Enhanced Prodigiosin Production in Serratia marcescens JNB5-1 by Introduction of a Polynucleotide Fragment into the pigN 3′ Untranslated Region and Disulfide Bonds into O-Methyl Transferase (PigF)
Enhanced Prodigiosin Production in Serratia marcescens JNB5-1 by Introduction of a Polynucleotide Fragment into the pigN 3′ Untranslated Region and Disulfide Bonds into O-Methyl Transferase (PigF)
复制标题
通过将多核苷酸片段引入pigN 3-非翻译区并将二硫键引入O-甲基转移酶(PigF)，增强粘质沙雷氏菌JNB5-1中灵菌红素的产量
DOI：
10.1128/aem.00543-21
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发表时间：
2021-09-01
影响因子：
4.4
通讯作者：
Rao，Zhiming
中科院分区：
文献类型：
--
作者：
Sun，Yang;Wang，Lijun;Rao，Zhiming
这项研究突出了一个有前途的策略，以提高mRNA/酶的稳定性，并增加生产使用从头PNF库和二硫键引入到蛋白质。PNF能延长靶基因mRNA的半衰期，有效地阻止其降解。粘质沙雷氏菌JNB 5 -1在30°C时高产灵菌红素，但在≥37°C时明显受到抑制。我们的初步结果表明，参与灵菌红素途径的O-甲基转移酶（PigF）和氧化还原酶（PigN）在S. marcescens JNB 5 -1在≥37°C时急剧下降。因此，在这项研究中，我们提高了mRNA的稳定性和蛋白质的生产，分别使用从头多核苷酸片段（PNF）和二硫键的引入，并观察其对灵菌红素生产的影响。我们的研究结果表明，在pigF和pigN的3′非翻译区添加PNF显著改善了这些基因的mRNA半衰期，导致转录和表达水平的增加。随后，在PigF中引入二硫键改善了PigF的热稳定性、pH稳定性和铜离子抗性。摇瓶发酵结果表明，工程菌的灵菌红素效价高于对照菌。marcescens菌株在30°C下的产量从5.36 g/L增加到8.65 g/L，比JNB 5 -1菌株增加了61.38%，并且在37°C下灵菌红素产量从0.38 g/L增加到0.78 g/L，显著增加了2.05倍。在这项研究中，我们发现PNF和二硫键的引入大大提高了pigF和pigN的表达和稳定性，从而有效地提高了S. marcescens在30和37°C。重要性这项研究强调了一个有前途的策略，以提高mRNA/酶的稳定性，并增加生产使用从头PNF库和二硫键引入到蛋白质。PNF能延长靶基因mRNA的半衰期，有效地阻止其降解。此外，PNF可以增加靶基因的相对强度而不影响其他基因的表达;因此，与其他调控元件如启动子相比，它可以减轻细胞负担。此外，我们通过向PigF中引入二硫键获得了具有改善的活性和稳定性的PigF变体。总的来说，我们在这里展示了一种新的方法，用于提高mRNA/酶的稳定性，使用PNF，这导致在S. marcescens在30°C。
This study highlights a promising strategy to improve mRNA/enzyme stability and to increase production using de novo PNF libraries and the introduction of disulfide bonds into the protein. PNFs could increase the half-life of target gene mRNA and effectively prevent its degradation. ABSTRACT In Serratia marcescens JNB5-1, prodigiosin was highly produced at 30°C, but it was noticeably repressed at ≥37°C. Our initial results demonstrated that both the production and the stability of the O-methyl transferase (PigF) and oxidoreductase (PigN) involved in the prodigiosin pathway in S. marcescens JNB5-1 sharply decreased at ≥37°C. Therefore, in this study, we improved mRNA stability and protein production using de novo polynucleotide fragments (PNFs) and the introduction of disulfide bonds, respectively, and observed their effects on prodigiosin production. Our results demonstrate that adding PNFs at the 3′ untranslated regions of pigF and pigN significantly improved the mRNA half-lives of these genes, leading to an increase in the transcript and expression levels. Subsequently, the introduction of disulfide bonds in pigF improved the thermal stability, pH stability, and copper ion resistance of PigF. Finally, shake flask fermentation showed that the prodigiosin titer with the engineered S. marcescens was increased by 61.38% from 5.36 to 8.65 g/liter compared to the JNB5-1 strain at 30°C and, significantly, the prodigiosin yield increased 2.05-fold from 0.38 to 0.78 g/liter at 37°C. In this study, we revealed that the introduction of PNFs and disulfide bonds greatly improved the expression and stability of pigF and pigN, hence efficiently enhancing prodigiosin production with S. marcescens at 30 and 37°C. IMPORTANCE This study highlights a promising strategy to improve mRNA/enzyme stability and to increase production using de novo PNF libraries and the introduction of disulfide bonds into the protein. PNFs could increase the half-life of target gene mRNA and effectively prevent its degradation. Moreover, PNFs could increase the relative intensity of target genes without affecting the expression of other genes; as a result, it could alleviate the cellular burden compared to other regulatory elements such as promoters. In addition, we obtained a PigF variant with improved activity and stability by the introduction of disulfide bonds into PigF. Collectively, we demonstrate here a novel approach for improving mRNA/enzyme stability using PNFs, which results in enhanced prodigiosin production in S. marcescens at 30°C.