title: Co-expression of phosphoenolpyruvate carboxykinase and nicotinic acid phosphoribosyltransferase for succinate production in engineered Escherichia coli.
abstract: 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 17.1 g L⁻¹ xylose and produced 15.5 g L⁻¹ succinate. Furthermore, anaerobic fermentation of corn stalk hydrolysate contained 30.1 g L⁻¹ xylose, 2.1 g L⁻¹ glucose and 1.5 g L⁻¹ arabinose, it produced a final succinate concentration of 17.2 g L⁻¹ with a yield of 0.94 g g⁻¹ total sugars.
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China. Electronic address: bioengine@njut.edu.cn.
authors.forename: Min
authors.initials: M
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China.
authors.forename: Liya
authors.initials: L
authors.lastname: Liang
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China.
authors.forename: Rongming
authors.initials: R
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Wan
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China.
authors.forename: Mingke
authors.initials: M
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China.
authors.forename: Hanwen
authors.initials: H
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China. Electronic address: majiangfeng@njut.edu.cn.
authors.forename: Jiangfeng
authors.initials: J
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China.
authors.forename: Kequan
authors.initials: K
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, Jiangsu, China.
authors.forename: Pingkai
authors.initials: P
authors.lastname: Ouyang
mesh_terms: D000693:Anaerobiosis; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D019149:Bioreactors; D003001:Cloning, Molecular; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D005285:Fermentation; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D006868:Hydrolysis; D007218:Industrial Microbiology / Q000379:methods*; D010430:Pentosyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010936:Plant Extracts / Q000378:metabolism; D018520:Plant Shoots; D013386:Succinates / Q000302:isolation & purification / Q000378:metabolism*; D014866:Waste Products; D014994:Xylose / Q000378:metabolism; D003313:Zea mays
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010936:Plant Extracts; D013386:Succinates; D014866:Waste Products; D014994:Xylose; D010430:Pentosyltransferases; D019764:Phosphoenolpyruvate Carboxykinase (ATP); C021864:nicotinate phosphoribosyltransferase
keywords: ATP; Corn stalk hydrolysate; NADH/NAD(+); NAPRTase; PEPCK; Succinate