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球孢白僵菌Beauveria bassiana是一种可寄生在多种昆虫上具较强致病性的真菌,已被广泛用作防治森林、农作物虫害的生物农药[1-2];它产生大量如非核糖体多肽、聚酮类等次生代谢产物,可抑制多种腐生或寄生线虫[3-4]。聚酮、非核糖体多肽及其杂合化合物如聚酮中的红霉素[5]、四环素[6];非核糖体多肽中的青霉素、头孢霉素[7-8];聚酮和非核糖体多肽的杂合化合物如他克莫司[9]、雷帕霉素[10]、博来霉素[11]、埃博霉素[12]等具有免疫抑制剂或抗肿瘤等生理活性[13]。迄今为止,球孢白僵菌中的聚酮和非核糖体多肽杂合型化合物仅有卵孢白僵菌素经基因敲除和异源表达鉴定的报道[3]。聚酮类和非核糖体肽是2类典型的小分子天然次生代谢化合物,分别由聚酮合酶(polyketide synthases, PKS)、非核糖体肽合成酶(nonribosomal peptide synthetases, NRPS)催化简单单体脂肪酸或氨基酸缩合形成[13-14]。真菌聚酮合酶主要属于Ⅰ型PKS[13, 15],其结构域通常包括酮体合成酶(ketosynthase, KS),酰基转移酶(acyltransferase, AT),脱水酶(dehydratase, DH),甲基转移酶(methyltransferase, MT),烯酯酰还原酶(enoylreductase, ER),酮体还原酶(ketoreductase, KR),酰基载体蛋白(acyl carier pretein, ACP)和末端释放酶等[13],短链醇脱氢酶(short chain dehydrogenase/reductase,SDR)家族在PKS中属末端释放酶[16],PKS中的SDR结构域对底物选择具一定的特异性[17];NRPS是一种由多模块组成的多酶复合体,由腺苷酰化结构域(adenylation domain, A),肽酰载体蛋白结构域(phosphopantetheine attachment site, PP)和缩合结构域(condensation domain, C)等3个核心结构域按照特定的时空顺序排列组成[18]。真菌PKS/NRPS酶蛋白复合物含有N端的PKS组件和C端的NRPS组件[19],可催化生成含有酰基和氨酰基构件的聚酮和氨基酸或肽类型化合物的生物合成[9, 20];同时聚酮和氨基酸或肽类化合物能够通过它们化学属性的混合,扩展其产物的生物活性,埃博霉素(epothilone)、雷帕霉素(rapamycin)和博来霉素(bleomycin)是这类混合分子中重要代表,编码这些化合物的PKS/NRPS基因大小均约10 kb[21]。随着高通量测序技术的发展,真菌基因组数据不断增加,基因组数据显示目前已通过结构鉴定、表型筛选和生物活性测定等传统方法分离的天然次生代谢产物仅为冰山一角,真菌基因组中尚有大量的天然次生代谢产物生物合成基因存在;该类基因大多数条件下处于沉默状态,其参与合成的化合物大多尚未被鉴定[22];应用基因组挖掘技术可将这些沉默的生物合成基因分离出来,再通过改变培养基配方和条件或进行遗传学修饰等方式诱导以激发它们全部的次生代谢潜力[23-24]。为了发掘球孢白僵菌中聚酮和非核糖体多肽杂合类化合物的生物合成基因,本研究采用基因组挖掘的方式从其基因组数据中获得1条PKS/NRPS基因(命名Bbpks2),通过生物信息学分析预测其潜在功能,尝试寻找该基因的表达条件,为最终确定其天然产物及对其异源表达奠定基础。
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[1] | 宋晓兵, 彭埃天, 程保平. 一株侵染柑橘木虱的球孢白僵菌的分离及鉴定[J]. 植物保护, 2017, 43(4): 139-144. doi: 10.3969/j.issn.0529-1542.2017.04.026 | SONG Xiaobing, PENG Aitian, CHENG Baoping. Isolation and identification of a Beauveria bassiana strain infecting Diaphorina citri[J]. Plant Prot, 2017, 43(4): 139-144. doi: 10.3969/j.issn.0529-1542.2017.04.026 |
[2] | 王月, 文斌, 马军. 球孢白僵菌(Beauveria bassiana)对寒地温室4种害虫防控研究[J]. 生物灾害科学, 2017, 40(4): 284-287. doi: 10.3969/j.issn.2095-3704.2017.04.62 | WANG Yue, WEN Bin, MA Jun. Control effects of Beauveria bassiana on 4 conservatory pests in frigid regions[J]. Biol Disaster Sci, 2017, 40(4): 284-287. doi: 10.3969/j.issn.2095-3704.2017.04.62 |
[3] | XIAO Guohua, YING Shenghua, ZHENG Peng. Genomic perspectives on the evolution of fungal entomopathogenicity in Beauveria bassiana[J]. Scic Rep, 2012, 2(): 483-. doi: 10.1038/srep00483 | |
[4] | 刘丹丹, 段玉玺, 陈立杰. Snf 907-76发酵液杀线虫活性测定及活性物质分离纯化[J]. 农药, 2007, 46(9): 644-646. doi: 10.3969/j.issn.1006-0413.2007.09.025 | LIU Dandan, DUAN Yuxi, CHEN Lijie. Determine nematicidal of ferment liquid of Snf 907-76 and isolation and purification nematocide[J]. Agrochemicals, 2007, 46(9): 644-646. doi: 10.3969/j.issn.1006-0413.2007.09.025 |
