Engineering Escherichia coli for robustly producing succinic acid and 1,4-butanediol together

Abstract

Biosynthetic succinic acid and 1,4-butanediol (1,4-BDO) are attractive due to their high quality, low cost, and environmental sustainability, yet their yields are limited by weak strain activity and complex metabolic pathways. In this study, an anaerobic succinate synthesis pathway for was constructed in Escherichia coli ATCC 8739 via the reductive tricarboxylic acid (rTCA) cycle. Anaerobic fluorescence-activated droplet sorting (aFADS) and adaptive evolution technologies were developed to screen the strain mutants with enhanced succinate production capabilities. We further developed an enzymatic reaction to generate 1,4-BDO from succinate using a carboxylic acid and aldehyde reductase fused enzyme (CAR-AKR). To increase the yields of succinic acid and 1,4-BDO, we screened frdA and CAR-AKR mutants by NAD(P)H dependent high-throughput screening platforms, and found that the frdA^C248F mutant could significantly reduce the accumulation of fumarate, and the MabCAR^{L284W/S394I/W428M}-SceAKR mutant could strengthen the catalytic efficiency of conversion from succinate to 1,4-BDO. By strengthening the cofactors and ATP regeneration, the final yields of succinic acid and 1,4-BDO reached 85.74 g/L and 4.62 g/L in anaerobic fed-batch fermentation.