Polycistronic gene expression in the model micro-organism Ustilago maydis
Polycistronic gene expression in the model micro-organism Ustilago maydis
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DOI：
10.1101/2020.05.06.081414
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发表时间：
2020-05
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通讯作者：
K. Müntjes;M. Philipp;Lisa Hüsemann;Nicole Heucken;S. Weidtkamp‐Peters;Kerstin Schipper;Matias D. Zurbriggen;M. Feldbrügge
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作者：
K. Müntjes;M. Philipp;Lisa Hüsemann;Nicole Heucken;S. Weidtkamp‐Peters;Kerstin Schipper;Matias D. Zurbriggen;M. Feldbrügge
真核微生物转录单顺反子mRNA以编码蛋白质。对于合成生物学方法如代谢工程，几种蛋白质在空间和时间上的精确共表达是有利的。一种直接的方法是应用病毒2A肽来设计真核生物中的合成多顺反子mRNA。在这里，我们建立了这样一个系统，在充分研究的模式微生物玉米黑粉菌。使用两种荧光报告蛋白，我们比较了五种病毒2A肽的活性。使用荧光显微镜在体内评价它们的活性，并使用荧光共振能量转移（FRET）进行验证。活性范围为20 - 100%，表现最好的2A肽是来自猪捷申病毒-1的P2 A。作为原理的证明，我们随着时间的推移有效地遵循调节的基因表达，并合成编码生物合成酶的三顺反子mRNA以产生甘露糖脂（MEL）。从本质上讲，我们评估了2A肽在体内，并证明了2A肽技术的适用性，为美国。玉米在基础科学和应用科学中的应用。
Eukaryotic microorganisms transcribe monocistronic mRNAs to encode proteins. For synthetic biological approaches like metabolic engineering, precise co-expression of several proteins in space and time is advantageous. A straightforward approach is the application of viral 2A peptides to design synthetic polycistronic mRNAs in eukaryotes. Here, we establish such a system in the well-studied model microorganism Ustilago maydis. Using two fluorescence reporter proteins, we compared the activity of five viral 2A peptides. Their activity was evaluated in vivo using fluorescence microscopy and validated using fluorescence resonance energy transfer (FRET). Activity ranged from 20 to 100% and the best performing 2A peptide was P2A from porcine teschovirus-1. As proof of principle, we followed regulated gene expression efficiently over time and synthesised a tri-cistronic mRNA encoding biosynthetic enzymes to produce mannosylerythritol lipids (MELs). In essence, we evaluated 2A peptides in vivo and demonstrated the applicability of 2A peptide technology for U. maydis in basic and applied science.