The Andorfer lab seeks to develop under-studied anaerobic enzyme classes as sustainable biocatalysts for producing value-added chemicals from inexpensive and abundant feedstocks, namely CO2 and hydrocarbons. Enzymes are widely-used catalysts across many industries, but many times, improvements to naturally occurring enzyme need to be made. Protein engineering techniques have allowed for the development of industrially-relevant biocatalysts that ultimately reduce synthetic waste and increase efficiency. There has been advancement over the last several decades in engineering biocatalysts; however, engineering efforts targeting anaerobic enzyme classes has been more limited. These enzymes use a range of cofactors, including both inorganic cofactors (e.g. 4Fe4S clusters) and/or organic cofactors (e.g. amino acid radicals) to enable challenging selective chemistry. We will explore new enzyme function through genome mining, elucidate molecular-level enzyme mechanism, and engineer these enzymes for non-native substrates and transformations.
We currently target enzymes that allow anaerobic microbes to activate components of crude oil (e.g. toluene, n-alkanes, and benzene) and subsequently use them as carbon sources for metabolism. The selective functionalization of relatively unactivated C–H bonds enabled by these enzymes could make them useful synthetically. Additionally, because anaerobic, crude-oil-polluted environments are recalcitrant to typical remediation methods, these enzymes could be useful for bioremediation efforts in anaerobic environments as well.
We will take two major approaches to study these crude oil degrading enzymes:
-Directed evolution of selective C–H functionalization biocatalysts for synthetically-useful transformations
-Structural and biochemical characterization of putative hydrocarbon-degrading enzymes to elucidate molecular-level mechanism
In addition to more traditional organic and molecular biology techniques, members of the group will use single particle cryogenic electron microscopy (cryo-EM), EPR spectroscopy, as well as anaerobic enzyme purifications and biochemical assays.