枯草芽孢杆菌rbsK基因和tkt基因的敲除对核黄素发酵的影响
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摘要
本课题的研究内容是通过在枯草芽孢杆菌中敲除rbsK基因,减少核黄素生产途径的副产物,进而考查对核黄素产量的影响。同时,敲除tkt基因即构建转酮酶缺陷菌株,使rbsK的敲除不至于引起其他副产物支路通量的增加,影响发酵结果。
rbsK基因在戊糖磷酸途径中表达核糖激酶,进一步催化D-核糖的生成。该基因的敲除理论上将造成D-核糖-5磷酸的积累。由于核黄素的生成需要两种直接前体物,其合成方程式为2DHPB+DARPP=Riboflavin,这两种前体物分别由核黄素生产的长途径和短途径提供,而D-核糖-5磷酸又是这两条途径必需的前体物。同时,为避免由于D-核糖生成支路的切断引起另一个方向酮糖生成支路通量的增大,减少不必要的副产物的生成,本实验设计方案敲除tkt基因。以rbsK基因的敲除为例:经PCR扩增得到的rbsK基因片段进行酶切,再与以同样内切酶切后的载体质粒pUC18在大肠杆菌中进行酶连,得到第一个重组质粒pUC18-rbsK;在pUC18-rbsK质粒的rbsK片段上选择合适的酶切位点进行酶切,再与同样酶切后的抗性基因(Spc、Kan抗性等)进行酶连,得到第二个重组质粒pUC18-rbsK-Spc或pUC18-rbsK-Kan,使rbsK基因不能完整表达而失活;构建成功的整合型敲除质粒pUC18-rbsK-Spc或pUC18-rbsK-Kan在枯草芽孢杆菌中双交换整合,利用插入的抗性基因对菌株进行抗性筛选,最终实现了在枯草芽孢杆菌中敲除rbsK基因的目的。同理,经一系列酶切、酶连、转化、筛选等操作后,在枯草芽孢杆菌中可实现tkt基因的敲除。
本实验主要在核黄素生产菌株RH33、RH44中敲除rbsK基因和tkt基因,基因操作完成后,对新构建的工程菌与出发菌株进行考察对比。首先利用LC-MS手段检测基因敲出前后菌株的相关代谢物,即D-核糖,一系列酮糖等的相对变化,结果显示,两个基因缺陷的菌株不再合成D-核糖,相关酮糖(以7磷酸-景天庚酮糖为检测目标)的产生量也有了较大程度的降低;摇瓶发酵后,发现两个基因的敲除对RH44的影响明显大于RH33。其中,两基因缺陷的工程菌RH44(R- Spcr)摇瓶发酵72 h后,核黄素产量最高可达到4.98 g,比出发菌株提高了约14.5%。
The purpose of this paper is increasing the precursor of riboflavin in Bacillus subtilis by knocking out rbsK gene, and then examining its yield. At the same time, the knockout of tkt gene causing Transketolase defecting that is constructed, so the knockout of rbsK cannot lead to flux increasing in other branches, which affecting the fermentation results.
rbsK gene expresses ribose kinase in pentose phosphate pathway, and further catalyzed the formation of D-ribose. Since the direct formation of riboflavin needs two precursors, that is 2DHPB + DARPP = Riboflavin, riboflavin produces by a long pathway and a short one, and D-ribose-5P is necessary for the two pathway precursors. Also, to avoid the flux increasing in other unwanted by-product pathway, we design to knock tkt gene out. Take the knockout of rbsK gene as example: rbsK gene fragments amplified by PCR obtain by digestion with the same end nuclease-off plasmid pUC18 in E. coli. Then we get the first recombinant plasmid pUC18- rbsK; in pUC18-rbsK the rbsK fragment of the selection which has suitable restriction sites were digested, and then linked with the same enzyme digested resistance gene (Spc, Kan resistance, etc.), there we get the second plasmid pUC18-rbsK-Spc or pUC18-rbsK-Kan, so rbsK gene can not express completely inactivation; Successfully built integrating plasmid pUC18-rbsK-Spc or pUC18-rbsK-Kan in Bacillus subtilis double exchange integration by inserting resistance gene express in Bacillus subtilis finally. Similarly, after a series of enzyme cutting, linking, transformation, screening operation, tkt gene can be knocked in Bacillus subtilis.
