Microsoft Word tb 牟慧艳-研究报告

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1 微生物学通报 Microbiology China Jan. 20, 2017, 44(1): DOI: /j.microbiol.china 研究报告牟慧艳恩拉霉素生产菌株的遗传改造 1 刘扬 1 王应东 1 刘宝爱 2 魏建军 2 张会图 ( ) ( ) 1* 摘要 : 目的提高杀真菌素链霉菌发酵生产恩拉霉素的产量方法利用定点突变技术, 对恩拉霉素生产菌株杀真菌素链霉菌 F1 中影响细胞次级代谢及抗生素合成的核糖体 S12 蛋白的编码基因 rpsl 进行改造, 将第 43 位的赖氨酸 (Lys) 分别替换为天冬酰胺 (Asn) 和精氨酸 (Arg), 并对改造菌株 L-M1(Asn43) 和 L-M2(Arg43) 的生长特性抗生素合成以及摇瓶发酵性能进行研究结果与野生型菌株相比, 改造菌株的生长特性及生理生化特性均发生了明显的改变 : 产孢周期明显缩短, 野生型菌株在 MS 培养基中,28 C 下需要培养 5 7 d 后才能产生孢子, 而在相同条件下, 改造菌株 3 d 后就能产生大量的孢子 ; 恩拉霉素产量相对提高, 摇瓶发酵条件下, 改造菌株 L-M1(Asn43) 和 L-M2(Arg43) 的恩拉霉素产量分别可达到 U/mL 和 U/mL, 与野生型菌株 F1 相比分别提高了 11.9% 和 22.1% 结论通过遗传改造, 恩拉霉素的产量得到了提高, 为其他位点的遗传改造提供了可行性关键词 : 恩拉霉素, 抗生素, 抗真菌素链霉菌, 遗传改造, 核糖体 S12 蛋白, 定点突变 Genetic modification of an enramycin producing strain MOU Hui-Yan 1 LIU Yang 1 WANG Ying-Dong 1 LIU Bao-Ai 2 WEI Jian-Jun 2 ZHANG Hui-Tu 1* (1. Laboratory of Enzyme and Applied Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin , China) (2. Tianjin Xinxing Veterinary Pharmaceutical Factory, Tianjin , China) Abstract: [Objective] To enhance the production of enramycin by Streptomyces fungicidicus. [Methods] In this study, the ribosomal S12 protein encoding gene rpsl which affects the secondary metabolism and biosynthesis of antibiotics in enramycin producing strain of S. fungicidicus F1 was changed through the method of site-directed mutation, during which the Lys43 was substituted for Asn and Arg, respectively. And then the growth characteristics, synthesis of antibiotic and flask fermentation performance of the modified strains were studied. [Results] The results showed that the growth characteristics and physiological and biochemical characteristics of the modified strains were significantly changed compared with the wild type strain. Sporulation cycle was shorten, spore was Foundation item: National Natural Science Foundation of China (No ) *Corresponding author: Tel/Fax: ; hzhang@tust.edu.en Received: January 19, 2016; Accepted: May 06, 2016; Published online ( May 24, 2016 基金项目 (No ) * 通讯作者 :Tel/Fax hzhang@tust.edu.cn 收稿日期 : ; 接受日期 : ; 优先数字出版日期 (

2 : 127 produced from wild type strain using MS medium under the condition of 28 C for 5 7 days, but the modified strains can produced large amounts of spore only after 3 days in the same conditions. Enramycin yield was relatively increased. Under the flask formation condition, the enramycin production of the modified strains of L-M1(Asn43) and L-M2(Arg43) can reach a maximum of U/mL and U/mL, respectively. Compared with the wild-type strain, it was improved by 11.9% and 22.1% separately. [Conclusion] Through the genetic modification, the yield of enramycin was improved, which might provide a feasibility for the modification of other sites. Keywords: Enramycin, Antibiotics, Streptomyces fungicidicus, Genetic modification, Ribosomal protein S12, Site-directed mutation (Enramycin) [1-2] 17 [3] [4] S. fungicidicus [5] 2004 Ochi RNA [6] 30S S12 (rpsl ) S Hesketh Ochi rpsl K88E A3(2) [7] 2000 Okamoto-Hosoya rpsl P91S A3(2) 7 10 [8] S12 (rpsl) S12 I (TPKKPNS ) II (RVKDLPGVR ) II 88 I S12 (rpsl ) 43 (Lys) (Asn) (Arg) 1 材料与方法 1.1 实验材料菌株质粒和引物 : 1 PCR 2

