Carotenogenesis in haloarchaea: from genetics to biotechnological and biomedical applications
- Autores: Micaela Giani Alonso
- Directores de la Tesis: Gloria Peiró Cabrera (dir. tes.), Rosa María Martínez Espinosa (dir. tes.)
- Lectura: En la Universitat d'Alacant / Universidad de Alicante ( España ) en 2023
- Idioma: inglés
- Número de páginas: 138
- Tribunal Calificador de la Tesis: Frutos C. Marhuenda Egea (presid.), Antonio Jesús Meléndez Martínez (secret.), Serena Rinaldo (voc.)
- Programa de doctorado: Programa de Doctorado en Ciencias Experimentales y Biosanitarias por la Universidad de Alicante
- Calificación: Sobresaliente Cum Laude
- Materias:
- Enlaces
- Tesis en acceso abierto en: RUA
- Resumen
- español
Los carotenoides son pigmentos sintetizados por una gran variedad de seres vivos, incluyendo hongos, algas, plantas y microorganismos. Son conocidos mayormente por sus propiedades antioxidantes, pero presentan otras propiedades potencialmente beneficiosas, tales como actividad inmunomodulatoria o efectos antitumorales. En la actualidad, hay una demanda creciente de productos de origen natural, y la síntesis microbiana de carotenoides se presenta como una alternativa interesante a la actual síntesis química. Las arqueas halófilas, como Haloferax mediterranei, producen el carotenoide bacteriorruberina (BR), cuya estructura a priori le confiere un gran potencial antioxidante.
El objetivo de este trabajo fue, en primer lugar, optimizar la síntesis de BR utilizando a Haloferax mediterranei como biofactoría y, en segundo lugar, explorar aplicaciones biomédicas de dichos extractos carotenoideos.
En una primera fase, a través de un estudio bioinformático, se identificaron los genes implicados en la síntesis de carotenoides en haloarqueas, los cuales están altamente conservados y se propuso una posible ruta de síntesis. A continuación, se exploró la respuesta de Haloferax mediterranei, en términos de síntesis de BR, a un estrés oxidativo generado por peróxido de hidrógeno. Haloferax mediterranei demostró una elevada tolerancia (hasta 25 mM) y se observó un aumento en la producción del carotenoide en respuesta a concentraciones crecientes del oxidante.
Como resultados del proceso de optimización, la combinación de una reducción en la concentración de sales inorgánicas (12.5%(p/v)), junto con una temperatura del 36,5% y elevada disponibilidad de carbono, en forma de glucosa o almidón (2,5% (p/v)), dio lugar a una de las concentraciones de BR más elevadas que se ha registrado en la literatura.
Además, se caracterizó el potencial antioxidante de los extractos obtenidos a través de cuatro técnicas distintas (ensayos de DPPH, FRAP, ABTS y decoloración del ß-caroteno), confirmando la elevada actividad de los extractos ricos en BR. Por otra parte, se detectó que las variaciones en la concentración de carbono en el medio de cultivo modulan la síntesis de carotenoides, alterando la composición de los extractos y sus propiedades.
Por último, la segunda fase de la tesis estuvo enfocada en la búsqueda de aplicaciones biomédicas. Por ese motivo, se determinó que los extractos ricos en BR producidos por Haloferax mediterranei eran capaces de inhibir las enzimas glucosidasa, amilasa y lipasa pancreática con mayor éxito que inhibidores comerciales, lo cual implica que podrían ser de gran utilidad para reducir el pico de glucosa posprandial en pacientes diabéticos; y la absorción de lípidos a nivel intestinal en pacientes con obesidad. Asimismo, en paralelo se evaluó el efecto de los carotenoides de Haloferax mediterranei en líneas celulares representativas de los subtipos de tumores de mama. Los resultados indicaron que el tratamiento tiene un efecto selectivo sobre las líneas celulares tumorales. Los subtipos más afectados son los tumores luminal A y B, aunque las líneas representativas de tumores triple negativo y aquellos con sobreexpresion de HER2 respondieron favorablemente al tratamiento, lo cual es muy prometedor.
