基因serA及sdaA对谷氨酸棒杆菌SYPS-062积累L-丝氨酸的影响分析
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摘要
本论文以实验室保藏的能利用糖质基质直接发酵产L-丝氨酸的谷氨酸棒杆菌(Corynebacterium glutamicum)SYPS-062为研究对象,以分子生物学技术为基础,运用重组DNA技术等分子手段L-丝氨酸代谢途径上的关键基因进行了分析,修饰,改造,构建了一株基因工程菌,并对该菌株进行了摇瓶发酵性能的初步研究。
3-磷酸甘油酸脱氢酶(3-PGDH)是L-丝氨酸的生物合成途径中的起始酶,其活性受到产物L-丝氨酸的反馈抑制作用。本论文运用PCR技术扩增出肽链C-末端缺失197个氨基酸的3-PGDH编码基因serA~(Δ591),通过在大肠杆菌中的表达测得其重组蛋白的比活力降低为原来的74.13%。比较了L-丝氨酸对基因serA~(Δ591)及serA表达的重组3-PGDH的反馈抑制作用,结果显示serA~(Δ591)基因编码的重组3-PGDH基本解除了高浓度L-丝氨酸对其酶活的抑制作用,为下一步研究工作奠定基础。
将基因serA~(Δ591)及serA在谷氨酸棒杆菌SYPS-062中进行了增强表达,与出发菌株相比,来源于重组菌SYPS-062(pJC1-tac-serA~(Δ591))的3-PGDH比活力提高了47.42%,L-丝氨酸产量提高了8.63%,来源于重组菌SYPS-062(pJC1-tac-serA)的3-PGDH比活力提高了67.38%,L-丝氨酸产量仅提高了1.84%,发酵结果表明增强基因serA~(Δ591)的表达更有利于L-丝氨酸产量的提高。L-丝氨酸脱水酶(Ser-DH)是由基因sdaA编码,催化L-丝氨酸降解为丙酮酸,本论
文利用交叉PCR获得基因sdaA同源片段,根据同源重组原理,将谷氨酸棒杆菌SYPS-062的关键基因sdaA进行敲除。sdaA基因缺失菌株和出发菌株发酵过程比较发现,sdaA基因缺失菌株生长缓慢,发酵周期延长,生物量降低了17.42%,L-丝氨酸积累量提高了3.61%,单位菌体产酸量提高了15.13%,耗糖速率量没有显著差异,以上结果表明改变L-丝氨酸代谢途径上关键基因的表达量确实能影响整个代谢流的分布。基于以上研究,本论文在基因sdaA缺失的基础上,增强了基因serA~(Δ591)的表达,构建了重组谷氨酸棒杆菌SYPS-062ΔsdaA(pJC1-tac-serA~(Δ591))。相比出发菌株,重组菌生长缓慢,发酵周期均延长,生物量降低了17.42%,发酵培养条件初步优化使其L-丝氨酸的积累量提高了23.18%,单位菌体产酸量(YP/X)提高了53.94%。同时考察了丙酮酸,玉米浆,酵母粉等对重组菌发酵产酸的影响,结果显示玉米浆促进菌体生长但不利于L-丝氨酸的积累,外源添加丙酮酸对菌株生长和产酸并未有显著影响,酵母粉的添加不利于菌株L-丝氨酸的积累。
由于微生物代谢网络自身存在着全局调控,从细胞代谢的全局来解析菌株的产酸机理,并对菌株进行合理的基因改造,构建出高产量的的L-丝氨酸基因工程菌株是未来发展的趋势。
This paper was focused on Corynebacterium glutamicum SYPS-062, a strain isolated from soil sample that can directly product L-serine from sugar as substance. Based on molecular biology techniques, using recombinant DNA technology and other means of molecular, we analyzed, modified the key enzyme genes of L-serine metabolic pathways in the strain and constructed a genetic engineering strain. The fermentation performance of the recombinant strain in shake flask was studied
3-Phosphoglycerate dehydrogenase (3-PGDH), coded by the gene serA, is the initial enzyme of the L-serine biosynthetic pathway with activity being inhibited by high concentrations of L-serine in C. glutamicum. Mutein of gene serA that 591 nucleic acid bases was truncated at the 3’-terminal end was constructed. When expressed in E. coli, mutein serA~(Δ591) showed a specific PGDH dehydrogenase activity of 1.092 U/mg protein. The activity reduced to 74.13% compared with the protein coded by gene serA but no longer being sensitive to the high concertration of L-serine.
