Exploring Codon Adjustment Strategies towards Escherichia coli-Based Production of Viral Proteins Encoded by HTH1, a Novel Prophage of the Marine Bacterium Hypnocyclicus thermotrophus
Open Access
- 23 June 2021
- Vol. 13 (7), 1215
- https://doi.org/10.3390/v13071215
Abstract
Marine viral sequence space is immense and presents a promising resource for the discovery of new enzymes interesting for research and biotechnology. However, bottlenecks in the functional annotation of viral genes and soluble heterologous production of proteins hinder access to downstream characterization, subsequently impeding the discovery process. While commonly utilized for the heterologous expression of prokaryotic genes, codon adjustment approaches have not been fully explored for viral genes. Herein, the sequence-based identification of a putative prophage is reported from within the genome of Hypnocyclicus thermotrophus, a Gram-negative, moderately thermophilic bacterium isolated from the Seven Sisters hydrothermal vent field. A prophage-associated gene cluster, consisting of 46 protein coding genes, was identified and given the proposed name Hypnocyclicus thermotrophus phage H1 (HTH1). HTH1 was taxonomically assigned to the viral family Siphoviridae, by lowest common ancestor analysis of its genome and phylogeny analyses based on proteins predicted as holin and DNA polymerase. The gene neighbourhood around the HTH1 lytic cassette was found most similar to viruses infecting Gram-positive bacteria. In the HTH1 lytic cassette, an N-acetylmuramoyl-L-alanine amidase (Amidase_2) with a peptidoglycan binding motif (LysM) was identified. A total of nine genes coding for enzymes putatively related to lysis, nucleic acid modification and of unknown function were subjected to heterologous expression in Escherichia coli. Codon optimization and codon harmonization approaches were applied in parallel to compare their effects on produced proteins. Comparison of protein yields and thermostability demonstrated that codon optimization yielded higher levels of soluble protein, but codon harmonization led to proteins with higher thermostability, implying a higher folding quality. Altogether, our study suggests that both codon optimization and codon harmonization are valuable approaches for successful heterologous expression of viral genes in E. coli, but codon harmonization may be preferable in obtaining recombinant viral proteins of higher folding quality.Keywords
Funding Information
- H2020 European Research Council (685778)
- Norges Forskningsråd (294363)
This publication has 122 references indexed in Scilit:
- Ultrafast Approximation for Phylogenetic BootstrapMolecular Biology and Evolution, 2013
- MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and UsabilityMolecular Biology and Evolution, 2013
- PHAST: A Fast Phage Search ToolNucleic Acids Research, 2011
- HMMER web server: interactive sequence similarity searchingNucleic Acids Research, 2011
- Codon usage: Nature's roadmap to expression and folding of proteinsBiotechnology Journal, 2011
- Bacteriophage endolysins: A novel anti-infective to control Gram-positive pathogensInternational Journal of Medical Microbiology, 2010
- trimAl: a tool for automated alignment trimming in large-scale phylogenetic analysesBioinformatics, 2009
- An Improved General Amino Acid Replacement MatrixMolecular Biology and Evolution, 2008
- MEGAN analysis of metagenomic dataGenome Research, 2007
- Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fusedProtein Science, 1999