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

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS ACS Synthetic Biology Pub 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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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.