According to the result before, gene serA~(Δ591) and serA were overexpressed respectively in C. glutamicum SYPS-062. It showed that the activity of 3-PGDH in C. glutamicum (pJC1-tac-serA~(Δ591)) and C. glutamicum (pJC1-tac-serA) increased by 47.42% and 67.38%. However, L-serine in C. glutamicum (pJC1-tac-serA~(Δ591)) was increased by 8.63% and increased by 1.84% in C. glutamicum (pJC1-tac-serA). It showed that the overexpressed of genes serA~(Δ591) was more conducive to the improvement of L-serine production. L-serine dehydratase (Ser-DH) catalyze the degradation of L-serine to pyruvateis ,it was encoded by the gene sdaA, on this paper we obtained the homologous fragment of gene sdaA homologous fragment by crossover-PCR, and the gene sdaA C. glutamicum SYPS-062 was deletion based on the principle of homologous recombination. When the gene sdaA coding SerDH was deletion, the unit cell L-serine production increased 15.13%, and there was no significant difference in the consumption of sugar.
According to the results above and the key position of L-serine in central metabolism, a strain as did the overexpression of gene serA~(Δ591) and the deletion of gene sdaA was constructed. As the change in the metabolism of L-serine in the recombinant C. glutamicum SYPS-062ΔsdaA(pJC1-tac-serA~(Δ591)), the recombine strain was resulted in a transient accumulation of L-serine increased 23.18% with a 53.94% improved in the unit cell by fermentation with the slower growth.
Due to the regulation of microbial metabolism network by itself, Analyzing the mechanism of L-serine producing from the cell metabolism, modifing the key genes of L-serine metabolic pathways, constructing a genetic engineering strain of high yields of L-serine is the future development trends.
3-磷酸甘油酸脱氢酶(3-PGDH)是L-丝氨酸的生物合成途径中的起始酶,其活性受到产物L-丝氨酸的反馈抑制作用。本论文运用PCR技术扩增出肽链C-末端缺失197个氨基酸的3-PGDH编码基因serA~(Δ591),通过在大肠杆菌中的表达测得其重组蛋白的比活力降低为原来的74.13%。比较了L-丝氨酸对基因serA~(Δ591)及serA表达的重组3-PGDH的反馈抑制作用,结果显示serA~(Δ591)基因编码的重组3-PGDH基本解除了高浓度L-丝氨酸对其酶活的抑制作用,为下一步研究工作奠定基础。
将基因serA~(Δ591)及serA在谷氨酸棒杆菌SYPS-062中进行了增强表达,与出发菌株相比,来源于重组菌SYPS-062(pJC1-tac-serA~(Δ591))的3-PGDH比活力提高了47.42%,L-丝氨酸产量提高了8.63%,来源于重组菌SYPS-062(pJC1-tac-serA)的3-PGDH比活力提高了67.38%,L-丝氨酸产量仅提高了1.84%,发酵结果表明增强基因serA~(Δ591)的表达更有利于L-丝氨酸产量的提高。L-丝氨酸脱水酶(Ser-DH)是由基因sdaA编码,催化L-丝氨酸降解为丙酮酸,本论
文利用交叉PCR获得基因sdaA同源片段,根据同源重组原理,将谷氨酸棒杆菌SYPS-062的关键基因sdaA进行敲除。sdaA基因缺失菌株和出发菌株发酵过程比较发现,sdaA基因缺失菌株生长缓慢,发酵周期延长,生物量降低了17.42%,L-丝氨酸积累量提高了3.61%,单位菌体产酸量提高了15.13%,耗糖速率量没有显著差异,以上结果表明改变L-丝氨酸代谢途径上关键基因的表达量确实能影响整个代谢流的分布。基于以上研究,本论文在基因sdaA缺失的基础上,增强了基因serA~(Δ591)的表达,构建了重组谷氨酸棒杆菌SYPS-062ΔsdaA(pJC1-tac-serA~(Δ591))。相比出发菌株,重组菌生长缓慢,发酵周期均延长,生物量降低了17.42%,发酵培养条件初步优化使其L-丝氨酸的积累量提高了23.18%,单位菌体产酸量(YP/X)提高了53.94%。同时考察了丙酮酸,玉米浆,酵母粉等对重组菌发酵产酸的影响,结果显示玉米浆促进菌体生长但不利于L-丝氨酸的积累,外源添加丙酮酸对菌株生长和产酸并未有显著影响,酵母粉的添加不利于菌株L-丝氨酸的积累。
由于微生物代谢网络自身存在着全局调控,从细胞代谢的全局来解析菌株的产酸机理,并对菌株进行合理的基因改造,构建出高产量的的L-丝氨酸基因工程菌株是未来发展的趋势。
