2020, Vol. 47扩展功能
文章信息
- 李娜, 冯杰, 冯娜, 刘艳芳, 张劲松
- LI Na, FENG Jie, FENG Na, LIU Yan-Fang, ZHANG Jing-Song
- 灵芝液态深层发酵三萜类化合物研究进展
- Research progress in submerged fermentation for triterpenes of Ganoderma
- 微生物学通报, 2020, 47(10): 3451-3469
- Microbiology China, 2020, 47(10): 3451-3469
- DOI: 10.13344/j.microbiol.china.191010
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文章历史
- 收稿日期: 2019-12-05
- 接受日期: 2020-03-24
- 网络首发日期: 2020-04-26
2. 上海海洋大学食品学院 上海 201306
2. College of Food Science, Shanghai Ocean University, Shanghai 201306, China
灵芝属于担子菌，在民间被认为具有延年益寿和保持活力的作用，是世界上最著名的药用真菌之一，作为中药已被广泛应用了数千年[1]。在灵芝的次级代谢产物中三萜是主要的活性成分，具有抗衰老、抗病毒、抗肿瘤、抗炎、降血压、降血糖、降血脂、免疫调节等药理活性[2-5]。灵芝三萜不仅可以从栽培生产出的子实体和灵芝孢子中获取，还可从发酵生产出的菌丝体或发酵液中获得[6]。目前，从灵芝子实体和孢子中分离出的三萜类化合物超过300种，包括灵芝酸、灵芝烯酸、灵芝醛、灵芝醇、赤灵酸、赤灵酮等；从灵芝菌丝体及发酵液中分离得到的三萜类化合物超过60种，其中也有灵芝酸类化合物，如灵芝酸T、S、Me、Mk、O等，但与灵芝子实体中的三萜结构不同，灵芝酸O是灵芝菌丝体中的特有化合物[7-10]。由于培育子实体耗时且栽培过程和环境条件不易掌控，难以满足社会市场对灵芝三萜质量的需求，因此，灵芝液态深层发酵作为高效生产灵芝三萜的方法受到了国内外学者的广泛关注[11]。近10年来，研究主要集中在高产菌株的筛选、培养基成分的改良、诱导剂的筛选、发酵工艺的优化以及灵芝三萜生物合成途径的研究等方面[12-15]。由于灵芝三萜生物合成途径仍然不清楚，因此，通过研究灵芝菌丝体发酵的最佳条件、筛选有效的外源添加物从而获得灵芝三萜高产的结果仍然是研究的热点。我们分别从灵芝三萜的生物合成、影响灵芝三萜生物合成的环境因素、外源添加物在提高灵芝三萜产量中的应用、灵芝三萜生物合成中的信号转导等四个方面，对近几年灵芝三萜的有关研究进行总结。
1 灵芝三萜生物合成灵芝三萜类化合物绝大部分是属于高度氧化的羊毛甾醇衍生物，包括灵芝酸类、醇类、醛类等。Hirotani等[16]利用同位素示踪方法发现，灵芝酸与其他萜类化合物类似，也是通过甲羟戊酸途径进行合成，其中甲羟戊酸被认为是唯一的前体。灵芝酸的生物合成途径大致可以分为三部分(图 1)：(1)上游阶段异戊烯基焦磷酸的合成；(2)上游阶段三萜环碳环系统的合成；(3)下游阶段环上复杂的官能团反应。其中，由于3-羟基-3-甲基戊二酰辅酶A还原酶参与的是不可逆反应，所以被认为是调控灵芝三萜生物合成的重要位点；鲨烯合成酶因其在代谢途径中位于法尼基焦磷酸到其他产物的分支上，特别是生成鲨烯这一重要的物质，所以该酶是灵芝三萜生物合成的一个关键酶；而羊毛甾醇合酶是催化2, 3-氧化鲨烯形成不同的灵芝酸骨架的关键酶，因此在灵芝三萜生物合成中也有重要的作用[18-19]。在灵芝三萜合成上游有11个酶被证实，并且已经完成了多个结构基因的克隆和鉴定，它们分别编码的是乙酰转移酶、3-羟基-3-甲基戊二酸辅酶A合酶、甲羟戊酸焦磷酸脱羧酶、3-羟基-3-甲基戊二酸辅酶A还原酶、鲨烯合成酶、鲨烯环氧化酶、法尼基焦磷酸合酶、羊毛甾醇合酶和异戊烯基焦磷酸异构酶[20-27]。目前灵芝三萜上游的反应过程已基本清楚，但缺乏对下游碳环骨架修饰形成过程的细节了解，其中细胞色素P450单加氧酶(cytochrome P450 monooxygenases，CYP450)被认为可以催化下游氧化反应，能进一步对碳环骨架进行修饰产生结构多样的三萜，已知与三萜生物合成相关的CYP450主要分布于CYP51簇、CYP71簇、CYP72簇、CYP85簇以及CYP86簇[28]。
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| 图 1 灵芝三萜生物合成基本途径[17] Figure 1 The basic pathway of biosynthesis of Ganoderma triterpenes[17] 注：(1) ACAT：乙酰乙酰辅酶A硫解酶；(2) HMGS：3-羟基-3-甲基戊二酰辅酶A合酶；(3) HMGR：3-羟基-3-甲基戊二酰辅酶A还原酶；(4) MVK：甲羟戊酸激酶；(5) MPK：磷酸甲羟戊酸激酶；(6) MVD：甲羟戊酸焦磷酸脱羧酶；(7) FPS：法尼基焦磷酸合酶；(8) FPPS：牻牛儿基焦磷酸合成酶；(9) SQS：鲨烯合成酶；(10) SE：鲨烯环氧酶；(11) LS：羊毛甾醇合酶；(12) 14α-LDM：甾醇14α-脱甲基酶.图中实线箭头表示已知的合成途径，虚线箭头表示猜测的合成途径. Note: (1) ACAT: Acetyl-CoA acetyltransferase; (2) HMGS: 3-hydroxy-3-methylglutaryl-CoA synthase; (3) HMGR: 3-hydroxy-3-methylglutaryl-CoA reductase; (4) MVK: Mevalonate kinase; (5) MPK: Phosphomevalonate kinase; (6) MVD: Pyrophosphomevalonate decarboxylase; (7) FPS: Farnesyl diphosphate synthases; (8) FPPS: Geranylgeranyl-PP synthase; (9) SQS: Squalene synthase; (10) SE: Squalene monooxygenase; (11) LS: Lanosterol synthase; (12) 14α-LDM: Lanosterol-14a-demethylase. Previously known pathways are indicated by solid arrows. Novel putative pathways proposed for Ganoderma are indicated by dotted arrows. |
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研究发现，灵芝三萜液态发酵的产量受培养基成分、发酵条件和外源添加物等多种环境因素的影响。
