Deciphering The Pathway-Specific Regulatory Network For Production Of Ten-Membered Enediyne Tiancimycins

Enediyne antibiotics are a class of highly toxic microbial natural products,which can be developed into warhead molecules for antibodydrug conjugates(ADCs),and have great drug prospects.Enediyne antibiotics can be divided into two types: 9-membered or 10-membered families.Anthraquinone-fused enediyne(AFE)are a subfamily of 10-membered ring enediynes.AFEs have unique structural features and potential as ADCs payloads.But the low fermentation yield of AFEs severely restricts their subsequent research and applications.Recent studies have shown that AFEs are likely to share a unified basic biosynthetic pathway.There are core genes involved in forming the backbone of AFEs,and multiple putative pathway-specific regulatory.Those genes are highly conserved in the biosynthetic gene clusters(BGCs)of AFEs.But pathway-specific regulatory genes how to control the synthesis of AFEs that is not well understood.Since the extremely strong cytotoxicity of AFEs is likely to force their production hosts to evolve strict regulatory mechanisms to control their synthesis.Analysis of the pathwayspecific regulatory network of AFEs will help us in-depth understanding of their biosynthetic mechanisms and provide insights into the development of AFEs.In this paper,Tiancimycins(TNMs)is a representative molecule of AFEs.TNMs was used as the research object.And five putative pathway-specific regulatory genes contained in the BGC(tnm)of the previously constructed high-yielding Streptomyces Streptomyces CB03234-S were investigated.We systematically studied and analyzed the regulatory network of TNMs biosynthesis through transcription analysis and EMSAs,which provided reference and guidance for understanding and applying the pathway-specific regulatory network of AFEs.First,we performed predicted protein sequence analysis of five putative pathway-specific regulatory genes in tnm that are tnm R1,tnm R2,tnm R3,tnm R4,and tnm R7.We found that only tnm R1,tnm R4,and tnm R7 encode regulators,while tnm R2 and tnm R3 encode kinases.At the same time,we constructed overexpression and knockout mutants of the above five genes respectively,and carried out fermentation verification.The results showed that tnm R2 and tnm R4 had nothing to do with the synthesis of TNMs.And tnm R1,tnm R3 and tnm R7 had significant effects on the production of TNMs: tnm R7 was a positive regulated gene,and knockout did not produce TNMs,but overexpression increased the production of TNMs by 1.3 times;tnm R1 was a negative regulator overexpression of tnm R1 gene did not produce TNMs,but the production of TNMs was also significantly reduced after knockout.While overexpression of tnm R3 had no obvious effect,but the performance of knockout mutants was similar to that of tnm R1 knockout mutants.Further biomass experiments showed that most of the mutants grew in the same way as CB03234-S,but the mutants knocked out tnm R1 or tnm R3 had significantly reduced biomass and were directly related to the production of TNMs,so Tnm R1 and Tnm R3 were likely to be composed of A special orphan two-component regulatory system to restrict TNMs synthesis.Subsequently,we performed sequence similarity network(SNN)analysis on putative pathway-specific regulatory genes contained in different AFE-BGCs and found that three classes represented by tnm R1,tnm R3 and tnm R7 were formed and conserved in AFE-BGCs,suggesting that AFEs are likely to possess similar pathwayspecific regulatory networks.To further dissect the pathway-specific regulatory network of TNMs,we determined that tnm can be divided into 11 transcriptional units by RTPCR,and then performed q RT-PCR and EMSAs analysis.The results show that Tnm R1 can inhibit the transcription of different types of proteins related to the biosynthesis of TNMs,including the core enzymes Tnm D/G/J/K1/K2 that form essential AFE intermediates and the important accessory proteins Tnm T2/S2/ which are responsible for the resistance and transport of Tnm T2/S2/T1/S1.In addition,Tnm R1 can also interfere with the transcription of the positive regulator Tnm R7,thereby downregulating the expression of another group of core enzymes Tnm N/O/P from a higher level.Based on the above results,we infer a pathway-specific regulatory network for TNMs: Tnm R3 phosphorylates Tnm R1 by sensing high levels of ATP during cell growth,making it tightly restrict the transcriptional expression of target genes and TNMs synthesis to avoid TNMs toxicity.The lethal effect ensures the normal growth of the host;the stagnation of growth in the middle and late stages of fermentation reduces the level of ATP,which leads to the failure of Tnm R3,while the binding ability of unphosphorylated Tnm R1 to the promoter region decreases,partially releasing the transcriptional expression of related genes to initiate TNMs Synthesis.This study revealed that a pathway-specific regulatory network composed of a special orphan two-component regulatory system Tnm R3/R1 and a positive regulator Tnm R7 finely and tightly controls the biosynthesis of TNMs.This pathway-specific regulatory network can be speculated as a common regulatory mechanism for existing AFEs,which will provide new insights into our deep understanding and manipulation of AFEs biosynthesis and contribute to the future development and clinical applications of AFEs.