Tikh, Ilya2014-02-142014-02-142013-12https://hdl.handle.net/11299/162529University of Minnesota Ph.D. dissertation. December 2013. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Claudia Schmidt-Dannert. 1 computer file (PDF); vii, 139 pages.Over billions of years photosynthetic organisms have refined the molecular machinery required for the capture and conversion of light into chemical energy. To date, much research has been devoted into harnessing this unique trait from photosynthetic organisms and utilizing them for ecologically clean production of valuable resources, such as alternatives to fossil fuels or commodity chemicals. Unfortunately, photosynthetic organisms are not always ideal host for the production of desired chemicals and are frequently difficult to engineer. In order to bypass those hurdles, this work focused on introducing the machinery responsible for the light-energy conversion into a nonphotosynthetic host. The supplementation of a heterologous host with the energy captured via the light-energy conversion could alleviate some of the host's metabolic burden and allow for greater yields of desired compounds. In order to achieve our goals, we set out to engineer functional expression of the bacterial reaction center from R. sphaeroides as well as the enzymes required for the production of bacteriochlorophyll into E. coli. For the first time we were able to demonstrate the expression of the reaction center complex as well as its primarily polar localization with E. coli cells. Furthermore, we characterized two previously poorly understood enzymes involved in the production bacteriochlorophyll, the 8-vinyl reductase (BciA) and the Mg protoporphyrin monomethylester cyclase (BchE). In the case of BciA, we showed that unexpectedly the BciA from R. sphaeroides was not functional when expressed in E. coli, unlike the BciA from C. tepidum. At the beginning of this work, BchE was the only enzyme involved in the biosynthesis of bacteriochlorophyll that has not been heterologously expressed and had no published biochemical or biophysical data. Through our efforts, we were able to demonstrate that BchE contained an oxygen sensitive 4Fe-4S cluster able to interact with SAM, the predicted co-factor. Additionally, for the first time, we showed the interaction of BchE with several intermediates of the bacteriochlorophyll biosynthetic pathways. Complementary to our efforts, we also produced a set of protein expression vectors for use in R. sphaeroides. R. sphaeroides is a photosynthetic organism which has been used extensively for the production of value added compounds and has the potential to be used for the production of membrane proteins. The novel vectors are BioBrickTM compatible and contain DsRed as a reporter protein driven by the photosynthetic puf promoter. We demonstrated that by selecting which section of the promoter was utilized in combination with various culture conditions, final reporter levels could be modulated. Reporter levels ranged from virtually undetectable to higher than what is present in E. coli when expression is driven from a constitutive lac promoter from the same vector backbone.en-US8-vinyl reductasePhotosynthesisPorphyrinReaction CenterSynthetic BiologyThesis or Dissertation