2.1 培养基组分对灵芝液态发酵三萜产量的影响培养基成分主要包括碳源、氮源、无机盐、生长因子等，不同培养基组分对灵芝液态发酵三萜产量的影响如表 1所示。碳源有葡萄糖、蔗糖、乳糖、麦芽糖、小麦粉、淀粉、玉米粉等。鲍锐等[29]分别以玉米粉、小麦粉、荞麦粉作为碳源探究了不同碳源对灵芝液体发酵的影响，结果发现当玉米粉作为碳源且浓度为7 g/L时，灵芝酸含量达到最大值23.54 mg/g，结合实际情况，优化培养基时使用了4 g/L的玉米粉。Cui等[30]分析了5种不同的碳源对灵芝菌丝体生长和次级代谢产物的影响，结果发现麦汁成本低、产量大，当添加41 g/L麦汁和18.9 g/L酵母提取物时，菌丝体生物量和灵芝酸含量分别达到18.7 g/L和0.93 g/L。氮源有硫酸铵、氯化铵、鱼粉蛋白胨、胰蛋白胨和酵母粉等。冯杰等[31]以沪农灵芝1号为供试菌株，探究了11种不同氮源对灵芝液体发酵产灵芝三萜的影响，结果显示当培养基中酵母自溶粉为2.76 g/L时，灵芝三萜的实际发酵产量提高到21.15 mg/g，比原工艺提高了6.22%。翟双星等[32]通过中心组合实验对3种总氮、氨基酸、游离氨基酸、水解氨基酸、微量元素及维生素等不同化学组成的酵母粉进行组合配比优化，得出向基础培养基中分别添加5.07、3.78、7.63 g/L三种不同酵母粉作为复合有机氮源时灵芝三萜的得率达到0.51 g/L，比添加单种酵母粉作为氮源时三萜产量有很大的提高。无机盐有Mg2+、K+、P5+、Fe2+、Mn2+等，鲍锐[33]研究了Cu2+、Ca2+对灵芝发酵产灵芝酸的影响，结果显示Cu2+、Ca2+浓度均在100 μmol/L时灵芝酸产量较高，分别达到0.208、0.187 g/L。生长因子有维生素B族，陈志玲等[34]通过向灵芝液态发酵培养基中添加不同浓度的VB1，结果显示当VB1添加量为1.0%时，菌丝体内灵芝三萜含量可高达23.56 mg/g。
| 菌种名称 Strain |
发酵规模 Fermentation scale |
研究内容 Research content |
菌丝体干重 Mycelium dry weight (g/L) |
三萜产量 GT production(g/L) |
参考文献 References |
| G. lucidum | 1 000 mL摇瓶 1 000 mL flask |
培养基玉米粉浓度4 g/L The concentration of corn flour was 4 g/L |
12.43 | 0.239 | [29] |
| 培养基玉米粉浓度7 g/L The concentration of corn flour was 7 g/L |
10.0 | 0.235 | |||
| 培养基小麦粉浓度10 g/L The concentration of wheat flour was 10 g/L |
10.20 | 0.200 | |||
| 培养基荞麦粉浓度7 g/L The concentration of buckwheat powder was 7 g/L |
11.64 | 0.227 | |||
| G. lucidum | 250 mL摇瓶 250 mL flask |
培养基中麦芽汁、酵母提取物含量分别为41、18.9 g/L、pH为5.4 The concentration of wort, yeast extract were 41, 18.9 g/L, respectively, pH 5.4 |
18.70 | 0.932 | [30] |
| G. lucidum G0119 | 500 mL摇瓶 500 mL flask |
培养基中葡萄糖、酵母自溶粉、磷酸二氢钾和七水硫酸镁的含量分别为31.06、2.76、1.77、1.99 g/L The concentration of glucose, autolyzed yeast, potassium dihydrogen phosphate and Epsom salt were 31.06, 2.76, 1.77, 1.99 g/L, respectively |
9.85 | 0.207 | [31] |
| G. lingzhi G0023 | 250 mL摇瓶 250 mL flask |
基础培养基中添加两种不同酵母粉浓度均为6.6 g/L The concentration of the two different kinds of yeast were 6.6 g/L |
− | 0.478 | [32] |
| 基础培养基中3种不同酵母粉添加量分别为5.07、3.78、7.63 g/L The concentration of the three yeast powders were 5.07, 3.78, 7.63 g/L, respectively |
0.514 | ||||
| G. lucidum | 1 000 mL摇瓶 1 000 mL flask |
培养基褪黑素浓度20 μmol/L The concentration of melotonin was 20 μmol/L |
− | 0.245 | [33] |
| 培养基硫酸铜浓度100 μmol/L The concentration of copper sulfate was 100 μmol/L |
0.208 | ||||
| 培养基氯化钙浓度100 μmol/L The concentration of calcium chloride was 100 μmol/L |
0.087 | ||||
| G. lucidum | 250 mL摇瓶 250 mL flask |
培养基VB添加量为1% The concentration of VB was 1% |
− | 0.236 | [34] |
| G. lucidum 5.26 | 10 L发酵罐 10 L fermenter |
培养基中葡萄糖、酵母提取物、柠檬酸含量分别为55、14、0.3 g/L The concentration of glucose, yeast, ferric acid were 55, 14, 0.3 g/L respectively |
25.70 | 0.639 | [35] |
| 300 L发酵罐 300 L fermenter |
23.90 | 0.670 | |||
| G. lucidum CCGMC 5.616 | 250 mL摇瓶 250 mL flask |
培养基中混合添加5 g/L的酵母提取物和5 g/L的蛋白胨, 初始葡萄糖浓度为50 g/L Combined addition of 5 g/L of yeast extract and 5 g/L of peptone and the initial concentration of glucose was 50 g/L in culture solution |
16.7 | 0.212 | [36] |
