A directed genome evolution method to enhance hydrogen production in Rhodobacter capsulatus
Nitrogenase-dependent H2 production by photosynthetic bacteria, such as Rhodobacter capsulatus, has been extensively investigated. An important limitation to increase H2 production using genetic manipulation is the scarcity of high-throughput screening methods to detect possible overproducing mutant...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2022-08-01
|
| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.991123/full |
| _version_ | 1818513384398651392 |
|---|---|
| author | Emma Barahona Elisa San Isidro Laura Sierra-Heras Inés Álvarez-Melcón Emilio Jiménez-Vicente José María Buesa Juan Imperial Luis M. Rubio Luis M. Rubio |
| author_facet | Emma Barahona Elisa San Isidro Laura Sierra-Heras Inés Álvarez-Melcón Emilio Jiménez-Vicente José María Buesa Juan Imperial Luis M. Rubio Luis M. Rubio |
| author_sort | Emma Barahona |
| collection | DOAJ |
| description | Nitrogenase-dependent H2 production by photosynthetic bacteria, such as Rhodobacter capsulatus, has been extensively investigated. An important limitation to increase H2 production using genetic manipulation is the scarcity of high-throughput screening methods to detect possible overproducing mutants. Previously, we engineered R. capsulatus strains that emitted fluorescence in response to H2 and used them to identify mutations in the nitrogenase Fe protein leading to H2 overproduction. Here, we used ultraviolet light to induce random mutations in the genome of the engineered H2-sensing strain, and fluorescent-activated cell sorting to detect and isolate the H2-overproducing cells from libraries containing 5 × 105 mutants. Three rounds of mutagenesis and strain selection gradually increased H2 production up to 3-fold. The whole genomes of five H2 overproducing strains were sequenced and compared to that of the parental sensor strain to determine the basis for H2 overproduction. No mutations were present in well-characterized functions related to nitrogen fixation, except for the transcriptional activator nifA2. However, several mutations mapped to energy-generating systems and to carbon metabolism-related functions, which could feed reducing power or ATP to nitrogenase. Time-course experiments of nitrogenase depression in batch cultures exposed mismatches between nitrogenase protein levels and their H2 and ethylene production activities that suggested energy limitation. Consistently, cultivating in a chemostat produced up to 19-fold more H2 than the corresponding batch cultures, revealing the potential of selected H2 overproducing strains. |
| first_indexed | 2024-12-11T00:00:33Z |
| format | Article |
| id | doaj.art-e2ab7100a6fe4a11b4b340fd9afbdf1e |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-11T00:00:33Z |
| publishDate | 2022-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-e2ab7100a6fe4a11b4b340fd9afbdf1e2022-12-22T01:28:28ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-08-011310.3389/fmicb.2022.991123991123A directed genome evolution method to enhance hydrogen production in Rhodobacter capsulatusEmma Barahona0Elisa San Isidro1Laura Sierra-Heras2Inés Álvarez-Melcón3Emilio Jiménez-Vicente4José María Buesa5Juan Imperial6Luis M. Rubio7Luis M. Rubio8Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), Madrid, SpainCentro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), Madrid, SpainDepartamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Madrid, SpainNitrogenase-dependent H2 production by photosynthetic bacteria, such as Rhodobacter capsulatus, has been extensively investigated. An important limitation to increase H2 production using genetic manipulation is the scarcity of high-throughput screening methods to detect possible overproducing mutants. Previously, we engineered R. capsulatus strains that emitted fluorescence in response to H2 and used them to identify mutations in the nitrogenase Fe protein leading to H2 overproduction. Here, we used ultraviolet light to induce random mutations in the genome of the engineered H2-sensing strain, and fluorescent-activated cell sorting to detect and isolate the H2-overproducing cells from libraries containing 5 × 105 mutants. Three rounds of mutagenesis and strain selection gradually increased H2 production up to 3-fold. The whole genomes of five H2 overproducing strains were sequenced and compared to that of the parental sensor strain to determine the basis for H2 overproduction. No mutations were present in well-characterized functions related to nitrogen fixation, except for the transcriptional activator nifA2. However, several mutations mapped to energy-generating systems and to carbon metabolism-related functions, which could feed reducing power or ATP to nitrogenase. Time-course experiments of nitrogenase depression in batch cultures exposed mismatches between nitrogenase protein levels and their H2 and ethylene production activities that suggested energy limitation. Consistently, cultivating in a chemostat produced up to 19-fold more H2 than the corresponding batch cultures, revealing the potential of selected H2 overproducing strains.https://www.frontiersin.org/articles/10.3389/fmicb.2022.991123/fullnitrogenaseflow cytometryhydrogenasebiological hydrogen productionhupAmutagenesis |
| spellingShingle | Emma Barahona Elisa San Isidro Laura Sierra-Heras Inés Álvarez-Melcón Emilio Jiménez-Vicente José María Buesa Juan Imperial Luis M. Rubio Luis M. Rubio A directed genome evolution method to enhance hydrogen production in Rhodobacter capsulatus Frontiers in Microbiology nitrogenase flow cytometry hydrogenase biological hydrogen production hupA mutagenesis |
| title | A directed genome evolution method to enhance hydrogen production in Rhodobacter capsulatus |
| title_full | A directed genome evolution method to enhance hydrogen production in Rhodobacter capsulatus |
| title_fullStr | A directed genome evolution method to enhance hydrogen production in Rhodobacter capsulatus |
| title_full_unstemmed | A directed genome evolution method to enhance hydrogen production in Rhodobacter capsulatus |
| title_short | A directed genome evolution method to enhance hydrogen production in Rhodobacter capsulatus |
| title_sort | directed genome evolution method to enhance hydrogen production in rhodobacter capsulatus |
| topic | nitrogenase flow cytometry hydrogenase biological hydrogen production hupA mutagenesis |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.991123/full |
| work_keys_str_mv | AT emmabarahona adirectedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT elisasanisidro adirectedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT laurasierraheras adirectedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT inesalvarezmelcon adirectedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT emiliojimenezvicente adirectedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT josemariabuesa adirectedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT juanimperial adirectedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT luismrubio adirectedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT luismrubio adirectedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT emmabarahona directedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT elisasanisidro directedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT laurasierraheras directedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT inesalvarezmelcon directedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT emiliojimenezvicente directedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT josemariabuesa directedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT juanimperial directedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT luismrubio directedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus AT luismrubio directedgenomeevolutionmethodtoenhancehydrogenproductioninrhodobactercapsulatus |