CONTROL OF PHOTOSYNTHETIC CARBON ALLOCATION BETWEEN PROTEINS, LIPIDS AND CARBOHYDRATES IN CYANOBACTERIA AND DIATOMS AND ITS IMPLICATIONS FOR BIOFUEL PRODUCTION

Abstract

Aquatic Microbial Oxygenic Phototrophs (AMOP's) capture carbon dioxide from the atmosphere and fix it into organic molecules used for growth and proliferation. However, the mechanisms that decide how photosynthetically fixed carbon is allocated between the major sinks (proteins, lipids, and carbohydrates) are not well understood, thus limiting the potential of AMOPs as biofuel factories. In the first three chapters of this dissertation several experiments are shown where the normal carbon distribution was genetically or environmentally perturbed in a model cyanobacterium (Synechococcus sp. strain PCC 7002) or in a diatom (Phaeodactylum tricornutum). The goal was to gain a better understanding of the mechanisms that control carbon allocation and to learn the impact of altered carbon compositions on biofuel productivities. In the last chapter the diatom strain was investigated for its hydrogen sulfide production capability.

In Chapter 1 the effects of glycogen synthase null mutants of Synechococcus sp. PCC 7002 on carbohydrate accumulation were explored under standard, nitrogen limited and hypersaline conditions. The hypothesis was that carbon from glycogen could be diverted into smaller sugars, converting these cells into soluble sugar factories. The results show that these mutants do increase their soluble sugar content and that under some conditions they spontaneously excrete soluble sugars to the medium confirming our hypothesis. Furthermore, under nitrogen limiting conditions evidence shows the existence of a currently unknown regulatory mechanism that directly links nitrogen recycling via pigment degradation and glycogen synthesis. In addition, a model predicts that about 39% of the photosynthetic capacity of this strain could be diverted to biofuel precursor production without affecting growth.

In Chapter 2 an ADP-glucose pyrophosphorylase null mutant of the same cyanobacterial strain was examined. This strain has severely impaired carbohydrate content but a larger soluble sugar fraction. The goal was to investigate what are the preferred substrates for autofermentation and hydrogen production in cyanobacteria. The results show that the lack of polymeric carbohydrates severely reduces respiratory and autofermentation catabolic rates and consequently implying that soluble sugars are not good substrates for hydrogen production. Furthermore, analysis of the intracellular metabolite pools during fermentation strongly suggests that glyceraldehyde-3-phosphate dehydrogenase is a metabolic choke point. Thus strains that relieve this choke point and have increased glycogen content should be pursued for enhanced biohydrogen production.

In Chapter 3 the model diatom Phaeodactylum tricornutum was used to explore the metabolic branch points involved in regulating the cellular lipid accumulation under conditions of nitrogen starvation. By analyzing the metabolite and mRNA pools of central carbon metabolism it was established the central role of the glutamine synthetase, glutamine oxoglutarate aminotransferase and glutamate dehydrogenase pathways on the switch between protein and lipid biosynthesis in diatoms. Furthermore, the more reducing environment may further facilitate lipid biosynthesis as a substrate and as a gene transcription regulator. In addition, the calculated average fluxes of carbon into proteins and lipids was correlated to their energetic cost and reveals that nitrogen deprivation, despite increasing cellular lipid content, is not a bioenergetically viable strategy for biofuel production.

Finally, in Chapter 4 the diatom P. tricornutum was screened for its hydrogen production capacity on a hydrogen electrode. Using metabolic inhibitors as well as light stimulation the results show that the current observed is not due to hydrogen but to some other interfering molecule. Hydrogen sulfide was demonstrated, for the first time, to be produced by axenic cultures of diatoms and to be detected by our home-built electrode.

Throughout this dissertation our understanding of how carbon is divided between proteins, lipids and carbohydrates was advanced and new strategies and regulatory mechanisms that are worth exploring in the future were identified. A more complete knowledge of this fundamental question will allow us to move closer to fully control the metabolism of photosynthetic microbes and potentially transform them in economically efficient cell factories for designer biofuels. The development of cost-efficient widespread biofuels would alleviate the strain put on our environment and international relations by the traditional fossil energy sources and thus benefit generations to come.