发酵条件主要包括初始pH、温度、供氧量、通气量、补料发酵策略等，发酵工艺的优化如表 2所示。Wang等[37]使用响应面方法对发酵的关键条件进行了优化，得出菌丝体三萜的最佳发酵pH为6.0，并在5 L发酵罐中验证，当发酵温度为30.1 ℃，灵芝三萜酸产量为291.0 mg/L，比未优化条件下得到的三萜产量提高了70.8%，将优化后的条件继续扩大到200 L发酵罐的生产规模，三萜产率达到47.9 mg/(L·d)。Fang等[38]在摇瓶中发酵灵芝，发现当初始pH值在3.5-7.0范围内变化时，对细胞生长和产物合成有显著影响，当初始pH为6.5时，菌丝体生物量为17.3 g/L，灵芝酸含量和总灵芝酸产量分别为12 mg/g和207.9 mg/L。Feng等[39]运用Logistic和Luedeking-Piret方程建立了菌丝生长和灵芝三萜的动力学模型，研究了在6 L发酵罐中23-32 ℃温度对灵芝G0119产三萜的影响，提出了提高三萜液体发酵生产的最佳温度控制策略为0-61 h在32 ℃培养，62-127 h之间将温度从31 ℃逐步降低到30 ℃，128 h后温度维持在29 ℃，三萜产量可达到0.27 g/L，比恒温29 ℃条件下提高了27.32%。Zhang等[40]研究了气相氧水平对灵芝液体静态培养中灵芝酸合成的影响，结果表明最佳氧气浓度为80%，在此条件下菌丝体生物量为29.8 g/L，总灵芝酸产量为1.43 g/L。Tang等[41]采用调控pH和溶解氧张力策略相结合的补料分批发酵工艺，研究发现在pH值为3.0培养4 d、pH值为4.5培养6 d后，继续在溶解氧张力为25%和10%时分别培养6 d的条件下能显著协同增强灵芝酸的产量，达到0.75 g/L。冯杰等[42]将灵芝液态深层发酵扩大到5 L发酵罐中，探究了在4、6、8和10 L/min等4种不同通气量对灵芝三萜合成的影响，得出在通气量为8 L/min时三萜含量最高，为23.3 mg/g。由于灵芝发酵过程中菌丝体生长和产三萜所需要的最佳搅拌速度不同，用传统的恒速搅拌并不能使灵芝三萜高产，因此Feng等[43]提出了两阶段搅拌速度的调控策略，不仅使三萜含量提高了36.48%，达到6.07 mg/g，而且还缩短了发酵时间，节约了成本。赵娜等[44]在灵芝发酵振荡阶段采用变转速调控手段，发现当转速由150 r/min变为100 r/min时，灵芝三萜产量提高，同时与静置培养相结合，通过两阶段培养的方法可使最终菌丝体三萜产量达到45.1 mg/g。冯杰等[45]在50 L发酵罐上分别对分批发酵、间歇式补料培养、恒速补料培养、变速补料培养和指数补料培养等5种培养方式对灵芝液态发酵产灵芝三萜的含量进行比较，结果表明相比其他培养方式，指数补料方式获得的菌丝体生物量和灵芝三萜含量最高，分别为17.68 g/L和45.8 mg/g。
| 菌种名称 Strain |
发酵规模 Fermentation scale |
研究内容 Research content |
菌丝体干重 Mycelium dry weight (g/L) |
三萜产量 GT production(g/L) |
参考文献 References |
| G. lucidum CCGMC 5.616 | 5 L发酵罐 5 L fermenter |
pH 6.0，转速161.9 r/min，温度30.1 ℃ pH 6.0, agitation speed 161.9 r/min and temperature 30.1 ℃ |
- | 0.291 | [37] |
| G. lucidum CCGMC 5.616 | 250 mL摇瓶 250 mL flask |
初始pH为6.5 Initial pH was 6.5 |
17.3 | 0.208 | [38] |
| G. lucidum G0119 | 6 L发酵罐 6 L fermenter |
0-61 h, 32 ℃培养，在62-127 h之间，温度从31 ℃逐步降低到30 ℃，128 h后，温度维持在29 ℃ From 0 to 61 h, culturing was performed at 32 ℃, the temperature was decreased stepwise from 31 to 30 ℃ between 62 and 127 h, after 128 h, temperature was maintained at 29 ℃ |
8.75 | 0.269 | [39] |
| G. lucidum CCGMC 5.616 | 250 mL摇瓶 250 mL flask |
气相氧浓度为80% The concentration of gaseous O2 level was 80% |
29.8 | 1.427 | [40] |
| G. lucidum CGMCC 5.616 | 5.5 L发酵罐 5.5 L fermenter |
pH为3.0培养4 d、pH为4.5培养6 d、溶解氧张力(dissolved oxygen tension，DOT)为25%培养6 d，DOT为10%培养6 d Incubate at pH 3.0 for 4 days, pH at 4.5 for 6 days, dissolved oxygen tension (DOT) at 25% for 6 days, and DOT at 10% for 6 days |
- | 0.755 | [41] |
| G. lucidum G0119 | 5 L发酵罐 5 L fermenter |
通气量为4 L/min Aeration rate was 4 L/min |
6.07 | 0.059 | [42] |
| 通气量为6 L/min Aeration rate was 6 L/min |
6.52 | 0.076 | |||
| 通气量为8 L/min Aeration rate was 8 L/min |
8.77 | 0.204 | |||
| 通气量为10 L/min Aeration rate was 10 L/min |
15.09 | 0.192 | |||
| G. lucidum G0119 | 6 L发酵罐 6 L fermenter |
搅拌速度为50 r/min Agitation speed was 50 r/min |
13.11 | 0.013 | [43] |
| 搅拌速度为100 r/min Agitation speed was 50 r/min |
13.48 | 0.052 | |||
| 搅拌速度为150 r/min Agitation speed was 150 r/min |
14.75 | 0.043 | |||
| 搅拌速度为200 r/min Agitation speed was 200 r/min |
14.90 | 0.029 | |||
| 搅拌速度：150 r/min (0-40 h)，100 r/min (40 h后) Agitation speed: 150 r/min (0-40 h)，100 r/min (after 40 h) |
14.16 | 0.086 | |||
| G. lingzhi G0023 | 5 L发酵罐 5 L fermenter |
