Impact of gene dosage on the production of lipase from Rhizopus chinensis CCTCC M201021 in Pichia pastoris.

State Key Laboratory of Food Science and Technology, the Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China.

Applied biochemistry and biotechnology 2013 Feb

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In this work, the high-level expression of the lipase r27RCL was achieved by optimization of the lipase gene copy number in the host strain Pichia pastoris. The copy number of the lipase gene proRCL from Rhizopus chinensis CCTCC M201021 was quantified by real-time quantitative polymerase chain reaction and a range of Mut(+) P. pastoris strains carrying one, three, five, and six copies of proRCL were obtained. The maximum lipase activity was achieved at 12,500 U/mL by the five-copy recombinant strain after 96 h of methanol induction in the 7-L fermenter. However, the enzyme activity of the six-copy recombinant strain decreased remarkably. By transcription analysis of proRCL, ERO1, and PDI, it suggested that unfolded protein response seemed to be triggered in the highest copy recombinant strain after 24 h. Thus, elaborate optimization of foreign gene dosage was very important for the high-level expression of foreign proteins in P. pastoris.

Citation

Chong Sha, Xiao-Wei Yu, Fei Li, Yan Xu. Impact of gene dosage on the production of lipase from Rhizopus chinensis CCTCC M201021 in Pichia pastoris. Applied biochemistry and biotechnology. 2013 Feb;169(4):1160-72