米曲霉基因工程技术的进展

doi:10.13560/j.cnki.biotech.bull.1985.2018-0252

摘要/Abstract

摘要： 丝状真菌米曲霉（Aspergillus oryzae）是发酵工业的重要菌株,具有强大的蛋白分泌能力和公认的食品安全性,可作为表达外源蛋白的细胞工厂。然而利用米曲霉分泌生产外源蛋白质常会受限于一些瓶颈问题：包括转录、翻译、蛋白质折叠、易位、降解、运输和分泌等。这些问题的存在导致米曲霉外源蛋白生产很难达到预期的效果。近年来,随着全基因组序列的破译,米曲霉生产相关基础研究以及基因工程技术都得到了较好的发展。如提高同源重组效率、选择性标记基因应用技术、染色体大片段删除技术、菌丝融合技术和DNA芯片技术等。这些技术的开发和建立为米曲霉工业生产应用提供了大量技术支持。针对米曲霉在外源表达蛋白中存在的瓶颈问题,主要通过以下几个方面进行了阐述：转化系统的优化和转化效率的提高、蛋白酶基因缺陷菌株的构建、融合表达策略,以及优化其外源表达系统来提高外源蛋白生产等。这些米曲霉生产相关基础研究以及基因工程技术的进步很大程度上提高了米曲霉的生产应用,并为今后米曲霉生产菌株的育种提供了更多有效的途径和研究空间。

关键词: 米曲霉, 细胞工厂, 外源蛋白生产, 基因工程技术

Abstract: The filamentous fungus Aspergillus oryzae is an important strain in fermentation industry. It has a strong capacity of secreting protein and recognized food safety,therefore is widely used as a cell factory for the expression of foreign proteins. However,the production and secretion of foreign proteins by A.oryzae are limited by a number of bottlenecks,including transcription,translation,protein folding,translocation,degradation,transport,and secretion,etc. The existence of these problems makes it difficult to achieve the desired target in the production of foreign proteins by A.oryzae. In recent years,with the decipherment of the whole genome sequence,the basic research and the genetic engineering technologies related to the production of A. oryzae have been well developed,for example,improvement of the homologous recombination efficiency,application of selectable marker genes,large chromosome deletion technology,hyphal fusion technique,DNA microarray,etc. The development and establishment of these technologies provided a great deal of technical support for the industrial production and application of A. oryzae. Regarding the bottlenecks in the heterologous protein expression of A. oryzae,this article mainly elaborated several aspects such as the optimization of genetic transformation system and the increase of transformation efficiency,the construction of protease gene-deleted strains,the fusion expression strategy,and the optimization of its exogenous expression system to improve the production of foreign proteins. These advances in basic research and genetic engineering technology have greatly improved the production and application of A.oryzae,and also provide more effective ways and research space for the breeding of A.oryzae production strains in the future.

Key words: Aspergillus oryzae, cell factory, productionof foreign protein, genetic engineering technology

张智敏, 庄淼, 金锋杰. 米曲霉基因工程技术的进展[J]. 生物技术通报, 2018, 34(9): 170-176.

ZHANG Zhi-min, ZHUANG Miao, JIN Feng-jie. Advances in Gene Engineering Technologies for Aspergillus oryzae[J]. Biotechnology Bulletin, 2018, 34(9): 170-176.

