Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, China.
Proc Natl Acad Sci U S A. 2010 Jun 22;107(25):11250-4. doi: 10.1073/pnas.1006085107. Epub 2010 Jun 7.
Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are widely used in the field of animal health, agriculture, and human health. Here we have adopted a practical approach to successfully improve avermectin production in an industrial overproducer. Transcriptional levels of the wild-type strain and industrial overproducer in production cultures were monitored using microarray analysis. The avermectin biosynthetic genes, especially the pathway-specific regulatory gene, aveR, were up-regulated in the high-producing strain. The upstream promoter region of aveR was predicted and proved to be directly recognized by sigma(hrdB) in vitro. A mutant library of hrdB gene was constructed by error-prone PCR and selected by high-throughput screening. As a result of evolved hrdB expressed in the modified avermectin high-producing strain, 6.38 g/L of avermectin B1a was produced with over 50% yield improvement, in which the transcription level of aveR was significantly increased. The relevant residues were identified to center in the conserved regions. Engineering of the hrdB gene can not only elicit the overexpression of aveR but also allows for simultaneous transcription of many other genes. The results indicate that manipulating the key genes revealed by reverse engineering can effectively improve the yield of the target metabolites, providing a route to optimize production in these complex regulatory systems.
阿维菌素及其类似物由放线菌链霉菌阿维链霉菌产生,广泛应用于动物健康、农业和人类健康领域。在这里,我们采用了一种实用的方法,成功地提高了工业高产菌中阿维菌素的产量。使用微阵列分析监测生产培养物中野生型菌株和工业高产菌的转录水平。阿维菌素生物合成基因,特别是途径特异性调节基因 aveR,在高产菌株中上调。预测并证明了 aveR 的上游启动子区域可被 sigma(hrdB) 在体外直接识别。通过易错 PCR 构建了 hrdB 基因的突变文库,并通过高通量筛选进行了选择。在改良的阿维菌素高产菌株中表达进化后的 hrdB 后,生产出了 6.38 g/L 的阿维菌素 B1a,产率提高了 50%以上,其中 aveR 的转录水平显著增加。鉴定出相关的残基集中在保守区域。hrdB 基因的工程化不仅可以引发 aveR 的过表达,还可以同时转录许多其他基因。结果表明,操纵反向工程揭示的关键基因可以有效地提高目标代谢物的产量,为优化这些复杂调控系统中的生产提供了一条途径。