Development of the “Applied Proteomics” Concept for Biotechnology Applications in Microalgae: Example of the Proteome Data in Nannochloropsis gaditana

Most of the marine ecosystems on our planet are still unknown. Among these ecosystems, microalgae act as a baseline due to their role as primary producers. The estimated millions of species of these microorganisms represent an almost infinite source of potentially active biocomponents offering unlimited biotechnology applications. This review considers current research in microalgae using the “omics” approach, which today is probably the most important biotechnology tool. These techniques enable us to obtain a large volume of data from a single experiment. The specific focus of this review is proteomics as a technique capable of generating a large volume of interesting information in a single proteomics assay, and particularly the concept of applied proteomics. As an example, this concept has been applied to the study of Nannochloropsis gaditana, in which proteomics data generated are transformed into information of high commercial value by identifying proteins with direct applications in the biomedical and agri-food fields, such as the protein designated UCA01 which presents antitumor activity, obtained from N. gaditana

Development of New Antiproliferative Compound against Human Tumor Cells from the Marine Microalgae Nannochloropsis gaditana by Applied Proteomics

Proteomics is a crucial tool for unravelling the molecular dynamics of essential biological processes, becoming a pivotal technique for basic and applied research. Diverse bioinformatic tools are required to manage and explore the huge amount of information obtained from a single proteomics experiment. Thus, functional annotation and protein-protein interactions are evaluated in depth leading to the biological conclusions that best fit the proteomic response in the system under study. To gain insight into potential applications of the identified proteins, a novel approach named "Applied Proteomics" has been developed by comparing the obtained protein information with the existing patents database. The development of massive sequencing technology and mass spectrometry (MS/MS) improvements has allowed the application of proteomics nonmodel microorganisms, which have been deeply described as a novel source of metabolites. Between them, Nannochloropsis gaditana has been pointed out as an alternative source of biomolecules. Recently, our research group has reported the first complete proteome analysis of this microalga, which was analysed using the applied proteomics concept with the identification of 488 proteins with potential industrial applications. To validate our approach, we selected the UCA01 protein from the prohibitin family. The recombinant version of this protein showed antiproliferative activity against two tumor cell lines, Caco2 (colon adenocarcinoma) and HepG-2 (hepatocellular carcinoma), proving that proteome data have been transformed into relevant biotechnological information. From Nannochloropsis gaditana has been developed a new tool against cancer-the protein named UCA01. This protein has selective effects inhibiting the growth of tumor cells, but does not show any effect on control cells. This approach describes the first practical approach to transform proteome information in a potential industrial application, named "applied proteomics". It is based on a novel bioalgorithm, which is able to identify proteins with potential industrial applications. From hundreds of proteins described in the proteome of N. gaditana, the bioalgorithm identified over 400 proteins with potential uses; one of them was selected as UCA01, "in vitro" and its potential was demonstrated against cancer. This approach has great potential, but the applications are potentially numerous and undefined

Biomasa de valorización de microalgas mediante enfoques clásicos y moleculares

La presente tesis doctoral ha sido desarrollada en el grupo de investigación de Microbiología del Instituto de Vitivinícolas y Agroalimentarias (IVAGRO) de la Universidad de Cádiz. Este trabajo, enmarcado dentro del proyecto nacional Producción de biocompuestos saludables de microalgas, A4HW (RTC-2016-4860-2), tiene como objetivo principal la revalorización de la biomasa de microalgas, mediante el desarrollo de nuevas aplicaciones en el campo de la biomedicina y la agroalimentación. El interés por el cultivo y las aplicaciones de las microalgas se ha incrementado de forma exponencial en estos últimos años. Este incremento se debe a sus potenciales aplicaciones industriales, y su capacidad para resolver problemas cruciales ocasionados por las actividades humanas, como por ejemplo las emisiones de efecto invernadero, la contaminación del agua, el agotamiento de los combustibles fósiles y la necesidad de nuevas terapias médicas. Este interés ha colocado a las microalgas como un elemento transversal de los esfuerzos para desarrollar nuevas herramientas biotecnológicas impulsadas por la iniciativa "Blue Growth" de la UE, teniendo como objetivo apoyar el crecimiento sostenible en los sectores marítimos marinos y costeros. Esta iniciativa viene a sumarse a las directrices de la economía circular. Esta tesis se centrará en determinar nuevos compuestos procedentes de la biomasa de microalgas mediante aproximaciones clásicas y moleculares, con aplicaciones en Agroalimentación y Biomedicina, así como el desarrollo de aproximaciones de “Proteómica Aplicada” que permitan determinar nuevas aplicaciones biotecnológicas a partir del proteoma de las microalgas. Una de las estrategias elegidas en la presente tesis para revalorizar la biomasa de microalgas, fue la extracción de carbohidratos de diferentes especies de microalgas (Arthrospira platensis (spirullina) (Endesa Generación, S. A.), Pyrocistis lúnula (CCAP 1131/1) y Nannochloropsis gaditana (Lubian CMP 572)., desarrollando un método para la extracción de azucares simples y polialcoholes, este método ha sido diseñado para facilitar su escalado industrial. Los datos obtenidos posicionan a las microalgas como una fuente alternativa de carbohidratos y polialcoholes, habiendo alcanzado niveles extractivos en torno al 50% de los obtenidos a partir de biomasa vegetal. Este desarrollo supondría la primera extracción de edulcorantes procedentes de organismos marinos (microalgas), orientados a la alta gastronomía. Durante el desarrollo de esta Tesis Doctoral se ha determinado por primera vez el proteoma de Nannochlropsis gaditana en condiciones industriales, comparando sus principales formas comerciales, alga fresca y atomizada. Este análisis nos permitió identificar 655 proteínas presentes en todas las réplicas que fueron categorizadas mediante ontología génica según su actividad biológica y su función molecular. Este análisis del proteoma se realizó con tres objetivos, obtener la primera aproximación proteómica de N. gaditana, analizando además la comparación entre sus formatos comerciales y por último, detectar proteínas con un potencial uso para desarrollar futuras aplicaciones biotecnológicas. Para demostrar la validez de las aproximaciones mediante proteómica aplicada y a partir del proteoma de N. gaditana, Se seleccionó una de las proteínas más prometedoras por su potencial aplicación industrial en el campo de la biomedicina. Se sintetizo y expreso la proteína UCA01 recombinante. Esta Proteína ha demostrado capacidad antitumoral, en dos líneas tumorales, adenocarcinoma de hígado (HepG2) y adenocarcinoma de colón (Caco-2). Así como su inocuidad en líneas celulares no tumorales. Este descubrimiento ha sido remitido a la oficina española de patentes y marcas siendo admitida a trámite con la referencia P201930775. Estos datos avalan la aplicación directa de la proteómica (“proteómica aplicada”), como herramienta de detección de proteínas con un potencial uso biotecnológico, así como su utilidad para la revalorización de la biomasa de microalgas. En resumen, esta tesis doctoral, proporciona el primer edulcorante alimenticio extraído del mar, además del primer estudio del proteoma de N. gaditana en condiciones industriales. El estudio de estos proteomas se ha convertido en una información de alto valor industrial, mediante la aplicación de un enfoque de proteómica aplicada, revelando multitud de posibles aplicaciones industriales potenciales, principalmente en el campo de la agroalimentación y de la biomedicina. Para demostrar la viabilidad del enfoque se ha demostrado la capacidad antiproliferativa de la proteína UCA01, obtenida a partir de N. gaditana en dos líneas tumorales, HepG2 (carcinoma del hígado), Caco-2 (adenocarcinoma de colón). Estos datos abren nuevas vías de mercado para lograr una valorización relevante de la biomasa microalgal

