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Cite: NAR/gkaa742 2020



PUL General Information

Literature Information

PUL ID

PUL0123

PubMed

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

Characterization method

fosmid library screen

Genomic accession number

MH106010.1

Nucelotide position range

59-36574

Substrate

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

Loci

FOS62_41_C11_0

Species

uncultured bacterium/77133

Degradation or Biosynthesis

degradation

Gene Name

Locus Tag

Protein ID

Gene Position

GenBank Contig Range

EC Number

- - MH106010_1 59 - 520 (-) MH106010.1:118-579 -
- - MH106010_2 842 - 1501 (+) MH106010.1:901-1560 -
- - MH106010_3 1575 - 1910 (+) MH106010.1:1634-1969 -
- - MH106010_4 1990 - 3147 (-) MH106010.1:2049-3206 -
- - MH106010_5 3226 - 5289 (-) MH106010.1:3285-5348 -
- - MH106010_6 5371 - 6891 (-) MH106010.1:5430-6950 -
- - MH106010_7 6983 - 8866 (-) MH106010.1:7042-8925 -
- - MH106010_8 8900 - 10744 (-) MH106010.1:8959-10803 -
- - MH106010_9 10744 - 13458 (-) MH106010.1:10803-13517 -
- - MH106010_10 13708 - 14148 (-) MH106010.1:13767-14207 -
- - MH106010_11 14258 - 15238 (+) MH106010.1:14317-15297 -
- - MH106010_12 15225 - 15470 (+) MH106010.1:15284-15529 -
- - MH106010_13 15416 - 16024 (+) MH106010.1:15475-16083 -
- - MH106010_14 16026 - 16814 (+) MH106010.1:16085-16873 -
- - MH106010_15 16848 - 18542 (-) MH106010.1:16907-18601 -
- - MH106010_16 18602 - 19435 (-) MH106010.1:18661-19494 -
- - MH106010_17 19448 - 20335 (-) MH106010.1:19507-20394 -
- - MH106010_18 20401 - 21732 (-) MH106010.1:20460-21791 -
- - MH106010_19 21809 - 22831 (-) MH106010.1:21868-22890 -
- - MH106010_20 23605 - 24504 (+) MH106010.1:23664-24563 -
- - MH106010_21 24467 - 24688 (+) MH106010.1:24526-24747 -
- - MH106010_22 24685 - 24924 (+) MH106010.1:24744-24983 -
- - MH106010_23 24921 - 25838 (-) MH106010.1:24980-25897 -
- - MH106010_24 25869 - 26195 (+) MH106010.1:25928-26254 -
- - MH106010_25 26597 - 26689 (+) MH106010.1:26656-26748 -
- - MH106010_26 26739 - 27170 (+) MH106010.1:26798-27229 -
- - MH106010_27 27261 - 29006 (+) MH106010.1:27320-29065 -
- - MH106010_28 29195 - 29587 (-) MH106010.1:29254-29646 -
- - MH106010_29 29624 - 29872 (-) MH106010.1:29683-29931 -
- - MH106010_30 30240 - 32741 (-) MH106010.1:30299-32800 -
- - MH106010_31 32750 - 32992 (-) MH106010.1:32809-33051 -
- - MH106010_32 33008 - 34336 (-) MH106010.1:33067-34395 -
- - MH106010_33 34426 - 34614 (-) MH106010.1:34485-34673 -
- - MH106010_34 34634 - 35020 (+) MH106010.1:34693-35079 -
- - MH106010_35 35013 - 35186 (+) MH106010.1:35072-35245 -
- - MH106010_36 35398 - 35685 (+) MH106010.1:35457-35744 -
- - MH106010_37 35663 - 36574 (+) MH106010.1:35722-36633 -

Cluster number

1

Gene name

Gene position

Gene type

Found by CGCFinder?

- 59 - 520 (-) CDS No
- 842 - 1501 (+) TC: gnl|TC-DB|P54176|1.C.113.1.1 Yes
- 1575 - 1910 (+) other Yes
- 1990 - 3147 (-) other Yes
- 3226 - 5289 (-) other Yes
- 5371 - 6891 (-) other Yes
- 6983 - 8866 (-) other Yes
- 8900 - 10744 (-) other Yes
- 10744 - 13458 (-) other Yes
- 13708 - 14148 (-) other Yes
- 14258 - 15238 (+) TC: gnl|TC-DB|P73329|3.A.1.141.1 Yes
- 15225 - 15470 (+) other Yes
- 15416 - 16024 (+) other Yes
- 16026 - 16814 (+) other Yes
- 16848 - 18542 (-) CAZyme: GH63 Yes
- 18602 - 19435 (-) TC: gnl|TC-DB|A5LBQ4|3.A.1.1.47 Yes
- 19448 - 20335 (-) other Yes
- 20401 - 21732 (-) other Yes
- 21809 - 22831 (-) TF: DBD-Pfam|LacI,DBD-SUPERFAMILY|0036955 Yes
- 23605 - 24504 (+) other Yes
- 24467 - 24688 (+) other Yes
- 24685 - 24924 (+) other Yes
- 24921 - 25838 (-) other Yes
- 25869 - 26195 (+) other Yes
- 26597 - 26689 (+) other Yes
- 26739 - 27170 (+) other Yes
- 27261 - 29006 (+) TC: gnl|TC-DB|D4ZZR3|9.B.34.1.3 Yes
- 29195 - 29587 (-) other Yes
- 29624 - 29872 (-) other Yes
- 30240 - 32741 (-) CAZyme: CBM67|GH78 Yes
- 32750 - 32992 (-) other Yes
- 33008 - 34336 (-) TC: gnl|TC-DB|Q9UEF7|8.A.49.1.1 Yes
- 34426 - 34614 (-) CDS No
- 34634 - 35020 (+) CDS No
- 35013 - 35186 (+) CDS No
- 35398 - 35685 (+) TF: DBD-Pfam|HTH_3,DBD-SUPERFAMILY|0043707 No
- 35663 - 36574 (+) CDS No

PUL ID

PUL0123

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.