Combinatorial Engineering of 1-Deoxy-D-Xylulose 5-Phosphate Pathway Using Cross-Lapping In Vitro Assembly (CLIVA) Method
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Zou-2013-Combinatorial Engine.pdf
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Author(s)
Zou, Ruiyang
Zhou, Kang
Stephanopoulos, Gregory
Too, Heng Phon
Date Issued
November 2013
Journal
PLoS ONE
Publisher
Public Library of Science
Citation
Zou, Ruiyang, Kang Zhou, Gregory Stephanopoulos, and Heng Phon Too. “Combinatorial Engineering of 1-Deoxy-D-Xylulose 5-Phosphate Pathway Using Cross-Lapping In Vitro Assembly (CLIVA) Method.” Edited by Jorg D. Hoheisel. PLoS ONE 8, no. 11 (November 5, 2013): e79557.
Version
Final published version
Abstract
The ability to assemble multiple fragments of DNA into a plasmid in a single step is invaluable to studies in metabolic engineering and synthetic biology. Using phosphorothioate chemistry for high efficiency and site specific cleavage of sequences, a novel ligase independent cloning method (cross-lapping in vitro assembly, CLIVA) was systematically and rationally optimized in E. coli. A series of 16 constructs combinatorially expressing genes encoding enzymes in the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway were assembled using multiple DNA modules. A plasmid (21.6 kb) containing 16 pathway genes, was successfully assembled from 7 modules with high efficiency (2.0 x 10[superscript 3] cfu/ µg input DNA) within 2 days. Overexpressions of these constructs revealed the unanticipated inhibitory effects of certain combinations of genes on the production of amorphadiene. Interestingly, the inhibitory effects were correlated to the increase in the accumulation of intracellular methylerythritol cyclodiphosphate (MEC), an intermediate metabolite in the DXP pathway. The overexpression of the iron sulfur cluster operon was found to modestly increase the production of amorphadiene. This study demonstrated the utility of CLIVA in the assembly of multiple fragments of DNA into a plasmid which enabled the rapid exploration of biological pathways.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
Singapore-MIT Alliance in Research and Technology (SMART)
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DOI of Published Version
https://doi.org/10.1371/journal.pone.0079557
