids encoding a polypeptide having UDP-N- acetylglucosamine pyrophosphorylase activity may be under the control of the promoter pTDH3. The said one or more recombinant nucleic acids encoding a polypeptide having UDP-N- acetylglucosamine pyrophosphorylase activity can originate or be derived from Saccharomyces cerevisiae, as shown in the examples herein.

[0303] A recombinant cell according to the invention, and in particular a recombinant yeast according to the invention, may comprise between 5 and 10 recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity. A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, may for example comprise 5, 7 or 8 recombinant nucleic acids encoding a polypeptide having UDP-N- acetylglucosamine pyrophosphorylase activity. Illustratively, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N- acetylglucosamine pyrophosphorylase activity may be inserted within the HIS3 gene and / or within the JLP1 gene and / or within the SAM3 gene of the recombinant cell, and in particular of the recombinant yeast, as it is shown in the examples herein.

[0304] In an embodiment of the invention, a recombinant cell, and in particular a recombinant yeast cell, of the invention comprises:

[0305] - between 5 and 10 recombinant nucleic acids encoding a polypeptide having UDP-N- acetylglucosamine pyrophosphorylase activity;

[0306] - said one or more recombinant nucleic acids encoding a polypeptide having UDP-N- acetylglucosamine pyrophosphorylase activity originating or being derived from Saccharomyces cerevisiae

[0307] - said one or more recombinant nucleic acids encoding a polypeptide having UDP-N- acetylglucosamine pyrophosphorylase activity being under the control of an inducible or repressible promoter that is functional in the recombinant cells of the invention, such as for example the inducible or repressible promoters pMET6 or pCUPl and / or under the control of the promoter pTDH3.

[0308] Recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 activity

[0309] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, may comprise one or more recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 (PGM1) activity.

[0310] A polypeptide having Phosphoglucomutase- 1 (PGM1) activity according to the invention means a polypeptide that converts Glucose-6-phosphate into the intermediate metabolite Glucose- 1- phosphate.

[0311] In an embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 (PGM1) activity are under the control of an inducible or repressible promoter that is functional in recombinant cells of the invention, such as for example the inducible or repressible promoters.

[0312] One or more of the recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 (PGM1) activity may be under the control of the promoter pPDCl. The one or more recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 (PGM1) activity may originate or be derived from Saccharomyces cerevisiae. as shown in the examples herein.

[0313] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, may comprise only one recombinant nucleic acid encoding a polypeptide having Phosphoglucomutase- 1 (PGM1) activity.

[0314] Illustratively, the one or more recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 activity may be inserted within the SAM3 gene of the recombinant cell, and in particular of the recombinant yeast, as it is shown in the examples herein.

[0315] In an embodiment of the invention, a recombinant cell, and in particular a recombinant yeast, of the invention comprises:

[0316] - only one recombinant nucleic acid encoding a polypeptide having Phosphoglucomutase- 1 activity;

[0317] - said recombinant nucleic acid encoding a polypeptide having Phosphoglucomutase- 1 activity originating or being derived from Saccharomyces cerevisiae,' and

[0318] - said recombinant nucleic acid encoding a polypeptide having Phosphoglucomutase- 1 activity being under the control of the promoter pPDCl.

[0319] Recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase activity

[0320] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, comprises one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase (UGP1) activity.

[0321] A polypeptide having UTP-glucose-1 -phosphate uridylyltransferase (UGP1) activity according to the invention means a polypeptide that converts the intermediate metabolite Glucose- 1 -Phosphate into the intermediate metabolite UDP-glucose.

[0322] In an embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase activity are under the control of an inducible or repressible promoter that is functional in recombiant cells of the invention, such as for example the inducible or repressible promoters pSAMl or pCUPl. One or more of the recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 - phosphate uridylyltransferase activity may be under the control of a promoter selected from the group consisting of pPDCl and pENO2.

[0323] The one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 - phosphate uridylyltransferase activity can originate or be derived from Saccharomyces cerevisiae. as shown in the examples herein.

[0324] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, can comprise between 5 and 10 recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase activity. A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, may for example comprise 5, 7 or 8 recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 - phosphate uridylyltransferase activity.

[0325] Illustratively, the one or more recombinant nucleic acids encoding a polypeptide having UTP- glucose-1 -phosphate uridylyltransferase activity gene may be inserted within the HIS3 gene and / or within the JLP1 gene and / or within the SAM3 gene of the recombinant cell, and in particular of the recombinant yeast, as it is shown in the examples herein.

[0326] In an embodiment of the invention, a recombinant cell, and in particular a recombinant yeast cell, of the invention comprises:

[0327] - between 5 and 10 recombinant nucleic acids encoding a polypeptide having UTP-glucose- 1 -phosphate uridylyltransferase activity;

[0328] - said one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose- 1 -phosphate uridylyltransferase activity originating or being derived from Saccharomyces cerevisiae,' and

[0329] - said one or more recombinant nucleic acids encoding a polypeptide having being under the control of an inducible or repressible promoter that is functional in the recombinant cells of the invention, such as for example the inducible or repressible promoters pSAMl or pCUPl and / or under the control of a promoter selected from the group consisting of pPDCl and pENO2. Recombinant nucleic acids encoding a polypeptide having Glucosamine-6-phosphate N- acetyltransferase activity

[0330] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, can comprise one or more recombinant nucleic acids encoding a polypeptide having Glucosamine-6-phosphate N-acetyltransferase (GNA1) activity.

[0331] A polypeptide having Glucosamine-6-phosphate N-acetyltransferase (GNA1) activity according to the invention means a polypeptide that can convert Glucosamine-6-phosphate into N-acetyl- glucosamine-6-phosphate.

[0332] In an embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having Glucosamine-6-phosphate N-acetyltransferase activity are under the control of an inducible or repressible promoter that is functional in the recombinant cells of the invention.

[0333] One or more of the recombinant nucleic acids encoding a polypeptide having Glucosamine-6- phosphate N-acetyltransferase activity may be under the control of the promoter pCWP2.

[0334] The said one or more recombinant nucleic acids encoding a polypeptide having Glucosamine-6- phosphate N-acetyltransferase activity may originate or be derived from Saccharomyces cerevisiae. as shown in the examples herein.

[0335] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, can comprise only one recombinant nucleic acid encoding a polypeptide having Glucosamine-6-phosphate N-acetyltransferase activity.

[0336] Illustratively, the one or more recombinant nucleic acids encoding a polypeptide having Glucosamine-6-phosphate N-acetyltransferase activity may be inserted within the SAM3 gene of the recombinant cell, and in particular of the recombinant yeast, as it is shown in the examples herein.

[0337] In an embodiment of the invention, a recombinant cell, and in particular a recombinant yeast, of the invention comprises:

[0338] - only one recombinant nucleic acid encoding a polypeptide having Glucosamine-6- phosphate N-acetyltransferase activity;

[0339] - said recombinant nucleic acid encoding a polypeptide having Glucosamine-6-phosphate N-acetyltransferase activity originating or being derived from Saccharomyces cerevisiae,' and

[0340] - said recombinant nucleic acid encoding a polypeptide having Glucosamine-6-phosphate N-acetyltransferase activity being under the control of the promoter pCWP2. Recombinant nucleic acids encoding a polypeptide having Phosphoacetylglucosamine mutase activity

[0341] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, can comprise one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity.

[0342] A polypeptide having phosphoacetylglucosamine mutase (PCM1) activity according to the invention means a polypeptide that can convert N-acetyl-glucosamine-6-phosphate N-acetyl- glucosamine-1 -phosphate.

[0343] In an embodiment, one or more of the recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity are under the control of an inducible or repressible promoter that is functional in the recombinant cells of the invention.

[0344] One or more of the recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may be under the control a promoter selected from the group consisting of pTEF3 and pTEFl.

[0345] The said one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity can originate or be derived from Saccharomyces cerevisiae. as shown in the examples herein.

[0346] A recombinant cell according to the invention, and in particular a recombinant yeast cell according to the invention, may comprise only one recombinant nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase activity.

[0347] Illustratively, the one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may be inserted within the SAM3 gene of the recombinant cell, and in particular of the recombinant yeast, as it is shown in the examples herein. In an embodiment of the invention, a recombinant cell, and in particular a recombinant yeast cell, of the invention comprises: only one recombinant nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase activity;

[0348] - said recombinant nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase activity originating or being derived from Saccharomyces cerevisiae,' and - said recombinant nucleic acid encoding a polypeptide having phosphoacetylglucosamine mutase activity being under the control of the promoter a promoter selected from the group consisting of pTEF3 and pTEFl.

[0349] HYALURONAN SYNTHASE (HASA)

[0350] The hyaluronan synthase enzyme is a protein which is described in the art for catalyzing the conversion of UDP-glucuronate or UDP-N-acetyl-glucose into hyaluronic acid. The hyaluronan synthase originating from Streptococcus zooepidemicus, Chlorella virus PBCV1, Xenopus laevis or Pasteurella multocida may be termed HASA.

[0351] A method implemented to measure the activity level of a polypeptide having hyaluronan synthase activity belongs to the general knowledge of the one skilled in the art.

[0352] In this regard, the one skilled in the art may advantageously refer to the method of colorimetric determination after treatment with concentrated sulfuric acid and carbazole described by Bitter and Muir (Analytical Biochemistry, 4, 330-334, 1962).

[0353] Preferred polypeptide having hyaluronan synthase activity in the present invention is an enzyme having an EC number of n° 2.4.1.212.

[0354] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may originate or be derived from organisms preferably selected in a group consisting of prokaryotic organisms and eukaryotic organisms. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may originate or be derived from archaebacteria. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may originate or be derived from bacteria, and especially from Streptococcus zooepidemicus (Sz) or from Pasteurella multocida (Pm), from Chlorella virus PBCV1 (Vir), Chlorella virus CviKl (Vir), Chlorella virus IL-5-2sl (Vir), Chlorella virus CZ-2 (Vir), Chlorella virus CVG-1 (Vir) or from Xenopus laevis (XI).

[0355] According to a yet preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may be selected from the group consisting of nucleic acid sequences having (i) at least 65%, advantageously at least 70%, preferably at least 80%, nucleic acid identity with a nucleic acid sequence as set forth as sequence 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 (XI), 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) a biological activity of the same nature as the nucleic acid sequence having, respectively, the nucleic acid sequence as set forth as sequence 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 (XI), 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).

[0356] A biological activity of the same nature regarding this sequence is, as previously explained, the capacity to code for a polypeptide that converts UDP-glucuronate or UDP-N-acetyl-glucose into hyaluronic acid.

[0357] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0358] As described herein, a nucleic acid sequence having at least 70% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0359] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses 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 the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0360] For the amino acid sequence of the polypeptide having hyaluronan synthase activity originating from Streptococcus zooepidemicus. Chlorella virus PBCV1, Xenopus laevis or Pasteurella mullocidct. the one skilled in the art may refer, respectively, to the accession numbers B4U0D4, Q84419, P13563, Q7BLV3, M1GZS8, M1H5V3, M1H2Q1 andMlHN86 in theUniProt database, or to SEQ ID NO: 8 (Vir), SEQ ID NO: 9 (Pm) SEQ ID NO: 10 (XI), 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) described herein, in particular to to SEQ ID NO: 8 (Vir), SEQ ID NO: 9 (Pm) SEQ ID NO: 10 (XI), SEQ ID NO:11 (Sz), SEQ ID NO: 105 (Vir), SEQ ID NO: 106 (Vir) or SEQ ID NO: 108 (Vir), and more particularly to SEQ ID NO: 8 (Vir), SEQ ID NO: 9 (Pm) SEQ ID NO: 10 (XI), SEQ ID NO: 11 (Sz).

[0361] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity may be nucleic acid(s) encoding 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 (XI), 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 also a biological activity of the same nature as the amino acid sequence set forth as SEQ ID NO: 8 (Vir), SEQ ID NO: 9 (Pm) SEQ ID NO: 10 (XI), 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). A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of UDP-glucuronate or UDP-N-acetyl-glucose into hyaluronic acid.

[0362] As described herein, an amino acid sequence having at least 50% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having 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% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0363] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses 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 said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

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

[0365] As above-mentioned, the expression level of the 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 herein after defined more in details, which are present in 5’ and 3’ position respectively of the one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity.

[0366] UDP-GLUCOSE DEHYDROGENASE (UDP-GlcDH OR HASB)

[0367] The UDP-Glucose dehydrogenase is a protein which is known in the art to catalyze the conversion of UDP-glucose into UDP-glucuronate. The UDP-Glucose dehydrogenase originating from the genome of Arabidopsis thaliana. Chlorella virus PBCV1 or Streptococcus zooepidemicus may be termed HASB.

[0368] A method implemented to measure the activity level of a polypeptide having UDP-Glucose dehydrogenase activity belongs to the general knowledge of the one skilled in the art.

[0369] In this regard, the one skilled in the art may advantageously refer to the method described by Oka and Jigami (FEBS Journal 273, 2645-2657, 2006).

[0370] Preferred polypeptide having UDP-Glucose dehydrogenase activity in the present specification is an enzyme having an EC number 1.1.1.22.

[0371] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase activity may originate or be derived from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase activity may originate or be derived from archaebacteria. In some preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase activity may originate or be derived from yeast, and especially from Arabidopsis thaliana, Chlorella virus PBCV1 or Streptococcus zooepidemicus haromyces.

[0372] According to a yet 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 a nucleic acid as 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) a biological activity of the same nature as the nucleic acid as 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 a polypeptide having UDP-Glucose dehydrogenase activity originating, respectively, from Arabidopsis thaliana (At), Chlorella virus PBCV1 (Vir) or Streptococcus zooepidemicus (Sz), that may herein also be collectively termed HASB.

[0373] A biological activity of the same nature regarding this sequence is, as previously explained, the capacity to code for a polypeptide that converts UDP -glucose into UDP-glucuronate.

[0374] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0375] As described herein, a nucleic acid sequence having at least 70% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequences, and also a biological activity of the same nature as the said reference nucleic acid sequences.

[0376] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses 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 the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequences.

[0377] For the amino acid sequence of the polypeptide having UDP-Glucose dehydrogenase activity from Arabidopsis thaliana, Chlorella virus PBCV1 or Streptococcus zooepidemicus, the one skilled in the art may refer to the accession numbers NP_173979.1, NP_048965 or KIS19289, respectively, in the UniProt database, or to the sequences as set forth in SEQ ID NO: 16 (At), SEQ ID NO. 17 (Vir) and SEQ ID NO: 18 (Sz) described herein.

[0378] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase activity may be nucleic acid(s) encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 55%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO: 16 (At), SEQ ID NO. 17 (Vir) and SEQ ID NO: 18 (Sz), and also a biological activity of the same nature as the amino acid sequence of SEQ ID NO: 16 (At), SEQ ID NO. 17 (Vir) and SEQ ID NO: 18 (Sz).

[0379] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of UDP-glucose into UDP-glucuronate.

[0380] As described herein, an amino acid sequence having at least 55% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0381] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses 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 said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0382] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0383] As above-mentioned, the expression level of the 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 herein after defined more in details, which are present in 5’ and 3’ position respectively of the one or more recombinant nucleic acids encoding a polypeptide having UDP-Glucose dehydrogenase activity.

[0384] HYALURONIDASE (HYAL)

[0385] The hyaluronidase enzyme is a protein which is described in the art for catalyzing the degradation of hyaluronic acid molecules into smaller hyaluronic acid molecules. The hyaluronidase originating from Cupiennius salei, Loxosceles intermedia, Hirudo nipponia, Bothrops atrox, Tityus serrulatus or Vespa magnifica may be termed HYAL.

[0386] The polypeptide having hyaluronidase activity of the invention may possess both a secretion signal and an anchoring signal or a secretion signal and no anchoring signal or a secretion-anchor signal with a dual secretion and anchoring function. When the encoded polypeptide having hyaluronidase activity possesses both a secretion signal and an anchoring signal, it may be termed HYAL-31 as represented in the examples. When the encoded polypeptide having hyaluronidase activity possesses a secretion signal and no anchoring signal, it may be termed HYAL-3 as represented in the examples.

[0387] A method implemented to measure the activity level of a polypeptide having hyaluronidase activity belongs to the general knowledge of the one skilled in the art.

[0388] In this regard, the one skilled in the art may advantageously monitor the molecular weight of the obtained hyaluronic acid on an agarose gel.

[0389] Preferred polypeptide having hyaluronidase activity in the present specification is an enzyme having an EC number of n° EC 3.2.1.35.

[0390] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may originate or be derived from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may originate or be derived from archaebacteria. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may originate or be derived from organisms preferably selected from yeasts. In some other preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may originate or be derived from Cupiennius salei (Csa), Loxosceles intermedia (Li), Hirudo nipponia (Hn), Bothrops atrox (Ba), Tityus serrulatus (Ts) or Vespa magnifica (Vm), and in particular from Cupiennius salei (Csa), Loxosceles intermedia (Li), Hirudo nipponia (Hn), Bothrops atrox (Ba) and Tityus serrulatus (Ts).

[0391] According to a yet 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%, preferably at least 80%, nucleic acid identity with a 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 a biological activity of the same nature 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 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) encode a polypeptide having hyluronidase activity, comprise a secretion signal and no anchoring signal, and originate or derive from Bothrops atrox, Loxosceles intermedia, Cupiennius salei, Tityus serrulatus, Hirudo nipponia or Vespa magnifica, respectively.

