Construction of a pyrF Gene Mutant of Clostridium sp. AWRP Using the Clostron Mutagenesis System

Abstract

Acetogenic bacteria uses the Wood-Ljungdahl pathway to fix either carbon monoxide (CO) or CO2 plus H2. These microorganisms have been receiving great attention for conversion of synthesis gas into industrially important feedstocks, including acetate, ethanol, and 2,3-butanediol (2,3-BDO). However, the typical production yield and titer obtained by these microorganisms are still low for economical production of such chemicals. Thus, metabolic engineering approaches are neededto further improve production performance. In this study, we developed basic genetic tools for Clostridium sp. AWRP, a newly isolated acetogenic species from a wetland at Ansan. As an example, we successfully disrupted the pyrF gene, encoding oritidine 5’-phosphate decarboxylase, in the AWRP strain using clostron mutagenesis. After transfer of the retargeted clostron plasmid via conjugation, the inactivation of the pyrF gene was both confirmed by erythromycin resistance andcolony PCR. As expected, the pyrF mutants showed the resistance to 5-fluoroorotic acid (FOA), which becomes toxic after converted to 5-fluorouracil by pyrF. These results suggest that the tools developed in this study can be used for metabolic engineering and physiological studies on Clostridium sp. AWRP.g acetate, ethanol, and 2,3-butanediol (2,3-BDO). However, the typical production yield and titer obtained by these microorganisms are still low for economical production of such chemicals. Thus, metabolic engineering approaches are neededto further improve production performance. In this study, we developed basic genetic tools for Clostridium sp. AWRP, a newly isolated acetogenic species from a wetland at Ansan. As an example, we successfully disrupted the pyrF gene, encoding oritidine 5’-phosphate decarboxylase, in the AWRP strain using clostron mutagenesis. After transfer of the retargeted clostron plasmid via conjugation, the inactivation of the pyrF gene was both confirmed by erythromycin resistance andcolony PCR. As expected, the pyrF mutants showed the resistance to 5-fluoroorotic acid (FOA), which becomes toxic after converted to 5-fluorouracil by pyrF. These results suggest that the tools developed in this study can be used for metabolic engineering and physiological studies on Clostridium sp. AWRP.