Heterologous Transformation and Expression of Hericium erinaceum Manganese Peroxidase 1 Gene in Aspergillus nidulans

YIN Li-wei; CHI Yu-jie

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Heterologous Transformation and Expression of Hericium erinaceum Manganese Peroxidase 1 Gene in Aspergillus nidulans

YIN Li-wei^1,2,
CHI Yu-jie^1,

1.
College of Forestry, Northeast Forestry University, Harbin 150040, Heilongjiang, China
2.
School of Life Science, Anqing Teachers College, Anqing 246011, Anhui, China

Received Date: 2013-01-16

Abstract

The recombinant plasmid pLB01/He-mnp1 which contains a gene encoding for manganese peroxidase (He-mnp1) from Hericium erinaceum CB1 was transformated into protoplasts of auxotrophic stain TN02A7 of Aspergillus nidulans by means of protoplast transformation method mediated by PEG/CaCl2 so as to enhance MnP production. A transformant stain TN02A7-He-mnp1 of A. nidulans was gained, the gene He-mnp1 was expressed under the control of alcohol dehydrogenase alcA(p) promoter. The transformant stain TN02A7-He-mnp1, auxotrophic stain TN02A7, wild stain of A. nidulans WJA01, and H. erinaceum CB1 were cultured under the same lignin condition and detected the MnP activity. The results showed that TN02A7-He-mnp1 could produce MnP activity in the absence and presence of heme, but the MnP activity was up to 38.31 U·L-1 on 96h with 0.05 g·L-1 heme which was 8.64 times higher than that without heme but less than that of H. erinaceum CB1, whereas TN02A7 and WJA01 could not produce MnP activity at any time, indicating that the gene He-mnp1 had been successfully transformed into TN02A7-He-mnp1 and expressed in lignin environment, and the heme was one of the restrictive factors for rescombinant mnp gene to express in A. nidulans. The study provides a new method to produce MnP and enhance MnP yield.
- Hericium erinaceum,
- manganese peroxidase,
- Aspergillus nidulans,
- transformation,
- expression

