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theses

Microalgae predominantly partition photosynthetically fixed carbon into proteins, starch and lipids. Of these, carbohydrates and lipids are desired as they are the precursors for biofuel production. Photosynthetic electron transport is closely coupled to carbon partitioning, thus frustrating efforts to substantially increase the yield of the desired terminal product without compromising photosynthetic fitness. The objective of this thesis was to investigate the role of starch biosynthesis with respect to photosynthesis and carbon partitioning in a model microalga Chlamydomonas reinhardtii. For this, I have used a series of starch deficient mutants together with nitrogen deprivation to modulate the normal carbon partitioning. Nitrogen deprivation is known to redirect biosynthesis away from proteins to starch and lipids, and towards lipids in mutants lacking starch biosynthesis genes. Starch-deficient mutants showed a 20-40% lower biomass accumulation under both nutrient replete and deplete conditions. Interrogation of the photosynthetic metabolic flux (water→PSII/PSI→ ATP & NADPH  3PG) revealed that above a threshold light intensity, starch deficiency attenuated NADPH reoxidation by the Calvin-Benson-Bassham (CBB) cycle, which attenuated water oxidation at PSII by product inhibition. Even with starch biosynthesis blocked in the starch-deficient mutant, a high gluconeogenic flux was maintained by redirecting carbon through the oxidative pentose phosphate (OPP) shunt and ultimately away from starch. Thus, upon loss of the ability to synthesize starch, water oxidation is attenuated to balance energy consumption, while the OPP shunt becomes the dominant pathway for repartitioning of the residual photosynthate that can be produced. The goal of the second part of the thesis was to use metabolic principles to develop a cyanobacterial mutant capable of attaining high H2 yield. Cyanobacteria catabolize the photosynthetically assimilated glycogen under anaerobic auto-fermentative conditions and produce hydrogen via the enzyme, hydrogenase. By sequentially enhancing the glycolytic rate together with the elimination of competing pathways in a euryhaline cyanobacterium, Synechococcus sp. PCC 7002 mutant with high hydrogenase gene expression, we were able to boost H2 production by 8-fold over the wild-type strain. Thus, my dissertation, addresses understanding how to control the metabolism of photosynthetic microbes and using targeted metabolic engineering to transform microalgae into efficient cell factories for biofuel production.

ETD_6924

Ph.D.

Includes bibliographical references

by Anagha Krishnan

doi:10.7282/T3WD42MQ

ETD doctoral

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