链球菌产透明质酸工程菌的构建研究
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摘要
透明质酸(hyaluronic acid, HA),又名玻璃酸,普遍存在于脊椎动物和某些细菌的荚膜,是由的D-葡萄糖醛酸和N-乙酰氨基葡萄糖以p-1,3和p-1,4糖苷键交连而成的高分子链状聚合物,具有良好的生物相容性和生物降解性,在医药和化妆品领域广泛应用,近年来透明质酸的需求与日俱增,表现出良好的市场潜力和广阔的发展前景。
目前,微生物发酵法已取代动物组织提取法成为透明质酸生产的主要方式。国内外发酵法生产透明质酸主要菌种为C组链球菌(Group C Streptococcus, GCS)的马链球菌马亚种和马链球菌兽疫亚种。马链球菌兽疫亚种与A组链球菌(Group A Streptococcus GAS)蛋白序列对比分析研究发现,该菌除没有链球菌致热外毒素外,几乎具有GAS业已证实的所有毒力因子。由于GCS没有宿主专嗜性,能引起多种动物,甚至人的链球菌病,因此用GCS发酵生产透明质酸,存在潜在安全风险,所以通过分子生物学的方法,构建透明质酸生产毒力基因缺失高产突变株,具有积极意义。
本实验从牛鼻粘膜样品中分离出一株产透明质酸链球菌,经16S rDNA基因序列相似性分析,鉴定为Streptococcus equi subsp. Zooepidemicus (马链球菌兽疫亚种),菌株在透明质酸发酵培养基中,37℃摇瓶培养,18小时左右透明质酸产量达到最高(0.96g/L)。以该菌为出发菌株,用温度敏感基因敲除载体系统pJR700,成功构建链球菌血红素受体(Streptecoccus Heme Receptor, Shr)基因缺失突变株。研究结果为进一步敲除其他毒力基因,构建无毒透明质酸生产基因工程菌打下了基础。
链球菌透明质酸合成酶是合成透明质酸的关键酶,其操纵子由hasA、hasB、hasC三个基因组成。本课题根据温度敏感载体pJR700在37℃能将同源基因整合到染色体并稳定遗传的特性,用该载体系统,构建携带hasABC基因的温度敏感性质粒载体pXL31,将该质粒载体转到血红素受体基因缺失突变株,获得含双拷贝hasABC基因工程菌。SDS-PAGE电泳证实,与血红素受体基因缺失突变株相比,工程菌分子量大约50KDa、47KDa和40KDa的三个蛋白高表达,工程菌高表达的这三个蛋白,理论上与马链球菌兽疫亚种hasA、hasB、hasC三个基因表达蛋白分子量相近；用Bitter-Muir方法测定菌株透明质酸含量结果显示,被测定的6株工程菌透明质酸产量比出发菌株提高范围在27-34%之间,其中一株菌株透明质酸产量达到1.28 g/L,比出发菌株提高34%。实验结果证实,用温度敏感基因载体系统pJR700增加染色体上基因的拷贝是可行的。这一方法研究成功,为通过基因工程的方法提高微生物代谢产物研究提供了一种新的选择。
Hyaluronic Acid, also known as Hyaluron or HA, was found naturally in the tissues of all vertebrates and in the capsule of some bacteria. HA is a high molecule linear polymer which composed of D-glucuronic acid and D-N-acetylglucosamine, linked via alternatingβ-1,4 andβ-1,3 glycosidic bonds. Because of its good biocompatibility and biodegradability, HA is usded wildly in pharmaceutical and cosmetic, and showing good market needs and broad development prospects in recent years.
Commercial HA is obtained by either chemical extraction from the rooster combs/umbilic-cords, or the microbial fermentation. The chemical extraction method is limited by resource availability. The microbial fermentation becomes the main processing for HA production. The main strain for HA production is the Streptococcus equi subsp. Zooepidemicus of Group C Streptococcus (GCS). Comparison vulunce factors of S. Zooepidemicus to Group A Streptococcus (GAS), the S. Zooepidemicus possess all the virulence factors which have been found in the GAS except streptococcal pyrogenic exotoxin (spe). The pathogenic GCS can infect different host and cause a variety of animals even human streptococcal disease. Therefore construction of the virulence gene deletion mutant is industrially emphasized recently for HA production.
In this study we isolated a strain which produced HA from ox snout, It was identified as Streptococcus equi subsp. Zooepidemicus by 16S rDNA gene sequence analysis. The HA titer was reached 0.96g/L in flask culture at 37℃and 18 hours.
