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We report the development of a new growth-coupled selection method for evolving NADPH-consuming enzymes based on the recycling of this redox cofactor. The method relies on a genetically modified Escherichia coli strain, which overaccumulates NADPH. This method was applied to the engineering of a carboxylic acid reductase (CAR) for improved catalytic activities on 2-methoxybenzoate and adipate. Mutant enzymes with up to 17-fold improvement in catalytic efficiency were identified from single-site saturated mutagenesis libraries. Obtained mutants were successfully applied to whole-cell conversions of adipate into 1,6-hexanediol, a C6 monomer commonly used in polymer industry.<\/jats:p>","DOI":"10.1021\/acssynbio.0c00290","type":"journal-article","created":{"date-parts":[[2020,6,27]],"date-time":"2020-06-27T03:47:21Z","timestamp":1593229641000},"page":"1632-1637","source":"Crossref","is-referenced-by-count":39,"title":["Engineering Carboxylic Acid Reductase (CAR) through\na Whole-Cell Growth-Coupled NADPH Recycling Strategy"],"prefix":"10.1021","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1084-4253","authenticated-orcid":true,"given":"Levi","family":"Kramer","sequence":"first","affiliation":[{"name":"University of Nebraska\ue5f8Lincoln , , , ,","place":["Lincoln, Nebraska, United States, 68588"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuan","family":"Le","sequence":"additional","affiliation":[{"name":"University of Nebraska\ue5f8Lincoln , , , ,","place":["Lincoln, Nebraska, United States, 68588"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marisa","family":"Rodriguez","sequence":"additional","affiliation":[{"name":"University of Nebraska\ue5f8Lincoln , , , ,","place":["Lincoln, Nebraska, United States, 68588"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6317-900X","authenticated-orcid":true,"given":"Mark A.","family":"Wilson","sequence":"additional","affiliation":[{"name":"University of Nebraska\ue5f8Lincoln , , , ,","place":["Lincoln, Nebraska, United States, 68588"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6983-9953","authenticated-orcid":true,"given":"Jiantao","family":"Guo","sequence":"additional","affiliation":[{"name":"University of Nebraska\ue5f8Lincoln , , , ,","place":["Lincoln, Nebraska, United States, 68588"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3826-1276","authenticated-orcid":true,"given":"Wei","family":"Niu","sequence":"additional","affiliation":[{"name":"University of Nebraska\ue5f8Lincoln , , , ,","place":["Lincoln, Nebraska, United States, 68588"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2020,6,26]]},"reference":[{"issue":"3","key":"2026081714210548800_cit1","doi-asserted-by":"publisher","first-page":"1874","DOI":"10.1128\/AEM.70.3.1874-1881.2004","article-title":"Nocardia sp. carboxylic\nacid reductase: cloning, expression, and characterization of a new\naldehyde oxidoreductase family","volume":"70","author":"He","year":"2004","journal-title":"Appl. Environ.\nMicrobiol."},{"issue":"1","key":"2026081714210548800_cit2","doi-asserted-by":"publisher","first-page":"478","DOI":"10.1074\/jbc.M607980200","article-title":"Reduction\nof carboxylic acids by Nocardia aldehyde oxidoreductase\nrequires a phosphopantetheinylated enzyme","volume":"282","author":"Venkitasubramanian","year":"2007","journal-title":"J.\nBiol. Chem."},{"key":"2026081714210548800_cit3","first-page":"425","article-title":"Biocatalytic reduction of carboxylic acids: mechanism\nand applications","volume-title":"Biocatalysis in the pharmaceutical\nand biotechnology industries","author":"Venkitasubramanian","year":"2007"},{"issue":"1","key":"2026081714210548800_cit4","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1073\/pnas.1216516110","article-title":"Carboxylic\nacid reductase is a versatile enzyme for the conversion of fatty acids\ninto fuels and chemical commodities","volume":"110","author":"Akhtar","year":"2013","journal-title":"Proc. Natl.\nAcad. Sci. U. 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A."},{"issue":"4","key":"2026081714210548800_cit5","doi-asserted-by":"publisher","first-page":"1089","DOI":"10.1002\/cctc.201300913","article-title":"Whole-cell carboxylate\nreduction for the synthesis of 3-hydroxytyrosol","volume":"6","author":"Napora-Wijata","year":"2014","journal-title":"ChemCatChem"},{"issue":"6","key":"2026081714210548800_cit6","doi-asserted-by":"publisher","first-page":"3753","DOI":"10.1021\/acscatal.6b00855","article-title":"One-pot cascade\nsynthesis of mono- and disubstituted piperidines and pyrrolidines\nusing carboxylic acid reductase (CAR), \u03c9-transaminase (\u03c9-TA),\nand Imine reductase (IRED) biocatalysts","volume":"6","author":"France","year":"2016","journal-title":"ACS\nCatal."