Effects of codon usage versus putative 5'-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm.
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.
Griswold
Karl E
KE
Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712, USA.
Mahmood
Nadir A
NA
Iverson
Brent L
BL
Georgiou
George
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003062:Codon / Q000235:genetics*; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics / Q000254:growth & development; D005453:Fluorescence; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry* / Q000235:genetics
D003062:Codon; D012333:RNA, Messenger; D002265:Carboxylic Ester Hydrolases; C019952:cutinase