Enhancing Transcription in Escherichia coli BW25113 and Pseudomonas Putida KT2440 Using Bacteriophage Lambda Anti-terminator Protein Q

Abstract

Functional characterization of metagenomic DNA often involves expressing heterologous DNA into genetically tractable microorganisms such as Escherichia coli. Expression of heterologous genes can suffer from limitations due to lack of recognition of foreign promoters or presence of intrinsic terminators on foreign DNA downstream of both the vector-based promoter and the transcription start site. Anti-terminator proteins are a possible solution to overcome this limitation. When bacteriophage lambda infects E. coli, it relies on the transcription machinery of the host to transcribe and express the phage DNA. Lambda genome encodes two anti-terminator proteins, namely N and Q (λQ), which regulate the expression of late early and late genes of the phage lambda, respectively. E. coli’s RNA polymerase recognizes the PR' promoter on the lambda genome and forms a complex with bacteriophage lambda anti-terminator Q, to overcome the terminator tR'. Here we show the use of protein Q to efficiently transcribe a gene cluster containing intrinsic terminators from Lactobacillus plantarum in Escherichia coli. In addition, we expand the use of anti-terminator protein Q by showing it can function in Pseudomonas putida KT2440. The results show higher expression of a fluorescent reporter located ~12.5 kbp downstream from the promoter, when the transcription is driven by PR' promoter in presence of protein Q.