Enabling Plasmid-Based Expression in Clostridium kluyveri Using a Biparental Methylation-Conjugation System

Clostridium kluyveri is a promising biocatalyst for producing medium-chain carboxylic acids (MCCAs) from waste-derived carbon via chain elongation. MCCAs are platform chemicals with diverse applications across agriculture, food, cosmetics, and fuels and could support tandem resource recovery and sustainable chemical production. However, host defense systems have hindered efforts to engineer C. kluyveri for improved product yields, control over chain length and selectivity, and production of non-native oleochemicals. Here, we report a streamlined, biparental methylation-conjugation system developed for C. kluyveri DSM555^T to bypass the organism's restriction-modification barriers and enable stable plasmid delivery. We use this system to demonstrate heterologous expression of two anaerobic fluorescent reporters: the Fluorescence-Activating and absorption-Shifting Tag (FAST) and an evolved Flavin-binding Fluorescent Protein (FbFP) from Pseudomonas putida. This system supports advances in the metabolic engineering of C. kluyveri and the broader adoption of genetic tools in chain elongating bacteria to expand the applications of anaerobic chain elongation in industrial biomanufacturing.