Liu Yong, Ren Chong-Yang, Wei Wen-Ping, You Di, Yin Bin-Cheng, Ye Bang-Ce
Laboratory of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering , East China University of Science and Technology , Shanghai 200237 , China.
Institute of Engineering Biology and Health, Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences , Zhejiang University of Technology , Hangzhou 310014 , Zhejiang , China.
ACS Synth Biol. 2019 May 17;8(5):1134-1143. doi: 10.1021/acssynbio.9b00024. Epub 2019 Apr 15.
The regulation of biosynthetic pathways is a universal strategy for industrial strains that overproduce metabolites. Erythromycin produced by Saccharopolyspora erythraea has extensive clinical applications. In this study, promoters of the erythromycin biosynthesis gene cluster were tested by reporter mCherry. The SACE_0720 ( eryBIV)-SACE_0721 ( eryAI) spacer was selected as a target regulatory region, and bidirectional promoters with dual single guide RNAs (sgRNAs) were knocked-in using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 method. qPCR results indicated that knock-in of Pj23119-PkasO, which replaced the native promoter, enabled biosynthetic gene cluster activation, with eryBIV and eryAI expression increased 32 and 79 times, respectively. High performance liquid chromatography results showed that, compared with the wild-type strain, the yield of erythromycin was increased (58.3%) in bidirectional promoter knock-in recombinant strains. On the basis of the activated strain Ab::Pj23119-PkasO, further investigation showed that CRISPR-based interference of sdhA gene affected erythromycin biosynthesis and cell growth. Finally, regulating the culture temperature to optimize the inhibition intensity of sdhA further increased the yield by 15.1%. In summary, this study showed that bidirectional promoter knock-in and CRISPR interference could regulate gene expression in S. erythraea. This strategy has potential application for biosynthetic gene cluster activation and gene regulation in Actinobacteria.
生物合成途径的调控是高产代谢物工业菌株的通用策略。糖多孢红霉菌产生的红霉素具有广泛的临床应用。在本研究中，通过报告基因mCherry对红霉素生物合成基因簇的启动子进行了测试。选择SACE_0720（eryBIV）-SACE_0721（eryAI）间隔区作为目标调控区域，并使用成簇规律间隔短回文重复序列（CRISPR）-Cas9方法敲入带有双单向导RNA（sgRNA）的双向启动子。定量PCR结果表明，敲入取代天然启动子的Pj23119-PkasO可激活生物合成基因簇，eryBIV和eryAI的表达分别增加了32倍和79倍。高效液相色谱结果表明，与野生型菌株相比，双向启动子敲入重组菌株中红霉素的产量提高了（58.3%）。在激活菌株Ab::Pj23119-PkasO的基础上，进一步研究表明基于CRISPR的sdhA基因干扰影响红霉素的生物合成和细胞生长。最后，调节培养温度以优化sdhA的抑制强度，使产量进一步提高了15.1%。总之，本研究表明双向启动子敲入和CRISPR干扰可调控糖多孢红霉菌中的基因表达。该策略在放线菌生物合成基因簇激活和基因调控方面具有潜在应用价值。