[0392] 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%, preferably at least 80%, nucleic acid identity with a 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 a biological activity of the same nature 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 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) encode a polypeptide having hyluronidase activity, comprise a secretion signal and an anchoring signal, and originate or derive from Cupiennius salei, Loxosceles intermedia, Hirudo nipponia, Bothrops atrox, Tityus serrulatus or Vespa magnifica, respectively.

[0393] In a particular 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 (i) having at least 65%, advantageously at least 70%, preferably at least 80%, nucleic acid identity with a 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) having a biological activity of the same nature 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).

[0394] In a particular 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 (i) at least 65%, advantageously at least 70%, preferably at least 80%, nucleic acid identity with a 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) having a biological activity of the same nature 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).

[0395] A biological activity of the same nature regarding this sequence is, as previously explained, the capacity to code for a polypeptide that catalyzes the degradation of hyaluronic acid molecules into smaller hyaluronic acid molecules.

[0396] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0397] As described herein, a nucleic acid sequence having at least 70% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0398] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses 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 the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0399] For the amino acid sequence of the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity from Cupiennius salei. Loxosceles intermedia, Hirudo nipponia. Bothrops atrox or Tityus serrulatus, the one skilled in the art may refer to the accession numbers A0A0S4JYH2, R4J7Z9, X4Y2L4, A0A2H4Z8F4 or P85841, respectively in the UniProt database, or to 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.

[0400] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be nucleic acid(s) encoding 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 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) which comprise a secretion signal and no anchoring signal, and also a biological activity of the same nature as the amino acid sequence 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).

[0401] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be nucleic acid(s) encoding polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 55%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO: 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) which comprise a secretion signal and an anchoring signal, and also a biological activity of the same nature 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).

[0402] In a particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be nucleic acid(s) encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 55%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO: 33 (Csa), SEQ ID NO: 37 (Li), SEQ ID NO: 35 (Hn), SEQ ID NO: 31 (Ba), or SEQ

[0403] ID NO: 39 (Ts), and also a biological activity of the same nature as the amino acid sequence of

[0404] SEQ ID NO: 33 (Csa), SEQ ID NO: 37 (Li), SEQ ID NO: 35 (Hn), SEQ ID NO: 31 (Ba), or SEQ

[0405] ID NO: 39 (Ts). In another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity may be nucleic acid(s) encoding a polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 55%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO: 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 a biological activity of the same nature 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).

[0406] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the degradation of hyaluronic acid molecules into smaller hyaluronic acid molecules.

[0407] As described herein, an amino acid sequence having at least 55% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0408] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses 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 said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0409] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0410] As above-mentioned, the expression level of the 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 herein after defined more in details, which are present in 5’ and 3’ position respectively of the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity.

[0411] GLUTAMINE SYNTHETASE (GLN1)

[0412] The glutamine synthetase enzyme is a protein which is described in the art for catalyzing the conversion of glutamate into glutamine. The glutamine synthetase originating from Saccharomyces cerevisiae may be termed GLN1.

[0413] A method implemented to measure the activity level of a polypeptide having glutamine synthetase activity belongs to the general knowledge of the one skilled in the art.

[0414] In this regard, the one skilled in the art may advantageously refer to the method described by Legrain et al. (1982) European Journal of Biochemistry 123, 611-616.

[0415] Preferred polypeptide having glutamine synthetase activity in the present invention is an enzyme having an EC number of n° EC 6.3.1.2.

[0416] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity may originate or be derived from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity may originate or be derived from archaebacteria. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity may originate or be derived from a yeast, and especially from Saccharomyces cerevisiae.

[0417] According to a yet preferred embodiment, 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%, preferably at least 80%, nucleic acid identity with the nucleic acid sequence as set forth as sequence SEQ ID NO: 96 (Sc), and (ii) a biological activity of the same nature as the nucleic acid sequence as set forth as sequence SEQ ID NO: 96 (Sc). The nucleic acid as set forth as sequence SEQ ID NO: 96 encodes a polypeptide having glutamine synthetase activity originating from Saccharomyces cerevisiae, that may also be termed GLN 1. A biological activity of the same nature regarding this sequence is, as previously explained, the capacity to code for a polypeptide that converts glutamate into glutamine, in particular through the consumption of one ATP and one NH .

[0418] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0419] As described herein, a nucleic acid sequence having at least 70% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0420] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses 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 the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0421] For the amino acid sequence of the polypeptide having glutamine synthetase activity originating from Saccharomyces cerevisiae. the one skilled in the art may refer to the accession number P32288 in the UniProt database, or to the sequence SEQ ID NO: 97 described herein.

[0422] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity may be nucleic acid(s) encoding 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 a biological activity of the same nature as the amino acid sequence of SEQ ID NO: 97.

[0423] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of glutamate into glutamine. As described herein, an amino acid sequence having at least 35% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having 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% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0424] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses 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 said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0425] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0426] As above-mentioned, the expression level of the polypeptide having glutamine synthetase activity in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5’ and 3’ position respectively of one or more recombinant nucleic acids encoding a polypeptide having glutamine synthetase activity.

[0427] GLUTAMATE SYNTHASE (GLT1)

[0428] The glutamate synthase enzyme is a protein which is described in the art for catalyzing the conversion of glutamine into glutamate. The glutamate synthase originating from Saccharomyces cerevisiae may be termed GLT1.

[0429] A method implemented to measure the activity level of a polypeptide having glutamate synthase activity belongs to the general knowledge of the one skilled in the art.

[0430] In this regard, the one skilled in the art may advantageously refer to the method described by Roon et al. (1974) Journal of bacteriology 118,89-95. Preferred polypeptide having glutamate synthase activity in the present invention is an enzyme having an EC number of n° EC 1.4.1.14 (SEQ ID NO: 98).

[0431] For the amino acid sequence of the polypeptide having glutamate synthase activity from Saccharomyces cerevisiae. the one skilled in the art may refer to the accession number QI 2680 in the UniProt database, or to the sequence SEQ ID NO: 99 described herein.

[0432] As above-mentioned, the expression level of a polypeptide having glutamate synthase activity may be reduced in a recombinant cell, and in particular in a recombinant yeast according to the invention as compared to the said cell, and in particular yeast, in its non-recombined form, i.e. at least one endogeneous gene of the recombinant cell, and in particular of the recombinant yeast, is disrupted.

[0433] GLUTAMINE-FRUCTOSE-6-PHOSPHATE AMIDOTRANSFERASE (GFA1)

[0434] The glutamine-fructose-6-phosphate amidotransferase enzyme is a protein which is described in the art for catalyzing the conversion of fructose-6-phosphate into glucosamine-6-phosphate. The glutamine-fructose-6-phosphate amidotransferase originating from Saccharomyces cerevisiae may be termed GFA1.

[0435] A method implemented to measure the activity level of a polypeptide having glutamine-fructose- 6-phosphate amidotransferase activity belongs to the general knowledge of the one skilled in the art.

[0436] In this regard, the one skilled in the art may advantageously refer to the method described by Shiga Shibatan and Hiroaki Kitazawa (Plant Biotechnology 26, 149-152, 2009).

[0437] Preferred polypeptide having glutamine-fructose-6-phosphate amidotransferase activity in the present invention is an enzyme having an EC number of n° EC 2.6.1.16.

[0438] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may originate or be derived from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may originate or be derived from archaebacteria. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may originate or be derived from organisms preferably selected from Bacillus subtilis, and yeasts. In some other preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may originate or be derived from a yeast, and especially from Saccharomyces cerevisiae.

[0439] According to a yet preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity may be selected from the group consisting of nucleic acid sequences having (i) at least 65%, advantageously at least 70%, preferably at least 80%, nucleic acid identity with the nucleic acid sequence as set forth as sequence SEQ ID NO: 47 (Sc), and (ii) a biological activity of the same nature as the nucleic acid sequence as set forth as sequence SEQ ID NO: 47 (Sc). The nucleic acid as set forth as sequence SEQ ID NO: 47 encodes a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity originating from Saccharomyces cerevisiae, that may also be termed GFA1.

[0440] 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%, preferably at least 80%, nucleic acid identity with the nucleic acid sequence as set forth as sequence SEQ ID NO: 48 or SEQ ID NO: 49, and (ii) a biological activity of the same nature as the nucleic acid sequence as set forth as sequence SEQ ID NO: 48 or SEQ ID NO: 49. The nucleic acid sequence as set forth as sequence SEQ ID NO: 48 or SEQ ID NO: 49 encodes a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity originating from Chlor ella virus PBCV1.

[0441] A biological activity of the same nature regarding this sequence is, as previously explained, the capacity to code for a polypeptide that converts fructose-6-phosphate into glucosamine-6- phosphate.

[0442] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0443] As described herein, a nucleic acid sequence having at least 70% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0444] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses 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 the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0445] For the amino acid sequence of the polypeptide having glutamine-fructose-6-phosphate amidotransferase activity originating from Saccharomyces cerevisiae. the one skilled in the art may refer to the accession number NP8012818 in the UniProt database, or to the sequence SEQ ID NO: 50 described herein.

[0446] For the amino acid sequence of the polypeptide having glutamine-fructose-6-phosphate amidotransferase activity originating from Chlorella virus PBCV1, the one skilled in the art may also refer to the accession number NP 048448 in the UniProt database, or to the sequence SEQ ID NO: 51 described herein.

[0447] 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 acid(s) encoding 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 with SEQ ID NO: 51, and also a biological activity of the same nature as the amino acid sequence of SEQ ID NO: 50 or with SEQ ID NO: 51.

[0448] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of fructose-6-phosphate into glucosamine-6-phosphate.

[0449] As described herein, an amino acid sequence having at least 35% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having 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% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0450] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses 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 said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0451] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0452] As above-mentioned, the expression level of the 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 herein after defined more in details, which are present in 5’ and 3’ position respectively of one or more recombinant nucleic acids encoding a polypeptide having glutamine-fructose-6-phosphate amidotransferase activity.

[0453] UDP-N-ACETYLGLUCOSAMINE PYROPHOSPHORYLASE (QRI1)

[0454] The UDP-N-acetylglucosamine pyrophosphorylase enzyme is a protein which is described in the art for catalyzing the conversion of N-acetyl-glucosmine-6-phosphate into UDP-N-acetyl-glucose. The UDP-N-acetylglucosamine pyrophosphorylase originating from Saccharomyces cerevisiae may be termed QRI1.

[0455] A method implemented to measure the activity level of a polypeptide having UDP-N- acetylglucosamine pyrophosphorylase activity belongs to the general knowledge of the one skilled in the art.

[0456] In this regard, the one skilled in the art may advantageously refer to the method described by Mio et al. (The Journal of Biological Chemistry, Col. 273, No 23, June 5, 1998, 14392-14397) except that UDP-N-acetyl-glucosamine is detected by LC MS / MS using a Synergi RP Fusion column. Preferred polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity in the present invention is an enzyme having an EC number of n° EC 2.7.7.23

[0457] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may originate or be derived from organisms preferably selected in a group consisting of prokaryotic organisms and eukaryotic organisms. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may originate or be derived from archaebacteria. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may originate or be derived from organisms preferably selected from Bacillus subtilis, and yeasts. In some other preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may originate or be derived from yeasts, and especially from Saccharomyces cerevisiae.

[0458] According to a yet preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may be selected from the group consisting of nucleic acid sequences having (i) at least 65%, advantageously at least 70%, preferably at least 80%, nucleic acid identity with a nucleic acid sequence as set forth as SEQ ID NO: 52, and (ii) a biological activity of the same nature as the nucleic acid sequence as 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, that may also be termed QRI1.

[0459] A biological activity of the same nature regarding this sequence is, as previously explained, the capacity to code for a polypeptide that converts N-acetyl-glucosmine-6-phosphate into UDP-N- acetyl-glucose.

[0460] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence. As described herein, a nucleic acid sequence having at least 70% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0461] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses 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 the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0462] For the amino acid sequence of the polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity originating from Saccharomyces cerevisiae. the one skilled in the art may refer to the accession number NP 010180 in the UniProt database, or to SEQ ID NO. 53 described herein.

[0463] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity may be nucleic acid(s) encoding polypeptide having an amino acid sequence selected from the group consisting of sequences having at least 35%, advantageously at least 45%, preferably at least 80%, amino acid identity with the amino acid sequence set forth as sequence SEQ ID NO. 53, and also a biological activity of the same nature as the amino acid sequence set forth as sequence SEQ ID NO. 53.

[0464] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of of N-acetyl-glucosmine-6-phosphate into UDP-N-acetyl- glucose.

[0465] As described herein, an amino acid sequence having at least 35% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having 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% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0466] As described herein, an amino acid sequence having at least 45% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having 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% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0467] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0468] As above-mentioned, the expression level of the 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 herein after defined more in details, which are present in 5’ and 3’ position respectively of the one or more recombinant nucleic acids encoding a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase activity.

[0469] PHOSPHOGLUCOMUTASE-1 (PGM1)

[0470] The Phosphoglucomutase- 1 enzyme is a protein which is described in the art for catalyzing the conversion of Glucose-6-phosphate into Glucose- 1 -phosphate. The Phosphoglucomutase- 1 originating from Saccharomyces cerevisiae may be termed PGM1.

[0471] A method implemented to measure the activity level of a polypeptide facing Phosphoglucomutase- 1 activity belongs to the general knowledge of the one skilled in the art.

[0472] In this regard, the one skilled in the art may advantageously refer to the method described by Tiwari and Bhat (Biochemical and Biophysical Research Communications 366, 340-345, 2008). Preferred polypeptide having Phosphoglucomutase- 1 activity in the present invention is an enzyme having an EC number of n° 5.4.2.2. According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 activity may originate from organisms preferably selected in a group consisting of prokaryotic organisms and eukaryotic organisms. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 activity may originate or be derived from archaebacteria. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 activity may originate or be derived from organisms preferably selected from bacteria. In a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 activity may originate or be derived from Saccharomyces cerevisiae.

[0473] According to an embodiment, the one or more recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 activity may be selected from the group consisting of nucleic acid sequences having (i) at least 80% nucleic acid identity with the nucleic acid sequence set forth as sequence SEQ ID NO: 54, which originates from Saccharomyces cerevisiae, and (ii) a biological activity of the same nature as the nucleic acid sequence set forth as sequence SEQ ID NO: 54.

[0474] A biological activity of the same nature regarding this sequence is, as previously explained, the capacity to code for a polypeptide that converts Glucose-6-phosphate into Glucose- 1 -phosphate. As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses 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 the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0475] For the amino acid sequence of the peptide having Phosphoglucomutase- 1 activity from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number NP33401 in the UniProt database, or to SEQ ID NO: 55 described herein.

[0476] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 activity may be nucleic acid(s) encoding polypeptide having an amino acid sequence selected from the group consisting of sequences having at least least 80% amino acid identity with the amino acid sequence set forth as sequence SEQ ID NO: 55, and also a biological activity of the same nature as the amino acid sequence set forth as sequence SEQ ID NO: 55. A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of Glucose-6-phosphate into Glucose- 1 -phosphate.

[0477] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0478] As above-mentioned, the expression level of the 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 herein after defined more in details, which are present in 5’ and 3’ position respectively of the one or more recombinant nucleic acids encoding a polypeptide having Phosphoglucomutase- 1 activity.

[0479] UTP-GLUCOSE-l-PHOSPHATE URIDYL YLTRANSFERASE (UGP1)

[0480] The UTP-glucose-1 -phosphate uridylyltransferase enzyme is a protein which is described in the art for catalyzing the conversion of Glucose- 1 -Phosphate into UDP-glucose. The UTP-glucose-1 - phosphate uridylyltransferase originating from Saccharomyces cerevisiae may be termed UGP1. A method implemented to measure the activity level of a polypeptide having UTP-glucose-1 - phosphate uridylyltransferase activity belongs to the general knowledge of the one skilled in the art.

[0481] In this regard, the one skilled in the art may advantageously refer to the method described by Roeben (J. Mol. Biol 364, 551-560, 2006).

[0482] Preferred polypeptide having UTP-glucose-1 -phosphate uridylyltransferase activity in the present invention is an enzyme having an EC number of n° 2.7.7.9.

[0483] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase activity may originate or be derived from organisms preferably selected in a group consisting of prokaryotic organisms and eukaryotic organisms. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase activity may originate or be derived from archaebacteria. In some embodiments, the one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase activity may originate or be derived from organisms preferably selected from bacteria. In a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UTP- glucose-1 -phosphate uridylyltransferase activity may originate or be derived from Saccharomyces cerevisiae.

[0484] According to a particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase activity may be selected from the group consisting of nucleic acid sequences having (i) at least 80% nucleic acid identity with a nucleic acid as set forth as sequence SEQ ID NO: 56, originating from Saccharomyces cerevisiae, and (ii) a biological activity of the same nature as the nucleic acid as set forth as sequence SEQ ID NO: 56.

[0485] A biological activity of the same nature regarding this sequence is, as previously explained, the capacity to code for a polypeptide that converts Glucose- 1 -phosphate into UDP-glucose.

[0486] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses 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 the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0487] For the amino acid sequence of the polypeptide having UTP-glucose-1 -phosphate uridylyltransferase activity originating from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number NP_32861 in the UniProt database, or to the sequence set forth as sequence SEQ ID NO: 57 described herein.

[0488] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase activity may be nucleic acid(s) encoding 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 a biological activity of the same nature as the amino acid sequence set forth as SEQ ID NO: 57.

