Abstract

Ansamitocin P-3 (AP-3) exhibits potent biological activities against various tumor cells. As an important drug precursor, reliable supply of AP-3 is limited by low fermentation yield. Although different strategies have been implemented to improve AP-3 yield, few have investigated the impact of efflux on AP-3 production. In this study, AP-3 efflux genes were identified through combined analysis of two sets of transcriptomes. The production-based transcriptome was implemented to search for efflux genes highly expressed in response to AP-3 accumulation during the fermentation process, while the resistance-based transcriptome was designed to screen for genes actively expressed in response to the exogenous supplementation of AP-3. After comprehensive analysis of two transcriptomes, six efflux genes outside the ansamitocin BGC were identified. Among the six genes, individual deletion of APASM_2704, APASM_6861, APASM_3193, and APASM_2805 resulted in decreased AP-3 production, and alternative overexpression led to AP-3 yield increase from 264.6 to 302.4, 320.4, 330.6, and 320.6 mg/L, respectively. Surprisingly, APASM_2704 was found to be responsible for exportation of AP-3 and another macro-lactam antibiotic pretilactam. Furthermore, growth of APASM_2704, APASM_3193, or APASM_2805 overexpression mutants was obviously improved under 300 mg/L AP-3 supplementation. In summary, our study has identified AP-3 efflux genes outside the ansamitocin BGC by comparative transcriptomic analysis, and has shown that enhancing the transcription of transporter genes can improve AP-3 production, shedding light on strategies used for exporter screening and antibiotic production improvement.

Key points

• AP-3-related efflux genes were identified by transcriptomic analysis.

• Deletion of the identified efflux genes led in AP-3 yield decrease.

• Overexpression of the efflux genes resulted in increased AP-3 production.

中文翻译：

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外生放线放线菌中安沙霉素P-3生产的外流鉴定和工程

摘要

Ansamitocin P-3（AP-3）对各种肿瘤细胞表现出强大的生物学活性。作为重要的药物前体，AP-3的可靠供应受到发酵产量低的限制。尽管已采用不同的策略来提高AP-3的产量，但很少有人研究外排对AP-3产量的影响。在这项研究中，通过对两组转录组的综合分析鉴定了AP-3外排基因。实施基于生产的转录组以搜索在发酵过程中响应AP-3积累而高度表达的外排基因，而基于抗性的转录组则被设计为筛选响应于外源补充AP-3而活跃表达的基因。 。在对两个转录组进行全面分析后，确定了安妥霉素BGC之外的六个外排基因。APASM_2704，APASM_6861，APASM_3193和APASM_2805导致AP-3产量减少，替代性过表达导致AP-3产量分别从264.6 mg / L增加到302.4、320.4、330.6和320.6 mg / L。出人意料的是，发现APASM_2704负责出口AP-3和另一种大内酰胺抗生素pretilactam。此外，APASM_2704，APASM_3193或APASM_2805的增长补充300 mg / L AP-3后，过表达突变体明显改善。综上所述，我们的研究通过比较转录组分析确定了安他霉素BGC之外的AP-3外排基因，并表明增强转运蛋白基因的转录可以改善AP-3的产生，为出口者筛选和抗生素生产改进策略提供了参考。

关键点

•通过转录组分析鉴定了与AP-3相关的外排基因。

•删除已鉴定的外排基因导致AP-3产量下降。

•外排基因的过表达导致AP-3产量增加。