随机诱变和基因组重排选育阿维拉霉素高产菌
|
摘要
以绿色产色链霉菌(Streptomyces viridoehrongenes 2-21)为原始出发菌株,依次进行常压等离子(atmospheric and room temperature, ARTP)和2轮~(137)Csγ诱变,从中筛选出高产阿维拉霉素的突变株,作为基因组重排的亲本菌株,以利福平为筛选剂,经过4轮原生质体递归融合,最终得到1株遗传稳定的高产阿维拉霉素重组菌株H_4-15。摇瓶发酵结果表明,阿维拉霉素产量可达到(2 155.48±7.81) mg/L,中试发酵结果表明,阿维拉霉素产量达到(2 356.44±6.34)mg/L,是原始出发菌株的3.47倍。高产菌株H_4-15发酵产物的LC-MS结果显示,其主要组分是阿维拉霉素A和阿维拉霉素B。综合表明,以利福平为筛选剂,结合传统随机诱变和基因组重排技术选育阿维拉霉素高产菌株,能大幅提升其生产能力,具有工业化生产的潜在价值。
Streptomyces viridoehrongenes 2-21 was used as an original strain and treated with atmospheric room temperature plasma(ARTP) and two rounds of ~(137)Csγ mutagenesis. Mutant strains with higher avilamycin productivity were screened and used as starting strains for genome shuffling. Using rifampicin as a selector, after four rounds of protoplast fusion, a high-yield-avilamycin-producing recombinant strain H_4-15 was obtained. The avilamycin yield reached(2 155.48±7.81) mg/L in the flask, and(2 356.44±6.34) mg/L after a pilot-scale fermentation, which was 3.47 times higher than that of the original strain. Besides, its main components were found to be avilamycin A and avilamycin B. In conclusion, combining traditional random mutagenesis and genome shuffling and using rifampicin as a selector can significantly improve the yield of avilamycin, and its future potential value was proved.
Streptomyces viridoehrongenes 2-21 was used as an original strain and treated with atmospheric room temperature plasma(ARTP) and two rounds of ~(137)Csγ mutagenesis. Mutant strains with higher avilamycin productivity were screened and used as starting strains for genome shuffling. Using rifampicin as a selector, after four rounds of protoplast fusion, a high-yield-avilamycin-producing recombinant strain H_4-15 was obtained. The avilamycin yield reached(2 155.48±7.81) mg/L in the flask, and(2 356.44±6.34) mg/L after a pilot-scale fermentation, which was 3.47 times higher than that of the original strain. Besides, its main components were found to be avilamycin A and avilamycin B. In conclusion, combining traditional random mutagenesis and genome shuffling and using rifampicin as a selector can significantly improve the yield of avilamycin, and its future potential value was proved.
引文
[1] CRISOLMARTíNEZ E,STANLEY D,GEIER M S,et al.Understanding the mechanisms of zinc bacitracin and avilamycin on animal production:linking gut microbiota and growth performance in chickens[J].Applied Microbiology & Biotechnology,2017,101(11):4 547-4 559.
[2] ARENZ S.Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(27):7 527-7 532.
[3] HO C,WONG Y T.Determination of avilamycin as dichloroisoeverninic acid in poultry and porcine muscles by isotope dilution liquid chromatography-tandem mass spectrometry[J].Analytical & Bioanalytical Chemistry,2013,405(26):8 633-8 643.
[4] FAULKNER D,CANTLEY M,WALKER M,et al.Evidence of non-extractable florfenicol residues:development and validation of a confirmatory method for total florfenicol content in kidney by UPLC-MS/MS[J].Food Additives & Contaminants,2016,33(6):983-994.
[5] 王学真,陈敏.温度和光照对阿维拉霉素稳定性的影响[J].中国畜牧兽医,2015,42(5):1 198-1 202.
[6] 邹君彪,姜平.阿维拉霉素的研究进展[J].国外畜牧学(猪与禽),2017,37(10):94-97.
