GlcN-6-P synthase is an important enzyme involving in amino sugar biosynthesis, catalyses the complex reaction resulting in D-glucosamine-6-phosphate and L-glutamate. Glucosamine is manufactured as a nutraceutical product with application in treatment of osteoarthritic conditions in animals and humans. Currently, glucosa,mine is produced by acid hydrolysis of chitin, but the process suffers from poor product yield and limited raw material. Therefore, a cost-effective. method for producing glucosamine is needed. The purpose of this work was construction of recombinant S. cerevisiae expressing glucosamine-6-phosphate synthase gene (GFAI) for production of N-acetylglucosamine and glucosamine. The recombinant plasmid pESC-His/GFAl was constructed by ligation reaction of the S. cerevisiae GFAI gene from cloning vector pCR2.I/GFA1 and the expression vc::crtor pESC-His after their cleaving with Xhol and BamHI. The gene in recombinant E. coli was verified by restriction digestion of the isolated plasmids with Xhol and BamHI. pESCHis/GFAI was transformed to competent S. cerevisiae KY117 by electroporation and recombinant colonies harboring pESC-His/GFAI were selected by PCR using specific primers Gal10 matching with expression vector, and total DNA from transformants as template. Using SC-His medium and inducer galactose, heterologous enzyme produced ITom the recombinant KY117 was checked at different periods of induction time. The obtained result showed that the S. cerevisiae GFA1 gene was expressed best after 48 hours induction in KY117.
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