核糖体工程技术选育阿维拉霉素高产菌株
|
摘要
阿维拉霉素是一种新型的代谢调节剂和消化促进剂,被广泛应用于国内外畜禽养殖中。菌种产素能力低、发酵工艺不成熟等因素限制了阿维拉霉素在国内的工业化生产。本研究通过核糖体工程技术,采用链霉素对实验室保藏的绿色产色链霉菌菌株(Streptomyces viridochromogenes gs 77)进行抗性选育,以获得阿维拉霉素高产突变菌株。经过药敏试纸法和高效液相色谱法(HPLC)筛选获得一株阿维拉霉素高产突变菌株S. viridochromogenes gs 77-54,摇瓶第8天时阿维拉霉素产量达到最大,为出发菌株的1.80倍,传代实验表明突变菌株高产性能遗传稳定。对突变前后菌株的形态特征和rpsl基因(编码核糖体蛋白S12)分析,结果显示突变菌株的气生菌丝长直形、较为粗壮,孢子为直径约1.1μm×0.6μm的椭圆形,与出发菌株存在明显区别。rpsl基因序列分析结果发现,该基因片段出现点突变,第356位的G突变为A,由精氨酸突变为谷氨酰胺。本研究筛选到阿维拉霉素高产突变菌株,为绿色产色链霉菌菌种选育提供了新的参考资料。
Avilamycin is widely used as a new type of metabolic regulator and digestive enhancer in livestock feeding. The low production capacity of the strain and the immature fermentation process limit the industrial of avilamycin in China. Ribosome engineering technique was used to carry out resistance breeding of the Streptomyces viridochromogenes gs 77 by streptomycin to obtain a high-yield mutant strain of avilamycin,. Through the drug sensitive test and high performance liquid chromatography, a high-yield avilamycin mutant S.viridochromogenes gs 77-54 was obtained. The yield of avilamycin reached the maximum at 8 th day of shake flask, and increased to 1.80 fold than that of the original strain. Passage experiments showed that the high-yield performance of mutant strains was genetically stable. rpsl gene(encoding the ribosomal protein S12) and the morphological characteristics of the strains parent and after the mutation were analyzed. The results showed that the aerial hyphae of the mutant strain were long and straight, and the spores were elliptical with a diameter of about 1.1 μm× 0.6 μm, which was significantly different from the starting strain. Sequence analysis of the rpsl gene revealed a point mutation in the gene fragment, the G mutation at position 356 was mutated A, which was mutated to glutamine by arginine. The avilamycin high-yield mutant strain was constructed successfully, which would provide a new idea for the breeding of S. viridochromogenes.
Avilamycin is widely used as a new type of metabolic regulator and digestive enhancer in livestock feeding. The low production capacity of the strain and the immature fermentation process limit the industrial of avilamycin in China. Ribosome engineering technique was used to carry out resistance breeding of the Streptomyces viridochromogenes gs 77 by streptomycin to obtain a high-yield mutant strain of avilamycin,. Through the drug sensitive test and high performance liquid chromatography, a high-yield avilamycin mutant S.viridochromogenes gs 77-54 was obtained. The yield of avilamycin reached the maximum at 8 th day of shake flask, and increased to 1.80 fold than that of the original strain. Passage experiments showed that the high-yield performance of mutant strains was genetically stable. rpsl gene(encoding the ribosomal protein S12) and the morphological characteristics of the strains parent and after the mutation were analyzed. The results showed that the aerial hyphae of the mutant strain were long and straight, and the spores were elliptical with a diameter of about 1.1 μm× 0.6 μm, which was significantly different from the starting strain. Sequence analysis of the rpsl gene revealed a point mutation in the gene fragment, the G mutation at position 356 was mutated A, which was mutated to glutamine by arginine. The avilamycin high-yield mutant strain was constructed successfully, which would provide a new idea for the breeding of S. viridochromogenes.
引文
蔡成平,王远山,郑裕国.2012.核糖体工程与微生物次级代谢产物合成[J].生物技术通报,5(9):52-58.(Cai C P,Wang Y S,Zheng Y G.2012.Ribosome engineering and microbial secondary metabolite synthesis production[J].Biotechnology Bulletin,5(9):52-58.)
