Abstract

Microalgal chloroplasts have a substantial potential as a sustainable alternative to conventional hosts for recombinant protein production, due to their photosynthetic ability. However, realization of microalgal chloroplast as a platform for the production of recombinant proteins has suffered from difficulties in genetic manipulation and development of molecular tools, including reporter proteins. Here, we investigated the suitability of a fluorescent protein, mCherry, as a reporter for quantitative in vivo monitoring of gene expression in the chloroplast of Chlamydomonas reinhardtii. By analyzing cell growth, the fluorescence intensity of a mCherry-expressing strain, as well as auto-fluorescence, under different photoautotrophic culture conditions, we demonstrated a strong correlation between the fluorescence intensity of mCherry expressed in the chloroplast and its protein expression level. In addition, we found that the supply of CO₂ and light energy can be an important factor for the synthesis of recombinant proteins in the microalgal chloroplast. Our results identified mCherry as a reliable and quantitative reporter for the study of gene expression in chloroplasts, which is essential for the biotechnological application of microalgal chloroplasts and for improved production of valuable recombinant proteins.

中文翻译：

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mCherry 蛋白作为莱茵衣藻叶绿体基因表达的体内定量报告基因

 抽象的

由于其光合作用能力，微藻叶绿体具有作为重组蛋白生产传统宿主的可持续替代品的巨大潜力。然而，微藻叶绿体作为重组蛋白生产平台的实现在遗传操作和分子工具（包括报告蛋白）的开发方面遇到了困难。在这里，我们研究了荧光蛋白 mCherry 作为报告基因对莱茵衣藻叶绿体中基因表达进行体内定量监测的适用性。通过分析不同光合自养培养条件下的细胞生长、表达 mCherry 的菌株的荧光强度以及自发荧光，我们证明了叶绿体中表达的 mCherry 的荧光强度与其蛋白表达水平之间存在很强的相关性。此外，我们发现CO ₂和光能的供应可能是微藻叶绿体中重组蛋白合成的重要因素。我们的结果表明，mCherry 是研究叶绿体基因表达的可靠定量报告基因，这对于微藻叶绿体的生物技术应用和提高有价值的重组蛋白的生产至关重要。