灵芝酸生物合成及代谢调控研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2021-0734

摘要/Abstract

摘要：

灵芝酸是药用灵芝菌的次级代谢产物,是灵芝菌的主要活性成分之一,已经在抗癌以及其他医药领域进行了深入研究。为了解决灵芝孢子、子实体生长周期长,生长环境要求高以及灵芝酸含量低等问题,灵芝菌深层液体发酵技术应运而生。尽管已经取得了一定的突破,但是发酵生产灵芝酸的低产量问题依然存在。灵芝酸生物合成途径的解析和调控对于解决发酵生产灵芝酸低产量问题意义重大,本文系统综述了近年来在灵芝酸生物合成和代谢调控方面的研究进展,并提出未来研究重点方向,为进一步通过基因工程手段和代谢通路调控手段实现灵芝酸的高浓度积累提供参考。

关键词: 灵芝酸, 合成代谢, 基因工程, 代谢调控

Abstract:

Ganoderic acid（GA）is the secondary metabolites from medicinal Ganoderma lucidum and main active component of it,which has been studied deeply in anti-cancer and other medical fields. However,due to the low yield of GAs,long growth cycle of spores and fruiting bodies,and high growth environment requirements,and the submerged liquid fermentation of G. Lucidum was developed. Although some breakthroughs have been made,the problem of low GAs yield is still existed. The analysis and regulation of GAs biosynthesis pathway is of significance for solving low yield in the fermentation of producing GAs. Therefore,this paper reviews the research progress of GAs biosynthesis and metabolic regulation,proposes the direction of future research,and provides a reference for further improving GAs yield through genetic engineering and metabolic pathway regulation.

Key words: ganoderic acids, synthesis metabolism, genetic engineering, metabolic regulation

袁恺, 何伟, 杨云丽, 朱威宇, 彭超, 安泰, 李丽, 周卫强. 灵芝酸生物合成及代谢调控研究进展[J]. 生物技术通报, 2021, 37(8): 46-54.

YUAN Kai, HE Wei, YANG Yun-li, ZHU Wei-yu, PENG Chao, AN Tai, LI Li, ZHOU Wei-qiang. Research Progress on Biosynthesis and Metabolic Regulation of Ganoderic Acids[J]. Biotechnology Bulletin, 2021, 37(8): 46-54.

