过表达LaeA激活海洋土曲霉中isoflavipucine类化合物的合成及其抗弧菌活性研究

Activating biosynthesis by overexpressing LaeA and anti-Vibrio activity of dihydroisoflavipucines of marine-derived Aspergillus terreus

丝状真菌土曲霉因产生结构独特且药理活性强烈的次级代谢产物而受到真菌学家和药学家的广泛关注。然而,在丝状真菌中,大部分次级代谢基因在常规实验室培养条件下表达量较低甚至沉默。本研究通过过表达全局性调控因子LaeA试图激活一株海洋来源土曲霉中的沉默基因簇以发现新次级代谢产物。结果表明,laeA基因过表达突变中未知产物基因簇被激活,指纹图谱分析发现了2个在野生菌株不存在的化合物吸收峰。质谱和核磁共振波谱分析确证了这2个化合物为dihydroisoflavipucine类小分子,特别是化合物1的产量在突变体中高达183 mg/L。此外,抗菌活性测试发现,这2个化合物对4种病原弧菌显示了强烈的活性,特别是化合物1抗弧菌活性更强,MIC低至16μg/mL。本研究提供了一条大量合成dihydroisoflavipucine类抗生素的新路线,并证明了次级代谢调控因子LaeA在丝状真菌中的功能是高度保守的,过表达LaeA是激活丝状真菌中沉默基因簇的有效手段。

The filamentous fungus Aspergillus terreus has attracted great attention from mycologists and pharmacognosists for its secondary metabolites(SMs)with distinctive chemical structures and excellent pharmacological activities.However,expression level of most of SM gene clusters is relatively low,or even silent under common lab culture condition in filamentous fungi.This study attempted activating the silent SM clusters by overexpressing the global regulator LaeA to mine new natural products with good biological activities.Our results showed that laeA overexpression resulted in the activation of a silent SM cluster without corresponding known compounds.NMR and MS analysis confirmed the compounds as dihydroisoflavipucines 1and 2,and the yield of 1 in the mutant reached a level of 183 mg/L.Antibacterial activity assay indicated that compound 1 showed stronger anti-Vibrio activity than 2 with a MIC of 16μg/mL.Our study provided a novel strategy to massively biosynthesize the antibiotic dihydroisoflavipucines by using marine-derived A.terreus,and further demonstrated that LaeA as a conserved protein played a crucial regulatory role in secondary metabolism in filamentous fungi.