{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,22]],"date-time":"2026-07-22T17:39:09Z","timestamp":1784741949755,"version":"3.55.0"},"reference-count":44,"publisher":"Wiley","issue":"12","license":[{"start":{"date-parts":[[2017,8,17]],"date-time":"2017-08-17T00:00:00Z","timestamp":1502928000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"name":"Grant-in-Aid","award":["16H04576"],"award-info":[{"award-number":["16H04576"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biotech &amp; Bioengineering"],"published-print":{"date-parts":[[2017,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:label\/><jats:p>Metabolic engineering of isopropyl alcohol (IPA)\u2010producing <jats:italic>Escherichia coli<\/jats:italic> strains was conducted along with <jats:sup>13<\/jats:sup>C\u2010metabolic flux analysis (MFA). A metabolically engineered <jats:italic>E. coli<\/jats:italic> strain expressing the <jats:italic>adc<\/jats:italic> gene derived from <jats:italic>Clostridium acetobutylicum<\/jats:italic> and the <jats:italic>IPADH<\/jats:italic> gene from <jats:italic>C. beijerinckii<\/jats:italic> did not produce IPA during its exponential growth phase in the aerobic batch culture. <jats:sup>13<\/jats:sup>C\u2010MFA was carried out, and revealed a deficiency in NADPH regeneration for IPA production in growth phase. Based on these findings, we used nitrogen\u2010starved culture conditions to reduce NADPH consumption for biomass synthesis. As a result, IPA yield was increased to 20% mol\/mol glucose. <jats:sup>13<\/jats:sup>C\u2010MFA revealed that the relative flux levels through the oxidative pentose phosphate (PP) pathway and the TCA cycle were elevated in nitrogen\u2010starved condition relative to glucose uptake rate. To prevent CO<jats:sub>2<\/jats:sub> release in the 6\u2010phosphogluconate dehydrogenase (6PGDH) reaction, metabolism of this <jats:italic>E. coli<\/jats:italic> strain was further engineered to redirect glycolytic flux to the glucose 6\u2010phosphate dehydrogenase (G6PDH) and Entner\u2010Doudoroff (ED) pathway. IPA yield of 55% mol\/mol glucose was achieved by combining the nitrogen\u2010starved culture condition with the metabolic redirection. The <jats:sup>13<\/jats:sup>C\u2010MFA data and intracellular NADPH levels obtained under these IPA production conditions revealed linear correlations between the specific IPA production rate and NADPH concentration, as well as between IPA yield and the pyruvate dehydrogenase (PDH) flux. Our results showed that <jats:sup>13<\/jats:sup>C\u2010MFA is a helpful tool for metabolic engineering studies, and that further improvement in IPA production by <jats:italic>E. coli<\/jats:italic> may be achieved by fine\u2010tuning the cofactor ratio and concentrations, as well as optimizing the metabolic pathways and culture conditions.<\/jats:p><\/jats:sec>","DOI":"10.1002\/bit.26390","type":"journal-article","created":{"date-parts":[[2017,7,29]],"date-time":"2017-07-29T07:36:39Z","timestamp":1501313799000},"page":"2782-2793","source":"Crossref","is-referenced-by-count":27,"title":["Metabolic engineering of isopropyl alcohol\u2010producing <i>Escherichia coli<\/i> strains with <sup>13<\/sup>C\u2010metabolic flux analysis"],"prefix":"10.1002","volume":"114","author":[{"given":"Nobuyuki","family":"Okahashi","sequence":"first","affiliation":[{"name":"Department of Bioinfomatic Engineering Graduate School of Information Science and Technology Osaka University Osaka Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fumio","family":"Matsuda","sequence":"additional","affiliation":[{"name":"Department of Bioinfomatic Engineering Graduate School of Information Science and Technology Osaka University Osaka Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katsunori","family":"Yoshikawa","sequence":"additional","affiliation":[{"name":"Department of Bioinfomatic Engineering Graduate School of Information Science and Technology Osaka University Osaka Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomokazu","family":"Shirai","sequence":"additional","affiliation":[{"name":"Synthetic Chemicals Laboratory Mitsui Chemicals Inc. 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