This paper was focused on Corynebacterium glutamicum SYPS-062, a strain isolated from soil sample that can directly product L-serine from sugar as substance. Based on molecular biology techniques, using recombinant DNA technology and other means of molecular, we analyzed, modified the key enzyme genes of L-serine metabolic pathways in the strain and constructed a genetic engineering strain. The fermentation performance of the recombinant strain in shake flask was studied
3-Phosphoglycerate dehydrogenase (3-PGDH), coded by the gene serA, is the initial enzyme of the L-serine biosynthetic pathway with activity being inhibited by high concentrations of L-serine in C. glutamicum. Mutein of gene serA that 591 nucleic acid bases was truncated at the 3’-terminal end was constructed. When expressed in E. coli, mutein serA~(Δ591) showed a specific PGDH dehydrogenase activity of 1.092 U/mg protein. The activity reduced to 74.13% compared with the protein coded by gene serA but no longer being sensitive to the high concertration of L-serine.
According to the result before, gene serA~(Δ591) and serA were overexpressed respectively in C. glutamicum SYPS-062. It showed that the activity of 3-PGDH in C. glutamicum (pJC1-tac-serA~(Δ591)) and C. glutamicum (pJC1-tac-serA) increased by 47.42% and 67.38%. However, L-serine in C. glutamicum (pJC1-tac-serA~(Δ591)) was increased by 8.63% and increased by 1.84% in C. glutamicum (pJC1-tac-serA). It showed that the overexpressed of genes serA~(Δ591) was more conducive to the improvement of L-serine production. L-serine dehydratase (Ser-DH) catalyze the degradation of L-serine to pyruvateis ,it was encoded by the gene sdaA, on this paper we obtained the homologous fragment of gene sdaA homologous fragment by crossover-PCR, and the gene sdaA C. glutamicum SYPS-062 was deletion based on the principle of homologous recombination. When the gene sdaA coding SerDH was deletion, the unit cell L-serine production increased 15.13%, and there was no significant difference in the consumption of sugar.
According to the results above and the key position of L-serine in central metabolism, a strain as did the overexpression of gene serA~(Δ591) and the deletion of gene sdaA was constructed. As the change in the metabolism of L-serine in the recombinant C. glutamicum SYPS-062ΔsdaA(pJC1-tac-serA~(Δ591)), the recombine strain was resulted in a transient accumulation of L-serine increased 23.18% with a 53.94% improved in the unit cell by fermentation with the slower growth.
Due to the regulation of microbial metabolism network by itself, Analyzing the mechanism of L-serine producing from the cell metabolism, modifing the key genes of L-serine metabolic pathways, constructing a genetic engineering strain of high yields of L-serine is the future development trends.
引文
1.王镜岩.生物化学[M]:上册.第3版.北京:高等教育出版社, 2002. 124-139
2.蒋滢.氨基酸的应用[M].北京:世界图书出版公司, 1996. 125
3.冯美卿,曹秀格,卢永辉. L-丝氨酸制备方法评述[J].氨基酸和生物资源, 2000, 22 (3): 42-44.
4. Tom J, Snell K, Marinus D, et al. L-Serine in disease and development. Biochem. J, 2003, 371:653–661.
5.蔡宇旸,吴梧桐,史燕东. SHMT基因工程菌的构建及高效表达[J].生物工程学报, 1996, 12(增刊): 28-33.
