Optimization of Promoters in Yeast by Genetic Engineering

• Main Authors: HUANG,YA-TING, 黃雅婷
• Other Authors: Ya-Ting Huang
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/p4cqgh

碩士 === 國立臺北科技大學 === 化學工程與生物科技系生化與生醫工程碩士班 === 105 === In most cases, production of heterologous protein in produced in E. coli through gene transfer. However, most of the valuable drug-derived proteins are originated from eukaryoties. When the eukaryotic protein-encoding gene is expressed in the prokaryotic system, there’s a chance that target proteins may loss their functions due to the absence of proper post-translational modification. Meanwhile such as these proteins are easy to form inclusion bodies, and are contaminted by endotoxins during protein extraction also happens oftenly. Therefore, using an eukaryotic system for protein expression is the major research trend. Yeasts, has the characteristics of eukaryotic cells, such as, post-translational modification, are easy to cultivate and operate, do not form inclusion bodies and endotoxin. This orgaism is getting quite popular for heterologous protein production in the industry. Yarrowia lipolytica is one of the most potential orgamsms among yeast because it secrets higher protein than traditional- Yeast Saccharomyces cerevisiae. Y. lipolytica is classified as a safe strain and has the ability to grow with low-cost carbon sources. Therefore, Yarrowia lipolytica is used for protein expression in this study. The purpose of this study is to construct a genetic tool that is easy to operate, and overexpress heterologous proteins in Y. lipolytica. The strategy of this experiment is to using the genetic engineering technology to recombine the upstream active sequence promoter (UAS1B) and the core promoters (LEU2 & TEF) and forme the recombinant promoter, which can be used to enhance the expression of the protein in yeast. The red fluorescent protein (DsRed-Express2) served as a reporter gene in this system. We used the SLEEP a heterologous protein to test this system for the estimating the potential use in the industrial. In order to improve the protein yield, our experimental strategy was to use the multiple-copy integration sites, Ylt1 retrotransposon. Ylt1 is flanked by long terminal repeats and exist inY. lipolytica CLIB122 chromosome. It is design as multiple integration sites to enhance the heterologous protein yield. We have been successfully construncted the recombinant promoter plasmid of pYLF4、pYLF5、pYLF6、pYLF7、pYLF8、pYLF9、pYLF10. Among them, the recombinant promoters of pYLF5, pYLF7 and pYLF10 have strong intensity. This study provides a system which is easy to operate and has high protein expression in Yeast, for food industry, the cosmetic industry, and biofuels to product the functional proteins.