两阶段培养：振荡阶段在1.5 d转速由150 r/min变为100 r/min，培养3 d；静置培养14 d Two-stage culture: agitation speed changed from 150 r/min to 100 r/ min in 1.5 days and cultured for 3 days in the oscillation culture phase; left to liquid static culture for 14 days |
20.3 | 0.926 | [44] |
| G. lucidum G0119 | 50 L发酵罐 50 L fermenter |
分批培养 Fed-batch culture |
10.67 | 0.243 | [45] |
| 间歇式补料培养 Batch feeding culture |
13.68 | 0.497 | |||
| 恒速补料培养 Constant speed feeding culture |
14.18 | 0.430 | |||
| 变数补料培养 Variable feeding culture |
15.26 | 0.588 | |||
| 指数补料培养 Exponential feeding culture |
17.68 | 0.810 | |||
| G. lucidum | 25 L发酵罐 25 L fermenter |
温度为30 ℃、通气比为1:0.75、搅拌速度为180 r/min条件下发酵80 h Fermentation at a temperature of 30 ℃, aeration rate of 1: 0.75, and an agitation speed of 180 r/min for 80 h |
- | 0.360 | [46] |
| G. lucidum CCGMC 5.616 | 250 mL摇瓶 250 mL flask |
在黑暗环境、0.94 W/m2白光、4.70 W/m2白光三阶段光辐照条件下分别培养2、6、10 d Three-stage light irradiation, incubate at dark environment for 2 days, 0.94 W/m2 white light irradiation for 6 days, and 4.70 W/m2 white light irradiation for 10 days |
- | 0.470 | [47] |
| G. lucidum CCGMC 5.616 | 250 mL摇瓶 250 mL flask |
初始氧传递系数为16.4 h-1 Initial KLa values of 16.4 h-1 |
11.35 | 0.246 | [48] |
| 初始氧传递系数为60.0 h-1 Initial KLa values of 60.0 h-1 |
13.85 | 0.280 | |||
| 初始氧传递系数为78.2 h-1 Initial KLa values of 78.2 h-1 |
15.62 | 0.339 | |||
| 初始氧传递系数为96.0 h-1 Initial KLa values of 96.0 h-1 |
13.75 | 0.450 | |||
| 注：GT：Ganoderma triterpenes；KLa：Volumetric oxygen transfer coefficient；-：文献未报道. Note: GT: Ganoderma triterpenes; KLa: Volumetric oxygen transfer coefficient；-: References unreported. | |||||
采用外源添加物对灵芝三萜的合成进行调控逐渐成为灵芝液态深层发酵调控的新策略和新手段，为进一步提高灵芝三萜含量提供了新的途径[49-50]。外源添加物能够促进信号化合物的合成，并增强细胞对生物和非生物胁迫的反应强度，随后调控灵芝三萜的合成[51]，外源添加物主要包括活性氧、金属离子、植物激素、P450酶诱导物以及其他外源添加物等。
3.1 活性氧(reactive oxygen species，ROS)在提高灵芝三萜产量中的应用冯杰等[52]通过添加氧载体正十二烷增大培养基中的溶氧量，从而提高灵芝三萜的产量，并对正十二烷的添加工艺进行优化，得出在5 L发酵罐发酵的25.63 h添加29.85 mL/L正十二烷的条件下，灵芝三萜产量比对照组提高了398%。You等[53]首次探究了在灵芝固体培养基中添加促氧化剂2, 4-二硝基氯苯(1-chloro-2, 4-dinitrobenzene，CDNB)或H2O2对灵芝酸生物合成的影响，发现总灵芝酸产量增加。Ren等[54]在灵芝发酵过程中添加5-8 mmol/L乙酸，实验结果表明灵芝酸含量增加，采用响应面法优化后，灵芝酸含量达到53.6 mg/g，比对照增加105%，其中中间代谢物羊毛甾醇和角鲨烯的干重也分别增加到47.13 μg/g和15.8 μg/g。You等[55]首次提出了灵芝酸的生物合成与真菌凋亡有关，在2 mmol/L阿斯匹林孵育的菌丝体中可以观察到活性氧的产生，随着阿斯匹林浓度增加到4 mmol/L，灵芝中的活性氧进一步增加，其结果表明，使用4 mmol/L阿司匹林诱导超过12 h可使总灵芝酸的产量达到最大，为53.85 mg/g，与对照组相比提高了2.8倍，但同时对菌丝体的生长也有一定的抑制作用。这些研究结果为ROS在灵芝中参与灵芝酸合成调控提供了依据。
3.2 金属离子在提高灵芝三萜产量中的应用金属离子在各种生物体的细胞生理和代谢过程中起着重要的作用。在植物中，通过添加金属离子来提高次级代谢产物的产量被认为是一种简单有效的策略，但该手段能否用于灵芝中提高灵芝酸物质的含量还有待进一步研究[56-58]。Tang等[59]研究了6种重金属离子对灵芝发酵的影响，发现银离子有利于二萜类物质的合成，而铜离子能提高三萜的产量，通过响应面分析得出在第4天添加2 mmol/L铜离子时灵芝三萜的含量达到30 mg/g。Zhu等[60]采用多铜离子添加、三阶段光照和碳氮源多脉冲培养相结合的方法开发了一种新的综合策略，结果显示不仅生物量得到了提高，而且总灵芝酸含量和产量分别高达41 mg/L和720.8 mg/L，对灵芝发酵大规模生产灵芝酸具有一定的指导意义。Li等[61]在灵芝发酵过程中将氮与钙离子添加相结合，结果表明灵芝酸T的含量能达到最大值为18.7 mg/g，比单独添加钙离子或氮分别提高2.1-4.2倍。Xu等[62-63]在灵芝发酵静置阶段添加NaCl、KCl和MnCl2时，发现添加钠离子与锰离子能使灵芝酸产量分别增加2.8倍和2.2倍，而钾离子对灵芝酸含量无明显变化，并且3种金属离子都对灵芝菌丝体生物量无明显影响，其中钠离子和锰离子对灵芝酸Mk、T、S、Me也有促进作用，其灵芝酸产量与对照组相比提高了1.78-4.30倍。张雪[64]研究发现灵芝发酵在热胁迫下，外源添加钙离子螯合剂EGTA使总灵芝酸、羊毛甾醇以及鲨烯含量与未处理菌株相比分别降低了22%、45%和41%，而回补CaCl2能基本恢复EGTA处理导致的总灵芝酸、鲨烯和羊毛留醇含量的降低，这些结果都证明在灵芝中胞内钙离子参与调控灵芝酸的合成。
