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This was done by a combination of gene deletions (genetic engineering) and metabolic evolution with over 2,000 generations of growth\u2010based selection. After deletion of the central anaerobic fermentation genes (<jats:italic>ldhA<\/jats:italic>, <jats:italic>adhE<\/jats:italic>, <jats:italic>ackA<\/jats:italic>), the pathway for malate and succinate production remained as the primary route for the regeneration of NAD+. Under anaerobic conditions, ATP production for growth was obligately coupled to malate dehydrogenase and fumarate reductase by the requirement for NADH oxidation. Selecting strains for improved growth co\u2010selected increased production of these dicarboxylic acids. Additional deletions were introduced as further improvements (<jats:italic>focA<\/jats:italic>, <jats:italic>pflB<\/jats:italic>, <jats:italic>poxB<\/jats:italic>, <jats:italic>mgsA<\/jats:italic>). The best succinate biocatalysts, strains KJ060(<jats:italic>ldhA<\/jats:italic>, <jats:italic>adhE<\/jats:italic>, <jats:italic>ackA<\/jats:italic>, <jats:italic>focA<\/jats:italic>, <jats:italic>pflB<\/jats:italic>) and KJ073(<jats:italic>ldhA<\/jats:italic>, <jats:italic>adhE<\/jats:italic>, <jats:italic>ackA<\/jats:italic>, <jats:italic>focA<\/jats:italic>, <jats:italic>pflB<\/jats:italic>, <jats:italic>mgsA<\/jats:italic>, <jats:italic>poxB<\/jats:italic>), produce 622\u2013733 mM of succinate with molar yields of 1.2\u20131.6 per mole of metabolized glucose. The best malate biocatalyst, strain KJ071(<jats:italic>ldhA<\/jats:italic>, <jats:italic>adhE<\/jats:italic>, <jats:italic>ackA<\/jats:italic>, <jats:italic>focA<\/jats:italic>, <jats:italic>pflB<\/jats:italic>, <jats:italic>mgsA<\/jats:italic>), produced 516 mM malate with molar yields of 1.4 per mole of glucose metabolized. Biotechnol. Bioeng. 2008;99: 1140\u20131153. \u00a9 2007 Wiley Periodicals, Inc.<\/jats:p>","DOI":"10.1002\/bit.21694","type":"journal-article","created":{"date-parts":[[2007,10,30]],"date-time":"2007-10-30T20:46:46Z","timestamp":1193777206000},"page":"1140-1153","source":"Crossref","is-referenced-by-count":314,"title":["Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of <i>Escherichia coli<\/i> C that produce succinate and malate"],"prefix":"10.1002","volume":"99","author":[{"given":"Kaemwich","family":"Jantama","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.J.","family":"Haupt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Spyros A.","family":"Svoronos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xueli","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J.C.","family":"Moore","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"K.T.","family":"Shanmugam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"L.O.","family":"Ingram","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2007,10,30]]},"reference":[{"key":"e_1_2_1_2_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.34.11.491"},{"key":"e_1_2_1_3_1","doi-asserted-by":"publisher","DOI":"10.1021\/bp060301y"},{"key":"e_1_2_1_4_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF01569920"},{"key":"e_1_2_1_5_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0308171100"},{"key":"e_1_2_1_6_1","doi-asserted-by":"publisher","DOI":"10.1042\/0264-6021:3520717"},{"key":"e_1_2_1_7_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.67.1.148-154.2001"},{"key":"e_1_2_1_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2005.09.006"},{"key":"e_1_2_1_9_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.120163297"},{"key":"e_1_2_1_10_1","doi-asserted-by":"crossref","first-page":"2351","DOI":"10.1128\/JB.181.8.2351-2357.1999","article-title":"The steady\u2010state internal redox state (NADH\/NAD) reflects the external redox state and is correlated with catabolicadaptation in Escherichia coli","volume":"181","author":"de Graef MR","year":"1999","journal-title":"J Bacteriol"},{"key":"e_1_2_1_11_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-007-1113-7"},{"key":"e_1_2_1_12_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.56.1.203"},{"key":"e_1_2_1_13_1","unstructured":"GlassnerDA DattaR.1992. 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