Evolution of pyruvate kinase-deficient Escherichia coli mutants enables glycerol-based cell growth and succinate production.

AIMS: The aim of this study was to engineer Escherichia coli strains that efficiently produce succinate from glycerol under anaerobic conditions after an aerobic growth phase. METHODS AND RESULTS: We constructed E. coli strain ss195 with deletions of pykA and pykF, which resulted in slow growth on glycerol as sole carbon source. This growth defect was overcome by the selection of fast-growing mutants. Whole-genome resequencing of the evolved mutant ss251 identified the mutation A595S in PEP carboxylase (Ppc). Reverse metabolic engineering by introducing the wild-type allele revealed that this mutation is crucial for the described phenotype. Strain ss251 and derivatives thereof produced succinate with high yields above 80% mol mol(-1) from glycerol under nongrowth conditions. CONCLUSIONS: The results show that during the aerobic growth of ss251, the formation of pyruvate proceeds via the proposed POMP pathway, starting with the carboxylation of PEP by Ppc. The resulting oxaloacetate is reduced by malate dehydrogenase (Mdh) to malate, which is then decarboxylated back to pyruvate by a malic enzyme (MaeA or MaeB). Mutation of ppc is crucial for fast growth of pykAF mutants on glycerol. SIGNIFICANCE AND IMPACT OF STUDY: An E. coli mutant that is capable of achieving high yields of succinate (a top valued-added chemical) from glycerol (an abundant carbon source) was constructed. The identified ppc mutation could be applied to other production strains that require strong PEP carboxylation fluxes.

Global transcription and metabolic flux analysis of Escherichia coli in glucose-limited fed-batch cultivations.

A time series of whole-genome transcription profiling of Escherichia coli K-12 W3110 was performed during a carbon-limited fed-batch process. The application of a constant feed rate led to the identification of a dynamic sequence of diverse carbon limitation responses (e.g., the hunger response) and at the same time provided a global view of how cellular and extracellular resources are used: the synthesis of high-affinity transporters guarantees maximal glucose influx, thereby preserving the phosphoenolpyruvate pool, and energy-dependent chemotaxis is reduced in order to provide a more economic "work mode." sigma(S)-mediated stress and starvation responses were both found to be of only minor relevance. Thus, the experimental setup provided access to the hunger response and enabled the differentiation of the hunger response from the general starvation response. Our previous topological model of the global regulation of the E. coli central carbon metabolism through the crp, cra, and relA/spoT modulons is supported by correlating transcript levels and metabolic fluxes and can now be extended. The substrate is extensively oxidized in the tricarboxylic acid (TCA) cycle to enhance energy generation. However, the general rate of oxidative decarboxylation within the pentose phosphate pathway and the TCA cycle is restricted to a minimum. Fine regulation of the carbon flux through these pathways supplies sufficient precursors for biosyntheses. The pools of at least three precursors are probably regulated through activation of the (phosphoenolpyruvate-)glyoxylate shunt. The present work shows that detailed understanding of the genetic regulation of bacterial metabolism provides useful insights for manipulating the carbon flux in technical production processes.

Hydantoinases and related enzymes as biocatalysts for the synthesis of unnatural chiral amino acids.

A cascade of hydantoinase, N-carbamoylase and hydantoinracemase can be used for the production of natural and unnatural chiral D- and L-amino acids from chemically synthesized hydantoin derivatives. Potentially, 100% conversion and 100% optically pure amino acids can be obtained at the same time if racemic substrates are used. Recent research activities concentrate on newly isolated or improved enzymes and include directed evolution techniques, structure elucidation, studies of fusion proteins and the use of specially designed whole cell biocatalysts.