3.3 植物激素在提高灵芝三萜产量中的应用由于植物激素在防御反应中发挥着关键性的作用，因此其在诱导研究中得到了广泛的应用；许多报道表明，植物防御反应的诱导依赖于茉莉酸、乙烯和水杨酸的信号通路之间的交叉效应，从而提供最佳的防御系统之一[51]。Ren等[65]首次使用茉莉酸甲酯作为诱导剂，并用均匀设计的统计方法对诱导条件进行优化后得出溶解在吐温-20的茉莉酸甲酯其最优添加量为254 μmol/L，GA (ganoderic acids)的产量高达45.2 mg/g，比对照组提高45.3%。乙烯是一种果实催熟激素，与果实衰老有关的生化和形态学变化有密切联系；Zhang等[66]发现在灵芝中使用15.9 mmol/L乙烯诱导灵芝细胞15.5 h后，能促进灵芝酸的产量达到33 mg/g，提高了90%，其中HMGR、SQS、OSC mRNA水平较对照组分别上调2.6、4.3、3.8倍。水杨酸(salicylic acid，SA)是一种在植物防御调控系统中起重要作用的小分子，外源SA的加入能使灵芝三萜产量增加；Ye等[67]在前人的研究基础上，将SA和钙离子作为联合诱导剂作用于灵芝液态发酵，能有效提高灵芝多糖和三萜的含量，与对照组相比，联合诱导使多糖和三萜含量分别增加9.02%和13.61%。
3.4 P450酶诱导物在提高灵芝三萜产量中的应用CYP450含有血红素，几乎存在于在所有的生物体中，是一个庞大而复杂的超家族，能参与多种初级和次级代谢反应，包括脂肪酸、甾醇、植物激素、萜类、黄酮类、信号分子和其他生物分子的产生[68-70]。
近两年，灵芝基因组测序引起了国内外学者的关注，Chen等[71]以Ganoderma lucidum CGMCC 5.0026为供试菌珠，鉴定了24个实体CYP基因簇，其中78个P450基因与羊毛甾醇合酶共表达，表明这78个P450很有可能参与灵芝下游的合成过程，其中16个与真菌CYP高度相似。徐晓兰[72]通过鉴定赤芝的全基因组数据库发现存在214个细胞色素氧化酶基因，可能与灵芝三萜下游的合成相关，其中具有真正P450功能的酶有195个，通过实时荧光定量PCR检测，得到有78个P450基因与SQS羊毛甾醇合酶共表达，可能在三萜生物合成中发挥作用，与Chen的研究结果一致。Liang等[73]首次探究了P450诱导物苯巴比妥对灵芝发酵的影响，结果表明在摇瓶培养转为静置培养的第5天添加100 μmol/L苯巴比妥为最优发酵条件，其中总灵芝酸含量达到41.4 mg/g，灵芝酸Mk、T、S和Me分别提高了47%、28%、36%和64%；同时，在苯巴比妥诱导下，发现关键中间体羊毛甾醇的积累减少。Nojoki等[74]发现P450诱导物利福平能有效提高灵芝酸的产量，并采用响应面法进行优化后得出在灵芝发酵第9天添加100 μmol/L利福平能使灵芝酸产量达到最大值为18.6 mg/g。
3.5 其他外源添加物在提高灵芝三萜产量中的应用Zhang等[75]通过两阶段培养的方式，以乳糖为底物添加纤维素酶后得到的灵芝酸含量高达1.33 g/L，未添加纤维素酶的对照组仅为0.78 g/L；控制添加时间发现，在第3天添加5 mg/L纤维素酶，灵芝酸的最高产量为1.61 g/L。刘高强等[76]首次在灵芝发酵中添加药用蜣螂虫粉，使灵芝胞外三萜含量达到0.30 g/L，有效地提高了三萜的产量，但由于蜣螂虫粉并不是一种纯物质，因此不能确定是哪种成分对灵芝三萜有促进作用，还有待进一步的研究。Feng等[77]比较了6种不同的外源添加物对灵芝菌丝体生长和三萜生物合成的影响，结果显示油酸的促进作用最为显著，并在6 L发酵罐进行验证，在添加30 mL/L油酸、诱导192 h后三萜产率达到1.08 g/L，证明此种三萜发酵生产技术和工艺具有较高的三萜产量和生产能力，可扩展用于工业生产。
有关活性氧、金属离子、植物激素、P450酶诱导物以及其他外源添加物对灵芝液态发酵三萜产量的影响如表 3所示。
| 菌种名称 Strain |
发酵规模 Fermentation scale |
诱导剂 Elicitors |
添加剂量 Added does |
诱导时效 Elicitation duration |
菌丝体干重 Mycelium dry weight (g/L) |
三萜产量 GT production(g/L) |
参考文献 References |
| G. lucidum G0119 | 500 mL摇瓶 500 mL flask |
正十二烷 N-dodecane |
29.85 mL/L | 25.63 h | - | 0.850 | [52] |
| 5 L发酵罐 5 L fermenter |
0.880 | ||||||
| G. lucidum CGMCC 5.616 | 250 mL摇瓶 250 mL flask |
铜离子 Copper ions |
2 mmol/L | 4 d | - | 0.348 | [59] |
| G. lucidum CGMCC 5.616 | 100 mL摇瓶 100 mL flask |
利福平 Rifampin |
100 mmol/L | 9 d | Inhibit | 0.186 | [74] |
| G. lucidum | 250 mL摇瓶 250 mL flask |
纤维素酶 Cellulose |
5 mg/L | 3 d | - | 1.608 | [75] |
| 2 L发酵罐 2 L fermenter |
5 mg/L | 12 d | - | 1.253 | |||
| G. lucidum | 500 mL摇瓶 500 mL flask |
蜣螂虫粉 Catharsius molossus |
5 g/L | - | 16.17 | 0.304 | [76] |
| G. lucidum G0119 | 250 mL摇瓶 250 mL flask |
油酸 Oleic acid |
30 mL/L | 0 h | 13.42 | 0.855 | [77] |
| 6 L发酵罐 6 L fermenter |
30 mL/L | 192 h | - | 1.076 | |||
| G. lucidum 5.534 | 1 000 mL摇瓶 1 000 mL flask |
黄芪 Astragalus membranaceus |
4 g/L | - | 13.15 | - | [78] |
| 甘草 Licorice |
6 g/L | - | 18.84 | 0.238 | |||
| 甘草 Licorice |
8 g/L | - | - | 0.653 | |||
| 山茱萸 Medical dogwood |
8 g/L | - | 19.95 | - | |||
| G. lucidum 01 | 500 mL摇瓶 500 mL flask |
亚油酸 Linoleic acid |
2 g/L | 7 d | Increase | 0.394 | [79] |
