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The use of glycerol as a carbon source for 2,3-BDO production in <jats:italic>Klebsiella pneumoniae<\/jats:italic> has been limited, since 1,3-propanediol (PDO) is generated during glycerol fermentation.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>In this study, the inactivation of the <jats:italic>budC<\/jats:italic> gene in <jats:italic>K. pneumoniae<\/jats:italic> increased the production rate of (<jats:italic>R,R<\/jats:italic>)-2,3-BDO from 21.92\u2009\u00b1\u20092.10 to 92.05\u2009\u00b1\u20091.20%. The major isomer form of <jats:italic>K. pneumoniae<\/jats:italic> (<jats:italic>meso<\/jats:italic>-2,3-BDO) was shifted to (<jats:italic>R,R<\/jats:italic>)-2,3-BDO. The purity of (<jats:italic>R,R<\/jats:italic>)-2,3-BDO was examined by agitation speed, and 98.54% of (<jats:italic>R,R<\/jats:italic>)-2,3-BDO was obtained at 500\u00a0rpm. However, as the cultivation period got longer, the purity of (<jats:italic>R,R<\/jats:italic>)-2,3-BDO declined. For this problem, a two-step agitation speed control strategy (adjusted from 500 to 400\u00a0rpm after 24\u00a0h) and over-expression of the <jats:italic>dhaD<\/jats:italic> gene involved in (<jats:italic>R,R<\/jats:italic>)-2,3-BDO biosynthesis were used. Nevertheless, the purity of (<jats:italic>R,R<\/jats:italic>)-2,3-BDO still gradually decreased over time. Finally, when pure glycerol was replaced with crude glycerol, the titer of 89.47\u00a0g\/L of (<jats:italic>R,R<\/jats:italic>)-2,3-BDO (1.69\u00a0g\/L of <jats:italic>meso<\/jats:italic>-2,3-BDO), productivity of 1.24\u00a0g\/L\/h, and yield of 0.35\u00a0g\/g consumed crude glycerol was achieved while maintaining a purity of 98% or higher.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>This study is meaningful in that it demonstrated the highest production and productivity among studies in that produced (<jats:italic>R,R<\/jats:italic>)-2,3-BDO with a high purity in <jats:italic>Klebsiella<\/jats:italic> sp. strains. In addition, to the best of our knowledge, this is the first study to produce (<jats:italic>R,R<\/jats:italic>)-2,3-BDO using glycerol as the sole carbon source.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12934-024-02480-4","type":"journal-article","created":{"date-parts":[[2024,7,23]],"date-time":"2024-07-23T18:22:50Z","timestamp":1721758970000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["High production of enantiopure (R,R)-2,3-butanediol from crude glycerol by Klebsiella pneumoniae with an engineered oxidative pathway and a two-stage agitation strategy"],"prefix":"10.1186","volume":"23","author":[{"given":"Min-Ho","family":"Jo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jung-Hyun","family":"Ju","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sun-Yeon","family":"Heo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chang-Bum","family":"Son","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ki Jun","family":"Jeong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Baek-Rock","family":"Oh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,7,23]]},"reference":[{"key":"2480_CR1","doi-asserted-by":"publisher","first-page":"48","DOI":"10.1186\/1754-6834-5-48","volume":"5","author":"JR Almeida","year":"2012","unstructured":"Almeida JR, F\u00e1varo LC, Quirino BF. 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