Abstract
BackgroundCitramalate, a chemical precursor to the industrially important methacrylic acid (MAA), can be synthesized using Escherichia coli overexpressing citramalate synthase (cimA gene). Deletion of gltA encoding citrate synthase and leuC 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 E. coli. This study investigated strategies to minimize acetate and maximize citramalate production in E. coli mutants expressing the cimA gene.ResultsKey knockouts that minimized acetate formation included acetate kinase (ackA), phosphotransacetylase (pta), and in particular pyruvate oxidase (poxB). Deletion of glucose 6-phosphate dehydrogenase (zwf) and ATP synthase (atpFH) aimed at improving glycolytic flux negatively impacted cell growth and citramalate accumulation in shake flasks. In a repetitive fed-batch process, E. coli gltA leuC ackA-pta poxB overexpressing cimA 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.ConclusionsThis study identified the gene deletions critical to reducing acetate accumulation during aerobic growth and citramalate production in metabolically engineered E. coli 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.
Highlights
Citramalate, a chemical precursor to the industrially important methacrylic acid (MAA), can be synthesized using Escherichia coli overexpressing citramalate synthase
Knockouts in the gltA, leuC and ackA genes coding for citrate synthase, 3-isopropylmalate dehydratase, and acetate kinase, respectively, were critical to achieving high citramalate yield [40]
Despite the deletion of acetate kinase, over 10 g/L acetate accumulated in a repetitive fed-batch process [40]
Summary
Citramalate, a chemical precursor to the industrially important methacrylic acid (MAA), can be synthesized using Escherichia coli overexpressing citramalate synthase (cimA gene). Acetate is an undesirable by-product potentially formed from pyruvate and acetyl-CoA, the precursors of citramalate during aerobic growth of E. coli. This study investigated strategies to minimize acetate and maximize citramalate production in E. coli mutants expressing the cimA gene. Some chemicals which cannot be synthesized exclusively by a biosynthetic route might be generated using hybrid approaches involving both biological and chemical synthesis. MAA can be synthesized via a hybrid route: biochemical production of citramalate from glucose [40], and subsequently transforming this compound chemically using base-catalyzed decarboxylation and dehydration [22]. Citramalate (or citramalic acid) is naturally found in the metabolic pathways of some anaerobic bacteria [8, 18, 32]
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