Role of regulatory proteins in carotenoid biosynthesis and metabolism in Fusarium fujikuroi

Abstract

La capacidad de los hongos filamentosos de adaptarse a cambios ambientales a cambios ambientales ha conducido al desarrollo de mecanismos sofisticados, como la producción de metabolitos secundarios que, a pesar de no ser esenciales para estos organismos, usualmente les supone una ventaja adaptativa. Dentro del género Fusarium, se encuentran diversas estirpes reconocidas como grandes patógenos de plantas, causando pérdidas económicas significativas a nivel global. Además de su virulencia y producción de micotoxinas, estos hongos producen metabolitos con interés industrial, como los carotenoides. F. fujikuroi produce un carotenoide particular llamado neurosporaxantina, que ha demostrado tener un gran poder antioxidante en estudios in vitro, y actividad provitamina A y buena absorción en ratones. Lo que le otorga un gran interés biotecnológico. Aunque la ruta bioquímica y los genes estructurales de esta producción están descritos, aspectos del mecanismo regulatorio aún permanecen sin resolver. Esta tesis contribuye principalmente a profundizar en la comprensión de reguladores involucrados en la síntesis de carotenoides y en el metabolismo secundario del hongo F. fujikuroi. Se esclarece el rol del complejo White-Collar en F. fujikuroi, confirmando su mecanismo de acción como un heterodímero en este hongo. Se profundiza en el estudio de la proteína CarS, previamente identificada en mutantes súper productores de carotenoides. La sobreexpresión de carS confirma su papel como represor de la síntesis de carotenoides, provocando una represión absoluta de los genes involucrados en la síntesis y de la propia producción de este metabolito. Además, la generación de perfiles metabólicos a partir de datos de GC-MS y HPLC contribuyó a comprender el impacto de CarS y la luz en el metaboloma de este hongo. Finalmente, se aborda el estudio de un gen que codifica un factor de transcripción, ubicado junto a los genes de la carotenogenesis en el genoma. Su deleción y sobreexpresión no tuvieron efecto sobre la carotenogénesis, y por el contrario, afectaron varios genes involucrados en el transporte y asimilación de compuestos nitrogenados, lo que sugiere una función reguladora en el metabolismo del nitrógeno.

Filamentous fungi are subject to many environmental changes that compromise their survival in nature. The ability to sense and transduce environmental signals has given them an advantage to prosper in most diverse ecological niches. For this reason, these organisms have developed molecular systems to respond adequately to a changing environment. An excellent example is found in the sophisticated regulatory mechanisms that fungi use to control the production of secondary metabolites, which includes complex photosensory systems and mechanisms specifically involved in nutrients availability sensing. Beyond its own adaptation, these machineries have an impact on plants and human lives. An outstanding example are Fusarium fungi, which comprise many phytopathogenic species responsible for agriculture damage and huge economic losses around the world. Because of its extreme complexity, the secondary metabolism of F. fujikuroi has been focus of study for several decades. The many secondary metabolites produced by this fungus includes gibberellin phytohormones, responsible of elongating symptoms of the bakanae plant disease, mycotoxins, such as fumonisin and fusarins, and pigments, such as bikaverin, fusaric acid, and carotenoids. Due to the industrial interest of carotenoids, we focus our attention on the regulation of their biosynthesis. The main carotenoid produced by this fungus is neurosporaxanthin, an unusual xanthophyll that has shown interesting antioxidant properties in vitro and good absorption and provitamin A activities in mice. The production of carotenoids is regulated primarily by light, but is also affected by other environmental signals, such as nitrogen starvation, oxidative stress, or heat stress. Currently, all structural genes and biosynthetic steps of the carotenoid pathway have been described, and some regulatory proteins have been investigated. However, most aspects of the regulatory mechanisms remain to be understood. This thesis investigates some aspects of the molecular mechanisms governing the production of carotenoids and other secondary metabolites. The first chapter delves into the impact of white-collar proteins WcoA and WcoB on carotenoid biosynthesis and secondary metabolism in the F. fujikuroi wild type strain IMI58289, elucidating the role of a putative WcoA/B complex in the fungus, and provides a comparative with the knowledge of WcoA function in F. fujikuroi FKMC1995 strain. The results confirmed the action of WcoA as a heterodimer with the WcoB protein, as indicates the same phenotype exhibited by mutants for both genes in most of the affected traits investigated. In addition, it showed regulatory differences between the functions of WcoA in IMI58289 and those formerly described in FKMC1995, suggesting a functional diversification of this class of regulatory protein. The following two chapters are focused on the regulatory roles of protein CarS. First, the overexpression of the encoding carS gene is described using two strategies: with the use of a constitutive promoter from the gpdA gene of A. nidulans, and with the Tet-on system, which allows external modulation of the expression of a target gene by using a promoter inducible by the addition of an antibiotic. In both cases, carS overexpression resulted in a total repression of the carotenoid pathway, indicating a fine modulation of carotenoid synthesis by the concentration of CarS in the cell and confirming its role as a repressor of the biosynthetic pathway. Former data indicated that the CarS protein influences the transcription of hundreds of F. fujikuroi genes, many of them involved in metabolism. For this reason, metabolomic techniques were employed in the third chapter to extend our understanding of CarS function through the study of the metabolic consequences of changing carS expression. This approach was based on GC-MS data revealed that the metabolome is not deeply impacted by light or carS mutation, but it is influenced by carotenoid production. This evidence was supported by correlation among carotenoid phenotype and the metabolic profile among the different mutants, revealing metabolic similarities between carS mutants and carS-overexpressing albino strains. In the last chapter, the function of a gene encoding a predicted transcription factor adjacent to the genes of the car cluster, and therefore suspected to be involved in the regulation of carotenoid biosynthesis, was investigated. The results indicated a lack of role of this gene in the regulation on carotenogenesis, but nutZ overexpression resulted in deregulation of genes related to transport or catabolic enzymes involved in nitrogen assimilation. This result, together with the low expression of nutZ in culture conditions with nitrate as the only N source, is consistent with a role of NutZ on nitrogen metabolism dependent on the presence of certain nitrogen sources, with implications in nitrogen-regulated secondary metabolism.