Gallic acid as biofilm inhibitor can improve transformation efficiency of Ruminiclostridium papyrosolvens.

Ruminiclostridium papyrosolvens is an anaerobic, mesophilic, and cellulolytic clostridia, promising consolidated bioprocessing (CBP) candidate for producing renewable green chemicals from cellulose, but its genetic transformation has been severely impeded by extracellular biofilm. Here, we analyzed the effects of five different inhibitors with gradient concentrations on R. papyrosolvens growth and biofilm formation. Gallic acid was proved to be a potent inhibitor of biofilm synthesis of R. papyrosolvens. Furthermore, the transformation efficiency of R. papyrosolvens was significantly increased when the cells were treated by the gallic acid, and the mutant strain was successfully obtained by the improved transformation method. Thus, inhibition of biofilm formation of R. papyrosolvens by using gallic acid will contribute to its genetic transformation and efficient metabolic engineering.

Development of an efficient ClosTron system for gene disruption in Ruminiclostridium papyrosolvens.

Ruminiclostridium papyrosolvens is an anaerobic, mesophilic, and cellulolytic clostridia, promising consolidated bioprocessing (CBP) candidate for producing renewable green chemicals from cellulose, but its metabolic engineering is limited by lack of genetic tools. Here, we firstly employed the endogenous xylan-inducible promoter to control ClosTron system for gene disruption of R. papyrosolvens. The modified ClosTron can be easily transformed into R. papyrosolvens and specifically disrupt targeting genes. Furthermore, a counter selectable system based on uracil phosphoribosyl-transferase (Upp) was successfully established and introduced into the ClosTron system, which resulted in plasmid curing rapidly. Thus, the combination of xylan-inducible ClosTron and upp-based counter selectable system makes the gene disruption more efficient and convenient for successive gene disruption in R. papyrosolvens. KEY POINTS: • Limiting expression of LtrA enhanced the transformation of ClosTron plasmids in R. papyrosolvens. • DNA targeting specificity can be improved by precise management of the expression of LtrA. • Curing of ClosTron plasmids was achieved by introducing the upp-based counter selectable system.