{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,28]],"date-time":"2026-02-28T08:58:57Z","timestamp":1772269137601,"version":"3.50.1"},"reference-count":43,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2024,8,20]],"date-time":"2024-08-20T00:00:00Z","timestamp":1724112000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Natural Science Foundation of China","award":["81673542"],"award-info":[{"award-number":["81673542"]}]},{"name":"National Natural Science Foundation of China","award":["KFB23077A"],"award-info":[{"award-number":["KFB23077A"]}]},{"name":"Fujian Agriculture and Forestry University","award":["81673542"],"award-info":[{"award-number":["81673542"]}]},{"name":"Fujian Agriculture and Forestry University","award":["KFB23077A"],"award-info":[{"award-number":["KFB23077A"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V\/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g\/L) of the product and providing both excellent productivity and yield values of 5.94 g\/L\/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.<\/jats:p>","DOI":"10.3390\/molecules29163934","type":"journal-article","created":{"date-parts":[[2024,8,22]],"date-time":"2024-08-22T06:28:51Z","timestamp":1724308131000},"page":"3934","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem"],"prefix":"10.3390","volume":"29","author":[{"given":"Jiming","family":"Zhang","sequence":"first","affiliation":[{"name":"College of Chemical Engineering, Huaqiao University, Xiamen 361021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3170-2024","authenticated-orcid":false,"given":"Hui","family":"Lin","sequence":"additional","affiliation":[{"name":"Institute of Edible Fungi, Fujian Academy of Agricultural Sciences, Fuzhou 350012, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chaosong","family":"Zheng","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bin","family":"Yang","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miao","family":"Liang","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yi","family":"Lin","sequence":"additional","affiliation":[{"name":"College of Chemical Engineering, Huaqiao University, Xiamen 361021, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7422-5438","authenticated-orcid":false,"given":"Liaoyuan","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,8,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1016\/j.biotechadv.2011.01.007","article-title":"Microbial 2,3-butanediol production: A state-of-the-art review","volume":"29","author":"Ji","year":"2011","journal-title":"Biotechnol. 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