3 128 微生物学通报 Microbiol. China 2017, Vol.44, No.1 表 1 菌株与质粒 Table 1 Strains and plasmids Strains and plasmids Characterizations Sources Escherichia coli JM109 Host strain Laboratory stock E. coli ET12567 puz8002 Km r Cml r Laboratory stock S. fungicidicus F1 Wild type Laboratory stock S. fungicidicusl-m1 Lys(43) of gene rpsl to Asn This study S. fungicidicusl-m2 Lys(43) of gene rpsl to Arg This study pucm-t Cloning vector Amp r Sangon Biotech pkc1139 Shuttle plasmid of Streptomyces and E. coli Apra r repts Laboratory stock 表 2 PCR 反应引物 Table 2 PCR primers Primers Primer sequence (5 3 ) Size (bp) rpslf1 GTGCCTACGATCCAGCAGCT 370 rpslr1 TTACTTCTCCTTCTTGGCGC 370 rpslf2 GCCGAGTTCGGCTTCTTCG rpslr2 ACAACCTGCAGGAGCACTCC S12F1 CCGAATTCTGAACGGCAAGGCGGTCGC S12R1 GCAAGCTTGCAGGTCAAGTGAAGTGGTA MrpsLF1 ACCACCCCGAACAAGCCGAA 778 MrpsLR1 TTCGGCTTGTTCGGGGTGGT 671 MrpsLF2 ACCACCCCGCGGAAGCCGAA 778 MrpsLR2 TTCGGCTTCCGCGGGGTGGT 671 Apra-F1 AGGTAGCTGTATGGGGTGTTCC 820 Apra-R1 CATGTGCGCCGATATAAACAT 培养基 : (E. coli) LB [5] (S. fungicidicus) MS (g/l) XYT [5] (g/l) ml/l ph (g/l) α ml/l 5.00 ml/l 0.50 ml/l ph 主要试剂和仪器 : DNA ( ) DNA ( ) PCR Eppendorf ( ) P230II 1.2 实验方法从杀真菌素链霉菌 F1 中克隆 rpsl 基因 : NCBI rpsl Vector NTI rpsl rpslf1/r1 (

4 : 129 2) PCR rpsl PCR 2 GC buffer 25 μl dntps (2.5 mmol/l) 4 μl (10 μmol/l) 2 μl 2 μl LATaq DNA polymerase (5 U/μL) 1 μl 50 μl PCR 95 C 5 min 95 C 30 s 55 C 30 s 72 C 90 s C 10 min PCR pucm-t E. coli JM109 rpsl rpslf2/r2 ( 2) PCR rpsl PCR pucm-t E. coli JM109 S12F1/R1( 2) rpsl K-rpsL PCR PCR 95 C 5 min 95 C 30 s 62 C 30 s 72 C 90 s C 10 min DNA 片段的回收酶切克隆转化质粒的提取及序列的测定 : DNA [9] 重叠 PCR: rpsl Vector NTI MrpsLF1/R1 MrpsLF2/R2 ( 2) PCR K-rpsL S12F1 MrpsLR1 S12R1 MrpsLF1 PCR P1 P2 PCR 2 GC buffer 10 μl dntps (2.5 mmol/l) 2 μl (10 μmol/l) 1 μl 1 μl LATaq DNA polymerase (5 U/μL) 0.2 μl 20 μl PCR 95 C 5 min 95 C 30 s 56 C 30 s 72 C 90 s C 10 min PCR P1 P2 3 5 S12F1/R1 PCR K-rpsL M1 M2 PCR M1 M2 pkc1139 pkc1139- L-M1 pkc1139-l-m2 ET 接合转移 : CaCl 2 puz8002 ET12567 PCR [10-11] 链霉菌发酵培养 : MS 28 C 7 d 1 cm 1 cm 28 C 200 r/min 48 h 50 ml/500 ml 20% 3 30 C 200 r/min 10 d HPLC 法测定恩拉霉素的产量 : (4% ) 30 min 1 h mg/l mg/l 10 ml r/min 5 min 10 ml 30 min 1 h r/min 10 min 4 C HPLC C 18 (4.6 mm 150 mm Φ=5 µm) 30 C 20 µl 267 nm :NaH 2 PO 4 (50 7 mmol/l)=3 ph ml/min 2 结果与分析 2.1 突变基因序列与原序列对比 PCR rpsl M1 M2 vector NTI 1 1 M1 M2 43

5 130 微生物学通报 Microbiol. China 2017, Vol.44, No.1 图 1 原 rpsl 基因与突变基因 M1 和 M2 氨基酸序列比对 Figure 1 Amino acid sequence alignment of rpsl and M1 and M2 2.2 突变菌株的构建与筛选 Apra-F1/Apra-R1 ( 2) PCR 2 pkc C MS 40 C 2 3 DNA MS 28 C C ( 3) PCR rpslf1/r1 S12 rpsl 1 M1 3 M 突变菌株产孢能力的考察 F1 M1 M2 MS 5 7 d 3 d 4 图 2 安普霉素抗性基因孢子 PCR 产物电泳图 Figure 2 Agarose gel electrophoresis of spore PCR production of Apra r M 10 kb marker 0 pkc M1 2 M2 3. Note: M: 10 kb marker; 0: pkc1139 control; 1: Type M1 positive trans-conjugant; 2: Type M2 positive trans-conjugant; 3: S. fungicidicus F1 control.

