Mingke Wu

Efficient succinic acid production from lignocellulosic biomass by simultaneous utilization of glucose and xylose in engineered Escherichia coli.

To enhance succinic acid formation during xylose fermentation in Escherichia coli, overexpression of ATP-forming phosphoenolpyruvate carboxykinase (PEPCK) from Bacillus subtilis 168 in an ldhA, pflB, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production. However, BA204 displays a low yield of glucose fermentation and sequential glucose-xylose utilization under regulation by the phosphotransferase system (PTS). To improve the capability of glucose fermentation and simultaneously consume sugar mixture for succinic acid production, a pflB, ldhA, ppc, and ptsG deletion strain overexpressing ATP-forming PEPCK, named E. coli BA305, was constructed. As a result, after 120 h fed-batch fermentation of sugarcane bagasse hydrolysate, the dry cell weight and succinic acid concentration in BA305 were 4.58 g L(-1) and 39.3 g L(-1), respectively.

Repetitive succinic acid production from lignocellulose hydrolysates by enhancement of ATP supply in metabolically engineered Escherichia coli.

In this study, repetitive production of succinic acid from lignocellulose hydrolysates by enhancement of ATP supply in metabolically engineered E. coli is reported. Escherichia coli BA305, a pflB, ldhA, ppc, and ptsG deletion strain overexpressing ATP-forming phosphoenolpyruvate (PEP) carboxykinase (PEPCK), produced a final succinic acid concentration of 83 g L(-1) with a high yield of 0.87 g g(-1) total sugar in 36 h of three repetitive fermentations of sugarcane bagasse hydrolysate. Furthermore, simultaneous consumption of glucose and xylose was achieved, and the specific productivity and yield of succinic acid were almost maintained constant during the repetitive fermentations.

Succinic acid production from sucrose by Actinobacillus succinogenes NJ113.

In this study, sucrose, a reproducible disaccharide extracted from plants, was used as the carbon source for the production of succinic acid by Actinobacillus succinogenes NJ113. During serum bottle fermentation, the succinic acid concentration reached 57.1g/L with a yield of 71.5%. Further analysis of the sucrose utilization pathways revealed that sucrose was transported and utilized via a sucrose phosphotransferase system, sucrose-6-phosphate hydrolase, and a fructose PTS. Compared to glucose utilization in single pathway, more pathways of A. succinogenes NJ113 are dependent on sucrose utilization. By changing the control strategy in a fed-batch culture to alleviate sucrose inhibition, 60.5g/L of succinic acid was accumulated with a yield of 82.9%, and the productivity increased by 35.2%, reaching 2.16g/L/h. Thus utilization of sucrose has considerable potential economics and environmental meaning.

Succinate production by metabolically engineered Escherichia coli using sugarcane bagasse hydrolysate as the carbon source.

Efficient biosynthesis of succinate from a renewable biomass resource by engineered Escherichia coli is reported in this paper. Fermentation of sugarcane bagasse hydrolysate by engineered E. coli BA204, a pflB, ldhA, and ppc deletion strain overexpressing the ATP-forming phosphoenolpyruvate carboxykinase from Bacillus subtilis 168, produced a final succinate concentration of 15.85 g L(-1) with a high yield of 0.89 g L(-1) total sugar under anaerobic conditions. During dual-phase fermentations, initial aerobic growth facilitated subsequent anaerobic succinate production, with a final succinate concentration of 18.88 g L(-1) and a yield of 0.96 g g(-1) total sugar after 24 h of anaerobic fermentation. The high succinate yield from sugarcane bagasse hydrolysate demonstrated a great potential application of renewable biomass as a feedstock for the economical production of succinate using metabolically engineered E. coli.

Progress of succinic acid production from renewable resources: Metabolic and fermentative strategies

Succinic acid is a four-carbon dicarboxylic acid, which has attracted much interest due to its abroad usage as a precursor of many industrially important chemicals in the food, chemicals, and pharmaceutical industries. Facing the shortage of crude oil supply and demand of sustainable development, biological production of succinic acid from renewable resources has become a topic of worldwide interest. In recent decades, robust producing strain selection, metabolic engineering of model strains, and process optimization for succinic acid production have been developed. This review provides an overview of succinic acid producers and cultivation technology, highlight some of the successful metabolic engineering approaches.

Co-expression of phosphoenolpyruvate carboxykinase and nicotinic acid phosphoribosyltransferase for succinate production in engineered Escherichia coli

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.

Performance and mechanism analysis of succinate production under different transporters in Escherichia coli

Succinic acid is a platform chemical with potential for bio-based synthesis. However, the production of bio-based succinate is limited because of insufficient succinate efflux capacity in the late stage of fermentation. In the present study, three different transporters, which have been reported to be responsible for C4-dicarboxylates transport, were employed for investigation of the transport capacity of succinate in Escherichia coli. After engineered strains were constructed, the fermentative production of succinic acid was studied in serum bottles and 3 L of fermentor. The results demonstrated that engineered strain showed better efflux capacity than control strain under high concentration of succinate. The highest production of succinate was 68.66 g/L, while the NCgl2130 transporter may be the best candidate for succinate export in E. coli. Further research showed that the expression levels and relative enzyme activities involved in the metabolic pathway all increased markedly, and the maximum activities of PPC, PCK, PYK, and MDH increased by 1.50, 1.38, 1.28, and 1.27-fold in recombinant E. coli AFP111/pTrc99a-NCgl2130, respectively. Moreover, the maximum level of intracellular ATP increased by 23.79% in E. coli AFP111/pTrc99a-NCgl2130. Taken together, these findings indicated that engineered transporters can improve succinate production by increasing key enzyme activities and intracellular ATP levels. To the best of thew authors’ knowledge, this is the first report on a mechanism to improve succinate production by engineered transporters. This strategy set up a foundation for improving the biosynthesis of other C4-dicarboxylates, such as fumaric acid and malic acid.