Aislamiento, caracterización y manipulación genética de microalgas marinas para la producción de compuestos de alto valor añadido

Abstract

Microalgae are a group of photosynthetic microorganisms with high attractiveness due its simple structure and unicellular nature, which facilitates its cultivation and genetic manipulation (Chapter 1). Nowadays, there is increasing interest in microalgae as a source of high-value compounds such as vitamins, antioxidants, lipids, proteins, etc. Moreover, the rapid growth rate outdoors makes them good candidates for bulk production of many compounds, including biodiesel, although its commercial applications are still limited. The best strategy to find microalgae well adapted to the local climatological conditions, able to simultaneously grow at high rates and produce different compounds of biotechnological interest is the selection of new autochthon strains. In chapter 2, the isolation and characterization of a new microalgae isolated from the marshlands of Odiel River is described. The new microalga belonging to the genus Picochlorum, is able to grow at a high growth rate and thrive with adverse conditions. The fatty acids profile of this microalga makes it a promising candidate for the production of biodiesel, and its high content in the carotenoids lutein and zeaxanthin indicates that the microalga could also be a good source of natural eye vitamin supplements. However, for an economically feasible biodiesel production based of this microalga, the lipid content should be significantly increased. In chapter 3 we have optimized the cultivation method of this microalga in order to improve the content of neutral lipids. The optimization of the operation mode, the initial nutrient content and the bioreactor diameter allowed increasing four-fold the final biomass of Picochlorum sp HMl. Furthermore, by mixotrophic and nutritional starvation conditions, the neutral lipids synthesis in the microalga was induced. Nitrate and phosphate starvation resulted in an increase of neutral lipids, of about 2.27 and 2-fold, respectively. With a two-step culture method, a lipid values up to 10 times higher than in the control culture could be achieved. The genetic manipulation of microalgae is a promising strategy claimed for many authors as the best approach to obtain production systems of compunds with commercial interest. Despite the biotechnological interest of microalgae, no robust and stable methods for genetic transformation of most microalga] strains exist and most of the work in this field has been carried out with a couple of strains. In Chapter 4 approaches for the genetic transformation of marine microalgae Tetraselmis suecica, Dunaliella salina and the recently identified Picochlorum sp HMl have been established. Transformation conditions for the three strains were optimized using the gene that confers resistance to the antibiotic paromomycin (APHVIII) and the gene that confers resistance to the antibiotic zeocin (BLE), both under the control of different heterologous promoters. Lipid metabolism is a network involving hundreds of proteins and many subcellular compartments, including plastids, endoplasmic reticulum and lipids granules. The acyl-CoA- diacylglycerol acyltransferase (DGAT) plays an important role in the triacylglycerols biosynthesis, being the last enzyme in the biosynthesis pathway. These lipids have a high value due to both their use in human and animal nutrition and as feedstock for the production of third generation biodiésel. In Chapter 5 the gene EpDGATl from the boraginaceae Echium pitardi was overexpressed in the model microalga Chlamydomonas reinhardtii, by transformation with a binary expression vector. Two obtained transformants were selected in which high gene expression was observed and a constitutive neutral lipids accumulation up 30% over that in the control culture. This is the first time that this gene is overexpressed in Chlamydomonas reinhardtii with satisfactory results, opening a promising line of study for the fatty acids biosynthesis pathway in this and other microalgae, as well as a valuable tool for the development of high added value products in microalgae.