Process for the production of hyaluronic acid in Escherichia coli or Bacillus megaterium
AssigneeFidia Farmaceutici S.P.A.
InventorsVincenza Corsa, Alessandro Negro, Sonia Bisicchia
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Abstract
A method of producing hyaluronic acid (HA) in Escherichia coli and Bacillus megaterium through episomal plasmid vectors wherein the gene is under the control of strong promoter T7, preferably under the control of strong promoter T7 of bacteriophage T7, and a system for the selection of stable bacterial strains producing high levels of hyaluronic acid, are provided.
Claims18
A process for the preparation of hyaluronic acid in Bacillus megaterium, comprising the following steps:
- (a) culturing transformed bacterial host cells of Bacillus megaterium, transformed in a stable way with the T7 RNA polymerase system under conditions suitable for the production of hyaluronic acid in the presence of xylose as an inductor, wherein said transformed bacterial host cells are characterised by being further transformed with:
- (i) at least one episomal plasmid vector comprising a sequence coding for the enzyme hyaluronan synthase and a sequence coding for the enzyme UDP-glucose dehydrogenase in tandem under the control of the strong inducible T7 promoter;
- or
- (ii) at least one episomal plasmid vector comprising a sequence coding for the enzyme hyaluronate synthase, a sequence coding for the enzyme UDP-glucose dehydrogenase, a sequence coding for the enzyme UDP-glucose pyrophosphorylase and a sequence coding for the enzyme glucose 6 phosphate isomerase, under the control of the strong inducible T7 promoter;
- (b) recovering hyaluronic acid from the culture medium,
- wherein the transformed bacterial host cells of Bacillus megaterium transformed in a stable way with the T7 RNA polymerase system and with plasmid vector (i) or (ii) able to produce hyaluronic acid of step a) are pre-selected on a xylose gradient;
- and
- the sequences coding for enzymes UDP-glucose dehydrogenase (tuaD), UDP-glucose pyrophosphorylase (gtaB) and glucose 6 phosphate isomerase (pgi) are obtained from Bacillus subtilis;
- and
- wherein the fermentation time is in the range between 80 and 160 hours and the product HA has a weight average molecular weight in the range 100-500 KDa or the fermentation time is in the range between 40 and 80 hours and the product HA has a weight average molecular weight in the range 500-1000 KDa or the fermentation time is in the range between 12 and 40 hours and the product HA has a weight average molecular weight in the range 1×106-3×106 D.
A plasmid vector comprising a strong inducible bacteriophage T7 promoter operationally linked to a sequence coding for a hyaluronan synthase enzyme and a sequence coding for a UDP-glucose dehydrogenase enzyme in tandem.
A plasmid vector comprising a strong inducible bacteriophage T7 promoter operationally linked to a sequence coding for a hyaluronate synthase enzyme, a sequence coding for a UDP-glucose dehydrogenase enzyme, a sequence coding for a UDP-glucose pyrophosphorylase enzyme and a sequence coding for a glucose 6 phosphate isomerase enzyme.
Dependencies are inferred from the claim text.