Engineering cytochrome P450 enzymes
Russ Komor, Phil Romero, Chris Snow, Eric Brustad, Ryan
Lauchli, Pedro Coelho, Yousong Ding
Cytochrome P450 enzymes (P450s) are found in nearly all organisms. These impressive iron heme- containing enzymes catalyze reactions that include selective hydroxylation of un-activated C-H bonds, desaturation of alkanes, de-alkylation of heteroatoms, and epoxidation of alkenes. We have employed P450s in directed evolution studies as a model for the evolutionary process, and have applied directed evolution to generate mutant P450s capable of functioning in unnatural conditions and for conducting novel reactions. We have engineered P450 enzymes recently for applications in alkane hydroxylation, carbohydrate synthesis, and the preparation of drug metabolites and bioactive compounds.
A model for evolution
P450 BM3 is a soluble, self-sufficient bacterial P450 which is easy to express in E. coli. This well-behaved enzyme has been our primary target for directed evolution studies for some years. This enzyme has proven to be highly 'evolvable': it readily accepts new substrates and exhibits new properties upon mutation. Our studies on P450 BM3 have allowed us to speculate on the properties that might make an enzyme (or other protein) more evolvable than another. For example, an enzyme that is a member of a functional diverse family is likely to be evolvable in the laboratory. And, higher thermostability usually enhances evolvability. It may also be that enzymes with non-polar active sites are more evolvable than enzymes with many polar or ionic residues in the active site. We have also dissected evolutionary lineages - the laboratory 'fossil record' - in order to observe how proteins change during adaptation.
Structure-guided protein recombination
Chris Snow, Phil Romero, Matt Smith
Recombination is a remarkably efficient method for searching protein sequence space due to the conservative nature of homologous substitutions. We have developed computational tools (SCHEMA energy function, RASPP) that use structural information to design chimeric protein libraries. These libraries are extremely diverse, with members differing by tens to hundreds of mutations, while still maintaining a high proportion of functional sequences. The combinatorial organization of these libraries permits the study of sequence-function relationships on a significantly larger scale than accessible by point mutagenesis. We are currently developing new methods to design and analyze these combinatorial protein libraries.
Cellulase Engineering
Chris Snow, Mary Farrow, Phil Romero, Russ Komor, Indira Wu, Matt Smith
Enzymatic conversion of cellulosic biomass to fuel has
potential to become a sustainable source of energy. Cellulases can
decompose cellulose into its component sugar molecules, which in
turn can be converted by microbes into fuel alcohols such as
ethanol or butanol. Although these enzymes are widely used in
industry, their use in fuel production is not yet efficient enough
to compete with traditional liquid fuel sources. The major
limitation of commercially viable cellulosic biofuels is the cost
of breaking down the biomass into sugars. Novel, thermostable
cellulases are expected to be exrremely active at high
temperatures; this will reduce the cost of enzymatic cellulose
hydrolysis. We focus on using a suite of protein engineering
techniques to improve cellulase performance at high
temperatures.
RIGHT: More stable cellulases have higher specific activities. From Comparison of family 9 cellulases from mesophilic and thermophilic bacteria, Florence Mingardon et al. Applied and Environmental Microbiology, 2011
Metabolic engineering for fuels and chemicals
Sabine Bastian, Yousong Ding, Chris Farwell, Tilman Flock, Marvin Kadisch, Xiang Liu, and Kersten Rabe.
Recent advances in biotechnology have made it possible to construct microorganisms harboring engineered metabolic pathways that convert renewable sugars into a wide array of chemicals and next-generation biofuels. However, limited sugar-to-product yields of these engineered strains pose a major obstacle to the viability of a microbial process. We are engineering enzymes to enable microbial production of fuels and chemicals.
An imbalance between the cofactor consumption of an engineered pathway and the reducing equivalents provided by the cell can limit productivity and yield. We solved this problem for an isobutanol pathway by engineering the cofactor dependencies of the enzymes to match the available cofactors. Other problems can arise if pathway enzymes depend on organic and inorganic cofactors, such as iron-sulfur clusters, which require entire metabolic pathways and significant energy inputs from the cell for their synthesis in vivo. Enzymes that use complex cofactors are also often difficult to over-express in recombinant host organisms. One strategy is to replace problematic enzymes with alternative, engineered enzymes.
We are also looking into the use of thermophilic hosts for fuels and chemicals. Thermophilic production hosts could solve a number of technological challenges for large-scale processes, such as cooling of the fermentor or contamination by other microbes. Thermophilic hosts might also enable direct separation of volatile compounds. We are creating appropriate thermoactive enzymes for the next generation of metabolically engineered thermophilic organisms.