Wu Hang, Wang Yansheng, Yuan Li, Mao Yongrong, Wang Weiwei, Zhu Lin, Wu Panpan, Fu Chengzhang, Müller Rolf, Weaver David T, Zhang Lixin, Zhang Buchang
Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei 230601, China.
CAS Key Laboratory of Pathogenic Microbiology & Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Synth Syst Biotechnol. 2016 Feb 16;1(1):39-46. doi: 10.1016/j.synbio.2016.01.004. eCollection 2016 Mar.
Erythromycin A is a widely used antibiotic produced by ; however, its biosynthetic cluster lacks a regulatory gene, limiting the yield enhancement via regulation engineering of . Herein, six TetR family transcriptional regulators (TFRs) belonging to three genomic context types were individually inactivated in A226, and one of them, SACE_3446, was proved to play a negative role in regulating erythromycin biosynthesis. EMSA and qRT-PCR analysis revealed that SACE_3446 covering intact N-terminal DNA binding domain specifically bound to the promoter regions of erythromycin biosynthetic gene , the resistant gene and the adjacent gene (encoding a long-chain fatty-acid CoA ligase), and repressed their transcription. Furthermore, we explored the interaction relationships of SACE_3446 and previously identified TFRs (SACE_3986 and SACE_7301) associated with erythromycin production. Given demonstrated relatively independent regulation mode of SACE_3446 and SACE_3986 in erythromycin biosynthesis, we individually and concomitantly inactivated them in an industrial WB. Compared with WB, the WBΔ and WBΔΔ mutants respectively displayed 36% and 65% yield enhancement of erythromycin A, following significantly elevated transcription of and . When cultured in a 5 L fermentor, erythromycin A of WBΔ and WBΔΔ successively reached 4095 mg/L and 4670 mg/L with 23% and 41% production improvement relative to WB. The strategy reported here will be useful to improve antibiotics production in other industrial actinomycete.
红霉素A是一种由 产生的广泛使用的抗生素；然而，其生物合成基因簇缺乏调控基因，限制了通过 调控工程提高产量。在此，属于三种基因组背景类型的六个四环素调控家族转录调节因子（TFRs）在 A226中被分别失活，其中一个，即SACE_3446，被证明在调节红霉素生物合成中起负作用。电泳迁移率变动分析（EMSA）和定量逆转录聚合酶链反应（qRT-PCR）分析表明，覆盖完整N端DNA结合结构域的SACE_3446特异性结合到红霉素生物合成基因 、抗性基因 和相邻基因 （编码一种长链脂肪酸辅酶A连接酶）的启动子区域，并抑制它们的转录。此外，我们探索了SACE_3446与先前鉴定的与红霉素产生相关的TFRs（SACE_3986和SACE_7301）的相互作用关系。鉴于已证明SACE_3446和SACE_3986在红霉素生物合成中具有相对独立的调节模式，我们在工业 WB中分别和同时使它们失活。与WB相比，WBΔ和WBΔΔ突变体的红霉素A产量分别提高了36%和65%， 和 的转录显著升高。当在5升发酵罐中培养时，WBΔ和WBΔΔ的红霉素A产量分别达到4095毫克/升和4670毫克/升，相对于WB产量提高了23%和41%。本文报道的策略将有助于提高其他工业放线菌抗生素的产量。