References

[1]	Hofrichter M. Review: lignin conversion by manganese peroxidase(MnP) [J]. Enzyme and Microbial Technology,2002,30(4):454-466
[2]	吴其威. 异源基因在丝状真菌系统中的克隆表达[J].生命科学,1993,5(2):19-21
[3]	闫培生, 罗信昌, 周启. 丝状真菌基因工程研究进展[J].生物工程进展,1999,19(1):36-41
[4]	Mayfield M B, Kishi K, Alic M, et al. Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium[J].Appl Environ Microbiol,1994,60(12):4303-4309
[5]	Gelpke M D S, Gambill M M, Cereghino G P L, et al. Homologous expression of recombinant lignin peroxidase in Phanerochaete chrysosporium[J].Appl Environ Microbiol,1999,65 (4):1670-1674
[6]	Singh D, Chen S. The white-rot fungus Phanerochaete chrysosporium: conditions for the production of lignin-degrading enzymes[J]. Appl Microbiol Biotechnol,2008,81(3):399-417
[7]	Whitwam R, Tien M. Heterologous expression and reconstitution of fungal Mn peroxidase[J].Arch Biochem Biophys,1996,333 (2):439-446
[8]	路福平. 木质素过氧化物酶和锰过氧化物酶基因的克隆及在甲醇毕赤酵母中的表达 [D].天津:天津科技大学, 2004
[9]	Larrondo L F, Lobos S, Stewart P J, et al. Isoenzyme Multiplicity and Characterization of Recombinant Manganese Peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium[J]. Applied and Environmental Microbiology,2001,67(5):2070-2075
[10]	Stewart P, Whitwam R E, Kersten P J, et al. Efficient Expression of a Phanerochaete chrysosporium Manganese Peroxidase Gene in Aspergillus oryzae[J].Applied and Environmental Microbiology,1996,62(3):860-864
[11]	Cortés-Espinosa D V,Absalón Á E, Sanchez N, et al. Heterologous Expression of Manganese Peroxidase in Aspergillus niger and Its Effect on Phenanthrene Removal from Soil[J].J Mol Microbiol Biotechnol,2011,21(3-4):120-129
[12]	Johnson T M, Li J K. Heterologous expression and characterization of an active lignin peroxidase from Phanerochaete chrysosporium using recombinant baculovirus[J].Arch Biochem Biophys,1991,291(2):371-378
[13]	Whitwam R E, Gazarian I, Tien M. Expression of fungal Mn peroxidase in E. coli and refolding to yield active enzyme[J]. Biochem Biophys Res Commun,1995,216(3):1013-1017
[14]	Doyle W A, Smith A T. Expression of lignin peroxidase H8 in Escherichia coli: folding and activation of the recombinant enzyme with Ca²⁺ and haem[J]. Biochem J,1996, 315:15-19
[15]	Gu L, Lajoie C, Kelly C.Expression of a Phanerochaete chrysosporium Manganese Peroxidase Gene in the Yeast Pichia pastoris[J].Biotechnol Prog,2003,19(5):1403-1409
[16]	Conesa A, Hondel C A M J J, Punt P J. Studies on the production of fungal peroxidases in Aspergillus niger[J].Appl Environ Microbiol.2000,66(7):3016-3023
[17]	Ruiz-dueńas F J, Martínez M J, Martínez Á T. Heterologous expression of Pleurotus eryngii peroxidase confirms its ability to oxidize Mn²⁺ and different aromatic substrates[J]. Applied and Environmental Microbiology,1999,65(10):4705-4707
[18]	Chen S, Song Y, Cao J, et al. Localization and function of calmodulin in live-cells of Aspergillus nidulans[J].Fungal Genetics and Biology,2010,47(3):268-278
[19]	Käfer E. Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations[J].Adv Genet,1977,19:131-133
[20]	Wang J, Hu H, Wang S, et al. The important role of Actinin-like protein (AcnA) in cytokinesis and apical dominance of hyphal cells in Aspergillus nidulans[J].Microbiology,2009,155(8):2714-2725
[21]	Wang G, Lu L, Zhang CY, et al. Calmodulin concentrates at the apex of growing hyphae and localizes to the Spitzenkorper in Aspergillus nidulans[J].Protoplasma,2006,228(4):159-166
[22]	曹进玲. 钙离子通道蛋白MidA在构巢曲霉中的功能特征研究.南京:南京师范大学,2011
[23]	胡美美. 偏肿革裥菌MnP基因在构巢曲霉中的转化与表达.哈尔滨:东北林业大学,2013
[24]	Romero B, Turner G, Olivas I, et al. The Aspergillus nidulans alcA promoter drives tightly regulated conditional gene expression in Aspergillus fumigatus permitting validation of essential genes in this human pathogen[J].Fungal Genetics and Biology,2003,40(2):103-114
[25]	池玉杰, 闫洪波. 红平菇木质素降解酶系统漆酶、锰过氧化物酶及木质素过氧化物酶的检测[J].林业科学,2009,45(12): 154-158
[26]	孟祥锋. 瑞氏木霉纤维二糖水解酶(CBH I)丝状真菌表达体系的构建.山东:山东大学,2010
[27]	Waring R B, May G S, Morris N R. Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes[J].Gene,1989,79(1):119-130
[28]	Gao L, Song Y, Cao J, et al. Osmotic stabilizer-coupled suppression of NDR defects is dependent on the calcium-calcineurin signaling cascade in Aspergillus nidulans[J].Cellular Signalling,2011,23(11):1750-1757
[29]	Jiang F, Kongsaeree P, Charron R, et al. Production and separation of manganese peroxidase from heme amended yeast[J]. Cultures.Biotechnology and Bioengineering,2008,99(3):540-549
[30]	Nayak T, Szewczyk E, Oakley C E, et al. A versatile and efficient gene-targeting system for Aspergillus nidulans[J].Genetics, 2006,172(3):1557-1566
[31]	Wollkow T D,Harris S D,Hamer J E. Cytokinesis in Aspergillus nidulans is controlled by cell size, nuclear positioning and mitosis[J].J Cell Sci,1996,109(8):2179-2188

