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

Title
Construction of a pyrF Gene Mutant of Clostridium sp. AWRP Using the Clostron Mutagenesis System
Author(s)
채철기; 이종민; 도미숙; 권수재; 김윤재; 임재규; 강성균; 이현숙
KIOST Author(s)
Lee, Joungmin(이종민)Kwon, Soo Jae(권수재)Kang, Sung Gyun(강성균)Lee, Hyun Sook(이현숙)
Publication Year
2018-10-04
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 needed to 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 and colony 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 needed to 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 and colony 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.
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/23105
Bibliographic Citation
2018 international Workshop on Deep-Sea Microbiology, 2018
Publisher
한국해양과학기술원
Type
Conference
Language
English
Publisher
한국해양과학기술원
Related Researcher
Research Interests

Metabolic engineering,Marine microbiology,대사공학,해양미생물

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