Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations
The bio-economy relies on microbial strains optimized for efficient large scale production of chemicals and fuels from inexpensive and renewable feedstocks under industrial conditions. The reduced one carbon compound methanol, whose production does not involve carbohydrates needed for the feed and f...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2019
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6595629/ https://www.ncbi.nlm.nih.gov/pubmed/31281294 http://dx.doi.org/10.3389/fmicb.2019.01313 |
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author | Belkhelfa, Sophia Roche, David Dubois, Ivan Berger, Anne Delmas, Valérie A. Cattolico, Laurence Perret, Alain Labadie, Karine Perdereau, Aude C. Darii, Ekaterina Pateau, Emilie de Berardinis, Véronique Salanoubat, Marcel Bouzon, Madeleine Döring, Volker |
author_facet | Belkhelfa, Sophia Roche, David Dubois, Ivan Berger, Anne Delmas, Valérie A. Cattolico, Laurence Perret, Alain Labadie, Karine Perdereau, Aude C. Darii, Ekaterina Pateau, Emilie de Berardinis, Véronique Salanoubat, Marcel Bouzon, Madeleine Döring, Volker |
author_sort | Belkhelfa, Sophia |
collection | PubMed |
description | The bio-economy relies on microbial strains optimized for efficient large scale production of chemicals and fuels from inexpensive and renewable feedstocks under industrial conditions. The reduced one carbon compound methanol, whose production does not involve carbohydrates needed for the feed and food sector, can be used as sole carbon and energy source by methylotrophic bacteria like Methylobacterium extorquens AM1. This strain has already been engineered to produce various commodity and high value chemicals from methanol. The toxic effect of methanol limits its concentration as feedstock to 1% v/v. We obtained M. extorquens chassis strains tolerant to high methanol via adaptive directed evolution using the GM3 technology of automated continuous culture. Turbidostat and conditional medium swap regimes were employed for the parallel evolution of the recently characterized strain TK 0001 and the reference strain AM1 and enabled the isolation of derivatives of both strains capable of stable growth with 10% methanol. The isolates produced more biomass at 1% methanol than the ancestor strains. Genome sequencing identified the gene metY coding for an O-acetyl-L-homoserine sulfhydrylase as common target of mutation. We showed that the wildtype enzyme uses methanol as substrate at elevated concentrations. This side reaction produces methoxine, a toxic homolog of methionine incorporated in polypeptides during translation. All mutated metY alleles isolated from the evolved populations coded for inactive enzymes, designating O-acetyl-L-homoserine sulfhydrylase as a major vector of methanol toxicity. A whole cell transcriptomic analysis revealed that genes coding for chaperones and proteases were upregulated in the evolved cells as compared with the wildtype, suggesting that the cells had to cope with aberrant proteins formed during the adaptation to increasing methanol exposure. In addition, the expression of ribosomal proteins and enzymes related to energy production from methanol like formate dehydrogenases and ATP synthases was boosted in the evolved cells upon a short-term methanol stress. D-lactate production from methanol by adapted cells overexpressing the native D-lactate dehydrogenase was quantified. A significant higher lactate yield was obtained compared with control cells, indicating an enhanced capacity of the cells resistant to high methanol to assimilate this one carbon feedstock more efficiently. |
format | Online Article Text |
id | pubmed-6595629 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-65956292019-07-05 Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations Belkhelfa, Sophia Roche, David Dubois, Ivan Berger, Anne Delmas, Valérie A. Cattolico, Laurence Perret, Alain Labadie, Karine Perdereau, Aude C. Darii, Ekaterina Pateau, Emilie de Berardinis, Véronique Salanoubat, Marcel Bouzon, Madeleine Döring, Volker Front Microbiol Microbiology The bio-economy relies on microbial strains optimized for efficient large scale production of chemicals and fuels from inexpensive and renewable feedstocks under industrial conditions. The reduced one carbon compound methanol, whose production does not involve carbohydrates needed for the feed and food sector, can be used as sole carbon and energy source by methylotrophic bacteria like Methylobacterium extorquens AM1. This strain has already been engineered to produce various commodity and high value chemicals from methanol. The toxic effect of methanol limits its concentration as feedstock to 1% v/v. We obtained M. extorquens chassis strains tolerant to high methanol via adaptive directed evolution using the GM3 technology of automated continuous culture. Turbidostat and conditional medium swap regimes were employed for the parallel evolution of the recently characterized strain TK 0001 and the reference strain AM1 and enabled the isolation of derivatives of both strains capable of stable growth with 10% methanol. The isolates produced more biomass at 1% methanol than the ancestor strains. Genome sequencing identified the gene metY coding for an O-acetyl-L-homoserine sulfhydrylase as common target of mutation. We showed that the wildtype enzyme uses methanol as substrate at elevated concentrations. This side reaction produces methoxine, a toxic homolog of methionine incorporated in polypeptides during translation. All mutated metY alleles isolated from the evolved populations coded for inactive enzymes, designating O-acetyl-L-homoserine sulfhydrylase as a major vector of methanol toxicity. A whole cell transcriptomic analysis revealed that genes coding for chaperones and proteases were upregulated in the evolved cells as compared with the wildtype, suggesting that the cells had to cope with aberrant proteins formed during the adaptation to increasing methanol exposure. In addition, the expression of ribosomal proteins and enzymes related to energy production from methanol like formate dehydrogenases and ATP synthases was boosted in the evolved cells upon a short-term methanol stress. D-lactate production from methanol by adapted cells overexpressing the native D-lactate dehydrogenase was quantified. A significant higher lactate yield was obtained compared with control cells, indicating an enhanced capacity of the cells resistant to high methanol to assimilate this one carbon feedstock more efficiently. Frontiers Media S.A. 2019-06-20 /pmc/articles/PMC6595629/ /pubmed/31281294 http://dx.doi.org/10.3389/fmicb.2019.01313 Text en Copyright © 2019 Belkhelfa, Roche, Dubois, Berger, Delmas, Cattolico, Perret, Labadie, Perdereau, Darii, Pateau, de Berardinis, Salanoubat, Bouzon and Döring. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Belkhelfa, Sophia Roche, David Dubois, Ivan Berger, Anne Delmas, Valérie A. Cattolico, Laurence Perret, Alain Labadie, Karine Perdereau, Aude C. Darii, Ekaterina Pateau, Emilie de Berardinis, Véronique Salanoubat, Marcel Bouzon, Madeleine Döring, Volker Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations |
title | Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations |
title_full | Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations |
title_fullStr | Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations |
title_full_unstemmed | Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations |
title_short | Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations |
title_sort | continuous culture adaptation of methylobacterium extorquens am1 and tk 0001 to very high methanol concentrations |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6595629/ https://www.ncbi.nlm.nih.gov/pubmed/31281294 http://dx.doi.org/10.3389/fmicb.2019.01313 |
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