Engineering Escherichia coli for Anaerobic Succinate Fermentation Using Corn Stover Hydrolysate as a Substrate

Abstract

Succinic acid is regarded as one of the most important platform chemicals used in materials science, chemistry, and food industrial applications. Currently, the main bottlenecks in the microbial succinate synthesis lie in the low titer, cofactor imbalance, and high production costs. To overcome these challenges, the reductive tricarboxylic acid cycle (TCA) and glucose uptake pathway were enhanced, increasing the titer of succinate to 4.31 g/l, 2.06-fold of the original strain. Furthermore, formate dehydrogenase from Candida boidinii was simultaneously overexpressed to increase the regeneration of NADH which was deficient in succinate synthesis under anaerobic condition. On this basis, the oxygen-responsive biosensor was used to replace the isopropyl-β-d-thiogalactoside (IPTG)-induction system, enabling strain to avoid the utilization of IPTG for succinate production. Using corn stover hydrolysate as the substrate, the optimum strain produced 60.74 g/l succinate in 5 L bioreactor. The engineered strain exhibited high succinate titer using biomass hydrolysate as substrate, significantly reduced the fermentation cost.

Keywords: Formate dehydrogenase; NADH; Succinate; isopropyl-β-d-thiogalactoside; reductive TCA pathway.

Fig. 1. Metabolic engineering for enhancing the metabolic flux of succinate production in E. coli B4.

(A) Schematic diagram of the succinate biosynthetic pathways from corn stover hydrolysate. TCA cycle, tricarboxylic acid cycle; G6P, glucose-6-phosphate; PEP: phosphoenolpyruvate; CIT, citrate; SUC, succinate; FUM, fumarate; MAL, malate; OAA, oxaloacetate; FNR, transcriptional regulator factor; The FNR dimers were activated under anaerobic conditions and subsequently activated downstream gene expression after binding upstream of the promoter P_FnrF8. (B) Increase succinate titer in the anaerobic phase by overexpressing genes. +: indicates the related genes were overexpressed. (C) The by-products levels of succinate producing strain after combined expression of genes. All experimental data were performed in triplicate, and error bars represent the standard deviation. One-way analysis of variance (ANOVA) was applied to check the significance of the data (*p < 0.05).

Fig. 2. Enhancing the regeneration of NADH by overexpression of fdh and addition of formate.

(A) The succinate titer and yield of E. coli B47 when addition of different concentrations of formate. (B) By-product levels of E. coli B47 when addition of different concentrations of formate. All experimental data were performed in triplicate, and error bars represent the standard deviation.

Fig. 3. Production of succinate by E. coli B47 and E. coli B48 using glucose or corn stover hydrolysate as substrate.

(A) The succinate titer and yield of E. coli B47 and E. coli B48 using glucose or corn stover hydrolysate. (B) Byproduct levels of E. coli B47 and E. coli B48 using glucose or corn stover hydrolysate. All experimental data were performed in triplicate, and error bars represent the standard deviation.

Fig. 4. Fed-batch fermentation of E. coli B48 using corn stover hydrolysate as substrate.