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

<i>Lactiplantibacillus plantarum</i> 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 <i>L. plantarum</i> 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 <i>de novo</i> assembly of shuttle vectors from <i>Escherichia coli</i> to <i>L. plantarum</i>. 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 <i>L. plantarum</i>. Using our toolbox, we were able to produce the aroma compounds linalool and geraniol in <i>L. plantarum</i> by extending its native mevalonate pathway with plant-derived monoterpenoid synthases.