Engineering central pathways for industrial-level (3R)-acetoin biosynthesis in Corynebacterium glutamicum

Abstract

Background: Acetoin, especially the optically pure (3S)- or (3R)-enantiomer, is a high-value-added bio-based platform chemical and important potential pharmaceutical intermediate. Over the past decades, intense efforts have been devoted to the production of acetoin through green biotechniques. However, efficient and economical methods for the production of optically pure acetoin enantiomers are rarely reported. Previously, we systematically engineered the GRAS microorganism Corynebacterium glutamicum to efficiently produce (3R)-acetoin from glucose. Nevertheless, its yield and average productivity were still unsatisfactory for industrial bioprocesses.Results: In this study, cellular carbon fluxes in the acetoin producer CGR6 were further redirected toward acetoin synthesis using several metabolic engineering strategies, including blocking anaplerotic pathways, attenuating key genes of the TCA cycle and integrating additional copies of the alsSD operon into the genome. Among them, the combination of attenuation of citrate synthase and inactivation of phosphoenolpyruvate carboxylase showed a significant synergistic effect on acetoin production. Finally, the optimal engineered strain CGS11 produced a titer of 102.45 g/L acetoin with a yield of 0.419 g/g glucose at a rate of 1.86 g/L/h in a 5 L fermenter. The optical purity of the resulting (3R)-acetoin surpassed 95%.Conclusion: To the best of our knowledge, this is the highest titer of highly enantiomerically enriched (3R)-acetoin, together with a competitive product yield and productivity, achieved in a simple, green processes without expensive additives or substrates. This process therefore opens the possibility to achieve easy, efficient, economical and environmentally-friendly production of (3R)-acetoin via microbial fermentation in the near future.

A Genome-Scale Metabolic Model of 2,3-Butanediol Production by Thermophilic Bacteria Geobacillus icigianus

“…Optimization results with the most benefit for 2,3-BTD production are presented in Table 3 , and show that reactions, which mostly require modifications are oxygen transport and citrate synthase (CS). It should be noted that reduction of the flux via CS, which was revealed by the LMOMA algorithm as one of the best solutions for 2,3-BTD production, was recently experimentally demonstrated by using a weak promoter for the gene encoding CS to increase the production of (3R)-acetoin, a precursor of 2,3-BTD in Corynebacterium glutamicum [ 39 ]. Some other reactions, which are predicted by this method for enhanced 2,3-BTD biosynthesis, are also related to TCA cycle and succinate production.…”

“…In silico predictions based on different optimization algorithms to accelerate biosynthesis of 2,3-BTD comprise genetic modifications related to some steps of the TCA cycle which result in a decrease in succinate production and cultivation of the strain in oxygen-limited conditions. Identified strategies of metabolic engineering for G.icigianus have been already experimentally demonstrated as capabilities for active 2,3-BTD biosynthesis in closely related species [ 39 , 40 , 42 , 43 , 44 , 45 ], thereby affirming a benefit of the computational modeling in biotechnological strain design.…”