光合细菌和重组大肠杆菌生物合成5-氨基乙酰丙酸的研究
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摘要
5-氨基乙酰丙酸(5-aminolevulinic acid，简称ALA)是生物合成四吡咯的前体，而四吡咯是构成生物体必不可少的物质(血红素，细胞色素，维生素B_(12))。对ALA的深入研究表明，ALA对人畜无毒性，在环境中易降解、无残留，是一种无公害的绿色农用化学品。在医学领域，ALA可作为抗癌药物的中间体，作为检验铅中毒的主要试剂，对吡咯紫质沉着症的临床诊断也有一定效果。1999年12月，美国FDA正式批准ALA为治疗皮肤癌前期的光动力药物，ALA在治疗其它表皮癌症中的应用也得到了越来越多科学家的兴趣。作为第二代光动力药，ALA的显著优点是副作用小、渗透性好、疗效确切、对疾病的适用范围广及价格低等。
国外已经研究了采用化学方法以及微生物发酵合成ALA的各种工艺路线。我国对化学方法以及微生物发酵法生产ALA的研究基本上仍属空白。
本论文对5—氨基乙酰丙酸的生物合成展开研究，主要包括光合细菌的筛选和诱变育种，光合细菌摇瓶发酵的研究，基因工程菌摇瓶条件和发酵罐上的研究。
我们利用van Niel培养基从杭州市四堡废水处理厂的活性污泥中分离得到四株光合细菌，选取其中产生ALA能力最佳的菌株(ALA产生能力为7.35mg／L)进行菌种鉴定，确定该菌株属红假单胞菌属。采用亚硝基胍，紫外线以及磷酸二乙酯和氯化锂协同作用对该菌株进行诱变处理，得到产量为21.2mg／L的菌株C-2。
对培养基组成和培养条件进行了初步优化，发现葡萄糖是最佳碳源、磷酸铵为最佳氮源。在培养基中加入的葡萄糖质量浓度为15g／L、磷酸铵质量浓度为2g／L、接种量为10％、100mL摇瓶装液量为20mL、调节初始pH7.0进行培养时，ALA的最高积累量可达24.9mg／L；进一步对前体的最佳添加量进行优化，发现甘氨酸的最佳添加浓度为100mmol／L、琥珀酸的最佳浓度为20mmol／L，同时加入脱水酶抑制剂乙酰丙酸20mmol／L时，ALA的最高产量达到38.9mg／L。
由于常规诱变工作量大，效率低。通过几轮诱变后发现进一步提高光合细菌产ALA的能力十分困难，于是本实验室构建了以E.coli DH5α为宿主的工程菌GT48，产ALA能力为19.8mg／L的，在摇瓶条件下对GT48菌株的培养特性进行了
浙江大学硕士学位论文
暴
摘要
初步考察，结果表明:最佳的初始pH为6.5，最佳诱导时间为稳定前期，适当
的葡萄搪浓度对ALA的积累有较大的促进作用。确定甘氨酸的加入对于ALA的积
累有一定效果。随后在高百特5L罐上研究了丑co了z’GT48的间歇培养规律。把产
ALA能力提高到47.smg/L。
为了进一步提高细胞密度及ALA产量，在5L高百特发酵罐中进行了流加培
养，探讨了不同流加培养基，发酵罐溶氧对大肠杆菌累积ALA的影响。最终确定
以恒定的流速向发酵罐中流加补料时，流加速度为smL/hr(相当于每小时加入
1g葡萄糖和0.59甘氨酸)，发酵罐装液量为3L，通气量为svvm，ALA的累积量
达到72.gmg/L。
本文工作只是对生物合成5一氨基己酞丙酸的初步探索，结果也不尽如人
意，但为5一氨基乙酞丙酸的深入研究打下了基础。
5-aminolevulinic acid (ALA) is the biosynthetic precursor of the tetrapyrroles, such as porphyr'in, heme, chlorophyll (CHL), and vitamin B12. It is reported that ALA has no toxicity, to either huraanbeing or animals, is degradable easily, and has no accumulation in environment; therefore, it is a green agricultural chemical. ALA can also be served as a pharmaceutical therapy fighting against cancer, and used as a chemical for detecting blood plumbum content. In December 1999 ALA was authorized by FDA as a photodynamic medicine to treat skin cancer in prophase. And it is drawing more and more attentions of scientists to apply it in the treatment of various epidermis cancers. As the second generation of photodynamic medicine, its outstanding advantages include less side effect, better curative effect and penetrability, lower price, etc.
