常压室温等离子体(ARTP)诱变选育高核酸酿酒酵母
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摘要
核糖核酸(RNA)是一类非常重要的生物分子,降解后得到的核苷酸、核苷及碱基具有广泛用途。酿酒酵母是目前生产RNA的主要食品级微生物。本研究采用常压室温等离子体(ARTP)技术进行酿酒酵母诱变育种,利用氯化钾敏感性筛选,多次反复诱变最终得到在以糖蜜为碳源的摇瓶试验中RNA含量为112 mg-RNA/g-DCW,提高了39%的突变菌株Y17aM3。经过对Y17aM3培养条件优化后,确定生产RNA最适接种量为10%,最适p H为5.5,最适温度为26℃,且传代稳定性良好。研究发现在最佳培养条件下,添加磷酸可使Y17aM3的RNA含量提高至119 mg-RNA/g-DCW;添加蛋白胨可使Y17aM3的RNA含量提高至122 mg-RNA/g-DCW。上述结果不仅证明ARTP诱变育种方法突变效果显著,可应用于工业微生物的选育,而且有助于降低基于核苷酸的食品添加剂的生产成本。
As an important biomolecule, ribonucleic acid(RNA) can be degraded into nucleotides, nucleosides and bases which exhibit a great potential in wide applications. Saccharomyces cerevisiae is widely used in food industry and generally regarded as safe are the preferred source of nucleic acid. In this study, the cells were repeatedly exposed under atmospheric and room temperature plasma(ARTP), with the aim to screen high-yield nucleic acid strains with potassium chloride sensitivity. A mutant strain Y17aM3 was successfully obtained. In shake flask test with molasses as carbon source, Y17aM3 contained 112 mg-RNA/g-DCW RNA, 39% higher than that of the untreated control. The optimal inoculation quantity of Y17aM3 to produce RNA was 10%.The optimal pH and temperature of fermentation was 5.5 and 26 ℃, respectively. When culturing with optimal condition, it was found that adding phosphoric acid increased RNA content to 119 mg-RNA/g-DCW, and adding peptone increased RNA content to 122 mg-RNA/g-DCW. The results not only demonstrate the effectiveness of using ARTP mutation strategy for the industrial microorganism breeding, but also can greatly reduce the production cost of nucleotide-based food additives.
As an important biomolecule, ribonucleic acid(RNA) can be degraded into nucleotides, nucleosides and bases which exhibit a great potential in wide applications. Saccharomyces cerevisiae is widely used in food industry and generally regarded as safe are the preferred source of nucleic acid. In this study, the cells were repeatedly exposed under atmospheric and room temperature plasma(ARTP), with the aim to screen high-yield nucleic acid strains with potassium chloride sensitivity. A mutant strain Y17aM3 was successfully obtained. In shake flask test with molasses as carbon source, Y17aM3 contained 112 mg-RNA/g-DCW RNA, 39% higher than that of the untreated control. The optimal inoculation quantity of Y17aM3 to produce RNA was 10%.The optimal pH and temperature of fermentation was 5.5 and 26 ℃, respectively. When culturing with optimal condition, it was found that adding phosphoric acid increased RNA content to 119 mg-RNA/g-DCW, and adding peptone increased RNA content to 122 mg-RNA/g-DCW. The results not only demonstrate the effectiveness of using ARTP mutation strategy for the industrial microorganism breeding, but also can greatly reduce the production cost of nucleotide-based food additives.