图/表 3

参考文献 51

[1]	赵艳. 灵芝关键成分三萜酸的深层发酵技术研究[D]. 长沙:中南林业科技大学, 2011.
	Zhao Y. Submerged fermentation of key medicinal components, triterpene acids from Ganoderma lucidum[D]. Changsha:Central South University of Forestry & Technology, 2011.
[2]	刘维, 虎虓真, 朱莉, 等. 灵芝三萜的研究与应用进展[J]. 食品科学, 2019, 40(5):309-315.
	Liu W, Hu XZ, Zhu L, et al. Recent progress in research and application of Ganoderma lucidum triterpenoids[J]. Food Sci, 2019, 40(5):309-315.
[3]	Kubota T, Asaka Y, Miura I, et al. Structures of ganoderic acid A and B, two new lanostane type bitter triterpenes from Ganoderma lucidum(FR. )KARST[J]. Helv Chim Acta, 1982, 65(2):611-619. doi: 10.1002/(ISSN)1522-2675 URL
[4]	Zhang W, Yu W, Ding X, et al. Self-assembled thermal gold nanorod-loaded thermosensitive liposome-encapsulated ganoderic acid for antibacterial and cancer photochemotherapy[J]. Artif Cells Nanomed Biotechnol, 2019, 47(1):406-419. doi: 10.1080/21691401.2018.1559177 URL
[5]	Liu Z, Li L, Xue B. Effect of ganoderic acid D on colon cancer Warburg effect:Role of SIRT3/cyclophilin D[J]. Eur J Pharmacol, 2018, 824:72-77. doi: 10.1016/j.ejphar.2018.01.026 URL
[6]	Sun ZW, Sun LB, Li WW. Effect of ganoderic acid on diethylnitrosamine-induced liver cancer in mice[J]. Trop J Pharm Res, 2021, 19(12):2639-2644. doi: 10.4314/tjpr.v19i12.23 URL
[7]	Chi B, Wang S, Bi S, et al. Effects of ganoderic acid A on lipopolysaccharide-induced proinflammatory cytokine release from primary mouse microglia cultures[J]. Exp Ther Med, 2018, 15(1):847-853.
[8]	Liang CY, Tian DN, Liu YZ, et al. Review of the molecular mechanisms of Ganoderma lucidum triterpenoids:Ganoderic acids A, C2, D, F, DM, X and Y[J]. Eur J Med Chem, 2019, 174:130-141. doi: 10.1016/j.ejmech.2019.04.039 URL
[9]	俞盈, 吴学谦, 熊科辉, 等. 灵芝三萜酸改善小鼠睡眠作用研究[J]. 食品工业科技, 2019, 40(10):297-301.
	Yu Y, Wu XQ, Xiong KH, et al. Hypnotic effects of Ganoderma lucidum triterpene acids in mice[J]. Sci Technol Food Ind, 2019, 40(10):297-301.
[10]	Hossain A, Radwan FF, Doonan BP, et al. A possible cross-talk between autophagy and apoptosis in generating an immune response in melanoma[J]. Apoptosis, 2012, 17(10):1066-1078. doi: 10.1007/s10495-012-0745-y pmid: 22847295
[11]	李丽, 杨云丽, 杨小凡, 等. 液体发酵生产灵芝三萜酸的过程调控研究进展[J]. 食品与发酵工业, 2021, 47(8):304-312.
	Li L, Yang YL, Yang XF, et al. Advances on process regulation of Ganoderma triterpene acids production by liquid fermentation[J]. Food Ferment Ind, 2021, 47(8):304-312.
[12]	Li HJ, He YL, Zhang DH, et al. Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum[J]. J Biotechnol, 2016, 227:35-40. doi: 10.1016/j.jbiotec.2016.04.017 URL
[13]	Shiao MS. Natural products of the medicinal fungus Ganoderma lucidum:occurrence, biological activities, and pharmacological functions[J]. Chem Rec, 2003, 3(3):172-180. doi: 10.1002/(ISSN)1528-0691 URL
[14]	Chen S, Xu J, Liu C, et al. Genome sequence of the model medicinal mushroom Ganoderma lucidum[J]. Nat Commun, 2012, 3:913. doi: 10.1038/ncomms1923 URL
[15]	Cai SQ, Xiao H, Wang XZ, et al. Bioconversion of a ganoderic acid 3-hydroxy-lanosta-8, 24-Dien-26-oic acid by a crude enzyme from Ganoderma lucidum[J]. Process Biochem, 2020, 95:12-16. doi: 10.1016/j.procbio.2020.05.002 URL
[16]	Yang C, Li W, Li C, et al. Metabolism of ganoderic acids by a Ganoderma lucidum cytochrome P450 and the 3-keto sterol reductase ERG27 from yeast[J]. Phytochemistry, 2018, 155:83-92. doi: 10.1016/j.phytochem.2018.07.009 URL
[17]	Song X, Xiao H, Luo SW, et al. Biosynjournal of squalene-type triterpenoids in Saccharomyces cerevisiae by expression of CYP505D13 from Ganoderma lucidum[J]. Bioresour Bioprocess, 2019, 6(1):1-10. doi: 10.1186/s40643-018-0235-3 URL
[18]	Li HJ, He YL, Zhang DH, et al. Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum[J]. J Biotechnol, 2016, 227:35-40. doi: 10.1016/j.jbiotec.2016.04.017 URL
