Application of a Cell-Free Synthetic Biology Platform for the Reconstitution of Teleocidin B and UK-2A Precursor Biosynthetic Pathways.

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS ACS Synthetic Biology Pub Date : 2024-10-29 DOI:10.1021/acssynbio.4c00560

Krishna Madduri, Deepa Acharya, Adam Lescallette, Jeremy McFadden, Paul Ketterer, Jade Bing, Babu Raman

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Abstract

We report the successful cell-free reconstitution of two natural product biosynthetic pathways of divergent complexity and structural classes. We first constructed the teleocidin biosynthetic pathway using our BY-2 (tobacco) cell-free protein synthesis (CFPS) system. We discovered a direct interaction between TleA and MbtH, and showed that the BY-2 system is capable of producing more than 80 mg/L teleocidin B-3 with cofactor supplementation and ∼20 mg/L with no cofactors supplemented, demonstrating the high metabolic activity of the system. We then extended our methodology and report the first successful cell-free biosynthesis of UK-2 diol (precursor to the commercially valuable secondary metabolite UK-2A) from simple building blocks by refactoring a complex pathway of 10 proteins in the wheat germ CFPS system. We show that plant CFPS systems are suitable for reconstructing pathways and identifying the functions of uncharacterized genes linked to biosynthetic gene clusters and rate-limiting biosynthetic steps.

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应用无细胞合成生物学平台重建远志苷 B 和 UK-2A 前体生物合成途径。

我们报告了两条天然产物生物合成途径的成功无细胞重组，它们的复杂性和结构类别各不相同。我们首先利用 BY-2（烟草）无细胞蛋白质合成（CFPS）系统构建了远志苷的生物合成途径。我们发现了 TleA 和 MbtH 之间的直接相互作用，并证明 BY-2 系统在补充辅助因子的情况下能产生超过 80 mg/L 的远志苷 B-3，而在不补充辅助因子的情况下则能产生 ∼20 mg/L，这表明该系统具有很高的代谢活性。随后，我们扩展了我们的方法，并报告了通过重构小麦胚芽 CFPS 系统中由 10 个蛋白质组成的复杂途径，首次成功地从简单的构建模块中无细胞生物合成了 UK-2 二醇（具有商业价值的次级代谢物 UK-2A 的前体）。我们的研究表明，植物 CFPS 系统适用于重建途径，并确定与生物合成基因簇和限速生物合成步骤相关的未定性基因的功能。

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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.