RESUMEN
Several chromosomally expressed AceE variants were constructed in Escherichia coli ΔldhA ΔpoxB ΔppsA and compared using glucose as the sole carbon source. These variants were examined in shake flask cultures for growth rate, pyruvate accumulation, and acetoin production via heterologous expression of the budA and budB genes from Enterobacter cloacae ssp. dissolvens. The best acetoin-producing strains were subsequently studied in controlled batch culture at the one-liter scale. PDH variant strains attained up to four-fold greater acetoin than the strain expressing the wild-type PDH. In a repeated batch process, the H106V PDH variant strain attained over 43 g/L of pyruvate-derived products, acetoin (38.5 g/L) and 2R,3R-butanediol (5.0 g/L), corresponding to an effective concentration of 59 g/L considering the dilution. The acetoin yield from glucose was 0.29 g/g with a volumetric productivity of 0.9 g/L·h (0.34 g/g and 1.0 g/L·h total products). The results demonstrate a new tool in pathway engineering, the modification of a key metabolic enzyme to improve the formation of a product via a kinetically slow, introduced pathway. Direct modification of the pathway enzyme offers an alternative to promoter engineering in cases where the promoter is involved in a complex regulatory network.
RESUMEN
Altering metabolic flux at a key branch point in metabolism has commonly been accomplished through gene knockouts or by modulating gene expression. An alternative approach to direct metabolic flux preferentially toward a product is decreasing the activity of a key enzyme through protein engineering. In Escherichia coli, pyruvate can accumulate from glucose when carbon flux through the pyruvate dehydrogenase complex is suppressed. Based on this principle, 16 chromosomally expressed AceE variants were constructed in E. coli C and compared for growth rate and pyruvate accumulation using glucose as the sole carbon source. To prevent conversion of pyruvate to other products, the strains also contained deletions in two nonessential pathways: lactate dehydrogenase (ldhA) and pyruvate oxidase (poxB). The effect of deleting phosphoenolpyruvate synthase (ppsA) on pyruvate assimilation was also examined. The best pyruvate-accumulating strains were examined in controlled batch and continuous processes. In a nitrogen-limited chemostat process at steady-state growth rates of 0.15 to 0.28 h-1, an engineered strain expressing the AceE[H106V] variant accumulated pyruvate at a yield of 0.59 to 0.66 g pyruvate/g glucose with a specific productivity of 0.78 to 0.92 g pyruvate/g cells·h. These results provide proof of concept that pyruvate dehydrogenase complex variants can effectively shift carbon flux away from central carbon metabolism to allow pyruvate accumulation. This approach can potentially be applied to other key enzymes in metabolism to direct carbon toward a biochemical product. IMPORTANCE Microbial production of biochemicals from renewable resources has become an efficient and cost-effective alternative to traditional chemical synthesis methods. Metabolic engineering tools are important for optimizing a process to perform at an economically feasible level. This study describes an additional tool to modify central metabolism and direct metabolic flux to a product. We have shown that variants of the pyruvate dehydrogenase complex can direct metabolic flux away from cell growth to increase pyruvate production in Escherichia coli. This approach could be paired with existing strategies to optimize metabolism and create industrially relevant and economically feasible processes.