竹黄伴生细菌激活竹红菌素生物合成的研究

Hypocrellins, naturally occurring perylene quinones isolated from fungal stroma (fruiting-body) of parasitic Shiraia bambusicola, are new type of non-porphyrin photodynamic therapy (PDT) agents. There are rich bacteria associated with fungal fruiting-body in many improtant medicnal fungi including S. bambusicola. It is of scientific significances to explore the role of associated bacteria on hypocrellin biosynthesis of S. bambusicola. The research on fungal-bacterial interaction (FBI) may result in unique contributions to a novel strategy for the production of PDT drugs. We will focus our research efforts on the higher hypocrellin-yielding strain of S. bambusicola and the bacteria associated with fungal fruiting body. The structure, distribution and diversity of the bacterial communities in the fruiting body will be investigated. In the co-culture system including in vitro fungal–bacterial confrontation bioassay and co-inoculation in submerged cultures, the strains of the associated bacteria to promote hypocrellin biosynthesis will be screened. The analysis on differential gene expression and the characterization of some enzymes responding to the living bacteria will shed the light on the pathway of hypocrellin biosynthesis. With the transcriptome mining technique, we wish to find the clues for activating silent fungal secondary metabolite gene clusters by associated bacteria and producing novel bioactive perylenequinones in S. bambusicola cultures . The methods for eliciting anamorphosis fermentation will be established for higher hypocrellin yields. We believe our ongoing results will be helpful in the application of associated bacteria on biotechnological production of hypocrellins and more new photosensitivity perylenequinones. Our research will give hints or theoretical basis of the modulation of associated bacteria on mycelium cultures and cultivation of important medicial mushirooms for the source of PDT drugs.

竹红菌素是从竹黄菌（Shiraia bambusicola）子座(子实体)中分离发现的新型光敏药物，真菌子实体中常具有丰富的伴生细菌，探讨伴生细菌对竹黄菌竹红菌素合成的调节作用，对挖掘细菌-真菌间相互关系具有重要的理论意义和应用价值。我们以课题组筛选和选育的竹黄菌及其伴生细菌为对象，分析伴生细菌的多样性和伴生特性，通过共培养体系考察伴生菌对竹红菌素合成的调节作用。通过基因差异表达分析，获得竹黄菌中对伴生菌诱导有响应的竹红菌素合成特异酶基因，完善竹红菌素合成途径。通过转录组挖掘，发现伴生菌激活的竹黄沉默基因簇和诱导产生的新型蒽醌类光敏剂；构建竹红菌素发酵生产的混合培养技术，为竹红菌素等真菌光敏药物的生物技术生产提供理论基础和参考。

竹红菌素是从竹黄菌（Shiraia）子座(子实体)中分离的新型光敏药物。本项目研究了竹黄菌子实体内伴生细菌对竹黄菌生长、发育及竹红菌素的生物合成的调控。分离得到子实体伴生细菌31株，芽胞杆菌属（Bacillus）和假单胞属（Pseudomonas）为优势种群，Chao指数（806.817）和Shannon指数（4.468）反映了丰富多样的竹黄伴生细菌。竹黄优势种群-芽胞杆属和芽孢杆菌属，它们对真菌竹红菌素的合成具有截然不同的作用，蜡样芽孢杆菌（B. cereus No. 1）完全抑制或分解竹红菌素的积累，而假单孢属分离株均表现出促进竹红菌素合成的功能。我们比较了活性伴生菌株（黄褐假单胞菌SB1和蜡样芽孢杆菌No. 1）与竹黄菌不同接触方式，发现主要作用方式是活菌直接接触。另一方面，伴生细菌也可通过挥发物产生显著的诱导作用。我们发现并解析了可以诱导竹红菌素的合成的黄褐假单胞菌胞外甘露聚糖。发现活性氧、eATP、Ca2+和NO信号通过信号互作参与了细菌-真菌共培养下细菌对竹红菌素合成的调控作用。在黄褐假单胞菌SB1的诱导下，28个注释到了竹红菌素合成基因簇且85%以上发生了上调，个别沉默基因甚至被SB1激活，如聚酮合酶PKS、FAD连接的氧化还原酶FAD及单氧酶活性Mono。SB1与竹黄菌共培养还可显著诱导竹红菌素转运相关基因的表达。蜡样芽孢杆菌No. 1与竹黄菌接触时，与HA合成和转运相关的基因均下调的，No. 1细菌对竹红菌甲素还有降解作用, 形成还原型的HAH2。基于伴生细菌调节竹红菌素的机制，我们发展了促进竹红菌素菌丝培养生产的生物技术，如细菌甘露聚糖EPS-1的诱导促进技术；基于细菌VOCs对竹黄菌HA产量的影响，我们建立了一种利用细菌挥发物的新型共培养生产体系。在优化的细菌-真菌活菌共培体系，竹红菌甲素总产量达最大值为325.87 mg/L，为单独培养对照组的3.20倍。共培后，真菌代谢物也具有显著差异。研究结果为理解伴生细菌与宿主真菌在生长发育与次生代谢诱导的生态关系及伴生细菌作用机制提供了帮助，同时也为提高竹红菌素产量的生物技术发酵提供了诱导和共培技术。