title: Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801.|Energy Biosciences Institute, Institute for Genomic Biology, Urbana, Illinois, 61801.
authors.forename: Meng
authors.initials: M
authors.lastname: Wang
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801.|Energy Biosciences Institute, Institute for Genomic Biology, Urbana, Illinois, 61801.
authors.forename: Sijin
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801. zhao5@illinois.edu.|Energy Biosciences Institute, Institute for Genomic Biology, Urbana, Illinois, 61801. zhao5@illinois.edu.|Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801. zhao5@illinois.edu.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D015966:Gene Expression Regulation, Fungal; D053263:Gene Regulatory Networks; D026901:Membrane Transport Proteins / Q000378:metabolism; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism
chemical_list: D026901:Membrane Transport Proteins; D014994:Xylose
keywords: directed evolution; gene circuit; metabolite-sensing/regulation; synthetic biology