Activating C4-dicarboxylate transporters DcuB and DcuC for improving succinate production.

Although many efforts had been performed to engineer Escherichia coli for succinate production, succinate efflux system had not been investigated as an engineering target for improving succinate production. In this work, four Dcu transporters, which had been reported to be responsible for C4-dicarboxylates transportation of E. coli, were investigated for their succinate efflux capabilities. These four dcu genes were deleted individually in a previously constructed succinate-producing strain to study their effects on succinate production. Deleting dcuA and dcuD genes had nearly no influence, while deleting dcuB and dcuC genes led to 15 and 11% decrease of succinate titer, respectively. Deleting both dcuB and dcuC genes resulted in 90% decrease of succinate titer, suggesting that DcuB and DcuC were the main transporters for succinate efflux and they functioned as independent and mutually redundant succinate efflux transporters. Furthermore, RBS library having strengths varied from 0.17 to 8.6 times of induced E. coli lacZ promoter was used to modulate dcuB and dcuC genes for improving succinate production. Modulating these two genes in combination led to 34% increase of succinate titer. To the best of knowledge, this was the first report about improving succinate production through engineering succinate efflux system.