Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (3): 546-552.doi: 10.3864/j.issn.0578-1752.2014.03.014

• HORTICULTURE • Previous Articles     Next Articles

Heterologous Expression of Taxadiene Synthase Gene in Ganoderma lucidum

 XIN  Yan-Hua-1, XIAO  Zhao-Yan-1, YOU  Lin-Feng-1, GUO  Li-Qiong-1, 2 , LIN  Jun-Fang-1, 2   

  1. 1、College of Food Science, South China Agriculture University, Guangzhou 510640;
    2、Institute of Biomass Energy, South China Agriculture University, Guangzhou 510640
  • Received:2013-04-18 Online:2014-02-01 Published:2013-05-28

Abstract: 【Objective】The objective of the study is to lay a theoretical foundation for the expression and production of taxol or its precursor products by using Ganoderma lucidum as a novel bioreactor.【Method】 For preparation of the G. lucidum protoplast, mycelium were collected and washed twice with 0.6 mol•L-1 mannitol, and enzymed with 1% lywallzyme at 30℃ for 3 h. Protoplasts were collected and suspended with 0.6 mol•L-1 mannitol, and the final concentration was adjusted to 1.0×108 cells/mL. For transformation of the exogenous gene (ts), the plasmid pgGIgpd-TS (1.0 μg) and pBgGI-hph (1.0 μg) were added in G. lucidum protoplast culture. Incubation on ice was performed for 30 min. Subsequently 0.5 mL of PEG was added and incubated at room temperature for 20 min. The transformed protoplasts were inoculated on the medium without hygromycin and cultivated 5-7 days at 25℃. After the colonies grew up, they were covered with the PDA semisolid medium with hygromycin. Genomic DNA and RNA were extracted from the mycelium of regenerated G. lucidum for transformant analyses. PCR and RT-PCR analyses were carried out for detection of the ts gene in putative transformants. For analysis of taxa-4(5),11(12)-diene, samples were analyzed on GC linked to a mass spectrometer using split-less injection. Temperature of the column was initially set and maintained at 100℃ for 1 min and was then increased to 300℃ at a rate of 8.0℃•min-1. The column was then maintained at this temperature for 2 min. 【Result】The putative transformants were obtained on the selective plates with hygromycin at 25℃. No colonies appeared on the nontransformed plates. This result indicated that the hph gene was transformed into the G. lucidum and obtained preliminary expression. The results showed that 4 out of 20 putative transformants were integrated into both ts and hph genes,with a co-transformation efficiency was 20%. RT-PCR results verified that all the 4 transformants had obtained the transcription of taxadiene synthase gene. Crude extracts from the G. lucidum were analyzed. It was found a GC peak at 16.762 min which was absent in the wild type control. The mass fragmentation pattern of this peak had the same diagnostic ions with taxa-4(5),11(12)-diene. The taxadiene was detected in the mycelia of transformed G. lucidum strain by GC-MS analysis.【Conclusion】This is the first report of the production of taxa-4(5),11(12)-diene by engineering a novel transformation system G. lucidum with taxadiene synthase gene. The authors proposed that G. lucidum is a promising host for the biotechnological production of precursors of paclitaxel, and the results for the study of molecular biology of G. lucidum is important.

Key words: Ganoderma lucidum , genetic transformation , taxadiene synthase , taxa-4(5) , 11(12)-diene , paclitaxel precursor

[1]Campos Ziegenbein F, Hanssen H P, Konig W A. Secondary metabolites from Ganoderma lucidum and Spongiporus leucomallellus. Phytochemistry, 2006, 67(2): 202-211.

[2]Liu Y W, Gao J L, Guan J, Qian Z M, Feng K, Li S P. Evaluation of antiproliferative activities and action mechanisms of extracts from two species of Ganoderma on tumor cell lines. Journal of Agricultural and Food Chemistry, 2009, 57(8): 3087-3093.

[3]Yue Q X, Cao Z W, Guan S H, Liu X H, Tao L, Wu W Y, Li Y X, Yang P Y, Liu X, Guo D A. Proteomics characterization of the cytotoxicity mechanism of ganoderic acid D and computer-automated estimation of the possible drug target network. Molecular & Cellular Proteomics, 2008, 7(5): 949-961.

[4]Stanley G, Harvey K, Slivova V, Jiang J, Sliva D. Ganoderma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-beta1 from prostate cancer cells. Biochemical and Biophysical Research Communications, 2005, 330(1): 46-52.

