Cloning and Expression of an Indigenous Mesophile Lipase and Evaluation of Bacillus Codon Translation in Pichia pastoris under Control of Two Different Promoters

Document Type : Original Article

Authors

National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

Abstract

Lipases are versatile biocatalysts with a wide range of application in food, dairy, leather, paper, pharmaceutical, and detergent industries. In this study, mesophilic lipase gene from an indigenous Bacillus pumilus F3, which its gene had been sequenced and identified previously, was cloned and expressed in methylotrophic yeast Pichia pastoris and codon translation of lipase gene was evaluated in P. pastoris under the control of‌ two different promoters (alcohol oxidase (AOX1) and glyceraldehid phosphate dehydrogenase (GAPDH) promoters). In addition, the expression conditions of recombinant lipase F3 was optimized in P. pastoris expression system using BMMY medium at pH 3, 26ºC, and in 0.75% methanol. The 648 bp lipase gene with natural signal peptid sequence from B. pumilus F3 and the codon optimized lipase gene were cloned and expressed in methylotrophic yeast P. pastoris. The lipase gene was excised from the recombinant plasmid with BamHI, EcoRI enzymes and ligated into linearized pPIC9 and pGAP9 with the same enzymes. The recombinant plasmids were confirmed by the PCR and restriction enzyme digestion. The Bgl II linearized Ppic9 and pGAP9 recombinant plasmids were introduced into the yeast P. pastoris GS115 genome by electroporation and confirmed by PCR. Lipase expressing yeast was cultivated in a 250-ml shaking flask containing expression medium. Expression of lipase gene was confirmed using p-nitrophenyl palmitate test and SDS-PAGE. Codon optimized lipase was being expressed as well as native gene and the expression level was low in both cases. Also, these results suggest that protein structure is more important than codon preference in the expression of proteins such as lipases.

Keywords