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Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System
Authors:Yoichiro Ito  Mamoru Yamanishi  Akinori Ikeuchi  Chie Imamura  Takashi Matsuyama
Institution:1. Matsuyama Research Group, TOYOTA Central Research and Development Laboratories Incorporation, Nagakute, Aichi, Japan.; 2. Biotechnology Laboratory, TOYOTA Central Research and Development Laboratories Incorporation, Nagakute, Aichi, Japan.; Virginia Tech, UNITED STATES,
Abstract:Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.
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