Development of a Golden Gate Assembly-Based Genetic Toolbox for Lactiplantibacillus plantarum and Its Application for Engineering Monoterpenoid Biosynthesis

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS ACS Synthetic Biology Pub Date : 2024-09-10 DOI:10.1021/acssynbio.4c00075

Xiangang Li, Pascal Y. Schönberg, Tabea Wucherpfennig, Christoph Hinze, Flavia Sulaj, Thomas Henle, Thorsten Mascher

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Abstract

Lactiplantibacillus plantarum is a food-grade lactic acid bacterium widely used in the food and beverage industry. Recently, this probiotic organism has been applied as a biofactory for the production of pharmaceutical and food-related compounds, but existing promoters and expression vectors for the genetic engineering of L. plantarum rely on inefficient cloning strategies and are usually not well-characterized. We therefore developed a modular and standardized Golden Gate Assembly-based toolbox for the de novo assembly of shuttle vectors from Escherichia coli to L. plantarum. A collection of the most relevant genetic parts, e.g., different origins of replication and promoters, was incorporated in our toolbox and thoroughly characterized by flow cytometry and the fluorescence assay. Standardized fusion sites allow combining the genetic part freely into a plasmid in one step. This approach allows for the high-throughput assembly of numerous constructs in a standardized genetic context, thus improving the efficiency and predictability of metabolic engineering in L. plantarum. Using our toolbox, we were able to produce the aroma compounds linalool and geraniol in L. plantarum by extending its native mevalonate pathway with plant-derived monoterpenoid synthases.

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为植物乳杆菌开发基于金门组装的遗传工具箱及其在单萜生物合成工程中的应用

植物乳杆菌（Lactiplantibacillus plantarum）是一种食品级乳酸菌，广泛应用于食品和饮料行业。最近，这种益生菌被用作生产药物和食品相关化合物的生物工厂，但现有的植物乳杆菌基因工程启动子和表达载体都依赖于低效的克隆策略，而且通常没有很好的表征。因此，我们开发了一个基于金门组装的模块化和标准化工具箱，用于从大肠杆菌到植物酵母的穿梭载体的从头组装。我们的工具箱收集了最相关的基因部分，如不同的复制起源和启动子，并通过流式细胞仪和荧光检测进行了全面鉴定。通过标准化的融合位点，只需一步就能将基因部分自由组合到质粒中。这种方法可以在标准化的基因环境中高通量地组装大量构建体，从而提高植物乳杆菌代谢工程的效率和可预测性。通过使用我们的工具箱，我们利用植物来源的单萜合成酶扩展了植物酵母的原生甲羟戊酸途径，从而在植物酵母中生产出芳樟醇和香叶醇。

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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.