[7] 章梅,宋元达,刘青,等.氧载体对绿色产色链霉菌发酵生产阿维拉霉素的影响[J].食品与机械,2018(2):42-45.
[8] 向荣华,詹晓北,朱莉,等.常压室温等离子体诱变绿色产色链霉菌及阿维拉霉素高产菌选育[J].中国抗生素杂志,2015,40(10):723-737.
[9] PARADIS M A,MCMILLAN E,BAGG R,et al.Efficacy of avilamycin for the prevention of necrotic enteritis caused by a pathogenic strain of Clostridium perfringens in broiler chickens[J].Avian Pathology,2016,45(3):365-369.
[10] 王庆龄.阿维拉霉素高产菌株选育[D].杭州:浙江工商大学,2015.
[11] WANG X F,LI Q Z,BAO T W,et al.In vitro rapid evolution of fungal immunomodulatory proteins by DNA family shuffling[J].Applied Microbiology & Biotechnology,2013,97(6):2 455-2 465.
[12] DAHIKAR S.Genome shuffling of Lactobacillus rhamnosus for improved production of Lactic acid[C].Congress of European Microbiologists,2015.
[13] ZHAO Y,JIANG C,YU H,et al.Genome shuffling of Aspergillus glaucus HGZ-2 for enhanced cellulase production[J].Applied Biochemistry & Biotechnology,2014,174(4):1 246-1 259.
[14] TIM S,MARTINA P N,STEFANIE V D B,et al.Large-scale robot-assisted genome shuffling yields industrial Saccharomyces cerevisiae,yeasts with increased ethanol tolerance[J].Biotechnology for Biofuels,2015,8(1):32-50.
[15] 姜丽艳,闫国栋,张宏宇,等.乳链菌肽高产菌株的推理选育[J].食品与机械,2009,25(4):38-40.
[16] 徐嫄,韩宁宁,于丽娜,等.微生物浊度法与管碟法测定吉他霉素效价的比较研究[J].中国兽药杂志,2016,50(11):31-33.
[17] 赵硕珍,张云峰,姬胜利,等.阿维拉霉素高产菌株的选育[J].中国生化药物杂志,2008,29(4):256-259.
[18] 齐香君,苟金霞,韩戌珺,等.3,5-二硝基水杨酸比色法测定溶液中还原糖的研究[J].纤维素科学与技术,2004,12(3):17-19.
[19] MERTZ J L,PELOSO J S,BARKER B J,et al.Isolation and structural identification of nine avilamycins[J].Journal of Antibiotics,1986,39(7):877-887.
[20] SAITO-SHIDA S,HAYASHI T,NEMOTO S,et al.Determination of total avilamycin residues as dichloroisoeverninic acid in porcine muscle,fat,and liver by LC-MS/MS[J].Food Chemistry,2018,249:84-90.
[21] LU Y,WANG L,MA K,et al.Characteristics of hydrogen production of an Enterobacter aerogenes,mutant generated by a new atmospheric and room temperature plasma (ARTP)[J].Biochemical Engineering Journal,2011,55(1):17-22.
[22] 胡向东,潘玲燕,叶茂,等.高产虾青素红法夫酵母菌株的选育和发酵参数优化[J].食品工业科技,2016,37(5):142-147.
[23] 刘芳,李晓荣,邹祥.阿维拉霉素高产菌株选育与发酵特性[J].西南大学学报(自然科学版),2012,34(11):143-147.
[24] 毛灵琪,李存治,陶兴无,等.利用基因组改组技术选育阿维拉霉素高产菌[J].生物技术通报,2015,31(5):54-60.
[2] ARENZ S.Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(27):7 527-7 532.
[3] HO C,WONG Y T.Determination of avilamycin as dichloroisoeverninic acid in poultry and porcine muscles by isotope dilution liquid chromatography-tandem mass spectrometry[J].Analytical & Bioanalytical Chemistry,2013,405(26):8 633-8 643.
[4] FAULKNER D,CANTLEY M,WALKER M,et al.Evidence of non-extractable florfenicol residues:development and validation of a confirmatory method for total florfenicol content in kidney by UPLC-MS/MS[J].Food Additives & Contaminants,2016,33(6):983-994.