6.克丽凤,张伟国,钱和. L-丝氨酸的生产及其应用[J].江苏食品与发酵, 2001(2): 19-22
7.柴多里,祁秋景,基木格等.丝氨酸的研究进展[J].化工科技市场, 2006, 59(5): 17-18
8. Cramer, E. Ueber die bestandtheile der seide[J]. J. Prakt. Chem, 1865, 96: 76-98
9. Simic P, Willuhn J, Sahm H, et al. Identification of glyA (encoding serine hydroxymethyltransferase) and its use together with the exporter ThrE to increase L-threonine accumulation by Corynebacterium glutamicum [J]. Appl Environ Microbiol, 2002, 68(7):3321–3327
10. Kubota K, Yokozeki K. Production of L-Serine from glycine by Corynebacterium glycinophilum and properties of Serine hydroxymethyltransferase, a key enzyme in L-Serine production. Ferment. Bioeng, 1989, 67:387–390.
11.卢发,张伟国.L-丝氨酸产生菌的分离筛选及发酵条件[J].食品与生物技术学报, 2005, 24(2): 48-51
12.张蓓.代谢工程[M].天津大学出版社, 2003. 40-50
13.李寅,曹竹安.微生物代谢工程:绘制细胞工厂的蓝图[J].化工学报, 2004, 55(10): 1573-1580.
14. Kauffman K J, Prakash P, Edwards J S. Advances in flux balance analysis. Curr Opin Biotech, 2003,14:1-61
15.赵学明,马红武,班睿.代谢工程的发展现状和未来.化学工程. 1997, 25(5): 57-59
16. Peters-Wendisch P, Stolz M, Etterich H, et al. Metabolic Engineering of Corynebacterium glutamicum for L-Serine Production[J]. Appl Environ Microbiol, 2005, 71(11): 7139-7144
17. ThompsomJ R, Bell J K,Bratt J , Grant GA , Banaszak L J . Vmax regulation through domain and subunit changes: The active form of phosphoglycerate dehydrogenase [J ] . Biochemistry,2005,(15) : 576325773
18. Netzer R, Peters-Wendisch P, Eggeling L, et al.. Cometabolism of a Nongrowth Substrate: L-Serine Utilization by Corynebacterium glutamicum [J]. Appl Environ Microbiol, 2004, 70(12): 7148–7155
19. Cossins E A, Chan P Y, Combepine G. One-carbon metabolism in neurospora crassa wild-type and in mutants partially deficient in serine hydroxymethyltransferase[J]. Biochem J, 1976, 160: 305-314.
20. Simic P, Willuhn J, Sahm H, et al. Identification of glyA (encoding serinehydroxymethyltransferase) and its use together with the exporter ThrE to increase L-threonine accumulation by Corynebacterium glutamicum [J]. Appl Environ Microbiol, 2002, 68(7):3321–3327
21.刘云,徐琪寿.生产氨基酸的基因工程菌研究进展[J].氨基酸和生物资源.1999,21 (1):45-49
22.张晓梅,窦文芳,许泓瑜,许正宏.产L-丝氨酸菌株SYPS-062的鉴定及碳源对发酵的影响[J].微生物学通报.2009,36(6):789-793
23.张晓娟,窦文芳,许泓瑜等.维生素对谷氨酸棒杆菌SYPS-062直接发酵合成L-丝氨酸的影响[J].中国生物工程杂志.2007,27(5):50-55
24.林琳,张晓梅,许正宏等.直接利用糖质原料产L-丝氨酸谷氨酸棒杆菌glyA基因序列分析.生物技术通报, 2008, 5: 176～180
25.任建洪,张晓梅,许正宏等.不同来源的谷氨酸棒杆菌氨基脱氧分支酸合成酶的活性分析,中国生物工程杂志,2009, 29 (8) : 57～61
26.徐国强,张晓梅,窦文芳,许泓瑜,许正宏.直接利用糖质原料生产L-丝氨酸菌株的选育[J].中国科技论文在线, 2008
27.谢玉清,罗淑萍,,茆军. L-丝氨酸产生菌c-11中serA基因的克隆与表达[J].新疆农业科学.2007,44(5):652-653
28.殷丽霞,崔春生,简子健等.大肠杆菌serB基因的克隆及其在黄色短杆菌中的表达[J].新疆农业科学.2006,43(2):96-99
29.魏东,谭慧林,杨海燕等.L-丝氨酸高产菌株的选育和摇瓶发酵条件优化[J].氨基酸和生物资源.2006,28(1):46-48
30.谭慧林,崔春生,杨海燕等.L-丝氨酸摇瓶条件的优化[J].新疆农业科学.2006,43(2):148-150
31. Stolz M, Peters-Wendisch P, Etterich H, et al. Reduced Folate Supply as a Key to Enhanced L-Serine Production by Corynebacterium glutamicum [J]. Appl Environ Microbiol, 2007, 73(3): 750-755
32. Suga M, Sugimoto M, Osumi T, Nakamatsu T, Hibino W, Ito M . Method for producing L-Serine by fermentation. 1999, EP 0 943 687 A2.
