The aim of the present work was to investigate the...
Erstellt am: 19. Juni 2025
Frage
The aim of the present work was to investigate the overexpression of the wysR gene in Streptomyces albulus var. wuyiensis strain CK‐15 based on the ΔwysR3 mutant strain including the effect on morphological development, wuyiencin production and antibacterial activity. At the same time, we report a new rapid method for producing genetically engineered strains for industrial production of wuyiencin.
Methods and Results
We developed a method to create a wysR overexpression strain based on the ΔwysR3 mutant strain by direct transformation. In this method, the desired gene fragment to be overexpressed was amplified by polymerase chain reaction (PCR) using Phusion High Fidelity DNA polymerase and fused with the linearized pSETC integrative plasmid by Gibson assembly. The resulting recombinant plasmid was transformed into ΔwysR3 mutant strain by the intergeneric conjugation method. The plasmid was then integrated into the chromosome and the resulting apramycin‐resistant overexpression strain was confirmed by PCR using the Apra‐F and Apra‐R primers. Finally, we successfully screened the genetically engineered strain with overexpression of wysR gene in ΔwysR3 mutant.
Conclusion
We can conclude that overexpression of wysR gene in ΔwysR3 mutant strain proved to be an effective strategy for significantly increasing wuyiencin production together with faster morphological development. Quantitative real‐time RT‐PCR analysis showed that wysR regulated wuyiencin biosynthesis by modulating other putative regulatory genes and bld, whi, chp, rdl and ram family genes are crucial for the morphological development.
Significance and Impact of the Study
Overexpression of wysR gene in the ΔwysR3 mutant strain named OoWysR strain may increase the efficiency in the industrial fermentation processes for wuyiencin production. The mechanism by which wysR overexpression promotes rapid sporulation and a high yield of wuyiencin production is likely related to modulation of other putative regulatory genes.翻译成中文
Frage
The aim of the present work was to investigate the overexpression of the wysR gene in Streptomyces albulus var. wuyiensis strain CK‐15 based on the ΔwysR3 mutant strain including the effect on morphological development, wuyiencin production and antibacterial activity. At the same time, we report a new rapid method for producing genetically engineered strains for industrial production of wuyiencin.
Methods and Results
We developed a method to create a wysR overexpression strain based on the ΔwysR3 mutant strain by direct transformation. In this method, the desired gene fragment to be overexpressed was amplified by polymerase chain reaction (PCR) using Phusion High Fidelity DNA polymerase and fused with the linearized pSETC integrative plasmid by Gibson assembly. The resulting recombinant plasmid was transformed into ΔwysR3 mutant strain by the intergeneric conjugation method. The plasmid was then integrated into the chromosome and the resulting apramycin‐resistant overexpression strain was confirmed by PCR using the Apra‐F and Apra‐R primers. Finally, we successfully screened the genetically engineered strain with overexpression of wysR gene in ΔwysR3 mutant.
Conclusion
We can conclude that overexpression of wysR gene in ΔwysR3 mutant strain proved to be an effective strategy for significantly increasing wuyiencin production together with faster morphological development. Quantitative real‐time RT‐PCR analysis showed that wysR regulated wuyiencin biosynthesis by modulating other putative regulatory genes and bld, whi, chp, rdl and ram family genes are crucial for the morphological development.
Significance and Impact of the Study
Overexpression of wysR gene in the ΔwysR3 mutant strain named OoWysR strain may increase the efficiency in the industrial fermentation processes for wuyiencin production. The mechanism by which wysR overexpression promotes rapid sporulation and a high yield of wuyiencin production is likely related to modulation of other putative regulatory genes.翻译成中文
Gedanke
Antwort
研究目的
本研究旨在基于 ΔwysR3 突变株，在 Streptomyces albulus var. wuyiensis CK-15 菌株中研究 wysR 基因的过表达，以及其对菌体形态发育、乌烟菌素（wuyiencin）产量和抗菌活性的影响。同时，我们报道了一种用于工业规模生产乌烟菌素的基因工程菌株的快速构建方法。
方法与结果
我们建立了一种在 ΔwysR3 突变株背景下直接转化构建 wysR 过表达菌株的方法。具体步骤如下：
- PCR 扩增
使用 Phusion 高保真 DNA 聚合酶扩增待过表达的基因片段。 - Gibson 组装
将扩增产物与线性化的整合质粒 pSETC 通过 Gibson 组装连接。 - 异源共轭转化
将重组质粒通过属间共轭方法转入 ΔwysR3 突变株。 - 染色体整合与筛选
质粒整合进染色体后，筛选获得氨普霉素（apramycin）抗性菌株，并使用引物 Apra-F/Apra-R 进行 PCR 验证。 - 成功获得工程菌
最终筛选到 wysR 基因过表达的 ΔwysR3 突变衍生菌株。
结论
在 ΔwysR3 突变株中过表达 wysR 基因显著提高了乌烟菌素产量，并加速了菌体形态发育。实时定量 RT-PCR（qRT-PCR）结果显示，wysR 通过调控其他潜在调控基因，以及 bld、whi、chp、rdl 和 ram 系列基因来调节乌烟菌素生物合成；这些基因同样对形态发育至关重要。
研究意义与影响
在 ΔwysR3 背景下过表达 wysR 基因所得工程菌株（命名为 OoWysR）有望在工业发酵过程中显著提高乌烟菌素生产效率。wysR 过表达促进快速孢子形成和高产乌烟菌素的机制，可能与其对其他潜在调控基因的调节作用有关。