ABSTRACT BOZDAG, AHMET. Investigation of Methanol and Formaldehyde Metabolism in Bacillus Methanolicus
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ABSTRACT BOZDAG, AHMET. Investigation of Methanol and Formaldehyde Metabolism in Bacillus methanolicus.(Under the direction of Prof. Michael C. Flickinger). Bacillus methanolicus is a Gram-positive aerobic methylotroph growing optimally at 50-53 °C. Wild-type strains of B. methanolicus have been reported to secrete 58 g/l of L- glutamate in fed-batch cultures. Mutants of B. methanolicus created via classical mutangenesis can secrete 37 g/l of L-lysine, at 50 °C. The genes required for methylotrophyin B. methanolicus are encoded by an endogenous plasmid, pBM19 in strain MGA3, except for hexulose phosphate synthase (hps) and phosphohexuloisomerase (phi) which are encoded on the chromosome.It is a promising candidate for industrial production of chemical intermediates or amino acids from methanol. B. methanolicus employs the ribulose monophospate (RuMP) pathway to assimilate the carbon derived from the methanol, but enzymes that dissimilate carbon are not identified, although formaldehyde and formate were identified as intermediates by 13C NMR. It is important to understand how methanol is oxidized to formaldehyde and then, to formate and carbon dioxide. This study aims to elucidate the methanol dissimilation pathway of B. methanolicus. Growth rates of B. methanolicus MGA3 were assessed on methanol, mannitol, and glucose as a substrate. B. methanolicus achieved maximum growth rate, µmax, when growing on 25 mM methanol, 0.65±0.007 h-1, and it gradually decreased to 0.231±0.004 h-1 at 2Mmethanol concentration which demonstrates substrate inhibition. The maximum growth rates (µmax) of B. methanolicus MGA3 on mannitol and glucose are 0.532±0.002 and 0.336±0.003 h-1, respectively. Spiking with 100 mM methanol did not cause any growth perturbation ofB. methanolicus MGA3 when grown on methanol, whereas, 50 mM methanol was enough to halt the growth when it was grown on mannitol. However, growth of B. methanolicus MGA3 on methanol was halted completely by a 1 mM formaldehyde spike, while mannitol grown B. methanolicus MGA3 can tolerate 2 mM formaldehyde addition. Formate did not have any apparent growth perturbations at tested levels (up to 2 mM). B. methanolicus MGA3 has a small genome, ~3.3 Mb, and contains 2,773 ORFs larger than 100 bp. Its genome encodes two methanol dehydrogenase(mdh) genes beside the mdh on pBM19. All of the RuMP pathway genes encoded on the pBM19 have counterparts encoded on the chromosome also. The genome contains putative tetrahydrofolate (THF)- linked enzymes for formaldehyde oxidation besides putative aldehyde dehydrogenase(aldh) genes. Two putative formate dehydrogenase (fdh) genes were also identified in the genome as were the genes of the Wood-Ljungdahl pathway genes.B. methanolicus strain PB1 was also found to carry a plasmid which we named as pBM20. The difference in the formaldehyde dissimilation pathway of strain PB1 was shown via Southern Blot. The PB1 strain also has 2-fold higher formaldehyde dehydrogenase and 3-fold higher formate dehydrogenase activity compared to strain MGA3. The methanol dissimilation pathway genes,- putative THF-linked formaldehyde oxidation genes, putative aldh genes, putative fdh gene- are all down regulated in response to spikes of methanol, formaldehyde and formate. However, the regulation of RuMP pathway genes were different in methanol vs. mannitol grown B. methanolicus MGA3 in that they were down regulated from already high levels in methanol grown cells whereas they were upregulated in mannitol grown cells. Formate is a more potent regulatory compound compared to methanol and formaldehyde. © Copyright 2012 by Ahmet Bozdag All Rights Reserved Investigation of Methanol and Formaldehyde Metabolism in Bacillus Methanolicus by Ahmet Bozdag A dissertation submitted to the Graduate Faculty of North CarolinaStateUniversity in partial fulfillment of the requirements for the degree of Doctor of Philosophy Microbiology Raleigh, North Carolina 2013 APPROVED BY: _______________________________ ______________________________ Dr. Michael C. Flickinger Dr. Michael R. Hyman Committee Chair ________________________________ ________________________________ Dr. Jose Manuel Bruno-Barcena Dr. Amy M. Grunden DEDICATION To my wife Turkan and daughter Vildan ii BIOGRAPHY Ahmet Bozdag was born in Besni, Southeast Turkey as the youngest of a large family. He moved to Gaziantep after elementary school, to seek a better education at the age of 11. He completed his high school and attended Middle East Technical University (METU) in Ankara. He taught science in middle and high schools in Adiyaman and Ankara while seeking a graduate degree at METU. Winning the Diversity Visa lottery, or Green Card, he moved to