傅路,裘娟萍.2012.核糖体工程改良微生物菌种特性的研究进展[J].科技通报,11(8):78-83.(Fu L,Qiu J P.2012.Research advance of strain improvement by ribosome engineering[J].Bulletin of Science and Technology,28(11):78-83.)
蒋立锐.1988.谷氨酰胺在代谢中的作用[J].氨基酸和生物资源,4:17-23.(Jiang L R.1988.The role of glutamine in metabolism[J].Amino Acids and Biological Resources,4:17-23.)
蒋顺进.2018.原生质体ARTP诱变选育阿维拉霉素高产菌株[J].中国抗生素杂志,43(7):831-836.(Jiang S J.2018.Protoplast mutagenesis breeding of Streptomyces viridochromogene producing high-yield avilamycin by ARTP[J].Chinese Journal of Antibiotics,43(7):831-836.)
李红梅,陈敏.2017.响应面优化绿色产色链霉菌发酵产阿维拉霉素[J].中国食品学报,17(9):109-115.(Li H M,Chen M.2017.Fermentation optimization of avilamycin using Streptomyces viridochromogenes by response surface methodology[J].Journal of Chinese Institute of Food Science and Technology,17(9):109-115.)
梁新乐,金英,陈敏,等.2010.阿维拉霉素高产突变株H15形态分化初步研究[J].核农学报,24(4):689-693.(Liang XL,Jin Y Y,Chen M,et al.2010.Preliminary research on morphological differentiation of avilamycin High-yield mutant strain H15[J].Acta Agriculturae Nucleatae Sinica,24(4):689-693.)
梁新乐,朱静,金英燕.2012.绿色产色链霉菌E-219菌落的形态分化[J].核农学报,26(3):466-470.(Liang X L,Zhu J,Jin Y Y.2012.Morphological differentiation of Streptomyces viridochromogenes E-219 on solid culture[J].Acta Agriculturae Nucleatae Sinica,26(3):466-470.)
刘玲,朱湘成,黄勇.2016.微生物核糖体工程在抗生素研发中的应用[J].中国感染控制杂志,15(5):355-359.(Liu L,Zhu X C,Huang Y.2016.Application of microbial ribosome engineering in the research and development of antibiotics[J].Chinese Journal of Infection Control,15(5):355-359.)
刘芳,李晓荣,邹祥.2010.阿维拉霉素生物合成与代谢调控研究进展[J].生物技术通报,12:25-30.(Liu F,Li X R,Zou X.2010.Advances on biosynthesis approach and metabolic regulation of avilamycin[J].Biotechnology Bulletin,12:25-30.)
罗林根,杨燕,魏慧,等.2016.须糖多孢菌Saccharopolyspora pogona的核糖体工程改造对丁烯基多杀菌素合成的影响[J].生物工程学报,32(2):259-263.(Luo L G,Yang Y,Wei H,et al.2016.Effect of ribosome engineering of Saccharopolyspora pogona on synthesis of spinosyl spinosyn[J].Chinese Journal of Biotechnology,32(2):259-263.)
毛灵琪,李存治,陶兴无,等.2015.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,31(5):54-60.(Mao L Q,Li C Z,Tao X W,et al.2015.Screening and breeding strains producing high-yield avilamycin by genome shuffling[J].Biotechnology Bulletin,31(5):54-60.)
王庆龄.2015.阿维拉霉高产菌株选育[D].硕士学位论文,浙江工商大学,导师:梁新乐,pp.20-26.(Wang Q L.2015.Breeding high avilamycin-producing by Streptomyces viridochromogenes[D].Thesis for M.S.,Zhejiang Gongshang University,Supervisor:Liang X L,pp.20-26.)
向荣华,詹晓北,朱莉,等.2015.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,40(10):732-737.(Xiang R H,Zhan X B,Zhu L,et al.2015.Screening of high yield avilamycin mutant of Streptomyces viridochromogene through atmospheric and room temperature plasma mutagenesis[J].Chinese Journal of Antibiotics,40(10):732-737.)
谢庶洁,肖静,徐俊.2009.微生物核糖体工程研究进展[J].微生物学报,49(8):981-986.(Xie S J,Xiao J,Xu J.2009.Advance in microbial ribosome engineering[J].Acta microbiologica Sinica,49(8):981-986.)
