三环倍半萜avermitilol合酶的发现、鉴定及其在无质粒谷氨酸棒杆菌中的异源生产

摘要(中文译文)

三环倍半萜是一类具有生物活性的天然产物，在药物、香料和可持续航空生物燃料等领域具有广泛应用。然而，已表征的三环倍半萜合酶（tSTS）数量仍然有限。为系统性地拓展这一酶类，我们开发了一套多步骤生物信息学工作流程，用于鉴定编码环状倍半萜合酶的基因，该流程整合了基于BLAST的同源筛选、保守基序验证、基于AlphaFold2的结构建模以及分子对接。该工作流程将初始的1063个tSTS候选酶逐步精炼至6个满足C1-C10距离标准的推定酶。将全部6个候选酶在过表达甲基赤藓醇4-磷酸途径基因模块的谷氨酸棒杆菌JP-2中进行异源表达，证实了其生物合成活性，产生了5种已知的环状倍半萜以及由来自Actinokineospora terrae的tSTS（AtTPS）产生的三环avermitilol，后者通过NMR波谱和高分辨电喷雾电离质谱进行了鉴定。为提高工业可行性，将化学诱导型启动子替换为组成型无前导序列合成σ启动子，并通过GGGGS连接肽将avermitilol合酶与法尼基焦磷酸合酶融合，增强了酶间中间体的局部浓度。此外，通过CRISPR相关转座子系统构建了无质粒、无抗生素选择压力的菌株，实现了avermitilol合酶表达盒的双重染色体整合，avermitilol产量达到81.52 mg/L。染色体整合菌株在连续传代中保持稳定生产，而基于质粒的菌株则表现出超过90%的生产力损失。在2 L生物反应器中对AVM-int02cp进行补料分批发酵，最终avermitilol滴度达到100.99 mg/L。本研究通过在一个微生物宿主中鉴定一个未表征的合酶基因，展示了针对三环倍半萜的序列和三维结构辅助的从基因发现到生产的工作流程。

Abstract

Tricyclic sesquiterpenes are bioactive natural products with broad applications in pharmaceuticals, fragrances, and sustainable aviation biofuels. However, the repertoire of characterized tricyclic sesquiterpene synthases (tSTSs) remains limited. To systematically expand this enzyme class, we developed a multi-step bioinformatics workflow to identify genes encoding for cyclic sesquiterpene synthase integrating BLAST-based homology filtering, conserved motif validation, AlphaFold2-based structural modeling, and molecular docking. This workflow progressively refined an initial set of 1063 tSTS candidates to six putative enzymes that satisfied a C1-C10 distance criterion. Heterologous expression of all six candidates in Corynebacterium glutamicum JP-2 overexpressing a methylerythritol 4-phosphate pathway gene module confirmed biosynthetic activity, yielding five known cyclic sesquiterpenes and tricyclic avermitilol produced by a tSTS (AtTPS) identified from Actinokineospora terrae by NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. To improve industrial feasibility, the chemical-inducible promoter was replaced with constitutive leaderless synthetic σ promoters, and fusion of avermitilol synthase with farnesyl pyrophosphate synthase via a GGGGS linker enhanced the local concentration of intermediates between enzymes. In addition, a plasmid-free, antibiotic selection-free strain was constructed via the CRISPR-associated transposons, enabling dual chromosomal integrations of the avermitilol synthase expression cassette and achieving 81.52 mg/L avermitilol production. The chromosomally integrated strain maintained stable production over serial passages, whereas the plasmid-based strain exhibited greater than 90% loss of productivity. Fed-batch fermentation of AVM-int02cp in a 2-L bioreactor achieved a final avermitilol titer of 100.99 mg/L. This work demonstrates a sequence- and 3D structure-assisted gene discovery-to-production workflow for a tricyclic sesquiterpene through the identification of an uncharacterized synthase gene in a microbial host.

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