In Vivo DNA Assembly in Yarrowia lipolytica.

IF 3.9 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-09-30 DOI:10.1021/acssynbio.5c00296

Wei Jiang, Ioannis Georgiadis, Tommaso Fumagalli, Shengbao Wang, Christina Vasileiou, Jonathan Dahlin, Irina Borodina

引用次数: 0

Abstract

The oleaginous yeast Yarrowia lipolytica is an important platform organism for biotechnology applications. In this study, we established an in vivo DNA assembly system leveraging CRISPR-Cas9 for efficient genomic integration of multiple DNA fragments into the genome of Y. lipolytica. Using the green fluorescent protein mNeonGreen as a model, we demonstrated 53% correct assembly of three DNA fragments with homology arms as short as 50 bp. The system was further validated by constructing 2-3 step biosynthetic pathways for pigments betaxanthin and betanin. To improve the homologous recombination efficiency of Y. lipolytica, we expressed S. cerevisiae RAD52 (ScRAD52) or a Cas9-hBrex27 fusion protein. While ScRAD52 expression impaired growth, the cas9-hBrex27 fusion enhanced integration efficiency, particularly for multifragment pathway assemblies. The in vivo assembly method simplifies pathway construction and gene overexpression in Y. lipolytica.

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脂性耶氏菌体内DNA组装。

产油酵母多脂耶氏酵母是生物技术应用的重要平台生物。在本研究中，我们利用CRISPR-Cas9建立了一个体内DNA组装系统，将多个DNA片段高效地整合到脂肪瘤Y. lipolytica的基因组中。使用绿色荧光蛋白mNeonGreen作为模型，我们证明了三个同源臂短至50 bp的DNA片段的正确组装率为53%。通过构建2-3步合成甜菜黄素和甜菜素的生物合成途径，进一步验证了该体系的有效性。为了提高Y. lipolytica的同源重组效率，我们表达了S. cerevisiae RAD52 （ScRAD52）或Cas9-hBrex27融合蛋白。虽然ScRAD52表达会损害生长，但cas9-hBrex27融合提高了整合效率，特别是对于多片段途径组件。体内组装方法简化了脂质体Y.的途径构建和基因过表达。

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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.