: 131 图 3 接合转化子转接划线平板 Figure 3 The switching streak plate of conjugation transformant A MS B MS. Note: A: MS medium without apramycin; B: MS medium with apramycin.

1% ( 3) S12 rpsl 43 43 20% 2.5 高产突变菌株遗传稳定性分析 L-M1-2 L-M2-1 4 C L-M1-2 1 5 100% 99.3% 98.2% 100.3% 99.1% L-M2-1 1 5 100% 97.4% 99.5% 99.3% 101.

6 : 131 图 3 接合转化子转接划线平板 Figure 3 The switching streak plate of conjugation transformant A MS B MS. Note: A: MS medium without apramycin; B: MS medium with apramycin. 图 4 突变菌株与野生菌株产孢能力的考察 Figure 4 The sporulation ability of modification strain and wild strain 2.4 突变菌株的发酵验证及 HPLC 分析 3 M1 4 M2 HPLC M U/mL 11.9% ( 3) M U/mL 22.1% ( 3) S12 rpsl % 2.5 高产突变菌株遗传稳定性分析 L-M1-2 L-M2-1 4 C L-M % 99.3% 98.2% 100.3% 99.1% L-M % 97.4% 99.5% 99.3% 101.2% 4 表 3 恩拉霉素产量对比一览表 Table 3 Enramycin yield comparison table Strain number Fermentation time (d) Enramycin yield (U/mL) Increase rate (%) F ±23 0 L-M ± L-M ± L-M ± L-M ± L-M ± L-M ± L-M ±

7 132 微生物学通报 Microbiol. China 2017, Vol.44, No.1 3 结论 S. fungicidicus F1 S12 rpsl 43 (Lys) (Asn) (Arg) L-M1 L-M2 F1 11.9% 22.1% 参考文献 [1] Higashide E, Hatano K, Shibata M, et al. Enduracidin, a new antibiotic. I. Streptomyces fungicidicus No. B5477, an enduracidin producing organism[j]. The Journal of Antibiotics, 1968, 21(2): [2] McCafferty DG, Cudic P, Frankel BA, et al. Chemistry and biology of the ramoplanin family of peptide antibiotics[j]. Biopolymers, 2002, 66(4): [3] Yin XH, Zabriskie TM. The enduracidin biosynthetic gene cluster from Streptomyces fungicidicus[j]. Microbiology, 2006, 152(Pt10): [4] Pedroso AA, Menten JF, Lambais MR, et al. Intestinal bacterial community and growth performance of chickens fed diets containing antibiotics[j]. Poultry Science, 2006, 85(4): [5] Wang JL, Xu MY, Shi TG, et al. UV mutagenesis for high-yield enramycin producing strains[j]. Industrial Microbiology, 2015, 45(5): (in Chinese),,,. [J]., 2015, 45(5): [6] Ochi K, Okamoto S, Tozawa Y, et al. Ribosome engineering and secondary metabolite production[j]. Advances in Applied Microbiology, 2004, 56: [7] Hesketh A, Ochi K. A novel method for improving Streptomyces coelicolor A3(2) for production of actinorhodin by introduction of rpsl (encoding ribosomal protein S12) mutations conferring resistance to streptomycin[j]. The Journal of Antibiotics, 1997, 50(6): [8] Okamoto-Hosoya Y, Sato TA, Ochi K. Resistance to paromomycin is conferred by rpsl mutations, accompanied by an enhanced antibiotic production in Streptomyces coelicolor A3(2)[J]. The Journal of Antibiotics, 2000, 53(12): [9] Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual[M]. 3rd Edition. New York: Cold Spring Harbor Laboratory Press, 2002 [10] Kieser T, Bibb MJ, Buttner MJ, et al. Practical Streptomyces Genetics[M]. Norwich: The John Innes Foundation, 2000 [11] Fang ZK, Hong WR, Yan LB, et al. Construction of the conjugal transfer system of Streptomyces aureofaciens[j]. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 2011, 40(5): (in Chinese),,,. [J]. :, 2011, 40(5):

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