Optimización de herramientas moleculares para la manipulación genética de microalgas y su aplicación en procesos de biofloculación

Abstract

Microalgae are a highly diversified group of eukaryotic and photosynthetic microorganisms with a relevant biotechnological potential because of they are producers of numerous bioproducts used in feed, cosmetic, pharmaceutical and bioenergy industry. The harvesting of the microalgal biomass becomes particularly important because it is a critical step which accounts for about 20-30% of the total production cost and its improvement is necessary. In this thesis, the optimization and the development of molecular tools are performed for genetic manipulation of microalgae and their further application for the improvement of bioflocculation methods to harvest microalgal biomass. In Chapter 2, using the genetic transformation developed for Chlamydomonas reinhardtii, the efficiency of the heterologous promoters of cauliflower mosaic virus 35S (CaMV 35S) and Agrobacterium nopaline synthase (NOS) genes was evaluated. The paromomycin resistance gene (APHV1II) was selected as marker gene. The transformation efficiency and the APHVIII transcript and protein levels were evaluated in a series of transformants for each promoter. Among the two heterologous promoters used, CaMVSSS and NOS, the highest transformation efficiencies and levels of APHVIII expression were found using the NOS promoter. Although the use of heterologous promoters has been a suitable technique for the expression of exogenous genes in microalgae, the search of endogenous promoters could be a feasible alternative to find a strong universal microalgal promoter. In Chapter 3, a nuclear genetic transformation in Chlamydomonas reinhardtii cells was carried out, using the APHVIII as a promoterless marker gene. The random insertions of the promoterless in the most robust selected transformants was demonstrated, allowing the identification of novel strong promoter sequences in microalgae. The inverse PCR technique allowed an easy determination of the genomic region, which precedes the marker gene. In most of the transformants analysed, the marker gene was inserted in intragenic regions and its expression relied on its adequate insertion in frame with native genes. In Chapter 4, the efficiency of the two heterologous promoters and APHVIII promoterless checked in Chapter 2 and 3, were also used for genetic transformation of the industrially important microalga Chlorella sorokinianci. Firstly, a cheap, simple and feasible method of electroporation was developed for this microalga, defining 2.5 kV of electric field strength and 3 electric pulses as the suitable parameters. And then, the efficiencies of the chosen promoters was analysed. The results showed the best efficiency values using the CaMV 35S promoter, in contrast with the results obtained for C. reinhardtii, confirming thus, that the heterologous promoters currently used are strongly specie-dependent. In the following chapters, physiological and molecular tools are developed and optimized to the improvement of bioflocculation methods for the microalgal biomass harvesting. In Chapter 5, the highly self-flocculating yeast Saccharomyces bayamis var. uvarum and its flocculating factors are used to induce flocculation in the two microalgae C. reinhardtii and Picochlorum sp. HM1. The addition of Saccharomyces and flocculating factors induced cell aggregation in both microalgal species studied, resulting in maximum recovery efficiency values of 95% and 75% for Chlamydomonas and Picochlorum respectively. In order to induce self-flocculating phenotypes in C. reinhardtii cells, in Chapter 6, a wild type strain of this chlorophyte was genetically transformed with a flocculin gene (FLOS) from the self-flocculating yeast Saccharomyces bayamis var. uvarum and, three Chlamydomonas transformants, CrFLOSll, CrFL0513 and CrFL0520, were identified having integrated the flocculin gene into their genome and exhibiting self-flocculating phenotypes.