title: Improved metagenome screening efficiency by random insertion of T7 promoters.
abstract: 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.
authors.affiliation: Department of BiologicalSciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea; Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, South Korea.
authors.forename: Yu Jung
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, South Korea.
authors.forename: Haseong
authors.initials: H
authors.lastname: Kim
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, South Korea.
authors.forename: Seo Hyeon
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, South Korea.
authors.forename: Eugene
authors.initials: E
authors.lastname: Rha
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, South Korea.
authors.forename: Su-Lim
authors.initials: SL
authors.lastname: Choi
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, South Korea.
authors.forename: Soo-Jin
authors.initials: SJ
authors.lastname: Yeom
authors.affiliation: Department of BiologicalSciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea.
authors.forename: Hak-Sung
authors.initials: HS
authors.lastname: Kim
authors.affiliation: Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, South Korea; Biosystems & Bioengineering, University of Science & Technology (UST), Daejeon 34113, South Korea. Electronic address: sglee@kribb.re.kr.
authors.forename: Seung-Goo
authors.initials: SG
authors.lastname: Lee
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004950:Esterases / Q000235:genetics / Q000378:metabolism; D008049:Lipase / Q000378:metabolism; D054892:Metagenome / Q000235:genetics*; D056186:Metagenomics / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*
chemical_list: D004950:Esterases; D008049:Lipase
keywords: Enforced-transcription; Esterase; Genetic diversity; High-efficiency screening; Lipase; Metagenomic screening