Some techniques to synthesize ALA by chemical and biological processes have been proposes abroad, however, they are still blankness in China.
In this work, the biological synthesis of ALA was studied. At first, the screen and mutating of photobacteria were performed to obtain high productive strain for ALA synthesizing, and the medium composition and fermentation conditions were optimized. Next, a strain of recombinant, E. coh' GT48, was applied for ALA production.
Four strains of photobacterium were screened from active sludge in a local wastewater treatment factory using van Niel medium. A strain with the highest ALA productivity among four strains was selected for further studied, which belongs to Rhodopseudomonas by an identification procedure. Treated by N-methyl-N' -nitro-N-nitrosoguanidine and ultraviolete, a mutant, C-2, was obtained with ALA productivity of 21.2 mg/L. After preliminary optimization in medium composition and cultivation conditions, the ALA productivity increased to 38. 9 mg/L.
A recombinant strain, E. coli GT48 was applied for ALA production, in which, a gene of ALA synthetase was cloned into a plasraid. The initial productivity of ALA was about 19.8mg/L. After preliminary optimization in medium composition and cultivation conditions, the ALA productivity increased to 47. 8mg/L in batch fermentation.
In order to improve the cell density and ALA productivity, The fed-batch fermentation was studied in 5L BET fermentor. The effects of medium composition in feed and dissolved oxygen on ALA production were evaluated, and the ALA productivity was increased up to 72. 9mg/L.
Although it is only a preliminary study of biological synthesis of ALA and the productivity is not as high as expected, this work lays the foundation for the further improvement in ALA production.
国外已经研究了采用化学方法以及微生物发酵合成ALA的各种工艺路线。我国对化学方法以及微生物发酵法生产ALA的研究基本上仍属空白。
本论文对5—氨基乙酰丙酸的生物合成展开研究，主要包括光合细菌的筛选和诱变育种，光合细菌摇瓶发酵的研究，基因工程菌摇瓶条件和发酵罐上的研究。
我们利用van Niel培养基从杭州市四堡废水处理厂的活性污泥中分离得到四株光合细菌，选取其中产生ALA能力最佳的菌株(ALA产生能力为7.35mg／L)进行菌种鉴定，确定该菌株属红假单胞菌属。采用亚硝基胍，紫外线以及磷酸二乙酯和氯化锂协同作用对该菌株进行诱变处理，得到产量为21.2mg／L的菌株C-2。
对培养基组成和培养条件进行了初步优化，发现葡萄糖是最佳碳源、磷酸铵为最佳氮源。在培养基中加入的葡萄糖质量浓度为15g／L、磷酸铵质量浓度为2g／L、接种量为10％、100mL摇瓶装液量为20mL、调节初始pH7.0进行培养时，ALA的最高积累量可达24.9mg／L；进一步对前体的最佳添加量进行优化，发现甘氨酸的最佳添加浓度为100mmol／L、琥珀酸的最佳浓度为20mmol／L，同时加入脱水酶抑制剂乙酰丙酸20mmol／L时，ALA的最高产量达到38.9mg／L。
由于常规诱变工作量大，效率低。通过几轮诱变后发现进一步提高光合细菌产ALA的能力十分困难，于是本实验室构建了以E.coli DH5α为宿主的工程菌GT48，产ALA能力为19.8mg／L的，在摇瓶条件下对GT48菌株的培养特性进行了
浙江大学硕士学位论文
暴
摘要
初步考察，结果表明:最佳的初始pH为6.5，最佳诱导时间为稳定前期，适当
的葡萄搪浓度对ALA的积累有较大的促进作用。确定甘氨酸的加入对于ALA的积
累有一定效果。随后在高百特5L罐上研究了丑co了z’GT48的间歇培养规律。把产
ALA能力提高到47.smg/L。
为了进一步提高细胞密度及ALA产量，在5L高百特发酵罐中进行了流加培
养，探讨了不同流加培养基，发酵罐溶氧对大肠杆菌累积ALA的影响。最终确定
以恒定的流速向发酵罐中流加补料时，流加速度为smL/hr(相当于每小时加入
1g葡萄糖和0.59甘氨酸)，发酵罐装液量为3L，通气量为svvm，ALA的累积量
达到72.gmg/L。
本文工作只是对生物合成5一氨基己酞丙酸的初步探索，结果也不尽如人
意，但为5一氨基乙酞丙酸的深入研究打下了基础。
