Efflux identification and engineering for ansamitocin P-3 production in Actinosynnema pretiosum
Efflux identification and engineering for ansamitocin P-3 production in Actinosynnema pretiosum
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Actinosynnema pretiosum 中安丝菌素 P-3 生产的外排鉴定和改造
DOI：
10.1007/s00253-020-11044-6
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Linquan Bai
中科院分区：
文献类型：
--
作者：
Xinran Wang;Jiianhua Wei;Yifan Xiao;Shuhui Luan;Xinjuan Ning;Linquan Bai
Ansamitocin P-3 (AP-3) 对多种肿瘤细胞表现出有效的生物活性。 AP-3作为重要的药物前体，发酵产率低，其可靠供应受到限制。尽管已经实施了不同的策略来提高 AP-3 产量，但很少有人研究外排对 AP-3 产量的影响。在本研究中，通过两组转录组的联合分析鉴定了 AP-3 外排基因。基于生产的转录组用于寻找在发酵过程中响应AP-3积累而高表达的外排基因，而基于抗性的转录组旨在筛选响应外源补充AP-3而活跃表达的基因。经过对两个转录组的综合分析，鉴定出了安丝菌素 BGC 之外的 6 个外排基因。在这六个基因中，APASM_2704、APASM_6861、APASM_3193和APASM_2805的单独缺失导致AP-3产量减少，而选择性过表达导致AP-3产量分别从264.6增加到302.4、320.4、330.6和320.6 mg/L。令人惊讶的是，APASM_2704被发现负责AP-3和另一种大内酰胺抗生素pretilactam的输出。此外，在300 mg/L AP-3补充下，APASM_2704、APASM_3193或APASM_2805过表达突变体的生长得到明显改善。总之，我们的研究通过比较转录组分析鉴定了安丝菌素 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提高 AP-3 的产量，为用于出口商筛选和抗生素生产改进的策略提供线索。要点： • 通过转录组分析鉴定了 AP-3 相关的外排基因。 • 删除已识别的外排基因会导致AP-3 产量下降。 • 外排基因的过度表达导致AP-3 产量增加。
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.
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影响因子：
14.9
作者：
Mitchell AL;Attwood TK;Babbitt PC;Blum M;Bork P;Bridge A;Brown SD;Chang HY;El-Gebali S;Fraser MI;Gough J;Haft DR;Huang H;Letunic I;Lopez R;Luciani A;Madeira F;Marchler-Bauer A;Mi H;Natale DA;Necci M;Nuka G;Orengo C;Pandurangan AP;Paysan-Lafosse T;Pesseat S;Potter SC;Qureshi MA;Rawlings ND;Redaschi N;Richardson LJ;Rivoire C;Salazar GA;Sangrador-Vegas A;Sigrist CJA;Sillitoe I;Sutton GG;Thanki N;Thomas PD;Tosatto SCE;Yong SY;Finn RD
通讯作者：
Finn RD
影响因子：
5.4
作者：
Sandoval NR;Papoutsakis ET
通讯作者：
Papoutsakis ET
影响因子：
3.4
作者：
D. Ng;H. K. Chin;Victor Wong
通讯作者：
D. Ng;H. K. Chin;Victor Wong
DOI：
10.1271/bbb1961.49.327
发表时间：
1985-02
期刊：
Agricultural and biological chemistry
影响因子：
--
作者：
K. Hatano;E. Mizuta;S. Akiyama;E. Higashide;Y. Nakao
通讯作者：
K. Hatano;E. Mizuta;S. Akiyama;E. Higashide;Y. Nakao
DOI：
--
发表时间：
2000
期刊：
--
影响因子：
--
作者：
T. Kieser
通讯作者：
T. Kieser