米尔贝霉素A3和A4高产菌的理性选育
|
摘要
目的应用理性选育理论,结合各种诱变手段,获得米尔贝霉素A4(1),A3(2)的高产菌种。方法以吸水链霉菌CGMCC7677为出发菌,通过紫外、甲基磺酸乙酯与常压室温等离子体等单独诱变或组合诱变,结合含甘氨酸、利福平、乙酸钠和3-溴丙酮酸的抗性平板进行筛选。结果通过多轮的筛选,得到米尔贝霉素突变株75-22和95-23。在不改变培养基组成和培养条件的情况下,75-22在50L发酵罐中培养360h,米尔贝霉素A4(1)的生产能力提高了5倍,米尔贝霉素A4(1)的含量也从70%提高至80%以上。95-23在50L发酵罐中培养360h,米尔贝霉素A3(2)的生产能力提高了10倍,米尔贝霉素A3(2)的含量提高至70%以上。结论主产单一组分菌种的获得,可进一步扩大米尔贝霉素的应用研究范围,也为米尔贝霉素的产业化提供了优良的菌种。
Objective To obtain high productivity of milbemycin A4(1) and milbemycin A3(2) using rational breeding and various kinds of mutation methods. Methods The strain Streptomyces sp. CGMCC 7677 was used as the original strain. After being mutated by a means of single or combination of UV, ethylmethane sulfonate, atmospheric and room temperature plasma(ARTP), strains werescreened in glycine-resistance, rifampicin-resistance, sodium acetateresistance, and 3-bromopyruvate tolerance plates. Results The mutants with single component of milbemycin 75-22 and 95-23 were obtained. The productivity of milbemycin A4(1) for 75-22 is 5 times improved compared with the initial strain CGMCC 7677 under the same fermentation conditions which run in 50 L fermenter for 360 h. The ratio of milbemycin A4(1) in total components increased from 70% to more than 80%. The productivity of milbemycin A3(2) for 95-23 is 10 times improved under the same conditions, and the ratio of milbemycin A3(2) in total components increased from 20% to more than 70%. Conclusion More research work and application could be performed base on the new high-yield single component strains that also supply good producing stains for industry production of milbemycin.
Objective To obtain high productivity of milbemycin A4(1) and milbemycin A3(2) using rational breeding and various kinds of mutation methods. Methods The strain Streptomyces sp. CGMCC 7677 was used as the original strain. After being mutated by a means of single or combination of UV, ethylmethane sulfonate, atmospheric and room temperature plasma(ARTP), strains werescreened in glycine-resistance, rifampicin-resistance, sodium acetateresistance, and 3-bromopyruvate tolerance plates. Results The mutants with single component of milbemycin 75-22 and 95-23 were obtained. The productivity of milbemycin A4(1) for 75-22 is 5 times improved compared with the initial strain CGMCC 7677 under the same fermentation conditions which run in 50 L fermenter for 360 h. The ratio of milbemycin A4(1) in total components increased from 70% to more than 80%. The productivity of milbemycin A3(2) for 95-23 is 10 times improved under the same conditions, and the ratio of milbemycin A3(2) in total components increased from 20% to more than 70%. Conclusion More research work and application could be performed base on the new high-yield single component strains that also supply good producing stains for industry production of milbemycin.
引文
[1]Takiguchi Y,Ono M,Muramatsu S,et al.Milbemycins,a new family of macrolide antibiotics-fermentation,isolation and physicochemical properties[J].J Antibiot,1980,33(10):1120-1127.
[2]陈小龙,郑裕国,沈寅初.生物农药米尔贝霉素的研究进展[J].农药,2003,42(4):5-9.
[3]王则,王相晶,向文胜.生物农药米尔贝霉素杀虫活性特性研究综述[J].世界农药,2009,31(1):13-15.
[4]林秀萍,刘永宏,李季伦.米尔贝霉素的产生菌、理化性质、生物学活性及应用研究进展[J].中国抗生素杂志,2013,38(4):314-322.
[5]Sadakane S,Tanaka K,Muraoka E,et al.Metabolic fates of milbemycin A3 and A4 in rats[J].J Pesticide Science,1992,17(3):147-154.
[6]Sadakane S,Tanaka K,Sato K,et al.Comparative metabolism of milbemycin A3 and A4 in rat-liver microsomes[J].J Pest Sci,1992,17(3):199-203.
[7]Wang X J,Yan Y J,Zhang B,et al.Genome sequence of the milbemycin-producing bacterium Streptomyces bingchenggensis[J].J Bacteriol,2010,192(17):4526-4527.
[8]Takiguchi Y,Ono M,Muramatsu S,et al.Milbemycins,a new family of macrolide antibiotics-fermentation,isolation and physicochemical properties of milbemycin-D,m i l b e m y c i n-E,m i l b e m y c i n-F,m i l b e m y c i n-G a n d milbemycin-H[J].J Antibiot,1983,36(5):502-508.
