초록
<P><B>Abstract</B></P><P><B>Aims</B></P><P>The aim of this study was to engineer <I>Escherichia coli</I> strains that efficiently produce succinate from glycerol under anaerobic conditions after an aerobic growth phase.</P><P><B>Methods and Results</B></P><P>We constructed <I>E.？coli</I> strain ss195 with deletions of <I>pykA</I> and <I>pykF</I>, 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<SUP>−1</SUP> from glycerol under nongrowth conditions.</P><P><B>Conclusions</B></P><P>The results show that during the aerobic growth of ss251, the formation of pyruvate proceeds <I>via</I> 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 <I>ppc</I> is crucial for fast growth of <I>pykAF</I> mutants on glycerol.</P><P><B>Significance and Impact of Study</B></P><P>An <I>E.？coli</I> 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 <I>ppc</I> mutation could be applied to other production strains that require strong PEP carboxylation fluxes.</P>