期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 113, 期 1, 页码 206-215出版社
WILEY
DOI: 10.1002/bit.25676
关键词
synthetic biology; gene circuit; metabolite-sensing; regulation; directed evolution
资金
- Energy Biosciences Institute
The development of high-throughput phenotyping tools is lagging far behind the rapid advances of genotype generation methods. To bridge this gap, we report a new strategy for design, construction, and fine-tuning of intracellular-metabolite-sensing/regulation gene circuits by repurposing bacterial transcription factors and eukaryotic promoters. As proof of concept, we systematically investigated the design and engineering of bacterial repressor-based xylose-sensing/regulation gene circuits in Saccharomyces cerevisiae. We demonstrated that numerous properties, such as induction ratio and dose-response curve, can be fine-tuned at three different nodes, including repressor expression level, operator position, and operator sequence. By applying these gene circuits, we developed a cell sorting based, rapid and robust high-throughput screening method for xylose transporter engineering and obtained a sugar transporter HXT14 mutant with 6.5-fold improvement in xylose transportation capacity. This strategy should be generally applicable and highly useful for evolutionary engineering of proteins, pathways, and genomes in S. cerevisiae. Biotechnol. Bioeng. 2016;113: 206-215. (c) 2015 Wiley Periodicals, Inc.
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