Evolution-Guided Biosynthesis of Terpenoid Inhibitors

Terpenoids, the largest and most structurally diverse group of natural products, include a striking variety of biologically active compounds, from flavors to medicines. Despite their well-documented biochemical versatility, the evolutionary processes that generate new functional terpenoids are poorly understood and difficult to recapitulate in engineered systems. This study uses a synthetic biochemical objectivea transcriptional system that links the inhibition of protein tyrosine phosphatase 1B (PTP1B), a human drug target, to the expression of a gene for antibiotic resistance in Escherichia coli (E. coli)to evolve a terpene synthase to produce enzyme inhibitors. Site saturation mutagenesis of poorly conserved residues on γ-humulene synthase (GHS), a promicuous enzyme, yielded mutants that improved fitness (i.e., the antibiotic resistance of E. coli) by reducing GHS toxicity and/or by increasing inhibitor production. Intriguingly, a combination of two mutations enhanced the titer of a minority producta terpene alcohol that inhibits PTP1Bby over 50-fold, and a comparison of similar mutants enabled the identification of a site where mutations permit efficient hydroxylation. Findings suggest that the plasticity of terpene synthases enables an efficient sampling of structurally distinct starting points for building new functional molecules and provide an experimental framework for exploiting this plasticity in activity-guided screens.萜类化合物（Terpenoids）是自然界中规模最大、结构多样性最丰富的天然产物类群，涵盖了种类繁多的生物活性化合物，从风味物质到药用成分均有涉及。尽管其生化多功能性已被充分研究，但生成新型功能萜类化合物的演化过程仍鲜为人知，且难以在工程化系统中复现。本研究采用一种合成生化筛选目标：将人类药物靶点蛋白酪氨酸磷酸酶1B（PTP1B）的抑制活性，与大肠杆菌（E. coli）内抗生素抗性基因的表达相偶联的转录系统，以此定向演化出可生成酶抑制剂的萜合酶（terpene synthase）。针对混杂型酶γ-蛇床烯合酶（GHS）上保守性较低的残基开展定点饱和诱变，最终获得了可提升宿主适应性的突变体——此类突变体可通过降低GHS的毒性，或同时提升抑制剂的产量，从而增强大肠杆菌的抗生素抗性。值得注意的是，两种突变的组合可将一种次要产物——一种可抑制PTP1B的萜类醇——的产量提升50倍以上；而通过对同类突变体的比较分析，还成功鉴定出一个可使突变体实现高效羟基化的位点。本研究结果表明，萜合酶的结构可塑性使其能够高效筛选结构各异的起始底物，以构建新型功能分子；同时也为在活性导向筛选中利用这一可塑性提供了实验框架。