仿突变生物合成调控对土曲霉C23-3次生代谢产物的影响

Effects of Simulated Mutational Biosynthetic Regulation on the Secondary Metabolites of Aspergillus terreus C23-3

【目的】丁内酯I是土曲霉(Aspergillus terreus)产生的一种具有多样生物活性的小分子天然产物,对其结构多样性拓展的探索具有重要意义。以一株高产丁内酯I的海洋来源土曲霉C23-3作为基础菌株,研究丁内酯前体小分子类似物和前体合成酶抑制剂对其次生代谢的影响。【方法】以海水马铃薯液体培养基为基础培养基,以3种前体小分子类似物和3种对羟基苯丙酮酸的合成酶抑制剂作为化学诱导剂,在静置发酵条件下进行菌株的化学调控培养。采用高效液相色谱、高效液相色谱-离子阱质谱联用及基于质谱的分子网络和数据库挖掘分析次生代谢产物的产量和多样性。【结果】多数前体小分子类似物和合成酶抑制剂对丁内酯I产量均有不同程度抑制,而高浓度的前体小分子类似物3,4-二羟基苯丙酮酸、两种合成酶抑制剂联用及这三者的联用表现出了较强的抑制效果;并且在丁内酯I的合成受到强烈抑制条件下,丁内酯类、土震素类、洛伐他汀类等多类次生代谢产物的合成也下降,但一种环肽类化合物产量超过10倍的大幅提升,可能是菌体对作为群体感应信号及全局性转录因子lae A诱导子的丁内酯I合成受到压制胁迫的一种应激反应,具体机制有待深入研究。【结论】丁内酯I的前体确为对羟基苯丙酮酸,3,4-二羟基苯丙酮酸及对羟基苯丙酮酸合成酶抑制剂可用作抑制丁内酯I合成的小分子工具,以用于仿突变生物合成的进一步探索以获得多样化丁内酯衍生物,也可诱导产生环肽类化合物。

【Objective】Butyrolactone I is a small molecular natural product of Aspergillus terreus with versatile bioactivities,thus the exploration of its structural diversity extension is significant.A marine-derived A.terreus strain C23-3 featuring producing butyrolactone I was used as the base strain to study the effects of butyrolactone I precursor analogs and precursor synthase inhibitors on its secondary metabolism.【Method】The strain was statically fermented under chemically regulative conditions established by seawater potato liquid medium supplied with three precursor small molecules and three 4-hydroxyphenylpyruvate synthase inhibitors.The high-performance liquid chromatography(HPLC),HPLC-ion trap mass spectrometry(MS),and MS based molecular networking together with database mining were used to further analyze the yields and diversity of secondary metabolites.【Result】The most of the precursor analogs and synthase inhibitors suppressed the synthesis of butyrolactone I to different extents.Moreover,the precursor analog 3,4-dihydroxyphenylpyruvate at a high dose,the combination of the two 4-hydroxyphenylpyruvate synthase inhibitors and the combination of these three demonstrated remarkable inhibitory effects.In addition,the global synthesis for sorts of secondary metabolites including butenoids,territrems,lovastatins,etc.,was downregulated when butyrolactone I was inhibited for production.However,the yield of a cyclopeptide was upregulated by more than ten folds under this situation,which was supposed to be a stress response to the synthetic suppression of butyrolactone I as a quorum sensing molecule and inducer of global transcriptive factor lae A.The detailed mechanism deserves further investigation.【Conclusion】The present study manifests that 4-hydroxyphenylpyruvate is the precursor of butyrolactone I and reveals that 3,4-dihydroxyphenylpyruvate and 4-hydroxyphenylpyruvate synthase inhibitors can be used as small molecule tools to suppress the production of butyrolactone I,which may be applied in further study on simulated mutational biosynthesis to generate diverse butyrolactone derivatives and induce the production of cyclopeptide.