This paper mailly introduces the knockout of rbsK gene and tkt gene in riboflavin producing strains RH33 and RH44, then we make some testing on the strains. First, some related metabolites in the strains constrcted before and after are examined, and we find D-ribose disappearing and Sedoheptulose-7P decreasing after the knockout of two genes. Then the strains ferment in flasks, and we find the effection of knocking out two strains is greater in RH44, which we get a strain with 4.98 g of riboflavin productin, 14.5% higher than before.
rbsK基因在戊糖磷酸途径中表达核糖激酶,进一步催化D-核糖的生成。该基因的敲除理论上将造成D-核糖-5磷酸的积累。由于核黄素的生成需要两种直接前体物,其合成方程式为2DHPB+DARPP=Riboflavin,这两种前体物分别由核黄素生产的长途径和短途径提供,而D-核糖-5磷酸又是这两条途径必需的前体物。同时,为避免由于D-核糖生成支路的切断引起另一个方向酮糖生成支路通量的增大,减少不必要的副产物的生成,本实验设计方案敲除tkt基因。以rbsK基因的敲除为例:经PCR扩增得到的rbsK基因片段进行酶切,再与以同样内切酶切后的载体质粒pUC18在大肠杆菌中进行酶连,得到第一个重组质粒pUC18-rbsK;在pUC18-rbsK质粒的rbsK片段上选择合适的酶切位点进行酶切,再与同样酶切后的抗性基因(Spc、Kan抗性等)进行酶连,得到第二个重组质粒pUC18-rbsK-Spc或pUC18-rbsK-Kan,使rbsK基因不能完整表达而失活;构建成功的整合型敲除质粒pUC18-rbsK-Spc或pUC18-rbsK-Kan在枯草芽孢杆菌中双交换整合,利用插入的抗性基因对菌株进行抗性筛选,最终实现了在枯草芽孢杆菌中敲除rbsK基因的目的。同理,经一系列酶切、酶连、转化、筛选等操作后,在枯草芽孢杆菌中可实现tkt基因的敲除。
本实验主要在核黄素生产菌株RH33、RH44中敲除rbsK基因和tkt基因,基因操作完成后,对新构建的工程菌与出发菌株进行考察对比。首先利用LC-MS手段检测基因敲出前后菌株的相关代谢物,即D-核糖,一系列酮糖等的相对变化,结果显示,两个基因缺陷的菌株不再合成D-核糖,相关酮糖(以7磷酸-景天庚酮糖为检测目标)的产生量也有了较大程度的降低;摇瓶发酵后,发现两个基因的敲除对RH44的影响明显大于RH33。其中,两基因缺陷的工程菌RH44(R- Spcr)摇瓶发酵72 h后,核黄素产量最高可达到4.98 g,比出发菌株提高了约14.5%。
The purpose of this paper is increasing the precursor of riboflavin in Bacillus subtilis by knocking out rbsK gene, and then examining its yield. At the same time, the knockout of tkt gene causing Transketolase defecting that is constructed, so the knockout of rbsK cannot lead to flux increasing in other branches, which affecting the fermentation results.
rbsK gene expresses ribose kinase in pentose phosphate pathway, and further catalyzed the formation of D-ribose. Since the direct formation of riboflavin needs two precursors, that is 2DHPB + DARPP = Riboflavin, riboflavin produces by a long pathway and a short one, and D-ribose-5P is necessary for the two pathway precursors. Also, to avoid the flux increasing in other unwanted by-product pathway, we design to knock tkt gene out. Take the knockout of rbsK gene as example: rbsK gene fragments amplified by PCR obtain by digestion with the same end nuclease-off plasmid pUC18 in E. coli. Then we get the first recombinant plasmid pUC18- rbsK; in pUC18-rbsK the rbsK fragment of the selection which has suitable restriction sites were digested, and then linked with the same enzyme digested resistance gene (Spc, Kan resistance, etc.), there we get the second plasmid pUC18-rbsK-Spc or pUC18-rbsK-Kan, so rbsK gene can not express completely inactivation; Successfully built integrating plasmid pUC18-rbsK-Spc or pUC18-rbsK-Kan in Bacillus subtilis double exchange integration by inserting resistance gene express in Bacillus subtilis finally. Similarly, after a series of enzyme cutting, linking, transformation, screening operation, tkt gene can be knocked in Bacillus subtilis.
This paper mailly introduces the knockout of rbsK gene and tkt gene in riboflavin producing strains RH33 and RH44, then we make some testing on the strains. First, some related metabolites in the strains constrcted before and after are examined, and we find D-ribose disappearing and Sedoheptulose-7P decreasing after the knockout of two genes. Then the strains ferment in flasks, and we find the effection of knocking out two strains is greater in RH44, which we get a strain with 4.98 g of riboflavin productin, 14.5% higher than before.
