Expansion of YALIcloneHR toolkit for Yarrowia lipolytica combined with Golden Gate and CRISPR technology

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biotechnology Letters Pub Date : 2023-12-08 DOI:10.1007/s10529-023-03444-1

Qi Shen, Fang Yan, Ya-Wen Li, Jian Wang, Jia Ji, Wen-Xin Yan, Dan-Chen He, Ping Song, Tian-Qiong Shi

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Abstract

Metabolic Engineering of yeast is a critical approach to improving the production capacity of cell factories. To obtain genetically stable recombinant strains, the exogenous DNA is preferred to be integrated into the genome. Previously, we developed a Golden Gate toolkit YALIcloneNHEJ, which could be used as an efficient modular cloning toolkit for the random integration of multigene pathways through the innate non-homologous end-joining repair mechanisms of Yarrowia lipolytica. We expanded the toolkit by designing additional building blocks of homologous arms and using CRISPR technology. The reconstructed toolkit was thus entitled YALIcloneHR and designed for gene-specific knockout and integration. To verify the effectiveness of the system, the gene PEX10 was selected as the target for the knockout. This system was subsequently applied for the arachidonic acid production, and the reconstructed strain can accumulate 4.8% of arachidonic acid. The toolkit will expand gene editing technology in Y. lipolytica, which would help produce other chemicals derived from acetyl-CoA in the future.

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结合 "金门 "和 CRISPR 技术，扩展用于脂溶性亚罗威氏菌的 YALIcloneHR 工具包

酵母代谢工程是提高细胞工厂生产能力的关键方法。要获得基因稳定的重组菌株，外源 DNA 最好能整合到基因组中。此前，我们开发了一个金门工具包 YALIcloneNHEJ，它可以作为一个高效的模块化克隆工具包，通过脂肪分解亚罗酵母的先天非同源末端连接修复机制随机整合多基因通路。我们利用 CRISPR 技术设计了更多的同源臂构件，从而扩展了该工具包。因此，重建的工具包被命名为 YALIcloneHR，设计用于基因特异性敲除和整合。为了验证该系统的有效性，我们选择了 PEX10 基因作为基因敲除的靶标。该系统随后被应用于花生四烯酸的生产，重建后的菌株可积累 4.8%的花生四烯酸。该工具包将扩展脂溶性酵母菌的基因编辑技术，这将有助于将来生产其他由乙酰-CoA衍生的化学物质。

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来源期刊

Biotechnology Letters 工程技术-生物工程与应用微生物

CiteScore

5.90

自引率

3.70%

发文量

108

审稿时长

1.2 months

期刊介绍： Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.