We successfully constructed the deletion shr mutants by the thermosensitive vector system of pJR700 in S. Zooepidemicus. The results showed that is possible that constructing Non-virulence gene-engineering strain for HA production by knockout of virulence genes using pJR700.
Streptococcus hyaluronic acid synthase (HAS) are the key enzyme for synthesis of HA in bacteria. The operon of HAS are composed of three genes which are has A, hasB and hasC. The thermosensitive vector system of pJR700 has the function, which integrate its carrying the homologue gene into chromosome of the streptococcus and the integrated homologue gene can transmit to generation. We constructed the thermosensitive delivery vector pLX31 which contain hasABC gene using pJR700, then transfered pLX31 into shr mutant of S. Zooepidemicus by electroporation, and obtain the gene engineering strain which contain two copies of hasA, hasB and hasC gene. The snalyses of SDS-PAGE indicated that the strain constructed can express exceed three protein, which molecular weight were 50KDa、47KDa and 40KDa, and in the same proteins of expression by hasA, hasB, and hasC of S. Zooepidemicus, respectively. The HA titer was increased high to 1.28g/L at 37℃and 18h in flask culture in two copies of hasABC strain. The hyaluronic acid productivity of gene-engineering strains were increased by 27 to 34% compared with the parental strain, shr mutant. Therefore, it is reasonable that we believe using pJR700 to increase the gene copy of streptococcus. Our method provides a new choice for microbial metabolic engineering research.
目前,微生物发酵法已取代动物组织提取法成为透明质酸生产的主要方式。国内外发酵法生产透明质酸主要菌种为C组链球菌(Group C Streptococcus, GCS)的马链球菌马亚种和马链球菌兽疫亚种。马链球菌兽疫亚种与A组链球菌(Group A Streptococcus GAS)蛋白序列对比分析研究发现,该菌除没有链球菌致热外毒素外,几乎具有GAS业已证实的所有毒力因子。由于GCS没有宿主专嗜性,能引起多种动物,甚至人的链球菌病,因此用GCS发酵生产透明质酸,存在潜在安全风险,所以通过分子生物学的方法,构建透明质酸生产毒力基因缺失高产突变株,具有积极意义。
本实验从牛鼻粘膜样品中分离出一株产透明质酸链球菌,经16S rDNA基因序列相似性分析,鉴定为Streptococcus equi subsp. Zooepidemicus (马链球菌兽疫亚种),菌株在透明质酸发酵培养基中,37℃摇瓶培养,18小时左右透明质酸产量达到最高(0.96g/L)。以该菌为出发菌株,用温度敏感基因敲除载体系统pJR700,成功构建链球菌血红素受体(Streptecoccus Heme Receptor, Shr)基因缺失突变株。研究结果为进一步敲除其他毒力基因,构建无毒透明质酸生产基因工程菌打下了基础。
链球菌透明质酸合成酶是合成透明质酸的关键酶,其操纵子由hasA、hasB、hasC三个基因组成。本课题根据温度敏感载体pJR700在37℃能将同源基因整合到染色体并稳定遗传的特性,用该载体系统,构建携带hasABC基因的温度敏感性质粒载体pXL31,将该质粒载体转到血红素受体基因缺失突变株,获得含双拷贝hasABC基因工程菌。SDS-PAGE电泳证实,与血红素受体基因缺失突变株相比,工程菌分子量大约50KDa、47KDa和40KDa的三个蛋白高表达,工程菌高表达的这三个蛋白,理论上与马链球菌兽疫亚种hasA、hasB、hasC三个基因表达蛋白分子量相近；用Bitter-Muir方法测定菌株透明质酸含量结果显示,被测定的6株工程菌透明质酸产量比出发菌株提高范围在27-34%之间,其中一株菌株透明质酸产量达到1.28 g/L,比出发菌株提高34%。实验结果证实,用温度敏感基因载体系统pJR700增加染色体上基因的拷贝是可行的。这一方法研究成功,为通过基因工程的方法提高微生物代谢产物研究提供了一种新的选择。
Hyaluronic Acid, also known as Hyaluron or HA, was found naturally in the tissues of all vertebrates and in the capsule of some bacteria. HA is a high molecule linear polymer which composed of D-glucuronic acid and D-N-acetylglucosamine, linked via alternatingβ-1,4 andβ-1,3 glycosidic bonds. Because of its good biocompatibility and biodegradability, HA is usded wildly in pharmaceutical and cosmetic, and showing good market needs and broad development prospects in recent years.
