杀粉蝶菌素A1生物合成基因簇中甲基转移酶PieB2的功能
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摘要
【目的】研究杀粉蝶菌素A1产生菌中甲基转移酶基因pieB2的功能。【方法】利用接合转移和同源重组双交换的方法,构建pieB2基因缺失突变株,以及利用接合转移的方法,构建回补菌株。通过高保真PCR克隆pieB2基因到表达载体pET28a上,构建质粒pJTU5997,转化入大肠杆菌E.coliBL21(DE3)/pLysE中诱导表达。利用高效液相色谱检测PieB2的体外酶活。【结果】获得了pieB2基因缺失的双交换突变株。发酵结果显示,该突变株不再产生杀粉蝶菌素A1,而是积累了一种脱甲基产物。N-末端融合组氨酸标签的PieB2在大肠杆菌中获得可溶性表达,通过体外催化证明了PieB2甲基转移酶的功能。【结论】体内遗传实验和体外生化实验证明了PieB2作为甲基转移酶在杀粉蝶菌素A1合成中的作用。
[Objective] In order to obtain piericidin intermediates of low toxicity by metabolic engineering,we studied the function of methyltransferase gene pieB2 in the biosynthetic cluster of piericidin A1.[Methods] The methyltransferase pieB2 gene disrupted Streptomyces piomogeues var.Hangzhouwanensis was constructedby double crossover recombination.The methyltransferase gene pieB2 was also PCR amplified and cloned into the plasmid p ET28 a for overexpressing N-(His)6-tag pieB2 in E.coli BL21(DE3)/p Lys E.The recombinant PieB2 was purified by affinity chromatography via AKTA FPLC system.The pieB2 catalyzed reactions were performed using SAM and demethyl-piericidinas substrates.[Results] The disruption mutant LQ-9 produced demethyl-piericidininstead of piereicidin A1,which was restored by in trans complementation of the pieB2 gene.The N-(His)6-tag pieB2 was expressed in E.coli in soluble form and was successfully purified via Ni~(2+) mediated affinity chromatography.In vitro biochemical experiments showed that PieB2 could convert demethyl-piericidininto piereicidin A1 in the presence of SAM.The demethyl-piericidin intermediat showed an attractive biological activities as well as piericidin A1.[Conclusion] We confirmed that pieB2 is function as a SAM dependent methyltransferase in the biosynthetic gene cluster of piericidin A1.
[Objective] In order to obtain piericidin intermediates of low toxicity by metabolic engineering,we studied the function of methyltransferase gene pieB2 in the biosynthetic cluster of piericidin A1.[Methods] The methyltransferase pieB2 gene disrupted Streptomyces piomogeues var.Hangzhouwanensis was constructedby double crossover recombination.The methyltransferase gene pieB2 was also PCR amplified and cloned into the plasmid p ET28 a for overexpressing N-(His)6-tag pieB2 in E.coli BL21(DE3)/p Lys E.The recombinant PieB2 was purified by affinity chromatography via AKTA FPLC system.The pieB2 catalyzed reactions were performed using SAM and demethyl-piericidinas substrates.[Results] The disruption mutant LQ-9 produced demethyl-piericidininstead of piereicidin A1,which was restored by in trans complementation of the pieB2 gene.The N-(His)6-tag pieB2 was expressed in E.coli in soluble form and was successfully purified via Ni~(2+) mediated affinity chromatography.In vitro biochemical experiments showed that PieB2 could convert demethyl-piericidininto piereicidin A1 in the presence of SAM.The demethyl-piericidin intermediat showed an attractive biological activities as well as piericidin A1.[Conclusion] We confirmed that pieB2 is function as a SAM dependent methyltransferase in the biosynthetic gene cluster of piericidin A1.
引文
[1]Hall C,Wu MJ,Crane FL,Takahashi H,Tamura S,Folkers K.Piericidin A:a new inhibitor of mitochondrial electron transport.Biochemical and Biophysical Research Communications,1966,25(4):373-377.
[2]Darrouzet E,Issartel JP,Lunardi J,Dupuis A.The 49-k Da subunit of NADH-ubiquinone oxidoreductase(Complex I)is involved in the binding of piericidin and rotenone,two quinone-related inhibitors.FEBS Letters,1998,431(1):34-38.
[3]Chang YJ,Chen WY,Huang CY,Liu HH,Wei PL.Glucoseregulated protein 78(GRP78)regulates colon cancer metastasis through EMT biomarkers and the NRF-2/HO-1 pathway.Tumour Biology,2015,36(3):1859-1869.
