Effects of codon usage versus putative 5 ′-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm

Abstract

Matching the codon usage of recombinant genes to that of the expression host is a common strategy for increasing the expression of heterologous proteins in bacteria. However, while developing a cytoplasmic expression system for Fusarium solani cutinase in Escherichia coli, we found that altering codons to those preferred by E. coli led to significantly lower expression compared to the wild-type fungal gene, despite the presence of several rare E. coli codons in the fungal sequence. On the other hand, expression in the E. coli periplasm using a bacterial PhoA leader sequence resulted in high levels of expression for both the E. coli optimized and wild-type constructs. Sequence swapping experiments as well as calculations of predicted mRNA secondary structure provided support for the hypothesis that differential cytoplasmic expression of the E. coli optimized versus wild-type cutinase genes is due to differences in 5 ′ mRNA secondary structures. In particular, our results indicate that increased stability of 5 ′ mRNA secondary structures in the E. coli optimized transcript prevents efficient translation initiation in the absence of the phoA leader sequence. These results underscore the idea that potential 5 ′ mRNA secondary structures should be considered along with codon usage when designing a synthetic gene for high level expression in E. coli.