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1 微生物学通报 Microbiology China Jan. 20, 2017, 44(1): DOI: /j.microbiol.china 研究报告阿维菌素生产菌的常压室温等离子体诱变育种及培养基优化 * 田萍萍曹鹏常传友胡栋张健高强 ( ) 摘要 : 目的通过诱变筛选技术选育阿维菌素高产突变株, 对其发酵培养基进行响应面优化, 提高阿维菌素产量方法采用常压室温等离子体 (ARTP) 诱变技术, 结合链霉抗性和卡那霉素抗性筛选法及 96 深孔板高通量筛选法, 筛选阿维菌素高产株在单因素实验的基础上, 应用响应面分析法对其发酵培养基进行优化, 最后确定最佳培养基配方结果获得一株遗传性状稳定的阿维菌素高产株 K-1A6, 其阿维菌素产量达到 4.22 g/l, 比出发菌株 9-39 提高了 23.4%, 在最佳培养基中阿维菌素产量达到 5.36 g/l, 较优化前提高了 27.01% 结论通过对阿维链霉菌 9-39 菌株进行 ARTP 诱变筛选及发酵培养基优化研究能显著提高阿维菌素的产量关键词 : 阿维菌素, 常压室温等离子体诱变技术, 抗性筛选, 高通量筛选, 响应面分析法 Atmospheric and room temperature plasma breeding and medium optimization of avermectin producing strains TIAN Ping-Ping CAO Peng CHANG Chuan-You HU Dong ZHANG Jian GAO Qiang * (Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin , China) Abstract: [Objective] To improve avermectin yield, mutants of Streptomyces avermitilis 9-39 were screened and the fermentation medium was optimized. [Methods] Atmospheric and room temperature plasma (ARTP) technology was used to treat the spores of S. avermitilis The method of streptomycin and kanamycin resistance combined with 96 deep-well plate screening was used for high-throughput screening of high-yielding mutants. Furthermore, based on single factor experiments, the fermentation medium for mutants was optimized by response surface methodology. [Results] A stable avermectin high-producing mutant K-1A6 was obtained and its avermectin production reached 4.22 g/l, 23.4% higher than that of the parent strain Under the optimized medium fermentation, the yield of avermectin reached 5.36 g/l, 27.01% higher than that before optimization. [Conclusion] The yield of avermectin can be increased by ARTP mutagenesis, screening and fermentation medium optimization. Foundation item: National Basic Research Program of China (973 Program) (No. 2013CB734004); National Natural Science Foundation of China (No , ) *Corresponding author: Tel: ; gaoqiang@tust.edu.cn Received: May 17, 2016; Accepted: July 25, 2016; Published online ( August 31, 2016 基金项目 (973 ) (No. 2013CB734004) (No ) * 通讯作者 :Tel gaoqiang@tust.edu.cn 收稿日期 : ; 接受日期 : ; 优先数字出版日期 (

2 : 151 Keywords: Avermectin, Atmospheric and room temperature plasma (ARTP), Resistance selection, High-throughput screening, Response surface methodology (Avermectin AVM) (Steptomyces avermitilis) [1] Satoshi Ōmura William C. Campbell 2015 [2] 100% [3] (Atmospheric and room temperature plasma ARTP) DNA 4 10 [4-5] ARTP [6-8] [9] [10-12] S. avermitilis 9-39 ARTP 1 材料与方法 1.1 材料菌种 : (Streptomyces avermitilis) 主要试剂和仪器 : CoCl 2 6H 2 O (NH 4 ) 2 SO 4 Na 2 MoO 4 2H 2 O MnSO 4 H 2 O PEG6000 CaCO 3 CH 3 CH 2 COONa B 1 ( 99.9%) Sigma ARTP-II Agilent 1200 (High Performance Liquid Chromatography HPLC) LRH-250A ZWYR-D 培养基的配制和培养方法 MS (g/l) Pa 20 min YMS (g/l) CoCl 2 6H 2 O ph Pa 20 min (g/l) CoCl 2 6H 2 O 0.03 ph Pa 25 min [13] (g/l) (NH 4 ) 2 SO CoCl 2 6H 2 O 0.02 Na 2 MoO 4 2H 2 O MnSO 4 H 2 O CaCO PEG CH 3 CH 2 COONa 2 mmol/l ph Pa 25 min B g/l

