Construction of a restriction deficient Clostridium kluyveri strain using the codAB counterselection system

Providing efficient regenerative production pathways for bulk chemicals as an alternative to the petrochemical synthesis will be inevitable for improving the carbon footprint and the transition to a circular economy. Gas-fermenting microbial co‑cultures possess great potential for such recycling processes as they can utilize synthesis gas generated as an industrial off-gas or by gasification of municipal or food waste. A synthetic co-culture between Clostridium carboxidivorans and Clostridium kluyveri performing carbon chain elongation produces medium-sized alcohols from synthesis gas. The performance of C. carboxidivorans in this co-culture is most efficient at high CO partial pressures, but growth of C. kluyveri is restricted at these CO levels. Therefore, further improvement of the process could be achieved by genetic adaption of C. kluyveri to high CO levels.

An in vivo methylation-based protocol for triparental conjugation was developed for C. kluyveri DSM555T, but tools for its genetic modification are still missing. To this end, the strain C. kluyveri was tested for its suitability for the codAB counter selection system and showed the necessary high 5‑fluorocytosine tolerance of up to 500 µg/ml and 5‑fluorouracil sensitivity below 30 µg/ml. We identified a suitable origin of replication to create a pseudo-suicide plasmid containing the codAB system favoring genomic integration via homologous recombination. The vector was transconjugated successfully and we were able to construct the first mutant strain of C. kluyveri lacking a dominant type II restriction/modification system. This 'domestication' should further increase the conjugation rates allowing to construct strains with improved CO tolerance.