[5] 王学真,陈敏.温度和光照对阿维拉霉素稳定性的影响[J].中国畜牧兽医,2015,42(5):1 198-1 202.
[6] 邹君彪,姜平.阿维拉霉素的研究进展[J].国外畜牧学(猪与禽),2017,37(10):94-97.
[7] 章梅,宋元达,刘青,等.氧载体对绿色产色链霉菌发酵生产阿维拉霉素的影响[J].食品与机械,2018(2):42-45.
[8] 向荣华,詹晓北,朱莉,等.常压室温等离子体诱变绿色产色链霉菌及阿维拉霉素高产菌选育[J].中国抗生素杂志,2015,40(10):723-737.
[9] PARADIS M A,MCMILLAN E,BAGG R,et al.Efficacy of avilamycin for the prevention of necrotic enteritis caused by a pathogenic strain of Clostridium perfringens in broiler chickens[J].Avian Pathology,2016,45(3):365-369.
[10] 王庆龄.阿维拉霉素高产菌株选育[D].杭州:浙江工商大学,2015.
[11] WANG X F,LI Q Z,BAO T W,et al.In vitro rapid evolution of fungal immunomodulatory proteins by DNA family shuffling[J].Applied Microbiology & Biotechnology,2013,97(6):2 455-2 465.
[12] DAHIKAR S.Genome shuffling of Lactobacillus rhamnosus for improved production of Lactic acid[C].Congress of European Microbiologists,2015.
[13] ZHAO Y,JIANG C,YU H,et al.Genome shuffling of Aspergillus glaucus HGZ-2 for enhanced cellulase production[J].Applied Biochemistry & Biotechnology,2014,174(4):1 246-1 259.
[14] TIM S,MARTINA P N,STEFANIE V D B,et al.Large-scale robot-assisted genome shuffling yields industrial Saccharomyces cerevisiae,yeasts with increased ethanol tolerance[J].Biotechnology for Biofuels,2015,8(1):32-50.
[15] 姜丽艳,闫国栋,张宏宇,等.乳链菌肽高产菌株的推理选育[J].食品与机械,2009,25(4):38-40.
[16] 徐嫄,韩宁宁,于丽娜,等.微生物浊度法与管碟法测定吉他霉素效价的比较研究[J].中国兽药杂志,2016,50(11):31-33.
[17] 赵硕珍,张云峰,姬胜利,等.阿维拉霉素高产菌株的选育[J].中国生化药物杂志,2008,29(4):256-259.
[18] 齐香君,苟金霞,韩戌珺,等.3,5-二硝基水杨酸比色法测定溶液中还原糖的研究[J].纤维素科学与技术,2004,12(3):17-19.
[19] MERTZ J L,PELOSO J S,BARKER B J,et al.Isolation and structural identification of nine avilamycins[J].Journal of Antibiotics,1986,39(7):877-887.
[20] SAITO-SHIDA S,HAYASHI T,NEMOTO S,et al.Determination of total avilamycin residues as dichloroisoeverninic acid in porcine muscle,fat,and liver by LC-MS/MS[J].Food Chemistry,2018,249:84-90.
[21] LU Y,WANG L,MA K,et al.Characteristics of hydrogen production of an Enterobacter aerogenes,mutant generated by a new atmospheric and room temperature plasma (ARTP)[J].Biochemical Engineering Journal,2011,55(1):17-22.
[22] 胡向东,潘玲燕,叶茂,等.高产虾青素红法夫酵母菌株的选育和发酵参数优化[J].食品工业科技,2016,37(5):142-147.
[23] 刘芳,李晓荣,邹祥.阿维拉霉素高产菌株选育与发酵特性[J].西南大学学报(自然科学版),2012,34(11):143-147.
[24] 毛灵琪,李存治,陶兴无,等.利用基因组改组技术选育阿维拉霉素高产菌[J].生物技术通报,2015,31(5):54-60.