RESUMO
The mitogen-activated protein kinase HOG1 (high-osmolarity glycerol response pathway) plays a crucial role in the response of yeast to hyperosmotic shock. Trichosporonoides oedocephalis produces large amounts of polyols (,e.g., erythritol and glycerol) in a culture medium. However, the effects of HOG1 gene knockout and environmental stress on the production of these polyols have not yet been studied. In this study, a To-HOG1 null mutation was constructed in T. oedocephalis using the loxP-Kan-loxP/Cre system as replacement of the targeted genes, and the resultant mutants showed much smaller colonies than the wild-type controls. Interestingly, compared with the wild-type strains, the results of shake-flask culture showed that To-HOG1 null mutation increased erythritol production by 1.44-fold while decreasing glycerol production by 71.23%. In addition, this study investigated the effects of citric acid stress on the T. oedocephalis HOG1 null mutants and the wild-type strain. When the supplementation of citric acid in the fermentation medium was controlled at 0.3% (w/v), the concentration of erythritol produced from the wild-type and To-HOG1 knockout mutant strains improved by 18.21% and 21.65%, respectively.
Assuntos
Basidiomycota/genética , Basidiomycota/metabolismo , Eritritol/biossíntese , Deleção de Genes , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Técnicas de Cultura Celular por Lotes , Ácido Cítrico/farmacologia , Fermentação , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Glicerol/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Fisiológico/genética , Transformação GenéticaRESUMO
The osmotolerant yeast, Trichosporonoides oedocephalis, is an excellent producer of erythritol, which has wide industrial applications. In this study, we developed an efficient transformation method for T. oedocephalis. To evaluate the T. oedocephalis transformation, we constructed a DNA fragment (loxP-Kan-loxP/Cre system) that was targeted to the mitogen-activated protein kinase HOG1 gene. Transformants were selected on plates containing G418 and response surface methodology was employed to obtain optimum transformation conditions. Optimal transformation could be achieved at an incubation time of 40 min, when the concentration of zymolyase-100T was 30 µg/mL, and when 100 mM CaCl2 was added to the mixture. The predicted optimal transformation efficiency was 133 transformants per µg of DNA. This novel method will facilitate studies in synthetic biology and metabolic engineering of T. oedocephalis.
