Molecular identification using ITS sequences and genome shuffling to improve 2-deoxyglucose tolerance and xylanase activity of marine-derived fungus, Aspergillus sp. NRCF5.

Abstract

During the screening of xylanolytic enzyme from marine-derived fungi isolated from the inner tissue of Egyptian soft coral Rhytisma sp., one strain, NRCF5, exhibited high enzyme activity with 0.1 % (w/v) antimetabolite 2-deoxyglucose (2DG) tolerance. This fungal strain was identified as Aspergillus sp. NRCF5 based on its morphological characteristics and internal transcribed spacer (ITS) sequences. The ITS region of hyperactive xylanolytic strain (NRCF5) was amplified, sequenced, and submitted to GenBank (accession no. JQ277356). To apply the fundamental principles of genome shuffling in breeding of xylanase-producing fungi, marine-derived fungus Aspergillus sp. NRCF5 was used as starting strain in this work and applied for induction of genetic variability using different combinations and doses of mutagens. Five mutants with high xylanase activity and 0.25 % (w/v) antimetabolite 2DG tolerance were obtained from the populations generated by the mutation of combination between ultraviolet irradiation (UV, 5 min) and N-methyl-N-nitro-N-nitrosoguanidine (NTG, 100 μg/ml) for 30 (UNA) and 60 (UNB)min as well as NTG (100 μg/ml) and ethidium bromide (250 μg/ml) for 30 (NEA) and 60 (NEB)min. Then, they were subjected for recursive protoplast fusion. Seven hereditarily stable recombinants with high xylanase activity and 1.0 % (w/v) 2DG tolerance were obtained by four rounds of genome shuffling. Among them, a high xylanase-producing recombinant, R4/31, was obtained, which produced 427.5 U/ml xylanase. This value is 6.13-fold higher than that of the starting strain NRCF5 and 2.48-fold higher than that of the parent strain (mutant NEA51). The subculture experiments indicated that the high producer of marine Aspergillus sp. R4/31 fusant was stable.