Steps toward optimization of ethanol production in the cyanobacterium Synechocystis PCC 6803

Dexter, Jason P.
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Development of renewable energy is rapidly embraced by our society and industry to achieve the economic growth goals, and to solve the world's energy and global warming crises. Currently most of the bioethanol production is from the fermentation of agricultural crops and residues. It is also agreed upon that the current US ethanol production approaches which primarily use com grain as feedstocks are neither cost-effective nor energy-efficient. Novel biological solutions for the production of biofuels with the potential to revolutionize biology-based energy production need to be developed. The primary objective of this work is to create a strain of Synechocystis sp. PCC 6803 that will form the foundation for an alternative method of ethanol production. Here, we report the success with the creation of a Synechocystis sp. PCC 6803 strain that can photoautotrophically convert C02 to bioethanol. Transformation was performed using a double homologous recombination system to integrate the pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh) genes from obligately ethanologenic Zymomonas mobilis into the Synechocystis 6803 chromosome under the control of the strong, light driven psbAII promoter. PCR based assay and ethanol production assay were used to screen for stable transformants. The system described here shows average yields during log phase growth of 5.2 mmol . OD730 unit -1 • liter -1 • day -1 with no required antibiotic/selective agent. Thus, the ethanol yields in our cyanobacterial system show a greater than three order of magnitude increase compared to initial results by Deng and Coleman of 54 nmol . OD730 unit -1 • liter -1 • day -1 (mmol vs. nmol) and later results of 0.0408 mmol of ethanol per mg of chlorophyll per day (2001). In addition, under controlled circumstances ethanol can be accumulated in the liquid media at a concentration on the order of 10 mM approximately 6 days after the inoculation. Also, we have modified the transformation platform with the addition of a multiple cloning site. Investigation was then made into the potential of MDR-like transmembrane proteins in increasing ethanol resistance by attempting to co-express the omrA transmembrane protein from the wine lactic acid bacteria Oenococcus oeni with the pdc/adh cassette. The results of these trials show that MDR-protein expression in Synechocystis will probably be detrimental to the cell, and not increase ethanol resistance. Finally, the effect of media ethanol concentration on cell growth was examined. It was shown that Synechocystis 6803 strains are readily capable of surviving exposure to 5 % (vol/vol) ethanol concentrations, although growth may be completely inhibited. In the above system growth would reinitiate after reduction of the ethanol concentration within the media via evaporative losses.
Thesis (M.S.)--University of Hawaii at Manoa, 2007.
Includes bibliographical references (leaves 66-68).
viii, 68 leaves, bound ill. 29 cm
Cyanobacteria -- Biotechnology, Biomass energy
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Theses for the degree of Master of Science (University of Hawaii at Manoa). Molecular Biosciences and Bioengineering; no. 4228
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