Abstract
To construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in Escherichia coli. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain ihfAL−-aidB+-ryfAM−-gadAH−, producing 30.0 g L−1 FFAs in fed-batch fermentation, the maximum titer in E. coli reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.
Highlights
To construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks
Identification of beneficial gene targets in the pathways related to free fatty acids (FFAs) metabolism using the CRISPR interference (CRISPRi) system
Genome-scale identification of beneficial gene targets is especially necessary for improving the production of desired products since microbial biosynthesis is subject to sophisticated interactions
Summary
To construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in sophisticated metabolic networks. Optimal combinatorial expression of 15 crucial genes in the FFA biosynthetic pathway with fadD deletion in E. coli BL21 resulted in a titer of 8.6 g L−1 FFAs in fed-batch cultivation with glucose as the carbon source[14] Despite these efforts, further improvement of FFAs production is hampered by the limited understanding of cellular rewiring mechanism and linkage between FFA biosynthesis and other cellular processes[9,22]. As an important supplementary method, omics analysis of differentially performing strains allows the acquisition of comprehensive data related to the mechanisms of cellular metabolism[10,32], providing additional available clues regarding candidate gene targets in complex intracellular interaction networks for enhanced biosynthesis of the desired product[11,33]
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