2017年4月23日，代谢工程领域顶级期刊 Metabolic Engineering《代谢工程》（影响因子：8.201）在线发表武汉大学药学院邓子新院士团队刘天罡教授课题组的最新研究成果《高效的萜类化合物底物供给平台助力释放萜类环化酶生物合成潜力》（Releasing the potential power of terpene synthases by a robust precursor supply platform）。
该项研究成果是刘天罡教授提出的“定向合成代谢”（Targeted Anabolism）理论技术体系的又一突破性研究成果。该工作将合成生物学和代谢工程的应用范围拓展到天然产物挖掘领域。这一新策略的提出使得萜类骨架化合物及其生物合成元件的挖掘速度取得了突破性的进展，可以在很大程度上避免对已知化合物的重复挖掘。同时也使得人们对萜类合成酶生物合成能力的认识提升到一个新的高度，具有广泛的应用性。
什么是萜类化合物
萜类化合物是指具有(C5H8)n通式以及其含氧和不同饱和程度的衍生物，可以看成是由异戊二烯或异戊烷以各种方式连结而成的一类天然化合物。萜类化合物在自然界中广泛存在，高等植物、真菌、微生物、昆虫、以及海洋生物，都有萜类成分的存在。萜类化合物是最重要的一类天然产物，是研究天然产物和开发新药的重要来源，青蒿素和紫杉醇都属于这一家族。
萜类化合物生物合成
目前研究人员对萜类化合物生物合成相关元件的挖掘还远远不足。其中，萜类环化酶（terpene cyclase）能够催化不同链长的异戊二烯单元合成单环或者多环的萜类化合物核心骨架，是萜类化合物结构多样性的基础。因而，挖掘新的萜类环化酶是拓展萜类化合物结构多样性的一个决定性步骤。
研究方法与结果
系统发育分析获得新酶
刘天罡教授课题组对丝状真菌来源的I型萜类环化酶进行系统发育分析，发现第三个（clade III）进化分支上的萜类环化酶具有底物及反应杂泛性（promiscuous）的潜质。研究人员从丝状真菌Fusarium graminearum J1-012基因组中筛选出两个沉默表达的位于 clade III进化分支的萜类环化酶FgMS和FgGS。
这两个酶具有空前的底物和反应杂泛性，能够以香叶基焦磷酸（GPP）、法尼基焦磷酸（FPP）、香叶基香叶基焦磷酸（GGPP）以及香叶基法尼基焦磷酸（GFPP）为底物合成多种类型的萜类化合物。且其中的FgMS是一个嵌合酶（chimeric enzyme），同时具有萜类环化酶和异戊烯基转移酶（prenyl transferase）结构域，能够以异戊烯焦磷酸（IPP）和烯丙基焦磷酸（DMAPP）为底物合成不同链长的萜类化合物。
利用新酶构建生物合成平台
随后，研究人员在前期对萜类化合物“定向合成代谢”深刻认识的基础上，构建了一个含有3个不同模块（底物供给模块，不同异戊二烯前体合成模块，萜类合酶模块）的萜类化合物组合生物合成平台，研究两个酶合成萜类化合物的潜力。最终结果显示，他们能够合成多达50种单萜、倍半萜、二萜以及二倍半萜化合物。对其中的12个产物进行鉴定并从中筛选出7个新的二萜和二倍半萜化合物，其中包含有3个新骨架化合物（图1）。显示了两个酶强大的萜类化合物合成能力以及该组合生物合成平台的高效性。
图1. 组合生物合成策略指导的具有底物及反应杂泛性萜类环化酶及新（骨架）产物的高效挖掘
酶工程进一步提升新酶合成能力
为了加速萜类化合物挖掘进程，研究人员对其中的FgMS进行同源建模，并对其活性口袋附近潜在的非保守氨基酸位点进行分析并开展理性的定点突变。体外研究结果显示，F65L和F159G位点的突变能够赋予FgMS新的产物合成能力，合成6个潜在新的二倍半萜化合物，这将在很大程度上丰富有限的二倍半萜化合物骨架的数量。
随后研究人员对其中1个主要产物进行发酵、纯化并鉴定其为新的二倍半萜类化合物。该组合生物合成结合酶的理性改造策略使得研究人员能够充分挖掘FgMS和FgGS的生物合成潜力。相较于传统的天然产物挖掘手段，发现的新（骨架）化合物的数量和效率得到了极大的提升。
此外，研究人员对通用蛋白数据库（UniProt）中真菌来源的I型萜类环化酶进行系统发育分析，从中筛选得到321个位于clade III的萜类环化酶，为后续具有潜在底物及反应杂泛性萜类环化酶及新产物的挖掘指明了方向。这将极大地加速新的萜类化合物生物合成元件及产物的挖掘进程并加深我们对该类萜类环化酶的认识。
该策略还可以快速推广到其它天然产物的挖掘进程中去，将有望颠覆传统的天然产物挖掘方式。论文的研究工作主要由博士生卞光凯和武汉臻智生物科技有限公司工程师韩以超（共一作）等完成。
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