Codon Optimization And Functional Expression Of Lip1 Gene From Yarrowia Lipolytica

Triacylglycerol hydrolases or lipases (EC3.1.1.3.), exists widely in various plant,animal and microorganisms, can catalyse hydrolysis, esterification, alcoholysis, transesterification and ester of reverse synthesis reactions, etc. Widely used in oil processing, foodstuff, textile, pharmaceutical, cosmetic, leather degreasing, washing and biological sensors, etc, has attracted a lot of attention. However, the catalyst---lipases have high price owing to its diffcult derivation. Therefore, the development of high efficiently expression lipase gene resources have become one of the research hot spots. In this paper, by following the information in NCBI, the lipase gene family lip1 - lip8 of Yarrowia lipolytica were amplified, and lip1 was successfully realized functional expression in Pichia pastoris for the first time. The main work and results were as follows:1. According to the lipase gene sequences of Y. lipolytica submitted to GeneBank, by applying bioinformatics to design primers, using Y. lipolytica genomic DNA as template, lipase genes lip1-lip8 were amplified. Axcept lip2,lip7 and lip8, there were no report on expression of other lipases so far. In this paper, we first realized functional expression of lip1 in P. pastoris GS115, which greatly enriched lipase gene resources.2. Because P. pastoris expression system exits codon bias, while lip1 contains low frequency usage codons, the eight amino acid sites of lip1 were optimized using overlap extensions PCR technology for the first time. The optimized gene Mlip1 was realized efficiently expression in P. pastoris. According to the bioinformatics analysis, lip1 contained no signal peptide, suggesting it is an intracellular lipase. Thus, in this paper we constructed constitutive expression vector pGAP9K with a strong promoter GAP so as to realize exocellular expression of lip1, which layed a solid foundation for further research of this lipase.3. Four expression vectors: pPIC9K-lip1,pPIC9K-Mlip1,pGAP9K-lip1 and pGAP9K-Mlip1 were built, and then were electrotransformated into P. pastoris GS115 to implement functional expression of lip1. High copy transformation recombinants were gained through lipase activity functional verification and G418 resistances screening. After shake flask fermentation, PNP ester was used as substrate for assay the activities of the supernatant. The results showed that the optimized gene had a higher expression level than the original one, and activity of the constitutive expression was superior to that of the inducible expression. Fermentation conditions of engineering strain GS115-pGAP9K-Mlip1 was preliminarily optimized the optimal YP (yeast extract + tryptone) content is 3 (YP), and the optimal carbon source is oleic acid.4. Preliminary analysis showed that the optimum substrate of Lip1 was p-nitrophenyl butyrate (C4), the optimal temperature and pH was 45℃and 8.5, respectively. After warm bath for 2 h at a constant temperature of 60℃, the residual activity of the lipase was 35.88%. Metal ion had significant influence on the lipase. Mg²⁺, Fe²⁺ and Zn²⁺ could stimulate lipase, while Cu²⁺,Ba²⁺,Mn²⁺ and EDTA inhibit its hydrolysis activity. In addition, the primary, secondary and tertiary structures of Lip1 were predicted and analyzed through bioinformatics, which provided a theoretical basis for the following study on protein purification and structure biology of Lip1.