En definitiva, la producción de carotenoides por Haloferax mediterranei puede ser optimizada con éxito a través de la modulación de las condiciones de cultivo, y los extractos obtenidos tienen gran potencial en la industria biotecnológica y biomédica.
- English
Carotenoids constitute a family of pigments which include more than 750 natural compounds synthesised by plants, algae, funghi and microorganisms (1). These coloured molecules are the source of the yellow, orange, and red hues of most organisms (2). Carotenoids are classified according to the number of carbons that make up their structure: 30, 40 and 50, giving rise to subtypes C30, C40 and C50, respectively (3). Chemically, carotenoids are hydrocarbon chains with conjugated double bonds. Among carotenoids, the carotenes are distinguished when the structure is composed only of carbon and hydrogen; and xanthophylls if they also present oxygen atoms. Although the main property that characterises carotenoids is their antioxidant function, various biological activities are attributed to them, such as provitamin A activity, modulation of gene expression, and anti-inflammatory and antitumour activities, among others (4,5).
Currently, the carotenoid market covers the pharmaceutical, cosmetic, nutraceutical, aquaculture and food sectors, both for humans and livestock (5).
Most carotenoid production is carried out by chemical synthesis or by isolation from plants (6). However, there is a growing demand from consumers from high- income countries for products of natural origin and a need for evolution at the biotechnological level to meet this demand (7). The synthesis of carotenoids using microorganisms as biofactories can be more efficient, more respectful to the environment and have a lower cost in comparison to the chemical synthesis or the isolation from other organisms. In addition, it is not limited by seasons, as in the case of production from plants (8). Consequently, the search for cost-effective and safe alternatives has aroused much interest in recent years (5,9–13).
Among the microorganisms currently used for the biotechnological production of carotenoids, yeasts, some bacteria, and microalgae stand out. For example, canthaxanthin (E161G) is a carotenoid approved as a food colorant obtained from the bacterium Bradyrhizobium sp. (14); and lycopene (E160D), which can be extracted from the species Blakeslea trispora (15). More recently, various studies show that halophilic microorganisms such as halophilic microalgae or haloarchaea may be good candidates for the biotechnological production of carotenoids. The use of Dunaliella salina as a producer of β-carotene should be highlighted (16–18). Haloferax mediterranei has positioned itself as a model for the study of haloarchaea for the characterization of various metabolic pathways, such as denitrification or the synthesis of polyhydroxyalkanoates (19–21). Hfx.
mediterranei is an obligate halophilic microorganism, belonging to the Archaea Domain, which requires salt concentrations between 1.5 and 4 M NaCl for its survival (22). It has a very versatile metabolism since it can use various carbon sources, such as carbohydrates, carboxylic acids or alcohols, among others. Hfx.
mediterranei is a heterotrophic facultative aerobe (23), capable of growing under microaerobiosis and anaerobiosis conditions thanks to desnitrification as respiratory pathway in the absence of oxygen (19). It inhabits hypersaline ecosystems where the salt concentration is higher than 10% and there is constant exposure to solar radiation (24). As a consequence, Hfx. mediterranei has developed various defense mechanisms to deal with the osmotic and oxidative stress present in these environments, such as ion accumulation (25), DNA repair mechanisms (26) and the production of the carotenoid bacterioruberin (BR) (13).
BR is a C50 carotenoid whose synthesis is exclusive to halophilic archaea, such as Hfx. mediterranei and some bacteria such as Salinibacter ruber (27). Its structure consists of a hydrocarbon chain that contains thirteen conjugated bonds and four hydroxyl groups that emerge from the terminal ends (28). These characteristics confer it a priori high antioxidant properties, which makes BR a compound of high biotechnological interest (29). However, few studies have evaluated the antioxidant capacity of this carotenoid (30–40) and there are hardly any references to its possible antitumour and immunomodulatory activity (37,41), relevant biological activities that could have significant applications in Biomedicine and Biotechnology in connection with pathologies such as cancer.
