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Under this regard, native methylotrophs have a large potential for converting methanol into value-added products but require targeted engineering approaches to enhance their performances and to widen their product spectrum. Here we use a systems-based approach to analyze and engineer <jats:italic>M. extorquens<\/jats:italic>\u00a0TK 0001 for production of glycolic acid. Application of constraint-based metabolic modeling reveals the great potential of <jats:italic>M.\u00a0extorquens<\/jats:italic> for that purpose, which is not yet described in literature. In particular, a superior theoretical product yield of 1.0 C-mol<jats:sub>Glycolic acid<\/jats:sub> C-mol<jats:sub>Methanol<\/jats:sub><jats:sup>\u22121<\/jats:sup> is predicted by our model, surpassing theoretical yields of sugar fermentation. Following this approach, we show here that strain engineering is viable and present 1st generation strains producing glycolic acid via a heterologous NADPH-dependent glyoxylate reductase. It was found that lactic acid is a surprising by-product of glycolic acid formation in <jats:italic>M.\u00a0extorquens<\/jats:italic>, most likely due to a surplus of available NADH upon glycolic acid synthesis. Finally, the best performing strain was tested in a fed-batch fermentation producing a mixture of up to total 1.2\u00a0g\u00a0L<jats:sup>\u22121<\/jats:sup> glycolic acid and lactic acid. Several key performance indicators of our glycolic acid producer strain are superior to state-of-the-art synthetic methylotrophs. The presented results open the door for further strain engineering of the native methylotroph <jats:italic>M. extorquens<\/jats:italic> and pave the way to produce two promising biopolymer building blocks from green methanol, i.e., glycolic acid and lactic acid.<\/jats:p>","DOI":"10.1186\/s12934-024-02583-y","type":"journal-article","created":{"date-parts":[[2024,12,23]],"date-time":"2024-12-23T05:11:02Z","timestamp":1734930662000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["A novel engineered strain of Methylorubrum extorquens for methylotrophic production of glycolic acid"],"prefix":"10.1186","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-7657-3307","authenticated-orcid":false,"given":"Katharina","family":"Dietz","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carina","family":"Sagstetter","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melanie","family":"Speck","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0456-4917","authenticated-orcid":false,"given":"Arne","family":"Roth","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2563-7561","authenticated-orcid":false,"given":"Steffen","family":"Klamt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-0509-5051","authenticated-orcid":false,"given":"Jonathan Thomas","family":"Fabarius","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,12,23]]},"reference":[{"key":"2583_CR1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2022.09.016","author":"J Philp","year":"2022","unstructured":"Philp J. 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