Abstract
The filamentous fungus Aspergillus oryzae, which has long been used for the production of Japanese traditional fermented foods, has been attracting attention as a promising host for recombinant protein production because of its safety and a high capacity for protein secretion. The production levels in A. oryzae of most nonfungal proteins of higher eukaryote origin are generally low compared to those of homologous or other fungal proteins. The reduced yields of heterologous proteins are also a common problem in other filamentous fungal species. Thus, there have been many attempts to improve the production yield of heterologous proteins, particularly those derived from higher eukaryotes. Consequently, several trials have successfully resulted in an increase in the production level of recombinant proteins. This review focuses on the various strategies for improving heterologous protein production in A. oryzae, mainly for improving transcription and translation efficiencies, mRNA stability, and protein secretion, as well as for preventing proteolytic degradation. Although these strategies are individually effective in improvement of protein yields, combination of these methods should be required to achieve the commercial production levels of heterologous proteins from A. oryzae. In addition to the efforts in productivity improvement from the molecular aspects, application of the solid-state fermentation process would be advantageous for heterologous protein production by A. oryzae.
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References
Bando H, Hisada H, Ishida H, Hata Y, Katakura Y, Kondo A (2011) Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture. Appl Microbiol Biotechnol 92:561–569
Berka RM, Ward M, Wilson LJ, Hayenga KJ, Kodama KH, Carlomagno LP, Thompson SA (1990) Molecular cloning and deletion of the gene encoding aspergillopepsin A from A. awamori. Gene (Amst) 86:153–162
Caddick MX, Jones MG, van Tonder JM, Le Cordier H, Narendja F, Strauss J, Morozov IY (2006) Opposing signals differentially regulate transcript stability in Aspergillus nidulans. Mol Microbiol 62:509–519
Carvalho ND, Arentshorst M, Kooistra R, Stam H, Sagt CM, van den Hondel CA, Ram AF (2010) Effects of a defective ERAD pathway on growth and heterologous protein production in Aspergillus niger. Appl Microbiol Biotechnol 89:357–373
Christensen T, Woeldike H, Boel F, Mortensen SB, Hjortshoej K, Thim L, Hansen M (1988) High level expression of recombinant genes in Aspergillus oryzae. Biotechnology 6:1419–1422
Conesa A, Jeenes D, Archer DB, van den Hondel CA, Punt PJ (2002) Calnexin overexpression increases manganese peroxidase production in Aspergillus niger. Appl Environ Microbiol 68:846–851
Davies RW (1994) Heterologous gene expression and protein secretion in Aspergillus. Prog Ind Microbiol 29:521–560 (In: Martinelli SD, Kinghorn JR (1994) Aspergillus: 50 years on. Elsevier, Amsterdam)
Fleissner A, Dersch P (2010) Expression and export: recombinant protein production systems for Aspergillus. Appl Microbiol Biotechnol 87:1255–1270
Gomi K, Akeno T, Minetoki T, Ozeki K, Kumagai C, Okazaki N, Iimura Y (2000) Molecular cloning and characterization of a transcriptional activator gene, amyR, involved in the amylolytic gene expression in Aspergillus oryzae. Biosci Biotechnol Biochem 64:816–827
Gouka RJ, Punt PJ, Hessing JG, van den Hondel CAMJJ (1996) Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains. Appl Environ Microbiol 62:1951–1957
Gouka RJ, Punt PJ, van den Hondel CAMJJ (1997a) Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post)transcriptional levels. Appl Environ Microbiol 63:488–497