[5] | DONADIO S, STAVER M J, MCALPINE J B. Modular organization of genes required for complex polyketide biosynthesis[J]. Science, 1991, 252(5006): 675-679. doi: 10.1126/science.2024119 | |
[6] | KIM E S, BIBB M J, BUTLER M J. Sequences of the oxytetracycline polyketide synthase-encoding otc genes from Streptomyces rimosus[J]. Gene, 1994, 141(1): 141-142. doi: 10.1016/0378-1119(94)90144-9 | |
[7] | AHARONOWITZ Y, COHEN G, MARTIN J F. Penicillin and cephalosporin biosynthetic genes:structure, organization, regulation, and evolution[J]. Ann Rev Microbiol, 1992, 46(): 461-495. doi: 10.1146/annurev.mi.46.100192.002333 | |
[8] | MARTIN F J. New aspects of genes and enzymes for beta-lactam antibiotic biosynthesis[J]. Appl Microbiol Biotechnol, 1998, 50(1): 1-15. | |
[9] | MOTAMEDI H, SHAFIEE A. The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506[J]. Eur J Biochem, 2010, 256(3): 528-534. | |
[10] | SCHWECKE T, APARICIO J F, MOLNAR I. The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin[J]. Proc Natl Acad Sci USA, 1995, 92(17): 7839-7843. doi: 10.1073/pnas.92.17.7839 | |
[11] | DU Liangcheng, SÁNCHEZ C, CHEN Mei. The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase[J]. Chem Biol, 2000, 7(8): 623-642. doi: 10.1016/S1074-5521(00)00011-9 | |
[12] | TANG Li, SHAH S, CHUNG L. Cloning and heterologous expression of the epothilone gene cluster[J]. Science, 2000, 287(5453): 640-642. doi: 10.1126/science.287.5453.640 | |
[13] | COX R J. Polyketides, proteins and genes in fungi:programmed nano-machines begin to reveal their secrets[J]. Org Biomol Chem, 2007, 5(13): 2010-2026. doi: 10.1039/b704420h | |
[14] | SESHIME Y, JUVVADI P R, KITAMOTO K. Aspergillus oryzae type Ⅲ polyketide synthase CsyA is involved in the biosynthesis of 3, 5-dihydroxybenzoic acid[J]. Bioorg Med Chem Lett, 2010, 20(16): 4785-4788. doi: 10.1016/j.bmcl.2010.06.119 | |
[15] | KROKEN S, GLASS N L, TAYLOR J W. Phylogenomic analysis of type Ⅰ polyketide synthase genes in pathogenic and saprobic ascomycetes[J]. Proc Natl Acad Sci USA, 2003, 100(26): 15670-15675. doi: 10.1073/pnas.2532165100 | |
[16] | SISKOS A P, BAERGA-ORTIZ A, BALI S. Molecular basis of Celmer's rules:stereochemistry of catalysis by isolated ketoreductase domains from modular polyketide synthases[J]. Chem Biol, 2005, 12(10): 1145-1153. doi: 10.1016/j.chembiol.2005.08.017 | |
[17] | BALI S, O'HARE H M, WEISSMAN K J. Broad substrate specificity of ketoreductases derived from modular polyketide synthases[J]. Org Biomol Chem, 2006, 7(3): 478-484. | |
