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The strain naturally overproduces and secretes L-glutamate, making it a promising platform for engineering pathways toward L-glutamate-derived amino acids such as L-proline, which has applications in nutrition, stress protection, and industry.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Heterologous expression of an osmotic stress\u2013responsive L-proline biosynthetic operon from the mesophile\n                      <jats:italic>Bacillus licheniformis<\/jats:italic>\n                      in the thermotolerant\n                      <jats:italic>B. methanolicus<\/jats:italic>\n                      strain MGA3 did not increase L-proline levels but instead led to accumulation of L-citrulline. This was likely due to heat sensitivity of pyrroline-5-carboxylate reductase (ProH), the last enzyme of the osmoregulatory L-proline biosynthetic route, and metabolic crosstalk between L-proline and L-arginine pathways operating in Bacilli. To overcome these limitations, a synthetic operon containing the native anabolic\n                      <jats:italic>proBA<\/jats:italic>\n                      and\n                      <jats:italic>proI<\/jats:italic>\n                      L-proline biosynthetic genes from\n                      <jats:italic>B. methanolicus<\/jats:italic>\n                      MGA3 was engineered to remove transcriptional T-box regulation and biochemical feedback inhibition of ProB enzyme activity. Expression of this engineered operon enhanced L-proline synthesis and triggered its secretion during methanol-based growth of\n                      <jats:italic>B. methanolicus<\/jats:italic>\n                      MGA3 at 50\u00b0 C. In fed-batch fermentation with methanol as carbon and energy source, extracellular L-proline levels reached 262\u2009\u00b1\u200920\u00a0mg L\u207b\n                      <jats:sup>1<\/jats:sup>\n                      after 40\u00a0h. During the fermentation process, a stepwise increase in medium osmolarity was observed, likely due to large-scale L-glutamate excretion, which impaired cellular growth.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      This study links osmolarity dynamics to methanol-based fermentation in\n                      <jats:italic>B. methanolicus<\/jats:italic>\n                      MGA3 and demonstrates its potential as a cell factory for L-proline and L-citrulline production. These findings support further strain optimization for producing value-added amino acids and highlight the relevance of methylotrophic thermophiles in sustainable biotechnology.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12934-026-03032-8","type":"journal-article","created":{"date-parts":[[2026,5,23]],"date-time":"2026-05-23T05:05:57Z","timestamp":1779512757000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Harnessing the thermotolerant methylotroph Bacillus methanolicus for methanol-based synthetic L-proline production"],"prefix":"10.1186","volume":"25","author":[{"given":"Christine","family":"Frank","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Virant","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gregor","family":"Kosec","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tamara","family":"Hoffmann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Oskar","family":"Zelder","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Max F.","family":"Felle","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erhard","family":"Bremer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,5,23]]},"reference":[{"key":"3032_CR1","doi-asserted-by":"publisher","first-page":"1575","DOI":"10.1016\/j.biortech.2017.05.065","volume":"245","author":"JH Lee","year":"2017","unstructured":"Lee JH, Wendisch VF. 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