[4]Fu Y,Lee AS.Glucose regulated proteins in cancer progression,drug resistance and immunotherapy.Cancer Biology&Therapy,2006,5(7):741-744.
[5]Hwang JH,Kim JY,Cha MR,Ryoo IJ,Choo SJ,Cho SM,Tsukumo Y,Tomida A,Shin-Ya K,Hwang YI,Yoo ID,Park HR.Etoposide-resistant HT-29 human colon carcinoma cells during glucose deprivation are sensitive to piericidin A,a GRP78 down-regulator.Journal of Cellular Physiology,2008,215(1):243-250.
[6]Little E,Ramakrishnan M,Roy B,Gazit G,Lee AS.The glucose-regulated proteins(GRP78 and GRP94):functions,gene regulation,and applications.Critical Reviews TM in Eukaryotic Gene Expression,1994,4(1):1-18.
[7]Chen CY,Fang QX,Lin KJ,Zhang MB.Studies on aphicidin:I.Identification of the producing streptomycete.Acta Microbiologica Sinica,1980,20(2):113-115.(in Chinese).陈传盈,方祺霞,林开江,张孟白.杀蚜素的研究I.杀蚜素产生菌的分离鉴定.微生物学报,1980,20(2):113-115.
[8]Chen CY,Huang WC,Lin KJ,Qian YY,Wang LY,Zhou WQ.Studies on aphicidin:Ⅱ.Isolation and characterization of aphcidin A.Acta Microbiologica Sinica,1981,21(2):192-196.(in Chinese).陈传盈,黄文彩,林开江,钱玉英,王龙英,周伟强.杀蚜素的研究Ⅱ.杀蚜素A的分离和鉴别.微生物学报,1981,21(2):192-196.
[9]Liu Q,Yao F,Chooi YH,Kang QJ,Xu W,Li YR,Shao YC,Shi YF,Deng ZX,Tang Y,You DL.Elucidation of piericidin A1 biosynthetic locus revealed a thioesterase-dependent mechanism ofα-pyridone ring formation.Chemistry&Biology,2012,19(2):243-253.
[10]Chen YL,Zhang WJ,Zhu YG,Zhang QB,Tian XP,Zhang S,Zhang CS.Elucidating hydroxylation and methylation steps tailoring piericidin A1 biosynthesis.Organic Letters,2014,16(3):736-739.
[11]He YL,Wang ZJ,Bai LQ,Liang JD,Zhou XF,Deng ZX.Two p HZ1358-derivative vectors for efficient gene knockout in Streptomyces.Journal of Microbiology and Biotechnology,2010,20(4):678-682.
[12]Xu H,Zhang YR,Yang J,Mahmud T,Bai LQ,Deng ZX.Alternative epimerization in C7N-aminocyclitol biosynthesis is catalyzed by Val D,a large protein of the vicinal oxygen chelate superfamily.Chemistry&Biology,2009,16(5):567-576.
[13]Mc Leod MP,Warren RL,Hsiao WWL,Araki N,Myhre M,Fernandes C,Miyazawa D,Wong W,Lillquist AL,Wang D,Dosanjh M,Hara H,Petrescu A,Morin RD,Yang G,Stott JM,Schein JE,Shin H,Smailus D,Siddiqui AS,Marra MA,Jones SJM,Holt R,Brinkman FSL,Miyauchi K,Fukuda M,Davies JE,Mohn WW,Eltis LD.The complete genome of Rhodococcus sp.RHA1 provides insights into a catabolic powerhouse.Proceedings of the National Academy of Sciences of the United States of America,2006,103(42):15582-15587.
[14]Kimura K,Nakayama S,Nakajima N,Yoshihama M,Miyata N,Kawanishi G.A new piericidin rhamnoside,3'-rhamnopiericidin A1.The Journal of Antibiotics,1990,43(10):1341-1343.
[15]Cano-Prieto C,García-Salcedo R,Sánchez-Hidalgo M,Bra?a AF,Fiedler HP,Méndez C,Salas JA,Olano C.Genome mining of Streptomyces sp.Tü6176:characterization of the nataxazole biosynthesis pathway.Chem Bio Chem,2015,16(10):1461-1473.
[2]Darrouzet E,Issartel JP,Lunardi J,Dupuis A.The 49-k Da subunit of NADH-ubiquinone oxidoreductase(Complex I)is involved in the binding of piericidin and rotenone,two quinone-related inhibitors.FEBS Letters,1998,431(1):34-38.
