Constitutive expression of lipase in Pichia pastoris and the high throughput screening method

Abstract:

[Objective] This study is aimed to constitutive express lipase in Pichia pastoris and establish an efficient method for high-throughput screening constitutive expression lipase gene in P. pastoris using olive oil-rhodamine B plate. [Methods] The GAP gene promoter was amplified from the plasmid of pGAPZαA and inserted into the inducible expression vector pPIC9K-proRCL resulting in constitutive expression of the lipase gene-proRCL. Based on the homologous recombinant region of AOX1 gene, proRCL from Rhizopus chinensis CCTCC M201021 was recombined into chromsome of P. pastoris GS115 by double-crossover integration event resulting in constitutive expression of the single-copy lipase gene from R. chinensis. [Results] The activity of lipase reached its peak (130 U/ml) after fermentation of 144 h. An efficient method was established for high-throughput screening constitutive expression lipase gene in P. pastoris using olive oil-rhodamine B plate. [Conclusion] The method was very convenient with advantages of shortened screening cycle from 12 d to 3 d without the interferences of multi-copy mutants. This method also established the foundation of the screening of library by directed evolution.