Dual-Regulation in Peroxisome and Cytoplasm toward Efficient Limonene Biosynthesis with Rhodotorula toruloides.

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2024-08-16 Epub Date: 2024-06-11 DOI:10.1021/acssynbio.4c00306

Qidou Gao, Yaqi Dong, Ying Huang, Sasa Liu, Xiaochun Zheng, Yiming Ma, Qingsheng Qi, Xue Wang, Zongbao Kent Zhao, Xiaobing Yang

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Abstract

Rhodotorula toruloides is a potential workhorse for production of various value-added chemicals including terpenoids, oleo-chemicals, and enzymes from low-cost feedstocks. However, the limited genetic toolbox is hindering its metabolic engineering. In the present study, four type I and one novel type II peroxisomal targeting signal (PTS1/PTS2) were characterized and employed for limonene production for the first time in R. toruloides. The implant of the biosynthesis pathway into the peroxisome led to 111.5 mg/L limonene in a shake flask culture. The limonene titer was further boosted to 1.05 g/L upon dual-metabolic regulation in the cytoplasm and peroxisome, which included employing the acetoacetyl-CoA synthase NphT7, adding an additional copy of native ATP-dependent citrate lyase, etc. The final yield was 0.053 g/g glucose, which was the highest ever reported. The newly characterized PTSs should contribute to the expansion of genetic toolboxes forR. toruloides. The results demonstrated that R. toruloides could be explored for efficient production of terpenoids.

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过氧物酶体和细胞质中的双重调控实现了 Rhodotorula toruloides 的高效柠檬烯生物合成。

红豆杉（Rhodotorula toruloides）是利用低成本原料生产各种高附加值化学品（包括萜类、油脂化学品和酶）的潜在主力军。然而，有限的遗传工具箱阻碍了它的代谢工程。在本研究中，首次对四种 I 型和一种新型 II 型过氧化物酶体靶向信号（PTS1/PTS2）进行了表征，并将其用于 R. toruloides 的柠檬烯生产。将生物合成途径植入过氧物酶体后，在摇瓶培养中可产生 111.5 mg/L 的柠檬烯。通过在细胞质和过氧物酶体中进行双代谢调节，包括使用乙酰乙酰-CoA 合成酶 NphT7、增加一个原生 ATP 依赖性柠檬酸裂解酶的拷贝等，柠檬烯的滴度进一步提高到 1.05 克/升。最终的产量为 0.053 克/克葡萄糖，这是迄今所报道的最高产量。新鉴定的 PTS 应有助于扩大 toruloides 的基因工具箱。研究结果表明，可以探索 R. toruloides 高效生产萜类化合物的途径。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.