New ‘synthetic biology’ or metabolic engineering approaches to producing fuels and chemicals from renewable resources requires rerouting reactants in a metabolic network or the bottom-up integration of totally new pathways into existing ones. This means transplanting enzymes into biochemical contexts possibly very different from their native ones. And what’s optimal for fuel production is hardly optimal for survival and fitness of the organism in which the enzymes evolved in nature. For example, a high titer of product is something that balanced metabolic enzyme networks usually try to evade. Many enzyme characteristics (e.g. stability, specific activity, cofactor usage, inhibition or regulation) have to be specifically tailored in order to optimize metabolic flux towards the desired fuel or chemical.
Enzyme engineering is a rapidly developing field, with loads of potential for green technology. We are developing new tools for protein engineering and using them to create new and improved catalysts for carbon fixation, sugar release from renewable polymers such as cellulose, and biosynthesis of fuels and chemicals.
Better cellulases: lignocellulose to sugars
Cellulose is abundant, but woefully underutilized as a source of renewable fuels and chemicals. Cellulases decompose cellulose into its component sugar molecules, which in turn can be converted by microbes into valuable products. The problem is cost; the natural enzymes are slow and therefore too expensive for low-value products like fuels and commodity chemicals. We are trying to create better cellulases using a suite of approaches, including recombination and random mutagenesis. We have already discovered cellulases that are highly thermostable and significantly more active at elevated temperatures. This project lets us develop and test new protein engineering approaches in the context of a highly relevant application.
Enzyme engineering to improve metabolic pathways
New ‘synthetic biology’ or metabolic engineering approaches to producing fuels and chemicals from renewable resources require rerouting reactants in a metabolic network or the bottom-up integration of totally new pathways into existing ones. This means transplanting enzymes into biochemical contexts possibly very different from their native ones. And what is optimal for fuel production is hardly optimal for survival and fitness of the organism in which the enzymes evolved in nature. For example, a high titer of product is something that balanced metabolic enzyme networks usually try to evade. Many enzyme characteristics (e.g. stability, specific activity, cofactor usage, inhibition, or regulation) have to be specifically tailored to optimize metabolic flux toward the desired fuel or chemical.
One particular problem our lab has focused on is nicotinamide cofactor usage. Cells use two distinct nicotinamide cofactors, NADH and NADPH, to donate and accept electrons for oxidative and reductive chemistry. Despite considerable structural similarity, most enzymes display exquisite specificity toward one or the other, allowing the cell to regulate whole sets of pathways by varying the relative rates of production of the cofactors. However, this regulation often runs counter to our goals as engineers, so the ability to interconvert enzymes from one specificity to another quickly and easily would be a powerful tool.
However, the phosphate which separates NADPH from NADH generally requires three or more side-chain interactions to bind (or not bind), making this a challenging problem for traditional single-mutation driven protein engineering techniques, such as site-saturation mutagenesis or error-prone PCR. For that reason, our current research focuses on developing structure- and sequence-based shortcuts to render this engineering task more straightforward. We have developed a simple recipe for the cofactor specificity reversal of ketol acid-reductoisomerases, and going forward hope to extend this to broader swathes of enzymes.
Thermostable enzymes for producing fuels and chemicals in thermophilic organisms
Thermophilic organisms have many potential advantages as hosts for producing fuels and chemicals: they can integrate well with product separation schemes, they are not susceptible to contamination by common mesophilic organisms, and there is the potential for higher catalytic activities and therefore production rates. We are currently investigating thermophilic pathways to produce isobutanol. We are exploring the genomes of different thermophilic bacterial strains for thermostable pathway enzymes. We are also using directed protein evolution to thermostabilize less-stable enzymes and optimize their activities in thermophilic hosts.
A key step in the isobutanol pathway is the decarboxylation of ketoisovalerate to isobutyraldehyde, a reaction which can be catalyzed by thiamine pyrophosphate-containing ketoacid decarboxylases. These enzymes are one focus of our engineering efforts. An interesting feature of these homodimeric proteins is the active site around the cofactor, which is comprised of amino acids from both subunits. Increasing the thermostability of this enzyme will enable its in vivo use at elevated temperatures. We are also establishing genetic tools for protein expression in thermophiles.