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Cite: NAR/gkaa742 2020



PUL General Information

Literature Information

PUL ID

PUL0121

PubMed

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

Characterization method

fosmid library screen

Genomic accession number

MH105972.1

Nucelotide position range

1-30126

Substrate

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

Loci

ORF1-ORF7

Species

uncultured bacterium/77133

Degradation or Biosynthesis

degradation

Gene Name

Locus Tag

Protein ID

Gene Position

GenBank Contig Range

EC Number

- - MH105972_1 1 - 669 (+) MH105972.1:2-670 -
- - MH105972_2 772 - 894 (-) MH105972.1:773-895 -
- - MH105972_3 899 - 1651 (+) MH105972.1:900-1652 -
- - MH105972_4 1644 - 2387 (+) MH105972.1:1645-2388 -
- - MH105972_5 2507 - 3214 (+) MH105972.1:2508-3215 -
- - MH105972_6 3211 - 4317 (+) MH105972.1:3212-4318 -
- - MH105972_7 4319 - 4798 (+) MH105972.1:4320-4799 -
- - MH105972_8 4800 - 6800 (+) MH105972.1:4801-6801 -
- - MH105972_9 6797 - 9007 (+) MH105972.1:6798-9008 -
- - MH105972_10 8997 - 10010 (-) MH105972.1:8998-10011 -
- - MH105972_11 10676 - 10912 (-) MH105972.1:10677-10913 -
- - MH105972_12 10926 - 11345 (-) MH105972.1:10927-11346 -
- - MH105972_13 11782 - 12012 (+) MH105972.1:11783-12013 -
- - MH105972_14 12758 - 13369 (-) MH105972.1:12759-13370 -
- - MH105972_15 13472 - 13840 (+) MH105972.1:13473-13841 -
- - MH105972_16 14112 - 14810 (-) MH105972.1:14113-14811 -
- - MH105972_17 14999 - 15298 (-) MH105972.1:15000-15299 -
- - MH105972_18 15341 - 15649 (-) MH105972.1:15342-15650 -
- - MH105972_19 15656 - 15880 (+) MH105972.1:15657-15881 -
- - MH105972_20 15880 - 16629 (+) MH105972.1:15881-16630 -
- - MH105972_21 17484 - 17948 (+) MH105972.1:17485-17949 -
- - MH105972_22 17897 - 19765 (-) MH105972.1:17898-19766 -
- - MH105972_23 19792 - 20019 (+) MH105972.1:19793-20020 -
- - MH105972_24 20093 - 20326 (-) MH105972.1:20094-20327 -
- - MH105972_25 20602 - 20928 (-) MH105972.1:20603-20929 -
- - MH105972_26 21035 - 21307 (+) MH105972.1:21036-21308 -
- - MH105972_27 21191 - 22279 (-) MH105972.1:21192-22280 -
- - MH105972_28 22279 - 23163 (-) MH105972.1:22280-23164 -
- - MH105972_29 23369 - 23518 (+) MH105972.1:23370-23519 -
- - MH105972_30 23652 - 25790 (-) MH105972.1:23653-25791 -
- - MH105972_31 25817 - 26563 (-) MH105972.1:25818-26564 -
- - MH105972_32 26694 - 26876 (-) MH105972.1:26695-26877 -
- - MH105972_33 26878 - 27885 (+) MH105972.1:26879-27886 -
- - MH105972_34 27839 - 28405 (-) MH105972.1:27840-28406 -
- - MH105972_35 28395 - 29060 (-) MH105972.1:28396-29061 -
- - MH105972_36 29482 - 30126 (+) MH105972.1:29483-30127 -

Cluster number

1

Gene name

Gene position

Gene type

Found by CGCFinder?

- 1 - 669 (+) TC: gnl|TC-DB|P0AEM9|3.A.1.3.10 Yes
- 772 - 894 (-) other Yes
- 899 - 1651 (+) TC: gnl|TC-DB|Q8YSA2|3.A.1.3.17 Yes
- 1644 - 2387 (+) TC: gnl|TC-DB|P45769|3.A.1.3.26 Yes
- 2507 - 3214 (+) other Yes
- 3211 - 4317 (+) STP: STP|Fer4,STP|Fer4 Yes
- 4319 - 4798 (+) other Yes
- 4800 - 6800 (+) other Yes
- 6797 - 9007 (+) other Yes
- 8997 - 10010 (-) CAZyme: GH13_23 Yes
- 10676 - 10912 (-) other Yes
- 10926 - 11345 (-) other Yes
- 11782 - 12012 (+) other Yes
- 12758 - 13369 (-) CAZyme: GH1 Yes
- 13472 - 13840 (+) CDS No
- 14112 - 14810 (-) CDS No
- 14999 - 15298 (-) CDS No
- 15341 - 15649 (-) CDS No
- 15656 - 15880 (+) CDS No
- 15880 - 16629 (+) CDS No
- 17484 - 17948 (+) CDS No
- 17897 - 19765 (-) CDS No
- 19792 - 20019 (+) CDS No
- 20093 - 20326 (-) CDS No
- 20602 - 20928 (-) STP: STP|AsnC_trans_reg No
- 21035 - 21307 (+) CDS No
- 21191 - 22279 (-) CDS No
- 22279 - 23163 (-) CDS No
- 23369 - 23518 (+) CDS No
- 23652 - 25790 (-) CDS No
- 25817 - 26563 (-) CDS No
- 26694 - 26876 (-) CDS No
- 26878 - 27885 (+) CDS No
- 27839 - 28405 (-) CDS No
- 28395 - 29060 (-) CDS No
- 29482 - 30126 (+) TC: gnl|TC-DB|Q8PW04|2.A.88.8.4 No

PUL ID

PUL0121

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.