红发夫酵母GGPP合成酶过量表达对虾青素产量的影响
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摘要
虾青素是一种类胡萝卜素,具有极强的淬灭自由基的能力并且还具有极强的着色能力,因此在医药,化妆品,食品及饲料行业都有广泛的应用前景。红发夫酵母的发酵生产虾青素,以培养时间短,不需光照而且可以进行高密度培养等优点得到广泛关注。
代谢工程方法改造菌种常用手段为敲除某些旁路的关键酶基因以减少碳的代谢分流,或者过表达某些关键酶基因。本文以野生型红法夫酵母As2.1557作为出发菌株,确定应该在上游基因编码的关键酶上入手,通过crtE基因的扩增,增加虾青素的前体物,进而考查其对红发夫酵母的生长及虾青素的产量带来的影响。
crtE基因编码牻牛儿基牻牛儿基焦磷酸合成酶(GGPP),GGPP是多种初生和次生代谢物的共同前体,为许多二萜、类胡萝卜素、叶绿素、醌和维生素E侧链等提供构建骨架。所以GGPP合酶能够起到调节碳流的作用,从而成为初生和次生代谢途径中的关键酶之一。
本实验通过同源重组的方法,构建了两个转化载体pSX-pc,pSX-gc。这两个载体均以rDNA为同源臂,分别将自带启动子的crtE基因,以及替换了强启动子gpd的crtE基因转化进红发夫酵母,并整合至其基因组上,以G418作为抗性筛选标记,最终筛选出成功扩增crtE基因的菌株。
通过测定其生长曲线,类胡萝卜素和虾青素的含量,发现,与野生菌株相比,对GGPP合成酶进行过表达后的转化株生长速度稍有下降;以替换gpd为启动子的crtE基因扩增载体改造的菌种GC,由于基因表达水平提高,从而使虾青素含量提升,最高达到了464.37μg/gDCW;以未替换启动子的crtE基因扩增载体改造的菌种PC,虾青素含量提升最高达到了503.22μg/gDCW。
Astaxanthin is a carotenoid, has strong ability to quench free radicals and also has a strong coloring ability, so in medicine, cosmetics, food and feed industry has a wide range of applications. Phaffia yeast fermentation of astaxanthin, in order to cultivate a short time, without light and can get the advantages of high-density culture attention.
Metabolic engineering methods for the transformation of a common means of knockout strains of certain key genes bypass to reduce the diversion of carbon metabolism or overexpression of certain key genes. This wild-type Phaffia yeast As2.1557 as the original strain, should determine the key enzyme in the upstream gene encoded on the start, through crtE gene amplification, increased astaxanthin precursors, and then examine its Phaffia yeast growth and astaxanthin production impact.
crtE gene coding based Mang Mang ox ox base pyrophosphate synthase (GGPP), GGPP is a variety of primary and secondary metabolites of a common precursor for many diterpenes, carotenoids, chlorophyll, and vitamin E quinone side chain, etc. to provide building skeleton. Therefore, GGPP synthase could play a role in regulating carbon flow in order to become primary and secondary metabolic pathways in one of the key enzymes.
In this study, the method by homologous recombination was constructed of two transformation vectors, pSX-pc and pSX-gc,. The two carriers are rDNA as homologous arms, respectively crtE own promoter gene, and the replacement of a strong promoter of crtE gpd gene was transformed into the yeast Phaffia, and integrated into its genome to G418 as anti- sexual selection marker, the ultimate success of selected gene amplification crtE strains.
By measuring its growth curve, carotenoids and astaxanthin content, found that compared with the wild strain, the overexpression of GGPP synthase for the conversion of strain after the growth rate declined slightly; to replace the gpd gene as the promoter crtE amplified strain vector transformation, due to increased gene expression, thereby enhancing astaxanthin content, up to a 464.37μg/gDCW; to not replace the promoter crtE gene amplification vector transformation strain, as integrated copies few more, GGPP synthase and a corresponding increase in the expression, enhance the astaxanthin content of the most significant to the 503.22μg/gDCW.
