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Cite: NAR/gkaa742 2020



PUL General Information

Literature Information

PUL ID

PUL0124

PubMed

31164449, mSystems. 2019 Jun 4;4(4):e00082-19. doi: 10.1128/mSystems.00082-19.

Characterization method

fosmid library screen

Genomic accession number

MH106116.1

Nucelotide position range

536-25765

Substrate

4-methylumbelliferyl 6-azido-6-deoxy-beta-D-galactoside

Loci

TolDC_32_D22_1

Species

uncultured bacterium/77133

Degradation or Biosynthesis

degradation

Gene Name

Locus Tag

Protein ID

Gene Position

GenBank Contig Range

EC Number

- - lcl|ORF5 448 - 1564 (+) MH106116.1:984-2100 -
- - lcl|ORF1 3489 - 5619 (+) MH106116.1:4025-6155 -
- - lcl|ORF18 5633 - 7244 (-) MH106116.1:6169-7780 -
- - lcl|ORF14 7932 - 8694 (-) MH106116.1:8468-9230 -
- - lcl|ORF2 9996 - 10623 (+) MH106116.1:10532-11159 -
- - lcl|ORF13 9018 - 10920 (-) MH106116.1:9554-11456 -
- - lcl|ORF11 11368 - 12697 (-) MH106116.1:11904-13233 -
- - lcl|ORF3 13317 - 14619 (+) MH106116.1:13853-15155 -
- - lcl|ORF17 12875 - 13526 (-) MH106116.1:13411-14062 -
- - lcl|ORF7 14627 - 15380 (+) MH106116.1:15163-15916 -
- - lcl|ORF6 16711 - 17359 (+) MH106116.1:17247-17895 -
- - lcl|ORF10 15424 - 17266 (-) MH106116.1:15960-17802 -
- - lcl|ORF9 17284 - 18583 (-) MH106116.1:17820-19119 -
- - lcl|ORF16 18563 - 19484 (-) MH106116.1:19099-20020 -
- - lcl|ORF4 19893 - 21297 (+) MH106116.1:20429-21833 -
- - lcl|ORF12 19776 - 21276 (-) MH106116.1:20312-21812 -
- - lcl|ORF8 21997 - 23005 (-) MH106116.1:22533-23541 -
- - lcl|ORF15 24863 - 25589 (-) MH106116.1:25399-26125 -

Cluster number

1

Gene name

Gene position

Gene type

Found by CGCFinder?

- 449 - 1564 (+) CDS No
- 3490 - 5619 (+) CDS No
- 5634 - 7244 (-) CAZyme: GH13|GH13_23 Yes
- 7933 - 8694 (-) STP: STP|CbiA Yes
- 9019 - 10920 (-) other Yes
- 9997 - 10623 (+) other Yes
- 11369 - 12697 (-) CAZyme: GH1 Yes
- 12876 - 13526 (-) other Yes
- 13318 - 14619 (+) other Yes
- 14628 - 15380 (+) other Yes
- 15425 - 17266 (-) other Yes
- 16712 - 17359 (+) other Yes
- 17285 - 18583 (-) TC: gnl|TC-DB|F4LLD9|2.A.1.15.18 Yes
- 18564 - 19484 (-) other Yes
- 19777 - 21276 (-) other Yes
- 19894 - 21297 (+) other Yes
- 21998 - 23005 (-) TC: gnl|TC-DB|A6VIJ1|2.A.119.1.3 Yes
- 24864 - 25589 (-) CDS No

PUL ID

PUL0124

PubMed

31164449, mSystems. 2019 Jun 4;4(4):e00082-19. doi: 10.1128/mSystems.00082-19.

Title

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

Author

Armstrong Z, Liu F, Kheirandish S, Chen HM, Mewis K, Duo T, Morgan-Lang C, Hallam SJ, Withers SG

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 beta-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-beta-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.