引文
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[16]杨依顺,丁玥,曹静,等.酿酒酵母发酵制备RNA的研究[J].药物生物技术,2013,4:310-313YANG Yi-shun,DING Yue,CAO Jing,et al.Studies on the preparation of RNA from Saccharomyces Cerevisiae[J].Pharmaceutical Biotechnology,2013,4:310-313
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[18]陈文明,郑国斌,姚娟,等.高产RNA酿酒酵母培养条件及发酵培养基的优化[J].中国调味品,2015,40(11):28-32CHEN Wen-ming,ZHENG Guo-bin,YAO Juan,et al.Optimization of culture conditions and fermentation medium of Saccharomyces cerevisiae of high-yield RNA[J].China Condiment,2015,40(11):28-32
[19]封晓霞,王雅琴.复合诱变和原生质体融合选育S-腺苷甲硫氨酸酿酒酵母高产菌株[J].北京化工大学学报(自然科学版),2011,38(6):87-92FENG Xiao-xia,WANG Ya-qin.Breeding of Saccharomyces cerevisiae giving an enhanced yield of S-adenosylL-methionine by composite mutagenesis and protoplast fusion[J].Journal of Beijing University of Chemical Technology(Nature Science),2011,38(6):87-92
[20]赵文若,李新江,包岩.生物统计学[M].吉林大学出版社,2016ZHAO Wen-ruo,LI Xin-jiang,BAO Yan.Biostatistics[M].Jilin University Press,2016
[21]Kim M.Determination of mutation rates of mating-type gene switching in Saccharomyces cerevisiae[D].Brandeis University,2009
[22]Jones E W,Pringle J R,Broach J R,et al.The Molecular and cellular biology of the yeast saccharomyces[M].Cole Spring Harbor Laboratory Press,1992:340-340
[23]Wang L,Chen X,Wu G,et al.Improvedε-poly-L-lysine production of Streptomyces,sp.FEEL-1 by atmospheric and room temperature plasma mutagenesis and streptomycin resistance screening[J].Annals of Microbiology,2015,65(4):2009-2017
[24]Vankudoth K R,Shilpa P,Rao K N,et al.Influence of carbon and nitrogen source on growth,don and niv production by two species of Fusarium isolated from finger millets[J].International Journal of Pharmacy&Pharmaceutical Sciences,2015,7(3):136-139
[25]Vranova V,Rejsek K,Formanek P.Proteolytic activity in soil:A review[J].Applied Soil Ecology,2013,70:23-32
[26]Zhang X,Zhang C,Zhou Q Q,et al.Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma(ARTP)and conventional mutagenesis[J].Applied Microbiology&Biotechnology,2015,99(13):5639
[2]Sasano Y,Kariya T,Usugi S,et al.Molecular breeding of Saccharomyces cerevisiae with high RNA content by harnessing essential ribosomal RNA transcription regulator[J].Amb Express,2017,7(1):32
[3]俞灿,郑国斌,姚娟,等.富含核糖核酸酿酒酵母的选育及其高密度发酵工艺[J].中国酿造,2016,35(12):66-71YU Can,ZHENG Guo-bin,YAO Juan,et al.Breeding and high density fermentation technology of Saccharomyces cerevisiae with high RNA content[J].China Brewing,2016,35(12):66-71
[4]任华景.核糖核酸高产菌株选育及其应用研究[D].南京工业大学,2011REN Hua-jing.Breeding high-yield strain for ribonucleic acid production its application[D].Nanjing University of Technology,2011
[5]Rollero S,Bloem A,Camarasa C,et al.Combined effects of nutrients and temperature on the production of fermentative aromas by Saccharomyces cerevisiae during wine fermentation[J].Applied Microbiology&Biotechnology,2015,99(5):2291
[6]张雪,张晓菲,王立言,等.常压室温等离子体生物诱变育种及其应用研究进展[J].化工学报,2014,65(7):2676-2684ZHANG Xue,ZHANG Xiao-fei,WANG Li-yan,et al.Recent progress on atmospheric and room temperature plasma mutation breeding technology and its applications[J].CIESC Journal,2014,65(7):2676-2684
[7]Dey A,Chroneos A,Braithwaite N S J,et al.Plasma engineering of graphene[J].Applied Physics Reviews,2016,3(2):666
[8]Zhang X,Zhang C,Zhou Q Q,et al.Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma(ARTP)and conventional mutagenesis[J].Applied Microbiology&Biotechnology,2015,99(13):5639