代谢工程方法改造菌种常用手段为敲除某些旁路的关键酶基因以减少碳的代谢分流,或者过表达某些关键酶基因。本文以野生型红法夫酵母As2.1557作为出发菌株,确定应该在上游基因编码的关键酶上入手,通过crtE基因的扩增,增加虾青素的前体物,进而考查其对红发夫酵母的生长及虾青素的产量带来的影响。
crtE基因编码牻牛儿基牻牛儿基焦磷酸合成酶(GGPP),GGPP是多种初生和次生代谢物的共同前体,为许多二萜、类胡萝卜素、叶绿素、醌和维生素E侧链等提供构建骨架。所以GGPP合酶能够起到调节碳流的作用,从而成为初生和次生代谢途径中的关键酶之一。
本实验通过同源重组的方法,构建了两个转化载体pSX-pc,pSX-gc。这两个载体均以rDNA为同源臂,分别将自带启动子的crtE基因,以及替换了强启动子gpd的crtE基因转化进红发夫酵母,并整合至其基因组上,以G418作为抗性筛选标记,最终筛选出成功扩增crtE基因的菌株。
通过测定其生长曲线,类胡萝卜素和虾青素的含量,发现,与野生菌株相比,对GGPP合成酶进行过表达后的转化株生长速度稍有下降;以替换gpd为启动子的crtE基因扩增载体改造的菌种GC,由于基因表达水平提高,从而使虾青素含量提升,最高达到了464.37μg/gDCW;以未替换启动子的crtE基因扩增载体改造的菌种PC,虾青素含量提升最高达到了503.22μg/gDCW。
Astaxanthin is a carotenoid, has strong ability to quench free radicals and also has a strong coloring ability, so in medicine, cosmetics, food and feed industry has a wide range of applications. Phaffia yeast fermentation of astaxanthin, in order to cultivate a short time, without light and can get the advantages of high-density culture attention.
Metabolic engineering methods for the transformation of a common means of knockout strains of certain key genes bypass to reduce the diversion of carbon metabolism or overexpression of certain key genes. This wild-type Phaffia yeast As2.1557 as the original strain, should determine the key enzyme in the upstream gene encoded on the start, through crtE gene amplification, increased astaxanthin precursors, and then examine its Phaffia yeast growth and astaxanthin production impact.
crtE gene coding based Mang Mang ox ox base pyrophosphate synthase (GGPP), GGPP is a variety of primary and secondary metabolites of a common precursor for many diterpenes, carotenoids, chlorophyll, and vitamin E quinone side chain, etc. to provide building skeleton. Therefore, GGPP synthase could play a role in regulating carbon flow in order to become primary and secondary metabolic pathways in one of the key enzymes.
In this study, the method by homologous recombination was constructed of two transformation vectors, pSX-pc and pSX-gc,. The two carriers are rDNA as homologous arms, respectively crtE own promoter gene, and the replacement of a strong promoter of crtE gpd gene was transformed into the yeast Phaffia, and integrated into its genome to G418 as anti- sexual selection marker, the ultimate success of selected gene amplification crtE strains.
By measuring its growth curve, carotenoids and astaxanthin content, found that compared with the wild strain, the overexpression of GGPP synthase for the conversion of strain after the growth rate declined slightly; to replace the gpd gene as the promoter crtE amplified strain vector transformation, due to increased gene expression, thereby enhancing astaxanthin content, up to a 464.37μg/gDCW; to not replace the promoter crtE gene amplification vector transformation strain, as integrated copies few more, GGPP synthase and a corresponding increase in the expression, enhance the astaxanthin content of the most significant to the 503.22μg/gDCW.
引文
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[2] Andrewes A.G., Borch G., Liaaen-Jensen S., et al. Animal carotenoids. 9. On the absolute configuration of astaxanthin and actinioerythrin. Acta Chem. Scand. 1974, B28: 730-736
[3] Andrewes A.G., Starr M.P. (3R, 3’R)-Astaxanthin from the yeast Phaffia rhAozyma. Phytochemistry. 1976, 15: 1009-1011
[4] Kurashige M. Inhibition of oxidative injury of biological membranes by astaxanthin. Physiol. Chem. Phys. Med. NMR 1990, 22: 27-38
[5] Schimidzu N. Carotenoids as singlet oxygen quenchers in marine organisms. Fish. Sci. 1996, 62: 134-137
[6] O’Connor I., O’Brien N. MAulation of UVA light-induced oxidative stress by beta-carotene, lutein and astaxanthin in cultured fibroblasts. J. Dermatol. Sci. 1998, 16: 226-230
[7]陈晓明,徐学明,富含虾青素的法夫酵母对金鱼体色的影响,中国水产科学,2004,(4):73-74
[8] Tanka T. Chemoprevention of mouse urinary bladder carcinogenesis by the naturally occurring carotenoid astaxanthin. Carcinogenesis. 1994, 15: 15-19