[0489] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of Glucose- 1 -phosphate into UDP-glucose.

[0490] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0491] As above-mentioned, the expression level of the 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 herein after defined more in details, which are present in 5’ and 3’ position respectively of the one or more recombinant nucleic acids encoding a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase activity.

[0492] GLUCOSAMINE-6-PHOSPHATE N-ACETYLTRANSFERASE (GNA1)

[0493] The Glucosamine-6-phosphate N-acetyltransferase enzyme is a protein which is described in the art for catalyzing the conversion of Glucosamine-6-phosphate into N-acetyl-glucosamine-6- phosphate. The Glucosamine-6-phosphate N-acetyltransferase originating from Saccharomyces cerevisiae may be termed GNAl.

[0494] A method implemented to measure the activity level of a polypeptide having Glucosamine-6- phosphate N-acetyltransferase activity belongs to the general knowledge of the one skilled in the art.

[0495] In this regard, the one skilled in the art may advantageously refer to the method described by Li et al. (Anal. Biochem. 370, 142-146, 2007).

[0496] Preferred polypeptide having Glucosamine-6-phosphate N-acetyltransferase activity in the present invention is an enzyme having an EC number of n° 2.3.1.4.

[0497] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having Glucosamine-6-phosphate N-acetyltransferase activity may originate or be derived from organisms preferably selected in a group consisting of prokaryotic organisms and eukaryotic organisms. In some preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having Glucosamine-6-phosphate N-acetyltransferase activity may originate or be derived from a yeast, and especially from Saccharomyces cerevisiae.

[0498] According to a particular embodiment, 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 a nucleic acid sequence as set forth as sequence SEQ ID NO: 58, and (ii) a biological activity of the same nature as the nucleic acid sequence as set forth as sequence SEQ ID NO: 58. The nucleic acid sequence as set forth as sequence SEQ ID NO: 58 encodes a polypeptide having Glucosamine-6-phosphate N-acetyltransferase activity originating from Saccharomyces cerevisiae. that may also be termed GNA1.

[0499] A biological activity of the same nature regarding this sequence is, as previously explained, the capacity to code for a polypeptide that converts Glucosamine-6-phosphate into N-acetyl- glucosamine-6-phosphate.

[0500] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses 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 the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0501] For the amino acid sequence of the polypeptide having Glucosamine-6-phosphate N- acetyltransferase activity originating from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number NP_116637 in the UniProt database, or to SEQ ID NO. 59 described herein.

[0502] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having Glucosamine-6-phosphate N-acetyltransferase activity may be nucleic acid(s) encoding 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 a biological activity of the same nature as the amino acid sequence set forth as SEQ ID NO. 59.

[0503] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of Glucosamine-6-phosphate into N-acetyl-glucosamine-6- phosphate.

[0504] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0505] As above-mentioned, the expression level of the 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 herein after defined more in details, which are present in 5’ and 3’ position respectively of the one or more recombinant nucleic acids encoding a polypeptide having Glucosamine-6-phosphate N- acetyltransferase activity.

[0506] PHOSPHOACETYLGLUCOSAMINE MUTASE (PCM1)

[0507] The phosphoacetylglucosamine mutase enzyme is a protein which is described in the art for catalyzing the conversion of N-acetyl-glucosamine-6-phosphate into N-acetyl-glucosamine-1- phosphate. The phosphoacetylglucosamine mutase originating from Saccharomyces cerevisiae may be termed PCM1.

[0508] A method implemented to measure the activity level of a polypeptide having phosphoacetylglucosamine mutase activity belongs to the general knowledge of the one skilled in the art.

[0509] In this regard, the one skilled in the art may advantageously refer to the method described by Bandini et al. (Molecular Microbiology 85(3), 513-534, 2012).

[0510] Preferred polypeptide having phosphoacetylglucosamine mutase activity in the present invention is an enzyme having an EC number of n° 5.4.2.3.

[0511] According to a preferred embodiment, the one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may originate or be derived from organisms preferably selected in a group consisting of prokaryotic organisms and eukaryotic organisms. In some preferred embodiments, the one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may originate or be derived from a yeast, and especially from Saccharomyces cerevisiae.

[0512] According to a particular embodiment, 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 a nucleic acid sequence set forth as SEQ ID NO: 60, and (ii) a biological activity of the same nature 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, that may also be termed PCM1. A biological activity of the same nature regarding this sequence is, as previously explained, the capacity to code for a polypeptide that converts N-acetyl-glucosamine-6-phosphate into N-acetyl- glucosamine-1 -phosphate.

[0513] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses 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 the said reference nucleic acid sequence, and also a biological activity of the same nature as the said reference nucleic acid sequence.

[0514] For the amino acid sequence of the polypeptide having phosphoacetylglucosamine mutase activity originating from Saccharomyces cerevisiae. the one skilled in the art may refer to the accession number NP_010856 in the UniProt database, or to SEQ ID NO. 61 described herein.

[0515] According to another particular embodiment, the one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity may be nucleic acid(s) encoding 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 a biological activity of the same nature as the amino acid sequence set forth as SEQ ID NO. 61.

[0516] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of N-acetyl-glucosamine-6-phosphate into N-acetyl- glucosamine-1 -phosphate.

[0517] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature as the said reference amino acid sequence.

[0518] As above-mentioned, the expression level of the 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 herein after defined more in detail, which are present in 5’ and 3’ position respectively of the one or more recombinant nucleic acids encoding a polypeptide having phosphoacetylglucosamine mutase activity. PROMOTERS

[0519] As disclosed herein, the expression of the genes of interest that have been genetically engineered for obtaining a recombinant cell according to the invention comprise appropriate regulatory sequences that are functional in the recombinant cells of the invention, and in particular in recombinant yeast cells of the invention, including in particular Saccharomyces cerevisiae.

[0520] Various promoters may be used for the desired expression of the coding sequences of interest.

[0521] Promoters according to the invention can be selected from the group consisting of the following promoters:

[0522] • pTDH3 (SEQ ID NO: 62),

[0523] • pTDH3.sk (SEQ ID NO: 63),

[0524] • pTDH3-l.Sba (SEQ ID NO: 64),

[0525] • pTDH3.Sar (SEQ ID NO: 65),

[0526] • pENO2 (SEQ ID NO: 66),

[0527] • pTEF3 (SEQ ID NO: 67),

[0528] • pTEFl (SEQ ID NO: 68),

[0529] • pTEFl .ago (SEQ ID NO: 69),

[0530] • pTEFl .sba (SEQ ID NO: 70),

[0531] • pPDCl (SEQ ID NO: 71),

[0532] • pCCW12 (SEQ ID NO: 72),

[0533] • pCCW12.Sm (SEQ ID NO: 73),

[0534] • pCCW12.sk (SEQ ID NO: 74),

[0535] • pCCW12.sba (SEQ ID NO: 75),

[0536] • pCCW12.sar (SEQ ID NO: 76),

[0537] • pNUP57 (SEQ ID NO: 77),

[0538] • pCCWlO.ago (SEQ ID NO: 78),

[0539] • pCWP2 (SEQ ID NO: 79),

[0540] • pRPLAl (SEQ ID NO: 80), and

[0541] • pCCW12.Spr (SEQ ID NO: 109).

[0542] Promoters more particularly interesting in the present invention may be selected from the group consisting of: pTDH3 (SEQ ID NO: 62), • pTDH3.sk (SEQ ID NO: 63),

[0543] • pTDH3-l.Sba (SEQ ID NO: 64),

[0544] • pTDH3.Sar (SEQ ID NO: 65),

[0545] • pENO2 (SEQ ID NO: 66),

[0546] • pTEF3 (SEQ ID NO: 67),

[0547] • pTEFl (SEQ ID NO: 68),

[0548] • pTEFl. ago (SEQ ID NO: 69),

[0549] • pTEFl .sba (SEQ ID NO: 70),

[0550] • pPDCl (SEQ ID NO: 71),

[0551] • pCCW12 (SEQ ID NO: 72),

[0552] • pCCW12.Sm (SEQ ID NO: 73),

[0553] • pCCW12.sk (SEQ ID NO: 74),

[0554] • pCCW12.sba (SEQ ID NO: 75),

[0555] • pCCW12.sar (SEQ ID NO: 76), and

[0556] • pCCW12.Spr (SEQ ID NO: 109).

[0557] Said promoters can in particular be selected from the group consisting of pTDH3, pTDH3-l.Sba, pTDH3.Sar, pENO2, pTEF3, pTEFl, pPDCl, pCCW12, pCCW12.Sm, pCCW12.sk, pCCW12.sba, and pCCW12.sar.

[0558] Alternatively, promoters interesting in the present invention may be selected from the group consisting of:

[0559] • pNUP57 (SEQ ID NO: 77), and

[0560] • pCCWlO.ago (SEQ ID NO: 78).

[0561] Other promoters of interest in the present invention may be:

[0562] • pCWP2 (SEQ ID NO: 79); and

[0563] • pRPLAl (SEQ ID NO: 80).

[0564] As previously mentioned, inducible or repressible promoters are promoters whose activity is controlled by the presence or absence of biotic or abiotic factors and also by the quantity of said factor. Accordingly, for some promoters, their activity will in particular be induced and thus increased when the quantity of a given factor increases or is increased, and, accordingly, the activity of these same promoters can be repressed and thus reduced when the quantity of said factor diminishes or is reduced. The quantity of said factor(s) in the culture medium of a recombinant yeast cell of the invention comprising inducible or repressible promoters can be decided and thus controlled by the man skilled in the art.

[0565] For example, increasing the quantity of copper in a culture medium of a recombinant yeast cell according to the invention comprising a pCUP-1 promoter will induce and thus increase transcription of the gene under the control of this promoter. On the contrary, reducing the quantity of copper in said culture medium will lead to a repression, and thus a reduced, transcription of the gene under the control of this promoter.

[0566] In another example, increasing the quantity of methionine in a culture medium of a recombinant yeast cell according to the invention comprising a pMET6 promoter will repress and thus decrease transcription of the gene under the control of this promoter. On the contrary, reducing the quantity of methionine in said culture medium will lead to an induced, and thus an increased, transcription of the gene under the control of this promoter.

[0567] For this reason, the following promoters are referred to in the present text as being inducible or repressible promoters.

[0568] According to a first embodiment, inducible or repressible promoters according to the invention may be selected from the group comprising promoters inducible or repressible with copper, promoters inducible or repressible with methionine and promoters inducible or repressible with threonine, and are in particular is CUP1 - copper inducible or repressible (SEQ ID NO: 81).

[0569] According to this embodiment, the inducible or repressible promoter according to the invention can in particular be pCUPl .

[0570] The activity of these promoters is thus induced by the increasing presence of methionine, copper or threonine as indicated above, and their activity diminishes, i.e. is repressed, when the quantity of methionine, copper or threonine is reduced.

[0571] According to a second embodiment, inducible or repressible promoters according to the invention may be selected from the group comprising promoters inducible or repressible with copper, promoters inducible or repressible with lysine and promoters inducible or repressible with methionine, and in particular selected from the group consisting of:

[0572] • pMET6 - methionine inducible or repressible (SEQ ID NO: 82),

[0573] • pMET25 - methionine inducible or repressible (SEQ ID NO: 83), and

[0574] • pSAMl - methionine inducible or repressible (SEQ ID NO: 84), 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 pSAMl.

[0575] The activity of these promoters is thus repressed by the increasing presence of methionine, copper, lysine or glucose as indicated above, and their activity increases, i.e. is induced, when the quantity of methionine, copper, lysine or glucose is reduced.

[0576] In a particular embodiment, inducible or repressible promoters according to the invention may be selected from the group comprising promoters inducible or repressible with copper, promoters inducible or repressible with glucose, promoters inducible or repressible with lysine, promoters inducible or repressible with methionine and promoters inducible or repressible with threonine.

[0577] In a more particular embodiment, the inducible or repressible promoter according to the invention may be selected from the group consisting of pCUPl, pMET6, pSAMl, and pMET25.

[0578] Synthetic promoters as described in Blazeck & Alper (2013) Biotechnol. J. 8 46-58 can also be used.

[0579] The promoters of the invention can originate from any organism from the Saccharomycetes class and can in particular originate from an organism selected from the group consisting of at least one of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces castelii, 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, .

[0580] The promoters of the invention can preferably originate from an organism selected from the group consisting of Saccharomyces cerevisiae (sc), Saccharomyces mikatae (Sm), Saccharomyces kudriavzevii (sk), Saccharomyces bayanus (sba), and Saccharomyces arboricola (Sar).

[0581] TERMINATORS

[0582] As it is disclosed herein, the expression of the genes of interest that have been genetically engineered for obtaining a recombinant cell according to the invention, and in particular a recombinant yeast according to the invention, comprise appropriate transcription terminator sequences that are functional in recombinant cells of the invention, and in particular in recombinant yeast cells of the invention, in particular in Saccharomyces cerevisiae.

[0583] Said transcription terminators, identical or different, may be found in literature Yamanishi et al, (2013) ACS synthetic biology 2, 337-347. Terminators more particularly interesting in the present invention may be selected from the group comprising:

[0584] • tTPIl from the gene encoding for the Triose Phosphate Isomerase (SEQ ID NO: 85),

[0585] • tMET25 from the gene encoding for the O-acetyl homoserine-O-acetyl serine sulfhydrylase (SEQ ID NO: 86),

[0586] • tDITl (SEQ ID NO: 87),

[0587] • tRPL3 (SEQ ID NO: 88),

[0588] • tRPL3.sm (SEQ ID NO: 89),

[0589] • tRPL3.sba (SEQ ID NO: 90),

[0590] • tRPL41B (SEQ ID NO: 91),

[0591] • tRPL15A (SEQ ID NO: 92),

[0592] • tRPL15A.sba (SEQ ID NO: 93),

[0593] • tIDPI (SEQ ID NO: 94),

[0594] • tTEFl.sba (SEQ ID NO: 95), and

[0595] • tTDH3 (SEQ ID NO: 100).

[0596] In particular, said terminator may be selected from the group consisting of tTPIl, tMET25, tDITl, tRPL3, tRPL3.sm, tRPL3.sba, tRPL41B, tRPL15A, tRPL15A.sba, tIDPI, tTEFl.sba and tTDH3. The terminators of the invention can originate from any organism from the Saccharomycetes class and can in particular originate from an organism selected from the group consisting of Saccharomyces cerevisiae and Saccharomyces Bayanus.

[0597] RECOMBINANT CELLS

[0598] Recombinant cells of the invention can be selected from the group consisting of yeasts and bacteria.

[0599] Recombinant cells of the invention, such as a recombinant host cell of the invention, are preferably recombinant yeast cells.

[0600] Generally, yeast can grow rapidly and can be cultivated at higher density as compared with bacteria, and does not require an aseptic environment in the industrial setting. Furthermore, yeast cells can be more easily separated from the culture medium compared to bacterial cells, greatly simplifying the process for product extraction and purification.

[0601] A recombinant cell of the invention, and in particular a recombinant yeast cell of the invention is preferably a Saccharomycetales cell. A recombinant cell of the invention, and in particular a recombinant yeast of the invention can in particular belong to the Saccharomyces genus, or to the Candida genus, or to the Kluyveromyces genus, or to the Ogataea genus, or to the Yarrowia genus, or to the Debaryomyces genus, or to the Ashbya genus.

[0602] A recombinant cell of the invention belonging to the Saccharomyces genus can be selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces castelli, Saccharomyces cariocanus, Saccharomyces kudriavzevii, Saccharomyces arboricolus, Saccharomyces pastorianus, Saccharomyces uvarum and Saccharomyces delbrueckii.

[0603] A recombinant cell of the invention belonging to the Candida genus can be selected from the group consisting of Candida albicans, Candida glabrata, Candida tropicalis, Candida dubliniensis, Candida parapsilosis, Candida lusitaniae and Candida guilliermondii.

[0604] A recombinant cell of the invention belonging to the Kluyveromyces genus can be selected from the group consisting of Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus, Kluyveromyces thermotoleren, Kluyveromyces dobzhanskii and Kluyveromyces wickerhamii.

[0605] A recombinant cell of the invention belonging to the Ogataea genus can 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.

[0606] A recombinant cell of the invention belonging to the Yarrowia genus can be selected from the group consisting of Yarrowia lypolytica, Yarrowia parophonii, Yarrowia galli, Yarrowia oslonensis, Yarrowia alimentaria, Yarrowia hollandica and Yarrowia yakushimensis.

[0607] A recombinant cell of the invention belonging to the Debaryomyces genus can be selected from the group consisting of Debaryomyces hansenii, Debaryomyces carsonii, Debaryomyces castellii, Debaryomyces marama, Debaryomyces occidentalis, Debaryomyces oviformis, Debaryomyces nepalensis, Debaryomyces coudertii, Debaryomyces udenii, Debaryomyces psychrosporus and Debaryomyces yamadae.

[0608] A recombinant cell of the invention belonging to the Ashbya genus can be selected from the group consisting of Ashbya gossypii and Ashbya aceri. A recombinant cell of the invention, and in particular a recombinant yeast cell of the invention, can in particular be 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, Kluyveromyces thermotolerens, Ogataea polymorpha, Yarrowia lypolytica, Debaryomyces hansenii, and Ashbya gossypii, and is preferably Saccharomyces cerevisiae.

