Reduction of wobble-position GC bases in Corynebacteria genes and enhancement of PCR and heterologous expression.
Corynebacteria codon usage exhibits an overall GC content of 67%, and a wobble-position GC content of 88%. Escherichia coli, on the other hand has an overall GC content of 51%, and a wobble-position GC content of 55%. The high GC content of Corynebacteria genes results in an unfavorable codon preference for heterologous expression, and can present difficulties for polymerase-based manipulations due to secondary-structure effects. Since these characteristics are due primarily to base composition at the wobble-position, synthetic genes can, in principle, be designed to eliminate these problems and retain the wild-type amino acid sequence. Such genes would obviate the need for special additives or bases during in vitro polymerase-based manipulation and mutant host strains containing uncommon tRNA's for heterologous expression. We have evaluated synthetic genes with reduced wobble-position G/C content using two variants of the enzyme 2,5-diketo-D-gluconic acid reductase (2,5-DKGR A and B) from Corynebacterium. The wild-type genes are refractory to polymerase-based manipulations and exhibit poor heterologous expression in enteric bacteria. The results indicate that a subset of codons for five amino acids (alanine, arginine, glutamate, glycine and valine) contribute the greatest contribution to reduction in G/C content at the wobble-position. Furthermore, changes in codons for two amino acids (leucine and proline) enhance bias for expression in enteric bacteria without affecting the overall G/C content. The synthetic genes are readily amplified using polymerase-based methodologies, and exhibit high levels of heterologous expression in E. coli.
Sanli
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G
Institute of Molecular Biophysics and Department of Chemistry, Florida State University, Tallahassee, 32306-3015, USA.
Blaber
S I
SI
Blaber
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M
D001482:Base Composition; D020029:Base Pairing; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D003596:Cytosine; D015870:Gene Expression / Q000187:drug effects; D005798:Genes, Bacterial; D006147:Guanine; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics*
D007544:Isopropyl Thiogalactoside; D006147:Guanine; D003596:Cytosine; D013401:Sugar Alcohol Dehydrogenases; C053163:2,5-diketo-D-gluconate 5-reductase