5-aminolevulinic acid (ALA) is the biosynthetic precursor of the tetrapyrroles, such as porphyr'in, heme, chlorophyll (CHL), and vitamin B12. It is reported that ALA has no toxicity, to either huraanbeing or animals, is degradable easily, and has no accumulation in environment; therefore, it is a green agricultural chemical. ALA can also be served as a pharmaceutical therapy fighting against cancer, and used as a chemical for detecting blood plumbum content. In December 1999 ALA was authorized by FDA as a photodynamic medicine to treat skin cancer in prophase. And it is drawing more and more attentions of scientists to apply it in the treatment of various epidermis cancers. As the second generation of photodynamic medicine, its outstanding advantages include less side effect, better curative effect and penetrability, lower price, etc.
Some techniques to synthesize ALA by chemical and biological processes have been proposes abroad, however, they are still blankness in China.
In this work, the biological synthesis of ALA was studied. At first, the screen and mutating of photobacteria were performed to obtain high productive strain for ALA synthesizing, and the medium composition and fermentation conditions were optimized. Next, a strain of recombinant, E. coh' GT48, was applied for ALA production.
Four strains of photobacterium were screened from active sludge in a local wastewater treatment factory using van Niel medium. A strain with the highest ALA productivity among four strains was selected for further studied, which belongs to Rhodopseudomonas by an identification procedure. Treated by N-methyl-N' -nitro-N-nitrosoguanidine and ultraviolete, a mutant, C-2, was obtained with ALA productivity of 21.2 mg/L. After preliminary optimization in medium composition and cultivation conditions, the ALA productivity increased to 38. 9 mg/L.
A recombinant strain, E. coli GT48 was applied for ALA production, in which, a gene of ALA synthetase was cloned into a plasraid. The initial productivity of ALA was about 19.8mg/L. After preliminary optimization in medium composition and cultivation conditions, the ALA productivity increased to 47. 8mg/L in batch fermentation.
In order to improve the cell density and ALA productivity, The fed-batch fermentation was studied in 5L BET fermentor. The effects of medium composition in feed and dissolved oxygen on ALA production were evaluated, and the ALA productivity was increased up to 72. 9mg/L.
Although it is only a preliminary study of biological synthesis of ALA and the productivity is not as high as expected, this work lays the foundation for the further improvement in ALA production.
引文
[1]朱章玉，俞吉安，林新志，李堃宝．光合细菌的研究及应用．上海：上海交通大学出版社，1991．28-189．
[2]Rosen, Arne, Larko, et al. Method for detecting cancer on skin of humans and mammals and arrangement for performing the method. US: 6421455,2002.
[3]Lentini, Peter J. Compositions useful in the phototherapeutic treatment of proliferative skin disorders. US:5885557,1999.
[4]Gierskcky, Karl E., Moan, et al. Esters of 5-aminolevulinic acid as photosensitizing agents in photochemotherapy US:6034267, 2000.
[5]戴秀莲．工业卫生与职业病，1989，15(6)：363．
[6]Rebeiz, Constantin A, Hopen, et al. Photodynamic herbicidal compositions using.delta.-aminolevulinic acid. US: 5286708, 1994.
[7]Rebeiz, Constantin A. Photodynamic herbicides. US: 5127938, 1992.
[8]Rebeiz, Constantin A. Photodynamic plant defoliants. US: 5321001, 1994.
[9]Rebeiz, Constantin A, Juvik, et al. Porphyric insecticides. US 5300526,1994.