[0609] In a particular embodiment, the recombinant yeast cell according to the invention is of the genus Saccharomyces, Kluyveromyces, or Eremothecium, more particularly is of the species selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces Marxianus, Ogataea polymorpha and Ashbya gossypii.

[0610] In an embodiment, a recombinant host cell of the invention is a yeast selected belonging to the Saccharomycetales order, in particular to the Saccharomycetaceae family, and is in particular selected from the group consisting of Yarrowia lipolitica, Kluyveromyces Marxianus, Ogataea polymorpha, Ashbya gossypii and Saccharomyces cerevisiae.

[0611] A recombinant cell of the invention can most preferably be a Saccharomyces cerevisiae cell.

[0612] As above-mentioned, a recombinant cell according to the invention has the ability to produce hyaluronic acid by insertion of the one or more recombinant nucleic acids according to the present invention. In certain embodiments, a recombinant yeast according to the invention has the ability to produce hyaluronic acid having a controlled size (controlled molecular weight) by insertion of the one or more recombinant nucleic acids according to the present invention.

[0613] Methods implemented to insert a specific DNA construct within a gene belong to the general knowledge of a man skilled in the art. A related method is described in more details in the herein after examples.

[0614] However, unexpected technical difficulties were encountered because the consequences of the insertion of DNA constructs within the genome of a cell, and in particular within the genome of a yeast, such as for example in the genome of Saccharomyces cerevisiae, are unpredictable. In particular, the survival rate of the cells, and in particular of the yeasts, and their ability to grow and produce the desired hyaluronic acid is also unpredictable. In order to obtain a recombinant cell, and in particular a recombinant yeast, of the invention, many different constructs were tested by the inventors in order to obtain a viable and efficient recombinant cell, and in particular yeast.

[0615] CULTURE CONDITIONS

[0616] The present invention also relates to the use of a recombinant cell of the invention, for the production of low molecular weight hyaluronic acid, in particular the low molecular weight hyaluronic acid as described in the second aspect of the present invention.

[0617] The present invention further relates to a method of producing low molecular weight hyaluronic acid (HA) comprising:

[0618] (a) cultivating a recombinant cell of the present invention in a cultivation medium for a time sufficient to produce the low molecular weight hyaluronic acid; and

[0619] (b) optionally isolating or recovering the hyaluronic acid (HA) from the recombinant cell and / or from the cultivation medium.

[0620] Typically, cells of the invention, and in particular yeasts of the invention, are grown at a temperature in the range of about 20 °C to about 37 °C, preferably at a temperature ranging from 27 °C to 34°C, in an appropriate culture medium.

[0621] Suitable growth media for cells of the invention, and in particular for yeasts of the invention, are common commercially prepared media such as broth that includes yeast nitrogen base, ammonium sulfate, and dextrose as the carbon / energy source or YPD Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular cell, and in particular yeast, will be known by one skilled in the art of microbiology or fermentation science. A particular medium that is suitable herein is the SY medium which comprises the following elements:

[0622] KH2PO4: 100 mM; MgSO47H2O: 2,8 mM; K2SO4: 11,5 mM; Na2SO4: l,l mM; NaCl: 2,6 mM; CaCl22H2O: 0,7 mM; CuSO45H2O: 15 pM; KI: 6 pM; FeCl3: 30 pM; ZnSO47H2O: 61 pM; MnSO4H2O: 25 pM; H2SO4: 110 pM; Panthotenic Acids hemicalcium salt: 42 pM; Thiamin hydrochloride: 59 pM; Pyridoxin hydrochloride: 49 pM; Myo-Inositol (CeHnOe): 555 pM; Nicotinic acid (CeHsNCh): 29 pM; D-Biotine: 0,82 pM; Ammonium citrate tribasic: 33 mM; and glucose or sucrose 2-30%. Carbon sources that may 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 unpurified mixtures from renewable feedstocks such as cheese whey permeate comsteep liquor, sugar beet molasses, and barley malt.

[0623] Nitrogen sources that may be included in the culture medium include peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium citrate and combinations thereof.

[0624] The culture medium may further comprise trace elements (e.g., metal salts), for example magnesium salts, cobalt salts and / or manganese salts; as well as growth factors such as amino acids, vitamins, growth promoters, and the like.

[0625] Examples of vitamins that may be included are Panthotenic Acids hemicalcium, Thiamin hydrochloride; Pyridoxin hydrochloride; Myo-Inositol; Nicotinic acid; D-Biotine, folic acid, p- aminobenzoique acid, riboflavin.

[0626] A culture medium of the invention may further include rare elements, such as CuSO45H2O, KI, FeCl3, ZnSO47H2O, MnSO4H2O, or H2SO4, MgCl2, CaCl2, NaCl, K2HPO4, KH2PO4, ZnCl, H3BO3, MnSO4, Na2MoO4.

[0627] The term “appropriate culture medium” is above-defined.

[0628] Examples of known culture media for a recombinant cell according to the present invention are known to the person skilled in the art, and are presented in the following publication D. Burke et al., Methods in yeast Genetics - A cold spring harbor laboratory course Manual (2000).

[0629] Suitable pH ranges for the fermentation may be between pH 3.0 to pH 7.5, where pH 4 to pH 6 is preferred as the initial condition.

[0630] As mentioned elsewhere in this specification, the pH value of the culture medium can be regulated during the cultivation step of a method of the invention in order to modulate the activity of the polypeptide facing hyaluronidase activity, which will impact the molecular weight of the hyaluronic acid molecules produced by the recombinant cell of the invention, and in particular by the recombinant yeast of the invention.

[0631] In particular, the pH of the culture medium can be modified according to the hyaluronic acid that is meant to be produced by the recombinant yeast. For example, the pH of the culture medium may remain at a pH of 4, of 5,5 or of 6 during the culture time. In a particular embodiment, the pH of the culture medium may change or be changed during the time length of the culture of the recombinant cell of the invention, in particular of the recombinant yeast of the invention. As previously mentioned, Saccharomyces cerevisiae acidifies the medium in which it is cultivated and thus reduces the pH of its culture medium. For example, the pH of the culture medium may begin at 6, may be taken down to 4 and then brought back up to 6. In another example, the pH of the culture medium may begin at 6, remain or be maintained at 6, and then may be lowered to 4.

[0632] In a particular embodiment, the pH of the culture medium may be modulated during the cultivating step (a) of a method of the invention so that at the end of the cultivating step of a method of the invention, the pH of the cultivation medium is the same as the pH at the beginning of said cultivation step (a).

[0633] In another embodiment, the pH of the culture medium may remain the same during the time length of the culture of the recombinant cell of the invention, and in particular the recombinant yeast cell of the invention.

[0634] Said time length of the culture of a recombinant cell according to the invention, and in particular of the recombinant yeast of the invention, can vary depending on the molecular weight of the hyaluronic acid of interest. The longer said time length, the lower the molecular weight of hyaluronic acid in a given culture medium of a recombinant cell of the invention, and in particular a recombinant yeast cell of the invention.

[0635] The time length of the culture time of a recombinant cell of the invention, and in particular a recombinant yeast cell in the present invention can be a period of from about 35 hours to about 60 hours, preferably from about 40 hours to about 55 hours, and is in particular about 52 hours. Fermentations may be performed under aerobic conditions or micro-aerobic conditions.

[0636] The amount of hyaluronic acid product in the fermentation medium can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC) or gas chromatography (GC).

[0637] The present process may employ a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation, the medium is inoculated with the desired organism or organisms, and fermentation is permitted to occur without adding anything to the system. Typically, however, a "batch" fermentation method or system is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as temperature, pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly up to the time when the fermentation is stopped. Within batch cultures cells progress through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.

[0638] A Fed-Batch system may also be used in the present invention. A Fed-Batch system is similar to a typical batch system with the exception that the carbon source substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression (e.g. glucose repression) is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2.

[0639] Batch and Fed-Batch culturing methods are common and well known in the art and examples may be found in Biotechnology: A Textbook of Industrial Microbiology, Crueger, Crueger, and Brock, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA, or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36, 227, (1992). Although the present invention is performed in batch mode it is contemplated that the method would be adaptable to continuous fermentation.

[0640] Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.

[0641] 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 will 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 a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.

[0642] It is contemplated that the present invention may be practiced using either batch, fed- batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for production.

[0643] In order to still improve the 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 an appropriate culture medium, such as above-mentioned, wherein the said culture medium comprises an optimal amount of carbon source, especially glucose or sucrose.

[0644] In preferred embodiments, the carbon source comprised in said optimal culture medium consists of glucose and / or sucrose. In preferred embodiments, the said optimal culture medium comprises 1% w / w or more glucose and / or sucrose, in particular comprises 5% w / w or more glucose and / or sucrose, in particular comprises 10% w / w or more glucose and / or sucrose, in particular comprises 15% w / w or more glucose and / or sucrose. In preferred embodiments, the said optimal culture medium comprises at most 40% w / w glucose, which includes at most 35% w / w glucose.

[0645] In a preferred embodiment, the method of the invention is carried out on an industrial scale.

[0646] More particularly, the cultivation medium of the method according to the 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.

[0647] The invention further relates to a method for producing hyaluronic acid as previously described, and comprising the steps of:

[0648] (a) culturing a recombinant cell of the invention in a culture medium; and

[0649] (b) recovering the hyaluronic acid from said culture medium, wherein the hyaluronic acid recovered in step (b) has a molecular weight controlled through the selection of:

[0650] - the nature and origin of the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity of the recombinant cell of the invention, and in particular of the recombinant yeast of the invention, and / or the nature and origin of the promoter controlling the expression of the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity of the recombinant cell of the invention, and in particular of the recombinant yeast of the invention, and / or

[0651] - the presence or absence of an anchoring signal associated to the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity of the recombinant cell of the invention, and in particular of the recombinant yeast of the invention, and / or

[0652] - the pH of the culture medium during the step of culturing the recombinant cell of the invention, and in particular of the recombinant yeast of the invention, and / or

[0653] - the duration of the culturing of the recombinant cell of the invention, and in particular of the recombinant yeast of the invention.

[0654] The present invention also relates to the use of a recombinant cell according to the invention, and in particular a recombinant yeast cell of the invention, for the production of low molecular weight hyaluronic acid, e.g. as described in the second aspect of the present invention.

[0655] The one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity of a recombinant cell of the invention can for example be selected from those originating or being derived from at least one of Cupiennius salei (Csa), Loxosceles intermedia (Li), Hirudo nipponia (Hn), Bothrops atrox (Ba), Tityus serrulatus (Ts) or Vespa magnifica (Vm), in particular from at least one of Cupiennius salei (Csa), Loxosceles intermedia (Li), Hirudo nipponia (Hn), Bothrops atrox (Ba) or Tityus serrulatus (Ts), more particularly from those of sequences set forth as sequences 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).

[0656] The one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity of a recombinant cell of the invention can for example be selected from those originating or being derived from at least one of Cupiennius salei (Csa), Loxosceles intermedia (Li), Hirudo nipponia (Hn), Bothrops atrox (Ba), Tityus serrulatus (Ts) or Vespa magnifica (Vm), in particular from Cupiennius salei (Csa), Loxosceles intermedia (Li), Hirudo nipponia (Hn), Bothrops atrox (Ba) or Tityus serrulatus (Ts), in particular from those of sequences set forth as sequences 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). The one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity of a recombinant cell of the invention can for example be placed under the control of a promoter selected from the group consisting of pCCW12.Spr, pTEFl, pCCW12, pCCW12.sba, pCCW12.Sar, pPDCl, pTEF3, pTDH3, pNUP57 and pCCWlO.ago.

[0657] In particular, the promoters of the one or more recombinant nucleic acids encoding a polypeptide having hyaluronidase activity of a recombinant cell of the invention, and in particular of a recombinant yeast of the invention, may originate or be derived from Saccharomyces Bayanus, Saccharomyces kudriavzevii, Saccharomyces mikalae, Saccharomyces arboricola or other saccharomycetales, or Abishia gossypii.

[0658] The secretion signal of the present invention may for example have:

[0659] - the nucleic acid sequence set forth as SEQ ID NO: 43; and / or

[0660] - the amino acid sequence set forth as SEQ ID NO: 44.

[0661] The anchoring signal of the present invention may for example have:

[0662] - the nucleic acid sequence set forth as SEQ ID NO: 45; and / or

[0663] - the amino acid sequence set forth SEQ ID NO: 46.

[0664] The secretion signal may be fused to the polypeptide having hyaluronidase activity by creating a chimeric nucleic acid starting by a nucleic acid sequence encoding the signal peptide followed by a recombinant nucleic acid encoding a polypeptide having hyaluronidase activity as previously defined.

[0665] The secretion signal and anchoring signal may be fused to the polypeptide having hyaluronidase activity by creating a chimeric nucleic acid starting by a nucleic acid sequence encoding the signal peptide followed by a recombinant nucleic acid encoding a polypeptide having hyaluronidase activity as previously defined, followed by a nucleic acid sequence encoding for the anchoring signal.

[0666] Such chimeric nucleic acid sequences may be obtained by techniques known by the man skilled in the art such as chemical synthesis of nucleic acids or any recombination techniques such as cloning or PCR.

[0667] Another aspect of the present invention relates to hyaluronic acid (HA) obtained or obtainable from a recombinant cell of the invention or from a method according to the invention.

[0668] A further aspect of the invention is a cultivation medium comprising the hyaluronic acid of the invention. The invention further relates to a composition comprising the hyaluronic acid according to the invention.

[0669] The invention also relates to an industrial product or a consumer product or a consumable comprising (i) the hyaluronic acid of the invention, (ii) the cultivation medium comprising hyaluronic acid of the invention or (iii) the composition comprising hyaluronic acid of the invention.

[0670] In particular, said industrial product or consumer product or consumable according to the invention can be a cosmetic product, a flavour product, a fragrance product, a food product, a food, a beverage, a texturant, a pharmaceutical composition, a dietary supplement, a nutraceutical, a cleaning product and / or a dental and / or an oral hygiene composition.

[0671] PURIFICATION OF HYALURONIC ACID

[0672] According to a specific aspect of the invention, the fermentative production of hyaluronic acid preferably comprises a step of isolation of the hyaluronic acid produced from the culture medium. Recovering the hyaluronic acid from the culture medium is a routine task for a man skilled in the art. It may be achieved by a number of techniques well known in the art including but not limiting to pervaporation, selective precipitation, filtration, centrifugation, spray drying, lyophilisation or liquid extraction. The expert in the field knows how to adapt parameters of each technique dependent on the characteristics of the material to be separated.

[0673] The yeast as model of a cell in the present invention is preferred in that the synthesized hyaluronic acid is / are entirely exported outside the cells, thus simplifying the purification process.

[0674] Gas stripping is achieved with a stripping gas chosen among helium, argon, carbon dioxide, hydrogen, nitrogen or mixture thereof.

[0675] Liquid extraction is achieved with organic solvent as the hydrophobic phase such as pentane, hexane, heptane or dodecane. Renewal solvents may also be used.

[0676] Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA molecules differing in their nucleotide sequences can be used to encode a given enzyme of the disclosure. The native DNA sequence encoding the biosynthetic enzymes described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA molecules of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure. In similar fashion, 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 a desired activity. The disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide. Furthermore, the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.

[0677] Described herein are specific genes and proteins useful in the methods, compositions and organisms of the disclosure; however, it will be recognized that absolute identity to such genes is not necessary. For example, changes in a particular gene or polynucleotide comprising a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically, such changes comprise conservative mutations and silent mutations. Such modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art.

[0678] Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or functionally equivalent polypeptides can also be used to clone and express the polynucleotides encoding such enzymes.

[0679] Techniques known to those skilled in the art may be suitable to identify additional homologous genes and homologous enzymes. Generally, analogous genes and / or analogous enzymes can be identified by functional analysis and will have functional similarities.

[0680] Techniques known to those skilled in the art may be suitable to identify analogous genes and analogous enzymes or any biosynthetic pathway genes, proteins, or enzymes, techniques may include, but are not limited to, cloning a gene by PCR using primers based on a published sequence of a gene / enzyme of interest, or by degenerate PCR using degenerate primers designed to amplify a conserved region among a gene of interest. Further, one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity. Techniques include examining a cell or cell culture 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), then isolating the enzyme with said activity through purification, determining the protein sequence of the enzyme through techniques such as Edman degradation, design of PCR primers to the likely nucleic acid sequence, amplification of said DNA sequence through PCR, and cloning of said 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 comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC. The candidate gene or enzyme may be identified within the above mentioned databases in accordance with the teachings herein.

[0681] DERIVATIVES

[0682] The term “hyaluronic acid”, as used herein, is also intended to cover derivatives of hyaluronic acid, such as but not limited to hyaluronate salts, acetylated, cationized or sulphated hyaluronic acid useful for applications in a cosmetic product, flavour a product, a fragrance product, a food product, a food, a beverage, a texturant, a pharmaceutical composition, a dietary supplement, a nutraceutical, a cleaning product and / or a dental and / or an oral hygiene composition or combinations thereof. Hyaluronic acid may be prepared as a composition.

[0683] FORMULATIONS AND PRODUCTS

[0684] Low molecular weight hyaluronic acid according to the second aspect of the invention, or that obtainable by or obtained by the method of the third aspect of the invention, may be incorporated into a formulation / product, such as a nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulation / product. Thus, the present invention provides a formulation (e.g. a cosmetic formulation) comprising said hyaluronic acid. The present invention also provides a product (e.g. a cosmetic product) comprising said composition. The present invention also provides the use of said composition in a nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulation / product.

