Improved metagenome screening efficiency by random insertion of T7 promoters

• 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.