原生质体ARTP诱变选育阿维拉霉素高产菌株
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摘要
目的筛选获得阿维拉霉素A含量高的菌株,进一步提高绿色产色链霉菌(Streptomyces viridochromogene)发酵阿维拉霉素的水平。方法对绿色产色链霉菌AVL4(S.viridochromogene AVL4)菌株原生质体制备及再生条件进行了优化,并在此基础上,采用ARTP诱变系统对其原生质体进行了诱变研究。结果 S.viridochromogene AVL4原生质体制备和再生的最佳条件为:以活化斜面转接至种子摇瓶培养24h的菌丝体为出发菌丝体,以SMM溶液作为渗透压稳定剂,采用12mg/m L的溶壁酶,30℃酶解反应60min。以原生质体为诱变对象,ARTP处理40s后的菌株经菌落形态初筛和摇瓶发酵复筛,获得一株编号S251的菌株,其摇瓶发酵阿维拉霉素的生物效价较出发菌株提高19.3%,其中,阿维拉霉素A占组分含量81%,较原始出发菌株AVL4含量提高6.4%,而且斜面连续转接五代遗传相对稳定。另外,成功实现变株S251的50L罐发酵验证,其产素水平最高达到4500U/m L,较对照菌株AVL4提高27.4%。结论 ARTP处理AVL4菌株原生质体,能够有效提高绿色产色链霉菌产阿维拉霉素的能力,获得的变株S251具有非常好的生产阿维拉霉素的工业化应用潜力。
Objective To screen the strains with high content of avilamycin A, and further improve the fermentation level of avilamycin produced by Streptomyces viridochromogene. Methods The protoplast preparation and regeneration conditions of AVL4(S. viridochromogene AVL4) strain were optimized by the single factor design method. On the basis of this, the protoplast was treated by the ARTP breeding system. Results The optimal condition for protoplast preparation and regeneration of AVL4 strains were as follows: the mycelium was cultured for 24 h as the starting mycelium, the SMM solution was used as osmotic pressure stabilizer, and the content of 12 mg/m L lywallzyme, and the reaction was carried out at 30℃ for 60 min. A mutant strain of number S251 was finally screened out and showed relatively high genetic stability after five subcultures. The avilamycin concentration by the mutant strain was increased by 19.3% compared with the original strain of AVL4 under the condition of shake flask fermentation, among which the content of avilamycin A was 81%, which was 6.4% higher than that of the original strain. In addition, the 50 L tank fermentation of the mutant strain S251 was successfully achieved, and its yield was up to 4500 U/m L, which was 27.4% higher than that of the control strain AVL4. Conclusion Protoplast mutagenesis by ARTP is an effective method to breed the S. viridochromogene and to improve the avilamycin production significantly. The strain of S. viridochromogene S251 has high yield of avilamycin and shows a very good potential for industrial application.
Objective To screen the strains with high content of avilamycin A, and further improve the fermentation level of avilamycin produced by Streptomyces viridochromogene. Methods The protoplast preparation and regeneration conditions of AVL4(S. viridochromogene AVL4) strain were optimized by the single factor design method. On the basis of this, the protoplast was treated by the ARTP breeding system. Results The optimal condition for protoplast preparation and regeneration of AVL4 strains were as follows: the mycelium was cultured for 24 h as the starting mycelium, the SMM solution was used as osmotic pressure stabilizer, and the content of 12 mg/m L lywallzyme, and the reaction was carried out at 30℃ for 60 min. A mutant strain of number S251 was finally screened out and showed relatively high genetic stability after five subcultures. The avilamycin concentration by the mutant strain was increased by 19.3% compared with the original strain of AVL4 under the condition of shake flask fermentation, among which the content of avilamycin A was 81%, which was 6.4% higher than that of the original strain. In addition, the 50 L tank fermentation of the mutant strain S251 was successfully achieved, and its yield was up to 4500 U/m L, which was 27.4% higher than that of the control strain AVL4. Conclusion Protoplast mutagenesis by ARTP is an effective method to breed the S. viridochromogene and to improve the avilamycin production significantly. The strain of S. viridochromogene S251 has high yield of avilamycin and shows a very good potential for industrial application.
引文
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[12]李青,张亮.管碟法测定苏云金杆菌上清液中增效物质含量的研究[J].微生物学通报,2002,29(3):73-74.
[13]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):732-737.
[2]Fernandez S A,Bizec B L,Ellis R.Avilamycin[J].Fao Jecfa Monographs,2009,24(4):320-323.
[3]Rodger H W,代建国.阿维拉霉素对肉仔鸡生产性能的影响[J].饲料研究,2001,(2):33-34.
[4]Roth F X.Influence of avilamycin and tylosin on retention and excretion of nitrogen in growing pigs[J].J Anim Physiol A Anim Nutr,2010,69(1-5):175-185.
[5]Sadeghi A A,Izadi W,Shawrang P,et al.A comparison of the effects of dietary ginger powder and avilamycin on growth performance and intestinal Salmonella count of challenged broiler chickens[J].Iran J Appl Anim Sci,2013,3(4):769-775.
[6]Wellenreiter R H,Mowrey D H,Stobbs L A,et al.Effects of avilamycin on performance of broiler chickens[J].Vet Ther,2000,1(2):118-124.
[7]陈永辉,刘波,曾兆国,等.阿维拉霉素的研究进展[J].饲料工业,2007,28(14):9-11.
[8]潘淑媛.阿维拉霉素对肉鸡盲肠产气梭状芽胞杆菌的数量及生长性能的作用[J].饲料研究,2002,(6):27-28.
[9]向荣华.阿维拉霉素生产菌的诱变及筛选[D].无锡:江南大学,2015.
[10]吴亦楠,邢新会,张翀,等.ARTP生物育种技术与装备研发及其产业化发展[J].生物产业技术,2017,(1):37-45.
[11]王旭东,赵静国.高效液相色谱法检测产绿链霉菌发酵液中肥拉霉素A的含量[J].化学与生物工程,2009,26(11):88-90.
[12]李青,张亮.管碟法测定苏云金杆菌上清液中增效物质含量的研究[J].微生物学通报,2002,29(3):73-74.
[13]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):732-737.