Protoplast mutation and genome shuffling induce the endophytic fungus Tubercularia sp. TF5 to produce new compounds.
Tubercularia sp. TF5 is an endophytic fungal strain isolated from the medicinal plant Taxus mairei. Previously, taxol has been detected in the fermentation products of this strain. However, it lost the capability of producing taxol after long-term laboratory culture. Herein, we tried to reactivate the production of taxol by protoplast mutations and genome shuffling. The protoplasts of Tub. sp. TF5 were prepared from its mycelia, and mutated by UV and NTG. The mutant strains regenerated from the mutated protoplasts were selected and classified into four groups on the basis of their phenotypes, the profile of their metabolites analyzed by TLC, MS, and bioassay data. Then, genome shuffling was subsequently carried out with eight mutant strains, with two representatives from each protoplast mutant group, and genome shuffling mutant strains were obtained and screened using the same screening procedure. Although taxol has not been detected in any mutant, two important mutants, M-741 and G-444 were selected for metabolites isolation and determination due to their phenotypes, and differences in TLC analysis result from TF5 and other mutants. Three new sesquiterpenoids, namely tuberculariols A-C (1-3), and a known dihydroisocoumarin (4) were obtained from M-741. Eighteen novel compounds were isolated from G-444, including five new sesquiterpenoids (5-9), two new dihydroisocoumarins (10, 11), one new tetralone (12), together with 10 known compounds (13-20, 1, and 2). The compounds isolated from the M-741 and G-444 were different in structure types and substitutions from those of TF5 (15, 21-29). The results showed, for the first time, that protoplast mutations and genome shuffling are efficient approaches to mining natural products from endophytic fungi. Understanding the mechanisms of unlocking the biosynthesis of new metabolites will facilitate the manipulation of the secondary metabolism in fungi.
Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, Xiamen Engineering Research Center of Marine Microbial Drug Discovery, Fujian Laboratory of Pharmaceutical Engineering, School of Life Sciences, Xiamen University, 422 South Siming Road, Xiamen, Fujian 361005, People's Republic of China.
Mingzi
M
Wang
Shaosong
S
Liu
Yaoyao
Y
Li
Ren
R
Xu
Chunhua
C
Lu
Yuemao
Y
Shen
D002681:China; D002855:Chromatography, Thin Layer; D003572:Cytochalasins / Q000302:isolation & purification / Q000378:metabolism; D032621:DNA Shuffling; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics*; D004224:Diterpenes / Q000302:isolation & purification / Q000378:metabolism; D005285:Fermentation; D016681:Genome, Fungal; D042822:Genomic Instability; D025301:Hyphae / Q000201:enzymology; D006999:Hypocreales / Q000235:genetics* / Q000378:metabolism*; D013058:Mass Spectrometry; D009154:Mutation; D017239:Paclitaxel / Q000378:metabolism; D010946:Plants, Medicinal / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism; D012717:Sesquiterpenes / Q000302:isolation & purification / Q000378:metabolism; D044024:Tetralones / Q000302:isolation & purification / Q000378:metabolism; D014466:Ultraviolet Rays
D003572:Cytochalasins; D004271:DNA, Fungal; D004224:Diterpenes; D012717:Sesquiterpenes; D044024:Tetralones; D017239:Paclitaxel