[0685] A “cosmetic product” is intended to mean any substance or mixture intended to be placed in contact with the external parts of the human body (epidermis, hair system, nails, lips and external genital organs) or with the teeth and the mucous membranes of the oral cavity with a view exclusively or mainly to cleaning them, perfuming them, changing their appearance, protecting them, keeping them in good condition, correcting body odours and / or combinations thereof.

[0686] A “substance” is intended to mean a chemical element and its compounds in the natural state or obtained by any manufacturing process, including any additive necessary to preserve its stability and any impurity deriving from the process used but excluding any solvent which may be separated without affecting the stability of the substance or changing its composition.

[0687] A “mixture” is intended to mean a mixture or solution composed of two or more substances. The nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulation / product may optionally further comprise pharmaceutically / veterinary / cosmetic (including cosmetic active) ingredients, such as excipients, carriers and mixtures thereof as appropriate.

[0688] “Cosmetic” or “cosmetic active ingredients” 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 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, wash off hair products, leave-on hair products, hair colorants (including but not limited to natural hair colorants) and / or combinations thereof), finished products and related technologies including, but not limited to components, incorporated, for example, into a cosmetic formulation (such as but not limited to natural colorants, preservatives, emulsifiers, anti-oxidants and the like which do not, for example, have an activity on the skin, hair, scalp and the like but play a role in the formulation of the finished product), delivery systems, marketing aids (such as, for example, coloured Unispheres™ applied to translucent formulations) and methods of making anything related thereto useful in / used for / intended for: application by rubbing, pouring, sprinkling, spraying or otherwise directly on or to a human or animal body and / or by placing in contact with the various external and / or on surface parts of a human or animal body (including but not limited to the skin, hair, body hair, the hair system, scalp, nails, lips, external genitalia, teeth, oral and / or nasal mucosa and the like); and / or application indirectly on or to a 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 (such as a capsule) or a delivery system (such as blends or formulations) applied to a textile; and / or cleansing, caring, cooling, beautifying, conditioning, treating, soothing, texturizing, promoting attractiveness, protecting, maintaining, improving, enhancing, altering and / or changing an external part and / or surface of a human or animal body (such as, but not limited to the scalp) or the aesthetic appearance of a human or animal body; and / or with a view to mainly cleaning or perfuming, or protecting or maintaining in good condition or combating body odour or changing the appearance of or correcting or repairing a state of imbalance in skin, oral mucosa, scalp or hair by providing a calming, healing, repairing, or revitalization, hydration of the skin or in order to provide relief to, lubricate, moisten, tone, heal, sterilize, relieve, correct and / or remedy states of dryness, irritation, injury or fatigue, and / or with a view to correcting pigmentation disorders or providing a non-pharmaceutical prevention and / or treatment of dandruff, acne, irritation and / or inflammation and the like and / or rebalancing the bacterial flora (such as, for example, the microbiome) on the surface of the skin (such as, for example, by promoting the level of beneficial bacterial flora on the skin surface) and / or for the purposes of keeping a human or animal body in good condition for health and / or wellbeing purposes and / or for improving the appearance of a human or animal body by, for example, improving the appearance of a product applied to a human or body; and / or providing a cosmetic and / or dermatological function and / or benefit with a biological activity benefit (but without affecting a body’s structure or function. For the avoidance of doubt, a cosmetic or cosmetic active ingredient or any part thereof may also qualify as a functional ingredient and / or nutraceutical.

[0689] “Functional ingredient” means a food ingredient or part of a food that provides medicinal or health benefits, including any of the following: a carotenoid, dietary fiber, fatty acid, saponin, antioxidant, flavonoid, isothiocyanate, phenol, polyphenol (such as resveratrol), plant sterol or stanol (phytosterols and phytostanols), a polyol, a prebiotic, a phytoestrogen, soy protein, sulfides / thiol, a vitamin, glucosamine, preservatives, hydration agents, edible gelling ingredients, edible gel mixes and gel compositions, long chain primary aliphatic saturated alcohols, colour agents, texturizing agents, emulsifiers and combinations thereof.

[0690] “Nutraceutical” means any and all natural, naturally occurring, sustainable, synthetically-produced and bio-synthetically-produced compounds, mixtures of compounds, functional ingredients, molecules, compositions, raw materials, and intermediates (including components and delivery devices (such as capsules) related thereto, delivery systems thereof (such as blends or formulations) and methods of making the foregoing) that are associated with health and / or cosmetic benefits, as well as improving or maintaining the appearance of the human body. For the avoidance of doubt, nutraceuticals includes compounds that can be used as supplements to food or beverage, whether a solid formulation, capsule, tablet, liquid formulation, solution or suspension.

[0691] Alternatively, the nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulation / product may consist or consist essentially of the composition of the invention. A cosmetic formulation / product may be an anti-aging formulation.

[0692] As used herein, references to pharmaceutically, veterinary or cosmetically acceptable excipients may refer to pharmaceutically, veterinary or cosmetically acceptable adjuvants, diluents and / or carriers as known to those skilled in the art.

[0693] By “pharmaceutically / veterinary / cosmetically acceptable” we mean that the additional components of the composition are generally safe, non-toxic, and neither biologically nor otherwise undesirable. For example, the additional components may be generally sterile and pyrogen free. Such components must be “acceptable” in the sense of being compatible with the composition of the invention and not deleterious to the recipients thereof. Thus, “pharmaceutically acceptable excipients” includes any compound(s) used in forming a part of the formulation that is intended to act merely as an excipient, i.e. not intended to have biological activity itself.

[0694] The nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulation / product may be in the form of a liquid or a solid.

[0695] Liquid dosage formulations / products for oral administration include solutions, emulsions, aqueous or oily suspensions, syrups and elixirs.

[0696] Formulations and products (e.g. pharmaceutical, veterinary or cosmetic formulations / products) described herein, such as those intended for oral administration, may be prepared according to methods known to those skilled in the art, such as by mixing the components of the formulation / product together.

[0697] The formulation or product (e.g. pharmaceutical, veterinary or cosmetic formulation / product) may contain one or more additional ingredients, such as pharmaceutical ingredients and excipients, such as sweetening agents, flavouring agents, colouring agents and preserving agents.

[0698] The formulation or product (e.g. pharmaceutical, veterinary or cosmetic formulation / product) may also contain one or more additional active ingredients, such as cosmetic or pharmaceutical active ingredients, such as hyaluronic acid, centella asiatica extract, peptides such as Matrixyl® and Argireline®, and mixtures thereof.

[0699] A formulation or product as disclosed herein may contain the active ingredient(s) in admixture with non-toxic pharmaceutically acceptable excipients (or ingredients). These excipients (or ingredients) may, for example, be: inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, maltodextrin or alginic acid; binding agents, for example, starch, gelatine or acacia; or lubricating agents, for example magnesium stearate, stearic acid, talc and mixtures thereof.

[0700] Liquid formulations or products (e.g. pharmaceutical, veterinary or cosmetic formulations / products) may be contained within a capsule, which may be uncoated or coated as defined above.

[0701] Moreover, 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.

[0702] Suitable cosmetic carriers are typically those that are suitable for topical administration to the outer surface of the human body, such as the skin and / or hair and / or scalp.

[0703] Typically, such carriers are dermatologically acceptable.

[0704] The phrase "dermatologically acceptable carrier" means that the carrier is suitable for topical application to the keratinous tissue, has good aesthetic properties, is compatible with the actives in the composition, and will not cause any unreasonable safety or toxicity concerns.

[0705] The carrier can be in a wide variety of forms. In some instances, the solubility or dispersibility of the components (e.g. extracts, sunscreen active, additional components) may dictate the form and character of the carrier. Non-limiting examples include simple solutions (e.g. aqueous or anhydrous), dispersions, emulsions, and solid forms (e.g. gels, sticks, flowable solids, or amorphous materials).

[0706] The dermatologically acceptable carrier may be in the form of an emulsion. An emulsion may be generally classified as having a continuous aqueous phase (e.g. oil-in-water and water-in-oil-in- water) or a continuous oil phase (e.g. water-in-oil or oil-in-water). The oil phase of the present invention may comprise silicone oils, non-silicone oils such as hydrocarbon oils, esters, ethers, and the like, and mixtures thereof. The aqueous phase typically comprises water and water-soluble ingredients (e.g. water-soluble moisturizing agents, conditioning agents, anti-microbials, humectants and / or other skin care actives). However, in some instances, the aqueous phase may comprise components other than water, including but not limited to water-soluble moisturizing agents, conditioning agents, antimicrobials, humectants and / or other water-soluble skin care actives. In some instances, the non-water component of the composition comprises a humectant, such as glycerin and / or other polyol(s). Emulsions may also contain an emulsifier. Emulsifiers may be non-ionic, anionic or cationic. The carrier may contain one or more dermatologically acceptable, hydrophilic diluents. As used herein, "diluent" includes materials in which the composition of the invention can be dispersed, dissolved, or otherwise incorporated. Hydrophilic diluents include water, organic hydrophilic diluents, such as lower monovalent alcohols (e.g., C1-C4), and low molecular weight glycols and polyols, including 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.

[0707] The cosmetic formulation / product may optionally include one or more additional ingredients commonly used in cosmetic compositions (e.g., colorants, skin tone agents, skin anti -aging agents, anti-inflammatory agents, sunscreen agents, combinations of these and the like), provided that the additional ingredients do not undesirably alter the anti-glycation benefits provided by the composition.

[0708] In some instances, it may be desirable to select skin tone agents that function via different biological pathways so that the actives do not interfere with one another, which could reduce the efficacy 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 response, and the like.

[0709] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those having ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Where used herein, the specific term “single” is limited to only “one.”

[0710] As utilized in accordance with the methods, compounds, and compositions of the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0711] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or when the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” The use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, or any integer inclusive therein. The term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term “at least one of X, Y, and Z” will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z.

[0712] The term “and / or” is understood to mean that all members of a group connected by the term “and / or” are represented both cumulatively with respect to each other in any combination, and alternatively with respect to each other. Exemplarily, for the expression “A, B and / or C”, the following disclosure is to be understood thereunder: i) (A or B or C), or ii) (A and B), or iii) (A and C), or iv) (B and C), or v) (A and B and C), or vi) (A and B or C), or vii) (A or B and C), or viii) (A and C or B).

[0713] As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. A range is intended to include any sub-range therein, although that sub-range may not be explicitly designated herein. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth. Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, a range of 1-1,000 includes, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150- 200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, and includes ranges of 1-20, 10-50, 50-100, 100-500, and 500-1,000. Reference to an integer with more (greater) or less than includes any number greater or less than the reference number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc. all the way down to the number one (1); and less than 10 includes 9, 8, 7, etc. all the way down to the number one (1).

[0714] The terms “increase,” “increasing,” "enhancing," or "enhancement" are defined as indicating a result that is greater in magnitude than a control number derived from analysis of a cohort, for example, the result can be a positive change of at least 5%, 10%, 20%, 30%, 40%, 50%, 80%, 100%, 200%, 300% or even more in comparison with the control number. Similarly, the terms “decrease,” “decreasing,” “lessening," or "reduction" are defined as indicating a result that is lesser in magnitude than a control number, for example, the result can be a negative change of at least 5%, 10%, 20%, 30%, 40%, 50%, 80%, 100%, 200%, 300% or even more in comparison with the control number.

[0715] As used in this specification and claims, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0716] When used herein “consisting of’ excludes any element, step, or ingredient not specified in the aspect, embodiment and / or claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect, embodiment and / or claim.

[0717] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0718] Throughout this application, the terms “about” or “approximately” are used to indicate that a value includes the inherent variation of error for the composition, the method used to administer the composition, or the variation that exists among the study subjects. As used herein the qualifiers “about” or “approximately” are intended to include not only the exact value, amount, degree, orientation, or other qualified characteristic or value, but are intended to include some slight variations due to measuring error, manufacturing tolerances, stress exerted on various parts or components, observer error, wear and tear, and combinations thereof, for example. The terms “about” or “approximately,” where used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass, for example, variations of ± 10%, or ± 5%, or ± 1%, or ± 0.1% (including all the values within this range) from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art. As used herein, the term “substantially” means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, the term “substantially” means that the subsequently described event or circumstance occurs at least 90% of the time, or at least 95% of the time, or at least 98% of the time.

[0719] As used herein any reference to "one embodiment" or "an embodiment" or "some embodiments" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment and may be included in other embodiments. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment and are not necessarily limited to a single or particular embodiment. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0720] As used herein, “pure” or “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other object species in the composition thereof), and particularly a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80% of all macromolecular species present in the composition, more particularly more than about 85%, more than about 90%, more than about 95%, or more than about 99%. The term “pure” or “substantially pure” also refers to preparations where the object species is at least 60% (w / w) pure, or at least 70% (w / w) pure, or at least 75% (w / w) pure, or at least 80% (w / w) pure, or at least 85% (w / w) pure, or at least 90% (w / w) pure, or at least 92% (w / w) pure, or at least 95% (w / w) pure, or at least 96% (w / w) pure, or at least 97% (w / w) pure, or at least 98% (w / w) pure, or at least 99% (w / w) pure, or 100% (w / w) pure.

[0721] Use of the word “we,” “us,” and / or “our” as a pronoun in the present disclosure refers generally to laboratory personnel, technicians, or other contributors who assisted in laboratory procedures and data collection and is not intended to represent an inventorship role by said laboratory personnel, technicians, or other contributors in any subject matter disclosed herein.

[0722] In addition, atoms making up the compounds of the present disclosure are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include13C and14C. It should be recognized that the particular anion or cation forming a part of any salt form of a compound provided herein is not critical, so long as the salt, as a whole, is acceptable from a flavour, fragrance and / or cosmetic perspective and / or is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0723] The term “carrier” as used herein, may also refer to a natural product or a product originating from nature that has been transformed or modified so that it is distinct from the natural product from which it originated, such as maltodextrin.

[0724] The amount of the composition disclosed herein present in nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulations or products will vary depending on the application.

[0725] Typically, the amount of composition disclosed herein that may be present in nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulations or products will be from about 0.001 to about 50% by weight, such as from about 0.01% to about 30% or from about 1% to about 20% of the nutraceutical, pharmaceutical, veterinary, oenological or cosmetic formulations or products, such as from about 0.01 to about 20%, or from about 0.1 to 10% or from about 1 to about 5% by weight of the formulation or product.

[0726] It is to be understood that this disclosure is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be 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 present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by the person skilled in the art. In accordance with the present disclosure there may be conventional molecular biology, microbiology, and recombinant DNA techniques employed which are within the skill of the art.

[0727] This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger,

[0728] H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0729] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety.

[0730] The examples and figures which follow are presented by way of illustration and without implied limitation of the invention.

[0731] The following numbered paragraphs define additional aspects of the present disclosure.

[0732] I. A recombinant cell producing hyaluronic acid (HA) wherein the recombinant cell comprises:

[0733] (a) one or more recombinant nucleic acids encoding a polypeptide having hyaluronan synthase activity;

[0734] (b) one or more recombinant nucleic acids encoding a polypeptide having UDP- Glucose dehydrogenase (UDP-GlcDH or HASB) activity;

[0735] (c) one or more recombinant nucleic acids, under the control of a pCCW12.Spr promoter, encoding a polypeptide having hyaluronidase activity wherein the polypeptide having hyaluronidase activity comprises a secretion signal so that hyaluronic acid, in particular of a desired molecular weight (HAMW) is produced by the recombinant cell, and

[0736] (d) (i) one or more recombinant nucleic acids encoding a polypeptide having a glutamine synthetase (GLN1) activity; and / or

[0737] (ii) one or more disrupted endogeneous nucleic acids encoding a glutamate synthase (GLT1).

[0738] 2. The recombinant cell according to paragraph 1, wherein the recombinant cell is a yeast cell, optionally belonging to the Saccharomyces genus, or to the Candida genus, or to the Kluyveromyces genus, or to the Ogataea genus, or to the Yarrowia genus, or to the Debaryomyces genus, or to the Ashbya genus.

[0739] 3. The recombinant cell according to paragraph 1 or 2, wherein the polypeptide having hyaluronidase activity further comprises an anchoring signal.

[0740] 4. The recombinant cell according to any one of paragraphs 1 to 3, wherein the hyaluronic acid has an average molecular weight of less than about 5 kDa. 5. The recombinant cell according to any one of paragraphs 1 to 4, wherein the hyaluronic acid has an average molecular weight of more than about 800 Da.

[0741] 6. The recombinant cell according to any one of paragraphs 1 to 5, wherein the hyaluronic acid has a median molecular weight of less than about 5 kDa, more particularly of less than about 3 kDa, e.g. about 2.8 kDa.

[0742] 7. The recombinant cell according to any one of paragraphs 1 to 6, wherein the nucleic acid encoding a polypeptide having a glutamine synthetase activity is obtained or derived from Saccharomyces cerevisiae.

[0743] 8. The recombinant cell according to any one of paragraphs 1 to 7, wherein the nucleic acid encoding a polypeptide having hyaluronidase activity is obtained or derived from at least one of Cupiennius salei. Loxosceles intermedia, Hirudo nipponia, Bothrops atrox or Tityus serrulatus.

[0744] 9. The recombinant cell according to any one of paragraphs 1 to 8, wherein the nucleic acid encoding a polypeptide having hyaluronan synthase activity is obtained or derived from Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviKl, Chlorella virus IL- 5-2sl, Chlorella virus CZ-2, Chlorella virus CVG-1, Xenopus laevis or Pasteurella mullocida, and is in particular obtained or derived from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviKl, Chlorella virus IL-5-2sl, Chlorella virus C7.-2, Chlorella virus CVG-1 o Xenopus laevis.

