谷氨酸棒杆菌天冬氨酸激酶基因的克隆和应用研究
|
摘要
运用传统育种手段筛选到谷氨酸棒杆YS的多重代谢突变组合菌株YS-C。该菌株为苏氨酸营养缺陷型，具有对赖氨酸的结构类似物AEC和AHV的抗性，并且能以琥珀酸为唯一碳源生长，能够耐受高浓度亮氨酸。突变株YS-C的赖氨酸产量为1.2g/l发酵液，是出发亲株YS的赖氨酸产量0.35g/l的3.42倍。运用PCR的方法从谷氨酸棒杆菌AEC抗性菌株GX染色体上扩增出天冬氨酸激酶基因，并构建重组穿梭质粒pELY。利用电转化的方法将重组穿梭质粒pELY转入多重代谢突变组合菌株YS-C，获得重组谷氨酸棒杆菌菌株：CZL。该菌的赖氨酸生产能力为6.94g/l，是YS-C菌株赖氨酸产量的5.78倍，是YS菌株赖氨酸产量的19.8倍。通过摇瓶发酵实验初步确定CZL的发酵条件为：初糖：150-180g/l，采用无机氮源和有机氮源配和使用，最佳组合是：硫酸铵质量浓度为55g/l；酵母膏质量浓度为15g/l，牛肉膏质量浓度为25g/l为最佳。在500ml的三角瓶装液量为30ml于往复式摇床上振荡培养，频率为115rpm，振幅78mm，温度30℃，培养时间48。优化摇瓶发酵条件后，重组菌的赖氨酸积累量可以达到20.8g/l。
A combined metabolic mutated strain (YS -C)was get by traditional filter method from Corynebacterium glutamicum(YS).This mutant is threonine auxotrop,AEC and AHV resistant,and it can grow in the medium which contants SCA as the only source of carbon,and the mutant can endure hight leveal of Leu .The mutanted strain can concentrate lysine.The concentration of lysine leveal was 1.20g/l. It was 3.42 times than the former strain YS.
Aspartokinase gene (lysC) from AEC resistant mutant Corynebacterium glutamicum GX was cloned and sequened.The lysC gene was recombinanted with the shuttle plasmid vector pEK to form recombinanted plasmid pELY. A recombine Corynebacterium glutamicum strain CZL which contains recombinanted plasmid pELY was transformated by eletric shock. Strain CZL can accumulate 6.94g/l lysine, which was 5.78 times than YS-C and 19.8 times than YS.
Shake-flask culture condition of lysine fermentation were studied with CZL .Appropriate feeding of organic niteogen sources was favorable for fermentation jn our experiment, the concentations of the yeast extract and beef extract were 15g/l and 25g/l .The initial (NH02SO4 and glucose concentrations suitable for fermentor production ranged form 55g/l and 150-180g/l respectively. The optimal condition of fermenation was 30' C, 48hours, and the vibration frequency was 115 rpm . 20g/l of Lys concentrtion was obtained under this condition.
A combined metabolic mutated strain (YS -C)was get by traditional filter method from Corynebacterium glutamicum(YS).This mutant is threonine auxotrop,AEC and AHV resistant,and it can grow in the medium which contants SCA as the only source of carbon,and the mutant can endure hight leveal of Leu .The mutanted strain can concentrate lysine.The concentration of lysine leveal was 1.20g/l. It was 3.42 times than the former strain YS.
Aspartokinase gene (lysC) from AEC resistant mutant Corynebacterium glutamicum GX was cloned and sequened.The lysC gene was recombinanted with the shuttle plasmid vector pEK to form recombinanted plasmid pELY. A recombine Corynebacterium glutamicum strain CZL which contains recombinanted plasmid pELY was transformated by eletric shock. Strain CZL can accumulate 6.94g/l lysine, which was 5.78 times than YS-C and 19.8 times than YS.
