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Here, transcriptome profiling demonstrated that 1004 genes were significantly regulated in the strain <jats:italic>R. toruloides<\/jats:italic> TO2 under methanol stress. Methanol impaired the function of membrane transport and subsequently weakened the utilization of glycerol, activities of the primary metabolism and functions of nucleus and ribosome. Afterwards the tolerance of TO2 to methanol was improved by using two\u2010round adaptive laboratory evolution (ALE). The final strain M2\u2010ale had tolerance up to 3.5% of methanol. <jats:sup>1<\/jats:sup>H NMR\u2010based metabolome analysis indicated that ALE not only improved the tolerance of M2\u2010ale to methanol but also tuned the carbon flux towards the biosynthesis of glycerolipid\u2010related metabolites. The biomass and lipid titer of M2\u2010ale reached 14.63\u00a0\u00b1\u00a00.45 g L<jats:sup>\u22121<\/jats:sup> and 7.06\u00a0\u00b1\u00a00.44 g L<jats:sup>\u22121<\/jats:sup> at 96 h in the crude glycerol medium, which increased up to 17.69% and 31.39%, respectively, comparing with TO2. Afterwards, an effective method for cell lysis was developed by combining sonication and enzymatic hydrolysis (So\u2010EnH). The lytic effect of So\u2010EnH was validated by using confocal imaging and flow cytometry. At last, lipid recovery rate reached 95.4\u00a0\u00b1\u00a02.7% at the optimized condition.<\/jats:p>","DOI":"10.1002\/biot.202300483","type":"journal-article","created":{"date-parts":[[2023,12,2]],"date-time":"2023-12-02T12:11:33Z","timestamp":1701519093000},"source":"Crossref","is-referenced-by-count":11,"title":["Application of adaptive laboratory evolution to improve the tolerance of <i>Rhodotorula<\/i> strain to methanol in crude glycerol and development of an effective method for cell lysis"],"prefix":"10.1002","volume":"19","author":[{"given":"Shiyu","family":"Wei","sequence":"first","affiliation":[{"name":"Key Laboratory of Metabolic Engineering and Biosynthesis Technology Ministry of Industry and Information Technology Nanjing University of Science and Technology  Nanjing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongyang","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Metabolic Engineering and Biosynthesis 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