United States with his ambition to seek a doctoral degree in microbiology. He earned his Master’s Degree at Georgia College & State University before attending the Microbiology department at NCSU during which he married to a beautiful woman, Turkan. He had his first child, a gorgeous baby girl Vildan just before completing his PhD degree. iii ACKNOWLEDGMENTS I would like to thank professor Michael C. Flickinger foremost, my thesis advisor, for accepting me into his laboratory and for his invaluable guidance. I would also like to thank my thesis committee members Drs. Jose Manuel Bruno-Barcena, Amy M. Grunden, Michael B. Goshe, and Michael R. Hyman. I would also like to thank my colleagues in the Flickinger laboratory, past and present. I would like to thank Dr. Thomas Pribyl for his partnership in the project, Dr. Jimmy Gosse for his ideas and discussions, Dr. Jessica Jenkins, Oscar I. Bernal and Mark Schulte for their friendship and ideas. I would like to thank German visiting students who helped me on the project along the way; Paul Simon, Fabian Gutmacher, Felix Hart and Elisa Degenkolbe. I would like to thank specially Dr. Claire Komives of San Jose State University for her help with sequencing theB. methanolicus genome. Also, I would like to thank Microbiology department staff members; Cindy Whitehead for reminding me everything I forgot and Robert Davis for his friendship. iv TABLE OF CONTENTS LIST OF TABLES ......................................................................................................... vi LIST OF FIGURES ....................................................................................................... vii CHAPTER 1 INTRODUCTION .................................................................................. 1 CHAPTER 2 GROWTH CHRACTERISTICS OF B. methanolicus ........................ 29 CHAPTER 3 GENOME SEQUENCE OF Bacillus methanolicus MGA3 AND ELUCIDATION OF CARBON DISSIMILATION PATHWAY ............................. 55 CHAPTER 4 ELUCIDATION OF C1 ASSIMILATION AND DISSIMILATION PATHWAYS IN B. methanolicus ................................................................................. 68 CHAPTER 5 REGULATION OF METHANOL AND FORMALDEHYDE METABOLISM BY MeOH, FORMALDEHYDE AND FORMATE ...................... 92 CHAPTER 6 FORMALDEHYDE AND FORMATE DEHYDROGENASES OF Bacilli .................................................................................................................. 136 CHAPTER 7 INVESTIGATION OF ANAEROBIC GROWTH OF Bacillus methanolicus MGA3....................................................................................................... 156 CHAPTER 8 STUDY CONCLUSIONS ...................................................................... 162 REFERENCES ............................................................................................................... 165 APPENDICES ................................................................................................................ 181 APPENDIX A Sequence of pBM20 .............................................................................. 182 APPENDIX B Annotation of Bacillus methanolicus MGA genome .......................... 191 APPENDIX CJournal Article Heggeset et al. 2012 .................................................... 365 v LIST OF TABLES Table 1.1 Distribution of carbon assimilation pathways in methylotrophs .............. 5 Table 1.2 Energetics of the RuMP pathway of formaldehyde fixation ................... 8 Table 2.1 Composition of trace metal solution ......................................................... 31 Table 2.2 Composition of vitamin stock solution ..................................................... 32 Table 3.1 Summary of B. methanolicus MGA3 genome sequencing ....................... 59 Table 4.1 List of primers used for cloning and DIG-labeled probes ........................ 75 Table 5.1 List of primers used for RT-qPCR ............................................................ 96 Table 6.1 List of primers used for cloning ................................................................ 146 Table 6.2 Formaldehyde dehydrogenase activities of crude cell extracts ................ 154 Table 6.3 Formate dehydrogenase activities of crude cell extracts .......................... 155 vi LIST OF FIGURES Figure 1.1 Four different pathways for one-carbon assimilation in methylotrophs .. 4 Figure 1.2 Comparison of C1 assimilation pathways by isocitratelyase-negative (A) and positive (B) methylotrophs ...................................................................... 10 Figure 1.3 Formaldehyde oxidation pathways by aerobic methylotrophic bacteria . 18 Figure 1.4 Specific CO2 production rates of B. methanolicus MGA3 and PB1 in 1-L fed- batch fermentation ................................................................................... 25 Figure