Site-directed mutagenesis of the quorum-sensing transcriptional regulator SinR affects the biosynthesis of menaquinone in Bacillus subtilis.
Site-directed mutagenesis of the quorum-sensing transcriptional regulator SinR affects the biosynthesis of menaquinone in Bacillus subtilis.
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群体感应转录调节因子 SinR 的定点诱变影响枯草芽孢杆菌中甲基萘醌的生物合成
DOI：
10.1186/s12934-021-01603-5
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发表时间：
2021-06-07
影响因子：
6.4
通讯作者：
Xue ZL
中科院分区：
文献类型：
--
作者：
Wu J;Li W;Zhao SG;Qian SH;Wang Z;Zhou MJ;Hu WS;Wang J;Hu LX;Liu Y;Xue ZL
甲基萘醌（Menaquinone，MK-7）是由枯草芽孢杆菌（Bacillussubtilis）产生的一种高价值的维生素K2。先前已经研究了用于促进MK-7产生的常见静态代谢工程方法。然而，这些方法导致有毒物质的积累并降低了产物产率。因此，动态调节的群体感应（QS）系统是一个很有前途的方法，实现产品合成和细胞growth.ResultsIn这项研究中，QS转录调节SinR，这起着重要的作用，生物膜形成和MK生产的同时，选择，其定点突变体构建。在这些突变体中，sinR敲除菌株（KO-SinR）的生物膜生物量增加了2.8倍相比，野生型。SinRquad使MK-7的产量最大（102.56 ± 2.84 mg/L）。为了解释该突变体如何调节MK-7合成的机制并找到增强MK-7合成的其他潜在调节剂，使用RNA-seq分析QS系统、生物膜形成和MK-7合成途径中的表达变化。结果表明，tapA、tasA和depsE的表达分别上调了9.79倍、0.95倍和4.42倍。因此，SinRquad形成的生物膜比BS 168更光滑、更光滑。在这种生物膜形态中，glpF、glpk和glpD的上调表达促进了甘油通过生物膜的流动。此外，NADH脱氢酶特别是dhA、sdhB、sdhCandglpD分别增加了1.01、3.93、1.87和1.11倍。NADH脱氢酶表达水平的增加表明电子传递系统产生了更多的电子。电超极化刺激合成的电子传递链组件，如细胞色素c和MK，以确保电子转移的效率。皱纹和光滑的生物膜形成了一个网络的相互连接的通道与低阻力的液体流动，这是有益的甘油的摄取，并促进代谢通量的四个模块的MK-7 synthesis pathway. Conclusions在这项研究中，我们首次报告，SinRquad具有显着的影响MK-7的合成，通过形成的皱纹和光滑的生物膜，上调大多数NADH脱氢酶的表达水平，并提供更高的膜电位以刺激电子传递系统中组分的积累。
BackgroundMenaquinone (MK-7) is a highly valuable vitamin K2produced byBacillus subtilis. Common static metabolic engineering approaches for promoting the production of MK-7 have been studied previously. However, these approaches caused an accumulation of toxic substances and reduced product yield. Hence, dynamic regulation by the quorum sensing (QS) system is a promising method for achieving a balance between product synthesis and cell growth.ResultsIn this study, the QS transcriptional regulator SinR, which plays a significant role in biofilm formation and MK production simultaneously, was selected, and its site-directed mutants were constructed. Among these mutants,sinRknock out strain (KO-SinR) increased the biofilm biomass by 2.8-fold compared to the wild-type. SinRquadmaximized the yield of MK-7 (102.56 ± 2.84 mg/L). To decipher the mechanism of how this mutant regulates MK-7 synthesis and to find additional potential regulators that enhance MK-7 synthesis, RNA-seq was used to analyze expression changes in the QS system, biofilm formation, and MK-7 synthesis pathway. The results showed that the expressions oftapA,tasAandepsEwere up-regulated 9.79-, 0.95-, and 4.42-fold, respectively. Therefore, SinRquadformed more wrinkly and smoother biofilms than BS168. The upregulated expressions ofglpF,glpk, andglpDin this biofilm morphology facilitated the flow of glycerol through the biofilm. In addition, NADH dehydrogenases especiallysdhA,sdhB,sdhCandglpD, increased 1.01-, 3.93-, 1.87-, and 1.11-fold, respectively. The increased expression levels of NADH dehydrogenases indicated that more electrons were produced for the electron transport system. Electrical hyperpolarization stimulated the synthesis of the electron transport chain components, such as cytochrome c and MK, to ensure the efficiency of electron transfer. Wrinkly and smooth biofilms formed a network of interconnected channels with a low resistance to liquid flow, which was beneficial for the uptake of glycerol, and facilitated the metabolic flux of four modules of the MK-7 synthesis pathway.ConclusionsIn this study, we report for the first time that SinRquadhas significant effects on MK-7 synthesis by forming wrinkly and smooth biofilms, upregulating the expression level of most NADH dehydrogenases, and providing higher membrane potential to stimulate the accumulation of the components in the electron transport system.
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影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
DOI：
10.4161/19381972.2014.968490
发表时间：
2014-01
期刊：
Dermato-endocrinology
影响因子：
--
作者：
Gröber U;Reichrath J;Holick MF;Kisters K
通讯作者：
Kisters K
通过营养因子和表面活性剂提高纳豆芽孢杆菌 R127 的 Menaquinone-7 产量
影响因子：
3
作者：
Hu, Xue-chao;Liu, Wei-ming;Huang, He
通讯作者：
Huang, He
影响因子：
5.4
作者：
Berenjian, Aydin;Mahanama, Raja;Dehghani, Fariba
通讯作者：
Dehghani, Fariba
影响因子：
46.9
作者：
Dahl, Robert H.;Zhang, Fuzhong;Keasling, Jay D.
通讯作者：
Keasling, Jay D.