Commercial HA is obtained by either chemical extraction from the rooster combs/umbilic-cords, or the microbial fermentation. The chemical extraction method is limited by resource availability. The microbial fermentation becomes the main processing for HA production. The main strain for HA production is the Streptococcus equi subsp. Zooepidemicus of Group C Streptococcus (GCS). Comparison vulunce factors of S. Zooepidemicus to Group A Streptococcus (GAS), the S. Zooepidemicus possess all the virulence factors which have been found in the GAS except streptococcal pyrogenic exotoxin (spe). The pathogenic GCS can infect different host and cause a variety of animals even human streptococcal disease. Therefore construction of the virulence gene deletion mutant is industrially emphasized recently for HA production.
In this study we isolated a strain which produced HA from ox snout, It was identified as Streptococcus equi subsp. Zooepidemicus by 16S rDNA gene sequence analysis. The HA titer was reached 0.96g/L in flask culture at 37℃and 18 hours.
We successfully constructed the deletion shr mutants by the thermosensitive vector system of pJR700 in S. Zooepidemicus. The results showed that is possible that constructing Non-virulence gene-engineering strain for HA production by knockout of virulence genes using pJR700.
Streptococcus hyaluronic acid synthase (HAS) are the key enzyme for synthesis of HA in bacteria. The operon of HAS are composed of three genes which are has A, hasB and hasC. The thermosensitive vector system of pJR700 has the function, which integrate its carrying the homologue gene into chromosome of the streptococcus and the integrated homologue gene can transmit to generation. We constructed the thermosensitive delivery vector pLX31 which contain hasABC gene using pJR700, then transfered pLX31 into shr mutant of S. Zooepidemicus by electroporation, and obtain the gene engineering strain which contain two copies of hasA, hasB and hasC gene. The snalyses of SDS-PAGE indicated that the strain constructed can express exceed three protein, which molecular weight were 50KDa、47KDa and 40KDa, and in the same proteins of expression by hasA, hasB, and hasC of S. Zooepidemicus, respectively. The HA titer was increased high to 1.28g/L at 37℃and 18h in flask culture in two copies of hasABC strain. The hyaluronic acid productivity of gene-engineering strains were increased by 27 to 34% compared with the parental strain, shr mutant. Therefore, it is reasonable that we believe using pJR700 to increase the gene copy of streptococcus. Our method provides a new choice for microbial metabolic engineering research.
引文
[1]Karl Meyer, John W Palmer. The polysaccharide of the vitreous humor. Biochem.1934, 107:629-634
[2]沈渤江.公鸡冠透明质酸的制备及其理化性质.中国医药工业杂志.1986,17(7)：291
[3]M.K. Cowman, S. Matsuoka. Experimental approaches to hyaluronan structure. Carbohydrate Research.2005,340:791-809
[4]Seott JE, Cummings C, Brass A, Chen Y. Seeondary and tertiary structures of hyaluronan in aqueous solution, investgated by rotary shadowing-election microscopey and computer simulation. Biochem.1991,274:699-705
[5]鲁念慈,谭天伟.透明质酸的制备及其应用.功能高分子学报.2001,14(3)：370-376
[6]Yamada T, Kawasaki T. Microbial synthesis of hyaluronan and chitin:New approaches. Biosci Bioeng.2005,99 (6):521-528
[7]郭学平,贺艳丽,孙茂利,张天民.透明质酸在保健品中的应用.中国生化药物杂志.2002,23(1)：49-51
[8]Balazs E A, Denlinger J L. Visco supplementation:a new concept in the treatment of Osteoarthritis. J Rheumatol Suppl.1993,39:3-9
[9]胡泽利.透明质酸在牙周组织创伤愈合过程中的作用.中国药业.2005,14(9)：22-23
[10]王文斌,顾其胜,吴萍.透明质酸钠—一种新型的防止术后组织粘连的高分子吸收生物医学材料.透析与人工器官.1997,8(1)：4
[11]贺艳丽,张霞,凌沛学,张天民.玻璃酸的生理功能.中国生化药物杂志.2001,22(3)： 156-158
[12]朱广华,方煜平.透明质酸制备方法及应用研究进展.中国医药工业杂志.1996,2(8)：382-384
[13]O'Regan M, Martini I, Crescenzi F, De Luca C, Lansing M. Molecular mechanisms and genetics of hyaluronan biosynthesis Int J Biol Macromol.1994,16(6):283-6
[14]Kendall FE, Heidelberger M, Dason MH. A serologically inactive polysaccharide streptococcus. Biochem.1937,118(1):61-69
[15]白绘宇,徐晶,李慧珺,杨逸群,刘晓亚.透明质酸的制备及应用研究进展.广东化工.2010,37(11)：243-244
[16]Roseman, et al. BioChem.1953.203:213
[17]Maclennan A P.Gen Microbio.1956,15:485
[18]Stoolmiller A C Dorfinan.BioChem.1969,244:236
[19]Woolcock JB. Gen Microbio.1974,85:372
[20]Sunghara, et al. Bio Chem.1979,254:6252
[21]Akasaka H, Seto S, Yanagi M, Fukushima S, Mitsui T. Industrial production of hyaluronic acid by Streptococcus zooepidemicus. Soc Cosmet Chem Japan.1988,22:35-42
[22]范红结,陆承平.马链球菌兽疫亚种毒力因子.中国人兽共患病学报.2006,22(3)： 927
[23]李瑾,焦翔.马链球菌兽疫亚种菌血症一例.上海医学检验杂志.1998,4：244.
