{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T04:03:36Z","timestamp":1780545816310,"version":"3.54.1"},"reference-count":27,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T00:00:00Z","timestamp":1774396800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T00:00:00Z","timestamp":1774396800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Biotech &amp; Bioengineering"],"published-print":{"date-parts":[[2026,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Microbial production of 1,3\u2010butanediol (1,3\u2010BDO) offers a renewable route to this versatile C\n                    <jats:sub>4<\/jats:sub>\n                    chemical. However, the low performance of CoA\u2010acylating butyraldehyde dehydrogenase (Bld), which contains a catalytic cysteine, limits efficient production in recombinant\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    (\n                    <jats:italic>E. coli<\/jats:italic>\n                    ). In this study, wild\u2010type\n                    <jats:italic>Clostridium saccharoperbutylacetonicum<\/jats:italic>\n                    Bld and its variant Bld* were biochemically characterized and engineered to improve conversion of 3\u2010hydroxybutyryl\u2010CoA (3\u2010HB\u2010CoA) to 3\u2010hydroxybutyraldehyde (3\u2010HBA). Enzyme activity was strongly reduced by the product, 3\u2010HBA, and this reduction was largely alleviated by added cysteine. To mitigate this interference, several noncatalytic cysteine residues in Bld* were substituted individually and in combination guided by multiple sequence alignment and machine\u2010learning\u2010based mutational prediction. The triple mutant C151N\/C189A\/C353L (designated CYS31) displayed ~30% higher specific activity without altering substrate affinity or selectivity. Incorporation of CYS31 into a 1,3\u2010BDO\u2010producing\n                    <jats:italic>E. coli<\/jats:italic>\n                    strain led to a corresponding ~30% increase in titer, indicating that enhanced in vitro kinetics translated to higher in vivo 1,3\u2010BDO production. These findings provide a more effective Bld variant for 1,3\u2010BDO production and demonstrate that non\u2010active\u2010site cysteine residues can be viable engineering targets when an aldehyde intermediate is involved.\n                  <\/jats:p>","DOI":"10.1002\/bit.70197","type":"journal-article","created":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T04:34:18Z","timestamp":1774499658000},"page":"1605-1614","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineering of CoA\u2010Acylating Butyraldehyde Dehydrogenase for Enhanced 1,3\u2010Butanediol Production in\n                    <i>Escherichia coli<\/i>"],"prefix":"10.1002","volume":"123","author":[{"given":"Seunghyun","family":"Cho","sequence":"first","affiliation":[{"name":"School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology Ulsan Republic of Korea"},{"name":"R&amp;D Center, ACTIVON Co. Ltd. Cheongju Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tayyab","family":"Islam","sequence":"additional","affiliation":[{"name":"School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology Ulsan Republic of Korea"},{"name":"R&amp;D Center, ACTIVON Co. Ltd. Cheongju Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sobia","family":"Islam","sequence":"additional","affiliation":[{"name":"School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology Ulsan Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junhak","family":"Lee","sequence":"additional","affiliation":[{"name":"School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology Ulsan Republic of Korea"},{"name":"R&amp;D Center, ACTIVON Co. Ltd. Cheongju Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sung Won","family":"Jung","sequence":"additional","affiliation":[{"name":"R&amp;D Center, ACTIVON Co. Ltd. Cheongju Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9777-7278","authenticated-orcid":false,"given":"Sunghoon","family":"Park","sequence":"additional","affiliation":[{"name":"School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology Ulsan Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,3,25]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1094\/asbcj-2015-0109-01"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkab1106"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/tx800465m"},{"key":"e_1_2_8_5_1","unstructured":"Global 1 3\u2010Butylene Glycol Market. n.d."},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.R700019200"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.25196\/abstract"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2023.129814"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2023.128911"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1080\/09168451.2014.891933"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiosc.2012.11.025"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2022.09.001"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.03172-16"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/msz179"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1039\/c3ee41405a"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2025.132147"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12257-016-0560-3"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0075554"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.1c00144"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms14316"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2018.04.013"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1818"},{"key":"e_1_2_8_23_1","doi-asserted-by":"crossref","unstructured":"Rives A. 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