Role of AtYap1 in the reactive oxygen species regulation of lovastatin production in Aspergillus terreus

Abstract

Lovastatin has great medical and economic importance, and its production in Aspergillus terreus is positively regulated at transcriptional level, by reactive oxygen species (ROS) generated during idiophase. To investigate the role of the transcription factor Yap1 in the regulation of lovastatin biosynthesis by ROS, an orthologue of yap1 was identified in A. terreus TUB F-514 and knocked down (silenced) by RNAi. Results confirmed that the selected knockdown strain (Siyap1) showed decreased yap1 expression in both culture systems (submerged and solid-state fermentation). Transformants showed higher sensitivity to oxidative stress. Interestingly, knockdown mutant showed higher ROS levels in idiophase and an important increase in lovastatin production in submerged and solid-state fermentations: 60 and 70% increase, respectively. Furthermore, sporulation also increased by 600%. This suggested that AtYap1 was functioning as a negative regulator of the biosynthetic genes, and that lack of AtYap1 in the mutants would be derepressing these genes and could explain increased production. However, we have shown that lovastatin production is proportional to ROS levels, so ROS increase in the mutants alone could also be the cause of production increase. In this work, when ROS levels were decreased with antioxidant, to the levels shown by the parental strain, the lovastatin production and kinetics were similar to the ones of the parental strain. This means that AtYap1 does not regulate lovastatin biosynthetic genes, and that production increase observed in the knockdown strain was an indirect effect caused by ROS increase. This conclusion is compared with studies on other secondary metabolites produced by other fungal species. KEY POINTS: • ROS regulates lovastatin biosynthesis at transcriptional level, in solid-state, and in submerged fermentations. • ATyap1 knockdown mutants showed important lovastatin production increases (60 and 70%) and higher ROS levels. • When ROS were decreased in the silenced mutant to the parental strain's level, lovastatin kinetics were identical to the parental strain's.

Keywords: Production and sporulation; ROS regulation of lovastatin genes; Regulation of secondary metabolism; yap1 knockdown.