초록
<P><B>Background</B></P><P>Citramalate, a chemical precursor to the industrially important methacrylic acid (MAA), can be synthesized using <I>Escherichia coli</I> overexpressing citramalate synthase (<I>cimA</I> gene). Deletion of <I>gltA</I> encoding citrate synthase and <I>leuC</I> encoding 3-isopropylmalate dehydratase were critical to achieving high citramalate yields. Acetate is an undesirable by-product potentially formed from pyruvate and acetyl-CoA, the precursors of citramalate during aerobic growth of <I>E. coli</I>. This study investigated strategies to minimize acetate and maximize citramalate production in <I>E. coli</I> mutants expressing the <I>cimA</I> gene.</P><P><B>Results</B></P><P>Key knockouts that minimized acetate formation included acetate kinase (<I>ackA</I>), phosphotransacetylase (<I>pta</I>), and in particular pyruvate oxidase (<I>poxB</I>). Deletion of glucose 6-phosphate dehydrogenase (<I>zwf</I>) and ATP synthase (<I>atpFH</I>) aimed at improving glycolytic flux negatively impacted cell growth and citramalate accumulation in shake flasks. In a repetitive fed-batch process, <I>E. coli gltA leuC ackA</I>-<I>pta poxB</I> overexpressing <I>cimA</I> generated 54.1 g/L citramalate with a yield of 0.64 g/g glucose (78% of theoretical maximum yield), and only 1.4 g/L acetate in 87 h.</P><P><B>Conclusions</B></P><P>This study identified the gene deletions critical to reducing acetate accumulation during aerobic growth and citramalate production in metabolically engineered <I>E. coli</I> strains. The citramalate yield and final titer relative to acetate at the end of the fed-batch process are the highest reported to date (a mass ratio of citramalate to acetate of nearly 40) without being detrimental to citramalate productivity, significantly improving a potential process for the production of this five-carbon chemical.</P>