Expression Of HS-L16Lipase Gene And Construction Of Vectors Containing Multiple Copies Of Expression Cassette

Lipases, which catalyse the hydrolysis, aminolysis, transesterification, esterification of long chain triglycerides, play an important role in the turnover of lipids which are water-insoluble compounds. Recently, microbial enzymes have got much more attention as the development of enzyme engineering technology. This is mainly due to that microbial enzymes are often more useful than enzymes derived from plants or animals because of the great variety of catalytic activities available, the high yields possible, ease of genetic manipulation, regular supply due to absence of seasonal fluctuations and rapid growth of microorganisms on inexpensive media. Microbial enzymes are also more stable than their corresponding plant and animal enzymes and their production is more convenient and safe.Wild type HS-L16(Penicillium crustosum) derived by our own lab is not suitable for large scale industrial application for its low lipase activity. Therefore, this article aims to construct a recombinant strain with lipase gene lip16from HS-L16and host strain X-33, which can express Hp16at a high level. The main work as follows:(1) Primers adding with restriction enzyme cutting sites were synthesized to clone Iip6from HS-L16, purified Up16fragment was then linked with expression vector pPICZaA to form the recombinant plasmid pPICZaA-lip16. The plasmid was integrated in the genome of X-33through electroporation. Positive colonies were cultivated in the BMMY induced with1%methanol. After the flask fermentation for96h, lipase activity reached the highest level for62.42U/mL. It was about10times higher than the lipase activity of wt-HS-L16which was only5.88U/mL. The result dedicated that Pichia pastorios eukaryotic expression system can be well used for the expression of heterologous lipase gene.(2) With the purpose of further improving the expression level of lip16in P. pastorios, vectors containing2&4copies of target gene expression cassettes were constructed respectively, based on that BamH Ⅰ&Bgl Ⅱ in the expression vector pPICZaA possess the same cohesive terminus. After integration and cultivation, the lipase activity of positive colonies was improved apparently. The activity of the double copies colony reached121.67U/mL at96h; it was2times higher than the single copy. And lipase activity of the strain containing four copies of Hp16(165.45U/mL) was2.6 times higher than single copy. Fluorescent quantitative PCR was carried out to measure the mRNA level of the target gene in the colonies with multiple copies. The result also showed that mRNA level of lip16was improved correspondingly with the copy number. It can be concluded that the strategy of multiple copies was efficient in the improvement of protein expression. In addition, experimental result also indicates that the total lip16expression level in P. pastorios integrant harboring4copies of the expression cassette was maintained over five generations, which attested to the stability of the lip16expression cassette in the P. pastoris genome.