[10]Rebeiz, Constantin A, Juvik, et al. Porphyric insecticides. US 5200427,1994.
[11]Tanaka, Takahashi, Hotta, et al. Method for promoting plant growth using 5-aminolevulinic acid or a salt thereof. US: 5298482, 1994.
[12]张一宾，周燚．5—氨基乙酰丙酸的植物生理活性[J]．世界农药，2000，22(3)：8-14．
[13]Rebeiz, Constantin A., Juvik, et al. Porphyric insecticides. US:5300526,1994.
[14]Rebeiz, Constantin A.,Juvik, et al. Porphyric insecticides. US:5200427,1993.
[15]Z'av yalov, Aronova, Makhova, et al. Synthesis of 5-aminolevulinic acid hydrochloride. Lzv. Akad. Nank SSSR. Ser. Khim.,1973, (3):657-658.
[16]Evans, Dacid A, Philip J. A simple route to α-aminoketones and related derivatives by dianion acylation reactoion. J.Chem. Soc. Common., 1978(17):752-754.
[17]Andreas Pfaltz, Saeed Anwar. Synthesis of α-aminoketones via readuction ofacyl cyanides. Tetrahedron Letter, 1984,25(28): 2977-2980.
[18]Haruhiko, Takeya, Tokhi, et al. Process for preparing 5-aminolevulinic acid. EP:607952, 1994.
[19]Kawakami H, Ebata T, Matsushita H. A new synthesis of 5-aminolevulinic acid. Agric. Biol. Chem., 1991,55(6):1687-1688.
[20]Gerand Descotes, Louis Cottier, Laurent Eynard, et al. Process for the preparation of N-acyl derivatives of 5-aminolevulinic acid, as well as the hydrochloride of the free acid .US:5344974,1994.
[21]L. Cottier, G. Descotes, L. Eymard, et al. Application to the synthesis of 5-aminolevulinic hydrochloride. Synthesis, 1995(3):303-306.
[22]Suzuki, Kojiro, Takeya, et al. Preparation of 5-aminolevulinic acid and
its intermediates. JP:0372450, 1991.
[23]Moens, Luc. Synthesis of an acid addition salt of delta-aminolevulinic acid from 5-bromo levulinic acid esters. US: 5907058, 1999.
[24]Z'av yalov, Zavozin. Synthesis of 5-amino-4-oxopentanoica acid hydrochloride, Akad. Nank SSSR. Ser. Khim.,1987, (18):1796-1799.
[25]Metcalf, Brian W, Adams, et al. Production of intermediates for enzyme inhibitors. US: 4325877,1982.
[26]Sasaki K, Watanabe, Nishizawa, et al. 5-Aminolevulinic acid production by Chiorella sp. during heterotrophic culture in the dark. World J. Microbiol. Biotechnol., 1995, 11: 361-362.
[27]Beale. The biosynthesis of δ-aminolevulinic acid in Chlorella. Plant Physiol., 1970,45: 504-596.
[28]Sasaki K, Tanaka, Nishizawa, et al. Enhanced production of 5-aminolevulinic acid by repeated addition of leculinic acid and supplement of precursors in photoheterotrophic culture of Rhodobacter sphaeroides. J. Ferment, Bioeng.,1991,71:403-406.
[29]刘如林．光合细菌及其应用．北京：中国农业科技出版社，1991．
[30]刘秀艳，叶敏，徐向阳．产生5—氨基乙酰丙酸(ALA)光合细菌生物学研究进展[J]．2000，20(5)：67-71．
[31]Dong lin et al. Journal Fermentation Bioengineering. 1989,68:88-91.
[32]Chen W, et al. Journal Bacteriology. 1994,175:2743-2746.
[33]Yoshiteru, Hashimoto, et al. Journal Fermentation Bioengineering. 1996,82:93-100.
[34]Toru Nakayashiki and Hachiro Inokuch, Genes Genet. Syst. 1996,71:237-241.
[35]Jill H. Zeilstera. Ryalls and Samuel Kaplan, Journal Bacteriology.1995,176:2760-2768.
[36]Mariet J. Van der Werf and J. Gregory zeikus, Applied and Enviromental Micro. 1996,38: 3560-3566.