3 152 微生物学通报 Microbiol. China 2017, Vol.44, No.1 [13] 1.3 诱变方法常压室温等离子体诱变方法 :ARTP-II 10 L/min 115 W 2 mm 10 μl /ml 诱变初筛 : 400 μl C 10 d 1 ml 20 min 0.22 μm HPLC 诱变复筛 : S. avermitilis 9-39 (CK) 15% 突变株稳定性试验 : YMS 分析方法阿维菌素的产量测定方法 :HPLC [14] 诱变致死曲线的测定 : /ml ARTP s 1.6 mg/l 0.4 mg/l MS 28 C 4 d (CFU) =( )/ 100% =/ 100% 抗性平板中链霉素和卡那霉素浓度的确定 : /ml MS 28 C 4 d 90% 95% 5% 1.5 发酵培养基的优化 Plackett-Burman 设计 : Plackett-Burman 11 ( 4 ) Minitab 16.0 Plackett-Burman 最陡爬坡试验 : Box-Behnken 试验 : ( 2) 表 1 Plackett-Burman 试验因素水平及编码 Table 1 Factors and levels of Plackett-Burman design Levels Code Factors X 1 CH 3 CH 2 COONa (mmol/l) X 2 PEG6000 (g/l) X 3 B 1 (g/l) X 5 Soya flour (g/l) X 6 Yeast extract (g/l) X 7 Na 2 MoO 4 2H 2 O (mg/l) X 9 MnSO 4 H 2 O (mg/l) X 10 CoCl 2 (mg/l) X 11 CaCO 3 (g/l) X 13 Corn starch (g/l) X 14 (NH 4 ) 2 SO 4 (mg/l) 表 2 Box-Behnken 试验因素水平及编码 Table 2 Factors and levels of Box-Behnken design Levels Code Factors X 1 Yeast extract (g/l) X 2 Soya flour (g/l) X 3 Corn starch (g/l) X 4 (NH 4 ) 2 SO 4 (g/l)

4 : 153 Minitab 结果与分析 2.1 抗性平板中链霉素和卡那霉素浓度的确定 mg/l 3% 1.6 mg/l 0.2 mg/l 85% 0.4 mg/l 3% 0.4 mg/l 2.2 ARTP 诱变 ARTP 诱变时间的确定 : 2 ARTP 30 s 35% 30 s ARTP 60 s 25% 60 s 链霉素抗性突变株初筛 : /ml ARTP 30 s 1.6 mg/l MS (73 ) (CK) 61.64% 15% 9 20% 图 1 链霉素 (A) 和卡那霉素 (B) 对阿维链霉菌的致死曲线 Figure 1 The lethality rate curves at different streptomycin (A) and kanamycin (B) concentrations 图 2 不同诱变时间抗性筛选的正突变率 Figure 2 Positive mutation rate at different mutagenesis times A B. Note: A: Streptomycin resistant screening; B: Kanamycin resistant screening.

154 微生物学通报 Microbiol. China 2017, Vol.44, No.1 2.2.3 卡那霉素抗性突变株初筛 : 1 10 8 /ml ARTP 60 s 0.

