J. Mol. Micro. Biotechnol. 3: 123-126
Reduction of Wobble-position GC Bases in
Corynebacteria Genes and Enhancement of PCR and Heterologous Expression
Gulsah Sanli, Sachiko I. Blaber and Michael Blaber
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.
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