Engineering Light-Energy Conversion into Nonphotosynthetic Hosts A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Ilya Tikh IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Claudia Schmidt-Dannert December 2013 © Ilya Tikh 2013 Acknowledgements First, I must thank Claudia for her guidance and patience over the years it took me figure out how to be a good scientist. Thank you for being a supportive mentor and not discarding some of my crazier ideas. I want to also thank all of the members of the Schmidt-Dannert lab, past and present, who have helped and supported me over the years. I am especially grateful to Ethan and Maureen, as both of you have shown tremendous patience with me and ultimately helped me to become the scientist I am today. I want to thank everyone in BTI for your thoughtful discussions and not minding occasional reagent borrowing. I am grateful to my chair, Dr. Michael Sadowsky and my committee members; Dr. Burckhard Seelig, Dr. Jeff Gralnick and Dr. Do-Hyung Kim for their guidance over the years. I was fortunate to start graduate school with some amazing peers who have become family over the years. Thank you for being there, good times and bad, Val, Lauren, Zach, Joel, Aaron and Eu Han. I want to thank Joni being patient with me and my continuisly evolving graduation timelines. Finally, I want to thank my family for their continuous and unwavering support over the last six years and for not asking me when I would graduate, too frequently. i Dedication To my grandparents ii Abstract 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 iii 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. iv Table of Contents Contents Acknowledgements .............................................................................................................. i Table of Contents ................................................................................................................ v List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii Chapter 1. ............................................................................................................................ 1 Chapter 2. .......................................................................................................................... 18 Chapter 3. .......................................................................................................................... 38 Chapter 4. .......................................................................................................................... 71 Chapter 5. ........................................................................................................................ 106 Conclusions and Future Directions. ................................................................................ 123 References ....................................................................................................................... 125 v List of Tables Table 1. Primers described in Chapter 2 ........................................................................... 23 Table 2. Primers described in Chapter 3. .......................................................................... 45 Table 3. Strains and plasmids used in Chapter 3. ............................................................. 45 Table 4. In vivo reduction of BChl pathway intermediates. ............................................. 54 Table 5. Primers used in Chapter 4. .................................................................................. 84 Table 6. N-terminus peptide sequence of the 60 kDa band from Figure 25A. ................. 89 Table 7. Primer utilized in Chapter 5. ............................................................................. 111 Table 8. Plasmids created in Chapter 5. .......................................................................... 113 vi List of Figures Figure 1. .............................................................................................................................. 3 Figure 2. .............................................................................................................................. 8 Figure 3. ............................................................................................................................ 10 Figure 4. ............................................................................................................................ 14 Figure 5. ............................................................................................................................ 17 Figure 6. ............................................................................................................................ 27 Figure 7. ............................................................................................................................ 30 Figure 8 ............................................................................................................................. 32 Figure 9. ............................................................................................................................ 41 Figure 10. .......................................................................................................................... 50 Figure 11. .......................................................................................................................... 52 Figure 12. .......................................................................................................................... 57 Figure 13. .......................................................................................................................... 57 Figure 14. .......................................................................................................................... 59 Figure 15. .......................................................................................................................... 60 Figure 16. .......................................................................................................................... 60 Figure 17. .......................................................................................................................... 61 Figure 18. .......................................................................................................................... 65 Figure 19. .........................................................................................................................