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137Csγ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H4-15。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H4-15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 137Csγ 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 H4-15 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.
[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.
