Interdisciplinary Program for Biochemical Engineering, Seoul National University, Seoul, South Korea.
School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, and Bioengineering Institute, Seoul National University, Seoul, South Korea.
Appl Microbiol Biotechnol. 2018 Apr;102(7):3243-3253. doi: 10.1007/s00253-018-8838-3. Epub 2018 Feb 17.
Most secondary metabolism in Actinobacteria is controlled by multi-layered, gene-regulatory networks. These regulatory mechanisms are not easily identified due to their complexity. As a result, when a strong transcriptional regulator (TR) governs activation of biosynthetic pathways of target antibiotics such as actinorhodin (ACT), additional enhancement of the biosynthesis is difficult in combination with other TRs. To find out any "synergistic transcriptional regulators (sTRs)" that show an additive effect on the major, often strong, transcriptional regulator (mTR), here, we performed a clustering analysis using the transcriptome datasets of an mTR deletion mutant and wild-type strain. In the case of ACT biosynthesis in Streptomyces coelicolor, PhoU (SCO4228) and RsfA (SCO4677) were selected through the clustering analysis, using AfsS (SCO4425) as a model mTR, and experimentally validated their roles as sTRs. Furthermore, through analysis of synergistic effects, we were able to suggest a novel regulation mechanism and formulate a strategy to maximize the synergistic effect. In the case of the double TR mutant strain (ΔrsfA pIBR25::afsS), it was confirmed that the increase of cell mass was the major cause of the synergistic effect. Therefore, the strategy to increase the cell mass of double mutant was further attempted by optimizing the expression of efflux pump, which resulted in 2-fold increase in the cell mass and 24-fold increase in the production of ACT. This result is the highest ACT yield from S. coelicolor ever reported.
放线菌中的大多数次级代谢产物受多层次的基因调控网络控制。由于其复杂性，这些调控机制不易识别。因此，当一个强转录调控因子（TR）控制着放线红菌素（ACT）等目标抗生素生物合成途径的激活时，与其他 TR 结合，进一步增强生物合成就变得困难。为了找出任何对主要转录调控因子（mTR）表现出附加效应的“协同转录调控因子（sTR）”，我们在这里使用 mTR 缺失突变体和野生型菌株的转录组数据集进行了聚类分析。在天蓝色链霉菌的 ACT 生物合成中，通过聚类分析选择了 PhoU（SCO4228）和 RsfA（SCO4677），并使用 AfsS（SCO4425）作为模型 mTR，实验验证了它们作为 sTR 的作用。此外，通过协同作用分析，我们能够提出一种新的调控机制，并制定了最大限度发挥协同作用的策略。在双 TR 突变株（ΔrsfA pIBR25::afsS）的情况下，证实细胞质量的增加是协同作用的主要原因。因此，通过优化外排泵的表达进一步尝试增加双突变体的细胞质量，导致细胞质量增加了 2 倍，ACT 的产量增加了 24 倍。这一结果是天蓝色链霉菌中报告的最高 ACT 产量。