[18] | CHALLIS G L, RAVEL J, TOWNSEND C A. Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains[J]. Chem Biol, 2000, 7(3): 211-224. | |
[19] | 黎志凤, NGUIMBIE, 李越中. 埃博霉素(Epothilones)的PKS/NRPS杂合基因簇[J]. 生物工程学报, 2003, 19(5): 511-515. doi: 10.3321/j.issn:1000-3061.2003.05.001 | LI Zhifeng, NGUIMBI E, LI Yuezhogn. The PKS/NRPS hetero-gene cluster of epothilones[J]. Chin J Biotechnol, 2003, 19(5): 511-515. doi: 10.3321/j.issn:1000-3061.2003.05.001 |
[20] | BOETTGER D, HERTWECK C. Molecular diversity sculpted by fungal PKS-NRPS hybrids[J]. Chem Biol Chem, 2013, 14(1): 28-42. doi: 10.1002/cbic.201200624 | |
[21] | MIYANAGA A, KUDO F, EGUCHI T. Protein-protein interactions in polyketide synthase-nonribosomal peptide synthetase hybrid assembly lines[J]. Nat Prod Rep, 2018, 14(1): 28-42. | |
[22] | BOK J W, HOFFMEISTER D, MAGGIOHALL L A. Genomic mining for Aspergillus natural products[J]. Chem Biol, 2006, 13(1): 31-37. | |
[23] | LIU Wencai, YANG Fan, ZHANG Ran. Production of polyketides with anthelmintic activity by the fungus Talaromyces wortmannii using one strain-many compounds (OSMAC) method[J]. Phytochem Lett, 2016, 18(): 157-161. doi: 10.1016/j.phytol.2016.10.006 | |
[24] | YUAN Chao, GUO Yuhua, WANG Haiying. Allelopathic polyketides from an endolichenic fungus Myxotrichum sp. by using OSMAC strategy[J]. Sci Rep, 2016, 6(): 19350-. doi: 10.1038/srep19350 | |
[25] | 季香云, 杨长举. 白僵菌的致病性与应用[J]. 中国生物防治学报, 2003, 19(2): 82-85. | JI Xiangyun, YANG Changju. Infection and application of Beauveria spp.[J]. Chin J Biol Control, 2003, 19(2): 82-85. |
[26] | WEBER T, BLIN K, DUDDELA S. antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters[J]. Nucleic Acids Res, 2015, 43(W1): W237-W243. doi: 10.1093/nar/gkv437 | |
[27] | ELEY K L, HALO L M, SONG Z. Biosynthesis of the 2-pyridone tenellin in the insect pathogenic fungus Beauveria bassiana[J]. Chem Biol Chem, 2007, 8(3): 289-297. doi: 10.1002/cbic.200600398 | |
[28] | SONG Z, COX R J, LAZARUS C M. Fusarin C biosynthesis in Fusarium moniliforme and Fusarium venenatum[J]. Chem Biol Chem, 2004, 5(9): 1196-1203. doi: 10.1002/cbic.200400138 | |
[29] | 原晓龙, 陈剑, 张传光. 长松萝中非核糖体肽合成酶NRPS基因克隆与鉴定[J]. 西部林业科学, 2016, 45(1): 14-20. | YUAN Xiaolong, CHEN Jian, ZHANG Chuanguang. The genetic clone and identification of non-ribosomal peptides synthetases (NRPS) from Usnea longissima[J]. J West China For Sci, 2016, 45(1): 14-20. |
[30] | HEMPHILL C F, SUREECHATCHAIYAN P, KASSACK M U. OSMAC approach leads to new fusarielin metabolites from Fusarium tricinctum[J]. J Antibiot, 2017, 70(6): 726-732. doi: 10.1038/ja.2017.21 |