Gouka RJ, Punt PJ, van den Hondel CAMJJ (1997b) Efficient production of secreted proteins by Aspergillus: progress, limitations and prospects. Appl Microbiol Biotechnol 47:1–11
Hama S, Tamalampudi S, Shindo N, Numata T, Yamaji H, Fukuda H, Kondo A (2008) Role of N-terminal 28-amino-acid region of Rhizopus oryzae lipase in directing proteins to secretory pathway of Aspergillus oryzae. Appl Microbiol Biotechnol 79:1009–1018
Hata Y, Ishida H, Ichikawa E, Kawato A, Suginami K, Imayasu S (1998) Nucleotide sequence of an alternative glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae. Gene 207:127–134
Hisada H, Tsutsumi H, Ishida H, Hata Y (2013) High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae. Appl Microbiol Biotechnol 97:761–766
Ishida H, Hata Y, Kawato A, Abe Y, Kashiwagi Y (2004) Isolation of a novel promoter for efficient protein production in Aspergillus oryzae. Biosci Biotechnol Biochem 68:1849–1857
Ishida H, Hata Y, Kawato A, Abe Y, Suginami K, Imayasu S (2000) Identification of functional elements that regulate the glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae. Curr Genet 37:373–379
Jacobs DI, Olsthoorn MM, Maillet I, Akeroyd M, Breestraat S, Donkers S, van der Hoeven RA, van den Hondel CA, Kooistra R, Lapointe T, Menke H, Meulenberg R, Misset M, Müller WH, van Peij NN, Ram A, Rodriguez S, Roelofs MS, Roubos JA, van Tilborg MW, Verkleij AJ, Pel HJ, Stam H, Sagt CM (2009) Effective lead selection for improved protein production in Aspergillus niger based on integrated genomics. Fungal Genet Biol 46:S141–S152
Jin FJ, Watanabe T, Juvvadi PR, Maruyama J, Arioka M, Kitamoto K (2007) Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae. Appl Microbiol Biotechnol 76:1059–1068
Kitano H, Kataoka K, Furukawa K, Hara S (2002) Specific expression and temperature-dependent expression of the acid protease-encoding gene (pepA) in Aspergillus oryzae in solid-state culture (Rice-Koji). J Biosci Bioeng 93:563–567
Kobayashi T, Abe K, Asai K, Gomi K, Juvvadi PR, Kato M, Kitamoto K, Takeuchi M, Machida M (2007) Genomics of Aspergillus oryzae. Biosci Biotechnol Biochem 71:646–670
Koda A, Minetoki T, Ozeki K, Hirotsune M (2004) Translation efficiency mediated by the 5′ untranslated region greatly affects protein production in Aspergillus oryzae. Appl Microbiol Biotechnol 66:291–296
Koda A, Bogaki T, Minetoki T, Hirotsune M (2005) High expression of a synthetic gene encoding potato alpha-glucan phosphorylase in Aspergillus niger. J Biosci Bioeng 100:531–537
Koda A, Bogaki T, Minetoki T, Hirotsune M (2006) 5′ Untranslated region of the Hsp12 gene contributes to efficient translation in Aspergillus oryzae. Appl Microbiol Biotechnol 70:333–336
Leroch M, Mernke D, Koppenhoefer D, Schneider P, Mosbach A, Doehlemann G, Hahn M (2011) Living colors in the gray mold pathogen Botrytis cinerea: codon-optimized genes encoding green fluorescent protein and mCherry, which exhibit bright fluorescence. Appl Environ Microbiol 77:2887–2897
Li XL, Skory CD, Ximenes EA, Jordan DB, Dien BS, Hughes SR, Cotta MA (2007) Expression of an AT-rich xylanase gene from the anaerobic fungus Orpinomyces sp. strain PC-2 in and secretion of the heterologous enzyme by Hypocrea jecorina. Appl Microbiol Biotechnol 74:1264–1275
Lugones LG, Scholtmeijer K, Klootwijk R, Wessels JG (1999) Introns are necessary for mRNA accumulation in Schizophyllum commune. Mol Microbiol 32:681–689
Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, Kusumoto K, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K, Horiuchi H, Kitamoto K, Kobayashi T, Takeuchi M, Denning DW, Galagan JE, Nierman WC, Yu J, Archer DB, Bennett JW, Bhatnagar D, Cleveland TE, Fedorova ND, Gotoh O, Horikawa H, Hosoyama A, Ichinomiya M, Igarashi R, Iwashita K, Juvvadi PR, Kato M, Kato Y, Kin T, Kokubun A, Maeda H, Maeyama N, Maruyama J, Nagasaki H, Nakajima T, Oda K, Okada K, Paulsen I, Sakamoto K, Sawano T, Takahashi M, Takase K, Terabayashi Y, Wortman JR, Yamada O, Yamagata Y, Anazawa H, Hata Y, Koide Y, Komori T, Koyama Y, Minetoki T, Suharnan S, Tanaka A, Isono K, Kuhara S, Ogasawara N, Kikuchi H (2005) Genome sequencing and analysis of Aspergillus oryzae. Nature (Lond) 438:1157–1161
Machida M, Yamada O, Gomi K (2008) Genomics of Aspergillus oryzae: learning from the history of koji mold and exploration of its future. DNA Res 15:173–183
Maruyama J, Kitamoto K (2008) Multiple gene disruptions by marker recycling with highly efficient gene-targeting background (DeltaligD) in Aspergillus oryzae. Biotechnol Lett 30:1811–1817
Minetoki T (2000) The expression system of heterologous genes in Aspergilli (in Japanese). Kagaku to Seibutsu 38:831–838
Minetoki T, Kumagai C, Gomi K, Kitamoto K, Takahashi K (1998) Improvement of promoter activity by the introduction of multiple copies of the conserved region III sequence, involved in the efficient expression of Aspergillus oryzae amylase-encoding genes. Appl Microbiol Biotechnol 50:459–467
Mizutani O, Kudo Y, Saito A, Matsuura T, Inoue H, Abe K, Gomi K (2008) A defect of LigD (human Lig4 homolog) for nonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae. Fungal Genet Biol 45:878–889
Mizutani O, Masaki K, Gomi K, Iefuji H (2012) Modified Cre-loxP recombination in Aspergillus oryzae by direct introduction of Cre recombinase for marker gene rescue. Appl Environ Microbiol 78:4126–4133
Morozov IY, Martinez MG, Jones MG, Caddick MX (2000) A defined sequence within the 3′ UTR of the areA transcript is sufficient to mediate nitrogen metabolite signalling via accelerated deadenylation. Mol Microbiol 37:1248–1257
Ohno A, Maruyama J, Nemoto T, Arioka M, Kitamoto K (2011) A carrier fusion significantly induces unfolded protein response in heterologous protein production by Aspergillus oryzae. Appl Microbiol Biotechnol 92:1197–1206
Okazaki F, Aoki JI, Tabuchi S, Tanaka T, Ogino C, Kondo A (2012) Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment VHH against EGFR. Appl Microbiol Biotechnol 96:81–88
Ozeki K, Kanda A, Hamachi M, Nunokawa Y (1996) Construction of a promoter probe vector autonomously maintained in Aspergillus and characterization of promoter regions derived from A. niger and A. oryzae genomes. Biosci Biotechnol Biochem 60:383–389
Punt PJ, Schuren FH, Lehmbeck J, Christensen T, Hjort C, van den Hondel CA (2008) Characterization of the Aspergillus niger prtT, a unique regulator of extracellular protease encoding genes. Fungal Genet Biol 45:1591–1599
Sato H, Toyoshima Y, Shintani T, Gomi K (2011) Identification of potential cell wall component that allows Taka-amylase A adsorption in submerged cultures of Aspergillus oryzae. Appl Microbiol Biotechnol 92:961–969
Scholtmeijer J, Wösten HAB, Springer J, Wessels JGT (2001) Effect of introns and AT-rich sequences on expression of the bacterial hygromycin B resistance gene in the basidiomycete Schizophyllum commune. Appl Environ Microbiol 67:481–483
Tamalanpudi S, Talukder MM, Hama S, Tanino T, Suzuki Y, Kondo A, Fukuda H (2007) Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica. Appl Microbiol Biotechnol 75:387–395
Tanaka M, Sakai Y, Yamada O, Shintani T, Gomi K (2011) In silico analysis of 3′-end-processing signals in Aspergillus oryzae using expressed sequence tags and genomic sequencing data. DNA Res 18:189–200
