RESUMEN
The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production.
Asunto(s)
Escherichia coli/enzimología , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Glicerol/metabolismo , Malatos/metabolismo , Aerobiosis , Bicarbonatos/metabolismo , Escherichia coli/crecimiento & desarrollo , Cinética , Malato Deshidrogenasa/metabolismo , Mutación/genética , Fosfoenolpiruvato Carboxilasa/metabolismoRESUMEN
Acetic acid is an abundant material that can be used as a carbon source by microorganisms. Despite its abundance, its toxicity and low energy content make it hard to utilize as a sole carbon source for biochemical production. To increase acetate utilization and isobutanol production with engineered Escherichia coli, the feasibility of utilizing acetate and metabolic engineering was investigated. The expression of acs, pckA, and maeB increased isobutanol production by up to 26%, and the addition of TCA cycle intermediates indicated that the intermediates can enhance isobutanol production. For isobutanol production from acetate, acetate uptake rates and the NADPH pool were not limiting factors compared to glucose as a carbon source. This work represents the first approach to produce isobutanol from acetate with pyruvate flux optimization to extend the applicability of acetate. This technique suggests a strategy for biochemical production utilizing acetate as the sole carbon source.