[37]Albert Neuberger et al. Biochem. J., 1973 136:477-490.
[38]Bindu RC, Vivekanandan M. Hormonal activities of 5-aminolevulinic acid in callus induction and microprogation. Plant Growth Regul, 1998, 26:15-18.
[39]Castelfranco PA, Beale SI. Chlorophyll biosynthesis: recent advances and areas of current interest. Annu Rev Plant Physiol, 1983, 34:241-278.
[40]Hfgen R, Axelsen KB, Kannangara G et al. A visible marker for antisense mRNA expression in plants:Inhibition of chlorophyll synthesis with a glutamate 1-semialdehyde aminotransferase antisense gene. Proc Natl Acad Sci USA, 1994,91:1726-1730.
[41]Huang DD, Wang WY. Chlorophyll biosynthesis in Chlamydomonas starts with the formation of glutamyl-Trna. J Biol Chem, 1986,261:13451-13455.
[42]Chen WM, Jahn D, O'Neill GP et al. Purification of the glutamyl-tRNA reductase from Chlamydomonas reinhardtii involved in δ-aminolevulinic acid formation during chlorophyll biosynthesis.J Biol Chem, 1990,
265:4058-4063.
[43]Von Wettstein D, Gough S, Kananagara CG. Clorophyll biosynthesis. Plant Cell, 1995,7:1039-1057.
[44]Beator J. Kloppstech K. The circadian oscillator coordinates the synthesis of apoproteins and their pigments during chloroplast development. Plant Physiol, 1993,103:191-196.
[45]Hansson M. Basic characterization of two barley Hem A promoter regions reveals stem-loop structures and suggests a regulatory role in Poaceae tetrapyrrole biosynthesis. Plant Physiol Bacterium, 2001,39:155-160.
[46]Masuda T, Takabe K, Ohta H et al. Enzymatic activities for the synthesis of chlorophyll in pigment-deficient variegated leaves of Euonymus japonicus. Plant Cell Physiol, 1996,137:481-487.
[47]Schmidt, Frank, Donn, et al. Method for producing transgenic plants with modified 5-aminolevulinic acid biosynthesis,method for identifying 5-aminolevulinic acid synthesis effectors. US:6603062,2003.
[48]Nishikawa, Seiji, Tanaka, et al. Microorganisms producing 5-aminolevulinic acid and processing for producing 5-aminolevulinic acid by using the same. US: 6342377,2002.
[49]刘秀艳．光合细菌利用工业有机废水产生5-氨基乙酰丙酸的研究．杭州：浙江大学硕士论文，2001．
[2]Rosen, Arne, Larko, et al. Method for detecting cancer on skin of humans and mammals and arrangement for performing the method. US: 6421455,2002.
[3]Lentini, Peter J. Compositions useful in the phototherapeutic treatment of proliferative skin disorders. US:5885557,1999.
[4]Gierskcky, Karl E., Moan, et al. Esters of 5-aminolevulinic acid as photosensitizing agents in photochemotherapy US:6034267, 2000.
[5]戴秀莲．工业卫生与职业病，1989，15(6)：363．
[6]Rebeiz, Constantin A, Hopen, et al. Photodynamic herbicidal compositions using.delta.-aminolevulinic acid. US: 5286708, 1994.
[7]Rebeiz, Constantin A. Photodynamic herbicides. US: 5127938, 1992.
[8]Rebeiz, Constantin A. Photodynamic plant defoliants. US: 5321001, 1994.
[9]Rebeiz, Constantin A, Juvik, et al. Porphyric insecticides. US 5300526,1994.
[10]Rebeiz, Constantin A, Juvik, et al. Porphyric insecticides. US 5200427,1994.
[11]Tanaka, Takahashi, Hotta, et al. Method for promoting plant growth using 5-aminolevulinic acid or a salt thereof. US: 5298482, 1994.
[12]张一宾，周燚．5—氨基乙酰丙酸的植物生理活性[J]．世界农药，2000，22(3)：8-14．
[13]Rebeiz, Constantin A., Juvik, et al. Porphyric insecticides. US:5300526,1994.