Deciphering the Dynamics of Signaling Cascades and Virulence Factors of B. cinerea during Tomato Cell Wall Degradation

The ascomycete Botrytis cinerea is one of the most relevant plant pathogenic fungi, affecting fruits, flowers, and greenhouse-grown crops. The infection strategy used by the fungus comprises a magnificent set of tools to penetrate and overcome plant defenses. In this context, the plant-pathogen communication through membrane receptors and signal transduction cascades is essential to trigger specific routes and the final success of the infection. In previous reports, proteomics approaches to B. cinerea signal transduction cascades changes in response to different carbon source and plant-based elicitors have been performed. Analyzing the secretome, membranome, phosphoproteome, and the phosphomembranome. Moreover, phenotypic changes in fungal biology was analyzed, specifically toxin production. To obtain the whole picture of the process and reveal the network from a system biology approach, this proteomic information has been merged with the phenotypic characterization, to be analyzed using several bioinformatics algorithms (GO, STRING, MCODE) in order to unravel key points in the signal transduction regulation crucial to overcome plant defenses, as well as new virulence/pathogenicity factors that could be used as therapeutic targets in the control of the gray mold rot disease. A total of 1721 and 663 exclusive or overexpressed proteins were identified under glucose (GLU) and deproteinized tomato cell walls (TCW), summarizing all of the protein identifications under phenotypic characterized stages. Under GO analysis, there are more biological process and molecular functions described in GLU, highlighting the increase in signaling related categories. These results agree with the high number of total identified proteins in GLU, probably indicating a more varied and active metabolism of the fungus. When analyzing only GO annotations related with signal transduction, it was revealed that there were proteins related to TOR signaling, the phosphorelay signal transduction system, and inositol lipid-mediated signaling, only under GLU conditions. On the contrary, calcium-mediated signaling GO annotation is only present between the proteins identified under TCW conditions. To establish a potential relationship between expressed proteins, cluster analyses showed 41 and 14 clusters under GLU and TCW conditions, confirming an increase in biological activity in GLU, where we identified a larger number of clusters related to transcription, translation, and cell division, between others. From these analyses, clusters related to signal transduction and clusters related to mycotoxin production were found, which correlated with the phenotypic characterization. The identification of the proteins encompassed in each condition and signal transduction cascade would provide the research community with new information about the B. cinerea infection process and potential candidates of pathogenicity/virulence factors, overcoming plant defenses, and new therapeutic targets.The present research was made possible due to the funding received from Universidad de Cadiz. Project: development of new proteomic approaches to B. cinerea to detect rapid changes in signaling cascades responsible for triggering the first steps of phytopathogenic infective processesPROTEOCAS (ref. PR2020-002)

Development of the “Applied Proteomics” Concept for Biotechnology Applications in Microalgae: Example of the Proteome Data in Nannochloropsis gaditana

Most of the marine ecosystems on our planet are still unknown. Among these ecosystems, microalgae act as a baseline due to their role as primary producers. The estimated millions of species of these microorganisms represent an almost infinite source of potentially active biocomponents offering unlimited biotechnology applications. This review considers current research in microalgae using the “omics” approach, which today is probably the most important biotechnology tool. These techniques enable us to obtain a large volume of data from a single experiment. The specific focus of this review is proteomics as a technique capable of generating a large volume of interesting information in a single proteomics assay, and particularly the concept of applied proteomics. As an example, this concept has been applied to the study of Nannochloropsis gaditana, in which proteomics data generated are transformed into information of high commercial value by identifying proteins with direct applications in the biomedical and agri-food fields, such as the protein designated UCA01 which presents antitumor activity, obtained from N. gaditana