Exploring codon context bias for synthetic gene design of a thermostable invertase in Escherichia coli

Pek, Han Bin; Klement, Maximilian; Ang, Kok Siong; Chung, Bevan Kai-Sheng; Ow, Dave Siak-Wei; Lee, Dong-Yup

doi:10.1016/j.enzmictec.2015.04.008

Cited by 13 publications

(4 citation statements)

References 38 publications

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“…Synonymous mutations may affect the regulation of protein expression at the stage of translation whereby certain codons are much preferred to code for a particular amino acid. This form of codon usage bias has been extensively described in Escherichia coli ( Sharp et al, 1988 ; Pek et al, 2015 ). Studying codon bias in P. aeruginosa , Grocock and Sharp (2002) concluded that a weak codon bias is present in P. aeruginosa and calculated relative synonymous codon usage (RSCU) values for each codon.…”

Section: Resultsmentioning

confidence: 99%

Small Colony Variants and Single Nucleotide Variations in Pf1 Region of PB1 Phage-Resistant Pseudomonas aeruginosa

et al. 2016

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Phage therapy involves the application of lytic bacteriophages for treatment of clinical infections but bacterial resistance may develop over time. Isolated from nosocomial infections, small colony variants (SCVs) are morphologically distinct, highly virulent bacterial strains that are resistant to conventional antibiotics. In this study, SCVs was derived from Pseudomonas aeruginosa exposed to the lytic bacteriophage PB1 and these cells were resistant to subsequent phage infection by PB1. To elucidate the mechanism of the SCV phage resistance, we performed phenotypic assays, DNA microarrays and whole-genome sequencing. Compared with wild-type P. aeruginosa, the SCV isolate showed impaired biofilm formation, decreased twitching motility, reduced elastase and pyocyanin production. The SCV is also more susceptible to the antibiotic ciprofloxacin and exhibited higher syrface hydrophobicity than the wild-type, indicative of changes to cell surface lipopolysaccharide (LPS) composition. Consistent with these results, transcriptomic studies of SCV revealed up-regulation of genes involved in O-specific antigen (OSA) biosynthesis, suggesting the regulation of surface moieties may account for phage resistance. Western blot analysis showed a difference in OSA distribution between the two strains. Simultaneously, genes involved in aromatic and branched chain amino acid catabolism were down-regulated. Whole genome sequencing of the SCV revealed multiple single nucleotide variations within the Pf1 prophage region, a genetic locus known to play a crucial role in biofilm formation and to provide survival advantage via gene transfer to a subpopulation of cells. Insights into phenotypic and genetic changes in SCV gained here should help direct future studies to elucidate mechanisms underpinning phage resistance, leading to novel counter resistance measures.

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Section: Resultsmentioning

confidence: 99%

Small Colony Variants and Single Nucleotide Variations in Pf1 Region of PB1 Phage-Resistant Pseudomonas aeruginosa

et al. 2016

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“…We provide evidence that bacterial promoters readily arise within open reading frames from synonymous mutations, and this lowers the previously held threshold for transcription initiation (and translation initiation), in agreement with a growing body of research showing that transcription initiation is a relatively promiscuous process in mycobacteria. In addition, codon context optimization has been reported to impact protein production to a greater extent than codon usage optimization alone in both prokaryotes and eukaryotes (47)(48)(49). Understanding the relationship between codon pairs and codon context more generally in thus necessary to improve recombinant protein research.…”

Section: Discussionmentioning

confidence: 99%

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria

Hegelmeyer

Previti

Andrade

et al. 2023

Preprint

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Each genome encodes some codons more frequently than their synonyms (codon usage bias), but codons are also arranged more frequently into specific pairs (codon pair bias). Recoding viral genomes and yeast or bacterial genes with non-optimal codon pairs has been shown to decrease gene expression. Gene expression is thus importantly regulated not only by the use of particular codons but by their proper juxtaposition. We therefore hypothesized that non-optimal codon pairing could likewise attenuate Mtb genes. We explored the role of codon pair bias by recoding Mtb genes (rpoB, mmpL3, ndh) and assessing their expression in the closely related and tractable model organism M. smegmatis. To our surprise, recoding caused the expression of multiple smaller protein isoforms from all three genes. We confirmed that these smaller proteins were not due to protein degradation, but instead issued from new transcription initiation sites positioned within the open reading frame. New transcripts gave rise to intragenic translation initiation sites, which in turn led to the expression of smaller proteins. We next identified the nucleotide changes associated with these new sites of transcription and translation. Our results demonstrated that apparently benign, synonymous changes can drastically alter gene expression in mycobacteria. More generally, our work expands our understanding of the codon-level parameters that control translation and transcription initiation.

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“…It is generally believed that codon context influences translational elongation rates and accuracy, by the compatibilities of adjacent tRNA molecules in the P- and A-sites on the surface of the large subunits of translating ribosomes [ 8 – 11 ]. Studies showed that codon context is more strongly related to translational efficiency than single codon usage [ 8 , 12 , 13 ]. However, CPB has been relatively ignored compared to CUB when considering for gene synthesis.…”

Section: Introductionmentioning

confidence: 99%

Codon pair optimization (CPO): a software tool for synthetic gene design based on codon pair bias to improve the expression of recombinant proteins in Pichia pastoris

Huang

Lin

et al. 2021

Microb Cell Fact

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Background Codon optimization is a common method to improve protein expression levels in Pichia pastoris and the current strategy is to replace rare codons with preferred codons to match the codon usage bias. However, codon-pair contexts have a profound effect on translation efficiency by influencing both translational elongation rates and accuracy. Until now, it remains untested whether optimized genes based on codon pair bias results in higher protein expression levels compared to codon usage bias. Results In this study, an algorithm based on dynamic programming was introduced to develop codon pair optimization (CPO) which is a software tool to provide simple and efficient codon pair optimization for synthetic gene design in Pichia pastoris. Two reporters (MT1-MMP E2C6 and ADAM17 A9B8 scFvs) were employed to test the effects of codon pair bias and CPO optimization on their protein expression levels. Four variants of MT1-MMP E2C6 and ADAM17 A9B8 for each were generated, one variant with the best codon-pair context, one with the worst codon-pair context, one with unbiased codon-pair context, and another optimized based on codon usage. The expression levels of variants with the worst codon-pair context were almost undetectable by Western blot and the variants with the best codon-pair context were expressed well. The expression levels on MT1-MMP E2C6 and ADAM17 A9B8 were more than five times and seven times higher in the optimized sequences based on codon-pair context compared to that based on codon usage, respectively. The results indicated that the codon-pair context-based codon optimization is more effective in enhancing expression of protein in Pichia pastoris. Conclusions Codon-pair context plays an important role on the protein expression in Pichia pastoris. The codon pair optimization (CPO) software developed in this study efficiently improved the protein expression levels of exogenous genes in Pichia pastoris, suggesting gene design based on codon pair bias is an alternative strategy for high expression of recombinant proteins in Pichia pastoris.

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Exploring codon context bias for synthetic gene design of a thermostable invertase in Escherichia coli

Cited by 13 publications

References 38 publications

Small Colony Variants and Single Nucleotide Variations in Pf1 Region of PB1 Phage-Resistant Pseudomonas aeruginosa

Small Colony Variants and Single Nucleotide Variations in Pf1 Region of PB1 Phage-Resistant Pseudomonas aeruginosa

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria

Codon pair optimization (CPO): a software tool for synthetic gene design based on codon pair bias to improve the expression of recombinant proteins in Pichia pastoris

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