| 硬脂酸 Stearic acid |
- | Inhibit | |||||
| 棕榈酸 Palmitic acid |
- | Inhibit | |||||
| G. lucidum SCIM0006 | 250 mL摇瓶 250 mL flask |
连翘水提物 The water extract from Forsythia suspensa |
0.4 g/L | 7 d | Increase | 0.303 | [80] |
| 枸杞子醇提物 Ethanol extracts from L. chinensis |
0.2 g/L | 10.38 | 0.238 | ||||
| G. lucidum CGMCC 5.616 | 250 mL摇瓶 250 mL flask |
蜣螂虫粉 Catharsius molossus |
0.2 g/L | - | - | 0.314 | [81] |
| 0.1 g/L | 0.259 | ||||||
| G. lucidum CGMCC 5.616 | 250 mL摇瓶 250 mL flask 500 mL摇瓶 500 mL flask |
马铃薯块茎 Tuber aestivum vittad |
60 mg/L | 14 d | - | 0.316 | [82] |
| G. lucidum ZG06 | 250 mL摇瓶 250 mL flask |
两性霉素 Amphotericin |
10 μg/mL | 72 h | 9.85 | 1.500 | [83] |
| G. lucidum 01 | 500 mL摇瓶 500 mL flask |
镨 Pr |
0.1 mmol/L | 7 d | - | 0.398 | [84] |
| 注：GT：Ganoderma triterpenes；-：文献未报道. Note: GT：Ganoderma triterpenes; -: References unreported. | |||||||
在灵芝中，由于不同诱导因子直接或间接地利用所有的信号成分诱导灵芝三萜生物合成，因此，了解外源添加物对灵芝三萜生物合成的信号转导通路对于优化其工业化生产具有重要意义，有助于揭示灵芝生长发育和代谢的信号调控途径，促进灵芝三萜含量的高产。有关灵芝三萜生物合成的信号转导主要集中在活性氧(reactive oxygen species，ROS)信号、钙离子信号、茉莉酸甲酯信号、一氧化氮信号、cAMP信号、膜流动性和磷脂信号等方面。
4.1 ROS信号调控灵芝三萜生物合成ROS包括超氧阴离子(O2-)、过氧化氢(H2O2)、单线态氧(O21)，防御反应重要事件是氧化暴发，当生物体内氧代谢的动态平衡遭到破坏就会导致ROS的产生，ROS还能引发对不同环境变化的机体防御功能，同时刺激代谢产物的积累[85-86]。其中，由于H2O2具有较高的化学反应活性和相对稳定性、可以跨膜运输、比其他活性氧分子的半衰期长等特性被认为是活性氧中最适合作为信号分子的一种物质[87]。
ROS通过烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NOX)对灵芝菌丝体进行诱导，该酶能催化烟酰胺腺嘌呤二核苷酸磷酸(NADPH)上的一个电子转移给氧气形成超氧阴离子，超氧阴离子在超氧化物歧化酶(SOD)的作用下生成H2O2[88]。用NOX抑制剂二苯基氯化碘盐或ROS清除剂抗坏血酸处理可以解除高温胁迫导致的ROS积累，这一结果表明NOX是活性氧信号转导过程中的一个重要物质[89]。在灵芝中，ROS可能对GT生物合成具有多方面的影响：ROS短期处理灵芝可诱导细胞生长、产孢子和灵芝酸的生物合成，而ROS长期孵育不仅可诱导细胞死亡，还可通过磷酸化Hog1和Fus3蛋白激活MAPK (mitogen-activated protein kinase)信号，从而触发GT生物合成；Slt2型MAPK基因的敲除导致菌丝生长缺陷和细胞内ROS含量降低，H2O2的加入可以恢复Slt2敲除菌株中下降的GA含量，进一步说明细胞内ROS水平通过灵芝的MAPK信号通路参与GT的生物合成[90]。有文献报道少量的ROS能起到信号分子的作用，但是热胁迫下ROS会大量积累导致氧胁迫，对生物体有一定的伤害[64]。不过生物体内存在选择性氧化酶(AOX)、SOD、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GPX)等抗氧化酶，能在一定程度上保护细胞不受氧化胁迫的损伤[91]。Li等[92]研究了GPX酶在真菌中尤其是大型担子菌中的作用，结果表明GPX在控制细胞内H2O2含量、菌丝分枝、抗氧化应激、胞质Ca2+含量和灵芝酸生物合成等方面具有重要作用，揭示了GPX与细胞内H2O2和Ca2+之间互相调节，进一步表明了ROS对真菌的生长发育和次生代谢具有复杂的影响，其中GPX起着重要的作用。
4.2 钙离子信号调控灵芝三萜生物合成Ca2+作为一种信号离子，具有多种用途，在生物体复杂的信号通路中起着交通枢纽的作用。在许多真菌中，Ca2+信号通路参与调控分生孢子的形成、形态分化和真菌致病性等生物过程[93]。有文献报道，每当生物体受到来自病原体和害虫的攻击，遭受干旱、盐、极端温度等主要的环境压力以及一些化学或激素刺激，都会引起细胞内Ca2+浓度的变化；钙信号的调控原理是多种刺激因素作用于质膜或内膜表面的受体，使细胞内的钙离子浓度发生变化，促使其作为第二信使将不同的胞外刺激转化为钙信号向胞内传递，从而调节多种生物学反应[94]。
Xu等[95]研究了钙调蛋白信号转导对灵芝酸生物合成的影响，实验发现在灵芝发酵过程中加入钙离子后灵芝生物合成基因和钙离子传感器的表达都上调。Wang等[94]研究发现，当Ca2+增加时，钙调蛋白亚单位A (CNA)通过结合CAM复合物被激活，进一步激活转录因子CRZ1与曲霉和酵母下游基因启动子中的钙调蛋白依赖反应元件(CDRE)基序结合。在灵芝中，还发现菌丝静态液体培养中Ca2+的加入增加了3个Ca2+传感器基因(CAM、CNA和CRZ1)的转录水平，触发了钙调素依赖的钙调神经素信号通路；徐轶宁[96]通过添加钙调磷脂酶抑制剂(环孢霉素A)发现总的粗灵芝酸含量下降，当回补Ca2+、Na+或Mn2+后，环孢素酶A对总灵芝酸的抑制作用在一定程度上又有解除，表明Ca2+、Na+或Mn2+调节灵芝酸的生物合成可能与钙调磷脂酶信号有关。钙调磷脂酶是钙信号下游的一种效应分子，是钙调素下游的重要靶蛋白之一，由一个催化亚基(CNA)和一个调节亚基(CNB)组成，属于丝/苏氨酸型蛋白磷酸酶家族。在灵芝发酵过程中加入Na+或Mn2+时，HMGR、SQS和LS的表达会上调，从而进一步表明添加Na+或Mn2+也是通过介导钙调磷酸酶信号转导的转录水平来提高灵芝酸产量[62-63]。
