Dhakal Dipesh, Eckhardt Campbell W, Jiang Yujia, Luesch Hendrik, Ding Yousong
Department of Medicinal Chemistry, Center for Natural Products, Drug Discovery and Development (CNPD3), University of Florida, Gainesville, Florida 32610, United States.
Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore 169857, Singapore.
ACS Synth Biol. 2025 Sep 2. doi: 10.1021/acssynbio.5c00390.
Heterologous expression of biosynthetic gene clusters (BGCs) is a powerful strategy for natural product (NP) discovery, yet achieving consistent expression across microbial hosts remains challenging. Here, we developed cross-phyla vector systems enabling the expression of BGCs from cyanobacteria and other bacterial origins in Gram-negative , Gram-positive , and two model cyanobacterial strains including unicellular PCC 6803 and filamentous sp. PCC 7120. Following validation using constitutive and inducible expression of the enhanced yellow fluorescent protein (eYFP), we applied these vectors to express the shinorine and violacein BGCs in all four hosts. Promoter tuning, substrate feeding, BGC refactoring, and inducible control enhanced NP production and mitigated host toxicity. Notably, we demonstrated that can serve as a chassis for cyanobacterial NP BGC expression. Our results provide versatile expression platforms for probing BGC function and accelerating natural product discovery from diverse cyanobacterial and other bacterial lineages.
生物合成基因簇（BGCs）的异源表达是天然产物（NP）发现的一种强大策略，但在不同微生物宿主中实现一致表达仍然具有挑战性。在此，我们开发了跨门载体系统，能够在革兰氏阴性菌、革兰氏阳性菌以及两种模式蓝藻菌株（包括单细胞的集胞藻PCC 6803和丝状的聚球藻属sp. PCC 7120）中表达来自蓝藻和其他细菌来源的BGCs。在使用增强型黄色荧光蛋白（eYFP）的组成型和诱导型表达进行验证后，我们应用这些载体在所有四种宿主中表达肌醇六磷酸和紫菌素BGCs。启动子调控、底物供应、BGC重构和诱导控制提高了NP产量并减轻了宿主毒性。值得注意的是，我们证明了[此处原文缺失相关内容]可作为蓝藻NP BGC表达的底盘。我们的结果为探究BGC功能和加速从不同蓝藻和其他细菌谱系中发现天然产物提供了通用的表达平台。
