常温常压等离子体诱变选育高产L-异亮氨酸谷氨酸棒杆菌
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摘要
以谷氨酸棒杆菌(Corynebacterium glutamicum)23798为原始菌株,对其进行常温常压等离子体(ARTP)诱变,以磺胺胍抗性和氨基酸与茚三酮特异显色为筛选标记,以期得到高产L-异亮氨酸的诱变谷氨酸棒杆菌,并对其遗传稳定性进行研究。结果表明,原始菌株23798经过ARTP诱变处理180 s后,经0.4 mg/m L磺胺胍抗性筛选、多孔板高通量筛选、发酵培养复筛,选育出一株高产L-异亮氨酸诱变谷氨酸棒杆菌(Corynebacterium glutamicum)B1。该菌株在摇瓶中发酵培养48 h,L-异亮氨酸产量达18.5 g/L,比原始菌株提高62.03%,且遗传性状稳定。
Using Corynebacterium glutamicum 23798 as original strain, the original strain was mutated by atmospheric and room temperature plasma(ARTP). Using the sulfaguanidine resistance and the characteristic color of amino acid and ninhydrine as selection markers, the high-yield L-isoleucine C. glutamicum mutant was obtained, and its genetic stability was studied. The results showed that after the original strain 23798 was mutated for 180 s by ARTP, a high-yield L-isoleucine C. glutamicum mutant strain B1 was bred by 0.4 mg/ml sulfaguanidine resistance screening, perforated plate high-throughput screening and fermentation rescreening. The fermentation of strain with stable genetic character was carried out in shake flask for 48 h,and the L-leucine yield reached 18.5 g/L, which was 62.03% higher than that of the original strain.
Using Corynebacterium glutamicum 23798 as original strain, the original strain was mutated by atmospheric and room temperature plasma(ARTP). Using the sulfaguanidine resistance and the characteristic color of amino acid and ninhydrine as selection markers, the high-yield L-isoleucine C. glutamicum mutant was obtained, and its genetic stability was studied. The results showed that after the original strain 23798 was mutated for 180 s by ARTP, a high-yield L-isoleucine C. glutamicum mutant strain B1 was bred by 0.4 mg/ml sulfaguanidine resistance screening, perforated plate high-throughput screening and fermentation rescreening. The fermentation of strain with stable genetic character was carried out in shake flask for 48 h,and the L-leucine yield reached 18.5 g/L, which was 62.03% higher than that of the original strain.
引文
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[22]陈倩,刘晶,张璐.纸层析法分离鉴定氨基酸实验的改进[J].化工管理,2017(12):25-26.
[23]丁松,黄和,胡燚.氨基酸分析研究进展[J].生物加工过程,2018,16(3):12-21.
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[2]WANG J,WEN B,XU Q Y,et al.Optimization of carbon source and glucose feeding strategy for improvement of L-isoleucine production by Escherichia coli[J].Biotechnol Biotechnol Equipment,2015,29(2):374-380.
[3]谢飞,崔国英,王翀,等.L-异亮氨酸高产菌种的选育研究[J].中国酿造,2014,33(11):98-100.
[4]李忠财.高产L-异亮氨酸谷氨酸棒杆菌的菌种选育及代谢优化[D].大连:大连工业大学,2016.
[5]SHI F,HUAN X J,WANG X Y,et al.Overexpression of NAD kinases improves the L-isoleucine biosynthesis in Corynebacterium glutamicum ssp.lactofermentum[J].Enzyme Microb Tech,2012,51(2):73-80.
[6]YIN L,HU X,XU D,et al.Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase L-isoleucine production in Corynebacterium glutamicum[J].Metab Eng,2012,14(7):542-550.
[7]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):37-45.
[8]TAN Y,FANG M,JIN L,et al.Culture characteristics of the atmospheric and room temperature plasma-mutated Spirulina platensis mutants in CO2aeration culture system for biomass production[J].J Biosci Bioeng,2015,120(4):438-443.
[9]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,2016,65(7):2676-2685.
[10]CAO S,ZHOU X,JIN W.Improving of lipid productivity of the oleaginous microalgae Chlorella pyrenoidosa via atmospheric and room temperature plasma(ARTP)[J].Bioresource Technol,2017,244(5):1400-1406.
[11]王方方,孙沛勇,银会娟,等.常压室温等离子体快速诱变酒精酵母及其突变株的特性研究[J].中国酿造,2013,32(10):117-120.
[12]孙小龙,付永前,徐睛,等.米根霉交替呼吸途径高活性突变株的选育及其对富马酸合成的影响[J].生物加工过程,2015,13(3):26-33.
[13]曹纲,刘云,郭金玲,等.高产酮基还原酶的乳酸克鲁维酵母选育及其整细胞催化特性[J].中国酿造,2018,37(5):42-28.
[14]BAO H,LIU R,LIANG L,et al.Succinic acid production from hemicellulose hydrolysate by an Escherichia coli mutant obtained by atmospheric and room temperature plasma and adaptive evolution[J].Enzyme Microb Tech,2014,66(7):10-15.
[15]宋文军.L-异亮氨酸高产菌的选育及其发酵条件研究[D].天津:天津科技大学,2004.
[16]谢兰香,沈加彬,罗磊,等.L-异亮氨酸产生菌的选育[J].氨基酸和生物资源,2013,35(4):60-63.
[17]LI Y J,WEI H,WANG T,et al.Current status on metabolic engineering for the production of L-aspartate family amino acids and derivatives[J].Bioresource Technol,2017,245(12):1588-1602.
[18]MUSTAFI N,ALEXANDER G,DIETRICH K,et al.The development and application of a single-cell biosensor for the detection of L-methionine and branched-chain amino acids[J].Metab Eng,2012,14(2):449-457.
[19]刘丽君.L-异亮氨酸产生菌的选育及发酵条件研究[D].吉林:吉林大学,2015.
[20]马文渐.改造谷氨酸棒杆菌生产L-异亮氨酸及PHA[D].无锡:江南大学,2018.
[21]郑明英,蔡友华,陆最青.常压室温等离子体快速诱变筛选高脯氨酸产率突变株[J].食品与发酵工业,2013,39(1):36-40.
[22]陈倩,刘晶,张璐.纸层析法分离鉴定氨基酸实验的改进[J].化工管理,2017(12):25-26.
[23]丁松,黄和,胡燚.氨基酸分析研究进展[J].生物加工过程,2018,16(3):12-21.
[24]葛祎楠,邹静,李斌板.栗大米清酒酒曲选择及氨基酸含量测定[J].中国酿造,2018,37(11):62-66.