9% Figure 3 图 3 链霉素抗性突变株和出发菌株 96 深孔板培养 10 d 阿维菌素产量 Avermectin production of the streptomycin resistant mutants and

5 154 微生物学通报 Microbiol. China 2017, Vol.44, No 卡那霉素抗性突变株初筛 : /ml ARTP 60 s 0.4 mg/l MS (CK) 71.15% 15% % Figure 3 图 3 链霉素抗性突变株和出发菌株 96 深孔板培养 10 d 阿维菌素产量 Avermectin production of the streptomycin resistant mutants and original strain in 96 deep-well plate by 10 d cultivation Figure 4 图 4 卡那霉素抗性突变株和出发菌株在 96 深孔板培养 10 d 的阿维菌素产量 Avermectin production of the kanamycin resistant mutants and original strain in 96 deep-well plate by 10 d cultivation

6 : 高产菌株复筛 : (CK) 5A S-2C3 S-2D2 5B K-1A6 K-1C 突变株遗传稳定性 : 4 S-2C3 S-2D2 K-1A6 K-1C (CK) 6 K-1C2 图 5 阿维菌素高产突变株的复筛 Figure 5 Rescreening of avermectin overproduction mutants AB. Note: A: Streptomycin resistant mutants; B: Kanamycin resistant mutants. 图 6 突变株 S-2C3 S-2D2 K-1A6 和 K-1C2 遗传稳定性 Figure 6 The genetic stability of the mutants S-2C3, S-2D2, K-1A6 and K-1C S-2C3 K-1A g/l 4.22 g/l % 23.4% K-1A6 2.3 发酵培养基的优化 Plackett-Burman 设计筛选显著性因素 : Plackett-Burman 1 3 Minitab 16.0 ( 4) 4 (NH 4 ) 2 SO 4 4 ( 1) 最陡爬坡试验 : Plackett-Burman g/l 3 3

7 156 微生物学通报 Microbiol. China 2017, Vol.44, No.1 表 3 Plackett-Burman 设计的各因素水平及响应表 Table 3 Experimental design and response results of Plackett-Burman design X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 Run serial number AVM (g/l) 表 4 Plackett-Burman 试验因素水平及效应分析 Table 4 Factor levels and effect estimates of Plackett-Burman design t P Code Factors t value P value Significant X 1 CH 3 CH 2 COONa (mmol/l) NS X 2 PEG6000 (g/l) NS X 3 B 1 (g/l) NS X NS X 5 Corn starch (g/l) X 6 Yeast extract (g/l) X 7 Na 2 MoO 4 2H 2 O (mg/l) NS X NS X 9 MnSO 4 H 2 O (mg/l) NS X 10 CoCl 2 (mg/l) NS X 11 CaCO 3 (g/l) NS X NS X 13 Soya flour (g/l) X 14 (NH 4 ) 2 SO 4 (g/l) X NS +(P<0.05) NS. Note: +: Significant (P<0.05); NS: Not significant.

8 : 157 表 5 最陡爬坡试验设计和结果 Table 5 Experimental design and results of steepest ascent experiments Serial number (X 1 ) Yeast extract (g/l) (X 2 ) Soya flour (g/l) (X 3 ) Corn starch (g/l) (X 4 ) (NH 4 ) 2 SO 4 (g/l) AVM (g/l) Box-Behnken 试验设计 : Minitab 16.0 Y= X X X X X 1 X X 1 X X 1 X X 2 X X 2 X X 3 X X X X X 2 4 ( 7) P R 2 =93.37% 8 P Y = X X X X 2 X X X 响应面交互作用分析及最佳培养基的确定 : 8 (X 2 ) (X 3 ) (P<0.05) Minitab 16.0 ( 7) g/l 4.1 g/l 16.5 g/l (NH 4 ) 2 SO g/l 5.36 g/l 5.44 g/l 4.22 g/l 27.01% 表 6 Box-Behnken 试验设计及响应 Table 6 Experimental design and response of Box-Behnken design Serial number X 1 X 2 X 3 X 4 AVM (g/l)