[5]Sliva D. Ganoderma lucidum in cancer research. Leukemia Research, 2006, 30(7): 767-768.

[6]Muller C I, Kumagai T, O'Kelly J, Seeram N P, Heber D, Koeffler H P. Ganoderma lucidum causes apoptosis in leukemia, lymphoma and multiple myeloma cells. Leukemia Research, 2006, 30(7): 841-848.

[7]Xu J W, Zhao W, Zhong J J. Biotechnological production and application of ganoderic acids. Applied Microbiology and Biotechnolog, 2010, 87(2): 457-466.

[8]Shieh Y H, Liu C F, Huang Y K, Yang J Y, Wu I L, Lin C H, Li S C. Evaluation of the hepatic and renal-protective effects of Ganoderma lucidum in mice. The American Journal of Chinese Medicine, 2001, 29(3/4): 501-507.

[9]郭溆, 孙超, 宋经元, 罗红梅, 陈士林. 灵芝三萜合成关键元件GLCYP450及其还原酶GLNADPH的基因克隆及共表达载体构建. 药学学报, 2013, 48(2): 206-210.

Guo S, Sun C, Song J Y, Luo H M, Chen S L. Construction of the coexpression vector containing key element GLCYP450 involved in Ganoderma triterpene biosynthesis and its reductase gene GLNADPH. Acta Pharmaceutica Sinica, 2013, 48(2): 206-210. (in Chinese)

[10]方星, 师亮, 徐颖洁, 赵明文. 灵芝甾醇14α-脱甲基酶基因的克隆及超量表达对三萜合成的影响. 菌物学报, 2011, 30(2): 242-248.

Fang X, Shi L, Xu Y J, Zhao M W. Cloning of a sterol 14á-demethylase gene and the effects of over-expression of the gene on biological synthesis of triterpenes in Ganoderma lucidum, Mycosystema, 2011, 30(2): 242-248. (in Chinese)

[11]Huang Q, Roessner C A, Croteau R, Scott A I. Engineering Escherichia coli for the synthesis of taxadiene, a key intermediate in the biosynthesis of taxol. Bioorganic & Medicinal Chemistry, 2001, 9(9): 2237-2242.

[12]Engels B, Dahm P, Jennewein S. Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production. Metabolic Engineering, 2008, 10(3/4): 201-206.

[13]Ajikumar P K, Xiao W H, Tyo K E, Wang Y, Simeon F, Leonard E, Mucha O, Phon T H, Pfeifer B, Stephanopoulos G. Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli. Science, 2010, 330(6000): 70-74.

[14]康林芝, 林俊芳, 黄秀琴, 郭丽琼, 梁科. 紫杉烯合酶基因遗传转化金针菇的研究. 食品工业科技, 2013, 34(2): 190-193.

Kang L Z, Lin J F, Huang X Q, Guo L Q, Liang K. Genetic transformation of Flammulina velutipes with taxadiene synthase gene. Science and Technology of Food Industry, 2013, 34(2): 190-193. (in Chinese)

[15]张银萍, 张舒, 汪洋, 李敏, 陈学平. 灵芝原生质体的高效制备及诱变. 安徽农业科学, 2012, 40(25): 12379-12381.

Zhang Y P, Zhang S, Wang Y, Li M, Chen X P. High preparation and mutagenesis of protoplast of Ganoderma lucidum. Journal of Anhui Agricultural Sciences, 2012, 40(25): 12379-12381. (in Chinese)

[16]李刚, 王强, 刘秋云, 李宝健. 利用PEG法建立药用真菌灵芝的转化系统. 菌物学报, 2004, 23(2): 255-261.

Li G, Wang Q, Liu Q Y, Li B J. Establishment of a transformation system of Ganoderma lucidum using PEG method. Mycosystema, 2004, 23(2): 255-261. (in Chinese)

[17]J萨姆布鲁克, DW拉塞尔. 分子克隆实验指南. 3版. 北京: 科学出版社, 2008.

Sambrook J, Russell D W. Molecular Cloning: a Laboratory Manual. 3rd ed. Beijing: Science Press, 2008. (in Chinese)

[18]Boghigian B A, Salas D, Ajikumar P K, Stephanopoulos G, Pfeifer B A. Analysis of heterologous taxadiene production in K- and B-derived Escherichia coli. Applied Microbiology and Biotechnology, 2012, 93(4): 1651-1661.

