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

Biotechnology and Bioengineering
Cong GaoLiming Liu

Abstract

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.

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Citations

Apr 6, 2018·International Journal of Molecular Sciences·Suhyung ChoByung-Kwan Cho
May 20, 2018·Applied and Environmental Microbiology·Hui LiuLiming Liu
May 16, 2019·FEMS Microbiology Letters·Belén Adiego-PérezJohn van der Oost
Feb 18, 2020·Biotechnology and Applied Biochemistry·Kerstin SchultenkämperVolker F Wendisch
Apr 28, 2020·Applied Microbiology and Biotechnology·Xiangyu JiChunbo Lou
Jun 13, 2020·Biotechnology and Bioengineering·Xiulai ChenLiming Liu
Aug 23, 2019·Nature Communications·Cong GaoLiming Liu
Sep 26, 2020·Applied Microbiology and Biotechnology·Li SunLong Liu
Aug 20, 2020·Microbial Cell Factories·Youming JiangMin Jiang
Sep 5, 2020·Microbial Cell Factories·Zhenquan LiuDawei Zhang
Nov 6, 2020·Metabolic Engineering·Dongdong ZhaoXueli Zhang
Apr 1, 2021·Biotechnology Advances·Maria Martin-PascualVitor A P Martins Dos Santos
Apr 6, 2021·Applied Microbiology and Biotechnology·Lihao JiLiming Liu

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