Subcellular compartmentalization is a crucial evolution characteristic of eukaryotic cells, providing inherent advantages for the construction of artificial biological systems to efficiently produce natural products. The establishment of an artificial protein transport system represents a pivotal initial step towards developing efficient artificial biological systems. Peroxisome has been demonstrated as a suitable subcellular compartment for the biosynthesis of terpenes in yeast. In this study, an artificial protein transporter ScPEX5* was firstly constructed by fusing the N-terminal sequence of PEX5 from S. cerevisiae and the C-terminal sequence of PEX5. Subsequently, an artificial protein transport system including the artificial signaling peptide YQSYY and its enhancing upstream 9 amino acid (9AA) residues along with ScPEX5* was demonstrated to exhibit orthogonality to the internal transport system of peroxisomes in S. cerevisiae. Furthermore, a library of 9AA residues was constructed and selected using high throughput pigment screening system to obtain an optimized signaling peptide (oPTS1*). Finally, the ScPEX5*-oPTS1* system was employed to construct yeast cell factories capable of producing the sesquiterpene α-humulene, resulting in an impressive α-humulene titer of 17.33 g/L and a productivity of 0.22 g/L/h achieved through fed-batch fermentation in a 5 L bioreactor. This research presents a valuable tool for the construction of artificial peroxisome cell factories and effective strategies for synthesizing other natural products in yeast.

中文翻译：

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构建酿酒酵母过氧化物酶体的正交运输系统以高效生产倍半萜


亚细胞区室化是真核细胞的关键进化特征，为构建人工生物系统高效生产天然产物提供了先天优势。人工蛋白质运输系统的建立代表了开发高效人工生物系统的关键第一步。过氧化物酶体已被证明是酵母中萜烯生物合成的合适亚细胞区室。在本研究中，首先通过将酿酒酵母 PEX5 的 N 端序列和 PEX5 的 C 端序列融合，构建了人工蛋白转运蛋白 ScPEX5*。随后，包括人工信号肽 YQSYY 及其增强的上游 9 个氨基酸 （9AA） 残基以及 ScPEX5* 的人工蛋白质转运系统被证明与酿酒酵母中过氧化物酶体的内部转运系统正交。此外，构建了一个 9AA 残基库，并使用高通量色素筛选系统进行选择，以获得优化的信号肽 （oPTS1*）。最后，采用 ScPEX5*-oPTS1* 系统构建能够生产倍半萜 α-humulene 的酵母细胞工厂，通过在 5 L 生物反应器中补料分批发酵，产生了令人印象深刻的 17.33 g/L α-humulene 滴度和 0.22 g/L/h 的生产率。这项研究为构建人工过氧化物酶体细胞工厂提供了有价值的工具，并为在酵母中合成其他天然产物提供了有效的策略。