[19]Anterola A, Shanle E, Perroud P F, Quatrano R. Production of taxa-4(5), 11(12)-diene by transgenic Physcomitrella patens. Transgenic Research, 2009, 18(4): 655-660.

[20]Besumbes O, Sauret-Gueto S, Phillips M A, Imperial S, Rodriguez- Concepcion M, Boronat A. Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene, the first committed precursor of Taxol. Biotechnology and Bioengineering, 2004, 88(2): 168-175.

[21]Kovacs K, Zhang L, Linforth R S, Whittaker B, Hayes C J, Fray R G. Redirection of carotenoid metabolism for the efficient production of taxadiene [taxa-4(5), 11(12)-diene] in transgenic tomato fruit. Transgenic Research, 2007, 16(1): 121-126.

[22]Williams D C, Wildung M R, Jin A Q, Dalal D, Oliver J S, Coates R M, Croteau R. Heterologous expression and characterization of a "Pseudomature" form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction. Archives of Biochemistry and Biophysics, 2000, 379: 137-146.

[23]仲亚. 灵芝全基因组精细图谱发布. 中医药管理杂志, 2012, 20(11): 1100.

Zhong Y. Ganoderma lucidum’ genome-wide map publishing. Journal of Traditional Chinese Medicine Management, 2012, 20(11): 1100. (in Chinese)

[24]Li P, Deng Y P, Wei X X, Xu J H. Triterpenoids from Ganoderma lucidum and their cytotoxic activities.  Journal of Asian Natural Products Research, 2013, 27: 17-22.

[25]Cheng C R, Yue Q X, Wu Z Y, Song X Y, Tao S J, Wu X H, Xu P P, Liu X, Guan S H, Guo D A. Cytotoxic triterpenoids from Ganoderma lucidum. Phytochemistry, 2010, 71: 1579-1585.

[26]韩军丽, 李振秋, 刘本叶, 王红, 李国凤, 叶和春. 植物萜类代谢工程. 生物工程学报, 2007, 23(4): 561-569.

Han J L, Li Z Q, Liu B Y, Wang H, Li G F, Ye H C. Metabolic engineering of terpenoids in plants. Chinese Journal of Biotechnology, 2007, 23(4): 561-569. (in Chinese)

[27]Hefner J, Rubenstein S M, Ketchum R E, Gibson D M, Williams R M, Croteau R. Cytochrome P450-catalyzed hydroxylation of taxa-4(5), 11(12)-diene to taxa-4(20), 11(12)-dien-5alpha-ol: the first oxygenation step in taxol biosynthesis. Chemistry & Biology, 1996, 3(6): 479-489.

[28]Schoendorf A, Rithner C D, Williams R M, Croteau R B. Molecular cloning of a cytochrome P450 taxane 10 beta-hydroxylase cDNA from Taxus and functional expression in yeast. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(4): 1501-1506.

[29]Wiebe M G. Stable production of recombinant proteins in filamentous fungi-problems and improvements. Mycologist, 2003, 17(3): 140-145.

[30]郭丽琼, 林俊芳, 熊盛, 陈守才. 抗冷冻蛋白基因遗传转化草菇的研究. 微生物学报, 2005, 45(1): 39-43.

Guo L Q, Lin J F, Xiong S, Chen S C. Transformation of Volvariella volvacea with a thermal hysteresis protein gene by particle bombardment. Acta Microbiologica Sinica, 2005, 45(1): 39-43. (in Chinese)

[31]张保龙, 倪万潮, 张天真, 郭旺珍, 徐英俊, 姚姝. 花粉管通道法转基因抗虫棉外源基因的整合方式. 江苏农业学报, 2004, 20(3): 144-148.

