Pyruvate Production by Escherichia coli by Use of Pyruvate Dehydrogenase Variants

Abstract

Altering metabolic flux at a key branchpoint 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 – 0.28 h−1, an engineered strain expressing the AceE[H106V] variant accumulated pyruvate at a yield of 0.59-0.66 g pyruvate/g glucose with a specific productivity of 0.78 – 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.

A Computational Framework to Identify Metabolic Engineering Strategies for the Co-Production of Metabolites

“…Although the deletion of pyruvate dehydrogenase has not been experimentally verified as yet, a similar study has been carried out in E. coli (Moxley and Eiteman, 2021). In this study, it has been shown that the deletion of the genes encoding pyruvate dehydrogenase improves pyruvate production (Moxley and Eiteman, 2021). In addition to pyruvate dehydrogenase, co-FSEOF was able to identify several other amplification targets, which can also improve the production of pyruvate and acetate.…”

“…The deletion of pyruvate dehydrogenase increases the production of pyruvate and acetate, as shown in Table 3. Although the deletion of pyruvate dehydrogenase has not been experimentally verified as yet, a similar study has been carried out in E. coli (Moxley and Eiteman, 2021). In this study, it has been shown that the deletion of the genes encoding pyruvate dehydrogenase improves pyruvate production (Moxley and Eiteman, 2021).…”

CRISPRi enables fast growth followed by stable aerobic pyruvate formation in Escherichia coli without auxotrophy

“…In particular, the production of isoprenoids in E. coli by the native MEP pathway has received considerable attention in the past years [ 13 , 14 ]. There is thus renewed interest in the construction of microbial strains capable of accumulating pyruvate [ 15 , 16 , 17 ].…”

“…Several studies have thus explored the possibility of throttling the flux from pyruvate to acetyl‐CoA to trigger the accumulation of pyruvate while maintaining a low level of flux through the TCA cycle to avoid acetate auxotrophy. The regulation of pyruvate dehydrogenase complex activity could be achieved by point mutations modulating catalytic activity [ 16 , 17 ]. Alternatively, gene expression was controlled by promoter engineering [ 9 ] or by regulated expression of antisense RNA [ 19 ].…”

“…Fed‐batch processes are usually the standard fermentation mode in white biotechnology but complementary techniques such as the partial decoupling of growth and production phases or in situ product removal are actively investigated [ 32 , 33 ]. Small carbon‐based molecules such as pyruvate require no nitrogen for product formation and nitrogen limitation is thus a simple method to achieve decoupling of growth and production [ 16 , 17 ]. Nitrogen‐limited processes are particularly attractive as nitrogen is a major component of biomass, nitrogen sources such as ammonia are usually cheap and easy to measure and the interplay of glucose and nitrogen metabolism in industrial hosts such as E. coli and S. cerevisiae is well characterized [ 34 , 35 ].…”