Abstract
The red yeast X. dendrorhous is one of the few natural sources of astaxanthin, a carotenoid used in aquaculture for salmonid fish pigmentation and in the cosmetic and pharmaceutical industries for its antioxidant properties. Genetic control of carotenogenesis is well characterized in this yeast; however, little is known about the regulation of the carotenogenesis process. Several lines of evidence have suggested that carotenogenesis is regulated by catabolic repression, and the aim of this work was to identify and functionally characterize the X. dendrorhous MIG1 gene encoding the catabolic repressor Mig1, which mediates transcriptional glucose-dependent repression in other yeasts and fungi. The identified gene encodes a protein of 863 amino acids that demonstrates the characteristic conserved features of Mig1 proteins, and binds in vitro to DNA fragments containing Mig1 boxes. Gene functionality was demonstrated by heterologous complementation in a S. cerevisiae mig1- strain; several aspects of catabolic repression were restored by the X. dendrorhous MIG1 gene. Additionally, a X. dendrorhous mig1- mutant was constructed and demonstrated a higher carotenoid content than the wild-type strain. Most important, the mig1- mutation alleviated the glucose-mediated repression of carotenogenesis in X. dendrorhous: the addition of glucose to mig1- and wild-type cultures promoted the growth of both strains, but carotenoid synthesis was observed only in the mutant strain. Transcriptomic and RT-qPCR analyses revealed that several genes were differentially expressed between X. dendrorhous mig1- and the wild-type strain when cultured with glucose as the sole carbon source. The results obtained in this study demonstrate that catabolic repression in X. dendrorhous is an active process in which the identified MIG1 gene product plays a central role in the regulation of several biological processes, including carotenogenesis.
Highlights
Most microorganisms can utilize a variety of substrates as carbon sources for growth; hierarchical mechanisms have been developed to preferentially utilize glucose over other carbon sources when this sugar is available
The expression of yeast genes involved in the use of alternative carbon sources other than glucose is usually inhibited by catabolic repression
Extracellular invertase activity was assayed during growth as this activity was not previously detected in a glucose-based medium, suggesting its catabolic repression [29], which occurs in other organisms [13]
Summary
Most microorganisms can utilize a variety of substrates as carbon sources for growth; hierarchical mechanisms have been developed to preferentially utilize glucose over other carbon sources when this sugar is available. The presence of glucose in the culture medium leads to rapid and dramatic repression of the expression of a large number of genes, including altered expression patterns of genes involved in the metabolism of alternative carbon sources. In the presence of glucose, this sugar is sensed at the cell membrane [9,10], activating an intracellular signaling cascade that leads to the activation of the Reg1-Glc7-phosphatase complex, which dephosphorylates Mig1 [11,12] In this state, Mig migrates to the nucleus where it binds to the promoters of target genes [1,13] and recruits a co-repressor complex formed by the proteins Tup and Cyc, which exerts transcriptional repression functions [8,14,15]. Mig is phosphorylated by the Snf kinase complex, causing it to lose its interaction with the Cyc8-Tup complex and be transported to the cytoplasm, releasing target gene repression [16]
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