基于herquline A 等天然产物生物合成的酚去芳构化生物催化体系的研究和应用

Dearomatization of phenols, which converts the planar, aromatic scaffolds into three-dimensional molecular architectures, is considered as a powerful strategy for synthesis of six-membered ring containing molecules. Organic chemists have developed several approaches for phenol dearomatization, which still suffer from poor regio- or stereo-selectivity. The use of biological catalysts for these conversions will be an option that worth considering. So far, most applicational examples for biological dearomatization are restricted to the use of dioxygenases existing in living microorganisms, as part of their primary metabolism, for a few types of substrates. Therefore, developing new types of biological phenol dearomatization processes would be critical for widening the biosynthetical applications. Dearomatized phenol moieties are widely existing in natural products from different sources. However, applications making use of these biosynthetic tools are rare. In the applicant’s previous publication on biosynthesis of herquline A, a dearomatized tyrosine derivative, a novel mode of phenol dearomatization was discovered, which involving two cooperating enzymes converting two phenols into a scaffold containing two crosslinked cyclohexenones. The involved enzymes HqlF and HqlC are proposed now for study on their biochemical details and for their potential applications. Explicitly, substrate promiscuity and specificity of the enzymes will be studied. Protein mutagenesis and regulatory approaches will also be used to optimize these conversions, to establish efficient biocatalytical process either in vitro or in vivo. Meanwhile, new examples in nature of this cooperating pattern of phenol dearomatization will be explored, using HqlC-HqlF pair as a thread. Biosynthesis of oxysporidinone, which should also involve a phenol dearomatization process like that in herquline A will be focused for this purpose. More dearomatization tools will be discovered during this mining strategies on natural product biosynthesis, which will lead to a widening pool of enzymes for bio-dearomatization of phenols.

酚的去芳构反应可以使平面的酚环结构一步生成三维环状结构，是制备多环分子的有效策略。去芳构的化学方法虽有相关报道，但是反应的区域选择性和立体选择性大都不理想。已知的酚去芳构的生物催化研究主要集中于微生物初级代谢中的双加氧酶，催化的化合物种类有限。因此发展新的酚去芳构催化体系，对拓展去芳构反应的生物催化应用至关重要。去芳构化天然产物在自然界中广泛存在，但相关的生物催化研究鲜有报道。申请人前期从去芳构天然产物herquline A的生物合成中鉴定了参与酚去芳构反应的酶HqlC和HqlF。本项目拟进一步研究HqlC和HqlF的生物催化功能，阐明底物杂泛性和酶的选择性，并通过定点突变和表达调控等手段，优化生物催化，构建高效的体内体外催化体系。同时，从oxysporidinone等具有酚去芳构单元的天然产物的生物合成中发掘新的功能酶，优化催化并构建高效应用体系，以进一步拓展酚去芳构反应的生物催化策略。

酚的去芳构化反应可以实现从芳香化合物平面分子到三维的取代环己烷/酮结构的转化，是合成多环系统等的重要合成策略。发展新型去芳构生物催化体系，对拓展去芳构反应类型和应用至关重要。本项目围绕天然产物生物合成途径中酚去芳构的生物催化反应开展了系列研究。在对herquline生物合成途径中催化去芳构反应的HqlC和HqlF的生物催化体系进行优化的研究中，通过定点突变等手段对HqlC和HqlF中与底物识别和催化效率相关的多个氨基酸位点进行优化，获得催化效率更高的突变株。同时，本项目还通过综合运用基因簇激活、基因敲除、构巢霉菌/酵母异源重构和体外酶反应等手段阐明了oxysporidinone生物合成中去芳构反应。此外，运用异戊烯基转移酶CdpC3PT的突变体F253G，构建了萘酚去芳构偕二异戊烯基化的生物催化体系。本项目开展新型去芳构生物催化剂的研究，拓展了酚去芳构反应的生物催化策略，为实现该类反应的高效生物催化奠定基础。