33. Peters-Wendisch P, Netzer R, Eggeling L. et al. 3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum: the C-terminal domain is not essential for activity but is required for inhibition by L-serine [J]. Appl Microbiol Biotechnol, 2002, 60(4): 437-441
34. Ho CL, Saito K (2001) Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana. Amino Acids 20:243–259
35. Ho CL, Noji M, Saito M, Saito K (1999) Regulation of serine biosynthesis in Arabidopsis. Crucial role of plastidic 3-phosphoglycerate dehydrogenase in non-photosynthetic tissues.J Biol Chem 274:397–402
36.张绪梅,郭长江,刘云等.大肠杆菌PGDH末端缺失突变体的构建及抗反馈抑制效应分析.[J].中国生物化学与分子生物学报.2006,22(10):806-810
37. Zhao G, Winkler M E. A novel alpha-ketoglutarate reductase activity of the serA encoded3-phosphoglycerate dehydrogenase of Escherichia coli K212 and its possible implications for human 22hydroxyglutaricaciduria. J Bacteriol , 1996 , 178(1) : 2322239
38.林琳.利用糖质原料产L-丝氨酸菌株SYPS-062的代谢机制初探[D]: [硕士学位论文].无锡:江南大学生物工程学院, 2008
39.任建洪.叶酸途径对谷氨酸棒杆菌SYPS-062积累L-丝氨酸的影响分析[D]: [硕士学位论文].无锡:江南大学生物工程学院, 2009
40. Volker F, bott M, Bernhard J, Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids. Current opinion in microbiology, 2006, 9: 268-274
41.谢承佳,何冰芳,李霜.基因敲除技术及其在微生物代谢工程方面的应用[J].生物加工过程, 2007, 5(3): 10-14
42. Tauch A, Pühler A, Kalinowski J. Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics. J Biotechnology, 2003, 104(1-3): 27-40
43.阮红, Eikmanns.B.谷氨酸棒杆菌基因缺失菌株的定点构建.微生物学报(Acta Microbiologica Sinica), 2002, 42(4): 458-464
44. Sukdeo N, Charles T. Application of crossover-PCR-mediateddeletion-insertion mutagenesis to analysis of the bdhA-xdhA2-xdhB2 mixed-function operon of Sinorhizobium meliloti. Arch Microbiol, 2003, 179:301–304.
45. J.Link A, Phillips D, M.Church G. Methods for Generating Precise Deletions and Insertions in the Genome of Wild-Type Escherichia coli: Application to Open Reading Frame Characterization. Journal of Biotechnology, 1997, 6228-6237.
46. Schafer A, Tauch A, Jager W, et al. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutumicum. Gene, 1994, 145:69-73.
47.余秉琦,沈微,诸葛健.适用于异源DNA高效整合转化的谷氨酸棒杆菌电转化法.中国生物工程杂志(China Biotechnology), 2005, 25(2):78-81
48.蔡晓凤,红平红球菌Rhodococcus erythropolis XP菌株的分子改造.山东大学,2007,51-6
49.韩雅珊.食品化学实验指导[M].北京:北京农业大学出版社, 1992. 19-23
50. Kalinowski J, Bathe B, Bartels d et al. The complete Corynebacterium glutamicum ATCC13032 genome sequence and its impact on the production of L-aspartate-derivrd amino acids and vitamins. Journal of Biptechnology, 2002, 104: 5-25
2.蒋滢.氨基酸的应用[M].北京:世界图书出版公司, 1996. 125
3.冯美卿,曹秀格,卢永辉. L-丝氨酸制备方法评述[J].氨基酸和生物资源, 2000, 22 (3): 42-44.
4. Tom J, Snell K, Marinus D, et al. L-Serine in disease and development. Biochem. J, 2003, 371:653–661.
5.蔡宇旸,吴梧桐,史燕东. SHMT基因工程菌的构建及高效表达[J].生物工程学报, 1996, 12(增刊): 28-33.
