萘醌-氧吲哚生物碱coprisidins生物合成途径的研究

Identification and characterization of the biosynthetic pathway of naphthoquinone-oxindole alkaloid coprisidins

摘要:

[目的] 新颖结构的天然萘醌-氧吲哚类生物碱coprisidins（A和B）分离自昆虫肠道相关链霉菌，具有预防癌症的活性。作为首例具有萘醌-氧吲哚骨架的生物碱，对其独特生物合成机理的研究可为II型聚酮类化合物生物合成途径提供新的认知。[方法] 本研究对coprisidins的产生菌Streptomyces sp.SNU607进行全基因组测序，并根据测序结果的生物信息学分析初步定位coprisidins的生物合成基因簇；通过基因敲除以及异源表达手段确定coprisidins的生物合成基因簇；基于体内遗传学实验与生物信息学分析初步推导coprisidins的生物合成途径。[结果] Streptomyces sp.SNU607中有23个基因簇可能参与次级代谢，其中4个基因簇与聚酮合酶（PKS）相关；通过基因敲除与异源表达实验，本研究证实1个II型PKS负责coprisidins的生物合成；基于生物信息学分析，我们推测copH/I/M/O/N构成了1个基因盒，并负责起始单元丁酰CoA的合成；KS_β（CopB）的序列比对表明coprisidins的II型PKS系统更倾向于合成C20的初始聚酮链。[结论] Coprisidins的萘醌-吲哚结构是由II型PKSs催化形成，我们推测丁酰CoA是coprisidins聚酮骨架的起始单元，在最小PKS、聚酮酶、环化酶的催化下先形成类似蒽环的四环系统，随后在后修饰酶与氧化重排的作用下生成萘醌-氧吲哚骨架。本研究为进一步探究萘醌-氧吲哚类生物碱的生物合成机制奠定了基础，同时增加了II型PKSs合成产物的结构多样性。

关键词:coprisidins;萘醌-氧吲哚类生物碱;芳香聚酮;生物合成基因簇;合成生物学

Abstract:

[Objective] Coprisidins (A and B) are structural unique naphthoquinone-oxindole alkaloids from an insect gut-associated Streptomyces with bioactivities for cancer prevention. As the first example of natural alkaloids with naphthoquinone-oxindole skeleton, the biosynthesis of coprisidins remains unclear. Exploration of the coprisidins biosynthetic mechanism would gain new insights into the biosynthesis of type II polyketide natural products. [Methods] The genome of coprisidin-producing strain Streptomyces sp. SNU607 was sequenced and in silico analysis revealed the biosynthetic potential. The coprisidins biosynthetic cluster was cloned and identified by the gene inactivation and heterologous expression experiments. A biosynthetic pathway for coprisidins was proposed according to the in vivo results and bioinformatics analysis. [Results] Genome analysis of Streptomyces sp. SNU607 revealed 23 gene clusters potentially for secondary metabolites with four of them related to polyketide synthases (PKSs). Gene inactivation and heterologous expression confirmed that a type II PKS gene cluster, which contains 30 putative open reading frames, is responsible for coprisidins biosynthesis. The copH/I/M/N/O genes could constitute a five gene cassette for the biosynthesis of butyryl start unit of coprisidins. Sequence alignment of KS_β (CopB) suggested that coprisidins are biosynthesized by a type II system with a nascent chain length of C20, which is afterward modified by a variety of accessory enzymes. [Conclusion] The rare naphthoquinone-oxindole backbone of coprisidins is assembled by the minimal type II PKS (CopB-C-A) utilizing a butyryl starter unit, along with ketoreductases and cyclases to form the anthracycline-like four ring system. Subsequently, tailoring enzymes and oxidative arrangement are proposed to shape the final naphthoquinone-oxindole skeleton. Considering the structure feature of coprisidins, this study would set the stage for biosynthetic mechanism studies of naphthoquinone-oxindole alkaloids and expand the structural diversity of products synthesized by type II PKSs.

Key words:coprisidins;naphthoquinone-oxindole alkaloid;aromatic polyketide;biosynthetic gene cluster;synthetic biology