In the last two decades, for various antioxidant compounds, including carotenoids, the ability to reduce the proliferation of tumour cells of different types has been demonstrated in vitro (42–44). For example, lycopene can repress the NF-kB pathway in lung and prostate cancer (45), and fucoxanthin inhibits proliferation in glioblastoma, colon, and melanoma, among others (46).
However, the lack of cohesion between studies regarding the dose used, cell lines and approach (preventive or therapeutic) makes it difficult to determine the mechanisms involved and the best strategy to use this type of compound in cancer treatment.
Cancer is the leading cause of death worldwide, with approximately 10 million deaths in 2020 (47). Specifically, breast cancer was diagnosed in 2,261,419 patients in 2020, being the most frequently diagnosed type of cancer in women (47). The presence or absence of hormone receptors, protein overexpression and/or amplification of the HER2 gene and the cell proliferation marker Ki67, determine the different phenotypes: Luminal A (RH+ HER2-), Luminal B (RH+, HER2±), Triple-Negative (RH-, HER2-) and HER2+ (RH- HER2+)(48). Although treatments are becoming more effective and the mortality rate is decreasing, Triple-Negative breast cancer, which accounts for 15-20% of tumours, is more aggressive and still has an unfavourable prognosis as well as a high recurrence rate (49). Tumour cells exhibit an alteration in redox homeostasis. There is a relationship between chronic inflammation, reactive oxygen species (ROS) production, and tumour necrosis factor-alpha (TNFα)-mediated signalling (50,51). It is widely known that prolonged exposure to ROS can lead to DNA damage and may be related to an increased risk of cancer (52,53). However, there is some controversy that ROS can dually stimulate carcinogenesis, senescence, and apoptosis in neoplastic cells (54).
At the beginning of this doctoral thesis, knowledge about the optimization of BR production and its potential biological activity was limited. Regarding the BR synthesis pathway, a carotenogenesis pathway proposal had been made in Haloarcula japonica (55) and in Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi and Natronomonas pharaonic (56). In terms of optimization, there was some consensus on the relevance of environmental conditions in inducing BR synthesis (57–60). And finally, regarding its possible beneficial effect, antimicrobial (40) and antiproliferative effects had been reported in liver cancer cells (37,61) and in other cell lines (41), as well as its usefulness in improving sperm motility after cryopreservation (36).
However, although some studies have partially evaluated the antioxidant activity of carotenoids from haloarchaea (30,31,33–39), the complete antioxidant activity profile of BR had not been explored to date nor its effect on breast cancer tumour cells representative of the different subtypes of this neoplasm.
Starting from this premise, the hypotheses of this doctoral thesis are: i) that the modification of temperature, salinity and carbon source can stimulate the production of BR and that this compound has great antioxidant potential; ii) in line with the biological activity exhibited by other carotenoids, BR can exert an antiglycemic and antilipidemic effect through the inhibition of enzymes implicated in the lipic and glucide metabolism, as well as an antitumour effect on breast cancer cells. Thereupon, this doctoral thesis entails an advance in the processes of optimisation of the production of BR for an after scaling, in the knowledge about the metabolic pathway called carotenogenesis, and in the search for biomedical applications for a said compound. The objectives of this research work are:
1. To conduct an extensive search on the knowledge to date of the carotenogenesis pathway and determine the main genes involved in the synthesis of BR in haloarchaea, particularly in Hfx. mediterranei.
2. To confirm the antioxidant role of BR as a defense mechanism against oxidative stress caused by an oxidizing agent, such as hydrogen peroxide, in Hfx.
mediterranei.
3. To optimize the production of carotenoids in the wild strain of Hfx. mediterranei R-4, through the modification of the conditions of the medium.
4. To explore potential applications in biomedicine of carotenoid extracts obtained from Hfx. mediterranei cells, specifically in the activity of different enzymes implicated in glucide and lipid metabolism, and in breast cancer cell lines.
- español
