{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T13:28:53Z","timestamp":1790256533570,"version":"4.1.0"},"reference-count":38,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2008,7,22]],"date-time":"2008-07-22T00:00:00Z","timestamp":1216684800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biotech &amp; Bioengineering"],"published-print":{"date-parts":[[2009,1]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p><jats:italic>Escherichia coli<\/jats:italic> strain PC09 (\u0394<jats:italic>xylB<\/jats:italic>, cAMP\u2010independent CRP (<jats:italic>crp<\/jats:italic>*) mutant) expressing an NADPH\u2010dependent xylose reductase from <jats:italic>Candida boidinii<\/jats:italic> (CbXR) was previously reported to produce xylitol from xylose while metabolizing glucose [Cirino et al. (2006) Biotechnol Bioeng 95(6): 1167\u20131176]. This study aims to understand the role of NADPH supply in xylitol yield and the contribution of key central carbon metabolism enzymes toward xylitol production. Studies in which the expression of CbXR or a xylose transporter was increased suggest that enzyme activity and xylose transport are not limiting xylitol production in PC09. A constraints\u2010based stoichiometric metabolic network model was used to understand the roles of central carbon metabolism reactions and xylose transport energetics on the theoretical maximum molar xylitol yield (xylitol produced per glucose consumed), and xylitol yields (<jats:italic>Y<\/jats:italic><jats:sub>RPG<\/jats:sub>) were measured from resting cell biotransformations with various PC09 derivative strains. For the case of xylose\u2010proton symport, omitting the Zwf (glucose\u20106\u2010phosphate dehydrogenase) or PntAB (membrane\u2010bound transhydrogenase) reactions or TCA cycle activity from the model reduces the theoretical maximum yield from 9.2 to 8.8, 3.6, and 8.0 mol xylitol (mol glucose)<jats:sup>\u22121<\/jats:sup>, respectively. Experimentally, deleting <jats:italic>pgi<\/jats:italic> (encoding phosphoglucose isomerase) from strain PC09 improves the yield from 3.4 to 4.0 mol xylitol (mol glucose)<jats:sup>\u22121<\/jats:sup>, while deleting either or both <jats:italic>E. coli<\/jats:italic> transhydrogenases (<jats:italic>sthA<\/jats:italic> and <jats:italic>pntA<\/jats:italic>) has no significant effect on the measured yield. Deleting either <jats:italic>zwf<\/jats:italic> or <jats:italic>sucC<\/jats:italic> (TCA cycle) significantly reduces the yield from 3.4 to 2.0 and 2.3 mol xylitol (mol glucose)<jats:sup>\u22121<\/jats:sup>, respectively. Expression of a xylose reductase with relaxed cofactor specificity increases the yield to 4.0. The large discrepancy between theoretical maximum and experimentally determined yield values suggests that biocatalysis is compromised by pathways competing for reducing equivalents and dissipating energy. The metabolic role of transhydrogenases during <jats:italic>E. coli<\/jats:italic> biocatalysis has remained largely unspecified. Our results demonstrate the importance of direct NADPH supply by NADP<jats:sup>+<\/jats:sup>\u2010utilizing enzymes in central metabolism for driving heterologous NADPH\u2010dependent reactions, and suggest that the pool of reduced cofactors available for biotransformation is not readily interchangeable via transhydrogenase. Biotechnol. Bioeng. 2009;102: 209\u2013220. \u00a9 2008 Wiley Periodicals, Inc.<\/jats:p>","DOI":"10.1002\/bit.22060","type":"journal-article","created":{"date-parts":[[2008,7,22]],"date-time":"2008-07-22T15:23:13Z","timestamp":1216740193000},"page":"209-220","source":"Crossref","is-referenced-by-count":75,"title":["Analysis of NADPH supply during xylitol production by engineered <i>Escherichia coli<\/i>"],"prefix":"10.1002","volume":"102","author":[{"given":"Jonathan W.","family":"Chin","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Reza","family":"Khankal","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caroline A.","family":"Monroe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Costas D.","family":"Maranas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick C.","family":"Cirino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2008,8,12]]},"reference":[{"key":"e_1_2_1_2_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.182.17.4934-4940.2000"},{"key":"e_1_2_1_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/msb4100050"},{"key":"e_1_2_1_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.21837"},{"key":"e_1_2_1_5_1","volume-title":"Escherichia coli and Salmonella: Cellular and molecular biology","author":"Bock A","year":"1996"},{"key":"e_1_2_1_6_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.02234-07"},{"key":"e_1_2_1_7_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-6968.2001.tb10892.x"},{"key":"e_1_2_1_8_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0337684100"},{"issue":"2","key":"e_1_2_1_9_1","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1016\/S0021-9258(18)65196-8","article-title":"Chemistry and properties of the 3\u2010acetylpyridine analogue of diphosphopyridine nucleotide","volume":"221","author":"Ciotti MM","year":"1956","journal-title":"J Biol Chem"},{"key":"e_1_2_1_10_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.21082"},{"key":"e_1_2_1_11_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M201691200"},{"key":"e_1_2_1_12_1","volume-title":"Tricarboxylic acid cycle and glyoxylate bypass","author":"Cronan JE","year":"1996"},{"key":"e_1_2_1_13_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.120163297"},{"key":"e_1_2_1_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0958-1669(00)00237-8"},{"key":"e_1_2_1_15_1","doi-asserted-by":"publisher","DOI":"10.1006\/mben.2001.0209"},{"key":"e_1_2_1_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/0304-4165(66)90149-8"},{"key":"e_1_2_1_17_1","doi-asserted-by":"publisher","DOI":"10.1515\/BC.1999.179"},{"key":"e_1_2_1_18_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.183.21.6384-6393.2001"},{"issue":"1","key":"e_1_2_1_19_1","doi-asserted-by":"crossref","first-page":"274","DOI":"10.1128\/jb.144.1.274-278.1980","article-title":"Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli","volume":"144","author":"Heller KB","year":"1980","journal-title":"J Bacteriol"},{"key":"e_1_2_1_20_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.185.24.7053-7067.2003"},{"key":"e_1_2_1_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiotec.2008.02.003"},{"key":"e_1_2_1_22_1","volume-title":"A short course in bacterial genetics: A laboratory manual and handbook for Escherichia coli and related bacteria","author":"Miller JH","year":"1992"},{"key":"e_1_2_1_23_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2004.06.002"},{"issue":"3","key":"e_1_2_1_24_1","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1042\/bj3260683","article-title":"NAD(P)H\u2010dependent aldose reductase from the xylose\u2010assimilating yeast Candida tenuis. 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