袁晖,张新宜,王欣荣.2014.抗性突变他克莫司高产菌株的选育[J].微生物学杂志,34(5):81-86.(Yuan H,Zhang X Y,Wang X R.2014.Breeding of resistant mutant tacrolimus-high-producing Streptomyces strain SIIA-9818[J].Journal of Microbiology,34(5):81-86.)
于志斌,朱天骄,崔承彬,等.2006.用核糖体工程技术二次开发海洋微生物菌株资源的研究[J].高技术通讯,16(11):1190-1194.(Yu Z B,Zhu T J,Cui C B,et al.2006.A practical approach for exploiting useless microbial strains from marine environment by ribosome-engineering technology[J].High Technology Letters,16(11):1190-1194.)
张丹丹.2012.阿维拉霉素产生菌的株的定向育种及机制研究[D].硕士学位论文,浙江工商大学,导师:梁新乐,pp.25-30.(Zhang D D.2012.Rational breeding of high aviromycin-producers in Streptomyces viridochromogenes and deciphering its evolution mechanism[D].Thesis for M.S.,Zhejiang Gongshang University,Supervisor:Liang XL,pp.25-30.)
张宪明,赵韶星,高颖,等.2001.药敏试纸简单制备法[J].山西医药杂志,30(5):447.(Zhang X M,Zhao Y X,Gao Y,et al.2001.Simple preparation of drug sensitive test strips[J].Shanxi Medical Journal,30(5):447.)
赵硕珍,张云峰,姬胜利,等.2008.阿维拉霉素高产菌株的选育[J].中国生化药物杂志,29(4):256-259.(Zhao S Z,Ji S L,et al.2008.Screening avilamycin-producing strain for high-yield[J].Chinese Journal of Biochemical Pharmaceutics,29(4):256-259.)
邹君彪,姜平.2017.阿维拉霉素的研究进展[J].国外畜牧学(猪与禽),37(10):95-97.(Zou B,Jiang P.2017.Research progres of avilamycin[J].Pigs and Poultry,37(10):95-97.)
朱传合,贺亚男,韩振林,等.2006.产绿链霉菌发酵生产Avilamycin菌丝结团问题研究[J].生物技术,27(4):22-25.(Zhu C H,He Y N,Han Z L,et al.2006.Reseraching the problem of mycelium clustering in the process of avilamycin by Streptomycin viridochromogenes(SV-1)[J].Biotechnology,27(4):22-25.)
Aarestrup F M,Seyfarth A M,Emborg H D,et al.2001.Effect of abolishment of the use of antimicrobial agents for growth promotion on occurrence of antimicrobial resistance in fecal enterococci from food animals in denmark[J].Antimicrob Agents Chemother,45(7):2054-2059.
Hofmann C,Boll R,Heitmann B.et al.2005.Genes encoding enzymes responsible for biosynthesis of l-lyxose and attachment of eurekanate during avilamycin biosynthesis[J].Chemistry and Biology,12(10):1137-1143.
Liu Z,Zhao X Q,Bai F G.2013.Production of xylanase by an alkaline-tolerant marine-derived Streptomyces viridochromogenes strain and improvement by ribosome engineering[J].Applied Microbiology and Biotechnology,97:4361-4368.
Lv X A,Jin Y Y,Li Y D.et al.2013.Genome shuffling of Streptomyces viridochromogenes for improved production of avilamycin[J].Applied Microbiology and Biotechnology,97(2):641-648.
Ochi,K.,Okamoto,S.,Tozawa,Y,et al.2004.Ribosome engineering and secondary metabolite production[J].Advances in Applied Microbiology,56(1):155-179.
Sharma D,Cukras A R,Rogers E J,et al.2007.Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome[J].Journal of Molecular Biology,374(4):1065-1076.
Weitnauer G,Muhlenweg A,Trefzer A,et al.2001.Biosynthesis of the orthosomycin antibiotic avilamycin A:Deductions from the molecular analysis of the avi biosynthetic gene cluster of Streptomyces viridochromogenes Tu57 and production of new antibiotics[J].Chemistry and Biology,8(6):569-581.
Zhu X C,Kong J Q,Yang H,et al.2018.Strain improvement by combined UV mutagenesis and ribosome engineering and subsequent fermentation optimization for enhanced6'-deoxy-bleomycin Z production[J].Applied Microbiology and Biotechnology,102:1651-1661.
