Triple-CRISPRi-mediated down-regulation of the shikimate pathway branch genes for enhancing 2-PE biosynthesis in Saccharomyces cerevisiae

IF 3 3区农林科学 Q2 FOOD SCIENCE & TECHNOLOGY European Food Research and Technology Pub Date : 2024-05-02 DOI:10.1007/s00217-023-04461-0

Shuaihu Fang, Xianghao Fan, Jinlan Li, Zijing Zhu, Pingping Huang, Xiaobao Qi

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Abstract

The 2-phenylethanol biosynthesis in Saccharomyces cerevisiae is limited by multiple-branch metabolism in the shikimate pathway. In this research, a total of 4 × 4 (sites × genes) guide sequences from four branch genes (TYR1, ARO8, AAT2 and ALD3) were designed. A single-gene down-regulation library of 4 × 4 Saccharomyces cerevisiae strains was constructed. By the assessment of gene expression level and 2-phenylethanol production, the optimal guide sequences of TYR1/AAT2/ALD3 were identified. On these bases, we first developed a high-yielding 2-phenylethanol strain carrying triple-CRISPRi system for simultaneous three branch repression. The INVScI-TYR1.AAT2.ALD3 successfully achieved the desired transcriptional repression effect and led to a 1.89-fold increase in 2-phenylethanol production compared to the starting strain. Triple-CRISPRi-mediated down-regulation of the shikimate pathway branch genes provided a convenient and efficient solution for the development of 2-phenylethanol high-yield Saccharomyces cerevisiae engineering strain.

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三重-CRISPRi-介导的莽草酸途径分支基因下调，促进酿酒酵母中 2-PE 的生物合成

酿酒酵母（Saccharomyces cerevisiae）的 2-苯乙醇生物合成受限于莽草酸途径中的多分支代谢。本研究设计了四个分支基因（TYR1、ARO8、AAT2 和 ALD3）共 4 × 4（位点 × 基因）的引导序列。构建了一个由 4 × 4 株酿酒酵母组成的单基因下调文库。通过评估基因表达水平和 2-苯乙醇产量，确定了 TYR1/AAT2/ALD3 的最佳引导序列。在此基础上，我们首先开发了一种携带三重 CRISPRi 系统的高产 2-苯乙醇菌株，可同时抑制三个分支。INVScI-TYR1.AAT2.ALD3成功实现了预期的转录抑制效果，使2-苯乙醇产量比初始菌株提高了1.89倍。三重-CRISPRi介导的莽草酸途径分支基因下调为2-苯乙醇高产酿酒酵母工程菌株的开发提供了一种便捷高效的解决方案。图文摘要

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来源期刊

European Food Research and Technology 工程技术-食品科技

CiteScore

6.60

自引率

3.00%

发文量

232

审稿时长

2.0 months

期刊介绍： The journal European Food Research and Technology publishes state-of-the-art research papers and review articles on fundamental and applied food research. The journal''s mission is the fast publication of high quality papers on front-line research, newest techniques and on developing trends in the following sections: -chemistry and biochemistry- technology and molecular biotechnology- nutritional chemistry and toxicology- analytical and sensory methodologies- food physics. Out of the scope of the journal are: - contributions which are not of international interest or do not have a substantial impact on food sciences, - submissions which comprise merely data collections, based on the use of routine analytical or bacteriological methods, - contributions reporting biological or functional effects without profound chemical and/or physical structure characterization of the compound(s) under research.