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ENGINEERING ACTINOBACILLUS SUCCINOGENES for SUCCINATE PRODUCTION- a FOCUS on SUCCINATE TRANSPORTERS and SMALL Rnas

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ENGINEERING ACTINOBACILLUS SUCCINOGENES for SUCCINATE PRODUCTION- a FOCUS on SUCCINATE TRANSPORTERS and SMALL Rnas ENGINEERING ACTINOBACILLUS SUCCINOGENES FOR SUCCINATE PRODUCTION- A FOCUS ON SUCCINATE TRANSPORTERS AND SMALL RNAs By Rajasi Virendra Joshi A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Microbiology and Molecular Genetics- Doctor of Philosophy 2017 ABSTRACT ENGINEERING ACTINOBACILLUS SUCCINOGENES FOR SUCCINATE PRODUCTION- A FOCUS ON SUCCINATE TRANSPORTERS AND SMALL RNAS By Rajasi Virendra Joshi An important aspect of any industrial scale bio-based production is the choice of biocatalyst used. Many commercially relevant microorganisms and industrial strains have been engineered to optimize the production of bio-based chemicals. One such chemical is succinate, listed as one of the top 12 building block chemicals from biomass by the US Department of Energy. Succinate is considered an important platform chemical as it has a number of applications and, most importantly, is a precursor to high-volume value-added commodity chemicals. Bio-based succinate is currently being produced at industrial scale levels using engineered microorganisms such as E. coli and S. cerevisiae. Actinobacillus succinogenes is one of the best natural succinate producers, which can grow on a wide variety of substrates, and, with the advance in genetic tools, can possibly be engineered for increased succinate production. Very few studies have focused on using succinate exporters as metabolic engineering targets for succinate production. Only a handful of studies have been carried out in E. coli and C. glutamicum with none whatsoever in A. succinogenes. With a combination of proteomics and transcriptomics we have identified candidate succinate transporters in A. succinogenes. Four of the top hits in our proteomics analysis were Asuc_1999, Asuc_0142, Asuc_2058 and Asuc_1990-91. To carefully tune the expression of these membrane proteins, we generated a library of promoters covering a large range of strengths below the strong, constitutive promoter (ppckA) we had been using. Some of the promoters were truncated versions of ppckA, and others were identified from our transcriptomics data. These promoters were tested using lacZ as the reporter gene in an A. succinogenes ∆lacZ background. Promoters ranged from ppckA as the highest down to pAsuc_0701 with a strength 209-fold lower than ppckA. The four succinate transporter candidates over-expressed under ppckA-92, a truncated version of ppckA, increased the succinate yield in glucose cultures compared to the control strain carrying the empty vector. Synthetic small RNAs (sRNAs) are another tool for metabolically engineering industrially relevant microorganisms. However, no sRNAs have been identified in A. succinogenes and only a few have been identified in other members of the Pasteurellaceae family. We identified sRNAs in A. succinogenes grown anaerobically on glucose and microaerobically on glycerol by RNA sequencing. We found 260 sRNAs in total, of which 39 were predicted by at least one of five computational programs. We validated 14 sRNAs identified from sequencing with RT-PCR. Additionally we identified probable Hfq-binding sRNAs through their Rho-independent terminators, a key feature of Hfq-binding sRNAs. Using additional characteristics of Hfq- binding sRNAs, we designed synthetic sRNAs targeting lacZ expression as a proof of concept. One plasmid-borne synthetic sRNA caused a 32% decrease in β-galactosidase activity in lactose- grown cultures. Using the same sRNAs scaffolds, we generated synthetic sRNAs that targeted the ackA and pta mRNAs to decrease the production of acetate, one of the major by-products of succinate production. One of the synthetic sRNAs targeting ackA caused a 14% decrease in the acetate yield of glucose-grown cultures. In summary, we have identified candidate succinate transporters and seen an increase in succinate production upon their overexpression. In the process, we have developed a promoter library for tunable expression of genes in A. succinogenes. We have also shown that sRNAs can be used as a tool for metabolic engineering in A. succinogenes, although additional studies are needed to make it more tunable and robust. This dissertation is dedicated to my entire family, especially my husband, Ashwin for his patience, constant encouragement and morale boosting pep talks. I thank my parents, sister, brother-in law and in-laws for always believing in me and supporting me throughout this journey. iv ACKNOWLEDGEMENTS This dissertation has been possible only because of the contributions of several people who have guided and helped me throughout these years. First and foremost, I would like to express my