We investigated the effect of codon optimization on the expression levels of heterologous proteins in Aspergillus 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 in A. 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 with A. 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 in A. 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.The filamentous fungus Aspergillus oryzae has been used in the production of fermented foods, such as sake, soy sauce, and miso (soybean paste), in Japan for over a thousand years. In addition, A. oryzae has the ability to secrete large amounts of proteins and has recently become a favorable host for recombinant protein production (5). By use of a series of classical random mutagenesis and screening procedures, hypersecretion mutants of heterologous proteins in aspergilli have been obtained (9, 49). However, the secretion yields of heterologous proteins are low compared to those of homologous proteins or proteins from closely related fungal species and generally do not exceed tens of milligrams per liter (16). In order to improve the expression levels of heterologous proteins, several trials have been conducted, and some strategies have been reported to be effective in increasing the level of heterologous protein production (39). These results provide information on how to increase the expression levels of heterologous genes; however, there is little information on the mechanisms hampering heterologous gene expression. In this study, we investigate the effect of codon optimization on heterologous gene expression. Such optimization has been effective in improving secretion levels of heterologous proteins in several hosts (19). Studies of heterologous gene expression through codon optimization have reported improved heterologous protein production in filamentous fungal species, such as Aspergillus awamori (2,15,35), Aspergillus niger (27, 35), Neurospora crassa (13, 35), and Trichoderma reesei (47). Based on studies of ...
