Cloning and Expression Analysis of Oxaloacetate Hydrolase（LeOAH1）Gene from Lentinula edodes

doi:10.13560/j.cnki.biotech.bull.1985.2019-1246

Abstract

Abstract: Oxaloacetate acetylhydrolase/Oxaloacetate hydrolase is one of the key enzymes for oxalate synthesis in Lentinula edodes. The LeOAH1 was cloned and its expression in vivo and in vitro was analyzed to lay the foundation for the functional research of LeOAH1. LeOAH1 was cloned from L. edodes L808 and bioinformatics analysis was conducted. The expression of LeOAH1 and the content of oxalic acid secreted by L. edodes under different pH conditions were respectively detected by RT-PCR and HPLC during the vegetative phase. In addition,a recombinant prokaryotic expression vector pET28a-oah1 was constructed and successfully expressed in Escherichia coli. The results showed that the length of gDNA of the LeOAH1 was 1 906 bp,the length of cDNA was 1 356 bp,encoding 451 amino acids,the protein molecular weight was about 48.9 kD,and the isoelectric point was 6.80. Bioinformatics analysis revealed that the enzyme was an unstable hydrophobic protein with four secondary structures of α-helix（40.08%）,random coil（34.59%）,extended chain（17.52%）,and β-sheet（7.10%）level structure;it was in the cytoplasm by subcellular localization. The sequence alignment results demonstrated that the enzyme had a conserved domain. The phylogenetic tree analysis suggested that the protein had the highest similarity with OAH from Japanese mushroom with 82% homology,followed by Pluteus cervinus with 64% homology. The RT-PCR and HPLC results showed that the expressions of LeOAH1 gene and the amount of oxalic acid secreted by L. edodes both increased with the increase of pH. The molecular weight of the induced protein in E. coli was in line with the predicted results. The gene characteristics and expression pattern of oxaloacetate hydrolase,as well as its relationship with oxalic acid secretion are preliminarily clarified.

Key words: Lentinula edodes, oxaloacetate hydrolase, gene cloning, bioinformatics analysis, prokaryotic expression

DUAN Ying-ce, HU Zi-yi, YANG Fan, LI Jin-tao, WU Xiang-li, ZHANG Rui-ying. Cloning and Expression Analysis of Oxaloacetate Hydrolase（LeOAH1）Gene from Lentinula edodes[J]. Biotechnology Bulletin, 2020, 36(9): 227-234.