sincere gratitude to my mentor, Dr. Claire Vieille for giving me the opportunity to work in her lab during my Master’s and for my PhD. I thank her for being a great mentor and for always taking out the time to discuss any idea, big or small, despite being busy with numerous other things. Her intellect, passion for science and positive attitude has always inspired and motivated me, especially during the more trying times of this project. I will always be indebted to her, for her guidance and thoughtful input throughout my PhD. I am thankful to all the members of the Vieille lab- Maris Laiveneiks, Dr. Bryan Schindler, Dr. Justin Beauchamp, Dr. Nikolas Mcpherson and Peggy Wolf for a great work atmosphere with lots of support and help. I am truly grateful for the time taken by Dr. Bryan Schindler to train me during my initial years in the lab even when he was busy with his own experiments. I am also grateful to Maris Laivenieks, who was always willing to help me, especially during my initial struggles while building a chemostat and mixing gas tanks. I would like to thank Dr. Nikolas McPherson for being an excellent lab member and for all the helpful discussions we had while working with Actinobacillus succinogenes during the course of both of our PhDs. I must acknowledge Dr. Justin Beauchamp for helping me with the intial RNAseq analysis. I wish to thank Barry Tigner, who has saved my experiments numerous times, by fixing last minute instrument fails. I must thank my committee members, Drs. Gemma Reguera, Michael Bagdasarian, Shannon Manning, Yair Shachar-Hill for their expert guidance and constructive feedback. I v would like to thank Dr. Donna Koslowsky for letting me use her lab and Laura Kirby, her grad student for helping me set up Northern blots. I would like to thank all the undergraduates who helped me with my project, Alex Yang, Jean Kim, Maeva Bottex, Matthias Schulz, Kanupriya Tiwari, Joel Tencer, Jacob Gibson, Aaron Walters, Jack Peleman and Corey Kennelly. It was a pleasure interacting with each of them and training them has made me a better researcher. I would like to thank Betty Miller, graduate secretary of Microbiology and Molecular Genetics for helping me navigate through the paperwork required and other assistance throughout these years. I am grateful for the lasting friendships I have made here at MSU. I feel fortunate to have had a strong support system in them, sharing the ups and downs of graduate life. vi TABLE OF CONTENTS LIST OF TABLES .......................................................................................................................... x LIST OF FIGURES ...................................................................................................................... xii Chapter 1 Introduction .................................................................................................................... 1 1.1 Bio-based succinate production ............................................................................................ 2 1.2 Substrates and feedstocks for succinate production .............................................................. 4 1.3 Applications for succinate and its derivatives ....................................................................... 4 1.4 Succinate-producing microorganisms ................................................................................... 5 1.4.1 Native succinate producers ............................................................................................. 5 1.4.2 Engineered succinate producers ................................................................................... 12 1.5 C4-dicarboxylate transporters ............................................................................................. 21 1.5.1 E. coli C4-dicarboxylate transporters ........................................................................... 21 1.5.2 C4-dicarboxylate transporters in other succinate producers ........................................ 23 1.6 sRNAs as synthetic regulators for metabolic engineering .................................................. 24 1.7 Identification of small RNAs in Pasteurelleaceae ............................................................... 28 1.8 Introduction to chapters ....................................................................................................... 28 REFERENCES .......................................................................................................................... 31 Chapter 2 Identification of succinate transporters in Actinobacillus succinogenes ...................... 42 2.1 Abstract ............................................................................................................................... 43 2.2 Introduction ........................................................................................................................
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