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Biotechnol Bioeng. 2018 Mar;115(3):661-672. doi: 10.1002/bit.26486. Epub 2017 Nov 22.

Engineering Escherichia coli for malate production by integrating modular pathway characterization with CRISPRi-guided multiplexed metabolic tuning.

Gao C1,2,3, Wang S1,2,3, Hu G1,2,3, Guo L1,2,3, Chen X1,2,3, Xu P4, Liu L1,2,3.

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State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.
International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
Chemical Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, Maryland.


The application of rational design in reallocating metabolic flux to overproduce desired chemicals is always restricted by the native regulatory network. Here, we demonstrated that in vitro modular pathway optimization combined with in vivo multiplexed combinatorial engineering enables effective characterization of the bottleneck of a complex biosynthetic cascade and improves the output of the engineered pathway. As a proof of concept, we systematically identified the rate-limiting step of a five-gene malate biosynthetic pathway by combinatorially tuning the enzyme loads of a reconstituted biocatalytic reaction in a cell-free system. Using multiplexed CRISPR interference, we subsequently eliminated the metabolic constraints by rationally assigning an optimal gene expression pattern for each pathway module. The present engineered strain Escherichia coli B0013-47 exhibited a 2.3-fold increase in malate titer compared with that of the parental strain, with a yield of 0.85 mol/mol glucose in shake-flask culture and titer of 269 mM (36 g/L) in fed-batch cultivation. The strategy reported herein represents a powerful method for improving the efficiency of multi-gene pathways and advancing the success of metabolic engineering.


CRISPRi; glyoxylate cycle; in vitro modular optimization; multiplexed combinatorial regulation

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