卡伍尔氏链霉菌合成农用抗生素bafilomycins的调控机制及其高产研究

Bafilomycins are promising lead compounds for the development of biopesticides and pharmaceuticals. In our previous study, bafilomycin B1 was found to be a potent agricultural antibiotic in druggability evaluation, showing promising development prospects as a new pesticide. However, the low fermentation efficiency has been a bottleneck in its industrial production. In order to rationally construct its high-yield genetically engineered strain, it is necessary to decipher the regulatory mechanism of bafilomycin biosynthesis in Streptomyces cavourensis. In this proposal, to clarify the regulatory mechanism of bafilomycin biosynthesis (especially pathway-specific regulation), based on the genomic DNA sequence and the bioinformatic analysis of the bafilomycin producing strain Streptomyces cavourensis NA4, we are planning to elucidate the roles of a series of regulatory genes employing gene inactivations and complementations, and combined qRT-PCR transcription analysis and HPLC-DAD-MS metabolic profiling. The molecular mechanism and regulatory network of the key or new regulatory genes will be elucidated using heterologous expression as well as EMSA and DNase I footprinting biochemical assays in vitro. Finally, on the basis of the above research, genetically engineered strains with highly enhanced production of bafilomycins will be constructed by a combined strategy including enhancing precursor supply, inactivation of negative regulatory genes, overexpression of positive regulatory genes and elimination of by-product reaction pathways. The implementation of this project will reveal the complex and unique pathway-specific regulatory network and the underlying mechanism of bafilomycin biosynthesis, and would help us to construct engineering strains to improve the bafilomycins production. At the same time, the high-yield engineering strains will lay the foundation for industrial application of bafilomycin B1 as a new bio-fungicidal agent.

Bafilomycins（BAF）类化合物是研发农药和医药的良好母体。前期农药成药性评价表明bafilomycin B1是有良好开发前景的候选农用抗生素，然而较低的发酵单位是限制其产业化生产的瓶颈，为了理性构建其高产菌株，必须明确其合成的调控机制。为此，在对链霉菌NA4合成BAF基因簇生物信息学分析的基础上，本项目拟采用体内基因敲除和回补，qRT-PCR检测各共转录单元的表达差异，LC-DAD-MS分析产物变化，推导各调控基因的调节作用；进一步对关键和新功能的调控基因进行异源表达，在体外使用EMSA、DNA足迹法等解析调控基因的分子机制。最终在此基础上，组合使用强化前体供应、改造调控基因和限速酶以及消除副产物反应途径等策略构建优质高产工程菌株。本项目的实施将揭示BAF合成复杂和独特的途径特异性调控机制，为理性设计、遗传改造宿主菌提供重要理论指导；同时高产菌株的构建将为其产业化奠定基础。

巴弗洛霉素（bafilomycin，BAF）是一类由I型聚酮合成酶装配合成的具有十六元大环内酯骨架的化合物，是研发农药和医药的良好母体。前期研究发现了深海来源的卡伍尔氏链霉菌NA4能产生BAF，然而较低的发酵产量限制了BAF产业化应用。为了在全面理解其生物合成调控机制的基础上理性构建BAF高产菌株，本项目通过基因灭活、代谢分析与生物信息学分析，发现了链霉菌NA4生物合成BAF关键的3个途径特异性正调控基因和3个具有负调控作用的基因或共转录本；系统阐明了bafG、bafI和bafW正调控基因联合调控BAF前体合成、骨架装配、后修饰单元组装的调控网络和协同调控机制；并发现了前体供体内不足是链霉菌NA4高产BAF的关键限制因子。在此基础上，通过消除多个前体共同来源乙酰CoA的竞争性代谢途径，强化了主要前体的供应，构建了2个高产菌株，使BAF产量分别达到原始菌株的9倍和6倍以上；进一步，复合过表达途径特异性正调控基因和强化前体供应策略，构建了3个高产工程菌株，BAF产量继续提高，最高达原始菌株的11倍以上；最后通过在培养基中添加甲基丙二酰CoA生成的原料丙酸钠或者缬氨酸，可使BAF产量再提高1-2倍。通过本研究揭示了链霉菌NA4合成BAF独特和复杂的调控机制，理性构建了多个高产菌株，为BAF产业化开发奠定了基础。