A plug-and-play pathway refactoring workflow for natural product research in Escherichia coli and Saccharomyces cerevisiae

Hengqian Ren, Pingfan Hu, Huimin Zhao

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)1847-1854
Number of pages8
JournalBiotechnology and bioengineering
Volume114
Issue number8
DOIs
StatePublished - Aug 2017

Keywords

  • biosynthetic gene clusters
  • natural products
  • pathway refactoring
  • synthetic biology

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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