Enabling Plasmid-Based Expression in <i>Clostridium kluyveri</i> Using a Biparental Methylation-Conjugation System

<i>Clostridium kluyveri</i> 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 <i>C. kluyveri</i> 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 <i>C. kluyveri</i> 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 <i>Pseudomonas putida</i>. This system supports advances in the metabolic engineering of <i>C. kluyveri</i> and the broader adoption of genetic tools in chain elongating bacteria to expand the applications of anaerobic chain elongation in industrial biomanufacturing.