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.
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Acknowledgements
We thank Dr. Michael Benedik for helpful discussion on the use of anti-terminator λQ.
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Supplementary Figure 1: E. coli rpoB homologs in other eubacteria. a An amino acid sequence alignment of the rpoB homologs from various genera highlighting the conserved flap-tip portion (red box) of the β-subunit where λQ is predicted to bind to the RNAP. b The amino acid similarity between E.coli rpoB sequence and the homologs in other genus, including accession numbers for the protein sequences used.
Supplementary Figure 2: Illustration showing insertion of the gene encoding λQ into the P. putida genome. The genes PP_3358-PP_3357-PP_3356 (coding for ech, vdh and fcs respectively) on P. putida chromosome are replaced by the gene encoding λQ with an RBS upstream of it using suicide vector pK18mobsacB.
Supplementary Figure 3: P. putida constructs used in the study. a Genome location showing presence of BxB1-attB site (yellow). b Insertion of the mKate2 (red) - KanR (purple) - Bxb1-attP (yellow) fragment (attP fragment) using phage integrase system. mKate2 does not carry a promoter upstream. c The heterologous DNA (black) with Plac inserted along with the attP fragment on the genome. d The heterologous DNA (black) with PR' inserted along with the attP fragment on the genome in absence of λQ. e The heterologous DNA (black) with PR' inserted along with the attP fragment on the genome in presence of λQ.
Supplementary Figure 4: No significant growth difference was observed between P. putida strains under study. The strains under study were cultured in M9 minimal media + Kanamycin for ~25 hours in TECAN plate reader. Every 15 minutes, absorbance was measured at 600nm (a) and fluorescence was measured using 588nm/633nm excitation/emission filters (b). All strains show similar growth but varied fluorescence. The growth curve represents an average of triplicates of 3 biological replicates. The error bars represent standard deviation.
Supplementary Table 1: List of strains used in this study.
Supplementary Table 2: List of plasmids used in this study.
Supplementary Table 3: List of promoter and gene sequences used in this study
Supplementary Table 4: Sequences of all the primers used in this study
Funding
This research was supported by Texas A&M President’s Excellence T3 X-grant. This work was in part supported by the Center for Bioenergy Innovation, U.S. DOE Bioenergy Research Center, supported by the Office of Biological and Environmental Research in the DOE Office of Science.
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KCK proposed, funded, and contributed to the analysis of the project. The Pseudomonas putida portion of the paper was funded and supervised by AMG. JAK performed the experiments, collected the data, prepared the figures, and wrote the paper with input from KCK and AMG All authors edited and approved the final version of the paper.
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Khan, J.A., Guss, A.M. & Kao, K.C. Enhancing transcription in Escherichia coli and Pseudomonas putida using bacteriophage lambda anti-terminator protein Q. Biotechnol Lett 44, 253–258 (2022). https://doi.org/10.1007/s10529-021-03206-x
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DOI: https://doi.org/10.1007/s10529-021-03206-x