9 158 微生物学通报 Microbiol. China 2017, Vol.44, No.1 表 7 Box-Behnken 试验回归模型方差分析 Table 7 Variance analysis for regression model of Box-Behnken design Source Degrees of freedom Sum of squares Mean square F F value P P value Model <0.001 Lack of fit Residual Pure error Cor total R 2 =93.37% R 2 adj =85.63% Table 8 表 8 Box-Behnken 试验回归系数显著性检验 Significant test for regression coefficients of Box-Behnken design Model Regression coefficients Standard errors t value P value Constant terms <0.001 X X <0.001 X <0.001 X X X X X X 1 X X 1 X X 1 X X 2 X X 2 X X 3 X t P 3 讨论图 7 淀粉液化液与黄豆饼粉交互影响阿维菌素产量的响应面图 Figure 7 The response surface plot for the effects of soya flour and corn starch on the yield of AVM ARTP [4,15-20] ARTP

10 : 159 [9,21-22] ARTP 96 ARTP K-1A g/l [23-27] (4.22 g/l) 27.01% [27] 致谢 : 感谢中国科学院微生物研究所张立新研究员对本研究的大力支持! 参考文献 [1] Burg RW, Miller BM, Baker EE, et al. Avermectins, new family of potent anthelmintic agents: producing organism and fermentation[j]. Antimicrobial Agents and Chemotherapy, 1979, 15(3): [2] Shen B. A New Golden Age of natural products drug discovery[j]. Cell, 2015, 163(6): [3] Liu ZJ, Wang J, Liu ZJ. The present status & analysis of the future market of abamectin[j]. Shandong Chemical Industry, 2005, 34(4): (in Chinese),,. [J]., 2005, 34(4): [4] van Gessel AFH, Hrycak B, Jasiński M, et al. Temperature and NO density measurements by LIF and OES on an atmospheric pressure plasma jet[j]. Journal of Physics D: Applied Physics, 2013, 46(9): [5] Bartis EAJ, Graves DB, Seog J, et al. Atmospheric pressure plasma treatment of lipopolysaccharide in a controlled environment[j]. Journal of Physics D: Applied Physics, 2013, 46(31): [6] Si XG, Guo G, Wang XX, et al. Rapid breeding of a high pyruvate production mutant by atmospheric room temperature plasma[j]. Science and Technology of Food Industry, 2014, 35(20): (in Chinese),,,. [J]., 2014, 35(20): [7] Jin LH, Fang MY, Zhang C, et al. Operating conditions for the rapid mutation of the oleaginous yeast by atmospheric and room temperature plasmas and the characteristics of the mutants[j]. Chinese Journal of Biotechnology, 2011, 27(3): (in Chinese),, 翀,. [J]., 2011, 27(3): [8] Wang LY, Huang ZL, Li G, et al. Novel mutation breeding method for Streptomyces avermitilis using an atmospheric pressure glow discharge plasma[j]. Journal of Applied Microbiology, 2010, 108(3): [9] Sun YW, Cui CB. Antibiotic-resistance mutation technique in microorganism breeding[j]. Journal of International Pharmaceutical Research, 2008, 35(3): (in Chinese),. [J]., 2008, 35(3): [10] Deepak V, Kalishwaralal K, Ramkumarpandian S, et al. Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology[j]. Bioresource Technology, 2008, 99(17): [11] Liu GQ, Wang XL. Optimization of critical medium components using response surface methodology for biomass and extracellular polysaccharide production by Agaricus blazei[j]. Applied Microbiology and Biotechnology, 2007, 74(1): [12] Cheng G, Xu JZ, Guo YF, et al. Breeding and fermentation optimization of L-arginine producing strains[j]. Microbiology China, 2016, 43(2): (in Chinese),,,. L- [J]., 2016, 43(2): [13] Gao H, Liu M, Liu JT, et al. Medium optimization for the production of avermectin B1a by Streptomyces avermitilis 14-12A using response surface methodology[j]. Bioresource Technology, 2009, 100(17): [14] Guo G, Tian PP, Tang D, et al. Metabolomics analysis between wild-type and industrial strains of Streptomyces avermitilis based on gas chromatography-mass spectrometry strategy[m]//zhang TC, Nakajima M, eds. Advances in Applied Biotechnology-Proceedings of the 2nd International Conference on Applied Biotechnology (ICAB 2014)-Volume II. Berlin Heidelberg: Springer, 2015, 333: [15] Shi XF, Ji ZC, Chang CY, et al. Rapid screening of a high yield γ-aminobutyric acid mutant by multifunction plasma mutagenesis system[j]. China Brewing, 2016, 35(4): (in Chinese)

