We developed a PyrF-based screening system to accomplish successive gene disruption in Clostridium cellulolyticum

[2013.11.5] We published a new paper on Appl. Microbiol. Biotechnol. to report a new genetic engineering tool in Clostridium cellulolyticum. In this study, we developed a PyrF-based screening system to accomplish successive gene disruption in C. cellulolyticum..

C. cellulolyticum produces biofuels and chemicals from lignocellulose and is a promising candidate for lignocellulose bioconversion and cellulosic ethanol production via consolidated bioprocessing. To overcome its natural drawbacks as low ethanol production and high feedback inhibition, we have constructed the genetic manipulation tools in previous work and have successfully achieved target gene disruption and expression in C. cellulolyticum H10, while the usual method for gene disruption is ClosTron system.

ClosTron is a targetron technique based on the group II intron Ll.LtrB, and has been wildly used in genetic engineering of mesophilic Clostridium. Plasmid curing is required before every step of gene disruption to eliminate the influence of plasmid incapacity. However, the frequency of plasmid curing in C. cellulolyticum is very low which results in long-time screening process and tedious work to accomplish one-step gene disruption, and the successive gene targeting will not achieved rapidly.

To solve the problem of plasmid curing, we developed a PyrF-based screening sytem to improve the ClosTron method. The screening system contains 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 is 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.

Dr. Gu-Zhen Cui is the first author. Dr. Ya-Jun Liu and Prof. Qiu Cui are the corresponding authors. 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).

NCBI PubMed 

Gu-Zhen Cui, Jie Zhang, Wei Hong, Chenggang Xu, Yingang Feng, Qiu Cui*, Ya-Jun Liu* (2014) Improvement of ClosTron for successive gene disruption in Clostridium cellulolyticum using a pyrF-based screening system. App. Microbiol. Biotechnol. 98(1), 313-323. [Full text (Publisher website)]