Hyaluronic acid-producing recombinant cells
The present invention relates to the field of bio-production of hyaluronic acid. There is a need in the art for hyaluronic acid production methods allowing its highly efficient synthesis and secretion. The solution proposed in the present invention is the use of a genetically modified cell comprising many modifications as described in the present text. The present invention further proposes methods allowing the bio-production of hyaluronic acid having a controlled molecular weight using the genetically modified cells of the invention.
1 . A recombinant host cell (i) or a Saccharomyces cerevisiae recombinant yeast cell (ii) producing hyaluronic acid (HA) wherein the recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii) 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 encoding a polypeptide having hyaluronidase activity wherein the polypeptide having hyaluronidase activity comprises a secretion signal so that hyaluronic acid-is produced by the recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii), wherein the hyaluronic acid produced by the recombinant host cell (i) is of a desired molecular weight (HAMW),
and wherein the polypeptide having hyaluronidase activity comprised in the recombinant host cell (i) further comprises an anchoring signal; 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).
2 . The S. cerevisiae recombinant yeast cell (ii) according to claim 1 wherein the hyaluronic acid is of the desired molecular weight (HAMW).
3 . The recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii) according to claim 1 , wherein the molecular weight of the HA is less than 50 kDa, or in a range of 20 kDa to 50 kDa.
4 . The recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii) according to claim 1 , wherein the molecular weight of the HA is greater than 50 kDa, or in a range of 50 kDa to 250 kDa or is greater than 100 kDa, or is in a range of 100 kDa to 1500 kDa.
5 . The recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii) according to claim 1 , wherein the nucleic acid encoding a polypeptide having a glutamine synthetase activity is obtained or derived from Saccharomyces cerevisiae.
6 . The recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii) according to claim 1 , 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 and Tityus serrulatus.
7 . The recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii) according to claim 1 , wherein the nucleic acid encoding a polypeptide having hyaluronan synthase activity is obtained or derived from Streptococcus zooepidemicus, Chlorella virus PBCV1 , Chlorella virus CviK1 , Chlorella virus IL-5-2s1, Chlorella virus CZ-2, Chlorella virus CVG-1, Xenopus laevis or Pasteurella multocida.
8 . The recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii) according to claim 1 , 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 and Streptococcus zooepidemicus.
9 . The recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii) according to claim 1 , wherein the recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii) further comprises a recombinant nucleic acid encoding one or more of:
(i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity; and/or
(ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity, and/or
(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.
10 . The recombinant host cell (i) according to claim 1 , wherein the recombinant host cell belongs to the Saccharomycesles order, and is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces castelli, Candida albicans, Candida glabrata, Candida tropicalis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polysporus, Kluyveromyces thermotolerens, Ogataea polymorpha, Yarrowia lypolytica, Debaryomyces hansenii , and Ashbya gossypii.
11 . A method of producing hyaluronic acid (HA) of a desired molecular weight (HAMW) comprising:
(a) cultivating the recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii) as defined in claim 1 in a cultivation medium for a time sufficient to produce hyaluronic acid (HA) of a desired molecular weight; and
(b) optionally isolating or recovering the hyaluronic acid (HA) from the recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii) and/or from the cultivation medium.
12 . The method according to claim 11 , wherein the HA has a molecular weight of from 20 kDa to 50 kDA, or from 20 kDa to 30 kDa, or from 30 kDa to 50 kDa, or from 50 kDa to 150 kDa, or from 150 kDa to 1500 kDa.
13 . The method according to claim 11 , wherein the recombinant host cell (i) or the S. cerevisiae recombinant yeast cell (ii) comprises at least one recombinant nucleic acid encoding one or more of:
(i) a polypeptide having glutamine-fructose-6-phosphate amidotransferase (GFA1) activity; and/or
(ii) a polypeptide having UDP-N-acetylglucosamine pyrophosphorylase (QRI1) activity, and/or
(iii) a polypeptide having Phosphoglucomutase-1 (PGM1) activity; and/or
(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 11 , wherein the time sufficient to produce hyaluronic acid (HA) of the desired molecular weight is a period of from 35 hours to 50 hours, or from 40 hours to 50 hours or is 48 hours.
15 . The method according to claim 11 , wherein the molecular weight of the hyaluronic acid is controlled by regulating the pH of the cultivation medium.
16 . The method according to claim 11 , wherein the method is carried out on an industrial scale where the cultivation medium is at least 100 L, in a range of 1000 L to 3000 L, or is 10,000 L, or is 100,000 L, or is 250,000 L.
17 . A method for producing hyaluronic acid comprising the steps of:
(a) culturing a recombinant host cell (i) or a S. cerevisiae recombinant yeast cell (ii) as defined in claim 1 in a culture medium; and
(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:
the nature and origin of the nucleic acid encoding the hyaluronidase of the recombinant yeast,
the nature and origin of the promoter controlling the expression of the nucleic acid encoding the hyaluronidase(s) of the recombinant yeast,
the presence of an anchoring and/or of a secretion signal associated with the encoded hyaluronidase(s) of the recombinant yeast,
the pH of the culture medium during the step of culturing the recombinant yeast, and/or
the duration of the culturing of the recombinant yeast.