产β-香树脂醇酿酒酵母细胞构建及高密度发酵
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摘要
该研究通过异源表达甘草来源β-香树脂醇合成酶(β-AS),成功在实验室前期保存的酿酒酵母底盘细胞中构β-香树脂醇的合成途径,实现了利用酿酒酵母生产β-香树脂醇,发酵质量浓度达5.97 mg·L~(-1)。通过进一步过表达酵母MVA途径的甲羟戊酸焦磷酸脱羧酶基因(ERG19),甲羟戊酸激酶基因(ERG12),3-羟基-3-甲基戊二酰-CoA合酶基因(ERG13),磷酸甲羟戊酸激酶基因(ERG8)和异戊烯二磷酸酯异构酶基因(IDI1),促进酵母代谢流走向β-香树脂醇合成方向,最终成功获得Y2-C2-4工程菌株,将β-香树脂醇浓度提高一倍,达到10.3 mg·L~(-1)。通过高密度发酵策略,该菌株其β-香树脂醇的产量可达到157.4 mg·L~(-1),相对于原始菌株提高了26倍,为公开报道基因工程酵母合成β-香树脂醇的较高产量。该研究不仅为β-香树脂醇生物合成奠定了基础,也为后期研究细胞色素氧化酶及糖基转移酶的挖掘提供优势底盘菌株。
In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.
In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.
引文
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[23] Shao Z, Zhao H, Zhao H. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways[J]. Nucleic Acids Res, 2009, 37(2): e16.
[24] Hayashi H, Huang P, Kirakosyan A, et al. Cloning and characterization of a cDNA encoding beta-amyrin synthase involved in glycyrrhizin and soyasaponin biosyntheses in licorice[J]. Biol Pharm Bull, 2001, 24(8): 912.
[25] 张根林. 酿酒酵母中β-香树脂醇合成途径的构建与调控[D].北京:北京理工大学, 2015.
[26] Petersen M, Abdullah Y, Benner J, et al.Evolution of rosmarinic acid biosynthesis[J]. Phytochemistry,2009,70(15):1663.
[2] Frota J T, Arruda B R, Melo T S D, et al. Antihyperglycemic and hypolipidemic effects of α, β-amyrin, a triterpenoid mixture from Protium heptaphyllum in mice[J].Lipids Health Dis, 2012, 11(1): 98.
[3] Pinto S A H, Pinto L M S, Cunha G M A, et al. Anti-inflammatory effect of α, β-amyrin, a pentacyclic triterpene from Protium heptaphyllum in rat model of acute periodontitis[J]. Inflammopharmacology, 2008, 16(1): 48.
[4] Simlai A, Rai A, Mishra S, et al. Antimicrobial and antioxidative activities in the bark extracts of Sonneratia caseolaris, a mangrove plant[J].Excli J, 2014, 13: 997.
[5] Ma S M, Garcia D E, Redding-Johanson A M, et al. Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases[J].Metab Eng, 2011, 13(5): 588.
[6] Zhou J, Du G, Jian C. Novel fermentation processes for manufacturing plant natural products[J].Curr Opin Bio Tech, 2014, 25(1): 17.
[7] Qamar W, Khan R, Khan A Q, et al. Alleviation of lung injury by glycyrrhizic acid in benzo(a)pyrene exposed rats: probable role of soluble epoxide hydrolase and thioredoxin reductase[J]. Toxicology, 2012, 291(1/3): 25.
[8] Arjumand W, Sultana S. Glycyrrhizic acid: a phytochemical with a protective role against cisplatin-induced genotoxicity and nephrotoxicity[J]. Life Sci, 2011, 89(13/14): 422.
[9] Seki H, Ohyama K, Sawai S, et al. Licorice β-amyrin 11-oxidase, a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin[J]. Proc Natl Acad Sci USA, 2008, 105(37): 14204.
[10] Seki H, Sawai S, Ohyama K, et al. Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin[J]. Plant Cell, 2011, 23(11): 4112.
[11] Ellington A A, Berhow Msingletary K W. Induction of macroautophagy in human colon cancer cells by soybean B-group triterpenoid saponins[J]. Carcinogenesis, 2005, 26(1): 159.
[12] Wei J, Liu H, Liu M, et al. Oleanolic acid potentiates the antitumor activity of 5-fluorouracil in pancreatic cancer cells[J]. Oncol Rep, 2012, 28(4): 1339.
[13] Paddon C J, Westfall P J, Pitera D J, et al. High-level semi-synthetic production of the potent antimalarial artemisinin[J]. Nature, 2013, 496(7446): 528.
[14] Ajikumar P K, Xiao W H, Tyo K E J, et al. Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli[J]. Science, 2010, 330(6000): 70.
[15] Zhou Y J, Wei G, Rong Q, et al. Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production[J]. J Am Chem Soc, 2012, 134(6): 3234.
[16] Guo J, Zhou Y J, Hillwig M L, et al. CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts[J]. Proc Natl Acad Sci USA, 2013, 110(29): 12108.
[17] Dai Z, Liu Y, Zhang X, et al. Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides[J].Metab Eng, 2013, 20(5): 146.
[18] Dai Z, Wang B, Liu Y, et al. Producing aglycons of ginsenosides in bakers′ yeast[J]. Sci Rep-UK, 2014, 4(3698): 3698.
[19] Stark R J M. The awesome power of yeast genetics-practical approaches and recipes for success[J].J Cell Sci, 2001, 114(14): 2551.
[20] Kirby J, Romanini D W, Paradise E M, et al. Engineering triterpene production in Saccharomyces cerevisiae-β-amyrin synthase from Artemisia annua[J].Febs J, 2008, 275(8): 1852.
[21] Leber R, Zenz R, Schr?ttner K, et al. A novel sequence element is involved in the transcriptional regulation of expression of the ERG1 (squalene epoxidase) gene in Saccharomyces cerevisiae[J].Febs J, 2001, 268(4): 914.
[22] Veen M, Stahl U, Lang C. Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae[J].Fems Yeast Res, 2003, 4(1): 87.
[23] Shao Z, Zhao H, Zhao H. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways[J]. Nucleic Acids Res, 2009, 37(2): e16.
[24] Hayashi H, Huang P, Kirakosyan A, et al. Cloning and characterization of a cDNA encoding beta-amyrin synthase involved in glycyrrhizin and soyasaponin biosyntheses in licorice[J]. Biol Pharm Bull, 2001, 24(8): 912.
[25] 张根林. 酿酒酵母中β-香树脂醇合成途径的构建与调控[D].北京:北京理工大学, 2015.
[26] Petersen M, Abdullah Y, Benner J, et al.Evolution of rosmarinic acid biosynthesis[J]. Phytochemistry,2009,70(15):1663.