rmCombi-OGAB for the Directed Evolution of a Biosynthetic Gene Cluster toward Productivity Improvement.

IF 3.9 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS ACS Synthetic Biology Pub Date : 2025-02-21 Epub Date: 2025-02-05 DOI:10.1021/acssynbio.4c00734

Naoki Miyamoto, Kentaro Hayashi, Naohisa Ogata, Naoyuki Yamada, Kenji Tsuge

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Abstract

Combinatorial Ordered Gene Assembly in Bacillus subtilis (Combi-OGAB) enables construction of combinatorial libraries of various genetic elements, such as promoters in a biosynthetic gene cluster (BGC), and screening of highly productive combinations from the library. The combinations are limited by the library design, and the selectable productivity is defined within the combination. To refine the selected BGC using conventional Combi-OGAB with expanded diversity, we devised a directed evolutionary method called as random mutagenesis with Combi-OGAB (rmCombi-OGAB), which includes random mutagenesis by error-prone PCR and Combi-OGAB. In the present study, Gramicidin S (GS)-producing plasmids were used to examine the utility of rmCombi-OGAB. GS plasmids, originally generated using conventional Combi-OGAB, were successfully evolved using rmCombi-OGAB. B. subtilis carrying the evolved plasmid with unpredictable mutations showed a 1.5-fold improvement in the GS productivity. We thus expect that rmCombi-OGAB can be applied to various BGCs for useful products, such as antibiotics, to improve their productivity.

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生物合成基因簇定向进化的rmcombo - ogab。

枯草芽孢杆菌的组合有序基因组装（Combi-OGAB）可以构建各种遗传元件的组合文库，如生物合成基因簇（BGC）中的启动子，并从文库中筛选高产组合。这些组合受到库设计的限制，可选择的生产率是在组合中定义的。为了利用传统的组合- ogab对所选的BGC进行改良，增加多样性，我们设计了一种定向进化方法，称为组合- ogab随机诱变（rmCombi-OGAB），其中包括易出错PCR和组合- ogab随机诱变。在本研究中，使用Gramicidin S （GS）产生质粒来检验rmcombbi - ogab的效用。GS质粒最初使用传统的combbi - ogab生成，使用rmcombbi - ogab成功进化。携带不可预测突变的进化质粒的枯草芽孢杆菌的GS产量提高了1.5倍。因此，我们期望rmcombo - ogab可以应用于各种bgc的有用产品，如抗生素，以提高它们的生产率。

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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.