{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T21:00:34Z","timestamp":1780088434180,"version":"3.54.0"},"reference-count":55,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2026,1,9]],"date-time":"2026-01-09T00:00:00Z","timestamp":1767916800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,1,9]],"date-time":"2026-01-09T00:00:00Z","timestamp":1767916800000},"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;amp; Bioengineering"],"published-print":{"date-parts":[[2026,4]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Isobutanol is a fusel alcohol that can be produced microbially for use as a biofuel or upgraded into sustainable aviation fuel (SAF). A key enzyme in the isobutanol biosynthetic pathway is 2\u2010ketoacid decarboxylase (KDC), which irreversibly decarboxylates 2\u2010ketoisovalerate (KIV) to yield isobutyraldehyde. However, many previously characterized KDC enzymes also act promiscuously on other 2\u2010ketoacids, (e.g., pyruvate) to produce a related aldehyde (e.g., acetaldehyde). This unwanted side reaction is especially important when isobutanol is produced in\n                    <jats:italic>Saccharomyces cerevisiae<\/jats:italic>\n                    (\n                    <jats:italic>S. cerevisiae<\/jats:italic>\n                    ) because it leads to pyruvate being diverted to ethanol. In order to make\n                    <jats:italic>S. cerevisiae<\/jats:italic>\n                    a strict isobutanologen, a KDC enzyme that is specific for KIV must be deployed. In this study, we used a combination of cell\u2010based and\n                    <jats:italic>in vitro<\/jats:italic>\n                    enzyme assays to investigate KDC substrate specificity, characterizing a large set of homologs for KIV, pyruvate, and phenylpyruvate (PPV) activity. A diverse range of substrate specificities was discovered, and some previously uncharacterized KDCs were revealed to have high KIV activity and low pyruvate activity. Multi\u2010site saturation mutagenesis (SSM) of one of these KDCs identified mutants with increased KIV activity, while maintaining low levels of pyruvate activity. In a KIV bioconversion experiment, bioprospected and engineered KDCs allowed similar KIV consumption to when using the previously characterized\n                    <jats:italic>Lactococcus lactis<\/jats:italic>\n                    KdcA, though with some ethanol also produced. The KDCs identified here show promise for production of isobutanol and other alcohols derived from 2\u2010ketoacids, and the dataset of newly characterized KDCs can inform future efforts to understand and engineer substrate specificity in KDCs.\n                  <\/jats:p>","DOI":"10.1002\/bit.70150","type":"journal-article","created":{"date-parts":[[2026,1,10]],"date-time":"2026-01-10T06:14:57Z","timestamp":1768025697000},"page":"1036-1049","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Engineering of 2\u2010ketoacid Decarboxylases for Production of Isobutanol and Other Fusel Alcohols in\n                    <i>Saccharomyces cerevisiae<\/i>"],"prefix":"10.1002","volume":"123","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0047-6127","authenticated-orcid":false,"given":"Joshua J.","family":"Dietrich","sequence":"first","affiliation":[{"name":"Department of Chemical and Biological Engineering University of Wisconsin\u2010Madison Madison Wisconsin USA"},{"name":"DOE Great Lakes Bioenergy Research Center University of Wisconsin\u2010Madison Madison Wisconsin USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maelia","family":"Dziedzic","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biological Engineering University of Wisconsin\u2010Madison Madison Wisconsin USA"},{"name":"DOE Great Lakes Bioenergy Research Center University of Wisconsin\u2010Madison Madison Wisconsin USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia","family":"Sun","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biological Engineering University of Wisconsin\u2010Madison Madison Wisconsin USA"},{"name":"DOE Great Lakes Bioenergy Research Center University of Wisconsin\u2010Madison Madison Wisconsin USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sri Harsha","family":"Adusumilli","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biological Engineering University of Wisconsin\u2010Madison Madison Wisconsin USA"},{"name":"DOE Great Lakes Bioenergy Research Center University of Wisconsin\u2010Madison Madison Wisconsin USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0420-4970","authenticated-orcid":false,"given":"Carla","family":"Gon\u00e7alves","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences Vanderbilt University Nashville Tennessee USA"},{"name":"Evolutionary Studies Initiative Vanderbilt University Nashville Tennessee USA"},{"name":"Associate Laboratory i4HB\u2014Institute for Health and Bioeconomy and UCIBIO\u2014Applied Molecular Biosciences Unit, Department of Life Sciences, NOVA School of Science and Technology Universidade NOVA de Lisboa Caparica Portugal"},{"name":"UCIBIO\u2010i4HB, Departamento de Ci\u00eancias da Vida, Faculdade de Ci\u00eancias e Tecnologia Universidade Nova de Lisboa Caparica Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5088-7461","authenticated-orcid":false,"given":"Chris Todd","family":"Hittinger","sequence":"additional","affiliation":[{"name":"DOE Great Lakes Bioenergy Research Center University of Wisconsin\u2010Madison Madison Wisconsin USA"},{"name":"Laboratory of Genetics, Center for Genomic Science Innovation, Wisconsin Energy Institute, J. F. Crow Institute for the Study of Evolution University of Wisconsin\u2010Madison Madison Wisconsin USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brian F.","family":"Pfleger","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biological Engineering University of Wisconsin\u2010Madison Madison Wisconsin USA"},{"name":"DOE Great Lakes Bioenergy Research Center University of Wisconsin\u2010Madison Madison Wisconsin USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,1,9]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-013-4758-4"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature06450"},{"key":"e_1_2_9_4_1","first-page":"182","volume-title":"Methods in Enzymology","author":"Becker D. 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