title: A plug-and-play pathway refactoring workflow for natural product research in Escherichia coli and Saccharomyces cerevisiae.
abstract: Pathway refactoring serves as an invaluable synthetic biology tool for natural product discovery, characterization, and engineering. However, the complicated and laborious molecular biology techniques largely hinder its application in natural product research, especially in a high-throughput manner. Here we report a plug-and-play pathway refactoring workflow for high-throughput, flexible pathway construction, and expression in both Escherichia coli and Saccharomyces cerevisiae. Biosynthetic genes were firstly cloned into pre-assembled helper plasmids with promoters and terminators, resulting in a series of expression cassettes. These expression cassettes were further assembled using Golden Gate reaction to generate fully refactored pathways. The inclusion of spacer plasmids in this system would not only increase the flexibility for refactoring pathways with different number of genes, but also facilitate gene deletion and replacement. As proof of concept, a total of 96 pathways for combinatorial carotenoid biosynthesis were built successfully. This workflow should be generally applicable to different classes of natural products produced by various organisms. Biotechnol. Bioeng. 2017;114: 1847-1854. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801.
authors.forename: Hengqian
authors.initials: H
authors.lastname: Ren
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801.
authors.forename: Pingfan
authors.initials: P
authors.lastname: Hu
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801.|Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois.
authors.forename: Huimin
authors.identifier: 0000-0002-9069-6739
authors.initials: H
authors.lastname: Zhao
mesh_terms: D001688:Biological Products / Q000302:isolation & purification / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics*; D002338:Carotenoids / Q000096:biosynthesis* / Q000235:genetics*; D003198:Computer Simulation; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D055442:Metabolome / Q000502:physiology; D008954:Models, Biological; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods; D057188:Workflow
chemical_list: D001688:Biological Products; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D002338:Carotenoids
keywords: biosynthetic gene clusters; natural products; pathway refactoring; synthetic biology