Metabolic and molecular approaches for the integrated production of bioenergetic precursors in microalgae

Abstract

Microalgae are photosynthetic microorganisms with a great ability of adapting to adverse conditions, developing regulation mechanisms to maintain optimal carbon/nitrogen ratios in the cell for survival. When these microorganisms are submitted to environmental stress conditions, the carbon initially devoted to the synthesis of structures is deviated towards the production of storage compounds. This doctoral thesis is framed within the response of the microalga Chlamydomonas reinhardtii to the absence of nitrogen in the culture medium. Under this nutrient deficiency, the assimilated carbon is relocated to the synthesis of the main reserve compounds: triacylglycerols (TAG) and starch. These compounds are of high interest due to their potential as precursors in the production of energetic compounds, such as biofuels or chemical building blocks in the industry. Other bioactive compounds that are synthetized by microalgae are carotenoids, widely used in pharmacological and pigment industry for their antioxidant properties. Therefore, scale-up processes where microalgae biomass is used for the production of several compounds for commercialization, in a sustainable and integrated way, imply to identify and enhance the key bottlenecks and difficulties in the production stage. To obtain starch and TAG, induction must be made under stress conditions, which limits cell growth because of the production of storage compounds at the expense of natural cell cycle and division. In this thesis, a first molecular approach is made for the enhancement of neutral lipids in Chlamydomonas reinhardtii, overexpressing a gene responsible for the increase of carbon flux upstream the specific neutral lipid pathway (Chapter 1). On the other hand, a second genetic engineering approach is followed, overexpressing a gene directly involved in TAG assembly (Chapter 2). In addition, the hypothesis whether the simultaneous overexpression of these genes, can synergistically improve the levels previously reached by carbon flux enhancement, is assessed (Chapter 3). Finally, the utilization of the microalgal biomass for the simultaneous production of carotenoids and starch under nitrogen starvation, and the use of starch for the production of chemical precursors is studied (Chapter 4). In Chapter 1, the effect of the overexpression of the endogenous gene, encoding the chloroplastic acetyl- CoA synthetase (ACS2), from Chlamydomonas reinhardtii, is presented. This enzyme is responsible for the direct assimilation of acetate into acetyl-CoA, which, among other metabolic pathways, feeds the route involved in fatty acid synthesis. The results show an increase in starch production under replete conditions, and a rise in acyl-CoA intermediates, which are further incorporated to TAG biosynthesis. Transfer of cultures to nitrogen depletion causes TAG production in the transformant in a greater proportion than the parental line, and higher transcript levels of the ACS2 gene as a consequence of its overexpression. In Chapter 2, the heterologous expression of the gene encoding a type-1 diacylglycerol acyltransferase, from the boraginaceae Echium pitardii (DGAT1) in Chlamydomonas reinhardtii, is studied. This enzyme catalyzes the last step of assembly of an acyl chain in a diacylglycerol backbone to yield a molecule of TAG. The transformant shows a higher amount of total fatty acids, and a significant rise of the neutral lipid fraction under nitrogen depletion conditions. A higher uptake of acetate from the medium, the presence of intracellular glycerol and a slower degradation of the photosynthetic apparatus compared to the parental line, are also observed. In Chapter 3, the generation of acs transformants, and the generation of double acs/dgat transformants from the single acs clones, is achieved with the aim of establishing a comparison between them. Results show a synergistic enhancement in the neutral lipid content in the double transformant, which simultaneously expresses both genes. In addition, the single acs and the double acs/dgat transformants present a higher amount of carotenoids under replete conditions, a higher content of starch in replete and N-depleted conditions, and a better evolution of photosynthetic efficiency throughout nutrient deficiency. Finally, in Chapter 4, the ability of Chlamydomonas reinhardtii to accumulate starch under nitrogen starvation is utilized for the production of the chemical precursors 5’-hydroxymethylfurfural (5’-HMF) and levulinic acid (LA). These intermediates are used in the industry for the synthesis of biofuels, additives, polymers or molecular imaging probes. The application of a Central Composite Design and Response Surface Methodology allowed to determine the influence of the operational conditions involved in the acidic hydrolysis of starch, firstly to glucose, and in a further step, to 5’-HMF and LA. The best temperature, acid percent, DMSO percent and reaction time conditions were selected for each of the three precursors. Moreover, the extraction of carotenoids, prior to hydrolysis, allows to set the basis for an integrated biorefinery process, aiming to produce several compounds of interest from a sole biomass.