4.3 茉莉酸甲酯(methyl jasmonate，MeJA)信号调控灵芝三萜生物合成MeJA信号通路被认为是许多植物次级代谢产物生物合成的一个完整信号，其中F-box蛋白COI1 (coronatine insensitive 1)起关键作用，与Skp1、Cullin和Rbx1蛋白结合形成SCFCOI1复合物[97-98]。JAZ (jasmonate zim-domain)蛋白是COI1的直接靶点，有研究表明JA-Ile (jasmonoyl-isoleucine)能诱导COI1和几种JAZ蛋白之间的相互作用，是目前唯一一个具有生物活性的JA[99-102]。虽然SCFCOI1对JAZ蛋白的泛素化还未得到验证，但茉莉酸的降解和转录因子的释放与这种相互作用确实有关，并且这些转录因子能调控特定的茉莉酸反应基因的表达，刺激特定次生代谢产物的产生[103-105]。在灵芝中能够激活MeJA信号级联中参与灵芝三萜生物合成基因表达转录因子的相关数据很少，因此植物中有关MeJA信号的报道有重要的参考价值。
对灵芝的全基因组转录组分析研究表明，MeJA诱导不仅调控GA的生物合成相关基因，还调控其他代谢途径的相关基因，如甘油代谢、ROS爆裂、丙酮酸代谢、乳酸代谢和鞘脂代谢等[106]。由于在植物中MeJA可以通过激活MAPK信号通路、活性氧信号或钙依赖蛋白激酶信号通路，从而诱导气孔闭合、单萜吲哚类生物碱和异戊二烯类生物合成，起到防御应答病原体的作用，因此得出MeJA诱导的灵芝三萜生物合成可能是通过激活ROS或钙依赖蛋白激酶信号通路发挥作用的假设[107-109]。Shi等[110]在灵芝菌丝培养过程中加入MeJA后发现不仅灵芝酸的含量增加，而且菌丝之间的距离也增加了约1.2倍，细胞内的ROS含量也有所增加，进一步研究表明ROS清除剂可以消除经过MeJA处理的灵芝菌丝体灵芝酸的生物合成以及菌丝的分枝，证实了MeJA对灵芝菌丝分枝和灵芝三萜合成的调控可能是通过ROS信号来实现的，其中NOX起了关键作用。
4.4 一氧化氮(nitric oxide，NO)信号调控灵芝三萜生物合成NO是一种自由扩散的膜渗透气体，有报道称NO是生物体内一种有效的信号分子，能作用于ROS信号分子的上游并对细胞中其他信号分子的生物合成进行调控，从而响应各种生物和非生物胁迫；NO有亲脂性，属于反应性氮(RNS)家族，能与多种靶点相互作用，不仅使基因重表达，而且能调节蛋白质的功能[111]。NO具有双重抗氧化或促氧化功能的作用，在促进灵芝发酵产灵芝酸过程中，NO能激活细胞内Ca2+通道，促使Ca2+流入细胞，其中NO与O2-的反应可以产生强氧化剂过氧化亚硝酸离子(ONOO-)，激活ROS信号通路[112]。NO与抗氧化剂谷胱甘肽之间的关系尤为密切，NO还可与还原型谷胱甘肽反应生成s-亚硝基谷胱甘肽(GSNO)[113]。Gu等[114]以硝普纳为NO供体，在灵芝液态深层发酵中添加外源性NO可以提高灵芝酸的含量以及生物合成相关基因的转录水平，在发酵72 h时添加5 mmol/L的硝普纳，灵芝酸含量与对照组相比提高了40.94%，其中NO可能直接作为基因表达调控因子在甲羟醛酸途径中诱导灵芝酸生物合成，而响应NO使灵芝酸产生也可能通过Ca2+和ROS的信号传递功能实现。在植物中，重金属胁迫表明NO的来源是未知的，这种分子随后通过促进一系列抗氧化酶的活性而发挥抗氧化作用；同样，外源NO能激活抗氧化剂SOD、POD和CAT，保护灵芝免受重金属镉的胁迫[111]。因此，NO信号调控灵芝三萜生物合成是通过涉及Ca2+和ROS信号转导通路完成的。
4.5 环腺苷单磷酸(cyclic adenosine monophosphate，cAMP)信号调控灵芝三萜生物合成cAMP是一种重要的信号分子，参与细胞外生物和非生物刺激的感知，并随后将这些信号转导到相应的反应。有文献报道，在灵芝发酵过程中加入适量的阿司匹林，能诱导细胞凋亡并显著增加灵芝酸的生物合成，这是由于细胞内ROS的暴发激活了灵芝中的cAMP信号[115]。越来越多的生理、生化和遗传学证据表明，cAMP在对诱导子处理或其他胁迫的反应中能调控细胞膜上阳离子通道，升高cAMP的作用是激活cAMP依赖蛋白激酶A，进而磷酸化下游靶蛋白，这些蛋白质包括酶、结构蛋白和转录因子，它们通过这一途径发出信号，从而产生无数种反应；除了钙离子外，cAMP是参与真菌各种生理活动的二级信使[87]。细胞内cAMP动力学主要与腺苷酸环化酶合成酶和磷酸二酯酶降解有关，与cAMP孵育后，灵芝细胞可激活腺苷酸循环活性或抑制磷酸二酯酶活性[115]。因此，咖啡因作为磷酸二酯酶的抑制剂、氟化钠(NaF)作为腺苷酸环化酶的激活剂用于提高灵芝细胞内cAMP水平，You等[115]用NaF、咖啡因或cAMP/IBMX (3-isobutyl-1- methylxanthine)处理菌丝时，能提高灵芝酸的产量，诱导真菌细胞凋亡，并且检测到SQS和LS基因表达下调，转录组分析表明，线粒体可能在cAMP诱导的细胞凋亡和灵芝酸生物合成中发挥重要作用。
4.6 膜流动性和磷脂信号调控灵芝三萜生物合成细胞膜能比较敏感地感知环境中的变化。在热胁迫条件下，由于灵芝细胞膜流动性的增加，NADPH氧化酶被激活，活性氧在热激信号转导途径中产生，发挥着第二信使的作用[116]。Liu等[117]通过质谱综合分析了热应激诱导的脂质重塑，结果表明，热胁迫条件下磷脂酸(phosphatidic acid，PA)积累显著增加，而磷脂酶D (phospholipase D，PLD)沉默菌株的遗传实验进一步表明，PA的积累依赖于热应激诱导PLD水解磷脂酰乙醇胺(phosphatidylethanolamine，PE)的能力；此外，PLD沉默菌株减少了部分由热应激诱导的灵芝酸的生物合成，而添加PA可以逆转该现象，这一研究结果表明PLD和PA参与热应激诱导的灵芝次生代谢的调控，阐明了灵芝如何通过磷脂重塑和积累次级代谢产物对热应激做出反应。Liu等[118]进一步研究了灵芝磷脂信号，在热应激条件下，灵芝中的磷脂酰肌醇(phosphatidylinositol，PI)在PI-4激酶的作用下转化为PI-4-磷酸，然后在PI-4-磷酸盐-5激酶的作用下转化为PI-4, 5-二磷酸，这一步骤与胞内Ca2+信号转导和灵芝酸生物合成有密切的联系，同时也表明磷脂信号和Ca2+信号之间存在相互作用，以响应环境高温的变化。有关膜流动性与磷脂信号的研究对进一步了解灵芝次生代谢物的合成有很大的启发作用。
有关活性氧ROS信号、钙离子信号、茉莉酸甲酯信号、一氧化氮信号、cAMP信号对灵芝液态发酵三萜产量的影响如表 4所示。
| 菌种名称 Strain |
诱导剂 Elicitors |
添加剂量 Added does(mmol/L) |
诱导时效 Elicitation duration |
信号组分 Signal components |
代谢物 Metabolites |
三萜产量 GT production(g/L) |
相关基因 Related gene |
作用机理 Mechanism of action |
参考文献 References |
| G. lucidum BCRC 36111 | CDNB | 0.4 | 4 d | Hog1 | Total GT | 4.60 | SQS, LS | ROS signal, MAPK signal | [53] |
| GA-24 | 0.32 | ||||||||
