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| Construction of Recombinant Xylose-utilizing Saccharomyces cerevisiae by Three-plasmid Co-transformation Combinatorial Screening Method |
| LIU Bao-li, LIU Gao-gang, LIN Qiu-hui, LI Bing-zhi, YUAN Ying-jin |
| School of Chemical Engineering, Key Laboratory of Systems Bioengineering(Ministry of Education), Tianjin University, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China |
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Abstract In the field of synthetic biology, quick construction of target metabolic pathways and rapid screening of combinatorial libraries is of great significance. A combinatorial screening method by co-transforming Saccharomyces cerevisiae with three plasmids was established and XR-XDH pathway was constructed in Saccharomyces cerevisiae as an application of the method. The gene expression cassettes of XR,XDH and XK were constructed efficiently using the Yeast Golden Gate (yGG) method. 100 recombinant strains with different promoter combinations were obtained through the three plasmids co-transformation system. Then the colonies were screened through spot assay on 2% SX plate and 16 colonies were chosen. In order to make the property more stable, the separate three gene modules of the corresponding colonies were assembled together to the expression vector pRS426, and then transformed to BY4741 to obtain the new recombinant strains. Oxygen-limited fermentation was carried out to test these strains. Among the 16 strains, Sc-LQH35(TDH3p-XR-ACS2t-FBA1p-XDH-ENO2t-PDC1p-XK-ASC1t) showed the highest products yield and fastest xylose utilization speed. Under oxygen-limited condition, xylitol and ethanol could accumulate to 7.14 g/L and 5.92 g/L separately when the medium contained 20 g/L xylose. The strain Sc-LQH39(TDH3p-XR-ACS2t-FBA1p-XDH-ENO2t-ZEO1p-XK-ASC1t) showed strong ability of producing xylitol on the oxygen-limited fermentation and xylitol yield could reach as high as 0.71 g/g. Three-plasmid co-transformation combinatorial screening method realized the flexible construction and rapid screening of the xylose-utilizing strains. Strains with high fermenting performance were obtained, and it showed that the method has great potential application in construction and screening of the recombinant strains.
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Received: 03 May 2016
Published: 25 December 2016
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