[9]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,2017,1:37-45WU Yi-nan,XING Xin-hui,ZHANG Chong,et al.Recent progress on atmospheric and room temperature plasma(ARTP)biobreeding technology,instrumentation and its industrialization[J].Biotechnology&Business,2017,1:37-45
[10]Herbert D,Phipps P J,Strange R E.Chapter III chemical analysis of microbial cells[J].Methods in Microbiology,1971,5:209-344
[11]赵海泉.微生物学实验指导[M].中国农业大学出版社,2014ZHAO Hai-quan.Experiment and guide for microbiology[M].China Agricultural University Press,2014
[12]杨瑞娟.高产酒率酿酒酵母单倍体的分离与杂交育种[D].浙江:浙江大学,2007YANG Rui-juan.Study on seperation of haploids from diploid cells of Saccharomyces cerevisiae and hybridization breeding of strains with high ethanol production[D].Zhejiang:Zhejiang University,2007
[13]侯丽华.酿酒酵母遗传操作方法研究及乙醇发酵高产菌株构建[D].天津:天津大学,2007HOU Li-hua.Development of new methods for genetic manipulation in Saccharomyces cerevisiae and construction of strains with improved ethanol yield[D].Tianjin:Tianjin University,2007
[14]赵宇,刘珊珊,陈叶福,等.ARTP诱变以及基因组重排筛选具有耐高温性能的酿酒酵母[J].现代食品科技,2017,33(11):37-41ZHAO Yu,LIU Shan-shan,CHEN Ye-fu,et al.Screening the Saccharomyces Cerevisiae with high temperature toleranceby ARTP mutagenesis and genome shuffling[J].Modern Food Science&Technology,2017,33(11):37-41
[15]李英英,杭海峰,庄英萍,等.常压常温等离子体(ARTP)诱变选育顶头孢霉高产菌[J].中国抗生素杂志,2017,42(11):945-950LI Ying-ying,HANG Hai-feng,ZHUANG Ying-ping,et al.High throughput screening of high-yield cephalosporin Cproducing strain by mutagenesis using atmospheric and room temperature plasma(ARTP)[J].Chinese Journal of Antibiotics,2017,42(11):945-950
[16]杨依顺,丁玥,曹静,等.酿酒酵母发酵制备RNA的研究[J].药物生物技术,2013,4:310-313YANG Yi-shun,DING Yue,CAO Jing,et al.Studies on the preparation of RNA from Saccharomyces Cerevisiae[J].Pharmaceutical Biotechnology,2013,4:310-313
[17]Barnett J A,Payne R W,Yarrow D.Yeasts:characteristics and identification,3rd edition[J].Pediatric Dermatology,2001,18(6):547-547
[18]陈文明,郑国斌,姚娟,等.高产RNA酿酒酵母培养条件及发酵培养基的优化[J].中国调味品,2015,40(11):28-32CHEN Wen-ming,ZHENG Guo-bin,YAO Juan,et al.Optimization of culture conditions and fermentation medium of Saccharomyces cerevisiae of high-yield RNA[J].China Condiment,2015,40(11):28-32
[19]封晓霞,王雅琴.复合诱变和原生质体融合选育S-腺苷甲硫氨酸酿酒酵母高产菌株[J].北京化工大学学报(自然科学版),2011,38(6):87-92FENG Xiao-xia,WANG Ya-qin.Breeding of Saccharomyces cerevisiae giving an enhanced yield of S-adenosylL-methionine by composite mutagenesis and protoplast fusion[J].Journal of Beijing University of Chemical Technology(Nature Science),2011,38(6):87-92
[20]赵文若,李新江,包岩.生物统计学[M].吉林大学出版社,2016ZHAO Wen-ruo,LI Xin-jiang,BAO Yan.Biostatistics[M].Jilin University Press,2016
[21]Kim M.Determination of mutation rates of mating-type gene switching in Saccharomyces cerevisiae[D].Brandeis University,2009
[22]Jones E W,Pringle J R,Broach J R,et al.The Molecular and cellular biology of the yeast saccharomyces[M].Cole Spring Harbor Laboratory Press,1992:340-340
[23]Wang L,Chen X,Wu G,et al.Improvedε-poly-L-lysine production of Streptomyces,sp.FEEL-1 by atmospheric and room temperature plasma mutagenesis and streptomycin resistance screening[J].Annals of Microbiology,2015,65(4):2009-2017
[24]Vankudoth K R,Shilpa P,Rao K N,et al.Influence of carbon and nitrogen source on growth,don and niv production by two species of Fusarium isolated from finger millets[J].International Journal of Pharmacy&Pharmaceutical Sciences,2015,7(3):136-139
[25]Vranova V,Rejsek K,Formanek P.Proteolytic activity in soil:A review[J].Applied Soil Ecology,2013,70:23-32
[26]Zhang X,Zhang C,Zhou Q Q,et al.Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma(ARTP)and conventional mutagenesis[J].Applied Microbiology&Biotechnology,2015,99(13):5639