Cloning of hyaluronan synthase (sz-hos) gene from Streptococcus zooepidemicus in Escherichia coli

Abstract

A 546-bp fragment of the sz-hasA gene encoding hyaluronan synthase (szHAS) from Streptococcus zooepidemicus (group C Streptococcus, GCS) was amplified by PCR with oligonucleotidesdesigned based on the conserved amino acid sequences of HASs from other organisms as primers. The entire sz-hasA gene was identified and cloned by Southern and colony hybridizations using this 546-bp fragment as a probe. Determination of the nucleotide sequence indicated that this gene encoded a protein with 417 amino acid residues (calculated molecular mass, 47.77 kDa). The amino acid sequence of szHAS was 74.2% identical to that of HAS from Strep. equisimilis. The overexpression of sz-hasA in Escherichia coli was detected by SDS-PAGE and confirmed by LC/MS-MS. To examine whether E. coli cells acquire an ability to synthesize hyaluronic acid (HA) upon transformation with plasmids bearing the sz-hasA gene, theplasmids pHAS-1 and pHAS-2, in which sz-hasA and sz-hasA with rare codon modifications at the 5′-terminus were ligated into the Ndel-BamHI sites of pET-28a, respectively, were constructed and used to transform E. coli BL21 (DE3), E. coli BL21 (codon+), and E. coli HMS 174 (DE3) plysS. Cultivation of these transformants, followed by induction for gene expression, revealed that the E. coli BL21 (codon+) transformants with pHAS-1 and E. coli HMS 174 (DE3) plysS transformants with pHAS-2 produced HA after 4 hr induction time at the maximum yield 16 μg/ml and 32.5 μg/ml, respectively. These are higher than the background levels in control bacteria, which were 5 μg/ml and 21.3 μg/ml, respectively.