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Astaxanthin is one of the most attractive carotenoids due to its high antioxidant activity and beneficial biological properties, while Xanthophyllomyces dendrorhous is one of its main microbial sources. Since astaxanthin is synthesized as a response to oxidative stress, several oxidative agents have been evaluated to increase X. dendrorhous astaxanthin yields. However, the extent of the stimulation is determined by the cellular damage caused by the applied oxidative agent. Phytohormones have also been reported as stimulants of astaxanthin biosynthesis acting directly on its metabolic pathway and indirectly promoting cellular resistance to reactive oxygen species. We reasoned that both oxidative agents and phytohormones lead to increased astaxanthin synthesis, but the latter could mitigate the drawbacks of the former. Thus, here, the stimulation on astaxanthin biosynthesis, as well as the cellular and transcriptional responses of wild type X. dendrorhous to phytohormones (6-benzylaminopurine, 6-BAP; abscisic acid, ABA; and indole-3-acetic acid, IAA), and oxidative agents (glutamate, menadione, H2O2, and/or Fe2+) were evaluated as a single or combined treatments. ABA and 6-BAP were the best individual stimulants leading to 2.24- and 2.60-fold astaxanthin biosynthesis increase, respectively. Nevertheless, the effect of combined 6-BAP and H2O2 led to a 3.69-fold astaxanthin synthesis increase (0.127 ± 0.018 mg astaxanthin/g biomass). Moreover, cell viability (> 82.75%) and mitochondrial activity (> 82.2%) remained almost intact in the combined treatment (6-BAP + H2O2) compared to control (< 52.17% cell viability; < 85.3% mitochondrial activity). On the other hand, mRNA levels of hmgR, idi, crtYB, crtR, and crtS, genes of the astaxanthin biosynthetic pathway, increased transiently along X. dendrorhous fermentation due to stimulations assayed in this study. Key points: • Combined 6-BAP and H2O2 is the best treatment to increase astaxanthin yields in X. dendrorhous. • 6-BAP preserves cell integrity under oxidative H2O2 stress conditions. • 6-BAP and H2O2 increase transcriptional responses of hmgR, idi, and crt family genes transiently. [ABSTRACT FROM AUTHOR]

Proteins with internal repeats (PIRs) are the second most abundant class of fungal cell wall resident proteins. In yeasts, PIRs preserve the stability of the cell wall under stressful conditions. They are characterized by conserved N-terminal amino acid sequences repeated in tandem (PIR motifs), and a cysteine (Cys)-rich C-terminal domain. PIRs have been identified in several filamentous fungi genomes; however, they have not been studied beyond yeasts. In this work, the diversity, evolution, and biological role of PIRs, with a particular focus on a new PIRs class, was addressed. Bioinformatic inference of PIRs in fungi indicated they were an innovation in Ascomycota. Predicted PIRs clustered in two main groups: classical yeasts PIRs (N-terminal PIR motifs; C-terminal Cys-rich domain), and PIRs from filamentous fungi with an inverted architecture (N-terminal Cys-rich domain; C-terminal PIR motifs), which could harbor additional glycosylphosphatidylinositol (GPI) addition-signals. As representatives of the second group, Neurospora crassa (Nc) PIR-1 (NCU04033) and PIR-2 (NCU07569) were studied. Confocal microscopy of eGFP-labeled Nc PIR-1 and Nc PIR-2 revealed they accumulate in apical plugs; additionally, PIR-1 requires the Kex2 processing site for correct maturation and harbors a predicted GPI modification signal. Moreover, Nc Δpir-1 and Δpir-2 single mutants showed a growth rate similar to that of Nc wild-type (WT), but the double mutant Nc Δpir-1/Δpir-2 grew significantly slower. Similarly, Nc Δpir-1 and Nc Δpir-2 were mildly sensitive to calcofluor white, although Nc Δpir-1/Δpir-2 double mutant was severely impaired. Despite the inverted architecture of Nc PIR-1 and Nc PIR-2, they maintain a role as cell wall stabilizers like classical yeast PIRs., (© 2025 Federation of European Biochemical Societies.)