[19]	Xu JW, Xu YN, Zhong JJ. Enhancement of ganoderic acid accumulation by overexpression of an N-terminally truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in the basidiomycete Ganoderma lucidum[J]. Appl Environ Microbiol, 2012, 78(22):7968-7976. doi: 10.1128/AEM.01263-12 URL
[20]	张宗源. 组蛋白乙酰化调控灵芝酸与灵芝多糖生物合成代谢的研究[D]. 福州:福建师范大学, 2017.
	Zhang ZY. Study on the regulation of histone acetylation on ganoderic acids and polysaccharide biosynthesis in Ganoderma lucidum[D]. Fuzhou:Fujian Normal University, 2017.
[21]	Fei Y, Li N, Zhang DH, et al. Increased production of ganoderic acids by overexpression of homologous farnesyl diphosphate synthase and kinetic modeling of ganoderic acid production in Ganoderma lucidum[J]. Microb Cell Factories, 2019, 18(1):115. doi: 10.1186/s12934-019-1164-3 URL
[22]	Ding YX, Ou-yang X, Shang CH, et al. Molecular cloning, characterization, and differential expression of a farnesyl-diphosphate synthase gene from the basidiomycetous fungus Ganoderma lucidum[J]. Biosci Biotechnol Biochem, 2008, 72(6):1571-1579. doi: 10.1271/bbb.80067 URL
[23]	Zhou JS, Ji SL, Ren MF, et al. Enhanced accumulation of individual ganoderic acids in a submerged culture of Ganoderma lucidum by the overexpression of squalene synthase gene[J]. Biochem Eng J, 2014, 90:178-183. doi: 10.1016/j.bej.2014.06.008 URL
[24]	Shang CH, Shi L, Ren A, et al. Molecular cloning, characterization, and differential expression of a lanosterol synthase gene from Ganoderma lucidum[J]. Biosci Biotechnol Biochem, 2010, 74(5):974-978. doi: 10.1271/bbb.90833 URL
[25]	Zhang DH, Li N, Yu X, et al. Overexpression of the homologous lanosterol synthase gene in ganoderic acid biosynjournal in Ganoderma lingzhi[J]. Phytochemistry, 2017, 134:46-53. doi: S0031-9422(16)30256-4 pmid: 27894599
[26]	Paddon CJ, Westfall PJ, Pitera DJ, et al. High-level semi-synthetic production of the potent antimalarial artemisinin[J]. Nature, 2013, 496(7446):528-532. doi: 10.1038/nature12051 URL
[27]	Guo J, Zhou YJ, Hillwig ML, et al. CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynjournal and enables heterologous production of ferruginol in yeasts[J]. PNAS, 2013, 110(29):12108-12113. doi: 10.1073/pnas.1218061110 URL
[28]	Dai Z, Wang B, Liu Y, et al. Producing aglycons of ginsenosides in bakers’ yeast[J]. Sci Rep, 2014, 4:3698. doi: 10.1038/srep03698 URL
[29]	Wang PP, Wei YJ, Fan Y, et al. Production of bioactive ginsenosides Rh2 and Rg3 by metabolically engineered yeasts[J]. Metab Eng, 2015, 29:97-105. doi: 10.1016/j.ymben.2015.03.003 URL
[30]	Wang WF, Xiao H, Zhong JJ. Biosynjournal of a ganoderic acid in Saccharomyces cerevisiae by expressing a cytochrome P450 gene from Ganoderma lucidum[J]. Biotechnol Bioeng, 2018, 115(7):1842-1854. doi: 10.1002/bit.v115.7 URL
[31]	Lan X, Yuan W, Wang M, et al. Efficient biosynjournal of antitumor ganoderic acid HLDOA using a dual tunable system for optimizing the expression of CYP5150L8 and a Ganoderma P450 reductase[J]. Biotechnol Bioeng, 2019, 116(12):3301-3311. doi: 10.1002/bit.v116.12 URL
[32]	Gu L, Zheng YM, Lian DH, et al. Production of triterpenoids from Ganoderma lucidum:Elicitation strategy and signal transduction[J]. Process Biochem, 2018, 69:22-32. doi: 10.1016/j.procbio.2018.03.019 URL
[33]	Gu L, Zhong X, Lian DH, et al. Triterpenoid biosynjournal and the transcriptional response elicited by nitric oxide in submerged fermenting Ganoderma lucidum[J]. Process Biochem, 2017, 60:19-26. doi: 10.1016/j.procbio.2017.05.029 URL
[34]	You BJ, Tien N, Lee MH, et al. Induction of apoptosis and ganoderic acid biosynjournal by cAMP signaling in Ganoderma lucidum[J]. Sci Rep, 2017, 7(1):318. doi: 10.1038/s41598-017-00281-x URL
[35]	Xu YN, Zhong JJ. Impacts of calcium signal transduction on the fermentation production of antitumor ganoderic acids by medicinal mushroom Ganoderma lucidum[J]. Biotechnol Adv, 2012, 30(6):1301-1308. doi: 10.1016/j.biotechadv.2011.10.001 URL