[24]孙永丽,李香兰,周春兰.马链球兽疫亚种引起化脓性脑膜炎1例分析.中国民康医学.2008,10(13)：20
[25]Nimrod A, Greenman B, Kanner D, Landsberg M, Beck Y. Method of producing high molecular weight sodium hyaluronate by fermentation of Streptococcus. US Patent 1988, 780(4):414
[26]Lars M. Blank, Philip Hugenholtz, Lars K.N. Evolution of the Hyaluronic Acid Synthesis (has) Operon in Streptococcus zooepidemicus and Other Pathogenic Streptococci. Applied Microbiology and Biotechnology.2008,67(1):13-22
[27]De Angelis Hyaluronan synthases:fascinating glycosyl- transferases from vertebrates bacterial pathogens, and algal viruses. Cell Mol Life Sci.1999,56:670-682.
[28]P.H. Weigel, V.C. Hascall and M. Tammi, Hyaluronan synthases. BiolChem.1997, 272:13997-14000.
[29]Dougherty BA, van de Rijn I. Molecular characterization of hasB from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP-glucose dehydrogenase activity. BiolChem.1993,268(10):7118-7124
[30]Crater DL, Dougherty BA, van de Rijn I. Molecular characterization of hasC from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP-glucose pyrophosphorylase activity. The Journal of biological chemistry.1996, 270(48):28676-28680
[31]Ashbaugh CD, Alberti S, Wessels MR. Molecular analysis of the capsule gene region of group A Streptococcus:the hasAB genes are sufficient for capsule expression. The Journal of Bacteriology.1998,180(18):4955-4959
[32]田平芳,曲凯,谭天伟.链球菌生物合成透明质酸的分子机理与基因工程菌构建进展.中国生物工程杂志.2008,28(4)：98-102
[33]Chien LJ, Lee CK. Hyaluronic acid production by recombinant Lactococcus lactis. Appl Microbiol Biotechnol.2007,77(2):339-346
[34]Huimin Yua, Gregory Stephanopoulos. Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid. Metabolic Engineering.2008,10(1):24-32
[35]Bill Widner, Regine Behr, Steve Von D.et.al. Hyaluronic Acid Production in Bacillus subtilis. Applied and Environmental Microbiology.2005,71(7):3747-3752
[36]Mao Z, Chen RR. Recombinant synthesis of hyaluronan by Agrobacterium sp. Biotechnol Prog.2007,23(5):1038-1042
[37]GravesMV, Burbank DE, Roth R, et al. Hyaluronan synthesis in virus PBVC-1 infected Chlorella-like green algae. Virology.1999,257:15-23.
[38]张容鹄,白洋,冯建成,温珍昌.链球菌合成透明质酸的研究进展.生物技术通报.2010,4：63-68
[40]郝宁,张晋宇,陈国强.兽疫链球菌中表达vgb 基因和透明质酸合成基因提高透明质酸产量.中国生物工程杂志.2005,25(6)：56-60
[41]Bitter T Muir H M.A modified uranic acid carbazole reaction. Analitical Biochemistry. 1962,4(4):330-334
[42]Bates CS, Montanez GE, Woods CR, Vincent RM, Eichenbaum Z. Identification and characterization of a Streptococcus pyogenes operon involved in binding of hemoproteins and acquisition of iron. Infect Immun.2003,71(3):1042-1055
[43]Morly Fisher, Ya-Shu Huang, Xueru Li, Kevin S. McIver, Chadia Toukoki, and Zehava Eichenbaum. Shr is a broad-spectrum surface receptor that contributes to adherence and virulence in Group A Streptococcus. Infection and Immunity.2008,76(11):5006-5015.
