Agena, E. M.; Badani, A. A.; Linder, B. G.; Gois, I. M.; Dempster, A. W.; Minton, N. P.; Mahadevan, R.; Lawson, C. E.

Clostridium kluyveri is a promising biocatalyst for producing medium-chain fatty acids (MCFAs) from waste-derived carbon via chain elongation. MCFAs are platform chemicals with diverse applications across agriculture, food, cosmetics, and fuels, and could support efforts towards tandem resource recovery and sustainable chemical production. However, genetic intractability has 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 DSM555T to bypass the organisms restriction-modification barriers and enable stable plasmid delivery. We use this system to demonstrate heterologous expression of the Fluorescence-Activated absorption-Shifting Tag (FAST), an anaerobic fluorescent reporter. This system supports advances in 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.