Abstract:
Shikimic acid (SA) is an important natural biochemical metabolite in plants and microorganisms that is known for antimicrobial and anti-inflammatory activities. Our project completed the metabolic engineering modification of Escherichia coli (E. coli) MG1655 to achieve SA accumulation. Among them, the YCY9 strain was able to produce 1875 mg/L of shikimic acid by continuous cultivation for 96 h at 10% inoculum, which greatly improved the target yield and provided the possibility of achieving mass production.
1. Overview of our design
We have identified strategies for metabolic engineering modifications E. coli based on genomic metabolic network modeling, which are divided into the following main points (See Design for more details):
1. Enhancement of PEP levels:
(1) blocking the PTS by knocking out the ptsG gene;
(2) blocking PEP
branching pathways,
including knocking out of ldhA, adhE, poxB, and pta genes;
2. Increase the E4P content: overexpression of tktA and talB genes;
3. Optimize the shikimic acid synthesis pathway: (1) overexpression of aroG, aroB, aroD, and aroE genes, enhance shikimic acid synthesis metabolic flux; (2) knockout of aroK and aroL genes to block the shikimic acid catabolic pathway;
4. In addition, we introduced glk and glf genes from Zymomonas mobilis (mainly by glk-glf integration into the ptsG locus)
Note: ptsG, glucose-specific EIICB protein components; glk, glucokinase; glf, glucose-promoting diffusion protein; tktA, transketonase; talB, transaldolase; ldhA, lactate dehydrogenase; adhE, alcohol dehydrogenase; poxB, pyruvate dehydrogenase; pta, phosphate transacetylase; aroG, DAHP synthase; aroB, 3-dehydroquinic acid synthetase; aroD, 3-dehydroquinic acid dehydratase; aroE, shikimic dehydrogenase; aroK/aroL, shikimic acid kinase.