6.克丽凤,张伟国,钱和. L-丝氨酸的生产及其应用[J].江苏食品与发酵, 2001(2): 19-22
7.柴多里,祁秋景,基木格等.丝氨酸的研究进展[J].化工科技市场, 2006, 59(5): 17-18
8. Cramer, E. Ueber die bestandtheile der seide[J]. J. Prakt. Chem, 1865, 96: 76-98
9. Simic P, Willuhn J, Sahm H, et al. Identification of glyA (encoding serine hydroxymethyltransferase) and its use together with the exporter ThrE to increase L-threonine accumulation by Corynebacterium glutamicum [J]. Appl Environ Microbiol, 2002, 68(7):3321–3327
10. Kubota K, Yokozeki K. Production of L-Serine from glycine by Corynebacterium glycinophilum and properties of Serine hydroxymethyltransferase, a key enzyme in L-Serine production. Ferment. Bioeng, 1989, 67:387–390.
11.卢发,张伟国.L-丝氨酸产生菌的分离筛选及发酵条件[J].食品与生物技术学报, 2005, 24(2): 48-51
12.张蓓.代谢工程[M].天津大学出版社, 2003. 40-50
13.李寅,曹竹安.微生物代谢工程:绘制细胞工厂的蓝图[J].化工学报, 2004, 55(10): 1573-1580.
14. Kauffman K J, Prakash P, Edwards J S. Advances in flux balance analysis. Curr Opin Biotech, 2003,14:1-61
15.赵学明,马红武,班睿.代谢工程的发展现状和未来.化学工程. 1997, 25(5): 57-59
16. Peters-Wendisch P, Stolz M, Etterich H, et al. Metabolic Engineering of Corynebacterium glutamicum for L-Serine Production[J]. Appl Environ Microbiol, 2005, 71(11): 7139-7144
17. ThompsomJ R, Bell J K,Bratt J , Grant GA , Banaszak L J . Vmax regulation through domain and subunit changes: The active form of phosphoglycerate dehydrogenase [J ] . Biochemistry,2005,(15) : 576325773
18. Netzer R, Peters-Wendisch P, Eggeling L, et al.. Cometabolism of a Nongrowth Substrate: L-Serine Utilization by Corynebacterium glutamicum [J]. Appl Environ Microbiol, 2004, 70(12): 7148–7155
19. Cossins E A, Chan P Y, Combepine G. One-carbon metabolism in neurospora crassa wild-type and in mutants partially deficient in serine hydroxymethyltransferase[J]. Biochem J, 1976, 160: 305-314.
20. Simic P, Willuhn J, Sahm H, et al. Identification of glyA (encoding serinehydroxymethyltransferase) and its use together with the exporter ThrE to increase L-threonine accumulation by Corynebacterium glutamicum [J]. Appl Environ Microbiol, 2002, 68(7):3321–3327
21.刘云,徐琪寿.生产氨基酸的基因工程菌研究进展[J].氨基酸和生物资源.1999,21 (1):45-49
22.张晓梅,窦文芳,许泓瑜,许正宏.产L-丝氨酸菌株SYPS-062的鉴定及碳源对发酵的影响[J].微生物学通报.2009,36(6):789-793
23.张晓娟,窦文芳,许泓瑜等.维生素对谷氨酸棒杆菌SYPS-062直接发酵合成L-丝氨酸的影响[J].中国生物工程杂志.2007,27(5):50-55
24.林琳,张晓梅,许正宏等.直接利用糖质原料产L-丝氨酸谷氨酸棒杆菌glyA基因序列分析.生物技术通报, 2008, 5: 176～180
25.任建洪,张晓梅,许正宏等.不同来源的谷氨酸棒杆菌氨基脱氧分支酸合成酶的活性分析,中国生物工程杂志,2009, 29 (8) : 57～61
26.徐国强,张晓梅,窦文芳,许泓瑜,许正宏.直接利用糖质原料生产L-丝氨酸菌株的选育[J].中国科技论文在线, 2008
27.谢玉清,罗淑萍,,茆军. L-丝氨酸产生菌c-11中serA基因的克隆与表达[J].新疆农业科学.2007,44(5):652-653
28.殷丽霞,崔春生,简子健等.大肠杆菌serB基因的克隆及其在黄色短杆菌中的表达[J].新疆农业科学.2006,43(2):96-99
29.魏东,谭慧林,杨海燕等.L-丝氨酸高产菌株的选育和摇瓶发酵条件优化[J].氨基酸和生物资源.2006,28(1):46-48
30.谭慧林,崔春生,杨海燕等.L-丝氨酸摇瓶条件的优化[J].新疆农业科学.2006,43(2):148-150
31. Stolz M, Peters-Wendisch P, Etterich H, et al. Reduced Folate Supply as a Key to Enhanced L-Serine Production by Corynebacterium glutamicum [J]. Appl Environ Microbiol, 2007, 73(3): 750-755
32. Suga M, Sugimoto M, Osumi T, Nakamatsu T, Hibino W, Ito M . Method for producing L-Serine by fermentation. 1999, EP 0 943 687 A2.
