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.

journal_name

Biotechnol Bioeng

authors

Ren H,Hu P,Zhao H

doi

10.1002/bit.26309

subject

Has Abstract

pub_date

2017-08-01 00:00:00

pages

1847-1854

issue

8

eissn

0006-3592

issn

1097-0290

journal_volume

114

pub_type

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