傅路,裘娟萍.2012.核糖体工程改良微生物菌种特性的研究进展[J].科技通报,11(8):78-83.(Fu L,Qiu J P.2012.Research advance of strain improvement by ribosome engineering[J].Bulletin of Science and Technology,28(11):78-83.)
蒋立锐.1988.谷氨酰胺在代谢中的作用[J].氨基酸和生物资源,4:17-23.(Jiang L R.1988.The role of glutamine in metabolism[J].Amino Acids and Biological Resources,4:17-23.)
蒋顺进.2018.原生质体ARTP诱变选育阿维拉霉素高产菌株[J].中国抗生素杂志,43(7):831-836.(Jiang S J.2018.Protoplast mutagenesis breeding of Streptomyces viridochromogene producing high-yield avilamycin by ARTP[J].Chinese Journal of Antibiotics,43(7):831-836.)
李红梅,陈敏.2017.响应面优化绿色产色链霉菌发酵产阿维拉霉素[J].中国食品学报,17(9):109-115.(Li H M,Chen M.2017.Fermentation optimization of avilamycin using Streptomyces viridochromogenes by response surface methodology[J].Journal of Chinese Institute of Food Science and Technology,17(9):109-115.)
梁新乐,金英,陈敏,等.2010.阿维拉霉素高产突变株H15形态分化初步研究[J].核农学报,24(4):689-693.(Liang XL,Jin Y Y,Chen M,et al.2010.Preliminary research on morphological differentiation of avilamycin High-yield mutant strain H15[J].Acta Agriculturae Nucleatae Sinica,24(4):689-693.)
梁新乐,朱静,金英燕.2012.绿色产色链霉菌E-219菌落的形态分化[J].核农学报,26(3):466-470.(Liang X L,Zhu J,Jin Y Y.2012.Morphological differentiation of Streptomyces viridochromogenes E-219 on solid culture[J].Acta Agriculturae Nucleatae Sinica,26(3):466-470.)
刘玲,朱湘成,黄勇.2016.微生物核糖体工程在抗生素研发中的应用[J].中国感染控制杂志,15(5):355-359.(Liu L,Zhu X C,Huang Y.2016.Application of microbial ribosome engineering in the research and development of antibiotics[J].Chinese Journal of Infection Control,15(5):355-359.)
刘芳,李晓荣,邹祥.2010.阿维拉霉素生物合成与代谢调控研究进展[J].生物技术通报,12:25-30.(Liu F,Li X R,Zou X.2010.Advances on biosynthesis approach and metabolic regulation of avilamycin[J].Biotechnology Bulletin,12:25-30.)
罗林根,杨燕,魏慧,等.2016.须糖多孢菌Saccharopolyspora pogona的核糖体工程改造对丁烯基多杀菌素合成的影响[J].生物工程学报,32(2):259-263.(Luo L G,Yang Y,Wei H,et al.2016.Effect of ribosome engineering of Saccharopolyspora pogona on synthesis of spinosyl spinosyn[J].Chinese Journal of Biotechnology,32(2):259-263.)
毛灵琪,李存治,陶兴无,等.2015.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,31(5):54-60.(Mao L Q,Li C Z,Tao X W,et al.2015.Screening and breeding strains producing high-yield avilamycin by genome shuffling[J].Biotechnology Bulletin,31(5):54-60.)
王庆龄.2015.阿维拉霉高产菌株选育[D].硕士学位论文,浙江工商大学,导师:梁新乐,pp.20-26.(Wang Q L.2015.Breeding high avilamycin-producing by Streptomyces viridochromogenes[D].Thesis for M.S.,Zhejiang Gongshang University,Supervisor:Liang X L,pp.20-26.)
向荣华,詹晓北,朱莉,等.2015.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,40(10):732-737.(Xiang R H,Zhan X B,Zhu L,et al.2015.Screening of high yield avilamycin mutant of Streptomyces viridochromogene through atmospheric and room temperature plasma mutagenesis[J].Chinese Journal of Antibiotics,40(10):732-737.)
谢庶洁,肖静,徐俊.2009.微生物核糖体工程研究进展[J].微生物学报,49(8):981-986.(Xie S J,Xiao J,Xu J.2009.Advance in microbial ribosome engineering[J].Acta microbiologica Sinica,49(8):981-986.)
