Enhancing transcription in Escherichia coli and Pseudomonas putida using bacteriophage lambda anti-terminator protein Q

Publication Date

2-1-2022

Document Type

Article

Publication Title

Biotechnology Letters

Volume

44

Issue

2

DOI

10.1007/s10529-021-03206-x

First Page

253

Last Page

258

Abstract

Functional characterization of metagenomic DNA often involves expressing heterologous DNA in genetically tractable microorganisms such as Escherichia coli. Functional expression of heterologous genes can suffer from limitations due to the lack of recognition of foreign promoters or presence of intrinsic terminators on foreign DNA between a 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 host transcription machinery to transcribe and express phage DNA. Lambda anti-terminator protein Q (λQ) regulates the expression of late-genes of phage lambda. E. coli RNA polymerase recognizes the PR' promoter on the lambda genome and forms a complex with λQ, to overcome the terminator tR'. Here we show the use of λQ to efficiently transcribe a capsular polysaccharide cluster, cps3, from Lactobacillus plantarum containing intrinsic terminators in Escherichia coli. In addition, we expand the use of anti-terminator λQ in Pseudomonas putida. The results show ~ fivefold higher expression of a fluorescent reporter located ~ 12.5kbp downstream from the promoter, when the transcription is driven by PR' promoter in presence of λQ compared to a lac promoter. These results suggest that λQ could be used in metabolic engineering to enhance expression of heterologous DNA.

Funding Sponsor

U.S. Department of Energy

Keywords

Anti-terminator protein Q, Escherichia coli, Heterologous DNA, Metagenomics, Pseudomonas putida

Department

Chemical and Materials Engineering

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