Succinate, a natural E. coli fermentation product, can serve as substrate for the production of many compounds currently derived from petroleum [112]. Although there have been numerous reports of succinate production by E. coli and other biocatalysts (for example [147,148]), these processes often involve undesirable nutritional supplementation, multiple steps and low product titers.

Due to our success, described above, in using a combination of directed engineering and metabolic evolution to design ethanol and lactic acid mi­crobial biocatalysts, we have used a similar approach to develop a succinate — producing microbial biocatalyst that attains high product titers in simple mineral salts media [149]. Directed engineering consisted of elimination of the lactate, acetate, and ethanol-forming pathways (IdhA, ackA, adhE), leaving succinate production as the primary route of NADH oxidation. The poor growth and fermentation of the resulting strain in mineral salts media were improved by metabolic evolution. Further directed engineering (focA, pflB, mgsA) reduced co-product formation. The resulting microbial biocat­alysts, KJ060 and KJ073 (poxB), produced nearly 700 mM succinate from glucose with a molar yield of 1.2-1.6; the maximum theoretical molar yield is 1.71 (Jantama et al., unpublished results). KJ060 and KJ073 produced 250 and 183 mM acetate, 39 and 118 mM malate, 0 and 5 mM pyruvate, and 2 and 0 mM lactate as co-products.

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