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Improved metagenome screening efficiency by random insertion of T7 promoters

Paper ID Volume ID Publish Year Pages File Format Full-Text
22652 43368 2016 7 PDF Available
Title
Improved metagenome screening efficiency by random insertion of T7 promoters
Abstract

•An enforced-transcription (FT) system was constructed using the EZ-Tn5 Transposon with T7 promoters.•The FT system significantly increased screening efficiency approximately 6.7-fold and had greater genetic diversity compared to the original metagenome library.•The FT technique shortened the expression timing of metagenomic lipolytic genes.•The FT technique has the potential to broaden our knowledge of unknown microbial species by expanding the range of detectable enzyme activities.

Metagenomes constitute a major source for the identification of novel enzymes for industrial applications. However, current functional screening methods are hindered by the limited transcription efficiency of foreign metagenomic genes. To overcome this constraint, we introduced the ‘Enforced Transcription’ technique, which involves the random insertion of the bi-directional T7 promoter into a metagenomic fosmid library. Then the effect of enforced transcription was quantitatively assessed by screening for metagenomic lipolytic genes encoding enzymes whose catalytic activity forms halos on tributyrin agar plates. The metagenomic library containing the enforced transcription system yielded a significantly increased number of screening hits with lipolytic activity compared to the library without random T7 promoter insertions. Additional sequence analysis revealed that the hits from the enforced transcription library had greater genetic diversity than those from the original metagenome library. Enhancing heterologous expression using the T7 promoter should enable the identification of greater numbers of diverse novel biocatalysts from the metagenome than possible using conventional metagenome screening approaches.

Keywords
Enforced-transcription; High-efficiency screening; Metagenomic screening; Genetic diversity; Esterase; Lipase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 230, 20 July 2016, Pages 47–53
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us