[0745] 10. The recombinant cell according to any one of paragraphs 1 to 9, 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, and in particular from Arabidopsis thaliana or Chlorella virus PBCV1.

[0746] 11. The recombinant cell according to any one of paragraphs 1 to 10, wherein the recombinant cell comprises at least one recombinant nucleic acid encoding one or more of:

[0747] (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity;

[0748] (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity;

[0749] (iii) a polypeptide having Phosphoglucomutase- 1 (PGM1) activity; and / or

[0750] (iv) a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase (UGP1) activity; and / or (v) a polypeptide having Glucosamine-6-phosphate N-acetyltransferase (GNA1) activity; and / or

[0751] (vi) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity.

[0752] 12. The recombinant cell according to any one of paragraphs 1 to 11, wherein the recombinant cell belongs to the Saccharomycetales order, and is in particular 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, Kluyveromyces thermotolerens, Ogataea polymorpha, Yarrowia lypolytica, Debaryomyces hansenii, and Ashbya gossypii, and is preferably Saccharomyces cerevisiae.

[0753] 13. A method of producing hyaluronic acid having i) a molecular weight of more than about 800 Da and less than about 5 kDa; and / or ii) a median molecular weight of less than about 5 kDa, more particularly of less than about 3 kDa, e.g. about 2.8 kDa, comprising:

[0754] (a) cultivating a recombinant cell as defined in any one of paragraphs 1 to 12 in a cultivation medium for a time sufficient to produce said hyaluronic acid; and

[0755] (b) optionally isolating or recovering the hyaluronic acid from the recombinant cell and / or from the cultivation medium.

[0756] 14. The method according to paragraph 13, wherein the recombinant cell comprises at least one recombinant nucleic acid encoding one or more of:

[0757] (i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity;

[0758] (ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity;

[0759] (iii) a polypeptide having Phosphoglucomutase- 1 (PGM1) activity;

[0760] (iv) a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase (UGP1) activity;

[0761] (v) a polypeptide having Glucosamine-6-phosphate N-acetyltransferase (GNA1) activity; and / or

[0762] (vi) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity.

[0763] 15. The method according to paragraph 13 or 14, wherein the recombinant cell is a member of the genus Saccharomyces, and in particular is Saccharomyces cerevisiae. 16. The method according to any one of paragraphs 13 to 15, wherein the time sufficient to produce the hyaluronic acid is a period of from about 35 hours to about 60 hours, preferably from about 40 hours to about 55 hours, in particular about 52 hours.

[0764] 17. The method according to any one of paragraphs 13 to 16, wherein the molecular weight of the hyaluronic acid is controlled by regulating the pH of the cultivation medium.

[0765] 18. Hyaluronic acid obtained or obtainable from a recombinant cell of any one of paragraphs 1 to 12 or from the method according to any one of paragraphs 13 to 17.

[0766] 19. A cultivation medium comprising the hyaluronic acid according to paragraph 18.

[0767] 20. A composition comprising the hyaluronic acid according to paragraph 18, and optionally a carrier.

[0768] 21. A composition according to paragraph 20, wherein said composition is a cosmetic preparation, and in particular a skin care composition.

[0769] 22. The composition according to paragraph 21, wherein the hyaluronic acid is present in an amount of 0.1 to 1.0% (w / v), more preferably 0.2 to 0.7% (w / v), e.g. about 0.5% (w / v), of the composition.

[0770] 23. Use of the composition of paragraph 21 or 22, or of the hyaluronic acid of paragraph 18, for anti-ageing treatment and / or anti-wrinkle treatment, more preferably for treatment of sleep wrinkles.

[0771] 24. A cosmetic method of skin treatment, comprising the step of applying to the skin an effective amount of the composition of paragraph 21 or 22, or of the hyaluronic acid of paragraph 18, wherein the skin treatment is preferably for anti-ageing treatment and / or anti-wrinkle treatment, and more preferably for treatment of sleep wrinkles.

[0772] 25. The use according to paragraph 23, or the method of paragraph 24, wherein the treatment leads to one or more of i) an increase in expression of DMNT3B; ii) a reduction in aberrant transcripts; iii) an increase in expression and activity of sirtuin-1; iv) an increase in expression and activity of PARP-1; v) an increase in expression and activity of telomerase; vi) an increase in production of type-1 collagen by fibroblasts.

[0773] EXAMPLES

[0774] The inventive concepts of the present disclosure will now be discussed in terms of several specific, non-limiting, examples. The examples described below, which include particular embodiments, will serve to illustrate the practice of the present disclosure, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of particular embodiments of the present disclosure only and are presented in the cause of providing what is believed to be a useful and readily understood description of procedures as well as of the principles and conceptual aspects of the inventive concepts.

[0775] Example 1 : Protocol for making a recombinant Saccharomyces cerevisiae strain according to the invention

[0776] All the hereinafter implemented recombinant Saccharomyces cerevisiae strains were constructed from standard strains using standard yeast molecular genetics procedure (Methods in yeast Genetics - A cold spring harbor laboratory course Manual (2000) by D. Burke, D. Dawson, T. Steams CSHL Press).

[0777] Cluster of the following-mentioned genes were integrated in recombinant yeast at once using the ability of yeast to efficiently recombine free DNA ends which have sequence homology.

[0778] In addition, for a better comprehension of following genotypes:

[0779] - jlpl, lypl, sam3, his3, leu2, trpl and ura3 are insertion sites.

[0780] - Lowercase letters mean that the considered gene is inactive, uppercase letters reflect an active gene.

[0781] - following a gene name means that the gene is interrupted by what follows (if more than one gene are inserted, they are noted in brackets []). The interruption of the gene is concomitant with an entire deletion of the coding sequence but preserves the promoter. In consequence the gene followed by is inactive and is noted in lowercase. If not specified, the transcription of the gene inserted is controlled by the promoter of the disrupted gene.

[0782] - “gene. KI” means that the gene originates from Kluyveromyces lactis.

[0783] More particularly, the coding sequences to be cloned were artificially synthetized. For heterologous sequences (non-yeast), the nucleic sequences were modified in order to obtain a synonymous coding sequence using the yeast codon usage. Using restriction enzyme and classical cloning technology, each synthetic sequence was cloned in between a transcription promoter and a transcription terminator. Each promoter sequence is preceded by a 50 to 200 nucleotide sequence homologous to the sequence of the terminator of the upstream gene. Similarly, the terminator of each gene (a gene comprising the promoter-coding sequence-terminator) is followed by sequences homologous to the gene immediately following. So that each of the unit to be integrated have a 50-200 nucleotide overlap with both the unit upstream and the unit downstream. For the first unit, the promoter is preceded by 50-200 nucleotides homologous to the yeast chromosome nucleotide for the locus in which it will be integrated. Similarly, for the last unit, the terminator is followed by 50-200 nucleotides homologous to the yeast chromosome nucleotide for the locus in which it will be integrated.

[0784] Each unit is then PCR amplified from the plasmids constructs, yielding X unit of linear DNA having overlapping sequences. At least one of this gene is an auxotrophic marker, in order to select for recombination event. All the linear fragments are transformed in the yeast at once, and a recombinant yeast cell is selected for the auxotrophy related to the marker used. The integrity of the sequence is then verified by PCR and sequencing.

[0785] Example 2: Production of Hyaluronic acid with an average molecular weight of less than about 5 kPa

[0786] A. Firstly, two recombinant strains are obtained: DA2886-5 and DA2886. Accordingly, these two strains are as follows:

[0787] DA2886-5: MAT-a / MAT-a, gltl ::[MET3.Sba, pCCW12.Spr-HYAL-31.Csa-tRPL41B.Sm, pCCW12.Sar-HYAL-3.Hn-tRPL15A, pTEFl.Ago-GLNl-tTDH3, pCCW12.Sba-HASB.vir- tRPL3, pCCW12.Sk-HASB-A.Vir-tTEFl.Sba, pCCW12-HASA-l.Vir-tRPL41B, pCCW12.Sm- HAS A-A. Vir-tRPL 15 A. Sba] / gltl ::[MET3. Sba, pCCW12.Spr-HYAL-31.Csa-tRPL41B.Sm, pCCW12.Sar-HYAL-3.Hn-tRPL15A, pTEFl.Ago-GLNl-tTDH3, pCCW12.Sba-HASB.vir- tRPL3, pCCW12.Sk-HASB-A.Vir-tTEFl.Sba, pCCW12-HASA-l.Vir-tRPL41B, pCCW12.Sm- HAS A- A. Vir-tRPL 15 A. Sba], hi s3 : : [tRPL3-UGP 1 -pS AMI , pMET6-QRH -tIDP 1 ,

[0788] HIS3]x3 / his3::[tRPL3-UGPl-pSAMl, pMET6-QRH-tIDPl, HIS3]x3, jlpl ::[LEU2.Sba, pTEF 1. Sba-HASB. Vir-tRPL3. Sm, pTDH3. Sk-HASB-A. Vir-tTEF 1. Sba, pTDH3-l . Sba-HAS A- 1. Vir-tRPL3. Sba, pTDH3. Sar-HAS A- A. Vir-tRPLl 5 A. Sba] / jlp 1 : : [LEU2. Sba, pTEF 1. Sba- HASB. Vir-tRPL3. Sm, pTDH3. Sk-HASB-A. Vir-tTEF 1 Sba, pTDH3-l . Sba-HASA-1. Vir- tRPL3.Sba, pTDH3. Sar-HAS A-A. Vir-tRPL 15 A. Sba], Ieu2 / leu2, met3Al / met3Al, sam3::[LEU2.Kl, pPDCl-PGMI-tIDPI, pTEFl.Ago-GFAl-tRPL15A, pENO2-UGPl-tRPL3, pCWP2-GNAl-tTPH, pTEFl-PCMl-tRPL41B, TRPl.Sba] / sam3::[LEU2.Kl, pPDCl-PGMl- tlDPl, pTEFl.Ago-GFAl-tRPL15A, pENO2-UGPl-tRPL3, pCWP2-GNAl-tTPH, pTEFl- PCMl-tRPL41B, TRPl.Sba], trpl / trpl, ura3::[tRPL3-MHPF.Ec-pPDCl, pTDH3-GDH-21.Eca- tlDPl, URA3]x2 / ura3::[tRPL3-MHPF.Ec-pPDCl, pTDH3-GDH-21.Eca-tIDPl, URA3]x2 D2886: MAT-a / MAT-a , gltl::[MET3.Sba, pCCW12.Spr-HYAL-31.Csa-tRPL41B.Sm, pCCW12.Sar-HYAL-3.Hn-tRPL15A, pTEFl.Ago-GLNl-tTDH3, pCCW12.Sba-HASB.vir- tRPL3, pCCW12.Sk-HASB-A.Vir-tTEFl.Sba, pCCW12-HASA-l.Vir-tRPL41B, pCCW12.Sm- HAS A-A. Vir-tRPL 15 A. Sba] / gltl ::[MET3. Sba, pCCW12.Spr-HYAL-31.Csa-tRPL41B.Sm, pCCW12.Sar-HYAL-31.Hn-tRPL15A, pTEFl.Ago-GLNl-tTDH3, pCCW12.Sba-HASB.vir- tRPL3, pCCW12.Sk-HASB-A.Vir-tTEFl.Sba, pCCW12-HASA-l.Vir-tRPL41B, pCCW12.Sm- HAS A- A. Vir-tRPL 15 A. Sba], hi s3 : : [tRPL3-UGP 1 -pS AMI , pMET6-QRH -tIDP 1 ,

[0789] HIS3]x3 / his3::[tRPL3-UGPl-pSAMl, pMET6-QRH-tIDPl, HIS3]x3, jlpl::[LEU2.Sba, pTEF 1. Sba-HASB. Vir-tRPL3. Sm, pTDH3. Sk-HASB-A. Vir-tTEF 1. Sba, pTDH3-l . Sba-HAS A- 1. Vir-tRPL3. Sba, pTDH3. Sar-HASA-A. Vir-tRPL 15 A. Sba] / j Ip 1 : : [pTEF 1. Sba-HASB . Vir- tRPL3. Sm, pTDH3. Sk-HASB-A. Vir-tTEF 1. Sba, pTDH3-l . Sba-HASA-1. Vir-tRPL3. Sba, pTDH3. Sar-HASA-A. Vir-tRPL 15 A. Sba], Ieu2 / leu2, met3Al / met3Al, sam3::[LEU2.Kl, pPDCl- PGMl-tlDPl, pTEFl.Ago-GFAl-tRPL15A, pENO2-UGPl-tRPL3, pCWP2-GNAl-tTPH, pTEFl-PCMl-tRPL41B, TRPLSba] / sam3::[LEU2.Kl, pPDCl-PGMI-tIDPI, pTEFl.Ago- GFAl-tRPL15A, pENO2-UGPl-tRPL3, pCWP2-GNAl-tTPH, pTEFl-PCMl-tRPL41B, ], trp 1 / trp 1 : : [pTDH3 -HYAL-31. Csa-tRPL3. Spr, TRP 1 ]x2, ura3 : : [tRPL3 -MHPF Ec-pPDC 1 , pTDH3 -GDH-21.Eca-tIDP 1 , URA3 ]x2 / ura3 : : [tRPL3 -MHPF Ec-pPDC 1 , pTDH3 -GDH-21 Eca- tlDPl, URA3]x2

[0790] HYAL-3 represents a nucleic acid sequence encoding a polypeptide having hyaluronidase activity associated with a secretion signal but no anchoring signal.

[0791] HAS A, HASA-1, HAS A- A, HASA2 and XHASA2 all represent a nucleic acid encoding a polypepyide having hyaluronan synthase activity. They differ from one another in that they are different reencoded versions of a nucleic acid sequence encoding the hyaluronan synthase enzymes. HASA-1 has the sequence SEQ ID NO: 1, HAS A- A has the sequence SEQ ID NO: 2, HASA2 has the sequence SEQ ID NO: 7 and XHASA2 has the sequence SEQ ID NO: 6.

[0792] HASB and HASB-A represent a nucleic acid sequence encoding a polypeptide having UDP- glucose 6-dehydrogenase activity. They differ from one another in that they are different reencoded versions of a nucleic acid sequence encoding the enzyme. HASB. At has the sequence SEQ ID NO: 12, HASB.Vir has the sequence SEQ ID NO: 13 and HASB-A. Vir has the sequence SEQ ID NO: 14. All these strains were incubated at O.D = 2 in SY medium. SY medium comprises the following elements:

[0793] KH2PO4: 100 mM; MgSO47H2O: 2.8 mM; K2SO4: 11.5 mM; Na2SO4: l.l mM; NaCl: 2.6 mM; CaCl22H2O: 0.7 mM; CuSO45H2O: 15 pM; KI: 6 pM; FeCl3: 30 pM; ZnSO47H2O: 61 pM; MnSO4H2O: 25 pM; H2SO4: HO pM; Panthotenic Acids hemicalcium salt: 42 pM; Thiamin hydrochloride: 59 pM; Pyridoxin hydrochloride: 49 pM; Myo-Inositol (CeHnOe): 555 pM; Nicotinic acid (C6H5NO2): 29 pM; D-Biotine: 0.82 pM; Ammonium citrate tribasic: 33 mM; and glucose or sucrose 2-30%.

[0794] Growth medium was recovered at 52h hours and assayed for hyaluronic acid content and quality. The medium contained about 80 - 83 g / L of hyaluronic acid. Hyaluronic acid was then recovered and purified according to a process including cell separation, ultrafiltration (2 kDa), continuous diafiltration (2 kDa), polishing and lyophilisation.

[0795] The size of hyaluronic acid molecules was characterized by performing a Size Exclusion Chromatography (SEC) using HA markers of known size. As can be seen in Figure 1, typically the Hyaluronic acid recovered from the culture medium has a median size of 2.8 KDa, and the Gaussian curve extends from about 800 Da to about 5 kDa.

[0796] The molecular weight is determined using size exclusion chromatography. In such technic, the product is injected on a column and is eluted with an invariant mobile phase. The heavier (bigger) the product, the earlier it is eluted (with a lower limit characteristic of the column) and conversely, the lighter (smaller) the product, the latter it is eluted (with an upper limit characteristic of the column). Then the product is characterized by the volume of mobile phase necessary to elute it, or by the retention (or elution) time, which is the time between the injection and the detection of the product at the output of the column. Then to determine the molecular weight of the product of interest, it is compared to the elution time of standards of known molecular weight. As in Figure 1, four standards have been run, which are different samples of hyaluronic acids which differ by their lengths (i.e. the number of monomer in the polymer) and hence their molecular weight. We used a 20-mer (MW of 3800 Da), a 14-mer (MW of 2660 Da), an 8-mer (MW of 1520 Da) and a 4-mer (MW of 760 Da). As can be seen in the figure, Mycronhyal (the particular sample obtained from the above-described production method) elutes from about the beginning of the Gaussian curve corresponding to the 20-mer (about 4500 Da) to the end of the Gaussian for the 8-mer (1500 Da). Example 3: Comparative examples for the production of Hyaluronic acid

[0797] In order to study the effect of the promoters controlling the expression of HYAL-31.Csa, three equivalent strains producing HA and expressing HYAL-31.Csa under the control of three different promoters were constructed.

