南极假丝酵母脂肪酶B在毕赤酵母中的高效表达及其在生物柴油生产中的应用

[Objective]In this study,Pichia Pastoris was used as host to construct a CALB-expressing engineering strain to achieve a large amount of effective secreted expression of CALB,and use it to catalyze the production of biodiesel from waste oil.[Method]Firstly,a CALB-expressing plasmid was constructed in vitro,which was a plasmid optimized with CALB codon.Then different P.pastoris were selected as hosts,and the plasmid was linearized and introduced into P.pastoris for secretion and expression by electroporation method.Then,seven different types of promoters were selected through literature review,and new plasmids were obtained by replacing promoter elements on the basis of the original plasmids,which were linearized and introduced into P.pastoris for secretion and expression.After culture in shake bottle,the supernatant was collected by centrifugation and lipase activity and protein concentration of CALB in the supernatant were compared to obtain the strongest promoter element.Then,the secretion performance of the five signal peptides was compared on the basis of the strongest promoter,and the optimal combination of promoter and signal peptide was obtained by comparing the lipase activity and protein concentration of CALB in the supernatant after shaking bottle culture.Then,recombinant P.pastoris containing 3 copies of calb gene were obtained by screening with high concentrations of antibiotics and cultured in fermenter.A number of culture conditions are optimized,including temperature,initial pH,and amount of inoculation.Finally,the expressed lipase CALB was used as a catalyst to catalyze the production of biodiesel from gutter oil,and the factors such as the amount of catalyst,water content,temperature and molar ratio of alcohol to oil were optimized.[Result]The expression effect of P.pastoris GS115 on CALB was slightly higher than that of P.pastoris X-33 on CALB.In the following experiments,P.pastoris GS115 was used as the host for CALB expression.The best combination of promoter and signal peptide for CALB expression was promoter PGCW14 and Mα signal peptide.The lipase activity increased by about 88%compared with that before the optimization of expression elements,and the protein content secreted into the extracellular cells also increased by nearly 80%.After the ten days of fermentation culture in BSM medium,the lipase activity and protein content after fermentation increased about 34 times and 20 times,respectively,compared with the original engineering strain.In addition,the obtained CALB supernatant was directly used to catalyze the production of biodiesel from gutter oil,and the free fatty acids were effectively converted.[Conclusion]In this study,the efficient expression of CALB was achieved by optimizing gene elements and culture conditions.In addition,the obtained CALB was used to catalyze the transesterification of acidified gutter oil with methanol,suggesting a promising pathway to convert waste or low quality of bio-oil feedstocks with high amount of free fatty acids into biodiesel by using recombinant CALB as catalyst.The results can provide with a good reference for efficient expression of CALB and enhancing lipase production in P.pastoris.It is supposed to bring with new possibility for the bio-production of other valuable proteins.