Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries
Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2013-09-01
|
| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2013.00282/full |
| _version_ | 1818276908411912192 |
|---|---|
| author | Mari eNyyssönen Huu M. Tran Huu M. Tran Ulas eKaraoz Claudia eWeihe Masood Z. Hadi Jennifer B. H. Martiny Adam C. Martiny Adam C. Martiny Eoin L. Brodie Eoin L. Brodie |
| author_facet | Mari eNyyssönen Huu M. Tran Huu M. Tran Ulas eKaraoz Claudia eWeihe Masood Z. Hadi Jennifer B. H. Martiny Adam C. Martiny Adam C. Martiny Eoin L. Brodie Eoin L. Brodie |
| author_sort | Mari eNyyssönen |
| collection | DOAJ |
| description | Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26 %) cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase families were detected. Using tetranucleotide frequency binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts. |
| first_indexed | 2024-12-12T22:53:07Z |
| format | Article |
| id | doaj.art-0613d62ed5964bcc9852286cb0e63a1f |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-12T22:53:07Z |
| publishDate | 2013-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-0613d62ed5964bcc9852286cb0e63a1f2022-12-22T00:09:00ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2013-09-01410.3389/fmicb.2013.0028256233Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic librariesMari eNyyssönen0Huu M. Tran1Huu M. Tran2Ulas eKaraoz3Claudia eWeihe4Masood Z. Hadi5Jennifer B. H. Martiny6Adam C. Martiny7Adam C. Martiny8Eoin L. Brodie9Eoin L. Brodie10Lawrence Berkeley National LaboratoryJoint BioEnergy Institute, Lawrence Berkeley National LaboratorySandia National LaboratoriesLawrence Berkeley National LaboratoryUniversity of California IrvineJoint BioEnergy Institute, Lawrence Berkeley National LaboratoryUniversity of California IrvineUniversity of California IrvineUniversity of California IrvineLawrence Berkeley National LaboratoryUniversity of California BerkeleyRecent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26 %) cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase families were detected. Using tetranucleotide frequency binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.http://journal.frontiersin.org/Journal/10.3389/fmicb.2013.00282/fullMetagenomicscarbon cyclinggene annotationmicrobial communitiesenzyme activityfunctional metagenomics |
| spellingShingle | Mari eNyyssönen Huu M. Tran Huu M. Tran Ulas eKaraoz Claudia eWeihe Masood Z. Hadi Jennifer B. H. Martiny Adam C. Martiny Adam C. Martiny Eoin L. Brodie Eoin L. Brodie Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries Frontiers in Microbiology Metagenomics carbon cycling gene annotation microbial communities enzyme activity functional metagenomics |
| title | Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries |
| title_full | Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries |
| title_fullStr | Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries |
| title_full_unstemmed | Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries |
| title_short | Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries |
| title_sort | coupled high throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries |
| topic | Metagenomics carbon cycling gene annotation microbial communities enzyme activity functional metagenomics |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2013.00282/full |
| work_keys_str_mv | AT marienyyssonen coupledhighthroughputfunctionalscreeningandnextgenerationsequencingforidentificationofplantpolymerdecomposingenzymesinmetagenomiclibraries AT huumtran coupledhighthroughputfunctionalscreeningandnextgenerationsequencingforidentificationofplantpolymerdecomposingenzymesinmetagenomiclibraries AT huumtran coupledhighthroughputfunctionalscreeningandnextgenerationsequencingforidentificationofplantpolymerdecomposingenzymesinmetagenomiclibraries AT ulasekaraoz coupledhighthroughputfunctionalscreeningandnextgenerationsequencingforidentificationofplantpolymerdecomposingenzymesinmetagenomiclibraries AT claudiaeweihe coupledhighthroughputfunctionalscreeningandnextgenerationsequencingforidentificationofplantpolymerdecomposingenzymesinmetagenomiclibraries AT masoodzhadi coupledhighthroughputfunctionalscreeningandnextgenerationsequencingforidentificationofplantpolymerdecomposingenzymesinmetagenomiclibraries AT jenniferbhmartiny coupledhighthroughputfunctionalscreeningandnextgenerationsequencingforidentificationofplantpolymerdecomposingenzymesinmetagenomiclibraries AT adamcmartiny coupledhighthroughputfunctionalscreeningandnextgenerationsequencingforidentificationofplantpolymerdecomposingenzymesinmetagenomiclibraries AT adamcmartiny coupledhighthroughputfunctionalscreeningandnextgenerationsequencingforidentificationofplantpolymerdecomposingenzymesinmetagenomiclibraries AT eoinlbrodie coupledhighthroughputfunctionalscreeningandnextgenerationsequencingforidentificationofplantpolymerdecomposingenzymesinmetagenomiclibraries AT eoinlbrodie coupledhighthroughputfunctionalscreeningandnextgenerationsequencingforidentificationofplantpolymerdecomposingenzymesinmetagenomiclibraries |