11 160 微生物学通报 Microbiol. China 2017, Vol.44, No.1,,,. γ-[j]., 2016, 35(4): [16] Cai C, Jiang T, Zheng ZJ, et al. Improved xylose utilization of Bacillus coagulans by atmospheric and room temperature plasma mutation for production of lactic acid[j]. Food Science, 2014, 35(1): (in Chinese),,,. L- [J]., 2014, 35(1): [17] Zhang GC, Jia SR, Yan L, et al. Mutation screening of a high bacterial cellulose-producing strain by plasma ion implantation[j]. Modern Food Science & Technology, 2010, 26(12): (in Chinese),,,. [J]., 2010, 26(12): [18] Kang FS, Yan B, Lü NQ, et al. Mutation breeding of Saccharomyces cerevisiae with low purine by atmospheric and room temperature plasma[j]. Modern Food Science and Technology, 2014, 30(2): (in Chinese),,,. ARTP [J]., 2014, 30(2): [19] Wang Q, Feng LR, Wei L, et al. Mutation breeding of lycopene-producing strain Blakeslea trispora by a novel atmospheric and room temperature plasma (ARTP)[J]. Applied Biochemistry and Biotechnology, 2014, 174(1): [20] Shen XJ, Zhang P, Shi YP. A mutant strain with high erythromycin yield obtained by using novel atmospheric and room temperature plasmas and UV mutation[j]. Chinese Journal of Veterinary Drug, 2015, 49(1): (in Chinese),,. [J]., 2015, 49(1): [21] Wang GJ, Inaoka T, Okamoto S, et al. A novel insertion mutation in Streptomyces coelicolor ribosomal S12 protein results in paromomycin resistance and antibiotic overproduction[j]. Antimicrobial Agents and Chemotherapy, 2009, 53(3): [22] Nishimura K, Hosaka T, Tokuyama S, et al. Mutations in rsmg, encoding a 16S rrna methyltransferase, result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2)[J]. Journal of Bacteriology, 2007, 189(10): [23] Zhang R, Xia HY, Xu QY, et al. Recombinational cloning of the antibiotic biosynthetic gene clusters in linear plasmid SCP1 of Streptomyces coelicolor A3(2)[J]. FEMS Microbiology Letters, 2013, 345(1): [24] Kim ES, Hong HJ, Choi CY, et al. Modulation of actinorhodin biosynthesis in Streptomyces lividans by glucose repression of afsr2 gene transcription[j]. Journal of Bacteriology, 2001, 183(7): [25] Chen JS, Liu M, Liu XT, et al. Interrogation of Streptomyces avermitilis for efficient production of avermectins[j]. Synthetic and Systems Biotechnology, 2016, 1(1): 7-16 [26] Chen JS, Liu M, Zhang LX. Avermectin, from winning the Nobel Prize to innovation in China [J]. Acta Microbiologica Sinica, 2016, 56(3): (in Chinese),,. [J]., 2016, 56(3): [27] Chen N, Wang YH, Chu J, et al. Effect of medium composition and feeding strategy on morphology of Streptomyces avermitilis in avermectin fermentation[j]. Journal of Huazhong Agricultural University, 2007, 26(4): (in Chinese),,,. [J]., 2007, 26(4):

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g 16 6 2018 11 Chinese Journal of Bioprocess Engineering Vol. 16 No. 6 Nov. 2018 doi 10. 3969 /j. issn. 1672-3678. 2018. 06. 004 1 2 1 1 1. 330200 2. 330006 4 Box-Behnken 1 25 g /ml 121 92 min 2 6. 284%