An expanded library of 76 integration sites for gene expression in Saccharomyces cerevisiae

IF 3.5 3区工程技术 Q2 ENGINEERING, CHEMICAL

AIChE Journal Pub Date : 2025-03-24 DOI:10.1002/aic.18818

Xiaolong Teng, Yuanwei Gou, Xuanwei Ding, Yueping Zhang, Di Gao, Yingjia Pan, Xiaolin Shen, Jiazhang Lian, Chun Li, Jinyu Fu, Shuobo Shi

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引用次数: 0

Abstract

Constructing efficient yeast cell factories involves introducing heterologous biosynthetic pathways and overexpressing key genes. Chromosomal integration of recombinant genes is preferred over episomal plasmids for greater stability during large-scale industrial cultivation. The expression of complex pathways in engineered microbes necessitates the activation of an increasing number of genes, a process limited by the availability of suitable integration sites. To address this challenge, we investigated 125 potential chromosomal sites in Saccharomyces cerevisiae by inserting mCherry using the CRISPR/Cas9 technique to evaluate their capacity to integrate and express heterologous genes. Subsequently, 76 sites were identified to support effective integration with genomic stability. Furthermore, to demonstrate the potential for multiplexed engineering, we successfully performed a one-step, four-locus integration of the β-carotene pathway using the characterized sites. The expanded integration sites are expected to be valuable for constructing yeast cell factories for applications in synthetic biology and metabolic engineering.

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酿酒酵母76个基因表达整合位点的扩展文库

构建高效的酵母细胞工厂需要引入异源生物合成途径和过表达关键基因。在大规模工业培养中，重组基因的染色体整合比表观质粒更稳定。工程微生物中复杂途径的表达需要激活越来越多的基因，这一过程受到合适整合位点的可用性的限制。为了解决这一挑战，我们利用CRISPR/Cas9技术，通过插入mCherry，研究了酿酒酵母中125个潜在的染色体位点，以评估它们整合和表达外源基因的能力。随后，鉴定出76个位点支持与基因组稳定性的有效整合。此外，为了证明多路工程的潜力，我们成功地利用这些特征位点对β-胡萝卜素途径进行了一步、四位点整合。扩展的整合位点有望在酵母细胞工厂的构建中应用于合成生物学和代谢工程。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

AIChE Journal 工程技术-工程：化工

CiteScore

7.10

自引率

10.80%

发文量

411

审稿时长

3.6 months

期刊介绍： The AIChE Journal is the premier research monthly in chemical engineering and related fields. This peer-reviewed and broad-based journal reports on the most important and latest technological advances in core areas of chemical engineering as well as in other relevant engineering disciplines. To keep abreast with the progressive outlook of the profession, the Journal has been expanding the scope of its editorial contents to include such fast developing areas as biotechnology, electrochemical engineering, and environmental engineering. The AIChE Journal is indeed the global communications vehicle for the world-renowned researchers to exchange top-notch research findings with one another. Subscribing to the AIChE Journal is like having immediate access to nine topical journals in the field. Articles are categorized according to the following topical areas: Biomolecular Engineering, Bioengineering, Biochemicals, Biofuels, and Food Inorganic Materials: Synthesis and Processing Particle Technology and Fluidization Process Systems Engineering Reaction Engineering, Kinetics and Catalysis Separations: Materials, Devices and Processes Soft Materials: Synthesis, Processing and Products Thermodynamics and Molecular-Scale Phenomena Transport Phenomena and Fluid Mechanics.