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Published on: June 27, 2017
Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering
Fuqiang Song1, Zhijie Qin2, Kun Qiu2
1Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China; Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China; Jiangsu Province Engineering Research Center of Food Synthetic Biotechnology, Jiangnan University, Wuxi, 214122, China.
Abstract:
Vitamin B5 [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (R)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel "PEACES" system by (1) expressing the NAD + kinase gene ppnk from Clostridium glutamicum and the NADP + -dependent gapCcae from Clostridium acetobutyricum and (2) knocking-out the endogenous sthA gene, which interacts with ilvC and panE in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products.
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