Abstract
Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes’ expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified ‘codon randomization’ strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.
Highlights
Protein biosynthesis based on cloned heterologous genes is often lower than expected due to a different cytoplasmic environment in the recombinant host
We have shown previously that decreasing the overall GC content, reduction of mRNA secondary structures, avoiding repetition of the same codons and other codon contexts obstacles lead to increased thermophile-derived RM.TaqII biosynthesis by approximately 10-fold [23]
In that approach the correction of GC content was performed by using codons preferred by E. coli, which generally have a lower GC% in comparison to genes originating from thermophiles, biasing for E. coli Ser codon UCU even though it has lower occurrence than Ser UCC
Summary
Protein biosynthesis based on cloned heterologous genes is often lower than expected due to a different cytoplasmic environment in the recombinant host. This problem especially concerns genes originating from thermophilic bacteria, which thrive at temperatures of 50–121 ̊C. Temperature and DNA stability imposes evolutionary adaptation pressures on the genomes, transcriptomes and proteomes of thermophilic bacteria. One of the observed characteristics is the increased GC content of their genomes as a function of growth temperature. As the differences in codon usage between species can adversely affect recombinant gene expression levels, various gene optimization strategies are used
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