Shake-flask culture condition of lysine fermentation were studied with CZL .Appropriate feeding of organic niteogen sources was favorable for fermentation jn our experiment, the concentations of the yeast extract and beef extract were 15g/l and 25g/l .The initial (NH02SO4 and glucose concentrations suitable for fermentor production ranged form 55g/l and 150-180g/l respectively. The optimal condition of fermenation was 30' C, 48hours, and the vibration frequency was 115 rpm . 20g/l of Lys concentrtion was obtained under this condition.
引文
[1] Kinoshita S. Vdakas, shimono MJ Gen Apple Microbil 1957 3:193-205
[2] 中国生物工程学会 中国生物技术产业发展报告2002 化学化工出版社 2003
[3] Sahm H. Eggeling L Natumis Snchaflen, 1999 86:33-38
[4] Weuelisch VF, DE Graaf AA, sahm H.et al-J Bacteriol, 2000 182:3088-3096
[5] Ikeda M. Katsuma R. Apple Environ Microbiol 1992.58:781-785
[6] Eggeling L. Amino Aciols 1994.6:261-272
[7] Gubler M Park SMjetten M et al Appl Microbiol Biotechncl 1994 40:857-863
[8] 阮红 Bernhard Eikmanns 微生物学报 2002 8:458-404
[9] 张克旭 氨基酸发酵工艺学 轻工业出版社 1997
[10] Eikmanns J. Eggeling L. Sham H Antonie Van leeuuenhock 1993 64:145-163
[11] Kalinowski Bachmann B Thierbach G, et al mol Gen Genet 1990 224:317-324
[12] Tosaka O, Takinamik Agric Biol Chem 1978 42:95-100
[13] 陶文昕 微生物发酵生理及遗传育种 轻工出版社 1997
[14] 刘阳剑 钝齿棒杆菌AK基因的克隆和序列分析 微生物学报 2002 8：395-399
[15] Jetten M, Follettic T. Sinskey J Appl Microbiol Biotechnol 1995 43:76-82
[16] J．萨姆布鲁克．E．F．弗里奇．T．曼尼阿蒂斯著 分子克隆实验指南 第二版 北京科学技术出版社 1992
[17] 石渡昭南 赖氨酸发酵[P] 日本特许公报 50 (874) 1975-01-02
[18] 北京大学生物系生化教研室 生物化学实验指导 北京高等教育出版社 1989
[19] 齐秀兰，阎浩林，L-赖氨酸产生菌钝齿棒杆菌高产突变株的选育 沈阳药科大学学报 1997，4，103-106
[20] Sahm H, Eggling L.Natumissnschaflen, 1999,86,33-38
[21] 阮红, Eikmanns.微生物学报, 2002, 42 (3): 326-330
[22] Hueck CJ,Hillen w. Mol Microbiol, 1995,15:395-401
[23] Kakuda H,Hosomo K, Shiroishi K, et al J Biochem, 1994,116:916-922
[24] 王红，齐秀兰，紫外诱变原生质体选育赖氨酸高产菌株，生物工程学报，1990，6(1)：32-38
[25] Steinbuchel A, SchlegelG Appl Microbiol Biotechnol, 1989,31:168-175
[26] 陈琪，李玲阁。微生物学报，1975，15(5)：1197-1204
[27] Shio I Miyajima R J Biotechnol 1969,65: 849-859
[28] 汪家政，范明，蛋白质技术手册．科学出版社，2001
[29] 卢圣栋，现代分子生物学实验技术．高教出版社，1998
[30] 张惠展，基因工程概论．华东理工大学出版社，2001
[31] J Kalinowski,J.Cremer, B.Bachmunn Mol Micro 1991, 5: 1197-1204
[32] J.H.Lu and C.C Liao Letters in Applied Microbiology 1997,24:211-213