[14]Rebeiz, Constantin A.,Juvik, et al. Porphyric insecticides. US:5200427,1993.
[15]Z'av yalov, Aronova, Makhova, et al. Synthesis of 5-aminolevulinic acid hydrochloride. Lzv. Akad. Nank SSSR. Ser. Khim.,1973, (3):657-658.
[16]Evans, Dacid A, Philip J. A simple route to α-aminoketones and related derivatives by dianion acylation reactoion. J.Chem. Soc. Common., 1978(17):752-754.
[17]Andreas Pfaltz, Saeed Anwar. Synthesis of α-aminoketones via readuction ofacyl cyanides. Tetrahedron Letter, 1984,25(28): 2977-2980.
[18]Haruhiko, Takeya, Tokhi, et al. Process for preparing 5-aminolevulinic acid. EP:607952, 1994.
[19]Kawakami H, Ebata T, Matsushita H. A new synthesis of 5-aminolevulinic acid. Agric. Biol. Chem., 1991,55(6):1687-1688.
[20]Gerand Descotes, Louis Cottier, Laurent Eynard, et al. Process for the preparation of N-acyl derivatives of 5-aminolevulinic acid, as well as the hydrochloride of the free acid .US:5344974,1994.
[21]L. Cottier, G. Descotes, L. Eymard, et al. Application to the synthesis of 5-aminolevulinic hydrochloride. Synthesis, 1995(3):303-306.
[22]Suzuki, Kojiro, Takeya, et al. Preparation of 5-aminolevulinic acid and
its intermediates. JP:0372450, 1991.
[23]Moens, Luc. Synthesis of an acid addition salt of delta-aminolevulinic acid from 5-bromo levulinic acid esters. US: 5907058, 1999.
[24]Z'av yalov, Zavozin. Synthesis of 5-amino-4-oxopentanoica acid hydrochloride, Akad. Nank SSSR. Ser. Khim.,1987, (18):1796-1799.
[25]Metcalf, Brian W, Adams, et al. Production of intermediates for enzyme inhibitors. US: 4325877,1982.
[26]Sasaki K, Watanabe, Nishizawa, et al. 5-Aminolevulinic acid production by Chiorella sp. during heterotrophic culture in the dark. World J. Microbiol. Biotechnol., 1995, 11: 361-362.
[27]Beale. The biosynthesis of δ-aminolevulinic acid in Chlorella. Plant Physiol., 1970,45: 504-596.
[28]Sasaki K, Tanaka, Nishizawa, et al. Enhanced production of 5-aminolevulinic acid by repeated addition of leculinic acid and supplement of precursors in photoheterotrophic culture of Rhodobacter sphaeroides. J. Ferment, Bioeng.,1991,71:403-406.
[29]刘如林．光合细菌及其应用．北京：中国农业科技出版社，1991．
[30]刘秀艳，叶敏，徐向阳．产生5—氨基乙酰丙酸(ALA)光合细菌生物学研究进展[J]．2000，20(5)：67-71．
[31]Dong lin et al. Journal Fermentation Bioengineering. 1989,68:88-91.
[32]Chen W, et al. Journal Bacteriology. 1994,175:2743-2746.
[33]Yoshiteru, Hashimoto, et al. Journal Fermentation Bioengineering. 1996,82:93-100.
[34]Toru Nakayashiki and Hachiro Inokuch, Genes Genet. Syst. 1996,71:237-241.
[35]Jill H. Zeilstera. Ryalls and Samuel Kaplan, Journal Bacteriology.1995,176:2760-2768.
[36]Mariet J. Van der Werf and J. Gregory zeikus, Applied and Enviromental Micro. 1996,38: 3560-3566.
[37]Albert Neuberger et al. Biochem. J., 1973 136:477-490.
[38]Bindu RC, Vivekanandan M. Hormonal activities of 5-aminolevulinic acid in callus induction and microprogation. Plant Growth Regul, 1998, 26:15-18.
[39]Castelfranco PA, Beale SI. Chlorophyll biosynthesis: recent advances and areas of current interest. Annu Rev Plant Physiol, 1983, 34:241-278.
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