[9] Tanka T. Chemoprevention of rat oral carcinogenesis by naturally occurring xanthophylls, astaxanthin and canthaxanthin. Cancer Res. 1995, 55: 4059-4064
[10] Tanka T. Suppression of azomethane-induced rat colon carcinogenesis by dietary administration of naturally occurring xanthophylls, astaxanthin and canthaxanthin during the postinitiation phase. Carcinogenesis, 1995, 16: 2957-2963
[11] Chew B. P. A comparison of the anticancer activities of dietaryβ-carotene, canthaxanthin and ataxanthin in mice in vivo. Anticancer Res. 1999, 19: 1846-1854
[12] Anderson M. MethA of inhibiting 5-αreductase with astaxanthin to prevent and treat benign prostate hyperplasia (BPH) and prostate cancer in human males. US Patent, 6277417, 2001
[13] Kurihara H. Contribution of the antioxidative property of astaxanthin to its protective effect on the promotion of cancer metastasis in mice treated with restraint stress. Life Sci. 2002, 70: 2509-2520
[14] Jyonouchi H. Effect of carotenoids on in vitro immunoglobulin prAuction by human peripheral bloA mononuclear cell: astaxanthin, a carotenoid without vitamin A activity, enhances in vitro immunoglobulin prAuction in response to a T-dependent stimulant and antigen. Nutr. Cancer. 1995, 23: 171-183
[15] Torrissen O.J., Christiansen R. Requirements for carotenoids in fish diets. J. Appl. Ichthyol., 1995, 11: 225-230
[16]Christiansen P., Glette J., et a1. Antioxidant statis and immunity in Atlantic salmon, Salmo sala L, fed semi-purified diets with and without astaxanthin supplementation. J. Fish Diseases, 1995, 18:317.
[17] Bjerkeng B., Johnson G. Frozen storage quality of rainbow trout (Oncorkynchus mykiss) as affected by oxygen, illumination and fillet pigment. J. FoA Sci, 1995, 60(2):284-290
[18] Tso M.O., Lam T.T. MethA of retarding and ameliorating central nervous system and eye damage. US Patent, 5527533, 1996
[19] Bennedsen M., Wang X. Treatment of H. pylori infected mice with antioxidant astaxanthin reduces gastric inflammation, bacterial load and mAulates cytokine release by splenocytes. Immunol Let, 1999, 70(3):185-189
[19] John A.B., Timothy D.L., Radheshyam K.J. Isolation of astaxanthin- overprAucing mutants of Phaffia rhAozyma. Biotechnology Letters. 1997, 19(2): 109-112
[20] An G.H., Bielich J., Auerbach R., et al. Isolation and characterization of carotenoids hyper prAucing mutants of yeast by flow cytometry and cell sorting. Biotechnology. 1991, 9: 70-73
[21] Nelis H.J., Leenheer A.P. Biochemistry of vitamins, pigments and growth factors, London: Elsevier Applied Science, 1989, 43-80
[22] Boussiba S. Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response. Physiol. Plant. 2000, 108:111-117
[23] Phaff H. J., Miller M. W., Yoneyama M., et al. A comparative study of the yeast florae associated with trees on the Japanese islands and on the west coast of North America. Fermentation Technology TAay, 1972, 12:759-774
[24] Andrewes A.G., Phaff H.J., Starr M.P. Carotenoids of Phaffia rhAozyma, a red-pigmented fermenting yeast. Phytochemistry, 1976, 15: 1003-1007
[25] Glolubev W.I. Perfect state of RhAomyces Dendrorhous (Phaffia rhAozyma). Yeast, 1995, 11: 101-110
[26] Vustin M.M., Belykh E.N., Kishilova S.A. Relationship between astaxanthin prAuction and the intensity of anabolic processes in the yeast Phaffia rhAozyma. Microbiology, 2004, 73(6): 643-649
[27] Yamane Y-I, Higashida K., Nakashimada Y., et al. Influence of oxygen andglucose on primary metabolism and astaxanthin prAuction by Phaffia rhodozyma in batch and fed-batch cultures: kinetic and stoichiometric analysis. Applied and Environmental Microbiology, 1997, 63(11): 4471-4478
[28]于孝东,姚为志等,虾青素的开发及其在水产养殖上的应用,江西水产科技,2004,03
[29]朱明军等,吴振强等,高产虾青素红发夫酵母选育研究进展,氨基酸和生物资源,2000,03
[30] Johson E.A., Villa T.G., Lewis M.J. Phaffia rhAozyma as an astaxanthin source in salmonid diets. Aquaculture, 1980, 20: 123-134
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