ScienceAsia 33 (2007): 389-395 |doi: 10.2306/scienceasia1513-1874.2007.33.389

Cloning of Hyaluronan Synthase (sz-has) Gene from Streptococcus zooepidemicus in Escherichia coli

Boonsri Jongsareejit,^a* Amaret Bhumiratana,^b Masaaki Morikawa^c and Shigenori Kanaya^c

 
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 oligonucleotides designed 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 SDSPAGE 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, the plasmids pHAS-1 and pHAS-2, in which sz-hasA and sz-hasA with rare codon modifications at the 5’-terminus were ligated into the NdeI-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.

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^a Department of Microbiology, Faculty of Science, Silpakorn University, Nakhon Pathom 73000, Thailand.
^b Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
^c Department of Material & Life Science, Graduate School of Engineering, Osaka University, Osaka 565- 0871, Japan.

* Corresponding author, E-mail: boonsri@su.ac.th

Received 20 Apr 2006, Accepted 17 Aug 2007