Engineering non-photosynthetic carbon fixation pathways for the production of small molecule targets

The development of sustainable synthesis methods for societally important chemical products which serve as fuels, industrial feedstocks and materials is a pressing global challenge. Many of these target molecules are currently produced from fossil fuel-derived starting materials, which is problematic due both to the impending exhaustion of these resources and the dire environmental consequences of unrestrained carbon dioxide release into the atmosphere. An attractive alternative is using carbon dioxide along with sustainable energy sources for the production of these chemical targets. This thesis explores the development of multiple sustainable synthesis platforms based on interfacing inorganic materials, which function well for solar energy capture and charge transfer, with autotrophic microorganisms, which serve as biocatalysts, able to fix carbon dioxide into value-added products using the energy provided by the inorganic components./nAn initial approach focused on the development of a hybrid bioinorganic device in which sustainable electrical and/or solar input drove production of hydrogen gas from water splitting using biocompatible inorganic catalysts. The hydrogen was then used by autotrphic microorganisms as a source of reducing equivalents for conversion of CO2 to the value-added chemical product methane. Employing platinum or a newly synthesized earth-abundant substitute, α-NiS, as biocompatible hydrogen evolution reaction (HER) electrocatalysts and Methanosarcina barkeri as a biocatalyst for CO2 fixation, robust and efficient electrochemical CO2 to CH4 conversion was demonstrated at up to 86% overall Faradaic efficiency for ≥7 d. Introduction of indium phosphide photocathodes and titanium dioxide photoanodes afforded a fully solar-driven system for methane generation from water and CO2, establishing that compatible inorganic and biological components can synergistically couple light-harvesting and catalytic functions for solar-to-chemical conversion./nA second device attempted to mimic the biosynthetic process of natural photosynthesis, where solar energy powers the reduction of CO2 to common biochemical building blocks, which are subsequently used for the synthesis of the complex mixture of molecular products that form biomass. The artificial photosynthesis system described here functions via a similar two-step process: A biocompatible high-surface-area silicon nanowire array harvests light energy to provide reducing equivalents to the obligately anaerobic acetogen, Sporomusa ovata, for the photoelectrochemical production of acetic acid with low overpotential (η < 200 mV), high Faradaic efficiency (up to 90%), and long-term stability (up to 200 h). The resulting acetate (∼6 g/L) can be activated to acetyl coenzyme A (acetyl-CoA) by strains of genetically engineered Escherichia coli and used as a building block for a variety of value-added chemicals, such as n-butanol, polyhydroxybutyrate (PHB) polymer, and three different isoprenoid natural products. Due to the local chemical environment within the nanowire array, the systems was also shown to perform well under aerobic conditions (21% O2), allowing the cultivation of an obligate anaerobe using oxygen-containing gas feedstocks for growth. This system demonstrated the power of interfacing biocompatible solid-state nanodevices with living systems in order to create programmable platforms for chemical synthesis entirely powered by sunlight./nInformed by the successes of the previous systems, a next generation device was envisioned in which the autotrophic microorganism, which was interfaced with the biocompatible inorganic components, is directly engineered to produce value-added products, offering the simplicity of a single organism system with the product scope of co-culture platforms. Towards this goal methods for metabolic engineering of Methanococcus maripaludis, a methanogenic archaea, have been developed. These efforts have chiefly focused on achieving robust expression of multienzyme heterologous pathways, a goal complicated by the incompletely understood translational machinery in this organism. For optimization the 3-hydroxybutyrate pathway was used, which results in production of a monomer unit, which can be polymerized into the bioplastic PHB. Robust expression of the full 3-hydroxybutyrate biosynthetic pathway was achieved using a strong Methanosarcina barkeri promoter. Activity of all pathway enzymes was demonstrated in cell lysates; however, no product formation was observed. It is currently hypothesized that this is due to a lack of appropriate redox cofactors, specifically NADH. The NADH pool observed in M. maripaludis was found only to be 3% of that observed in E. coli. Current efforts are underway to enlarge this cofactor pool and incorporate it into the redox metabolism of the organism./nThis work has demonstrated the power of hybrid biological-inorganic devices for the sustainable synthesis of value-added products. Systems have been developed that are fueled using sustainable sources of electric current or directly by illumination. A variety of product has been produced, both native products as well as more complex products, using a co-culture system. Efforts are ongoing to develop metabolic engineering tools for the unique class of organisms amenable to integration with inorganic materials. These engineering efforts will pave the way for future simplified devices with expanded product scopes.

Advisor: Chang, Michelle CY