[Burkholderia cepacia lipase gene modification and its constitutive and inducible expression in Pichia pastoris]

To achieve fast, safe and stable expression of Burkholderia cepacia lipase in Pichia pastoris.

We first amplified B. cepacia lipase gene, and then analyzed the codon usage of B. cepacia and Pichia, lipase gene signal peptide with bioinformatics methods. On this basis, we applied the overlap PCR to modify the lipase gene and finally got the optimized gene with Pichia codon usage and lower G + C content. Subsequently, we cloned the optimized and wild lipase gene into vector pGAPZalpha and pPIC9K, respectively. As a result, constitutive expression vector pGAPlipW, pGAPlipO and inducible expression vector pPIClipW, pPIClipO were obtained. Finally, we electroporated these expression vectors into GS115, and therefore, got a series of engineering strains. After fermentation and NTA resin purification, the enzymatic properties of lipase were studied.

The lipase activities of pPIClipW, pPIClipO, pGAPlipW and pGAPlipO were 37.8 U/mL, 129.5 U/mL, 40.2 U/mL, and 184.3 U/mL, respectively. The optimized lipase activity increased 4.6-fold. Enzymatic properties study showed that the optimal temperature and pH was 60 degrees C and 9.0, respectively. The lipase was rather stable at 40 degrees C - 65 degrees C and pH 6.0-pH10.0.

After overlap PCR modification, the lipase expression efficiency in Pichia was significantly increased, which indicates that the overlap PCR modification is a potential strategy for lipase overexpression. The GAP promoter is more appropriate than the AOX1 promoter for the B. cepacia lipase expression. Additionally, the recombinant lipase whose enzymatic properties were identical to the wild type satisfies the needs of industrial application.