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Heterologous Transformation and Expression of Hericium erinaceum Manganese Peroxidase 1 Gene in Aspergillus nidulans

1. College of Forestry, Northeast Forestry University, Harbin 150040, Heilongjiang, China

2. School of Life Science, Anqing Teachers College, Anqing 246011, Anhui, China

Received Date: 2013-01-16

Abstract: The recombinant plasmid pLB01/He-mnp1 which contains a gene encoding for manganese peroxidase (He-mnp1) from Hericium erinaceum CB1 was transformated into protoplasts of auxotrophic stain TN02A7 of Aspergillus nidulans by means of protoplast transformation method mediated by PEG/CaCl2 so as to enhance MnP production. A transformant stain TN02A7-He-mnp1 of A. nidulans was gained, the gene He-mnp1 was expressed under the control of alcohol dehydrogenase alcA(p) promoter. The transformant stain TN02A7-He-mnp1, auxotrophic stain TN02A7, wild stain of A. nidulans WJA01, and H. erinaceum CB1 were cultured under the same lignin condition and detected the MnP activity. The results showed that TN02A7-He-mnp1 could produce MnP activity in the absence and presence of heme, but the MnP activity was up to 38.31 U·L-1 on 96h with 0.05 g·L-1 heme which was 8.64 times higher than that without heme but less than that of H. erinaceum CB1, whereas TN02A7 and WJA01 could not produce MnP activity at any time, indicating that the gene He-mnp1 had been successfully transformed into TN02A7-He-mnp1 and expressed in lignin environment, and the heme was one of the restrictive factors for rescombinant mnp gene to express in A. nidulans. The study provides a new method to produce MnP and enhance MnP yield.

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Reference (31)