[33] Sunita Kochhar Henry Pahlus. Microbiology 1996,142:1635-1639
[34] Jun Sun Ilse Smets Jan Van Impe Appl and Environmental Micro Biology 2001,8:3350-3357
[35] Berhard J.Eikmanns,Markus Metzger. Appl Microbiol Biotechnol 1991 34:617-622
[36] 那淑敏，贾盘兴 棒杆菌宿主中质粒不稳定性的研究 生物工程学报 1994 10：234-238
[37] 邓兆鑫，马楚平．谷氨酸棒杆菌嵌合质粒的组建生物工程学报 1987 3：183-188
[2] 中国生物工程学会 中国生物技术产业发展报告2002 化学化工出版社 2003
[3] Sahm H. Eggeling L Natumis Snchaflen, 1999 86:33-38
[4] Weuelisch VF, DE Graaf AA, sahm H.et al-J Bacteriol, 2000 182:3088-3096
[5] Ikeda M. Katsuma R. Apple Environ Microbiol 1992.58:781-785
[6] Eggeling L. Amino Aciols 1994.6:261-272
[7] Gubler M Park SMjetten M et al Appl Microbiol Biotechncl 1994 40:857-863
[8] 阮红 Bernhard Eikmanns 微生物学报 2002 8:458-404
[9] 张克旭 氨基酸发酵工艺学 轻工业出版社 1997
[10] Eikmanns J. Eggeling L. Sham H Antonie Van leeuuenhock 1993 64:145-163
[11] Kalinowski Bachmann B Thierbach G, et al mol Gen Genet 1990 224:317-324
[12] Tosaka O, Takinamik Agric Biol Chem 1978 42:95-100
[13] 陶文昕 微生物发酵生理及遗传育种 轻工出版社 1997
[14] 刘阳剑 钝齿棒杆菌AK基因的克隆和序列分析 微生物学报 2002 8：395-399
[15] Jetten M, Follettic T. Sinskey J Appl Microbiol Biotechnol 1995 43:76-82
[16] J．萨姆布鲁克．E．F．弗里奇．T．曼尼阿蒂斯著 分子克隆实验指南 第二版 北京科学技术出版社 1992
[17] 石渡昭南 赖氨酸发酵[P] 日本特许公报 50 (874) 1975-01-02
[18] 北京大学生物系生化教研室 生物化学实验指导 北京高等教育出版社 1989
[19] 齐秀兰，阎浩林，L-赖氨酸产生菌钝齿棒杆菌高产突变株的选育 沈阳药科大学学报 1997，4，103-106
[20] Sahm H, Eggling L.Natumissnschaflen, 1999,86,33-38
[21] 阮红, Eikmanns.微生物学报, 2002, 42 (3): 326-330
[22] Hueck CJ,Hillen w. Mol Microbiol, 1995,15:395-401
[23] Kakuda H,Hosomo K, Shiroishi K, et al J Biochem, 1994,116:916-922
[24] 王红，齐秀兰，紫外诱变原生质体选育赖氨酸高产菌株，生物工程学报，1990，6(1)：32-38
[25] Steinbuchel A, SchlegelG Appl Microbiol Biotechnol, 1989,31:168-175
[26] 陈琪，李玲阁。微生物学报，1975，15(5)：1197-1204
[27] Shio I Miyajima R J Biotechnol 1969,65: 849-859
[28] 汪家政，范明，蛋白质技术手册．科学出版社，2001
[29] 卢圣栋，现代分子生物学实验技术．高教出版社，1998
[30] 张惠展，基因工程概论．华东理工大学出版社，2001
[31] J Kalinowski,J.Cremer, B.Bachmunn Mol Micro 1991, 5: 1197-1204
[32] J.H.Lu and C.C Liao Letters in Applied Microbiology 1997,24:211-213
[33] Sunita Kochhar Henry Pahlus. Microbiology 1996,142:1635-1639
[34] Jun Sun Ilse Smets Jan Van Impe Appl and Environmental Micro Biology 2001,8:3350-3357
[35] Berhard J.Eikmanns,Markus Metzger. Appl Microbiol Biotechnol 1991 34:617-622
[36] 那淑敏，贾盘兴 棒杆菌宿主中质粒不稳定性的研究 生物工程学报 1994 10：234-238
[37] 邓兆鑫，马楚平．谷氨酸棒杆菌嵌合质粒的组建生物工程学报 1987 3：183-188