[44]李尧, 蓝小玲, 李学如, 郭泰林,姚宁,江南屏,任瑶瑶.一种构建马链球菌兽疫亚种血红素受体基因缺失突变株的方法.微生物学报.2010,50(6)：822-827
[2]沈渤江.公鸡冠透明质酸的制备及其理化性质.中国医药工业杂志.1986,17(7)：291
[3]M.K. Cowman, S. Matsuoka. Experimental approaches to hyaluronan structure. Carbohydrate Research.2005,340:791-809
[4]Seott JE, Cummings C, Brass A, Chen Y. Seeondary and tertiary structures of hyaluronan in aqueous solution, investgated by rotary shadowing-election microscopey and computer simulation. Biochem.1991,274:699-705
[5]鲁念慈,谭天伟.透明质酸的制备及其应用.功能高分子学报.2001,14(3)：370-376
[6]Yamada T, Kawasaki T. Microbial synthesis of hyaluronan and chitin:New approaches. Biosci Bioeng.2005,99 (6):521-528
[7]郭学平,贺艳丽,孙茂利,张天民.透明质酸在保健品中的应用.中国生化药物杂志.2002,23(1)：49-51
[8]Balazs E A, Denlinger J L. Visco supplementation:a new concept in the treatment of Osteoarthritis. J Rheumatol Suppl.1993,39:3-9
[9]胡泽利.透明质酸在牙周组织创伤愈合过程中的作用.中国药业.2005,14(9)：22-23
[10]王文斌,顾其胜,吴萍.透明质酸钠—一种新型的防止术后组织粘连的高分子吸收生物医学材料.透析与人工器官.1997,8(1)：4
[11]贺艳丽,张霞,凌沛学,张天民.玻璃酸的生理功能.中国生化药物杂志.2001,22(3)： 156-158
[12]朱广华,方煜平.透明质酸制备方法及应用研究进展.中国医药工业杂志.1996,2(8)：382-384
[13]O'Regan M, Martini I, Crescenzi F, De Luca C, Lansing M. Molecular mechanisms and genetics of hyaluronan biosynthesis Int J Biol Macromol.1994,16(6):283-6
[14]Kendall FE, Heidelberger M, Dason MH. A serologically inactive polysaccharide streptococcus. Biochem.1937,118(1):61-69
[15]白绘宇,徐晶,李慧珺,杨逸群,刘晓亚.透明质酸的制备及应用研究进展.广东化工.2010,37(11)：243-244
[16]Roseman, et al. BioChem.1953.203:213
[17]Maclennan A P.Gen Microbio.1956,15:485
[18]Stoolmiller A C Dorfinan.BioChem.1969,244:236
[19]Woolcock JB. Gen Microbio.1974,85:372
[20]Sunghara, et al. Bio Chem.1979,254:6252
[21]Akasaka H, Seto S, Yanagi M, Fukushima S, Mitsui T. Industrial production of hyaluronic acid by Streptococcus zooepidemicus. Soc Cosmet Chem Japan.1988,22:35-42
[22]范红结,陆承平.马链球菌兽疫亚种毒力因子.中国人兽共患病学报.2006,22(3)： 927
[23]李瑾,焦翔.马链球菌兽疫亚种菌血症一例.上海医学检验杂志.1998,4：244.
[24]孙永丽,李香兰,周春兰.马链球兽疫亚种引起化脓性脑膜炎1例分析.中国民康医学.2008,10(13)：20
[25]Nimrod A, Greenman B, Kanner D, Landsberg M, Beck Y. Method of producing high molecular weight sodium hyaluronate by fermentation of Streptococcus. US Patent 1988, 780(4):414
[26]Lars M. Blank, Philip Hugenholtz, Lars K.N. Evolution of the Hyaluronic Acid Synthesis (has) Operon in Streptococcus zooepidemicus and Other Pathogenic Streptococci. Applied Microbiology and Biotechnology.2008,67(1):13-22
[27]De Angelis Hyaluronan synthases:fascinating glycosyl- transferases from vertebrates bacterial pathogens, and algal viruses. Cell Mol Life Sci.1999,56:670-682.
[28]P.H. Weigel, V.C. Hascall and M. Tammi, Hyaluronan synthases. BiolChem.1997, 272:13997-14000.
[29]Dougherty BA, van de Rijn I. Molecular characterization of hasB from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP-glucose dehydrogenase activity. BiolChem.1993,268(10):7118-7124
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