ABSTRACT LIAN, HONG. Transcriptomic and Physiological Analysis of a Recombinant Pyrococcus furiosus Strain Metabolically Engineered to Produce 3-Hydroxypropionate from CO2 and Maltose. (Under the direction of Dr. Robert M. Kelly). As a new paradigm for renewable fuels, Electrofuels expand the boundaries of traditional biofuels that rely on photosynthetic carbon fixation. Electrofuels could overcome the drawback inherent to biofuels approaches via direct utilization of CO2 to bypass energy intermediate sugar, more efficient non-photosynthetic CO2 fixation pathways and durable low potential energy sources. Here, we aim to metabolically engineer a hyperthermophilic archeaon, P. furiosus strain COM1, as a host for production of desired liquid fuels or valuable chemicals (e.g., 3-hydroxypropionate) by incorporation of the 3HP/4HB cycle from the thermoacidophile M. sedula. Sub-pathway 1 (SP1) of the 3-hydroxypropionate/4- hydroxybutyrate (3HP/4HB) cycle is composed of the heteromultimeric acetyl- CoA/propionyl-CoA carboxylase (E1) (encoded by Msed_0147, Msed_0148, and Msed_1375), malonyl-CoA reductase (E2) (encoded by Msed_0709), and malonic semialdehyde reductase (E3) (encoded by Msed_1993). These three enzymes sequentially - convert HCO3 and acetyl-CoA into 3-hydroxypropionate (3-HP). To achieve our goal, the in vitro study of SP1 was first performed to confirm all enzyme functions individually, and that if assembled could convert acetyl-CoA to 3-HP. The genes encoding these enzymes were produced by heterologous expression in E. coli or P. furiosus. In this process, the rate- limiting carboxylase in carbon fixation and its accessory enzyme biotin protein ligase (BPL) captured our specific attention, as did carbonic anhydrase (CA), the enzyme catalyzing conversion of CO2 to bicarbonate as substrate for carboxylase. Therefore, Msed_0390, and Msed_2010 were identified in the M. sedula genome to encode a functional CA and BPL. The in vitro work provided the basis for incorporating CA and BPL by metabolic engineering into recombinant P. furiosus to facilitate 3-HP/4-HB cycle function for production of fuels and chemicals. All three enzymes (E1, E2 and E3) were produced in recombinant form, with E2 and E3 in E. coli, and the three subunits encoding E1 in P. furiosus. Recombinant forms of M. sedula BPL and CA were also produced in E. coli. Formation of 3-HP was demonstrated in vitro by the assembly of recombinant E1, E2 and E3, using either acetyl- CoA or malonyl-CoA as substrate. The five M. sedula enzymes mentioned above were then metabolically engineered into P. furiosus strain COM1 to enable production of 3- hydroxypropionate (3-HP) from maltose and CO2. P. furiosus was grown in a 2-liter fermentor at 95°C until late exponential phase, at which time the temperature was reduced to 72°C to initiate 3-HP production. The addition of genes encoding M. sedula CA and biotin BPL, led to two-fold higher 3-HP titers than in their absence. Furthermore, CO2 mass transfer was found to be rate-limiting, as 10-fold higher 3-HP concentrations (from 0.3 to 3 mM) were obtained when sparging and agitation rates were increased from 15 mL/min to 50 mL/min, and 250 rpm to 400 rpm, respectively. Transcriptomic analysis using a hybrid oligonucleotide microarray revealed that the M. sedula genes were among the most highly transcribed in the recombinant P. furiosus genome, both at 2.5 h and 40 h after the temperature shift. Monovalent cation /H+ antiporter subunits were found up-regulated in MW76, presumably involved in the export of 3-HP to maintain cytosolic homeostasis to prevent intracellular acidification. From 2.5 h to 40 h, the P. furiosus COM1 transcriptome showed significant down-regulation of genes involved in energy metabolism, amino acid biosynthesis, DNA metabolism and protein synthesis, while MW76 maintained relatively high cellular activity. Up-regulation of biotin synthesis genes in MW76 was also noted at 40h, presumably to provide sufficient biotin for biotinylation of the M. sedula carboxylase subunit. The results of this study will be critical in designing further genetic refinements to the MW76 strain and to coordinate these changes to bioprocessing strategies. © Copyright 2014 Hong Lian All Rights Reserved Transcriptomic and Physiological Analysis of a Recombinant Pyrococcus furiosus Strain Metabolically Engineered to Produce 3-Hydroxypropionate from CO2 and Maltose by Hong Lian A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Chemical Engineering Raleigh, North Carolina 2014 APPROVED BY: _______________________________ ______________________________ Robert M. Kelly Balaji M. Rao Committee Chair _______________________________ _______________________________ David F. Ollis Amy M. Grunden BIOGRAPHY