Regulation of the cutinases expressed by Aspergillus nidulans and evaluation of their role in cutin degradation
Four cutinase genes are encoded in the genome of the saprophytic fungus Aspergillus nidulans, but only two of them have proven to codify for active cutinases. However, their overall roles in cutin degradation are unknown, and there is scarce information on the regulatory effectors of their expression. In this work, the expression of the cutinase genes was assayed by multiplex qRT-PCR in cultures grown in media containing both inducer and repressor carbon sources. The genes ancut1 and ancut2 were induced by cutin and its monomers, while ancut3 was constitutively expressed. Besides, cutin induced ancut4 only under oxidative stress conditions. An in silico analysis of the upstream regulatory sequences suggested binding regions for the lipid metabolism transcription factors (TF) FarA for ancut1 and ancut2 while FarB for ancut3. For ancut4, the analysis suggested binding to NapA (the stress response TF). These binding possibilities were experimentally tested by transcriptional analysis using the A. nidulans mutants ANΔfarA, ANΔfarB, and ANΔnapA. Regarding cutin degradation, spectroscopic and chromatographic methods showed similar products from ANCUT1 and ANCUT3. In addition, ANCUT1 produced 9,10-dihydroxy hexadecanoic acid, suggesting an endo-cleavage action of this enzyme. Regarding ANCUT2 and ANCUT4, they produced omega fatty acids. Our results confirmed the cutinolytic activity of the four cutinases, allowed identification of their specific roles in the cutinolytic system and highlighted their differences in the regulatory mechanisms and affinity towards natural substrates. This information is expected to impact the cutinase production processes and broaden their current biotechnological applications.
KeywordsCutinase Expression Carbon catabolite repression Cutin degradation Transcription factors Oxidative stress Aspergillus nidulans
We want to thank Dr. Xóchitl Pérez Martínez for her valuable comments and the support given to this project, Adriana López Calderón for the production of ANCUT3, Cynthia Bastida for the production and identification of ANCUT4, and Augusto Castro-Rodríguez for his assistance in the synthesis and purification of cutin. Dr. José Correa (IPN) performed the UHPLC MS/MS analysis. We appreciate the contribution of C. Warden and María Elena Sánchez-Salazar in the review of the English manuscript. EBG received a CONACYT scholarship as a student from the Biochemical Sciences graduate program. Funds from CONACYT 153500, PAPIIT IN217414, and PAIP 5000-9095 are also acknowledged. CSP wishes to acknowledge funding from the European Research Council through grant ERC-2014-CoG-647928, and to Fundação para a Ciência e Tecnologia through grant UID/Multi/04551/2013 (Research unit GREEN-it “Bioresources for Sustainability”). I.M. is grateful to Fundação para a Ciência e a Tecnologia (FCT), Portugal, for the fellowships SFRH/BPD/110841/2015.
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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