Leveraging Extreme Thermoacidophily for Bio-based Chemicals

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Sulfur compounds and carbon dioxide are generated in copious amounts as byproducts of natural gas and petroleum refining processes, which creates significant challenges in their safe handling and economical disposal. Fortunately, microorganisms have much to offer toward the development of environmentally-sound and energy-efficient processes to recycle these industrial waste streams. Sulfur and carbon dioxide also happen to be potential sources of energy and carbon for microorganisms called thermoacidophilic archaea that grow in extremely hot, acidic environments. One potential approach to dealing with the excess industrial sulfur and carbon dioxide is to engineer the metabolic pathways of these archaea such that they use the byproducts to produce chemicals of commercial value. This project will apply recently developed tools for genetically engineering extreme thermoacidophiles to create novel microorganisms. The study will examine the hypothesis that inserting sulfur oxidation genes from one organism into another will result in a new microorganism capable of converting carbon dioxide and sulfur into industrially-relevant organic chemicals. Engineering sulfur oxidation genes into microorganisms that naturally thrive under extreme conditions has many potential benefits. The ability to grow at hot and acidic conditions minimize risk of contamination by undesirable microorganisms and enables the bioprocess to be operated much like a conventional chemical process. The high heat tolerance of the microorganisms is also advantageous in recovering biochemical products with low boiling points. To broaden the educational impact and inclusiveness of this study, faculty and students at the North Carolina School for the Deaf (NCSD) will participate through biotechnology-based teaching modules. The modules will incorporate closed-captioning and other features to facilitate educational objectives for the deaf and hearing-impaired. Modules will be demonstrated on-site at NCSD. Additionally, the NCSU Biotechnology Program will host visiting middle school and high school students from NCSD for a day-long laboratory experience that will also familiarize them with higher educational opportunities. The objective of this project is to metabolically engineer strains of a model thermoacidophilic archaeon, Sulfolobus acidocaldarius DSM639, to produce chemicals from carbon dioxide (CO2) and reduced, inorganic sulfur compounds (RISCs). Pangenomic analysis of the extremely thermoacidophilic archaea revealed the presence of a novel CO2 fixation cycle and key genes that can be recruited to enable S. acidocaldarius to oxidize sulfur. These unique biological characteristics will be exploited in the metabolic engineering of the naturally heat- and acid-tolerant organism. Metabolic engineering approaches will be informed by fundamental assessments of wild-type and recombinant S. acidocaldarius physiology. The specific objectives of this project are: (1) Create and characterize engineered S. acidocaldarius DSM639 strains that utilize CO2 as a carbon source and RISCs as energy sources. S. acidocaldarius encodes a cycle for CO2 fixation and an incomplete pathway for sulfur oxidation that can be repaired with genes recruited from chemolithoautotrophic Sulfolobales. (2) Demonstrate that a functional, biosynthetic, thermophilic pathway for acetone can be engineered into strains of S. acidocaldarius DSM639. Genes/enzymes have been identified that can be recruited from other thermophilic microorganisms to enable S. acidocaldarius to produce an industrial chemical from simple sugars. (3) Demonstrate that engineered strains of S. acidocaldarius DSM639 can produce acetone from CO2 as a carbon source and RISCs as energy sources. The goal is to obtain acetone production under chemolithoautotrophic conditions. (4) Establish, at bioreactor-scale, that engineered S. acidocaldarius DSM639 strains can produce acetone that can be recovered through in situ distillation. T-x-y information indicates that acetone-water mixtures form a two-phase system at aqueous acetone concentrations that will facilitate recovery as a process intensification step. The project will demonstrate the utility of extreme thermoacidophiles as versatile metabolic engineering platforms, presenting a unique approach toward bio-based chemical production from an industrial waste stream.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.