[0798] The three strains are as follows:

[0799] YA5569: MAT-a, his3::[pSAMl-UGPl- tRPL3, pMET6-QRH-tIDPl, HIS3]x5, jlpl ::[LEU2.Kl, pCUPl-HASA-l.Sz-tRPL41B, pCUPl-UGPl-tRPL3, pCUPl-QRH-tIDPI, pPDCl-UGPl- tTPIl, pTDH3-QRIl-tMET25, pCCW12-HASB.At-tRPL15A], leu2, lypl::[pCWP2-HYAL- 3 LCsa-tRPL15A, pCCW12.Sba-HASB.vir-tRPL3, pCCW12.Sk-HASB-A.Vir-tTEFl.Sba, pCCW12-HASA-l.Vir-tRPL41B, pCCW12.Sm-HASA-A.Vir-tRPL15A.Sba], sam3::[LEU2.Kl, pPDCl-PGMI-tIDPI, pTEFl.Ago-GFAl-tRPL15A, pENO2-UGPl-tRPL3, pCWP2-GNAl- tTPIl, pTEFl-PCMI-tIDPI, pCCW12-XHASA2.Xl-tRPL3, pTDH3-QRH-tIDPl], trpl ::[tRPL3- HASB.Vir-pMET6, pMET25-HASA-l.Vir-tIDPl, TRPl]x2

[0800] YA5326-3: MAT-a, canl-100, his3::[pSAMl-UGPl- tRPL3-pMET6-QRH-tIDPl- HIS3]x5, jlpl::[LEU2.Kl, pCUPl-HASA2.Sz-tRPL41B, pCUPl-UGPl-tTPIl, pTDH3-QRH-tMET25, pCC W 12-HASB . At-tRPL 15 A], leu2, ly p 1 : : [pCC W 10. ago-HYAL-31. C sa-tRPL 15 A, pCC W 12. Sk-HASB - A. Vir-tTEF 1. Sba, pCCW 12-H AS A- 1. Vir-tDIT 1 , pCC W 12. Sba-HASB . vir- tRPL3, pCCW12.Sm-HASA-A.Vir-tRPL15A.Sba], sam3::[LEU2.Kl, pPDCl-PGMI-tIDPI, pTEFl-GFAl-tRPL15A, pENO2-UGPl-tRPL3, pCWP2-GNAl-tTPIl, pTEF3-PCMl-tIDPl, pCC W 12-XHAS A2. Xl-tRPL3 , pTDH3 -QRI 1 -tIDP 1 ] , trp 1 : : [pMET6-HASB . Vir-tRPL3 , pMET25-HASA-l.Vir-tIDPl- TRPl]x2

[0801] YA6178: MAT-a, gltl ::[MET3.Sba, pTDH3-HYAL-31.Csa-tRPL15A, pTEFl.Ago-GLNl- tTDH3, pCCW12.Sba-HASB.vir-tRPL3, pCCW12.Sm-HASA-A.Vir-tRPL15A.Sba], his3::[pSAMl-UGPl-tRPL3, pMET6-QRH-tIDPl, HIS3]x3, jlpl::[LEU2.Sba-loxP, pTEFl. Sba- HASB. Vir-tRPL3. Sm, pTDH3. Sk-HASB-A. Vir-tTEF 1 Sba, pTDH3-l . Sba-HASA-1. Vir- tRPL3.Sba, pTDH3.Sar-HASA-A.Vir-tRPL15A.Sba], leu2, met3Al, sam3::[LEU2.Kl, pPDCl- PGMl-tlDPl, pTEFl.Ago-GFAl-tRPL15A, pENO2-UGPl-tRPL3, pCWP2-GNAl-tTPH, pTEFl-PCMl-tRPL41B, TRPl.Sba-loxP], trpl, ura3::[ pPDCl-MHPF.Ec- tRPL3, pTDH3- GDH-21.Eca-tIDPl, URA3]x2

[0802] The above three strains were incubated in 25 ml of SY medium buffered with MES 0,1 M pH 5.5 in a baffled Erlenmeyer for 48 hours at 28°C under vigorous agitation. Growth medium was recovered at 48 hours and assayed for hyaluronic acid content and quality. In order to determine the size of the HA produced, an aliquot of the medium was loaded and ran on a agarose gel subsequently coloured with ”Stains-AH” (sigma Aldrich CAS Number 7423-31- 6).

[0803] It was observed that YA5569 produces HA showing a molecular weight of about 20 kDa to 50 kDa. YA5326-3 produces HA showing a molecular weight 50 kDa or comprised between 50 kDa and 250 kDa. YA6178 produces HA showing a molecular weight of about 20 kDa.

[0804] This observation on the strains not comprising the hyaluronidase expressed under the promoter pCCW12.spr demonstrates the critical role and effect of this particular promoter in determining the molecular weight of the produced HA, i.e. remarkably shifting it to the lower range of less than 5 kDa.

[0805] Example 4: Skin penetration of Micronhyal

[0806] Material and methods

[0807] Skin explants culture and preparation

[0808] Human skin explants were prepared and kept in survival medium (MIL215001, Bio-predic) for 24 hours at 37 °C and 5% CO2. The next day, Micronhyal at 3% (w / v) in sterile water was topically applied and incubated for 8 hours at 37 °C and 5% CO2 before skin penetration analysis. The untreated condition did not receive any treatment. After the end of incubation, the skin surface was cleaned in order to eliminate any excess of the product. The skin explants were then frozen at -80 °C and cut longitudinally using a cryotome with a thickness of 20 pm. For each explant, three tissue sections were selected and deposited on a CaF2 support for Raman imaging analysis for a total of 3 Raman images per condition. Four other adjacent sections of 7 pm thickness were prepared for an Hematoxylin & Eosin staining.

[0809] Raman micro-imaging

[0810] Raman images have a size of Y: 10 pm / X: 100 pm with a step of 5 pm in X and 5 pm in Y. Each Raman image has 3 Y spectra and 22X spectra (66 spectra per image).

[0811] - Laser wavelength: 660 nm

[0812] - Objective: 100 X, long focal length with a numerical aperture 0.75

[0813] - Acquisition time: 30 seconds

[0814] - Accumulation: IX

[0815] - Spectral range: 600 to 3100 cm'1 - Grafting: 950T

[0816] - Confocal Hole: 300 gm

[0817] - Slit width: 150 gm (spectral resolution 6.5 cm’1)

[0818] - Step in X: 5 pm, Step in Y: 5 pm

[0819] In order to ensure reproducibility of the measurements, before each use, the Raman spectrometer is calibrated with silicon which gives a Raman peak at 520.7 cm’1. Continuous control of the laser power at the sample level is achieved. A pre-processing of Raman images was made by eliminating aberrant spectra (fluorescence, burning, saturation), correcting the baseline, applying a spectral smoothing and despike and a spectral normalization.

[0820] The processing of corrected data maps was performed based on least squares fitting method that operates with Matlab software. This method involves mathematical modelling of reference spectra in the overall spectral image to determine the contribution and distribution of these spectra within the image.

[0821] Results

[0822] It was demonstrated that Micronhyal has a good skin penetration profile and is able to reach 100pm of depth (Figure 2). Coefficient of fitting was measured and mapped on skin section to illustrate where the molecule was identified according skin depth and its level of intensity (Blue for low detection and red expressed high detection).

[0823] Example 5: DMNT3B expression

[0824] DMNT3B has been identified as a key enzyme involved in DNA ageing which is related to longevity. It plays an active role in DNA methylation and controlling the quality of transcription. Indeed, DMNT3B expression and activity is significantly decreased during ageing, contributing to accumulated truncated transcripts and subsequently the increase of non-functional proteins. We defined that modulating this epigenetic mechanism controlling ageing mediated-gene expression changes can be an innovative approach for boosting longevity of skin cells and limiting ageing signs development.

[0825] Materials and methods

[0826] Cell culture and treatment

[0827] Young skin cells were incubated during 72 hours in absence of any treatment as untreated control. Aged skin cells after excessive replicative senescence induction (passage 4) were incubated during 72 hours in absence (untreated “aged” control at passage 4) or in presence of reference product or increasing concentrations of test compound, which were Micronhyal 0.005 mg / ml and O.Olmg / ml. Test compounds preparation

[0828] The test compound named Micronhyal was directly solubilized at 1 mg / ml in incubation medium. The solution obtained was then diluted in the incubation medium in order to reach the different concentrations described above.

[0829] Measurement of DNMT3B expression

[0830] At the end of the incubation period, nuclear proteins were extracted from the cell monolayer. DMNT3B was then quantified using a sensitive and specific ELISA kit.

[0831] Protein quantification for data normalization

[0832] Proteins contained in the cell lysates (nuclear fraction) were quantified using a spectro- colorimetric method (Bradford method).

[0833] Results

[0834] A dose-dependent significant increase of DMNT3B expression with Micronhyal was observed (Figure 3). It was observed that Micronhyal increased DNMT3B expression 12% and 27% compared to the prematurely aged control, when used in concentration of 5 pg / ml and 10 pg / ml, respectively.

[0835] Example 6: Quality of Transcription

[0836] We demonstrated that Micronhyal significantly increases the expression of DMNT3B in aged skin cells. DMNT3B is directly involved in DNA methylation, which regulates the correct transcription of genes. During ageing, its level of expression and activity reduces, leading to truncated transcription, also called cryptic transcription. This mechanism triggers cryptic ageing which is caused by accumulation of truncated transcript and related non-functional proteins. After proving the restoration of normal DMNT3 expression in the context of aged skin cells, we wanted to know if this can lead to an improvement of the quality of the transcription by quantifying the truncated / cryptic transcripts. As a result, we demonstrated that cryptic transcription is significantly overcome as observed by reduction of truncated transcripts number (isoforms) after treatment with micronhyal on aged skin cells, reaching the same level as young one.

[0837] Materials and methods

[0838] Cell treatment

[0839] Skin cells culture and treatment was performed according to Example 2. Cryptic transcription analysis

[0840] For the sequencing study, an Iso-Seq® protocol was used to capture aberrant transcripts resulting from cryptic transcription. This protocol involves the selection of all polyadenylated RNA, as aberrant transcripts resulting from cryptic transcription have followed the classical maturation steps. Then, a sequencing is performed using PacBio technology, which enables the acquisition of full-length transcripts. Subsequently, the different isoforms of transcripts produced by control and prematurely aged keratinocytes were compared using bioinformatics analysis.

[0841] Results

[0842] The sequencing study revealed a significantly higher number of aberrant transcripts in prematurely aged keratinocytes (+36%*), which are transcripts that can lead to the production of dysfunctional proteins. These findings suggest that the dysregulation of transcriptional fidelity in prematurely aged keratinocytes is associated with the generation of aberrant transcripts, contributing to the production of dysfunctional proteins (Figure 4).

[0843] As a part of the mechanisms involved in longevity of cells, we identified diverse nuclear pathways to target. Genomic instability is the delicate balance between DNA damages (mutation, deletion, breakage etc.) and DNA repair mechanisms. Among them, PARP family (Poly ADP ribose polymerase) plays an essential role. PARP-1 is the most important member and plays dominant roles in DNA repair pathways. It has been known for a long time that PARP is critical for single strand break (SSB) repair and base excision repair (BER) pathways. Boosting the expression or activity of PARP1 is a way to decelerate cell ageing and preserve genomic integrity.

[0844] Telomere attrition, a type of DNA damage at the end of chromosomes (telomeres), contributes to ageing. Replicative DNA polymerases are unable to complete the telomere regions. As a result, shortening of telomere regions after several cell division is observed, which induces cell ageing (senescence). Reverse transcriptase activity of telomerase (an active ribonucleoprotein), can elongates telomeres and maintain their adequate length. Therefore, reactivating telomerase expression and activity is a way to decelerate ageing.

[0845] Epigenetic mechanisms like Histone acetylation involving deacetylase or acetyl transferase enzymes are key for controlling the correct gene expression. Epigenetic mechanisms are very dynamically influenced by environmental factors controlling the gene expression. Ageing is associated with loss of control of this mechanism, inducing uncontrolled regulation of gene expression. Among the key players, Sirtuin-1 (SIRT-1) is a deacetylase enzyme that regulates and stabilizes chromatin structure. They modulate the activity of telomerase to maintain telomere length and integrity. Restoration of SIRT-1 expression or activity is a way to control gene expression and limit deceleration of ageing.

[0846] In order to demonstrate the impact of Micronhyal on improvement of cell longevity, we evaluated its impact on PARP1, Sirtuin-1 and finally Telomerase activity.

[0847] Example 7: Sirtuin-1 expression and activity

[0848] We worked on aged skin cells (from a 67-year-old donor) in order to evaluate firstly the Sirtuin-1 expression and activity in presence of Micronhyal.

[0849] We observed that Micronhyal can significantly increase both expression and activity of sirtuin-1, which contributes to restoring the normal gene expression and related functions and consequently preserving the integrity of telomeres.

[0850] Materials and methods

[0851] Normal human Keratinocytes from the donor were incubated for 24 hours in absence (control) or in presence of reference product or increasing concentrations of test compound, which was Micronhyal 0.005 mg / ml and 0.01 mg / ml.

[0852] Test compounds preparation

[0853] The test compound named Micronhyal was directly solubilized at Img / ml in incubation medium. The solution obtained was then diluted in the incubation medium in order to reach the different concentrations described above.

[0854] Measurement of Total Sirtuins activity

[0855] At the end of the incubation period, nuclear proteins were extracted from cell monolayer. Total sirtuins activity was then quantified using a sensitive and specific enzymatic activity measurement kit (Abeam kit under the reference AB 156915).

[0856] Measurement of SIRT-1 expression

[0857] At the end of the incubation period, nuclear proteins were extracted from cell monolayer. SIRT-1 expression was then quantified using a sensitive and specific ELISA kit (Abeam kit under the reference AB 171573).

[0858] Results

[0859] After 24h of incubation we observed a dose-dependent and significant increase of Siturin-1 expression, up to +136%. In parallel, activity measurement indicated a significant increase in SIRT1 activity, up to 19%, in a dose-dependent manner. This data shows the ability of Micronhyal in restoring a normal epigenetic control of gene expression through Sirtuin-1 (Figure 5).

[0860] Example 8: PARP-1 expression and activity

[0861] We also showed that Micronhyal significantly increase expression and activity of PARP-1, which is involved in DNA repair mechanism.

[0862] Materials and methods

[0863] Cell culture and treatment was performed according to Example 5.

[0864] Measurement of P ARP activity

[0865] At the end of the incubation period, nuclear proteins were extracted from the cell monolayer. PARP activity was then quantified using a sensitive and specific enzymatic activity measurement kit (R&D system kit under the reference 4677-096-K-104).

[0866] Measurement of PARP-1 expression

[0867] At the end of the incubation period, nuclear proteins were extracted from the cell monolayer. PARP-1 expression was then quantified using a sensitive and specific ELISA kit (Abeam kit under the reference AB285289).

[0868] Results

[0869] After 24h of incubation we observed a dose-dependent and significant increase in PARP1 expression up to +57%. In parallel, activity measurement showed a significant increase in PARP1 activity, up to 52%, in a dose-dependent manner. These data indicate that Micronhyal induces repairing DNA damages through PARP-1 pathway (Figure 6).

[0870] Example 9: Telomerase expression and activity

[0871] Finally, we evidenced that Telomerase expression and activity were significantly boosted by treatment with Micronhyal.

[0872] Materials and methods

[0873] For this study, normal human keratinocytes are cultured in the absence ("basal control") or presence of the active ingredient MAD034 [10 pg / ml Micronhyal] over 2 passages between P2 and P4, i.e. a contact time of 5 days (120 h). After this incubation time at 37 °C + 5% CO2, the cell pellets are collected and stored at -80 °C until DNA extraction is carried out using the DNeasy kit (Qiagen) and qPCR analysis is performed.

[0874] During this study, the control and the sample are tested in quadruplicate. Results

[0875] Our results demonstrated a significant increase in Telomerase expression (up to 30%) and its activity (by +11%) in an aged skin cell model. Our result evidenced that Micronhyal protects against Telomere shortening by reactivate Telomerase activity on aged skin cells (Figure 7).

[0876] Example 10: DNA protection and production of type I collagen

[0877] Our data demonstrated that Micronhyal significantly increases the expression and activity of key players involved in DNA protection mechanism as a proof of performant property.

[0878] On top of this activity, we also demonstrated that Micronhyal can stimulate the production of type I collagen from fibroblasts.

[0879] Material and methods

[0880] Evaluation of Anti -ageing Activity

[0881] The aim of this study was to evaluate the anti-ageing activity of the pigment of the invention. The anti-ageing activity was evaluated by the quantification of type I pro-collagen release in premature aged condition, which was mimicked by chemical treatment (H2O2). Treatment with H2O2 is largely described in the literature to induce cells’ premature ageing through the induction of senescence. In the present study, this condition was used as the “aged” condition.

[0882] Cell Culture and Treatment

[0883] The cell culture was done on primary cells isolated from biopsies. Normal Human Dermal Fibroblasts (NHDFs) were seeded in a 96-wells black plate with glass bottom at 10’000 cells per well in triplicate. The cells were incubated for 48 hours in complete medium (DMEM medium, Gibco) supplemented with 10% fetal calf serum (FCS, Biowest) and 1% antibiotics (Sigma- Aldrich) at 37 °C with 5% CO2.

[0884] At the end of the incubation, the cells intended for the “aged” condition were stressed by treatment with hydrogen peroxide (H2O2, Sigma-Aldrich) at 500 pM for 2 hours at 37 °C with 5% CO2.

