Xiangya International Academy of Translational Medicine, Central South University, Changsha, 410013, China.
The Affiliated Nanhua Hospital, Department of Pharmacy, Institute of Clinical Pharmacy, Hengyang Medical School, University of South China, Hengyang, 421002, China.
Microb Cell Fact. 2024 May 4;23(1):128. doi: 10.1186/s12934-024-02399-w.
Anthraquinone-fused enediynes (AFEs) are excellent payloads for antibody-drug conjugates (ADCs). The yields of AFEs in the original bacterial hosts are extremely low. Multiple traditional methods had been adopted to enhance the production of the AFEs. Despite these efforts, the production titers of these compounds are still low, presenting a practical challenge for their development. Tiancimycins (TNMs) are a class of AFEs produced by Streptomyces sp. CB03234. One of their salient features is that they exhibit rapid and complete cell killing ability against various cancer cell lines.
In this study, a combinatorial metabolic engineering strategy guided by the CB03234-S genome and transcriptome was employed to improve the titers of TNMs. First, re-sequencing of CB03234-S (Ribosome engineered mutant strains) genome revealed the deletion of a 583-kb DNA fragment, accounting for about 7.5% of its genome. Second, by individual or combined inactivation of seven potential precursor competitive biosynthetic gene clusters (BGCs) in CB03234-S, a double-BGC inactivation mutant, S1009, was identified with an improved TNMs titer of 28.2 ± 0.8 mg/L. Third, overexpression of five essential biosynthetic genes, including two post-modification genes, and three self-resistance auxiliary genes, was also conducted, through which we discovered that mutants carrying the core genes, tnmE or tnmE10, exhibited enhanced TNMs production. The average TNMs yield reached 43.5 ± 2.4 mg/L in a 30-L fermenter, representing an approximately 360% increase over CB03234-S and the highest titer among all AFEs to date. Moreover, the resulting mutant produced TNM-W, a unique TNM derivative with a double bond instead of a common ethylene oxide moiety. Preliminary studies suggested that TNM-W was probably converted from TNM-A by both TnmE and TnmE10.
Based on the genome and transcriptome analyses, we adopted a combined metabolic engineering strategy for precursor enrichment and biosynthetic pathway reorganization to construct a high-yield strain of TNMs based on CB03234-S. Our study establishes a solid basis for the clinical development of AFE-based ADCs.
蒽醌融合烯二炔（AFE）是抗体药物偶联物（ADC）的优秀有效载荷。原始细菌宿主中 AFE 的产量极低。已经采用了多种传统方法来提高 AFE 的产量。尽管做出了这些努力，但这些化合物的生产滴度仍然很低，这对它们的开发构成了实际挑战。天蚕霉素（TNMs）是由链霉菌 sp. CB03234 产生的一类 AFE。它们的一个突出特点是对各种癌细胞系表现出快速和完全的细胞杀伤能力。
在这项研究中，我们采用了一种组合代谢工程策略，该策略以 CB03234-S 基因组和转录组为指导，以提高 TNMs 的产量。首先，对 CB03234-S（核糖体工程突变株）基因组进行重新测序，发现其基因组中有一个 583-kb 的 DNA 片段缺失，约占其基因组的 7.5%。其次，通过单独或组合失活 CB03234-S 中的七个潜在前体竞争生物合成基因簇（BGCs），我们鉴定出一个双 BGC 失活突变体 S1009，其 TNMs 产量提高到 28.2 ± 0.8 mg/L。第三，我们还通过过表达五个必需生物合成基因，包括两个后修饰基因和三个自身抗性辅助基因，发现携带核心基因 tnmE 或 tnmE10 的突变体表现出增强的 TNMs 生产能力。在 30-L 发酵罐中，TNMs 的平均产量达到 43.5 ± 2.4 mg/L，比 CB03234-S 提高了约 360%，是迄今为止所有 AFE 中最高的产量。此外，所得突变体产生了 TNM-W，这是一种独特的 TNM 衍生物，具有一个双键而不是常见的环氧乙烷部分。初步研究表明，TNM-W 可能是由 TnmE 和 TnmE10 共同作用，从 TNM-A 转化而来的。
基于基因组和转录组分析，我们采用了一种组合代谢工程策略，通过前体富集和生物合成途径重组来构建基于 CB03234-S 的 TNMs 高产菌株。我们的研究为 AFE 为基础的 ADC 的临床开发奠定了坚实的基础。