袁晖,张新宜,王欣荣.2014.抗性突变他克莫司高产菌株的选育[J].微生物学杂志,34(5):81-86.(Yuan H,Zhang X Y,Wang X R.2014.Breeding of resistant mutant tacrolimus-high-producing Streptomyces strain SIIA-9818[J].Journal of Microbiology,34(5):81-86.)
于志斌,朱天骄,崔承彬,等.2006.用核糖体工程技术二次开发海洋微生物菌株资源的研究[J].高技术通讯,16(11):1190-1194.(Yu Z B,Zhu T J,Cui C B,et al.2006.A practical approach for exploiting useless microbial strains from marine environment by ribosome-engineering technology[J].High Technology Letters,16(11):1190-1194.)
张丹丹.2012.阿维拉霉素产生菌的株的定向育种及机制研究[D].硕士学位论文,浙江工商大学,导师:梁新乐,pp.25-30.(Zhang D D.2012.Rational breeding of high aviromycin-producers in Streptomyces viridochromogenes and deciphering its evolution mechanism[D].Thesis for M.S.,Zhejiang Gongshang University,Supervisor:Liang XL,pp.25-30.)
张宪明,赵韶星,高颖,等.2001.药敏试纸简单制备法[J].山西医药杂志,30(5):447.(Zhang X M,Zhao Y X,Gao Y,et al.2001.Simple preparation of drug sensitive test strips[J].Shanxi Medical Journal,30(5):447.)
赵硕珍,张云峰,姬胜利,等.2008.阿维拉霉素高产菌株的选育[J].中国生化药物杂志,29(4):256-259.(Zhao S Z,Ji S L,et al.2008.Screening avilamycin-producing strain for high-yield[J].Chinese Journal of Biochemical Pharmaceutics,29(4):256-259.)
邹君彪,姜平.2017.阿维拉霉素的研究进展[J].国外畜牧学(猪与禽),37(10):95-97.(Zou B,Jiang P.2017.Research progres of avilamycin[J].Pigs and Poultry,37(10):95-97.)
朱传合,贺亚男,韩振林,等.2006.产绿链霉菌发酵生产Avilamycin菌丝结团问题研究[J].生物技术,27(4):22-25.(Zhu C H,He Y N,Han Z L,et al.2006.Reseraching the problem of mycelium clustering in the process of avilamycin by Streptomycin viridochromogenes(SV-1)[J].Biotechnology,27(4):22-25.)
Aarestrup F M,Seyfarth A M,Emborg H D,et al.2001.Effect of abolishment of the use of antimicrobial agents for growth promotion on occurrence of antimicrobial resistance in fecal enterococci from food animals in denmark[J].Antimicrob Agents Chemother,45(7):2054-2059.
Hofmann C,Boll R,Heitmann B.et al.2005.Genes encoding enzymes responsible for biosynthesis of l-lyxose and attachment of eurekanate during avilamycin biosynthesis[J].Chemistry and Biology,12(10):1137-1143.
Liu Z,Zhao X Q,Bai F G.2013.Production of xylanase by an alkaline-tolerant marine-derived Streptomyces viridochromogenes strain and improvement by ribosome engineering[J].Applied Microbiology and Biotechnology,97:4361-4368.
Lv X A,Jin Y Y,Li Y D.et al.2013.Genome shuffling of Streptomyces viridochromogenes for improved production of avilamycin[J].Applied Microbiology and Biotechnology,97(2):641-648.
Ochi,K.,Okamoto,S.,Tozawa,Y,et al.2004.Ribosome engineering and secondary metabolite production[J].Advances in Applied Microbiology,56(1):155-179.
Sharma D,Cukras A R,Rogers E J,et al.2007.Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome[J].Journal of Molecular Biology,374(4):1065-1076.
Weitnauer G,Muhlenweg A,Trefzer A,et al.2001.Biosynthesis of the orthosomycin antibiotic avilamycin A:Deductions from the molecular analysis of the avi biosynthetic gene cluster of Streptomyces viridochromogenes Tu57 and production of new antibiotics[J].Chemistry and Biology,8(6):569-581.
Zhu X C,Kong J Q,Yang H,et al.2018.Strain improvement by combined UV mutagenesis and ribosome engineering and subsequent fermentation optimization for enhanced6'-deoxy-bleomycin Z production[J].Applied Microbiology and Biotechnology,102:1651-1661.