[36]	Xu YN, Xia XX, Zhong JJ. Induction of ganoderic acid biosynjournal by Mn²⁺ in static liquid cultivation of Ganoderma lucidum[J]. Biotechnol Bioeng, 2014, 111(11):2358-2365. doi: 10.1002/bit.25288 URL
[37]	Xu YN, Xia XX, Zhong JJ. Induced effect of Na(+)on ganoderic acid biosynjournal in static liquid culture of Ganoderma lucidum via calcineurin signal transduction[J]. Biotechnol Bioeng, 2013, 110(7):1913-1923. doi: 10.1002/bit.24852 URL
[38]	Zhang G, Ren A, Shi L, et al. Functional analysis of an APSES transcription factor(GlSwi6)involved in fungal growth, fruiting body development and ganoderic-acid biosynjournal in Ganoderma lucidum[J]. Microbiol Res, 2018, 207:280-288. doi: 10.1016/j.micres.2017.12.015 URL
[39]	Wang S, Shi L, Hu Y, et al. Roles of the Skn7 response regulator in stress resistance, cell wall integrity and GA biosynjournal in Ganoderma lucidum[J]. Fungal Genet Biol, 2018, 114:12-23. doi: 10.1016/j.fgb.2018.03.002 URL
[40]	Wu FL, Zhang G, Ren A, et al. The pH-responsive transcription factor PacC regulates mycelial growth, fruiting body development, and ganoderic acid biosynjournal in Ganoderma lucidum[J]. Mycologia, 2016, 108(6):1104-1113.
[41]	Zhu J, Wu FL, Yue SN, et al. Functions of reactive oxygen species in apoptosis and ganoderic acid biosynjournal in Ganoderma lucidum[J]. FEMS Microbiol Lett, 2019, 366(23):fnaa015.
[42]	李雄标. 灵芝促原活化蛋白激酶MAPK4和转录因子SWI6的功能研究[D]. 南京:南京农业大学, 2014.
	Li XB. Functional analysis of mapkinase MAPK₄ and transcription factor SWI₆ in Ganoderma lucidum[D]. Nanjing:Nanjing Agricultural University, 2014.
[43]	Huang W, Shang YF, Chen PL, et al. MrpacC regulates sporulation, insect cuticle penetration and immune evasion in Metarhizium robertsii[J]. Environ Microbiol, 2015, 17(4):994-1008. doi: 10.1111/1462-2920.12451 pmid: 24612440
[44]	Wu CG, Tian JL, Liu R, et al. Ornithine decarboxylase-mediated production of putrescine influences ganoderic acid biosynjournal by regulating reactive oxygen species in Ganoderma lucidum[J]. Appl Environ Microbiol, 2017, 83(20):e01289-17. DOI: 10.1128/aem.01289-17. doi: 10.1128/aem.01289-17
[45]	Ren A, Shi L, Zhu J, et al. Shedding light on the mechanisms underlying the environmental regulation of secondary metabolite ganoderic acid in Ganoderma lucidum using physiological and genetic methods[J]. Fungal Genet Biol, 2019, 128:43-48. doi: 10.1016/j.fgb.2019.03.009 URL
[46]	Tripathi L, Wu LP, Chen JC, et al. Synjournal of Diblock copolymer poly-3-hydroxybutyrate-block-poly-3-hydroxyhexanoate[PHB-b-PHH_x]by a β-oxidation weakened Pseudomonas putida KT2442[J]. Microb Cell Factories, 2012, 11(1):1-11. doi: 10.1186/1475-2859-11-1 URL
[47]	贺望兴. 灵芝全局性调控基因LaeA的克隆及其在灵芝酸生物合成过程中的差异表达[D]. 福州:福建师范大学, 2015.
	He WX. Molecular cloning and differential gene expression of the global regulator LaeA in ganoderic acid biosynthesis process[D]. Fuzhou:Fujian Normal University, 2015.
[48]	孙超, 胡鸢雷, 徐江, 等. 灵芝:一种研究天然药物合成的模式真菌[J]. 中国科学:生命科学, 2013, 43(6):447-456.
	Sun C, Hu YL, Xu J, et al. Ganoderma lucidum:an emerging medicinal model fungus for study of the biosynjournal of natural medicines[J]. Sci Sin:Vitae, 2013, 43(6):447-456.
[49]	张梦薇, 陈美榕, 刘舒雯, 等. LaeA在丝状真菌次级代谢、生长发育和其他重要生物过程中的作用[J]. 菌物学报, 2020, 39(3):548-555.
	Zhang MW, Chen MR, Liu SW, et al. The roles of LaeA in secondary metabolism, growth and development and other important biological processes of filamentous fungi:a review[J]. Mycosystema, 2020, 39(3):548-555.
[50]	蓝丽雯. DNA甲基化调控灵芝酸生物合成代谢的研究[D]. 福州:福建师范大学, 2016.
	Lan LW. Study on the regulation of DNA methylation on ganoderic acids biosynthesis in Ganoderma lucidum[D]. Fuzhou:Fujian Normal University, 2016.
[51]	张宗源, 蒋咏梅, 章文贤. 组蛋白乙酰化对灵芝生长、灵芝多糖和灵芝酸生物合成的影响[J]. 中国农业科学, 2020, 53(3):632-641.
	Zhang ZY, Jiang YM, Zhang WX. Effects of histone acetylation on Ganoderma lucidum growth, polysaccharide and ganoderic acid biosynjournal[J]. Sci Agric Sin, 2020, 53(3):632-641.

