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 DSM555T 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.