Effect of adaptive laboratory evolution of engineered Escherichia coli in acetate on the biosynthesis of succinic acid from glucose in two-stage cultivation.

Escherichia coli MLB (MG1655 ΔpflB ΔldhA), which can hardly grow on glucose with little succinate accumulation under anaerobic conditions. Two-stage fermentation is a fermentation in which the first stage is used for cell growth and the second stage is used for product production. The ability of glucose consumption and succinate production of MLB under anaerobic conditions can be improved significantly by using acetate as the solo carbon source under aerobic condition during the two-stage fermentation. Then, the adaptive laboratory evolution (ALE) of growing on acetate was applied here. We assumed that the activities of succinate production related enzymes might be further improved in this study. E. coli MLB46-05 evolved from MLB and it had an improved growth phenotype on acetate. Interestingly, in MLB46-05, the yield and tolerance of succinic acid in the anaerobic condition of two-stage fermentation were improved significantly. According to transcriptome analysis, upregulation of the glyoxylate cycle and the activity of stress regulatory factors are the possible reasons for the elevated yield. And the increased tolerance to acetate made it more tolerant to high concentrations of glucose and succinate. Finally, strain MLB46-05 produced 111 g/L of succinic acid with a product yield of 0.74 g/g glucose. SYNOPSIS.

Efficient biosynthesis of 3-hydroxypropionic acid from ethanol in metabolically engineered Escherichia coli.

Engineering microbial cell factories to convert CO2-based feedstock into chemicals and fuels provide a feasible carbon-neutral route for the third-generation biorefineries. Ethanol became one of the major products of syngas fermentation by engineered acetogens. The key building block chemical 3-hydroxypropionic acid (3-HP) can be synthesized from ethanol by the malonyl-CoA pathway with CO2 fixation. In this study, the effect of two ethanol consumption pathways on 3-HP synthesis were studied as well as the effect of TCA cycle, gluconeogenesis pathway, and transhydrogenase. And the 3-HP synthesis pathway was also optimized. The engineered strain synthesized 1.66 g/L of 3-HP with a yield of 0.24 g/g. Furthermore, the titer and the yield of 3-HP increased to 13.17 g/L and 0.57 g/g in the whole-cell biocatalysis system. This study indicated that ethanol as feedstock had the potential to synthesize 3-HP, which provided an alternative route for future biorefinery.