引文
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[2]黄冈日报调研:广济药业领跑世界核黄素生产行业2010-1-19
[3]广济药业吊装世界最大容量的医药发酵罐中国医药信息2003-08-22
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[2]黄冈日报调研:广济药业领跑世界核黄素生产行业2010-1-19
[3]广济药业吊装世界最大容量的医药发酵罐中国医药信息2003-08-22
[4] Vitreschak AG, Rodionov DA, Mironov AA, et al. Regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation. Nucleic Acids Research. 2002, 30:3141-3151.
[5] Stahmann KP, Revuelta JL, Seulberger H. Three biotechnical processes using Ashbya gossypii, Candida famata or Bacillus subtilis compete with chemical riboflavin production. Appl Microbiol Biotechnol. 2000, 53:509-516.
[6]经济合作与发展组织.生物技术在工业可持续发展中的应用.北京,科学技术文献出版社, 2005.
[7] Kasler B, Sahm H, Stahmann KP, et al. Riboflavin-production process by means of micro-organisms with modified isocitratlyase activity. US Patent:5976844, 1999.
[8] Forster C, Santos MA, Ruffert S, et al. Physiological consequence of disruption of the VMA1 gene in the riboflavin overproducer Ashbya gossypii. J Biol Chem, 1999, 274(14):9442-9448.
[9] Bigelis R. Industrial products of biotechnology: application of gene technology. In: Rehm HJ, Reed G (eds) Biotechnology, 1989, 7b:243-256.
[10] Heefner DL, Weaver CA, Yarus MJ, et al. Method for producing riboflavin with Candida famata. US Patent 5,164,303, 1992.
[11] Stefan Herz, Sabine Eberhardt, Adelbert Bacher. Biosynthesis of riboflavin in plants. The ribA gene of Arabidopsis thaliana specifies a bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase. Phytochemistry 53 (200) 723-731
[12] Foor F, Brown G M. Purification and Properties of Guanosine Triphosphate Cyclohydrolase II. J Biol Chem, 1975, 250:3545-3551.
[13] Bacher W. Biosynthesis of Riboflavin in Bacillus subtilis. J Bacterial, 1978, 134 (2): 476-482.
[14] Richter G, Fischer M, Krieger C, et al. Biosynthesis of Riboflavin:Characterization of the Bifunctional Deaminase-Reductase of E. coli and Bacillus subtilis. J Bacterial, 1997, 179 (6):2022-2028.
[15] Shavlovskii G M. Enzyme Activity in the Second Step of Flavingensis of Reductase in the Yeast P. guilliermondii Mikrobiologiya,1981, 50 (6):1008-1011
[16] Volk R, Bacher A. Studies on the 4-Carbon Precursor in the Biosynthesis of Riboflavin. J Biol Chem, 1990, 265 (32):19479-19485.
[17] Nakajima K. Possibility of Diacetyl and Related Compounds at 4-carbon Compound Necessary for the Foemation of Riboflavin in A. gossyii. Acta Vitaminal Enzymal, 1984, 6 (4):271-282.
[18] Schott K, Ladenstein R, Konig A. The Lumazine Synthase-Riboflavin Synthase Complex of Bacillus subtilis. J Bio Chem, 1990, 265 (21):12686-12689.
[19] Plaut G W. Biosynthesis of Water-Soluble Vitamins. Annu Rev Biochem,1974, 43:899-922.
[20] Humbelin M , Griesser V , Keller T, et al . GTP cyclohydrolase II and 3,4- dihydroxy-2-butanone 4-phosphate synthase are rate-limiting enzymes in riboflavin synthesis of an industrial Bacillus subtilis strain used for riboflavin production. J Ind Microbiol Biotechnol , 1999 , 2: 1-7.
[21] Johannes Kaiser, Nicholas Schramek, Biosynthesis of vitamin B2.An essential zinc ion at the catalytic site of GTP cyclohydrolase II. Eur. J. Biochem. 269, 5264–5270 (2002) _ FEBS 2002
[22] Burrows R B. Presence in E.coli of Deaminase and Reductase Involved in Biosynthesis of Riboflavin. J Bacteriol, 1978, 136 (2):657-667.
[23] Ludwig H C, Lottspeich F, Henschen A, et al. Heavy Riboflavin Synthase of Bacillus subtilis. J Biol Chem, 1987, 262 (3):1016-1021.
[24] Nishikawa H, Momose H, Shiio I. Regulation of Purine nucleotide synthesis in Bacillus subtilis. J Biochem, 1976, 62(1):92-98.
[25]张会图,姚斌,范云六,核黄素基因工程研究进展,中国生物工程杂志,2004,24(12):32-38.
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