METABOLIC ENGINEERING OF THE ARCHAEON Methanosarcina acetivorans FOR PRODUCTS FROM METHANE
Open Access
- Author:
- Mcanulty, Michael Justin
- Graduate Program:
- Chemical Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 30, 2016
- Committee Members:
- Thomas K. Wood, Dissertation Advisor/Co-Advisor
Thomas K. Wood, Committee Chair/Co-Chair
Manish Kumar, Committee Member
Esther Winter Gomez, Committee Member
Stephen John Knabel, Outside Member - Keywords:
- Metabolic engineering
methanogen
anaerobic oxidation of methane
acetate
lactate
microbial fuel cell
flavins
consortia - Abstract:
- Metabolic engineering involves the manipulation of microbial metabolism through genetic engineering and has applications in industry for producing valuable products. While metabolic engineering can entail the insertion of an entirely novel heterologous pathway, processes can be simplified by mainly relying on the host’s natural metabolism, where applicable. In this work, we utilize metabolic engineering involving one or two added reaction steps for converting methane to the value-added products acetate, lactate, and electricity, and for secreting flavins as an economical solution for producing phosphorylated flavins. To create the first organism that grows anaerobically on methane as a pure culture, we engineered the methanogen (archaeon that produces methane) Methanosarcina acetivorans to produce methyl-coenzyme M reductase (Mcr) from the metagenome of a consortium in the Black Sea; Mcr catalyzes the first step in anaerobic methane consumption or the last step in methanogenesis. The host’s entire methanogenic metabolic pathway was thus reversed to allow for growth on methane as the main carbon source and production of acetate as a by-product. The methane-consuming strain was engineered further to produce the stereospecific product L-lactate from methane by having the strain produce 3-hydroxybutyryl-CoA dehydrogenase (Hbd) from Clostridium acetobutylicum. Production of Mcr from the Black Sea consortium was also used to generate electricity from methane for the first time by designing a consortium that includes the exoelectrogen (microbe naturally capable of generating an electrical charge) Geobacter sulfurreducens and methane acclimated sludge isolated from a secondary anaerobic digester. The flavin transporter YeeO from Escherichia coli for use as a transporter of phosphorylated flavins was also characterized. Overall, this dissertation describes the research that unlocks the potential of biological anaerobic methane conversion by demonstrating the production of acetate, lactate, and electricity.