| H2O2 | 1.6 | 1 h | Hog1, Fus3 | Total GT | 8.60 | ||||
| GA-24 | 0.48 | ||||||||
| G. lucidum HG | Acetic acid | 5 | 32 h | - | Total GT | 39.80 | HMGR, SQS | ROS signal | [54] |
| 8.21 | 22.68 h | Total GT | 55.20 | ||||||
| G. lucidum BCRC36111 | Aspirin | 4 | 1 d | MAPKp38, Hog1 | Total GT | 53.85 | SQS, LS | Apoptosis signaling | [55] |
| G. lucidum CGMCC 5.616 | Ca2+ | 10 | 14 d | - | GA-T | 18.70 | HMGR, SQS, LS | Calcineurin signal transduction | [61] |
| 8 d | |||||||||
| G. lucidum CGMCC 5.616 | Na+ | 100 | 2 d | CAM, CAN, CRZ1, ENA1, SOCE, Ca2+- ATPase | GA-Mk | 17.96 | HMGR, SQS, LS | Na+/Ca2+ exchanger, Ca2+ signal transduction | [62] |
| GA-T | 14.15 | ||||||||
| GA-S | 5.19 | ||||||||
| GA-Me | 6.15 | ||||||||
| K+ | 100 | 0 d | GA-Mk | 4.59 | |||||
| GA-T | 3.44 | ||||||||
| GA-S | 1.87 | ||||||||
| GA-Me | 2.24 | ||||||||
| G. lucidum CGMCC 5.616 | Mn2+ | 10 | 2 d | CAM, CAN, CRZ, PMR1 | GA-Mk | 6.97 | HMGR, SQS, LS | Mn2+/Ca2+ exchanger, Ca2+ signal transduction | [63, 96] |
| GA-T | 10.29 | ||||||||
| GA-S | 5.83 | ||||||||
| GA-Me | 5.39 | ||||||||
| G. lucidum HG | Methyl jasmonate | 0.05 | 6 d | AAO, NBP, CDC, CAL, CAT, VMP, HD, GLS, HK, CAT, RHO | Total GT | 40.00 | HMGR, SQS, MVD, FPS | ROS signal, MAPK signal, Ca2+ signal transduction | [65, 109] |
| 0.1 | 37.20 | ||||||||
| 0.15 | 37.40 | ||||||||
| 0.234 | 45.20 | ||||||||
| G. lucidum HG | Ethylene | 15.9 | 15.5 h | ACS | Total GT | 33.00 | HMGR, SQS, OSC | - | [66] |
| G. lucidum CGMCC 5.616 | Phenobarbital | 0.1 | 5 d | - | Total GT | 41.40 | SQS, LS | P450 inducer | [73] |
| GA-Mk | 1.04 | ||||||||
| GA-T | 2.48 | ||||||||
| GA-S | 1.17 | ||||||||
| GA-Me | 0.77 | ||||||||
| G. lucidum CGMCC 5.616 | CaCl2 | 10 | 2 d | CAM, CAN, CRZ1 | Total GT | 71.12 | HMGR, SQS, LS | Calcineurin signal transduction | [95] |
| GA-Mk | 1.91 | ||||||||
| GA-T | 11.94 | ||||||||
| GA-S | 2.33 | ||||||||
| GA-Me | 3.03 | ||||||||
| G. lucidum GIM5.250 | Sodium nitroprusside | 5 | 72 h | CAM, CATP, GPX, POD, SOD | Total GT | 118.50 | ACAT, SE, HMGR, HMGS | NO signal pathway | [114] |
| G. lucidum BCRC 36111 | Caffeine | 80 | 4 d | cAMP | Total GT | 11.09 | SQS, LS | cAMP signal | [115] |
| GA-24 | 0.43 | ||||||||
| NaF | 20 | Total GT | 13.72 | ||||||
| GA-24 | 0.75 | ||||||||
| cAMP+15 IBMX | 40 | Total GT | 10.09 | ||||||
| GA-24 | 0.36 | ||||||||
| G. lucidum HG | Salicylic acid | 0.2 | 24 h | - | GA-A | 2.29 | HMGR, SQS | - | [119] |
| 注：GT：Ganoderma triterpenes；NaF：Sodium fluoride；IBMX：3-Isobutyl-1-methylxanthine；-：文献未报道. Note: GT：Ganoderma triterpenes; NaF: Sodium fluoride; IBMX: 3-Isobutyl-1-methylxanthne; -: References unreported. | |||||||||
灵芝三萜不仅具有广泛的生理活性和良好的应用前景，而且市场对灵芝三萜的需求量也在持续增加，虽然灵芝菌丝体液态深层发酵技术对提高灵芝三萜的产量有一定的帮助，但还是不能满足实际生产的需要，制约了灵芝三萜的应用。结合国内外学者对灵芝三萜的研究结果，我们认为灵芝三萜发酵的研究需要进一步加强几个方面的工作：(1)利用转录组学、蛋白组学和代谢组学相结合的手段对灵芝三萜的生物合成途径进行全面解析，阐明灵芝三萜生物合成下游的具体途径；(2)对灵芝三萜合成的诱导策略和信号转导网络进行研究，了解三萜的代谢通路，找到有效提高灵芝三萜产量的调控方法；(3)进一步研究外源物质对促进灵芝三萜合成的方法和机制，建立有效提高灵芝三萜含量的添加工艺。通过解决灵芝三萜发酵的基础研究和应用基础研究，将为灵芝三萜的进一步开发利用奠定良好的技术基础。
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