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

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

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.

Details

Original languageEnglish
Pages (from-to)2764-2779
Number of pages16
JournalACS synthetic biology
Volume13
Issue number9
Publication statusPublished - 20 Sept 2024
Peer-reviewedYes

External IDs

PubMed 39254046

Keywords

Keywords

  • cloning toolbox, golden gate assembly, lactic acid bacteria, metabolic engineering, mevalonate pathway, monoterpenoid