title: Codon optimization increases steady-state mRNA levels in Aspergillus oryzae heterologous gene expression.
abstract: 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.
authors.affiliation: Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Tokuoka
authors.forename: Mizuki
authors.initials: M
authors.lastname: Tanaka
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Ono
authors.forename: Shinobu
authors.initials: S
authors.lastname: Takagi
authors.forename: Takahiro
authors.initials: T
authors.lastname: Shintani
authors.forename: Katsuya
authors.initials: K
authors.lastname: Gomi
mesh_terms: D001236:Aspergillus oryzae / Q000235:genetics* / Q000378:metabolism*; D003062:Codon; D015870:Gene Expression; D008969:Molecular Sequence Data; D026723:Polyadenylation; D020871:RNA Stability; D012331:RNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D003062:Codon; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins
references.citation: Biotechnology (N Y). 1995 May;13(5):498-503
references.pmid: 9634791
references.citation: Genes Dev. 1994 Nov 1;8(21):2641-52
references.pmid: 7958922
references.citation: Plant Physiol. 1998 Aug;117(4):1445-61
references.pmid: 9701600
references.citation: J Biosci Bioeng. 2002;94(4):285-98
references.pmid: 16233305
references.citation: Int Arch Allergy Immunol. 1997 May-Jul;113(1-3):102-4
references.pmid: 9130494
references.citation: Appl Microbiol Biotechnol. 2007 May;75(2):387-95
references.pmid: 17225093
references.citation: Science. 2002 Mar 22;295(5563):2258-61
references.pmid: 11910109
references.citation: Protein Expr Purif. 2002 Feb;24(1):18-24
references.pmid: 11812218
references.citation: Mol Microbiol. 2004 Jun;52(5):1437-50
references.pmid: 15165245
references.citation: Protein Expr Purif. 2006 May;47(1):249-57
references.pmid: 16403645
references.citation: Eukaryot Cell. 2004 Aug;3(4):1036-48
references.pmid: 15302836
references.citation: Curr Genet. 1998 Feb;33(2):151-6
references.pmid: 9506904
references.citation: Appl Microbiol Biotechnol. 2004 Jul;65(1):74-83
references.pmid: 15221230
references.citation: J Biosci Bioeng. 2005 Nov;100(5):531-7
references.pmid: 16384792
references.citation: Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):14055-60
references.pmid: 10570197
references.citation: Appl Environ Microbiol. 2001 Jan;67(1):481-3
references.pmid: 11133486
references.citation: Biotechnology (N Y). 1991 Oct;9(10):976-81
references.pmid: 1368725
references.citation: Appl Environ Microbiol. 1997 Feb;63(2):488-97
references.pmid: 9023927
references.citation: Nature. 2005 Dec 22;438(7071):1105-15
references.pmid: 16372000
references.citation: Nature. 2005 Dec 22;438(7071):1157-61
references.pmid: 16372010
references.citation: Tsitologiia. 2003;45(1):94-100
references.pmid: 12683242
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Genetics. 1998 May;149(1):37-44
references.pmid: 9584084
references.citation: Science. 2002 Mar 22;295(5563):2262-4
references.pmid: 11910110
references.citation: Clin Exp Allergy. 2002 Jul;32(7):1048-53
references.pmid: 12100052
references.citation: Gene. 2005 Nov 21;361:13-37
references.pmid: 16213112
references.citation: Clin Exp Allergy. 1995 Oct;25(10):1000-6
references.pmid: 8556554
references.citation: RNA. 2005 Oct;11(10):1485-93
references.pmid: 16131587
references.citation: Clin Exp Allergy. 2001 Jan;31(1):116-24
references.pmid: 11167959
references.citation: Plant Physiol. 2005 Jul;138(3):1457-68
references.pmid: 15965016
references.citation: Biosci Biotechnol Biochem. 1999 Apr;63(4):703-9
references.pmid: 10361683
references.citation: Nucleic Acids Res. 1992 Dec 25;20(24):6707-12
references.pmid: 1480491
references.citation: Agric Biol Chem. 1991 May;55(5):1233-8
references.pmid: 1368682
references.citation: Nucleic Acids Res. 2002 Apr 15;30(8):1851-8
references.pmid: 11937640
references.citation: Nature. 2005 Dec 22;438(7071):1151-6
references.pmid: 16372009
references.citation: Trends Biotechnol. 2002 May;20(5):200-6
references.pmid: 11943375
references.citation: Trends Biotechnol. 2004 Jul;22(7):346-53
references.pmid: 15245907
references.citation: Plant Physiol. 1998 Aug;117(4):1433-43
references.pmid: 9701599
references.citation: Appl Environ Microbiol. 1996 Jun;62(6):1951-7
references.pmid: 8787393
references.citation: J Mol Biol. 1981 Sep 25;151(3):389-409
references.pmid: 6175758
references.citation: Biochem Biophys Res Commun. 2004 Jan 2;313(1):89-96
references.pmid: 14672702
references.citation: Eukaryot Cell. 2008 Jan;7(1):28-37
references.pmid: 17766461
references.citation: Protein Eng. 1992 Dec;5(8):827-9
references.pmid: 1287665
references.citation: Protein Expr Purif. 2002 Oct;26(1):96-105
references.pmid: 12356476
references.citation: Appl Microbiol Biotechnol. 1997 Jan;47(1):1-11
references.pmid: 9035405
references.citation: J Allergy Clin Immunol. 1992 Jan;89(1 Pt 1):95-102
references.pmid: 1730842
references.citation: Protein Sci. 1998 Jun;7(6):1415-22
references.pmid: 9655346
references.citation: FEMS Microbiol Lett. 2000 Sep 1;190(1):13-9
references.pmid: 10981683
references.citation: Biotechnol Bioeng. 2003 Aug 5;83(3):249-59
references.pmid: 12783481
references.citation: Curr Opin Biotechnol. 1995 Oct;6(5):494-500
references.pmid: 7579660