ENGINEERING METABOLIC PATHWAYS IN THE GREEN ALGA Chlamydomonas reinhardtii TO ENHANCE BIOFUEL PRODUCTION
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Date
2020-01-20
Type of Work
Department
Biological Sciences
Program
Biological Sciences
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Distribution Rights granted to UMBC by the author.
This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu
This item may be protected under Title 17 of the U.S. Copyright Law. It is made available by UMBC for non-commercial research and education. For permission to publish or reproduce, please see http://aok.lib.umbc.edu/specoll/repro.php or contact Special Collections at speccoll(at)umbc.edu
Abstract
Microalgae are a diverse group of microorganisms that can synthesize a wide variety of products such. Presently, cultivating microalgae and converting it to biofuel is too costly to compete with fossil fuels. Costs could be reduced by genetically engineering metabolic pathways to increase growth rates and lipid content. However, some genetic engineering techniques developed for lab strains of microalgae are not generally accessible. Furthermore, it is not yet clear which metabolic pathways are the most promising targets for growth rate and lipid content enhancement. This thesis work addressed each of these issues. First, to test the idea that the regeneration stage of the Calvin cycle is rate limiting for algal growth, I introduced FBP/SBPase, a Calvin cycle regeneration stage gene, into the chloroplast of the model alga Chlamydomonas reinhardtii to overcome what is believed to be a bottleneck step in carbon fixation. I show that the growth rate for C. reinhardtii does not increase with expression of the FBP/SBPase transgene, indicating that if there is a bottleneck in carbon fixation, the enzyme encoded by this transgene cannot relieve it. Next, I developed a method of evaluating electroporation parameters by electroporating fluorescent dyes into microalgae and imaging with flow cytometry and fluorescence microscopy. I used this method to evaluate electroporation parameters for the industrially relevant strain Chlorella vulgaris. To make gene-editing easier to implement for microalgae, I modified an existing C. reinhardtii CRISPR/Cas9 method by replacing a complex electroporation waveform with a simple exponential waveform. Finally, I used my modified electroporation protocol to test the importance of two genes that are critical in the synthesis of triacylglycerols (TAGs) in C. reinhardtii, diacylglycerol acyltransferase 1 and 2 (CrDGTT1 and CrDGTT2). I knocked out both genes and measured the TAG accumulation and TAG fatty acid profile of the knockout strains and compared the effect of the knockout to previously published RNAi knockdown of the same genes. The overall TAG accumulation of both knockdown strains was equivalent to the wild type, in contrast to the knockdown strains. However, the fatty acid profile of the CrDGTT1 knockout was markedly different to both the wild type and the CrDGTT2 knockout, suggesting a significant genetic compensation could be occurring in the CrDGTT1 knockout. An unexpected decrease in cell size was observed for both knockout strains, opening interesting possibilities for future research. Cumulatively, the work reported in this dissertations provides important insights that advance our understanding of algal growth and TAG accumulation that will help other researchers in the microalgae biofuel community.