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Applied and Environmental Microbiology, November 2008, p. 6538-6546, Vol. 74, No. 21
0099-2240/08/$08.00+0 doi:10.1128/AEM.01354-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Codon Optimization Increases Steady-State mRNA Levels in Aspergillus oryzae Heterologous Gene Expression
,
Masafumi Tokuoka,1,
Mizuki Tanaka,1
Kazuhisa Ono,2
Shinobu Takagi,3
Takahiro Shintani,1 and
Katsuya Gomi1*
Laboratory of Bioindustrial Genomics, Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan,1 Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan,2 Novozymes Japan Ltd., CB-6, Makuhari Techno Garden, 1-3 Nakase, Mihama-ku, Chiba 261-8501, Japan3
Received 17 June 2008/ Accepted 3 September 2008
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.
* Corresponding author. Mailing address: Laboratory of Bioindustrial Genomics, Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. Phone: 81-22-717-8901. Fax: 81-22-717-8902. E-mail: gomi{at}biochem.tohoku.ac.jp
Published ahead of print on 12 September 2008.
Supplemental material for this article may be found at http://aem.asm.org/.
Present address: Noda Institute of Industrial Sciences, 399 Noda, Noda 278-0037, Japan.
Applied and Environmental Microbiology, November 2008, p. 6538-6546, Vol. 74, No. 21
0099-2240/08/$08.00+0 doi:10.1128/AEM.01354-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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