信号转导调控通路 Signal transduction regulatory pathway	作用方式 Method of operation	代谢水平变化 Changes in metabolic levels	灵芝酸含量变化 Changes in ganoderic acid content	文献 Reference
钙调磷脂酶信号	添加10 mmol/L Ca²⁺	cam、cna、crz1、hmgr、sqs、lss转录水平上调	GAs含量增加3.7倍;灵芝酸MK、灵芝酸T、灵芝酸S和灵芝酸Me分别增加2.6倍、4.5倍、3.2倍和3.8倍	[35]
	添加10 mmol/L Mn²⁺	cam、cna、crz1、pmr1、hmgr、sqs、lss转录水平上调	GAs含量增加2.2倍	[36]
	添加100 mmol/L Na⁺	hmgr转录水平上调20倍,sqs上调109倍,lss上调4倍	GAs含量增加2.8倍	[37]
活性氧信号	通过基因工程获得Glswi6沉默菌株	NAHD氧化酶活性下降48%,hmgr、sqs、lss转录水平均下调	GAs含量降低25%	[38]
	在Glswi6沉默菌株发酵过程中回补1 mmol/L H₂O₂	NAHD氧化酶活性和hmgr、sqs、lss转录水平恢复至野生型菌株水平	GAs含量恢复至野生型菌株水平	[38]
	通过基因工程获得Glskn7沉默菌株Skn7i-5和Skn7i-7	H₂O₂含量上升73.7%和76.4%,ROS水平是原始菌株的1.78和1.82倍,hmgr转录水平上升56%、52%,sqs上调49%、53%,lss上调95%、93%	GAs含量分别提高52.1%和55.9%	[39]
	在Glskn7沉默菌株发酵过程中回补加入5 mmol/L N-乙酰半胱氨酸	--	GAs含量降低至与野生型菌株持平	[39]
	通过基因工程获得PacC沉默菌株	胞内SOD、CAT、Gpx、Apx等抗氧化酶活性下降幅度超过40%,ROS水平上升幅度超过2倍	GAs含量明显提升	[40]
	当转录因子PacC沉默时,回补1 mmol/L NAC 和 2 mmol/L维生素C时	--	GAs含量降低至与出发菌株持平	[41]
细胞壁完整性信号	通过基因工程获得MAPK4沉默菌株	hmgr、sqs、lss转录水平下调	GAs含量最大降低40%	[42]
茉莉酮酸（JA）及其衍生物信号	添加254 μmol/L的MeJA	fps、sqs、hmgr、mvd、se转录水平上调	GAs含量提高45.3%	[32]
NO信号	添加5 mmol/L的NO供体	sqs转录水平上调2.43倍	灵芝三帖含量提高40.94%	[33]
cAMP信号	添加80 mmol/L咖啡因	尽管检测结果显示hmgr、sqs、lss转录水平下调,但是GAs的生物合成代谢表现出正向结果,目前作者仍在探究其中原因	GAs含量增长3.6倍	[34]

