- Philip Romero
Engineering of an acyl-CoA reductase for improved substrate specificity
Fatty alcohols are promising alternatives for petrochemicals in synthesis of a variety of compounds, such as fuels, cosmetics, and surfactants. Medium chain length fatty alcohols are of special interest because they have higher selling prices than biofuels. Medium length fatty alcohols can be produced in microbes such as E. Coli by reversing the beta-oxidation pathway and overexpressing an acyl-CoA reductase (ACR), which forms fatty alcohols from an acyl-CoA. Unfortunately, many ACRs have low activity on medium length substrates, making production of medium length fatty alcohols challenging. An additional challenge is that ACRs are not often selective, resulting products with widely distributed lengths. I plan on using protein engineering and optimization strategies to develop an ACR that is capable of selectively producing medium chain fatty alcohols. In order to do this I will construct an enzyme library using an algorithm used for designing chimeric protein libraries (SCHEMA). The library design portion of the project is complete, and assembling variants from the library will be done by Golden Gate assembly. Designing the library involved creating homology models of the ACR domains three two-domain acyl-coA reductase enzymes. These models, along with a multiple sequence alignment, were then used to predict ideal crossover points for shuffling the homolgous sequences using SCHEMA. I am currently developing an assay for detection of medium chain fatty alcohols by GC-FID, which I will use to compare different variants from my library and screen for improved activity and selectivity.