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 <i>Escherichia coli</i>
Riboflavin analogs lacking one methyl group (7α or 8α) 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 pheno...
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2024-12-01
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| author | Farshad La-Rostami Alexandra Scharf Chenyang Albert Nils Wax Marina Creydt Boris Illarionov Adelbert Bacher Stefan Weber Markus Fischer |
| author_facet | Farshad La-Rostami Alexandra Scharf Chenyang Albert Nils Wax Marina Creydt Boris Illarionov Adelbert Bacher Stefan Weber Markus Fischer |
| author_sort | Farshad La-Rostami |
| collection | DOAJ |
| description | Riboflavin analogs lacking one methyl group (7α or 8α) 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 <i>E. coli</i> strain auxotrophic for riboflavin. As a result, the riboflavin requirement of the <i>E. coli</i> strain was reduced ~10-fold in the presence of 7,8-didemethyl-riboflavin. The whole genome sequencing of <i>E. coli</i> strains isolated from three experiments revealed two mutation hotspots: <i>lpd</i>A coding for the flavoenzyme dihydrolipoyl dehydrogenase (LpdA), and <i>omp</i>F coding for the major outer membrane protein. In order to investigate the essentiality of flavin’s methyl groups to LpdA, the wild type and mutant variants of <i>lpd</i>A were cloned. At least two <i>lpd</i>A mutants increased the fitness of <i>E. coli</i>, 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. |
| format | Article |
| id | doaj-art-fcaa5e05a70d409b8a28b4a8daec7ead |
| institution | Kabale University |
| issn | 1420-3049 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | MDPI AG |
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| series | Molecules |
| spelling | doaj-art-fcaa5e05a70d409b8a28b4a8daec7ead2024-12-27T14:42:34ZengMDPI AGMolecules1420-30492024-12-012924589110.3390/molecules29245891Adaptive 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 <i>Escherichia coli</i>Farshad La-Rostami0Alexandra Scharf1Chenyang Albert2Nils Wax3Marina Creydt4Boris Illarionov5Adelbert Bacher6Stefan Weber7Markus Fischer8Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, GermanyHamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, GermanyHamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, GermanyHamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, GermanyHamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, GermanyHamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, GermanyTUM School of Natural Sciences, Technical University of Munich, Boltzmannstraße 10, 85748 Garching, GermanyInstitute of Physical Chemistry, Albert-Ludwigs-Universität Freiburg, Albertstraße 21, 79104 Freiburg, GermanyHamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, GermanyRiboflavin analogs lacking one methyl group (7α or 8α) 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 <i>E. coli</i> strain auxotrophic for riboflavin. As a result, the riboflavin requirement of the <i>E. coli</i> strain was reduced ~10-fold in the presence of 7,8-didemethyl-riboflavin. The whole genome sequencing of <i>E. coli</i> strains isolated from three experiments revealed two mutation hotspots: <i>lpd</i>A coding for the flavoenzyme dihydrolipoyl dehydrogenase (LpdA), and <i>omp</i>F coding for the major outer membrane protein. In order to investigate the essentiality of flavin’s methyl groups to LpdA, the wild type and mutant variants of <i>lpd</i>A were cloned. At least two <i>lpd</i>A mutants increased the fitness of <i>E. coli</i>, 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.https://www.mdpi.com/1420-3049/29/24/5891riboflavin analogsisoalloxazineflavoproteinsadaptive laboratory evolutionwhole genome sequencing |
| spellingShingle | Farshad La-Rostami Alexandra Scharf Chenyang Albert Nils Wax Marina Creydt Boris Illarionov Adelbert Bacher Stefan Weber Markus Fischer 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 <i>Escherichia coli</i> Molecules riboflavin analogs isoalloxazine flavoproteins adaptive laboratory evolution whole genome sequencing |
| 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 <i>Escherichia coli</i> |
| title_full | 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 <i>Escherichia coli</i> |
| title_fullStr | 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 <i>Escherichia coli</i> |
| title_full_unstemmed | 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 <i>Escherichia coli</i> |
| title_short | 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 <i>Escherichia coli</i> |
| title_sort | 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 i escherichia coli i |
| topic | riboflavin analogs isoalloxazine flavoproteins adaptive laboratory evolution whole genome sequencing |
| url | https://www.mdpi.com/1420-3049/29/24/5891 |
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