Methods for producing hyaluronan in a recombinant host cell

A method for producing a hyaluronic acid, comprising:

• (a) cultivating a Bacillus host cell under conditions suitable for production of the hyaluronic acid, wherein the Bacillus host cell comprises an artificial operon comprising a short “consensus” amyQ promoter having the sequence TTGACA for the “-35” region and TATAAT for the “-10” region operably linked to a hyaluronan synthase encoding sequence, a UDP-glucose 6-dehydrogenase encoding sequence, and a UDP-glucose pyrophosphorylase encoding sequence;
• wherein the hyaluronan synthase encoding sequence is (i) a nucleic acid sequence encoding a polypeptide comprising SEQ ID NO:
• 95;
• or (ii) a nucleic acid sequence which hybridizes under high stringency conditions with SEQ ID NO:
• 94 or its full-length complement;
• wherein the UDP-glucose 6-dehydrogenase encoding sequence is (i) a nucleic acid sequence encoding a polypeptide comprising SEQ ID NO:
• 12;
• or (ii) a nucleic acid sequence which hybridizes under high stringency conditions with SEQ ID NO:
• 11 or its full-length complement;
• wherein the UDP-glucose pyrophosphorylase encoding sequence is (i) a nucleic acid sequence encoding a polypeptide comprising SEQ ID NO:
• 22;
• or (ii) a nucleic acid sequence which hybridizes under high stringency conditions with SEQ ID NO:
• 21 or its full-length complement;
• and
• wherein high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, and washing three times each for 15 minutes using 2×SSC, 0.2% SDS preferably at least at 65° C.;
• and
• (b) recovering the hyaluronic acid from the cultivation medium.