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A plug‐and‐play pathway refactoring workflow for natural product research in Escherichia coli and Saccharomyces cerevisiae
Author(s) -
Ren Hengqian,
Hu Pingfan,
Zhao Huimin
Publication year - 2017
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26309
Subject(s) - code refactoring , synthetic biology , saccharomyces cerevisiae , plasmid , computational biology , escherichia coli , biology , workflow , natural product , gene , cloning (programming) , genetics , computer science , biochemistry , programming language , database , software
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.