Improvement of ClosTron for successive gene disruption in Clostridium cellulolyticum using a pyrF-based screening system
Clostridium includes a number of species, such as thermophilic Clostridium thermocellum and mesophilic Clostridium cellulolyticum, producing biofuels and chemicals from lignocellulose, while genetic engineering is necessary to improve wild-type strains to fulfill the requirement of industrialization. ClosTron system is widely used in the gene targeting of Clostridium because of its high efficiency and operability. However, the targetron plasmid present in cell hinders the successive gene disruption. To solve this problem, a pyrF-based screening system was developed and implemented in C. cellulolyticum strain H10 in this study for efficient targetron plasmid curing. The screening system was composed of a pyrF-deleted cell chassis (H10ΔpyrF) constructed via homologous recombination and a PyrF expression cassette located in a targetron plasmid containing an erythromycin resistance gene. With the screening system, the gene targeting could be achieved following a two-step procedure, including the first step of gene disruption through targetron transformation and erythromycin selection and the second step of plasmid curing by screening with 5-fluoroorotic acid. To test the developed screening system, successive inactivation of the major cellulosomal exocellulase Cel48F and the scaffoldin protein CipC was achieved in C. cellulolyticum, and the efficient plasmid curing was confirmed. With the assistance of the pyrF-based screening system, the targetron plasmid-cured colonies can be rapidly selected by one-plate screening instead of traditional days' unguaranteed screening, and the successive gene disruption becomes accomplishable with ClosTron system with improved stability and efficiency, which may promote the metabolic engineering of Clostridium species aiming at enhanced production of biofuels and chemicals.
KeywordsClostridium cellulolyticum ClosTron Targetron Plasmid curing PyrF
We thank Dr. Weihong Jiang and Dr. Sheng Yang from the Institute of Plant Physiology and Ecology, Shanghai, People's Republic of China for providing plasmid pSY6. We thank Dr. Yin Li and Dr. Hongjun Dong from the Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China for the helpful discussion. This work was supported by the National Basic Research Program of China (973 Program, grant 2011CB707404), the Key Technologies R&D Program from the Ministry of Science and Technology of China (grant 2011BAD22B02), and the Instrument Developing Project of the Chinese Academy of Sciences (grant no. YZ201138).
- Lambowitz AM, Zimmerly S (2011) Group II introns: mobile ribozymes that invade DNA. Cold Spring Harb Perspect Biol 3(8):a003616Google Scholar
- Li Y, Tschaplinski TJ, Engle NL, Hamilton CY, Rodriguez M Jr, Liao JC, Schadt CW, Guss AM, Yang Y, Graham DE (2012) Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations. Biotechnol Biofuels 5:2PubMedCentralPubMedCrossRefGoogle Scholar
- Pavlostathis SG, Miller TL, Wolin MJ (1988) Fermentation of insoluble cellulose by continuous cultures of Ruminococcus albus. Appl Environ Microb 54:2655–2659Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Tripathi SA, Olson DG, Argyros DA, Miller BB, Barrett TF, Murphy DM, McCool JD, Warner AK, Rajgarhia VB, Lynd LR, Hogsett DA, Caiazza NC (2010) Development of pyrF-based genetic system for targeted gene deletion in Clostridium thermocellum and creation of a pta mutant. Appl Environ Microbiol 76:6591–6599PubMedCentralPubMedCrossRefGoogle Scholar