CRISPR/Cas9-Based Genome Editing for Protein Expression and Secretion in Kluyveromyces lactis

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2024-06-13 DOI:10.1021/acssynbio.4c00157

Lingtong Liao, Xiuru Shen, Zhiyu Shen, Guocheng Du, Jianghua Li* and Guoqiang Zhang*,

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Abstract

The budding yeast Kluyveromyces lactis has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of K. lactis as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to K. lactis. Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene KU80 and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the KlΔKU80 strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of TMED10 and HSP90 to characterize the extracellular secretion of lactase in K. lactis. Generally, genome editing is a powerful tool for constructing K. lactis cell factories for protein and chemical production.

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基于 CRISPR/Cas9 基因组编辑的乳酸克鲁维酵母菌蛋白质表达和分泌。

乳酸克鲁维酵母（Kluyveromyces lactis）已成为工业生物技术中一种前景广阔的微生物底盘。然而，由于缺乏高效的分子遗传操作工具和策略，K. lactis 作为生物制造平台的发展受到了阻碍。在本研究中，我们开发了一种基于 CRISPR/Cas9 的基因组编辑方法并将其应用于 K. lactis。通过破坏非同源末端连接相关基因 KU80 并进行一系列流程优化，单基因编辑效率提高到了 80%。随后，研究人员根据不同的 sgRNA 递送模式探索了 CRISPR/Cas9 系统，以实现多基因同步编辑。借助颜色指示器，KlΔKU80 菌株的两个和三个基因的编辑效率分别达到了 73.3% 和 36%。此外，CRISPR/Cas9系统还被用于多位点整合以提高乳糖酶的产量，以及组合敲除TMED10和HSP90以鉴定乳糖酶在K.lactis中的胞外分泌特性。一般来说，基因组编辑是构建乳酸菌细胞工厂以生产蛋白质和化学品的有力工具。

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

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.