Co-expression of phosphoenolpyruvate carboxykinase and nicotinic acid phosphoribosyltransferase for succinate production in engineered Escherichia coli
2014
Jiang, Min | Chen, Xu | Liang, Liya | Liu, Rongming | Wan, Qing | Wu, Mingke | Zhang, Hanwen | Ma, Jiangfeng | Chen, Kequan | Ouyang, Pingkai
Succinate is not the dominant fermentation product from xylose in wild-type Escherichia coli K12. E. coli BA 203 is a lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), and phosphoenolpyruvate (PEP)-carboxylase (ppc) deletion strain. To increase succinate accumulation and reduce byproduct formation, engineered E. coli BA204, in which ATP-forming PEP-carboxykinase (PEPCK) is overexpressed in BA203, was constructed and produced 2.17-fold higher succinate yield. To further improve the biomass and the consumption rate of xylose, nicotinic acid phosphoribosyltransferase (NAPRTase), a rate limiting enzyme in the synthesis of NAD(H), was also overexpressed. Thus, co-expression of PEPCK and NAPRTase in recombinant E. coli BA209 was investigated. In BA209, the pck gene and the pncB gene each have a trc promoter, hence, both genes are well expressed. During a 72-h anaerobic fermentation in sealed bottles, the total concentration of NAD(H) in BA209 was 1.25-fold higher than that in BA204, and the NADH/NAD⁺ ratio decreased from 0.28 to 0.11. During the exclusively anaerobic fermentation in a 3-L bioreactor, BA209 consumed 17.1gL⁻¹ xylose and produced 15.5gL⁻¹ succinate. Furthermore, anaerobic fermentation of corn stalk hydrolysate contained 30.1gL⁻¹ xylose, 2.1gL⁻¹ glucose and 1.5gL⁻¹ arabinose, it produced a final succinate concentration of 17.2gL⁻¹ with a yield of 0.94gg⁻¹ total sugars.
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