参考文献

[1] Abe K, Gomi K, Hasegawa F, et al.Impact of Aspergillus oryzae genomics on industrial production of metabolites[J]. Mycopathologia, 2006, 162(3):143.
[2] Galagan JE, Calvo SE, Cuomo C, et al.Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae[J]. Nature, 2005, 438(7071):1105-1115.
[3] 银超. 曲霉工业菌种基因组测序及比较基因组研究[D]. 广州:华南理工大学, 2014.
[4] Uchima CA, Tokuda G, Watanabe H, et al.Heterologous expression and characterization of a glucose-stimulated β-glucosidase from the termite Neotermes koshunensis in Aspergillus oryzae[J]. Appl Microbiol Biotechnol, 2011, 89(6):1761-1771.
[5] Murphy RA, Power RFG.Expression of an α-galactosidase from Saccharomyces cerevisiae, in Aspergillus awamori, and Aspergillus oryzae[J]. Journal of Industrial Microbiology & Biotechnology, 2002, 28(2):97.
[6] Bartling S, vanden Hombergh JP, Olsen O, et al. Expression of an Erwinia pectate lyase in three species of Aspergillus[J]. Current Genetics, 1996, 29(5):474-481.
[7] Vongsangnak W, Olsen P, Hansen K, et al.Improved annotation through genome-scale metabolic modeling of Aspergillus oryzae[J]. Bmc Genomics, 2008, 9(1):245.
[8] 张田, 唐克轩. 丝状真菌的遗传工程研究进展[J]. 上海交通大学学报:农业科学版, 2010, 28(5):480-486.
[9] Mattern IE, Unkles S, Kinghorn JR, et al.Transformation of Aspergillus oryzae using the A. niger pyrG gene[J]. Molecular & General Genetics Mgg, 1987, 210(3):460-461.
[10] Kitamoto N, Kimura T, Kito Y, et al.The nitrate reductase gene from a shoyu koji mold, Aspergillus oryzae KBN616[J]. Biosci Biotechnol Biochem, 1995, 59(9):1795-1797.
[11] Yoon J, Maruyama J, Kitamoto K.Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins[J]. Appl Microbiol Biotechnol, 2011, 89(3):747.
[12] Jin FJ, Maruyama JI, Juvvadi PR, et al.Development of a novel quadruple auxotrophic host transformation system by argB, gene disruption using adeA, gene and exploiting adenine auxotrophy in Aspergillus oryzae[J]. FEMS Microbiol Lett, 2004, 239(1):79-85.
[13] Takahashi T, Masuda T, Koyama Y.Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae[J]. Mol Genet Genomics, 2006, 275(5):460-470.
[14] Takahashi T, Jin FJ, Sunagawa M, et al.Generation of large chromosomal deletions in koji molds Aspergillus oryzae and Aspergillus sojae via a loop-out recombination[J]. Appl Environ Microbiol, 2008, 74(24):7684-7693.
[15] Takahashi T, Jin FJ, Koyama Y.Nonhomologous end-joining deficiency allows large chromosomal deletions to be produced by replacement-type recombination in Aspergillus oryzae[J]. Fungal Genetics & Biology, 2009, 46(11):815-824.
[16] Jin FJ, Takahashi T, Utsushikawa M, et al.A trial of minimization of chromosome 7 in Aspergillus oryzae by multiple chromosomal deletions[J]. Mol Genet Genom, 2010, 283(1):1-12.
[17] Hara S, Jin FJ, Takahashi T, et al.A further study on chromosome minimization by protoplast fusion in Aspergillus oryzae[J]. Mol Genet Genomics, 2012, 287:177-187.
[18] Jin FJ, Takahashi T, Machida M, et al.Identification of bHLH-type transcription regulator gene by systematically deleting large chromosomal segments in Aspergillus oryzae[J]. Appl. Environ Microbiol, 2009, 75:5943-5951.
[19] Wada R, Jin FJ, Koyama Y, et al.Efficient formation of heterokaryotic sclerotia in the filamentous fungus Aspergillus oryzae[J]. Appl Microbiol Biotechnol, 2014, 98(1):325-334.
[20] Fleiβner A, Dersch P.Expression and export:recombinant protein production systems for Aspergillus[J]. Appl Microbiol Biotechnol, 2010, 87(4):1255-1270.
[21] Yoder WT, Lehmbeck J.Heterologous expression and protein secretion in filamentous fungi[M]. Advances in Fungal Biotechnology for Industry, Agriculture, and Medicine. Springer US, 2004:201-219.
[22] Jin FJ, Maruyama J, Juvvadi PR, et al.Adenine auxotrophic mutants of Aspergillus oryzae:development of a novel transformation system with triple auxotrophic hosts[J]. Biosci Biotechnol Biochem, 2004, 68:656-662.
[23] Jin FJ, Watanabe T, Juvvadi PR, et al.Double disruption of the proteinase genes, tppA, and pepE, increases the production level of human lysozyme by Aspergillus oryzae[J]. Appl Microbiol Biotechnol, 2007, 76(5):1059-1068.
[24] Yoon J, Kimura S, Maruyama J, et al.Construction of quintuple protease gene disruptant for heterologous protein production in Aspergillus oryzae[J]. Appl Microbiol Biotechnol, 2009, 82(4):691-701.
[25] Yoon J, Aishan T, Maruyama J, et al.Enhanced production and secretion of heterologous proteins by the filamentous fungus Aspergillus oryzae via disruption of vacuolar protein sorting receptor gene Aovps10[J]. Appl Environ Microbiol, 2010, 76:5718-5727.
[26] Nemoto T, Watanabe T, Mizogami Y, et al.Isolation of Aspergillus oryzae mutants for heterologous protein production from a double proteinase gene disruptant[J]. Appl Microbiol Biotechnol, 2009, 82(6):1105-1114.
[27] Yaver DS, Lamsa M, Munds R, et al.Using DNA-tagged mutage-nesis to improve heterologous protein production in Aspergillus oryzae[J]. Fungal Genetics & Biology, 2000, 29(1):28.