References

[1] Jarosz-Wilkolazka A, Gadd GM.Oxalate production by wood-rotting fungi growing in toxic metal-amended medium[J]. Chemosphere, 2003, 52(3):541-547.
[2] Dutton MV, Evans CS .Oxalate production by fungi:Its role in pathogenicity and ecology in the soil environment[J]. Canadian Journal of Microbiology, 1996, 42(9):881-895.
[3] Liang X, Liberti D, Moyi LI, et al.Oxaloacetate acetylhydrolase gene mutants of Sclerotinia sclerotiorum do not accumulate oxalic acid, but do produce limited lesions on host plants[J]. Molecular Plant Pathology, 2015, 16(6). 559-571.
[4] Pedersen H, Christensen B, Hjort C, et al.Construction and characterization of an oxalic acid nonproducing strain of Aspergillus niger[J]. Metabolic Engineering, 2000, 2(1):34-41.
[5] Kobayashi K, et al.Oxalic acid production by citric acid-producing Aspergillus niger overexpressing the oxaloacetate hydrolase gene oahA[J]. J Ind Microbiol Biotechnol, 2014, 41(5):749-756.
[6] Watanabe T, et al.Oxalate efflux transporter from the brown rot fungus Fomitopsis palustris[J]. Appl Environ Microbiol, 2010, 76(23):7683-7690.
[7] 周桥胜. 球孢白僵菌pH响应转录因子BbPacC的功能研究[D].重庆:西南大学, 2015.
Zhou QS.Characterization of pH-responsive transcription factor, BbPacC, in the entomopathogenic fungus Beauveria bassiana[D]. Chongqing:Southwest University, 2015.
[8] Munir E, et al.A physiological role for oxalic acid biosynthesis in the wood-rotting basidiomycete Fomitopsis palustris[J]. Proc Natl Acad Sci USA, 2001, 98(20):11126-11130.
[9] 伏贝贝. 改造酿酒酵母乙酰辅酶A合成途径及其在香叶醇生产中的应用[D]. 济南:齐鲁工业大学, 2018.
Fu BB.Engineering acetyl-CoA synthetic pathway and its application in geraniol production in Saccharomyces cerevisiae[D]. Jinan:Qilu University of Technology, 2018.
[10] Kubicek CP, Schreferl-Kunar G, Wöhrer W.Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger[J]. Appl Environ Microbiol, 1988, 54(3):633-637.
[11] Akamatsu Y, Shimada M.Partial purification and characterization of glyoxylate oxidase from the brown-rot basidiomycete Tyromyces palustris[J]. Phytochemistry, 1994, 37(3):649-653.
[12] 段应策, 胡姿仪, 张瑞颖. 草酸是香菇调控环境pH促进菌丝生长的重要因素[C]. 多彩菌物美丽中国——中国菌物学会2019年学术年会论文摘要, 2019.
Duan YC, Hu ZY, Zhang RY.The oxalate is involved in regulating ambient pH to improve mycelial growth for Lentinula edodes[C].Colorful Fungi Beautiful China-Abstract of papers presented at the 2019 Academic Annual Meeting of Mycological Society of China, 2019.
[13] 王竹青, 陈云, 杨玉婷. 甘蔗抗坏血酸过氧化物酶基因(ScAPX)的克隆及表达分析[J]. 农业生物技术学报, 2015, 23(2):170-180.
Wang ZQ, Chen Y, Yang YT.Cloning and expression analysis of peroxidase gene(ScAPX1)from sugarcane[J]. Journal of Agricultural Biotechnology, 2015, 23(2):170-180.
[14] Guggiari M, Bloque R, Aragno M.Experimental calcium-oxalate crystal production and dissolution by selected wood-rot fungi[J]. Int Biodeterior Biodegradation, 2011, 65(6):803-809.
[15] 侯志浩, 赵梦然, 邬向丽. 热胁迫下糙皮侧耳实时荧光定量PCR内参基因的选择[J]. 食用菌学报, 2019 (3):11-18.
Hou ZH, Zhao MR, Wu XL.Selection of reference genes for real-time quantitative PCR of Pleurotus ostreatus under heat stress[J]. Acta Edulis Fungi, 2019(3):11-18.
[16] Lenz H, et al.Partial purification and some properties of oxalacetase from Aspergillus niger[J]. Eurjbiochem, 1976, 1:225-236.
[17] Balmforth AJ, Thomson A.Isolation and characterization of glyoxylate dehydrogenase from the fungus Sclerotium rolfsii[J]. Biochemical Journal, 1984, 218(1):113-118.
[18] Mäkelä M, Galkin S, Hatakka A.Production of organic acids and oxalate decarboxylase in lignin-degrading white rot fungi[J]. Enzyme and Microbial Technology, 2002, 30(4):542-549.
[19] Han Y, Joosten HJ, Niu W.Oxaloacetate Hydrolase, the C-C bond lyase of oxalate secreting fungi[J]. Journal of Biological Chemistry, 2007, 282(13):9581-9590.
[20] Gombert AK, Veiga T, Puig-Martinez M.Functional characterization of the oxaloacetase encoding gene and elimination of oxalate formation in the β-lactam producer Penicillium chrysogenum[J]. Fungal Genet Biol, 2011, 48(8):831-839.
[21] Munir E, Yoon JJ, Tokimatsu T.New role for glyoxylate cycle enzymes in wood-rotting basidiomycetes in relation to biosynthesis of oxalic acid[J]. J Wood Sci, 2001, 47(5):368-373.
[22] Tokimatsu T, Nagai Y, Hattori T.Purification and characteristics of a novel cytochrome c dependent glyoxylate dehydrogenase from a wood-destroying fungus Tyromyces palustris[J]. FEBS Letters, 1998, 437(1-2):117-121.
[23] Narayanan B, Niu W, Joosten HJ.Structure and function of 2, 3-dimethylmalate lyase, a PEP mutase/isocitrate lyase superfamily member[J]. J Mol Biol, 2009, 386(2):486-503.
[24] Lu Z, Feng X, Song L, et al.Diversity of function in the isocitrate lyase enzyme superfamily:The Dianthus caryophyllus petal death protein cleaves α-keto and α-hydroxycarboxylic acids[J]. Biochemistry, 2005, 44(50):16365-16376.
[25] Pedersen H, Hjort C, Nielsen J.Cloning and characterization of oah, the gene encoding oxaloacetate hydrolase in Aspergillus niger[J]. Molecular and General Genetics, 2000, 263(2):281-286.
[26] Rascle C, Dieryckx C, Dupuy JW, et al.The pH regulator PacC:a host-dependent virulence factor in Botrytis cinerea[J]. Environmental Microbiology Reports, 2018, 10(5):555-568.
[27] Alkan N, et al.Virulence regulation of phytopathogenic fungi by pH[J]. Antioxid Redox Signal, 2013, 9:1012-1025.
[28] Jarosz-Wilkoazka A, Marcin G.Organic acids production by white rot basidiomycetes in the presence of metallic oxides[J]. Canadian Journal of Microbiology, 2006, 52(8):779-785.
[29] Kuan IC, Tien M.Stimulation of Mn peroxidase activity:A poss-ible role for oxalate in lignin biodegradation[J]. Proc National Acad Sci USA, 1993, 90(4):1242-1246.
[30] Guimarães RL, Stotz HU.Oxalate Production by Sclerotinia sclerotiorum deregulates guard cells during infection[J]. Plant Physiology, 2004, 136(3):3703-3711.