| Yarrowia lipolytica is one of the non-conventional yeasts. It is generally regarded as safe (GRAS) and can produce various kinds of metabolites and proteins for industrial applications by using cheap industrial raw materials as substrates. Since 1960s, this yeast has been applied in different industries. Its expression/secretion system has many advantages, such as exogenous genes easy to integrate into its genome, high expression level of heterogeneous proteins, post translational process of the expressed proteins, high density fermentation and underglycosylation. The unique property and better protein expression/secretion system make Y. lipolytica become one of the most potential yeasts in research and industrial application. However, the lack of basic research and imperfect expression/secretion system hinder its further application. Lipase family is one of the unique characteristics of Y. lipolytica, which may explain the particular taxonomic status of the strain to some extent. Improvement of the existed expression/secretion system may bring broader application of Y. lipolytica.Y. lipolytica CGMCC 2.1405 was presented by Professor Ma (IMCAS, the institute of microbiology, Chinese Aca iemy of Sciences), whose lipase cDNAs (lip1, lip3, lip4, lip5) were cloned by rea ersed PCR with total RNA. The lipases'properties were characterized after being expressed and purified. Lip4 and Lip5 were proved to be lipase, but Lipl and Lip3 were esterase. To find new lipase, bioinformatics was employed to predict possible lipase genes from Y. lipolytica genome. Then, E. coli expression system was introduced to construct a small gene library to screen new lipase gene. One new lipase gene lip9 was identified. The new lipase was expressed, purified and examined with their enzymatic properties. All lipases (except lip2) in Y. lipolytica cells were located by using Enhanced Green Fluorecence Protein and Confocal laser scanning microscopy. They all was distributed inside the cell except Lip8, which was cell wall-bound lipase. Suc2 (Saccharomyces cerevisia) and mel(Pseudomonas maltophilia) were applied as selective markers to construct new Y. lipolytica expression/secretion vectors. They were confirmed to be existed stably in recombinants yeast genome, and with which human Rho oncogene was successfully expressed. Moreover, these two systems can lay a solidated foundation for eliminating environmental pollution and less-expensive industrial applications of the yeast. The main work and innovations were summarized as follows:(1) The cDNAs of Lipl, Lip3, Lip4, and Lip5 were cloned by Reverse Transcription PCR, and inserted into Pichia pastoris expression/secretion vectors. The expressed proteins were purified by Ni-NTA affinity chromatography. The enzymatic properties suggested that Lip1 and Lip3 were esterases not lipases, while Lip4 and Lip5 were lipase.(2) Bioinformatics was used to analyze Y. lipolytica genome sequences and found 18 putative lipase genes. They were all cloned and inserted into E. coli expression vector pET-28a (+), then the expressed proteins were screened for hydrolyzing activities toward p-nitrophenyl-palmitate. One new lipase gene was indentified. To characterize its enzymatic properties, this gene was functionally expressed in Pichia pastoris GS115 and purified by Ni-NTA affinity chromatography. The result indicated that the protein preferred to hydrolyze long-chain esters, and it also could hydrolyze olive oil, which confirmed that it was lipase, and nominated as Lip9. In addition, a new gene clone method was well established here.(3) Suc2 was cloned as an auxotroph selective marker gene to construct Y. lipolytica new expression/secretion vectors pINA1297-a-TS and pINA1297-TS. Comparative results implied that promoter pTEF was more suitable for industry application. With this new system, human Rho oncogene was successfully expressed.(4) Based on the vector pINA1297-a-TS and using EGFP, protein-location vector were constructed, with which the lipases of Y. lipolytica were all (except lip2) located for the first time. They were all distributed inside the cell except Lip8, which was cell wall-bound, and could eluted by PBS buffer after ten times. This implied that it was non-covalent binding to cell wall.(5) Tyrosinase encoded gene mel of Pseudomonas maltophilia was for the first time used in the construction of yeast expression/secretion vectors. Assembly PCR was applied to synthesize mel gene. Then, Over-lap PCR was used for placing mel downstream of the promoter pTEF and signal sequences XPR2pre, and upstream of terminator LIP2t, resulting in a new Y. lipolytica expression/secretion system (containing vector pINA1297-a-MS and pINA1297-a-MS), which can screen the recombinants by the effect of melanin. The vectors can exist in the Y. lipolytica genome stably. This demonstrated the new expression/secretion yeast system was successfully constructed.
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