Next, we attempted to the development of transformation system. We isolated a uracil auxotroph (strain CFP3) derived from strain CF-2612, which is a cellulase hyper-producing mutant originated from strain Y-94, and cloned a wild-type pyrF gene encoding orotate phosphoribosyl transferase (OPRTase) from Y-94. OPRTase activity was not detected in strain CFP3, which had one nucleotide substitution in its pyrF gene. The wild-type pyrF gene restored the defective growth of CFP3 on uracil-free medium, and PCR and Southern analyses revealed that wild-type pyrF was integrated into the genome. These results indicated that a transformation system for A. cellulolyticus with the pyrFgene as a selection marker was successfully developed.
These results will provide a set of instructions or suggestions about the improvements in cellulolytic enzyme production in A. cellulolyticus.