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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (8): 59-68    DOI: 10.13523/j.cb.20180808
技术与方法     
具有双重抗生素抗性的ε-聚赖氨酸高产菌株选育及生理特性 *
赵俊杰,张龙,王靓,陈旭升,毛忠贵()
江南大学工业生物技术教育部重点实验室 无锡 214122
Breeding and Physiological Characteristics of ε-Polylysine High-Producing Strain with Double Antibiotic Resistance
Jun-jie ZHAO,Long ZHANG,Liang WANG,Xu-sheng CHEN,Zhong-gui MAO()
Key Lab of Industrial Biotechnology of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China
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摘要：

以ε-聚赖氨酸产量为1.60g/L的Streptomyces albulus M-Z18为出发菌株,利用核糖体工程技术选育具有双重抗生素抗性的ε-聚赖氨酸高产菌株,并对高产菌株和出发菌株的生理生化性能进行比较。通过链霉素诱变成功选育出了1株遗传稳定的ε-聚赖氨酸产生菌S.albulus S-7,ε-聚赖氨酸产量为2.03g/L;对S.albulus S-7叠加巴龙霉素,获得1株遗传稳定的具有双重抗性的ε-聚赖氨酸产生菌S.albulus SP-14,ε-聚赖氨酸产量为2.37g/L,比出发菌株S.albulus M-Z18的ε-聚赖氨酸产量增加了48.10%。使用链霉素和巴龙霉素选育具有双重抗生素抗性的ε-聚赖氨酸高产菌株是一种有效的手段。
关键词： 发酵工程;  生理生化性能;  双重抗生素抗性;  ε-聚赖氨酸    
Abstract:

Streptomyces albulus M-Z18 with 1.60g/L yield of ε-Poly-L-lysine was used as initial strain to screen high-yield mutant,and the physiological and biochemical properties of high-yield mutant were compared with M-Z18.The ribosomal engineering technology was used to breed the high-yield mutant with double antibiotic resistance. A genetically stable strain named S-7, was successfully screened by streptomycin mutagenesis. The ε-Poly-L-lysine yield of S-7 was 2.03g/L. S-7 was used as initial strain to screen mutant with paromomycin,and obtained SP-14, with 2.37g/L yield of ε-Poly-L-lysine. The use of streptomycin and paromomycin to breed high-yield mutant with high antibiotic resistance is an effective method.
Key words: Fermentation engineering    Physiological and biochemical property    Double drug resistance    ε-Poly-L-lysine
收稿日期: 2018-04-02 出版日期: 2018-09-11
ZTFLH:  Q813  
基金资助: 江苏省产学研合作前瞻性联合研究(BY2016022-25)
通讯作者: 毛忠贵     E-mail: maozg@jiangnan.edu.cn
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引用本文:

赵俊杰,张龙,王靓,陈旭升,毛忠贵. 具有双重抗生素抗性的ε-聚赖氨酸高产菌株选育及生理特性 *[J]. 中国生物工程杂志, 2018, 38(8): 59-68.

Jun-jie ZHAO,Long ZHANG,Liang WANG,Xu-sheng CHEN,Zhong-gui MAO. Breeding and Physiological Characteristics of ε-Polylysine High-Producing Strain with Double Antibiotic Resistance. China Biotechnology, 2018, 38(8): 59-68.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180808    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I8/59

图1  S.albulus M-Z18在不同浓度链霉素和巴龙霉素的固体平板上的生长情况
筛选过程 突变数(M) 正突变数(P) 总菌落数(T) 突变率RM(%) 正突变率RP(%)
链霉素初筛 45 26 112 40.18 57.78
链霉素复筛 42 23 112 37.50 54.76
双重抗性初筛 19 7 61 31.15 36.84
双重抗性复筛 18 6 61 29.51 31.58
表1  突变结果统计
筛选方法 出发菌株 筛选浓度 平均产量(g/L) 最高产量(g/L)
筛选链霉素抗性菌株 M-Z18(1.60g/L) 链霉素:7~10mg/L 1.95±0.10 2.03±0.10
筛选双重抗性菌株 S-7(2.03g/L) 巴龙霉素:4.5~6.3mg/L 2.30±0.10 2.37±0.10
表2  筛选抗性菌株结果统计
图2  S.albulus S-7在不同浓度巴龙霉素的固体平板上的生长
图3  S.albulus M-Z18(a)和SP-14(b)在固体平板上的菌丝以及孢子形态差异
图4  S.albulus M-Z18(a)和SP-14(b)在摇瓶中发酵过程中不同时间点菌体形态比较(100×)
菌株编号 出发菌 筛选抗生素浓度
(mg/L)		 ε-PL产量
(g/L)		 抗性(mg/L)
Str Par
M-Z18 出发菌 1.60 6.00 9.00
S-3 M-Z18 6.00(链霉素) 2.03 7.00 15.00
S-7 M-Z18 6.00(链霉素) 2.03 7.00 15.00
S-47 M-Z18 6.00(链霉素) 2.02 7.00 15.00
SP-1 S-7 10.50(巴龙霉素) 2.20 25.00 20.00
SP-14 S-7 7.50(巴龙霉素) 2.37 40.00 28.00
表3  菌株抗性
图5  S.albulus M-Z18和SP-14在不同培养基中发酵ε-PL产量对比
图6  S.albulus M-Z18和SP-14摇瓶自然发酵比较
图7  S.albulus M-Z18和SP-14在摇瓶发酵过程中的酶活力比较
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