[9]Wang X J,Wang X C,Xiang W S.Improvement of milbemycin-producing Streptomyces bingchenggensis by rational screening of ultraviolet-and chemically induced mutants[J].World J Microl Biotech,2009,25(6):1051-1056.
[10]Zhang B X,Wang X J,Xiang W S.Optimization of fermentation medium for enhanced production of milbemycin by a mutant of Streptomyces bingchenggensis BC-X-1 using response surface methodology[J].African J Biotech,2011,10(37):7225-7234.
[11]Atsushi A R F,Toshio N.Antibiotic substances:US,3950360[P].1976-4-13.
[12]夏焕章,刘晓辉,任丹,等.单组分妥布霉素产生菌的快速筛选[J].微生物学杂志,2003,23(1):4-6.
[13]Gu C K,Wang G Y,Mai S A,et al.ARTP mutation and genome shuffling of ABE fermentation symbiotic system for improvement of butanol production[J].Appl Microl Biotech,2017,101(5):2189-2199.
[14]Ren F,Chen L,Tong Q Y.Highly improved acarbose production of actinomyces through the combination of ARTP and penicillin susceptible mutant screening[J].World J Microl Biotechem,2017,33(16):1-7
[15]郑玲辉,洪云,陈晓静,等.奥利万星中间体A82846B的菌种选育与培养基优化[J].中国医药工业杂志,2015,46(5):462-466.
[16]曾常茜,吴思宇.3-溴丙酮酸抗肿瘤作用及机制研究[J].中国新药杂志,2009,18(9):793-796
[17]Feng X L,Wang P,Liu Q H,et al.Glycolytic inhibitors2-deoxyglucose and 3-bromopyruvate synergize with photodynamic therapy respectively to inhibit cell migration[J].J Bioenerg Biomembra,2015,47(3):189-197.
[2]陈小龙,郑裕国,沈寅初.生物农药米尔贝霉素的研究进展[J].农药,2003,42(4):5-9.
[3]王则,王相晶,向文胜.生物农药米尔贝霉素杀虫活性特性研究综述[J].世界农药,2009,31(1):13-15.
[4]林秀萍,刘永宏,李季伦.米尔贝霉素的产生菌、理化性质、生物学活性及应用研究进展[J].中国抗生素杂志,2013,38(4):314-322.
[5]Sadakane S,Tanaka K,Muraoka E,et al.Metabolic fates of milbemycin A3 and A4 in rats[J].J Pesticide Science,1992,17(3):147-154.
[6]Sadakane S,Tanaka K,Sato K,et al.Comparative metabolism of milbemycin A3 and A4 in rat-liver microsomes[J].J Pest Sci,1992,17(3):199-203.
[7]Wang X J,Yan Y J,Zhang B,et al.Genome sequence of the milbemycin-producing bacterium Streptomyces bingchenggensis[J].J Bacteriol,2010,192(17):4526-4527.
[8]Takiguchi Y,Ono M,Muramatsu S,et al.Milbemycins,a new family of macrolide antibiotics-fermentation,isolation and physicochemical properties of milbemycin-D,m i l b e m y c i n-E,m i l b e m y c i n-F,m i l b e m y c i n-G a n d milbemycin-H[J].J Antibiot,1983,36(5):502-508.
[9]Wang X J,Wang X C,Xiang W S.Improvement of milbemycin-producing Streptomyces bingchenggensis by rational screening of ultraviolet-and chemically induced mutants[J].World J Microl Biotech,2009,25(6):1051-1056.
[10]Zhang B X,Wang X J,Xiang W S.Optimization of fermentation medium for enhanced production of milbemycin by a mutant of Streptomyces bingchenggensis BC-X-1 using response surface methodology[J].African J Biotech,2011,10(37):7225-7234.
[11]Atsushi A R F,Toshio N.Antibiotic substances:US,3950360[P].1976-4-13.
[12]夏焕章,刘晓辉,任丹,等.单组分妥布霉素产生菌的快速筛选[J].微生物学杂志,2003,23(1):4-6.
[13]Gu C K,Wang G Y,Mai S A,et al.ARTP mutation and genome shuffling of ABE fermentation symbiotic system for improvement of butanol production[J].Appl Microl Biotech,2017,101(5):2189-2199.
[14]Ren F,Chen L,Tong Q Y.Highly improved acarbose production of actinomyces through the combination of ARTP and penicillin susceptible mutant screening[J].World J Microl Biotechem,2017,33(16):1-7
[15]郑玲辉,洪云,陈晓静,等.奥利万星中间体A82846B的菌种选育与培养基优化[J].中国医药工业杂志,2015,46(5):462-466.
[16]曾常茜,吴思宇.3-溴丙酮酸抗肿瘤作用及机制研究[J].中国新药杂志,2009,18(9):793-796
[17]Feng X L,Wang P,Liu Q H,et al.Glycolytic inhibitors2-deoxyglucose and 3-bromopyruvate synergize with photodynamic therapy respectively to inhibit cell migration[J].J Bioenerg Biomembra,2015,47(3):189-197.