Nonribosomal peptide synthetases represent potential platforms for the design and engineering of structurally complex peptides. While previous focus has been centred mainly on bacterial systems, fungal synthetases assembling drugs like the antifungal echinocandins, the antibacterial cephalosporins or the anthelmintic cyclodepsipeptide (CDP) PF1022 await in-depth exploitation. As various mechanistic features of fungal CDP biosynthesis are only partly understood, effective engineering of NRPSs has been severely hampered. By combining protein truncation, in trans expression and combinatorial swapping, we assigned important functional segments of fungal CDP synthetases and assessed their in vivo biosynthetic capabilities. Hence, artificial assembly line components comprising of up to three different synthetases were generated. Using Aspergillus niger as a heterologous expression host, we obtained new-to-nature octa-enniatin (4 mg L–1) and octa-beauvericin (10.8 mg L–1), as well as high titers of the hybrid CDP hexa-bassianolide (1.3 g L–1) with an engineered ring size. The hybrid compounds showed up to 12-fold enhanced antiparasitic activity against Leishmania donovani and Trypanosoma cruzi compared to the reference drugs miltefosine and benznidazole, respectively. Our findings thus contribute to a rational engineering of iterative nonribosomal assembly lines.
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机译：非核糖体肽合成酶代表了结构复杂肽的设计和工程化的潜在平台。虽然以前的关注点主要集中在细菌系统上，但真菌合成酶等组装药物如抗真菌棘球and素，抗菌头孢菌素或驱虫性环二肽（CDP）PF1022仍在等待深入开发。由于仅部分地了解了真菌CDP生物合成的各种机制特征，因此严重阻碍了NRPS的有效工程化。通过结合蛋白质截断，反式表达和组合交换，我们分配了真菌CDP合成酶的重要功能部分，并评估了它们在体内的生物合成能力。因此，产生了由多达三个不同合成酶组成的人工组装线组件。使用黑曲霉作为异源表达宿主，我们获得了新到性质的八-enniatin（4 mg L –1 ）和八-beauvericin（10.8 mg L –1 ），以及具有设计环大小的高滴度的杂化CDP六联亚苄基内酯（1.3 g L –1 ）。与参考药物miltefosine和benznidazole相比，杂合化合物显示出对杜氏利什曼原虫和克氏锥虫的抗寄生虫活性提高了12倍。因此，我们的发现有助于合理设计迭代式非核糖体装配线。
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