Transcriptome-guided target identification of the TetR-like regulator SACE_5754 and engineered overproduction of erythromycin in Saccharopolyspora erythraea
Transcriptome-guided target identification of the TetR-like regulator SACE_5754 and engineered overproduction of erythromycin in Saccharopolyspora erythraea
复制标题
转录组引导的 TetR 样调节因子 SACE_5754 的靶点识别和红霉素糖多孢菌中红霉素的过量生产
DOI：
10.1186/s13036-018-0135-2
复制
发表时间：
2019-01-24
影响因子：
5.6
通讯作者：
Zhang，Buchang
中科院分区：
文献类型：
--
作者：
Wu，Hang;Chu，Zuling;Zhang，Buchang
背景：红霉素A（Erythromycin A，Er-A）是一种重要的抗生素，广泛应用于人类医学。研究红霉素生物合成相关转录调控因子及其靶基因，对于通过工程改造S. ericraea.Results相关调控元件获得红霉素高产菌株具有重要意义。SACE_5754间接抑制了ferycluster的转录，并且不能调节自身及其邻近基因SACE_5753。进行与EMSA和qRT-PCR偶联的RNA-seq以鉴定SACE_5754的靶标，并证实SACE_0388（编码丙酮酸、水双激酶）、SACE_3599（编码抗生素抗性大环内酯糖基转移酶）和SACE_6149（编码FAD结合单加氧酶）的转录被SACE_5754直接抑制。利用DNA酶I足迹法和EMSA法证明了SACE_5754的共有回文序列TYMAGG-n2/n4/n11-KKTKRA（Y：C/T，M：A/C，K：T/G，R：A/G）是SACE_5754结合所必需的。在SACE_5754、SACE_0388和SACE_6149这三个靶基因中，对红霉素的产量有正向影响。SACE_5754中SACE_0388或SACE_6149的过表达进一步增加Er-A的产量。通过改造工业菌株。SACE_5754/pIB 139 -0388和SACE_6149中Er-A的产量分别比WB高42%和30%。SACE_5754中SACE_0388和SACE_6149的共过表达导致Er-A产量相对于WB增加64%。在5L发酵罐中，WB菌株SACE_5754/pIB 139 -0388-6149的Er-A产量为4998 mg/L，比WB菌株提高了48%。结论：我们已经鉴定了一个TFR，SACE_5754，它是红霉素生物合成的负调控因子，并且SACE_5754及其靶基因SACE_0388和SACE_6149的工程改造导致了野生型和工业菌株中红霉素产量的提高。erythraeastrains.该策略对于调控转录调控因子及其作用靶点以提高工业放线菌抗生素产量具有重要意义。
Background:Erythromycin A (Er-A) produced by the actinomyceteSaccharopolyspora erythraeais an important antibiotic extensively used in human medicine. Dissecting of transcriptional regulators and their target genes associated with erythromycin biosynthesis is crucial to obtain erythromycin overproducer strains through engineering of relevant regulatory elements inS. erythraea.Results:Here, we identified a TetR family transcriptional regulator (TFR), SACE_5754, negatively controlling erythromycin production. SACE_5754 indirectly repressed the transcription oferycluster and cannot regulate itself and its adjacent geneSACE_5753. RNA-seq coupled with EMSAs and qRT-PCR was performed to identify the targets of SACE_5754, and confirmed that transcription ofSACE_0388(encoding a pyruvate, water diknase),SACE_3599(encoding an antibiotic resistance macrolide glycosyltransferase) andSACE_6149(encoding a FAD-binding monooxygenase) were directly repressed by SACE_5754. A consensus palindromic sequence TYMAGG-n2/n4/n11-KKTKRA (Y: C/T, M: A/C, K: T/G, R: A/G) was proved to be essential for SACE_5754 binding using DNase I footprinting and EMSAs. During the three target genes of SACE_5754,SACE_0388andSACE_6149exhibited the positive effect on erythromycin production. Overexpression of eitherSACE_0388orSACE_6149in ∆SACE_5754further increased the Er-A production. By engineering the industrial strainS. erythraeaWB with deletion ofSACE_5754combined with overexpression of eitherSACE_0388orSACE_6149, Er-A production in WB∆SACE_5754/pIB139-0388 and WB∆SACE_5754/pIB139-6149 was successively increased by 42 and 30% compared to WB. Co-overexpression ofSACE_0388andSACE_6149in WB∆SACE_5754resulted in enhanced Er-A production by 64% relative to WB. In a 5-L fermenter, WB∆SACE_5754/pIB139-0388-6149 produced 4998 mg/L Er-A, a 48% increase over WB.Conclusion:We have identified a TFR, SACE_5754, as a negative regulator of erythromycin biosynthesis, and engineering ofSACE_5754and its target genes,SACE_0388andSACE_6149, resulted in enhanced erythromycin production in both wild-type and industrialS. erythraeastrains. The strategy demonstrated here may be valuable to facilitate the manipulation of transcriptional regulators and their targets for production improvement of antibiotics in industrial actinomycetes.