Construction of a novel anaerobic pathway in Escherichia coli for propionate production

Abstract

Background: Propionate is widely used as an important preservative and important chemical intermediate for synthesis of cellulose fibers, herbicides, perfumes and pharmaceuticals. Biosynthetic propionate has mainly been produced by Propionibacterium, which has various limitations for industrial application.

Results: In this study, we engineered E. coli by combining reduced TCA cycle with the native sleeping beauty mutase (Sbm) cycle to construct a redox balanced and energy viable fermentation pathway for anaerobic propionate production. As the cryptic Sbm operon was over-expressed in E. coli MG1655, propionate titer reached 0.24 g/L. To increase precursor supply for the Sbm cycle, genetic modification was made to convert mixed fermentation products to succinate, which slightly increased propionate production. For optimal expression of Sbm operon, different types of promoters were examined. A strong constitutive promoter Pbba led to the highest titer of 2.34 g/L. Methylmalonyl CoA mutase from Methylobacterium extorquens AM1 was added to strain T110(pbba-Sbm) to enhance this rate limiting step. With optimized expression of this additional Methylmalonyl CoA mutase, the highest production strain was obtained with a titer of 4.95 g/L and a yield of 0.49 mol/mol glucose.

Conclusions: With various metabolic engineering strategies, the propionate titer from fermentation achieved 4.95 g/L. This is the reported highest anaerobic production of propionate by heterologous host. Due to host advantages, such as non-strict anaerobic condition, mature engineering and fermentation techniques, and low cost minimal media, our work has built the basis for industrial propionate production with E. coli chassis.

Keywords: Methylmalonyl CoA mutases; Novel fermentation pathway; Propionate; Sbm operon; Succinate.

Fig. 1

Engineering of an anaerobic propionate fermentation pathway in E. coli. Bold arrows indicate engineered pathway; stars indicate deleted genes; pck* was a mutated form of the pck in the promoter region to increase its expression

Fig. 2

Propionate and other fermentation products of engineered strains with sbm operon under different promoters. Strains in glass screw-cap tubes with NBS (20 g/L glucose) media were cultivated aerobically in the dark at 37 °C, until the cultures reached at an OD600 of 0.5. Then cultures were added with appropriate concentrations of IPTG and cultivated anaerobically in dark for 72 h. All experiments were performed in triplicate

Fig. 3

The effects of expression of M. extorquens AM1 Methylmalonyl CoA mutase on propionate production. Strains were cultivated aerobically in glass screw-cap tubes in dark at 37 °C, until the cultures reached at an OD600 of 0.5. Then IPTG with appropriate concentration was added and cells were cultivated anaerobically in the dark for 72 h at 30 °C. All experiments were performed in triplicate

Fig. 4

Improving production of propionate by a series of engineering strategies and manipulations