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Abstract
Citramalic acid (citramalate) is a five-carbon hydroxy-dicarboxylic acid that serves as precursor for the methacrylic acid synthesis from renewable resources. Methacrylic polymers are widely used in many fields such as construction, vehicles, furniture, medical material, and lighting equipment. The primary goal of this research is to improve the microbial production of citramalate by metabolic engineering and fermentation process development. Citramalate production was investigated in numerous engineered strains of Escherichia coli that were overexpressing the citramalate synthase gene, cimA. Knockouts of gltA, citrate synthase, leuC, 3-isopropylmalate dehydratase and ackA, acetate kinase significantly increased citramalate accumulation compared to the control strain. A fed-batch process in a controlled fermenter using the strain MEC499/pZE12-cimA (MG1655 gltA leuC ackA) accumulated 46.5 g/L citramalate in 132 h with a yield of 0.63 g/g. This amounts to greater than 75% of the theoretical maximum yield from glucose of 0.82 g/g. This process also generated about 10 g/L acetate with a yield of 0.14 g/g. Strain MEC568/pZE12-cimA (MG1655 gltA leuC ackA-pta poxB) reduced acetate formation yield to less than 0.04 g/g from glycerol in a fed-batch process. Identical citramalate production was achieved over 31 g/L when using either purified or crude glycerol at yields exceeding 0.50 g citramalate per g glycerol in 132 h. Glutamate was previously supplemented in the medium for the gltA knockout strain, due to the essential role in intracellular acetyl CoA levels. This resulted in a correspondingly enhanced the citramalate production. Taking this into consideration, further protein engineering of E. coli citrate synthase (F383M variant) reduced but not eliminated the enzyme activity. Cell growth was restored with decreased citrate synthase activity using glucose as sole carbon source. In addition, citramalate production was dramatically increased by 125% compared to the control strain containing the native citrate synthase in the batch fermentation. Over 60 g/L citramalate with the yield 0.53 g citramalate per g glucose was achieved in a fed-batch process with exponential feeding strategy in 132 h using MEC626/pZE12-cimA (gltA-F383M).