{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T18:51:53Z","timestamp":1789411913479,"version":"build-2803163510"},"reference-count":40,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2024,12,13]],"date-time":"2024-12-13T00:00:00Z","timestamp":1734048000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Hans-Fischer-Gesellschaft","award":["459493567"],"award-info":[{"award-number":["459493567"]}]},{"name":"DFG, German Research Foundation","award":["459493567"],"award-info":[{"award-number":["459493567"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Riboflavin analogs lacking one methyl group (7\u03b1 or 8\u03b1) can still serve as a surrogate for riboflavin in riboflavin-deficient microorganisms or animals. The absence of both methyl groups at once completely abolishes this substitution capability. To elucidate the molecular mechanisms behind this phenomenon, we performed an adaptive laboratory evolution experiment (in triplicate) on an E. coli strain auxotrophic for riboflavin. As a result, the riboflavin requirement of the E. coli strain was reduced ~10-fold in the presence of 7,8-didemethyl-riboflavin. The whole genome sequencing of E. coli strains isolated from three experiments revealed two mutation hotspots: lpdA coding for the flavoenzyme dihydrolipoyl dehydrogenase (LpdA), and ompF coding for the major outer membrane protein. In order to investigate the essentiality of flavin\u2019s methyl groups to LpdA, the wild type and mutant variants of lpdA were cloned. At least two lpdA mutants increased the fitness of E. coli, and when 7,8-didemethyl-flavin was added to the growth medium, the increase was significant. To the best of our knowledge, an adaptive laboratory evolution experiment running in triplicate as a tool for the identification of mutation hotspots in the genome of microorganisms exposed to metabolic stress challenges is described here for the first time.<\/jats:p>","DOI":"10.3390\/molecules29245891","type":"journal-article","created":{"date-parts":[[2024,12,13]],"date-time":"2024-12-13T10:34:12Z","timestamp":1734086052000},"page":"5891","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Adaptive Laboratory Evolution of Flavin Functionality Identifies Dihydrolipoyl Dehydrogenase as One of the Critical Points for the Activity of 7,8-Didemethyl-Riboflavin as a Surrogate for Riboflavin in Escherichia coli"],"prefix":"10.3390","volume":"29","author":[{"given":"Farshad","family":"La-Rostami","sequence":"first","affiliation":[{"name":"Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandra","family":"Scharf","sequence":"additional","affiliation":[{"name":"Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenyang","family":"Albert","sequence":"additional","affiliation":[{"name":"Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8362-2160","authenticated-orcid":false,"given":"Nils","family":"Wax","sequence":"additional","affiliation":[{"name":"Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2868-2838","authenticated-orcid":false,"given":"Marina","family":"Creydt","sequence":"additional","affiliation":[{"name":"Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Boris","family":"Illarionov","sequence":"additional","affiliation":[{"name":"Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adelbert","family":"Bacher","sequence":"additional","affiliation":[{"name":"TUM School of Natural Sciences, Technical University of Munich, Boltzmannstra\u00dfe 10, 85748 Garching, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4090-7435","authenticated-orcid":false,"given":"Stefan","family":"Weber","sequence":"additional","affiliation":[{"name":"Institute of Physical Chemistry, Albert-Ludwigs-Universit\u00e4t Freiburg, Albertstra\u00dfe 21, 79104 Freiburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7243-4199","authenticated-orcid":false,"given":"Markus","family":"Fischer","sequence":"additional","affiliation":[{"name":"Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,12,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Hille, R.M.S., and Palfey, B. 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