[0885] Cells were then rinsed twice with PBS (Gibco) and incubated for 72 hours in basal medium (DMEM medium without FCS) supplemented with 1% antibiotics with the following conditions:

[0886] The actives were diluted in culture medium. Skin cells untreated and cultivated with basal medium were used as a negative control. Cells were then incubated for 72 hours at 37 °C, 5% CO2. At the end of the culture, a collagen I immunostaining assay was performed as de-scribed below.

[0887] Collagen I Immunostaining

[0888] Cells were fixed for 5 minutes with 2% paraformaldehyde (PAF), rinsed with PBS and permeabilized for 15 minutes with 2% PAF + 0.5% Triton X100. Cells were then rinsed again with PBS and non-specific sites were saturated for 1 hour with a saturation solution (3% bovine serum albumin + 0.1% Tween 20). Cells were incubated overnight at 4 °C with a primary anticollagen I antibody from rabbit (Abeam) diluted at 1 :500 in saturation solution diluted 1 : 10.

[0889] On the next day, the primary antibody was removed and the cells were rinsed three times with washing solution (PBS + 0.3% Triton XI 00) and two times with PBS before being incubated for 2 h 30 min at room temperature with a secondary anti-rabbit antibody Alexa Fluor 488 diluted at 1 :500 and Hoechst 33342 diluted 1 :5000 in saturation solution diluted 1 : 10. After secondary antibody incubation, the antibody solution was removed and cells were rinsed two times with PBS. Pictures were taken with PICO Cell Reporter Xpress (Molecular Devices) in DAPI and Green channels in each well. Automatized cell scoring was performed, and the % of cells expressing collagen I was compared for each of the conditions.

[0890] Statistical Analysis

[0891] A Shapiro-Wilk normality test was performed to evaluate whether the data follow the Gaussian Law. The results did not follow the Gaussian Law.

[0892] As a consequence, a non-parametric statistical analysis was performed by Kruskal-Wallis ANOVA followed by Mann Whitney U test. Results were considered significant with p<0.05 with *, p<0.01 with ** and p<0.001 with ***.

[0893] Results

[0894] Using premature ageing skin model induced by oxidative stress in vitro, we demonstrated that Micronhyal is able to restore collagen I neosynthesis as observed by the significant increase of collagen I, up +48%. These results demonstrated anti-ageing activity of Micronhyal by boosting collagen I, the main collagen found in the skin (Figure 8). Example 11 : specificity of Micronhyal on DNMT3B expression

[0895] This example is on demonstration of the specificity of Micronhyal on DNMT3B expression in comparison to other HA grades (Primalhyal ultrafiler (Acetylated HA), Primalhyal 50 (low MW HA) and Primalhyal 300 (intermediate MW HA)).

[0896] Material & Methods

[0897] Cells culture and treatment

[0898] Cell culture and treatment was identical to the same in Example 2, and the test compounds were Micronhyal (0.005 and O.Olmg / ml), PUF Img / ml, P50 and P300: 5mg / ml.

[0899] Test compounds preparation, Measurement of DNMT3B expression, and Protein quantification for data normalization was performed in the same way as in Example 2. Data were expressed in quantity of DNMT3D expression per quantity of total protein and mg of HA.

[0900] Results

[0901] We compared different grade of HA with Micronhyal on DNMT3B expression in order to know if this activity is specific to Micronhyal or not. Each HA was tested at the highest non cytotoxic concentration and expression of DMNT3B is expressed related to mg of HA for each product. Our data demonstrated that DMNT3B is drastically expressed by Micronhyal while other HA grades showed very weak impact. Interestingly, Micronhyal was significantly better than each HA tested grades.

[0902] Our result showed that the impact of HA on DMNT3B is specific to Micronhyal and not found with other type of HA grade (Figure 9).

[0903] Example 12: clinical evaluation

[0904] This Example relates to clinical evaluation on the anti-ageing and sleep wrinkles properties of Micronhyal.

[0905] 1. Aim of the study

[0906] The aim of the study was to evaluate the anti-aging properties and sleep wrinkles of the active ingredient Micronhyal at 0.5% compared to a placebo with twice-daily application of a cream on the face after 28 and 56 days on panellists with fine lines and wrinkles. It was measured by:

[0907] • Illustrative photos with a camera, and

[0908] • Illustration photos and facial analysis were taken by Visia CR 2.3® In fact, wrinkles due to gravity are oriented downwards. Conversely, sleep wrinkles are oriented laterally due to compressive forces (pillow / head weight). See Anson G, et al., Aesthet Surg J. 2016 (PMID: 27329660), which is incorporated herein by reference in its entirety.

[0909] For this study, two analyzes were carried out: i) global wrinkle focus on the entire wrinkle; and ii) focus on sleep wrinkles.

[0910] Two products have been tested, as described in the table below.

[0911] Table 1. The products used for clinical evaluation.

[0912] 2. Study description

[0913] 2.1. Study characteristics

[0914] The study was a double-blind and randomized study. The assessment was based on an intra-subject comparison by the comparison of the results obtained with 0.5% Micronhyal cream (active product) and the cream placebo on face.

[0915] The measurements were taken in a controlled-atmosphere room 22±2 °C and 50±10%.

[0916] Subjects were recruited from Givaudan Active Beauty’s database. The study was performed in healthy subjects who met all of the inclusion criteria and exclusion criteria presented below.

[0917] All subjects received verbal and written information concerning the study. This information emphasized that participation in the study is voluntary and that the subject may withdraw from the study at any time and for any reason. All subjects were given the opportunity to ask questions about the study and were given sufficient time to consider their participation before consenting.

[0918] The subject’s written informed consent to participate in the study was obtained prior to any study- related procedure being carried out.

[0919] 2.2. Population description

[0920] 40 volunteers (aged between 51 and 74, mean age: 61.4 ± 6.9 years) with fine lines and wrinkles on the face and corresponding to the inclusion and exclusion criteria previously defined in the protocol were involved in the study. In order to preserve the anonymity, each subject was identified by a subject code and a subject number. The subject code consisted of the GP code (Givaudan Panellist) followed by a number representing the volunteer.

[0921] Subjects were identified by their subject numbers, subject code, gender and date of birth only.

[0922] 2.3. Inclusion and exclusion criteria, study limits

[0923] 2.3.1. Inclusion criteria

[0924] Subjects must satisfy all of the following criteria for inclusion in the study.

[0925] • Be a woman for working on standardized population because ageing signs are not the same according to gender.

[0926] • Be aged between 50 to 75 years.

[0927] • Northern European origin for working on standardized population because ageing signs are not the same according to ethnic origin (Phototyp I, II or III).

[0928] • Have wrinkles and fine lines on the face.

[0929] • Be a healthy, mobile volunteer.

[0930] • Subj ect having received fair and complete information on the methods of implementing the study and, in particular, on the investigation methods and their consequences, and, on the other hand, complies with the requirements of the Data Protection Act.

[0931] • Subject having given written informed consent both to carrying out the study and to implementing the processing of personal data that it involves.

[0932] • Cooperative subject, informed of the need for tests and the duration of controls in order to fully follow the study implementation methods established by Givaudan

[0933] • Have signed the non-opposition form.

[0934] • Understand French, so as to be able to read the documents made available, and freely comply with the instructions provided as a way to verify the capacity to take decision by themselves (with a particular attention exceptionally for volunteers aged over 65 years considered as elderly population).

[0935] 2.3.2. Exclusion criteria

[0936] Any subjects meeting any of the following exclusion criteria were excluded from the study.

[0937] • Have omitted to sign the non-opposition form. • Present skin problems in the area of interest (i.e., face and neck) that could alter the research results that could alter the research results such as irritation, redness, itching, related to skin disorders (eczema, atopic dermatitis, ...).

[0938] • Be pregnant or breast-feeding because our ingredient is not verifying for this type of population.

[0939] • Present symptoms of recent intense exposure to the sun or to UV radiation like sunburn, redness etc.

[0940] • Have been treated by a dermatologist in the areas of interest over the last 6 months (for example, this care can include the application of self-tanning products, peeling, depigmentation or the injection of cosmetic products).

[0941] • Present a chronic or acute affection and / or use a topical or systemic treatment that the principal investigator believes could influence the research results.

[0942] • Have a systemic disease or any dermatitis that the principal investigator considers likely to influence the research results.

[0943] • Present a history of allergy or hypersensitivity reactions to one of the ingredients in the product being studied.

[0944] • Be incapable of following the requirements of the protocol.

[0945] • Have participated in or have the intention of participating in a study that could interfere with the proposed research.

[0946] • Not having applied anti-aging / anti -wrinkle cream to the face and neck in the 15 days preceding the start of the study.

[0947] 2.4. Product application

[0948] The volunteers applied a pressure of product (placebo cream or active cream) twice a day (morning and evening) to the face and neck for 56 days).

[0949] The application of products was randomized:

[0950] • 20 volunteers applied a cream containing Micronhyal at 0.5%;

[0951] • 20 volunteers applied a placebo cream.

[0952] 2.5. INCI formulas

[0953] The INCI formula is as follows: AQUA / WATER, CETYL ALCOHOL, GLYCERYL STEARATE, PEG-75 STEARATE, CETETH-20, STEARETH-20, ISODECYL NEOPENT ANO ATE, PENTYLENE GLYCOL, CAPRYLIC / CAPRIC TRIGLYCERIDE, ± MICRONHYAL, CITRIC ACID, DIMETHICONE, SODIUM BENZOATE, AMMONIUM ACRYLOYDIMETHYLTAURATE / BEHENETH-25 METHACRYLATE CROSSPOLYMER, FRAGANCE.

[0954] 3. Study protocol

[0955] From Time DO to D56:

[0956] First check-up at DO (Weeks 38 and 39):

[0957] • Verification that the volunteer fully understands the study.

[0958] • Signing of the agreement by the volunteer.

[0959] • Acclimatization of 15 minutes in the tempered room at 22±2 °C and 50±10%.

[0960] • Measurements taken using Visia CR 2.3® and illustrative photos with a camera (with and without pillow).

[0961] • Products distribution and application of cream.

[0962] - Check-up at D28 (Weeks 42 and 43):

[0963] • Acclimatization of 15 minutes in the tempered room at 22±2 °C and 50±10%.

[0964] • Measurements taken using Visia CR 2.3® and illustrative photos with a camera (with and without pillow).

[0965] - Check-up at D56 (Weeks 46 and 47):

[0966] • Acclimatization of 15 minutes in the tempered room at 22±2 °C and 50±10%.

[0967] • Measurements taken using Visia CR 2.3® and illustrative photos with a camera (with and without pillow).

[0968] 4. Data analysis

[0969] The following data was analyzed: 4.1. Calculation formula

[0970] Comparison Before / After

[0971] A = TZti - TZtO

[0972] A% = TZti -TZtO X100

[0973] TZtO

[0974] With TZ: value obtained on the zone treated by the tested product tO: before product application ti: at each measurement time after product application

[0975] Comparison Product / Placebo

[0976] AA = A Pdt- A Pla

[0977] AA% = A% Pdt- A% Pla with: Pdt: value obtained on the zone treated by the tested product Pla: value obtained on the zone treated by the placebo tO: before product application ti: at each measurement time after product application

[0978] 4.2. Statistical method

[0979] Comparison Before / After

[0980] Comparison Product / Placebo

[0981] The software used for statistical analysis was GraphPad.

[0982] 5. Methods

[0983] 5.1. Vista CR 2.3®

[0984] Through Visia CR 2.3® from Canfield imaging systems, digital photography of the face was done at different times with repositioning at DO. The control of the repositioning takes place directly on data...

Claims

CLAIMS1. A recombinant yeast cell producing hyaluronic acid (HA) wherein the recombinant yeast cell comprises:(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;(c) one or more recombinant nucleic acids, under the control of a pCCW12.Spr promoter, encoding a polypeptide having hyaluronidase activity wherein the polypeptide having hyaluronidase activity comprises a secretion signal so that hyaluronic acid, in particular 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 a glutamine synthetase (GLN1) activity; and / or(ii) one or more disrupted endogeneous nucleic acids encoding a glutamate synthase (GLT1); wherein said recombinant yeast cell belongs to the Saccharomyces genus, or to the Candida genus, or to the Kluyveromyces genus, or to the Ogataea genus, or to the Yarrowia genus, or to the Debaryomyces genus, or to the Ashbya genus.

2. The recombinant cell according to claim 1, wherein the polypeptide having hyaluronidase activity further comprises an anchoring signal.

3. The recombinant cell according to claim 1 or 2, wherein the hyaluronic acid has an average molecular weight of less than about 5 kDa.

4. The recombinant cell according to any one of claims 1 to 3, wherein the hyaluronic acid has an average molecular weight of more than about 800 Da.

5. The recombinant cell according to any one of claims 1 to 4, wherein the hyaluronic acid has a median molecular weight of less than about 5 kDa, more particularly of less than about 3 kDa, e.g. about 2.8 kDa.

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

7. The recombinant cell according to 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 Cupiennius salei. Loxosceles intermedia, Hirudo nipponia, Bothrops atrox or Tityus serrulatus.

8. The recombinant cell according to 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 CviKl, Chlor ella virus IL-5-2sl , Chlor ella virus CZ-2, Chlorella virus CVG-1, Xenopus laevis or Pasteurella mullocida, and is in particular obtained or derived from at least one of Streptococcus zooepidemicus, Chlorella virus PBCV1, Chlorella virus CviKl, Chlorella virus IL-5 -2s 1, Chlorella virus C7.-2, Chlorella virus CVG-1 or Xenopus laevis.

9. The recombinant cell according to 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, and in particular from Arabidopsis thaliana or Chlorella virus PBCV1.

10. The recombinant cell according to any one of claims 1 to 9, wherein the recombinant cell comprises at least one recombinant nucleic acid encoding one or more of:(i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity;(ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity;(iii) a polypeptide having Phosphoglucomutase- 1 (PGM1) activity; and / or(iv) a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase (UGP1) activity; and / or(v) a polypeptide having Glucosamine-6-phosphate N-acetyltransferase (GNA1) activity; and / or(vi) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity.

11. The recombinant cell according to any one of claims 1 to 10, wherein the recombinant cell belongs to the Saccharomycetales order, and is in particular 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, Kluyveromycespolysporus, Kluyveromyces thermotolerens, Ogataea polymorpha, Yarrowia lypolylica, Debaryomyces hansenii. and Ashbya gossypii, and is preferably Saccharomyces cerevisiae.

12. A method of producing hyaluronic acid having i) a molecular weight of more than about 800 Da and less than about 5 kDa; and / or ii) a median molecular weight of less than about 5 kDa, more particularly of less than about 3 kDa, e.g. about 2.8 kDa, comprising:(a) cultivating a recombinant cell as defined in any one of claims 1 to 11 in a cultivation medium for a time sufficient to produce said hyaluronic acid; and(b) optionally isolating or recovering the hyaluronic acid from the recombinant cell and / or from the cultivation medium.

13. The method according to claim 12, wherein the recombinant cell comprises at least one recombinant nucleic acid encoding one or more of:(i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity;(ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity;(iii) a polypeptide having Phosphoglucomutase- 1 (PGM1) activity;(iv) a polypeptide having UTP-glucose-1 -phosphate uridylyltransferase (UGP1) activity;(v) a polypeptide having Glucosamine-6-phosphate N-acetyltransferase (GNA1) activity; and / or(vi) a polypeptide having phosphoacetylglucosamine mutase (PCM1) activity.

14. The method according to claim 12 or 13, wherein the recombinant cell is a member of the genus Saccharomyces, and in particular is Saccharomyces cerevisiae.

15. The method according to any one of claims 12 to 14, wherein the time sufficient to produce the hyaluronic acid is a period of from about 35 hours to about 60 hours, preferably from about 40 hours to about 55 hours, in particular about 52 hours.

16. The method according to any one of claims 12 to 15, wherein the molecular weight of the hyaluronic acid is controlled by regulating the pH of the cultivation medium.

17. Hyaluronic acid obtained or obtainable from a recombinant cell of any one of claims 1 to 11 or from the method according to any one of claims 12 to 16.

18. A cultivation medium comprising the hyaluronic acid according to claim 17.

19. A composition comprising the hyaluronic acid according to claim 17, and optionally a carrier.

20. A composition according to claim 19, wherein said composition is a cosmetic preparation, and in particular a skin care composition.

21. The composition according to claim 20, wherein the hyaluronic acid is present in an amount of 0.1 to 1.0% (w / v), more preferably 0.2 to 0.7% (w / v), e.g. about 0.5% (w / v), of the composition.

22. Use of the composition of claim 20 or 21, or of the hyaluronic acid of claim 17, for anti-ageing treatment and / or anti-wrinkle treatment, more preferably for treatment of sleep wrinkles.

23. A cosmetic method of skin treatment, comprising the step of applying to the skin an effective amount of the composition of claim 20 or 21, or of the hyaluronic acid of claim 17, wherein the skin treatment is preferably for anti-ageing treatment and / or anti-wrinkle treatment, and more preferably for treatment of sleep wrinkles.

24. The use according to claim 22, or the method of claim 23, wherein the treatment leads to one or more of i) an increase in expression of DMNT3B; ii) a reduction in aberrant transcripts; iii) an increase in expression and activity of sirtuin-1; iv) an increase in expression and activity of PARP- 1; v) an increase in expression and activity of telomerase; vi) an increase in production of type-1 collagen by fibroblasts.

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