Codon Optimization Increases Steady-State mRNA Levels in Aspergillus oryzae Heterologous Gene Expression

We investigated the effect of codon optimization on the expression levels of heterologous proteins inAspergillus oryzae , using the mite allergen Der f 7 as a model protein. A codon-optimized Der f 7 gene was synthesized according to the frequency of codon usage inA. oryzae by recursive PCR. Both native and optimized Der f 7 genes were expressed under the control of a high-level-expression promoter with their own signal peptides or in a fusion construct withA. oryzae glucoamylase (GlaA). Codon optimization markedly increased protein and mRNA production levels in both nonfused and GlaA-fused Der f 7 constructs. For constructs with native codons, analysis by 3′ rapid amplification of cDNA ends revealed that poly(A) tracts tended to be added within the coding region, producing aberrant mRNAs that lack a termination codon. Insertion of a termination codon between the carrier GlaA and native Der f 7 proteins in the GlaA fusion construct resulted in increases in mRNA and secreted-carrier-GlaA levels. These results suggested that mRNAs without a termination codon as a result of premature polyadenylation are degraded, possibly through the nonstop mRNA decay pathway. We suggest that codon optimization inA. oryzae results in elimination of cryptic polyadenylation signals in native Der f 7, thereby circumventing the production of truncated transcripts and resulting in an increase in steady-state mRNA levels.