信号转导调控通路 Signal transduction regulatory pathway	作用方式 Method of operation	代谢水平变化 Changes in metabolic levels	灵芝酸含量变化 Changes in ganoderic acid content	文献 Reference
钙调磷脂酶信号	添加10 mmol/L Ca²⁺	cam、cna、crz1、hmgr、sqs、lss转录水平上调	GAs含量增加3.7倍;灵芝酸MK、灵芝酸T、灵芝酸S和灵芝酸Me分别增加2.6倍、4.5倍、3.2倍和3.8倍	[35]
	添加10 mmol/L Mn²⁺	cam、cna、crz1、pmr1、hmgr、sqs、lss转录水平上调	GAs含量增加2.2倍	[36]
	添加100 mmol/L Na⁺	hmgr转录水平上调20倍,sqs上调109倍,lss上调4倍	GAs含量增加2.8倍	[37]
活性氧信号	通过基因工程获得Glswi6沉默菌株	NAHD氧化酶活性下降48%,hmgr、sqs、lss转录水平均下调	GAs含量降低25%	[38]
	在Glswi6沉默菌株发酵过程中回补1 mmol/L H₂O₂	NAHD氧化酶活性和hmgr、sqs、lss转录水平恢复至野生型菌株水平	GAs含量恢复至野生型菌株水平	[38]
	通过基因工程获得Glskn7沉默菌株Skn7i-5和Skn7i-7	H₂O₂含量上升73.7%和76.4%,ROS水平是原始菌株的1.78和1.82倍,hmgr转录水平上升56%、52%,sqs上调49%、53%,lss上调95%、93%	GAs含量分别提高52.1%和55.9%	[39]
	在Glskn7沉默菌株发酵过程中回补加入5 mmol/L N-乙酰半胱氨酸	--	GAs含量降低至与野生型菌株持平	[39]
	通过基因工程获得PacC沉默菌株	胞内SOD、CAT、Gpx、Apx等抗氧化酶活性下降幅度超过40%,ROS水平上升幅度超过2倍	GAs含量明显提升	[40]
	当转录因子PacC沉默时,回补1 mmol/L NAC 和 2 mmol/L维生素C时	--	GAs含量降低至与出发菌株持平	[41]
细胞壁完整性信号	通过基因工程获得MAPK4沉默菌株	hmgr、sqs、lss转录水平下调	GAs含量最大降低40%	[42]
茉莉酮酸（JA）及其衍生物信号	添加254 μmol/L的MeJA	fps、sqs、hmgr、mvd、se转录水平上调	GAs含量提高45.3%	[32]
NO信号	添加5 mmol/L的NO供体	sqs转录水平上调2.43倍	灵芝三帖含量提高40.94%	[33]
cAMP信号	添加80 mmol/L咖啡因	尽管检测结果显示hmgr、sqs、lss转录水平下调,但是GAs的生物合成代谢表现出正向结果,目前作者仍在探究其中原因	GAs含量增长3.6倍	[34]