Hong Lian was born in Zhangzhou, a small city in the South of China. In 2004, she was enrolled into East China University of Science and Technology (ECUST) and started her journey of being a bio-engineer. ECUST is where people in China produced antibiotics via fermentation for the first time. In the college, she had a chance to contact respected professors and outstanding fellows. In Shanghai, she experienced the viability of this city for its internationality. She wanted to learn more advanced technology and knowledge in the United States. After graduation and earning her Bachelors of Science in Engineering in Biochemical Engineering, she headed to North Carolina State University where she was happy to become a member of the Electrofuels team, working on saving the world from the energy crisis. In the lab, she has become friends with bugs, such as E. coli, M. sedula, and P. furiosus---though these bugs had given her some tough times, finally they show their characteristics as loyal and excellent friends. ii ACKNOWLEDGMENTS I would like to thank Dr. Kelly for allowing me the opportunity to study under his guidance. I would also like to thank fellow members of the Kelly group who have offered me so much. I would like to thank US DOE ARPA-E projects to fund the research we are doing in our lab. Especially thank our ARPA-E team, Dr. Yejun Han, Dr. Aaron Hawkins, and Andrew Loder. Also, I want to thank Dr. Arpan Mukherjee for the help of microarray experiments. iii TABLE OF CONTENTS LIST OF TABLES ................................................................................................................. ix LIST OF FIGURES ............................................................................................................... xi Chapter 1: Biological Conversion of Carbon Dioxide and Hydrogen into Liquid Fuels and Industrial Chemicals ........................................................................................................1 The rise of electrofuels .............................................................................................................2 What is an Electrofuel and why it is advantageous over traditional Biofuels? .........................2 Electrofuels: State of the art .......................................................................................................3 3HP/4HB cycle and Metallosphaera sedula ..............................................................................6 Pyrococcus furiosus as electrofuel host .....................................................................................8 Carboxylases ............................................................................................................................9 Classification and functions of carboxylases ............................................................................9 Comparison of autotrophic carboxylases and their connection to pathway physiology .........10 Application of carboxylases in synthetic biology ...................................................................13 Biotin protein ligase, an accessory enzyme to acetyl-CoA carboxylase ...........................14 Carbon dioxide capture ........................................................................................................15 CO2 concentrating mechanisms and carbonic anhydrase in RubisCO-based carbon fixation .....................................................................................................................................15 Novel CO2-concentrating mechanisms and carbonic anhydrase for Electrofuels ...................17 3-hydroxypropionic acid: A valuable intermediate in the 3HP/4HB cycle .....................19 3-hydroxypropionic acid, a valuable industrial chemical ........................................................19 Chemical method for 3-HP production ....................................................................................20 Biological routes for 3-HP production ....................................................................................20 iv Summary .................................................................................................................................25 REFERENCES .......................................................................................................................27 Chapter 2: Carbonic Anhydrase (CA) from the Extremely Thermoacidophilic Archaeon Metallosphaera sedula (Msed_0390, CAMS) Represents a New Sub-class of β- Carbonic Anhydrases ............................................................................................................46 ABSTRACT ............................................................................................................................47