[28] Zhu L, Nemoto T, Yoon J, et al.Improved heterologous protein production by a tripeptidyl peptidase gene(AosedD)disruptant of the filamentous fungus Aspergillus oryzae[J]. Journal of General & Applied Microbiology, 2012, 58(3):199-209.
[29] Zhu L, Maruyama J, Kitamoto K.Further enhanced production of heterologous proteins by double-gene disruption(ΔAosedD ΔAovps10)in a hyper-producing mutant of Aspergillus oryzae[J]. Appl Microbiol Biotechnol, 2013, 97(14):6347.
[30] Ohno A, Maruyama J, Nemoto T, et al.A carrier fusion significantly induces unfolded protein response in heterologous protein production by Aspergillus oryzae[J]. Appl Microbiol Biotechnol, 2011, 92(6):1197-1206.
[31] Lombrana M, Moralejo F, Pinto RJ.Modulation of Aspergillus awamori thaumatin secretion by modification of bipA gene expression[J]. Appl Environ Microbiol, 2004, 70(9):5145-5152.
[32] Moralejo FJ, Watson AJ, Jeenes DJ, et al.A defined level of protein disulfide isomerase expression is required for optimal secretion of thaumatin by Aspegillus awamori[J]. Mol Genet Genom, 2001, 266(2):246-253.
[33] Yoon J, Kikuma T, Maruyama J, et al.Enhanced production of bovine chymosin by autophagy deficiency in the filamentous fungus Aspergillus oryzae[J]. PLoS One, 2013, 8(4):e62512.
[34] Hoang HD, Maruyama J, Kitamoto K.Modulating endoplasmic reticulum-Golgi cargo receptors for improving secretion of carrier-fused heterologous proteins in the filamentous fungus Aspergillus oryzae[J]. Appl Environ Microbiol, 2015, 81(2):533-543.
[35] Gasser B, Mattanovich D.Antibody production with yeasts and filamentous fungi:on the road to large scale?[J]. Biotechnology Letters, 2007, 29(2):201.
[36] Maras M, Van DI, Contreras R, et al.Filamentous fungi as production organisms for glycoproteins of bio-medical interest[J]. Glycoconjugate Journal, 1999, 16(2):99-107.
[37] Ohno A, Maruyama J, Nemoto T, et al.A carrier fusion significantly induces unfolded protein response in heterologous protein production by Aspergillus oryzae[J]. Appl Microbiol Biotechnol, 2011, 92(6):1197-1206.
[38] Tsuchiya K, Nagashima T, Yamamoto Y, et al.High level secretion of calf chymosin using a glucoamylase-prochymosin fusion gene in Aspergillus oryzae[J]. Biosci Biotechnol Biochem, 1994, 58(5):895-899.
[39] Nemoto T, Watanabe T, Mizogami Y, et al.Isolation of Aspergillus oryzae mutants for heterologous protein production from a double proteinase gene disruptant[J]. Appl Microbiol Biotechnol, 2009, 82(6):1105-1114.
[40] Ham A, Ezzat SM, Houseny AM.Improved production of kojic acid by mutagenesis of Aspergillus flavus HAk1 and Aspergillus oryzae HAk2 and their potential antioxidant activity[J]. Biotech, 2017, 7(5):276.
[41] 胡杰, 潘力, 罗立新, 等. 米曲霉孢子原生质体复合诱变及高活力蛋白酶菌株选育[J]. 食品工业科技, 2007(5):116-118.
[42] Murthy PS, Kusumoto KI.Acid protease production by Aspergillus oryzae, on potato pulp powder with emphasis on glycine releasing activity:A benefit to the food industry[J]. Food & Bioproducts Processing, 2015, 96:180-188.
[43] Wang S, Duan M, Liu Y, et al.Enhanced production of fructosyltransferase in Aspergillus oryzae by genome shuffling[J]. Biotechnology Letters, 2016, 39(3):391-396.
[44] Ogasawara H, Obata H, Hata Y, et al.Crawler, a novel Tc1/mariner-type transposable element in Aspergillus oryzae transposes under stress conditions[J]. Fungal Genetics & Biology, 2009, 46(6-7):441-449.
[45] Jin FJ, Hara S, Sato A, et al.Discovery and analysis of an active long terminal repeat-retrotransposable element in Aspergillus oryzae[J]. Journal of General & Applied Microbiology, 2014, 60(1):1-6.
[46] Ehrlich KC, Mack BM.Comparison of expression of secondary metabolite biosynthesis cluster genes in Aspergillus flavus, A. parasiticus, and A. oryzae[J]. Toxins, 2014, 6(6):1916.
[47] Takeda I, Umemura M, Koike H, et al.Motif-independent prediction of a secondary metabolism gene cluster using comparative genomics:application to sequenced genomes of Aspergillus and ten other filamentous fungal species[J]. DNA Research, 2014, 21(4):447-457.
[48] Marui J, Ohashikunihiro S, Ando T, et al.Penicillin biosynthesis in Aspergillus oryzae and its overproduction by genetic engineering[J]. Journal of Bioscience & Bioengineering, 2010, 110(1):8-11.
[49] Yamada R, Yoshie T, Wakai S, et al.Aspergillus oryzae -based cell factory for direct kojic acid production from cellulose[J]. Microbial Cell Factories, 2014, 13(1):71.
[50] Sanchez JF, Somoza AD, Keller NP, et al.Advances in Aspergillus secondary metabolite research in the post-genomic era[J]. Natural Product Reports, 2012, 29(3):351.
[51] Amare MG, Keller NP.Molecular mechanisms of Aspergillus flavus secondary metabolism and development[J]. Fungal Genetics & Biology, 2014, 66(3):11.
[52] Ichinose S, Tanaka M, Shintani T, et al.Improved α-amylase production by Aspergillus oryzae after a double deletion of genes involved in carbon catabolite repression[J]. Appl Microbiol Biotechnol, 2014, 98(1):335-343.