33. Peters-Wendisch P, Netzer R, Eggeling L. et al. 3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum: the C-terminal domain is not essential for activity but is required for inhibition by L-serine [J]. Appl Microbiol Biotechnol, 2002, 60(4): 437-441
34. Ho CL, Saito K (2001) Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana. Amino Acids 20:243–259
35. Ho CL, Noji M, Saito M, Saito K (1999) Regulation of serine biosynthesis in Arabidopsis. Crucial role of plastidic 3-phosphoglycerate dehydrogenase in non-photosynthetic tissues.J Biol Chem 274:397–402
36.张绪梅,郭长江,刘云等.大肠杆菌PGDH末端缺失突变体的构建及抗反馈抑制效应分析.[J].中国生物化学与分子生物学报.2006,22(10):806-810
37. Zhao G, Winkler M E. A novel alpha-ketoglutarate reductase activity of the serA encoded3-phosphoglycerate dehydrogenase of Escherichia coli K212 and its possible implications for human 22hydroxyglutaricaciduria. J Bacteriol , 1996 , 178(1) : 2322239
38.林琳.利用糖质原料产L-丝氨酸菌株SYPS-062的代谢机制初探[D]: [硕士学位论文].无锡:江南大学生物工程学院, 2008
39.任建洪.叶酸途径对谷氨酸棒杆菌SYPS-062积累L-丝氨酸的影响分析[D]: [硕士学位论文].无锡:江南大学生物工程学院, 2009
40. Volker F, bott M, Bernhard J, Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids. Current opinion in microbiology, 2006, 9: 268-274
41.谢承佳,何冰芳,李霜.基因敲除技术及其在微生物代谢工程方面的应用[J].生物加工过程, 2007, 5(3): 10-14
42. Tauch A, Pühler A, Kalinowski J. Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics. J Biotechnology, 2003, 104(1-3): 27-40
43.阮红, Eikmanns.B.谷氨酸棒杆菌基因缺失菌株的定点构建.微生物学报(Acta Microbiologica Sinica), 2002, 42(4): 458-464
44. Sukdeo N, Charles T. Application of crossover-PCR-mediateddeletion-insertion mutagenesis to analysis of the bdhA-xdhA2-xdhB2 mixed-function operon of Sinorhizobium meliloti. Arch Microbiol, 2003, 179:301–304.
45. J.Link A, Phillips D, M.Church G. Methods for Generating Precise Deletions and Insertions in the Genome of Wild-Type Escherichia coli: Application to Open Reading Frame Characterization. Journal of Biotechnology, 1997, 6228-6237.
46. Schafer A, Tauch A, Jager W, et al. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutumicum. Gene, 1994, 145:69-73.
47.余秉琦,沈微,诸葛健.适用于异源DNA高效整合转化的谷氨酸棒杆菌电转化法.中国生物工程杂志(China Biotechnology), 2005, 25(2):78-81
48.蔡晓凤,红平红球菌Rhodococcus erythropolis XP菌株的分子改造.山东大学,2007,51-6
49.韩雅珊.食品化学实验指导[M].北京:北京农业大学出版社, 1992. 19-23
50. Kalinowski J, Bathe B, Bartels d et al. The complete Corynebacterium glutamicum ATCC13032 genome sequence and its impact on the production of L-aspartate-derivrd amino acids and vitamins. Journal of Biptechnology, 2002, 104: 5-25