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Metabolic and physiological engineering of photosynthetic microorganisms for the synthesis of bioenergy feedstocks: development, characterization, and optimization
Work, Victoria H.
Work, Victoria H.
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2014
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2014
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2015-10-08
Abstract
Biological processes are the reason for Earth's hydrocarbon reservoirs and its oxygenated atmosphere. Life would be different today if not for the advent of photosynthetic carbon reduction by cyanobacteria 3 billion years ago. Oxygenic photoautotrophy uses carbon dioxide (CO2) from the air, the radiant energy of sunlight, and reductive potential abstracted from water to derive structural carbon. The simplicity of this concept is rivaled by the complexity of its constituent mechanisms, but this has not inhibited the vast diversity of plantlife throughout the ages. Photosynthetic organisms thrive in most photic environments in unicellular and multicellular forms. It is testament to evolution that anthropologic efforts now seek to understand these life forms for their biosynthetic, photophysiological, and adaptive properties. Of particular interest is the possibility to direct the metabolism of CO2-reducing organisms into bioproducts of practical significance in modern human lifestyle. One perception is that combustible molecules could be synthesized by these organisms, recovered for conversion to a fuel source, and once utilized as such, the products (CO2 and water) could be reconverted into the same reduced carbon compounds by the same organisms. Taking the model of carbon neutrality to its fruition is as sensitive, multifaceted, and profoundly complex an endeavor as bringing the concept of photosynthesis into reality. The characterization of photosynthetic microorganisms (PSMs) has been widely pursued for over 50 years, and the study of photosynthesis even longer. Still requiring much clarity, research has begun to manipulate photosynthetic metabolism for desired effects. This thesis defines the physiologies of two distinct classes of PSMs, green algae and cyanobacteria, under conditions to assess the strains' capabilities and adaptations toward bioenergy-related productivities. The green alga Chlamydomonas reinhardtii was genetically engineered to eliminate one of its major biomolecular constituents, polyglucan carbohydrates, such as starch and amylose. The purpose was to determine the possibility of reallocating fixed carbon into another basic component, fatty acid-containing lipids such as triacylglycerol (TAG). The results were mildly favorable, but partitioning was drastically altered when the ability to properly synthesize starch was reintroduced by complementation. Effects of nitrogen deprivation, a known starch- and lipid-accumulation trigger, were assessed, but significantly, complemented mutants accumulated greater amounts of both starch and storage lipid during nutrient replete cultivation than wildtype or starchless strains during nitrogen stress. This hyperaccumulation phenotype is promising for the possibility of tuning photosynthetic metabolism to the synthesis of specific molecules. The cyanobacterium Synechococcus sp. PCC 7002 was likewise modified for the interruption of glucose activation to higher glucans and was also engineered for the secretion of fatty acids. Carboxylated hydrocarbons of medium chain length such as lauric acid (C12) are drop-in fuel precursors that require minimal processing to derive the combustible product. When conferred with a C12-secreting capability, this organism dedicated 10% of its fatty acid portfolio to lauric acid, most of which was released from the cell into the culture medium without further persuasion. Though eliminating higher carbohydrates did not change the amount of C12 generated, a small increase in total fatty acyl lipids was observed. Aside from a severe decrease in reducing carbohydrate content, the most dramatic effects of removing this important pathway occurred in photosynthesis and during nitrogen deprivation. Rearrangements were observed in electron transport from photosystem II and through the plastoquinone pool, and the photoprotective abilities of this organism are illustrated by wildtype levels of O2 being generated by the inhibited strain despite a lower growth rate. When nitrogen starved, a buildup of metabolic precursors resulted in organic acids being secreted into the culture medium, which are also valuable biocommodities. Synechococcus sp. PCC 7002 is a robust platform for metabolic engineering and physiological investigation, and it may be emerging as a feedstock organism for targeted bioproducts. The task of re-engineering photosynthetic metabolism can be likened to domesticating an agricultural plant. We can begin the process, but its outcome will be dictated by the ancient biology on which it is based. The results of this work can be progressively adjusted in the pursuit of renewable and sustainable energy sources, an endeavor that appears to be a viable possibility. To those that photosynthesize, we salute you.
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