Lignocellulosic biomass is an abundant renewable feedstock that can be used to produce biofuels and platform chemicals. However, its industrial valorization is a challenge due to its natural recalcitrance. Clostridium cellulovorans is a mesophilic cellulolytic bacterium that has received special attention from the scientific community regarding enzymatic hydrolysis of cellulose and the production of n-butanol by consolidated bioprocessing after genetic engineering. However, the lack of reliable genetic tools prevents a precise understanding of the strain's physiology and makes its biotechnological potential underexplored. Genetic modification and genomic engineering have failed due to the low transformability and the small number of characterized synthetic biology parts available for this organism. Here, we describe a genetic system comprising rationally designed deletion plasmid that allows strain engineering by the allelic-coupled exchange method. Two key features in the deletion plasmid were remarkably important. First, a suitable clostridial "pseudo-suicide" origin of replication was identified, allowing plasmid transconjugation and further integration into the chromosome. Second, an efficient counterselection method was established. The codBA counterselection method is based on the presence of the codBA cassette in the deletion plasmid and the use of the 5-fluorocytosine (5-FC) as the counterselective compound. The codBA cassette from C. ljungdahlii under the control of the PclpB promoter was used and determination of the minimum inhibitory concentration (MIC) of 5-FC was performed. The constructed deletion plasmids also contained flanking regions of the locus targeted for deletion. Transconjugants were cultivated on solid medium containing reduced thiamphenicol. Thereafter, potential integrants were cultivated on solid medium containing 5-FC. The confirmation of the mutation was performed via PCR. These results demonstrated the ability to obtain a scarless chromosomal mutant and contributed to expanding the molecular tool kit for C. cellulovorans.

Keywords: Clostridia, cellulolytic microorganisms, genetic engineering, synthetic biology tools