High-Throughput Recovery and Characterization of Metagenome-Derived Glycoside Hydrolase-Containing Clones as a Resource for Biocatalyst Development

ABSTRACT Functional metagenomics is a powerful tool for both the discovery and development of biocatalysts. This study presents the high-throughput functional screening of 22 large-insert fosmid libraries containing over 300,000 clones sourced from natural and engineered ecosystems, characterization...

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Autores principales:	Zachary Armstrong, Feng Liu, Sam Kheirandish, Hong-Ming Chen, Keith Mewis, Tianmeng Duo, Connor Morgan-Lang, Steven J. Hallam, Stephen G. Withers
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	CAZymes environmental genomics glycosidases high-throughput characterization high-throughput screening metagenomics Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/18d3a1734b504475b3208bfe2e32a771
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spelling	oai:doaj.org-article:18d3a1734b504475b3208bfe2e32a7712021-12-02T19:46:18ZHigh-Throughput Recovery and Characterization of Metagenome-Derived Glycoside Hydrolase-Containing Clones as a Resource for Biocatalyst Development10.1128/mSystems.00082-192379-5077https://doaj.org/article/18d3a1734b504475b3208bfe2e32a7712019-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00082-19https://doaj.org/toc/2379-5077ABSTRACT Functional metagenomics is a powerful tool for both the discovery and development of biocatalysts. This study presents the high-throughput functional screening of 22 large-insert fosmid libraries containing over 300,000 clones sourced from natural and engineered ecosystems, characterization of active clones, and a demonstration of the utility of recovered genes or gene cassettes in the development of novel biocatalysts. Screening was performed in a 384-well-plate format with the fluorogenic substrate 4-methylumbelliferyl cellobioside, which releases a fluorescent molecule when cleaved by β-glucosidases or cellulases. The resulting set of 164 active clones was subsequently interrogated for substrate preference, reaction mechanism, thermal stability, and optimal pH. The environmental DNA harbored within each active clone was sequenced, and functional annotation revealed a cornucopia of carbohydrate-degrading enzymes. Evaluation of genomic-context information revealed both synteny and polymer-targeting loci within a number of sequenced clones. The utility of these fosmids was then demonstrated by identifying clones encoding activity on an unnatural glycoside (4-methylumbelliferyl 6-azido-6-deoxy-β-d-galactoside) and transforming one of the identified enzymes into a glycosynthase capable of forming taggable disaccharides. IMPORTANCE The generation of new biocatalysts for plant biomass degradation and glycan synthesis has typically relied on the characterization and investigation of one or a few enzymes at a time. By coupling functional metagenomic screening and high-throughput functional characterization, we can progress beyond the current scale of catalyst discovery and provide rapid annotation of catalyst function. By functionally screening environmental DNA from many diverse sources, we have generated a suite of active glycoside hydrolase-containing clones and demonstrated their reaction parameters. We then demonstrated the utility of this collection through the generation of a new catalyst for the formation of azido-modified glycans. Further interrogation of this collection of clones will expand our biocatalytic toolbox, with potential application to biomass deconstruction and synthesis of glycans.Zachary ArmstrongFeng LiuSam KheirandishHong-Ming ChenKeith MewisTianmeng DuoConnor Morgan-LangSteven J. HallamStephen G. WithersAmerican Society for MicrobiologyarticleCAZymesenvironmental genomicsglycosidaseshigh-throughput characterizationhigh-throughput screeningmetagenomicsMicrobiologyQR1-502ENmSystems, Vol 4, Iss 4 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	CAZymes environmental genomics glycosidases high-throughput characterization high-throughput screening metagenomics Microbiology QR1-502
spellingShingle	CAZymes environmental genomics glycosidases high-throughput characterization high-throughput screening metagenomics Microbiology QR1-502 Zachary Armstrong Feng Liu Sam Kheirandish Hong-Ming Chen Keith Mewis Tianmeng Duo Connor Morgan-Lang Steven J. Hallam Stephen G. Withers High-Throughput Recovery and Characterization of Metagenome-Derived Glycoside Hydrolase-Containing Clones as a Resource for Biocatalyst Development
description	ABSTRACT Functional metagenomics is a powerful tool for both the discovery and development of biocatalysts. This study presents the high-throughput functional screening of 22 large-insert fosmid libraries containing over 300,000 clones sourced from natural and engineered ecosystems, characterization of active clones, and a demonstration of the utility of recovered genes or gene cassettes in the development of novel biocatalysts. Screening was performed in a 384-well-plate format with the fluorogenic substrate 4-methylumbelliferyl cellobioside, which releases a fluorescent molecule when cleaved by β-glucosidases or cellulases. The resulting set of 164 active clones was subsequently interrogated for substrate preference, reaction mechanism, thermal stability, and optimal pH. The environmental DNA harbored within each active clone was sequenced, and functional annotation revealed a cornucopia of carbohydrate-degrading enzymes. Evaluation of genomic-context information revealed both synteny and polymer-targeting loci within a number of sequenced clones. The utility of these fosmids was then demonstrated by identifying clones encoding activity on an unnatural glycoside (4-methylumbelliferyl 6-azido-6-deoxy-β-d-galactoside) and transforming one of the identified enzymes into a glycosynthase capable of forming taggable disaccharides. IMPORTANCE The generation of new biocatalysts for plant biomass degradation and glycan synthesis has typically relied on the characterization and investigation of one or a few enzymes at a time. By coupling functional metagenomic screening and high-throughput functional characterization, we can progress beyond the current scale of catalyst discovery and provide rapid annotation of catalyst function. By functionally screening environmental DNA from many diverse sources, we have generated a suite of active glycoside hydrolase-containing clones and demonstrated their reaction parameters. We then demonstrated the utility of this collection through the generation of a new catalyst for the formation of azido-modified glycans. Further interrogation of this collection of clones will expand our biocatalytic toolbox, with potential application to biomass deconstruction and synthesis of glycans.
format	article
author	Zachary Armstrong Feng Liu Sam Kheirandish Hong-Ming Chen Keith Mewis Tianmeng Duo Connor Morgan-Lang Steven J. Hallam Stephen G. Withers
author_facet	Zachary Armstrong Feng Liu Sam Kheirandish Hong-Ming Chen Keith Mewis Tianmeng Duo Connor Morgan-Lang Steven J. Hallam Stephen G. Withers
author_sort	Zachary Armstrong
title	High-Throughput Recovery and Characterization of Metagenome-Derived Glycoside Hydrolase-Containing Clones as a Resource for Biocatalyst Development
title_short	High-Throughput Recovery and Characterization of Metagenome-Derived Glycoside Hydrolase-Containing Clones as a Resource for Biocatalyst Development
title_full	High-Throughput Recovery and Characterization of Metagenome-Derived Glycoside Hydrolase-Containing Clones as a Resource for Biocatalyst Development
title_fullStr	High-Throughput Recovery and Characterization of Metagenome-Derived Glycoside Hydrolase-Containing Clones as a Resource for Biocatalyst Development
title_full_unstemmed	High-Throughput Recovery and Characterization of Metagenome-Derived Glycoside Hydrolase-Containing Clones as a Resource for Biocatalyst Development
title_sort	high-throughput recovery and characterization of metagenome-derived glycoside hydrolase-containing clones as a resource for biocatalyst development
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/18d3a1734b504475b3208bfe2e32a771
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High-Throughput Recovery and Characterization of Metagenome-Derived Glycoside Hydrolase-Containing Clones as a Resource for Biocatalyst Development

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