Rouse, Daniel P.2011-07-142011-07-142011-05https://hdl.handle.net/11299/109210University of Minnesota M.S. thesis. May 2011. Major: Microbial Engineering. Advisor: Friedrich Srienc. 1 computer file (PDF); xii, 44 pages, appendix p. 132-144.With the world’s petroleum reserves being depleted at a rapid rate, vast pollution caused by the inability of nature to break down modern day plastics, and global warming greatly affecting the Earth’s climate, green alternatives to current industrial practices are becoming more necessary now than ever before. One possible environmentally friendly alternative is the production of poly 3-hydroxyalkanoates (PHAs), biodegradable polymers naturally synthesized by many types of bacteria. PHB is the most common and easiest to produce PHA, but the polymer is quite brittle and holds little commercial value. However, research has shown that a wide variety of monomers can be incorporated into the polymer chains giving a vast array of physical and material properties to the polymer, some with the potential to be very valuable. With the advances in molecular biology techniques and DNA sequencing technology, metabolic engineering has become a quick and powerful tool for constructing recombinant bacteria with gene sets capable of performing tasks previously unattainable. The main focus of this thesis was to construct a recombinant strain of Ralstonia eutropha capable of producing medium chain length (mcl) PHAs using soybean oil as the sole carbon source. Two approaches were used to accomplish this goal. In one approach the mcl-PHA polymerase gene phaC1 from Pseudomonas oleovorans was integrated into the genome of R. eutropha in place of the native phaC PHB polymerase. Another approach tested was to construct a plasmid based expression system using homologous recombination in yeast to express an engineered mcl-PHA operon containing the P. oleovorans phaC1 in conjunction with the phaJ from P. oleovorans or the fox2 gene from S. cerevisiae on a polycistronic gene cassette in a mutated strain of R. eutropha rendered unable to synthesize PHAs naturally. Both recombinant systems were tested for mcl-PHA production in fed batch bioreactors using minimal media supplemented with soybean oil. GC-MS analysis of cell samples taken from the reactor experiments indicated the recombinant organism harboring the integrated phaC1 gene accumulated trace amounts of mcl-PHAs, mostly in the form of 3-hydroxyoctanoate, but the plasmid expression systems showed much higher accumulation of mcl-PHAs with constituents of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydecenoate. This demonstrated the ability of the recombinant R. eutropha to accumulate mcl-PHAs when grown on fatty acids. It was also demonstrated that R. eutropha has the ability to grow to high cell densities using soybean oil as the sole carbon source, and that foreign gene expression is possible in R. eutropha using plasmid based systems and through gene integrations.en-USMicrobial EngineeringThesis or Dissertation