High Expression of LIP1 in Pichia pastoris

BEI Jin-Long, WANG Jin-Wen, WANG Xun-Zhang*, LONG Qin-Xing, YANG Lin, DENG Ying-Yu

( The State Key Laboratory for Biological Control, Biopharmaceutical Center, Zhongshan University, Guangzhou 510275, China )

Abstract The gene of LIP1, the most important isoenzyme of Candida rugosa lipase (CRL), was artificially synthesized according to its mature peptide sequence. It consisted of 20 codons of preference in Pichia pastoris. The artificial gene was cloned into methanol-inducible expression vector pPICZaA, and constitutive expression vector pGAPZaA, respectively. The linearized recombinant plasmids were transformed into chromosome of Pichia pastoris SMD1168H strain by electroporation. The abilities of expressing LIP1 in both transformed yeasts had been compared, and the yeast transformed with pGAPZaA was more efficient than pPICZaA. A recombinant yeast strain named CHT-II expressed LIP1 constitutively, and was the most efficient one. Some enzymetic properties of the recombinant LIP1 were also determined. CHT-II secreted LIP1 into supernate at a level of 2.00´105 u/L after 72 h (the cells had been transferred to fresh culture medium at the 24th h). After optimizing the conditions for high cell-density fermentation, the selected yeast strain could secrete LIP1 into supernate at a level of 1.395´106 u/L after 72 h. The results indicated that this modification of lip1 gene was successful.

Key words artificial synthesis; CRL; LIP1; Pichia pastoris; constitutive expression

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