Zhang B L, Ni W C, Zhang T Z, Guo W Z, Xu Y J, Yao S. Integration patterns of foreign gene in the transgenic Bt cotton via pollen tube pathway. Jiangsu Journal of Agricultural Sciences, 2004, 20(3): 144-148. (in Chinese)
[1] ZHAO HaiXia,XIAO Xin,DONG QiXin,WU HuaLa,LI ChengLei,WU Qi. Optimization of Callus Genetic Transformation System and Its Application in FtCHS1 Overexpression in Tartary Buckwheat [J]. Scientia Agricultura Sinica, 2022, 55(9): 1723-1734.
[2] DING Xi,ZHAO KaiXi,WANG YueJin. Expression of Stilbene Synthase Genes from Chinese Wild Vitis quinquangularis and Its Effect on Resistance of Grape to Powdery Mildew [J]. Scientia Agricultura Sinica, 2021, 54(2): 310-323.
[3] ZHANG ZongYuan,JIANG YongMei,ZHANG WenXian. Effects of Histone Acetylation on Ganoderma lucidum Growth, Polysaccharide and Ganoderic Acid Biosynthesis [J]. Scientia Agricultura Sinica, 2020, 53(3): 632-641.
[4] LIU MengQi,WU FengYing,WANG YueJin. Expression of Stilbene Synthase Gene and Resistance to Powdery Mildew Analysis of Chinese Wild Vitis quinquangularis [J]. Scientia Agricultura Sinica, 2019, 52(14): 2436-2449.
[5] YE MingWang, ZHANG ChunZhi, HUANG SanWen. Construction of High Efficient Genetic Transformation System for Diploid Potatoes [J]. Scientia Agricultura Sinica, 2018, 51(17): 3249-3257.
[6] ZHANG ZhongXiao, WANG HongYan, WANG KaiYun, WANG Dong, JIANG LiLi. Induction Effect of Sharp Eyespot of Wheat and the Effect of Wheat Growth After Ganoderma lucidum Polysaccharides (GLP) Seed Dressing [J]. Scientia Agricultura Sinica, 2018, 51(1): 96-104.
[7] LI Fang, DENG Zi-niu, ZHAO Ya, LI Da-zhi, DAI Su-ming. Construction and Transformation of RNAi Vector for Citrus tristeza virus Gene p23 [J]. Scientia Agricultura Sinica, 2016, 49(20): 3927-3933.
[8] NING Yu-bo, WANG Hong-yan, QIAO Kang, LIU Xiu-mei, WANG Kai-yun. Induced Resistance by Polysaccharides Isolated from Ganoderma lucidum in Tomato Against Gray Mold [J]. Scientia Agricultura Sinica, 2016, 49(11): 2103-2112.
[9] LIU Rui, ZHANG Huan-Huan, CHEN Zhi-Xiong, SHAHID Muhammad Qasim, FU Xue-Lin, LIU Yao-Guang, LIU Xiang-Dong, LU Yong-Gen. Development of Drought-Tolerant Rice Germplasm by Screening and Transforming TAC Clones of Oryza officinalis Wall. [J]. Scientia Agricultura Sinica, 2014, 47(8): 1445-1457.
[10] WANG Hai-Yan, LI Bao-Hua, ZHANG Qing-Ming, LI Gui-Fang, DONG Xiang-Li, WANG Cai-Xia. Transformation of Agrobacterium tumefaciens-Mediated Colletotrichum gloeosporioides and Identification of Transformants [J]. Scientia Agricultura Sinica, 2013, 46(9): 1799-1807.
[11] DU Kun, GAO Ya-Nan, KONG Yue-Qin, FAN Yun, WANG You-Ping. Effects of OsLTP Gene on Salt Tolerance of Transgenic Brassica napus [J]. Scientia Agricultura Sinica, 2013, 46(13): 2625-2632.
[12] XIE Chao, YANG Qing, SHI Ce, JUE Deng-Wei, ZHU Yan-Ping, LIU Shui-Ping. Cloning and Analysis of StLTPa7 from Solanum torvum [J]. Scientia Agricultura Sinica, 2012, 45(8): 1505-1512.
[13] WU Li, LI Yan-Jun, ZHANG Xin-Yu, WANG Ya-Qin, SUN Jie. Functional Analysis of Cyclophilin (GhCYP1) in Gossypium hirsutum [J]. Scientia Agricultura Sinica, 2012, 45(17): 3624-3631.
[14] YAN Li-Min, WANG Xiao-Ming, XU Rong-Qi, DONG Fang-Yang, LI Hong-Jie. Root and Leaf Infection as Revealed by Autofluorescent Reporter Protein GFP Labeled Bipolaris sorokiniana in Wheat [J]. Scientia Agricultura Sinica, 2012, 45(17): 3506-3514.
[15] FAN Su-Jie, WU Jun-Jiang, CHEN Chen, WANG Xin, JIANG Liang-Yu, WANG Jin-Sheng, LI Wen-Bin, XU Peng-Fei, ZHANG Shu-Zhen. Isolation and Functional Analysis of Resistance Gene SDR1 to Phytophthora sojae in Soybean [J]. Scientia Agricultura Sinica, 2012, 45(11): 2139-2146.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!