Tanaka M, Tokuoka M, Shintani T, Gomi K (2012) Transcripts of a heterologous gene encoding mite allergen Der f 7 are stabilized by codon optimization in Aspergillus oryzae. Appl Microbiol Biotechnol 96:1275–1282
Te'o VS, Cziferszky AE, Bergquist PL, Nevalainen KM (2000) Codon optimization of xylanase gene xynB from the thermophilic bacterium Dictyoglomus thermophilum for expression in the filamentous fungus Trichoderma reesei. FEMS Microbiol Lett 190:13–19
Tokuoka M, Tanaka M, Ono K, Takagi S, Shintani T, Gomi K (2008) Codon optimization increases steady-state mRNA levels in Aspergillus oryzae heterologous gene expression. Appl Environ Microbiol 74:6538–6546
Tsuboi H, Koda A, Toda T, Minetoki T, Hirotsune M, Machida M (2005) Improvement of the Aspergillus oryzae enolase promoter (P-enoA) by the introduction of cis-element repeats. Biosci Biotechnol Biochem 69:206–208
Tsuchiya K, Nagashima T, Yamamoto Y, Gomi K, Kitamoto K, Kumagai C, Tamura G (1994) High level secretion of calf chymosin using a glucoamylase-prochymosin fusion gene in Aspergillus oryzae. Biosci Biotechnol Biochem 58:895–899
Valkonen M, Ward M, Wang H, Penttilä M, Saloheimo M (2003) Improvement of foreign-protein production in Aspergillus niger var. awamori by constitutive induction of the unfolded-protein response. Appl Environ Microbiol 69:6979–6986
van den Hombergh JPTW, Sollewijn Gelpke MD, van de Vondervoort PJI, Buxton FP, Visser J (1997) Disruption of three acid proteases in Aspergillus niger: effects on protease spectrum, intracellular proteolysis, and degradation of target proteins. Eur J Biochem 247:605–613
van Gemeren IA, Punt PJ, Drint-Kuyvenhoven A, Broekhuijsen MP, van’t Hoog A, Beijersbergen A, Verrips CT, van den Hondel CA (1997) The ER chaperone encoding bipA gene of black aspergilli is induced by heat shock and unfolded proteins. Gene (Amst) 198:43–52
Ward OP (2012) Production of recombinant proteins by filamentous fungi. Biotechnol Adv 30:1119–1139
Ward M, Wilson LJ, Kodama KH, Rey MW, Berka RM (1990) Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion. Bio/Technology 8:435–440
Wiebe MG, Karandikar A, Robson GD, Trinci AP, Candia JL, Trappe S, Wallis G, Rinas U, Derkx PM, Madrid SM, Sisniega H, Faus I, Montijn R, van den Hondel CA, Punt PJ (2001) Production of tissue plasminogen activator (t-PA) in Aspergillus niger. Biotechnol Bioeng 76:164–174
Yabuki M, Fukui S (1970) Presence of binding site for α-amylase and of masking protein for this site on mycelial cell wall of Aspergillus oryzae. J Bacteriol 104:138–144
Yamanishi M, Ito Y, Kintake R, Imamura C, Katahira S, Ikeuchi A, Moriya H, Matsuyama T (2013) A genome-wide activity assessment of terminator regions in Saccharomyces cerevisiae provides a “Terminatome” toolbox. ACS Synth Biol 2:337–347
Yoon J, Kimura S, Maruyama J, Kitamoto K (2009) Construction of quintuple protease gene disruptant for heterologous protein production in Aspergillus oryzae. Appl Microbiol Biotechnol 82:691–701
Yoon J, Aishan T, Maruyama J, Kitamoto K (2010) Enhanced production and secretion of heterologous proteins by the filamentous fungus Aspergillus oryzae via disruption of vacuolar protein sorting receptor gene Aovps10. Appl Environ Microbiol 76:5718–5727
Yoon J, Maruyama J, Kitamoto K (2011) Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins. Appl Microbiol Biotechnol 89:747–759
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Tanaka, M., Gomi, K. (2014). Strategies for Increasing the Production Level of Heterologous Proteins in Aspergillus oryzae . In: Anazawa, H., Shimizu, S. (eds) Microbial Production. Springer, Tokyo. https://doi.org/10.1007/978-4-431-54607-8_14
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