沉香特征性成分2-(2-苯乙基)色酮的生源途径解析及关键酶的鉴定

Agarwood, a rare medicinal herb frequently used in Chinese medicine, is a resinous heartwood produced by Aquilaria sinensis in response to external stimulus. Naturally, the formation of agarwood is occasional and takes a long-time period, resulting in its lack of resources and extremely expensive, which seriously affects the clinical applications of agarwood-containing medicines and the sustainable developments of related industries. As the major and characteristic components of agarwoods, 2- (2-phenylethyl) chromones were accordingly assigned as index components in the Chinese Pharmacopoeia for the evaluation of the quality of agarwood. However, the biosynthesis of 2-(2-phenylethyl) chromones are currently kept unknown. We have already elucidated that 1,5-diphenyl-1,3-pentanedione was a biosynthetic precursor of 2- (2-phenylethyl) chromone, and the key enzyme involved in the biosynthesis of this important precursor was also successfully elucidated. The present project would focus on the characterization of the biogenetic pathway and other key enzymes involved in the biosynthesis of 2- (2-phenylethyl) chromones using biotransformation of labeled substrate, transcriptome sequencing, and metabolomic analysis. Clarification of the biogenetic pathway and key enzymes involved in the biosynthesis of 2- (2-phenylethyl) chromone are definitely advantageous to understand the molecular mechanism of agarwood-formation, which would be conducive for the process regulation and intervention of agarwood-formation, and thus be beneficial to resolve the problem of agarwood resource. Additionally, characterization of the key enzymes would be profoundly meaningful to the further studies on the synthetic biology of the biologically important 2- (2-phenylethyl) chromone.

沉香为临床常用名贵中药，是白木香在受外界刺激后，缓慢形成的含树脂的木材。自然条件下，沉香的形成非常困难，造成其资源匮乏，价格昂贵，严重影响含沉香类中药的临床应用及相关产业的可持续性发展。2-（2-苯乙基）色酮为沉香的特征性成分，也是《中国药典》中用于评价沉香质量的指标性成分，其生物合成机制目前尚不清楚。我们前期研究发现1,5-二苯基-1,3-戊二酮是2-（2-苯乙基）色酮的生物合成前体，并成功鉴定了合成该重要前体的关键酶。本项目拟在此基础上，利用悬浮细胞转化标记底物、比较转录组测序及代谢组学分析等技术，继续解析2-（2-苯乙基）色酮的生源途径，挖掘、克隆并鉴定其生物合成关键酶。项目研究不仅为解释沉香形成的生物学机制，干预与调控结香过程提供理论依据，对解决沉香资源问题具有重要意义；而且将丰富天然产物的生物合成研究方面的内容，为深入开展2-（2-苯乙基）色酮的合成生物学研究奠定基础。

沉香为常用名贵中药，是白木香在受外界刺激后，缓慢形成的含树脂木材。自然条件下，沉香的形成非常困难，造成其资源匮乏，价格昂贵，严重影响含沉香类中药的临床应用及相关产业的可持续发展。苯乙基色酮为沉香的特征性成分，也是其功效和香味的主要物质，其生物合成机制目前尚不清楚。本项目研究中，首先利用新鲜的白木香组织，建立了稳定的愈伤组织及悬浮细胞培养体系；发现150mM氯化钠诱导的愈伤组织及悬浮细胞合成苯乙基色酮的能力最强，从氯化钠处理的愈伤组织中鉴定出苯乙基色酮类成分40多个，从氯化钠诱导的悬浮细胞中分离鉴定代谢产物18个，其中新的苯乙基色酮类成分1个。在此基础上，通过对转录组测序数据的挖掘，成功发现能够催化生成1,5-二苯基-1,3-戊二酮的聚酮合酶PECPS，利用悬浮细胞对F原子标记的1,5-二苯基-1,3-戊二酮进行转化，鉴定出10个F原子取代的苯乙基色酮，证明1,5-二苯基-1,3-戊二酮为苯乙基色酮生物合成的关键前体，破解了苯乙基色酮的生物合成途径，同时也阐明了三种常见苯乙基色酮母核的生源关系。将PECPS在本氏烟中瞬时表达、在白木香愈伤组织中敲低表达，证明了PECPS的体内生物学功能；通过解析PECPS及其4个关键突变体蛋白的晶体结构，阐明了PECPS独特的催化机制。进一步对转录组数据进行挖掘，筛选出34条可能参与苯乙基色酮生物合成的候选基因，对其进行克隆表达及酶活性分析，成功发现由1,5-二苯基-1,3-戊二酮前体合成苯乙基色酮母核的关键催化酶，同时还克隆鉴定出多个与苯乙基色酮生物合成相关的催化酶。研究结果为利用合成生物学手段制备结构多样的苯乙基色酮类成分和精准调控白木香的结香过程奠定基础。