High-throughput protein characterization by complementation using DNA barcoded fragment libraries
Abstract Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we...
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| Format: | Article |
| Language: | English |
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Springer Nature
2024-10-01
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| Series: | Molecular Systems Biology |
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| Online Access: | https://doi.org/10.1038/s44320-024-00068-z |
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| author | Bradley W Biggs Morgan N Price Dexter Lai Jasmine Escobedo Yuridia Fortanel Yolanda Y Huang Kyoungmin Kim Valentine V Trotter Jennifer V Kuehl Lauren M Lui Romy Chakraborty Adam M Deutschbauer Adam P Arkin |
| author_facet | Bradley W Biggs Morgan N Price Dexter Lai Jasmine Escobedo Yuridia Fortanel Yolanda Y Huang Kyoungmin Kim Valentine V Trotter Jennifer V Kuehl Lauren M Lui Romy Chakraborty Adam M Deutschbauer Adam P Arkin |
| author_sort | Bradley W Biggs |
| collection | DOAJ |
| description | Abstract Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we used complementation of auxotrophs and DNA barcode sequencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains of Escherichia coli to identify genes that complement missing biochemical activity. We recovered 41% of expected hits, with effectiveness ranging per source genome, and observed success even with distant E. coli relatives like Bacillus subtilis and Bacteroides thetaiotaomicron. Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein. Among the unexpected function identified was a sulfate uptake transporter, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. We also identified instances of cross-feeding wherein protein overexpression and nearby non-auxotrophic strains enabled growth. Altogether, Coaux-Seq’s utility is demonstrated, with future applications in ecology, health, and engineering. |
| format | Article |
| id | doaj-art-be2f0052335c41e5b844bbca96e0a1aa |
| institution | Kabale University |
| issn | 1744-4292 |
| language | English |
| publishDate | 2024-10-01 |
| publisher | Springer Nature |
| record_format | Article |
| series | Molecular Systems Biology |
| spelling | doaj-art-be2f0052335c41e5b844bbca96e0a1aa2024-11-10T12:48:31ZengSpringer NatureMolecular Systems Biology1744-42922024-10-0120111207122910.1038/s44320-024-00068-zHigh-throughput protein characterization by complementation using DNA barcoded fragment librariesBradley W Biggs0Morgan N Price1Dexter Lai2Jasmine Escobedo3Yuridia Fortanel4Yolanda Y Huang5Kyoungmin Kim6Valentine V Trotter7Jennifer V Kuehl8Lauren M Lui9Romy Chakraborty10Adam M Deutschbauer11Adam P Arkin12Environmental Genomics and Systems Biology Division, Lawrence Berkeley National LaboratoryEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National LaboratoryDepartment of Bioengineering, University of California-BerkeleyDepartment of Bioengineering, University of California-BerkeleyDepartment of Bioengineering, University of California-BerkeleyEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National LaboratoryDepartment of Bioengineering, University of California-BerkeleyEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National LaboratoryEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National LaboratoryEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National LaboratoryEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National LaboratoryEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National LaboratoryEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National LaboratoryAbstract Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we used complementation of auxotrophs and DNA barcode sequencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains of Escherichia coli to identify genes that complement missing biochemical activity. We recovered 41% of expected hits, with effectiveness ranging per source genome, and observed success even with distant E. coli relatives like Bacillus subtilis and Bacteroides thetaiotaomicron. Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein. Among the unexpected function identified was a sulfate uptake transporter, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. We also identified instances of cross-feeding wherein protein overexpression and nearby non-auxotrophic strains enabled growth. Altogether, Coaux-Seq’s utility is demonstrated, with future applications in ecology, health, and engineering.https://doi.org/10.1038/s44320-024-00068-zDNA BarcodingFunctional GenomicsHigh-throughput CharacterizationProtein Annotation |
| spellingShingle | Bradley W Biggs Morgan N Price Dexter Lai Jasmine Escobedo Yuridia Fortanel Yolanda Y Huang Kyoungmin Kim Valentine V Trotter Jennifer V Kuehl Lauren M Lui Romy Chakraborty Adam M Deutschbauer Adam P Arkin High-throughput protein characterization by complementation using DNA barcoded fragment libraries Molecular Systems Biology DNA Barcoding Functional Genomics High-throughput Characterization Protein Annotation |
| title | High-throughput protein characterization by complementation using DNA barcoded fragment libraries |
| title_full | High-throughput protein characterization by complementation using DNA barcoded fragment libraries |
| title_fullStr | High-throughput protein characterization by complementation using DNA barcoded fragment libraries |
| title_full_unstemmed | High-throughput protein characterization by complementation using DNA barcoded fragment libraries |
| title_short | High-throughput protein characterization by complementation using DNA barcoded fragment libraries |
| title_sort | high throughput protein characterization by complementation using dna barcoded fragment libraries |
| topic | DNA Barcoding Functional Genomics High-throughput Characterization Protein Annotation |
| url | https://doi.org/10.1038/s44320-024-00068-z |
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