[1]	Hofrichter M. Review: lignin conversion by manganese peroxidase(MnP) [J]. Enzyme and Microbial Technology,2002,30(4):454-466
[2]	吴其威. 异源基因在丝状真菌系统中的克隆表达[J].生命科学,1993,5(2):19-21
[3]	闫培生, 罗信昌, 周启. 丝状真菌基因工程研究进展[J].生物工程进展,1999,19(1):36-41
[4]	Mayfield M B, Kishi K, Alic M, et al. Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium[J].Appl Environ Microbiol,1994,60(12):4303-4309
[5]	Gelpke M D S, Gambill M M, Cereghino G P L, et al. Homologous expression of recombinant lignin peroxidase in Phanerochaete chrysosporium[J].Appl Environ Microbiol,1999,65 (4):1670-1674
[6]	Singh D, Chen S. The white-rot fungus Phanerochaete chrysosporium: conditions for the production of lignin-degrading enzymes[J]. Appl Microbiol Biotechnol,2008,81(3):399-417
[7]	Whitwam R, Tien M. Heterologous expression and reconstitution of fungal Mn peroxidase[J].Arch Biochem Biophys,1996,333 (2):439-446
[8]	路福平. 木质素过氧化物酶和锰过氧化物酶基因的克隆及在甲醇毕赤酵母中的表达 [D].天津:天津科技大学, 2004
[9]	Larrondo L F, Lobos S, Stewart P J, et al. Isoenzyme Multiplicity and Characterization of Recombinant Manganese Peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium[J]. Applied and Environmental Microbiology,2001,67(5):2070-2075
[10]	Stewart P, Whitwam R E, Kersten P J, et al. Efficient Expression of a Phanerochaete chrysosporium Manganese Peroxidase Gene in Aspergillus oryzae[J].Applied and Environmental Microbiology,1996,62(3):860-864
[11]	Cortés-Espinosa D V,Absalón Á E, Sanchez N, et al. Heterologous Expression of Manganese Peroxidase in Aspergillus niger and Its Effect on Phenanthrene Removal from Soil[J].J Mol Microbiol Biotechnol,2011,21(3-4):120-129
[12]	Johnson T M, Li J K. Heterologous expression and characterization of an active lignin peroxidase from Phanerochaete chrysosporium using recombinant baculovirus[J].Arch Biochem Biophys,1991,291(2):371-378
[13]	Whitwam R E, Gazarian I, Tien M. Expression of fungal Mn peroxidase in E. coli and refolding to yield active enzyme[J]. Biochem Biophys Res Commun,1995,216(3):1013-1017
[14]	Doyle W A, Smith A T. Expression of lignin peroxidase H8 in Escherichia coli: folding and activation of the recombinant enzyme with Ca²⁺ and haem[J]. Biochem J,1996, 315:15-19
[15]	Gu L, Lajoie C, Kelly C.Expression of a Phanerochaete chrysosporium Manganese Peroxidase Gene in the Yeast Pichia pastoris[J].Biotechnol Prog,2003,19(5):1403-1409
[16]	Conesa A, Hondel C A M J J, Punt P J. Studies on the production of fungal peroxidases in Aspergillus niger[J].Appl Environ Microbiol.2000,66(7):3016-3023
[17]	Ruiz-dueńas F J, Martínez M J, Martínez Á T. Heterologous expression of Pleurotus eryngii peroxidase confirms its ability to oxidize Mn²⁺ and different aromatic substrates[J]. Applied and Environmental Microbiology,1999,65(10):4705-4707
[18]	Chen S, Song Y, Cao J, et al. Localization and function of calmodulin in live-cells of Aspergillus nidulans[J].Fungal Genetics and Biology,2010,47(3):268-278
[19]	Käfer E. Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations[J].Adv Genet,1977,19:131-133
[20]	Wang J, Hu H, Wang S, et al. The important role of Actinin-like protein (AcnA) in cytokinesis and apical dominance of hyphal cells in Aspergillus nidulans[J].Microbiology,2009,155(8):2714-2725
[21]	Wang G, Lu L, Zhang CY, et al. Calmodulin concentrates at the apex of growing hyphae and localizes to the Spitzenkorper in Aspergillus nidulans[J].Protoplasma,2006,228(4):159-166
[22]	曹进玲. 钙离子通道蛋白MidA在构巢曲霉中的功能特征研究.南京:南京师范大学,2011
[23]	胡美美. 偏肿革裥菌MnP基因在构巢曲霉中的转化与表达.哈尔滨:东北林业大学,2013
[24]	Romero B, Turner G, Olivas I, et al. The Aspergillus nidulans alcA promoter drives tightly regulated conditional gene expression in Aspergillus fumigatus permitting validation of essential genes in this human pathogen[J].Fungal Genetics and Biology,2003,40(2):103-114
[25]	池玉杰, 闫洪波. 红平菇木质素降解酶系统漆酶、锰过氧化物酶及木质素过氧化物酶的检测[J].林业科学,2009,45(12): 154-158
[26]	孟祥锋. 瑞氏木霉纤维二糖水解酶(CBH I)丝状真菌表达体系的构建.山东:山东大学,2010
[27]	Waring R B, May G S, Morris N R. Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes[J].Gene,1989,79(1):119-130
[28]	Gao L, Song Y, Cao J, et al. Osmotic stabilizer-coupled suppression of NDR defects is dependent on the calcium-calcineurin signaling cascade in Aspergillus nidulans[J].Cellular Signalling,2011,23(11):1750-1757
[29]	Jiang F, Kongsaeree P, Charron R, et al. Production and separation of manganese peroxidase from heme amended yeast[J]. Cultures.Biotechnology and Bioengineering,2008,99(3):540-549
[30]	Nayak T, Szewczyk E, Oakley C E, et al. A versatile and efficient gene-targeting system for Aspergillus nidulans[J].Genetics, 2006,172(3):1557-1566
[31]	Wollkow T D,Harris S D,Hamer J E. Cytokinesis in Aspergillus nidulans is controlled by cell size, nuclear positioning and mitosis[J].J Cell Sci,1996,109(8):2179-2188

Heterologous Transformation and Expression of Hericium erinaceum Manganese Peroxidase 1 Gene in Aspergillus nidulans

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通讯作者: 陈斌, bchen63@163.com

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Heterologous Transformation and Expression of Hericium erinaceum Manganese Peroxidase 1 Gene in Aspergillus nidulans

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