[3]Chang YJ,Chen WY,Huang CY,Liu HH,Wei PL.Glucoseregulated protein 78(GRP78)regulates colon cancer metastasis through EMT biomarkers and the NRF-2/HO-1 pathway.Tumour Biology,2015,36(3):1859-1869.
[4]Fu Y,Lee AS.Glucose regulated proteins in cancer progression,drug resistance and immunotherapy.Cancer Biology&Therapy,2006,5(7):741-744.
[5]Hwang JH,Kim JY,Cha MR,Ryoo IJ,Choo SJ,Cho SM,Tsukumo Y,Tomida A,Shin-Ya K,Hwang YI,Yoo ID,Park HR.Etoposide-resistant HT-29 human colon carcinoma cells during glucose deprivation are sensitive to piericidin A,a GRP78 down-regulator.Journal of Cellular Physiology,2008,215(1):243-250.
[6]Little E,Ramakrishnan M,Roy B,Gazit G,Lee AS.The glucose-regulated proteins(GRP78 and GRP94):functions,gene regulation,and applications.Critical Reviews TM in Eukaryotic Gene Expression,1994,4(1):1-18.
[7]Chen CY,Fang QX,Lin KJ,Zhang MB.Studies on aphicidin:I.Identification of the producing streptomycete.Acta Microbiologica Sinica,1980,20(2):113-115.(in Chinese).陈传盈,方祺霞,林开江,张孟白.杀蚜素的研究I.杀蚜素产生菌的分离鉴定.微生物学报,1980,20(2):113-115.
[8]Chen CY,Huang WC,Lin KJ,Qian YY,Wang LY,Zhou WQ.Studies on aphicidin:Ⅱ.Isolation and characterization of aphcidin A.Acta Microbiologica Sinica,1981,21(2):192-196.(in Chinese).陈传盈,黄文彩,林开江,钱玉英,王龙英,周伟强.杀蚜素的研究Ⅱ.杀蚜素A的分离和鉴别.微生物学报,1981,21(2):192-196.
[9]Liu Q,Yao F,Chooi YH,Kang QJ,Xu W,Li YR,Shao YC,Shi YF,Deng ZX,Tang Y,You DL.Elucidation of piericidin A1 biosynthetic locus revealed a thioesterase-dependent mechanism ofα-pyridone ring formation.Chemistry&Biology,2012,19(2):243-253.
[10]Chen YL,Zhang WJ,Zhu YG,Zhang QB,Tian XP,Zhang S,Zhang CS.Elucidating hydroxylation and methylation steps tailoring piericidin A1 biosynthesis.Organic Letters,2014,16(3):736-739.
[11]He YL,Wang ZJ,Bai LQ,Liang JD,Zhou XF,Deng ZX.Two p HZ1358-derivative vectors for efficient gene knockout in Streptomyces.Journal of Microbiology and Biotechnology,2010,20(4):678-682.
[12]Xu H,Zhang YR,Yang J,Mahmud T,Bai LQ,Deng ZX.Alternative epimerization in C7N-aminocyclitol biosynthesis is catalyzed by Val D,a large protein of the vicinal oxygen chelate superfamily.Chemistry&Biology,2009,16(5):567-576.
[13]Mc Leod MP,Warren RL,Hsiao WWL,Araki N,Myhre M,Fernandes C,Miyazawa D,Wong W,Lillquist AL,Wang D,Dosanjh M,Hara H,Petrescu A,Morin RD,Yang G,Stott JM,Schein JE,Shin H,Smailus D,Siddiqui AS,Marra MA,Jones SJM,Holt R,Brinkman FSL,Miyauchi K,Fukuda M,Davies JE,Mohn WW,Eltis LD.The complete genome of Rhodococcus sp.RHA1 provides insights into a catabolic powerhouse.Proceedings of the National Academy of Sciences of the United States of America,2006,103(42):15582-15587.
[14]Kimura K,Nakayama S,Nakajima N,Yoshihama M,Miyata N,Kawanishi G.A new piericidin rhamnoside,3'-rhamnopiericidin A1.The Journal of Antibiotics,1990,43(10):1341-1343.
[15]Cano-Prieto C,García-Salcedo R,Sánchez-Hidalgo M,Bra?a AF,Fiedler HP,Méndez C,Salas JA,Olano C.Genome mining of Streptomyces sp.Tü6176:characterization of the nataxazole biosynthesis pathway.Chem Bio Chem,2015,16(10):1461-1473.