The Effects Of Salt Stress And Sexual Development On Product Biosynthesis In Marine-derived Aspergillus Glaucus

Aspergillus glaucus HB1-19 is a typical salt-tolerance marine-derived filamentous fungus. The previous study showed that artificial seawater promoted its asexual development and antitumor aspergiolide A biosynthesis. So it was inferred that asexual development might facilitate aspergiolide A biosynthesis. As development of filamentous fungus is usually closely related with secondary metabolism, this study aims to explore relationships among salt stress, development and aspergiolide A biosynthesis to find new methods for production enhancement.Degenerate PCR and genome walking were performed to clone the gene sequences of AgfluG^ AgbrlA、AgabaA、AgwetA and AgvosA. Three types of glucose minimal medium dissolved with deionized water (MM+DIH2O), artificial seawater (MM+ASW), sodium chloride solution of isotonic artificial seawater (MM+NaCl), respectively, were used to study the effects of salt stress on A. glaucus under dark condition. MM+NaCl effectively improved asexual development and generated a large number of green conidiospores. However, MM+DIH2O was suitable for sexual development with yellow cleistothecia production. Expression analysis by qRT-PCR revealed that expression of AgfluG、AgbrlA、AgabaA、 AgwetA and AgvosA in MM+ASW were slightly higher than that in the MM+DIH2O, while the MM+NaCl were substantially highest, especially in the early phase of cell growth. The production of aspergiolide A in solid fermentation medium dissolved with artificial seawater was the highest, whereas that with NaCl was the lowest. This phenomenon was conflict with previous hypothesis. We thought that production improvement of aspergiolide A in artificial seawater might be due to the effects of metal ions in artificial seawater on secondary metabolism rather than asexual development enhancement.In order to further analyze relationship between development and aspergiolide A biosynthesis in A. glaucus, AgB14 strain (genotype alcA(p)::AgbrlA) was constructed by homologous integration. The alcA promoter is repressed under glucose, constitutive expression under glycerol and induced expression under ethanol or threonine in A. nidulans. We hoped to explore the relationship between development and aspergiolide A biosynthesis by regulating non-expression and overexpression of aexual development related gene under alcA promoter control. The qRT-PCR results showed that alcA promoter could not effectively start in AgB14 strain under different conditions. The reason was probably that alc A promoter in A. nidulans did not work well with AlcR regulatory protein in A. glaucus. AgB14 strain produced only sexual cleistothecium but no asexual conidiophore when AgbrlA not expressing, which indicated that AgbrlA was a key gene to control asexual conidiophores formation in A. glaucus. This result was consistent with that in A. nidulans. Moreover, the production of aspergiolide A in the AgB14 strain showed remarkable higher than that in the WT in three different conditions when AgB14 strain only produced sexual cleistothecium. These results indicated that not sexual development but asexual development facilitated aspergiolide A biosynthesis in marine-derived Aspergillus glaucus, which also provided a new regulation idea of aspergiolide A improvement.