Enabling Plasmid-based Expression in Clostridium kluyveri using a Biparental Methylation-Conjugation System

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. Lay SummaryWe developed a streamlined method for genetically modifying Clostridium kluyveri, a promising strain for industrial anaerobic biomanufacturing, and used it to express a fluorescent protein useful for downstream applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/657078v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@11e2a58org.highwire.dtl.DTLVardef@16e1099org.highwire.dtl.DTLVardef@103c72forg.highwire.dtl.DTLVardef@10a1fce_HPS_FORMAT_FIGEXP M_FIG C_FIG