},{"issue":"5","key":"2026081714210548800_cit7","doi-asserted-by":"publisher","first-page":"944","DOI":"10.1002\/bit.25860","article-title":"Characterizing and predicting carboxylic\nacid reductase activity for diversifying bioaldehyde production","volume":"113","author":"Moura","year":"2016","journal-title":"Biotechnol. 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Biotechnol."},{"issue":"3","key":"2026081714210548800_cit17","doi-asserted-by":"publisher","first-page":"1201","DOI":"10.1021\/jacs.8b11561","article-title":"Biocatalytic N-alkylation of amines using either primary\nalcohols or carboxylic acids via reductive aminase cascades","volume":"141","author":"Ramsden","year":"2019","journal-title":"J. Am. Chem. Soc."},{"issue":"9","key":"2026081714210548800_cit18","doi-asserted-by":"publisher","first-page":"975","DOI":"10.1038\/nchembio.2434","article-title":"Structures of carboxylic\nacid reductase reveal domain dynamics underlying catalysis","volume":"13","author":"Gahloth","year":"2017","journal-title":"Nat. Chem. 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Biol."},{"issue":"6","key":"2026081714210548800_cit31","doi-asserted-by":"publisher","first-page":"352","DOI":"10.1016\/j.ymben.2008.09.001","article-title":"Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with\na NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways","volume":"10","author":"Martinez","year":"2008","journal-title":"Metab.\nEng."},{"issue":"5","key":"2026081714210548800_cit32","doi-asserted-by":"publisher","first-page":"504","DOI":"10.1016\/j.ymben.2012.07.002","article-title":"A selection platform for carbon chain elongation using\nthe CoA-dependent pathway to produce linear higher alcohols","volume":"14","author":"Machado","year":"2012","journal-title":"Metab. Eng."},{"issue":"12","key":"2026081714210548800_cit33","doi-asserted-by":"publisher","first-page":"1449","DOI":"10.1007\/s10295-013-1335-x","article-title":"Improvement of NADPH\nbioavailability in Escherichia coli by replacing\nNAD+-dependent glyceraldehyde-3-phosphate dehydrogenase GapA with\nNADP+-dependent GapB from Bacillus subtilis and addition\nof NAD kinase","volume":"40","author":"Wang","year":"2013","journal-title":"J. Ind. Microbiol. Biotechnol."},{"issue":"2","key":"2026081714210548800_cit34","doi-asserted-by":"publisher","first-page":"173","DOI":"10.1038\/nbt.3763","article-title":"Lipid production\nin Yarrowia lipolytica is maximized by engineering\ncytosolic redox metabolism","volume":"35","author":"Qiao","year":"2017","journal-title":"Nat. Biotechnol."},{"issue":"2","key":"2026081714210548800_cit35","doi-asserted-by":"publisher","first-page":"247","DOI":"10.1111\/j.1574-6968.2001.tb10892.x","article-title":"Metabolic flux response\nto phosphoglucose isomerase knock-out in Escherichia coli and impact of overexpression of the soluble transhydrogenase UdhA","volume":"204","author":"Canonaco","year":"2001","journal-title":"FEMS Microbiol. Lett."},{"issue":"8","key":"2026081714210548800_cit36","doi-asserted-by":"publisher","first-page":"6613","DOI":"10.1074\/jbc.M311657200","article-title":"The soluble and membrane-bound transhydrogenases\nUdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli","volume":"279","author":"Sauer","year":"2004","journal-title":"J. Biol. 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Comput.\nChem."}],"container-title":["ACS Synthetic Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acssynbio.0c00290","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/asbcd6\/article-pdf\/9\/7\/1632\/5929857\/sb0c00290.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/asbcd6\/article-pdf\/9\/7\/1632\/5929857\/sb0c00290.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T19:00:58Z","timestamp":1786993258000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/asbcd6\/article\/9\/7\/1632\/592005\/Engineering-Carboxylic-Acid-Reductase-CAR-through"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,6,26]]},"references-count":39,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2020,6,26]]},"published-print":{"date-parts":[[2020,7,17]]}},"URL":"https:\/\/doi.org\/10.1021\/acssynbio.0c00290","relation":{},"ISSN":["2161-5063"],"issn-type":[{"value":"2161-5063","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,6,26]]}}}