Progress 01/01/06 to 12/31/08
Outputs
OUTPUTS: For this project we have engineered a series of cytochrome P450 enzymes that catalyze the oxidation of small alkanes and alkenes to alcohols and epoxides, and do so with high activity and high efficiency for coupling to the NADPH cofactor. Because the catalysts are highly coupled, reducing equivalents are not wasted by the enzyme, and whole-cell reactions using these enzymes are more efficient in converting substrate to product. These enzymes have been licensed to Codexis, Inc. for commercialization for production of industrial chemicals. This laboratory also disseminates results via its well-used web site: http://www.che.caltech.edu/groups/fha/. Arnold group researchers have discussed these results at the 2007 annual meeting of the American Chemical Society, the 2007 Enzyme Engineering meeting (where Arnold received the Genencor Award in Enzyme Engineering), and at numerous universities. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The Arnold lab reported the engineering of a proficient P450 propane monooxygenase (P450PMOR2) that catalyzes the subterminal hydroxylation of propane with in vivo activities comparable to those of naturally-occurring alkane monooxygenases from alkanotrophic organisms (Fasan et al. (2007) Angew. Chem. Int. Ed. 46: 8414-8418). Bioconversions were carried out expressing the P450 enzyme in E. coli and feeding the bacteria with propane (substrate), oxygen (co-substrate), and glucose as energy source in lab-scale bioreactors. Overall, these studies led to the identification of a set of gene deletions that induce a 240% increase in product per glucose molar yield in P450-dependent transformation in E. coli. These investigations also enabled us to identify in the pta-ackA genes the next target for knock-out engineering and in the citrate synthase enzyme a potential bottleneck in the efficient oxidation to produce NADPH. We also demonstrated a versatile chemoenzymatic strategy for selective fluorination.
Publications
- Fasan, R., Chen, M. M., Crook, N. C., and Arnold, F. H. (2007). Engineered Alkane-Hydroxylating Cytochrome P450(BM3) Exhibiting Native-like Catalytic Properties, AngewChemie, 46: 8414-8418.
- Rentmeister, A., Arnold, F. H., and Fasan, R. (2008). Chemo-enzymatic Fluorination of Unactivated Organic Compounds, Nature Chemical Biology, on-line doi:10.1038/nchembio.128
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Progress 01/01/07 to 12/31/07
Outputs
For this project we have engineered a series of cytochrome P450 enzymes that catalyze the oxidation of small alkanes and alkenes to alcohols and epoxides, and do so with high activity and high efficiency for coupling to the NADPH cofactor. Because the catalysts are highly coupled, reducing equivalents are not wasted by the enzyme, and whole-cell reactions using these enzymes are more efficient in converting substrate to product. These enzymes have been licensed to Codexis, Inc. for commercialization for production of industrial chemicals. A patent application has been filed. This laboratory also disseminates results via its well-used web site: http://www.che.caltech.edu/groups/fha/. Arnold group researchers have discussed these results at the 2007 annual meeting of the American Chemical Society, the 2007 Enzyme Engineering meeting (where Arnold received the Genencor Award in Enzyme Engineering), and at numerous universities.
Impacts
We have engineered a P450BM3 variant that converts propane to propanol with high efficiency in the utilization of the NADPH cofactor usng a domain based protein engineering strategy to separately evolve the heme, FAD, and FMN domains of a variant of cytochrome P450 of Bacillus megaterium. In parallel with ongoing efforts to increase activity via mutagenesis of the P450 heme domain, we targeted mutations to the FAD and FMN domains and screened for improved activity on dimethyl ether (DME). Resulting beneficial mutations were further optimized by saturation mutagenesis. In a final step, a set of beneficial reductase domain mutations was fused to the best heme domain of variant. The most active variant isolated showed 45,800 total turnovers on propane to produce 2- and 1-propanol in a 9:1 ratio. Coupling efficiency was increased to 98.2%. Whole-cell bioconversions using resting cells were carried out in a 100 mL bioreactor in nitrogen-free, glucose containing minimal
medium. A propane/air (ratio 1:1) mixture served as substrate and oxidant feed. Activities of up to 120 U per cell dry weight (cdw) were reached with cell densities under 1 g cell dry weight per liter. When the cells were fed with pure oxygen (propane/oxygen ratio 1:1), activities up to 180 were achieved. At higher cell densities (up to 4 g per L), propanol concentrations of 15 mM were reached.Higher propanol concentrations up to 30 mM did not result in product inhibition. Overoxidation to acetone was not detected. 52% of the initial P450PMOR2 were correctly folded at the end of the experiment. This suggested that limitations were caused by the host cell and not by the biocatalyst. In fact, resuspending the cells in fresh media restored 40 - 60% of the initial activity.
Publications
- No publications reported this period
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Progress 01/01/06 to 01/01/07
Outputs
During this granting period, whole-cell catalysis experiments were performed in a fermentor to determine the efficiency and total activity of cells containing engineered P450BM3 enzymes for the conversion of propane to propanol. Two engineered P450BM3 mutants were chosen for the bioconversion experiment. 4E10 has two active site mutations, while 19A12 contains 18 mutations in the heme domain and 2 mutations in the reductase domain. The initial catalytic rate of 19A12 is about 10 times higher than that of 4E10, but coupling between propane oxidation and cofactor oxidation is lower in the former. Total turnovers sustained by 19A12 are about twice those obtained with 4E10. 4E10 has stability and expression levels comparable to wild-type enzyme, while 19A12 shows much reduced stability and lower expression levels. The goal was to compare the catalytic efficiency of these mutants in vivo and determine how in vitro properties relate to activities measured in vivo.
Preliminary experiments evaluated how minimal medium influenced 4E10 and 19A12 expression levels. Parameters such as E. coli strain, inducer (IPTG) concentration, and harvesting time were also varied to define optimal expression conditions. Using the most favorable conditions, cultures of 19A12- and 4E10-expressing cells were grown on a 500 mL scale. Cell suspensions were used for the bioconversion of propane on a 250 mL scale at 30 deg C. Samples were removed from the reaction vessels at different times and propanol and enzyme concentrations were determined. Activities for 19A12 and 4E10 were calculated to be 10-15 and 4-8 U / g cdw, respectively, within the first 90 minutes. These activities correspond to rates of 100-150 mol product / mol P450 / min for 19A12 and 30-40 mol product / mol P450 / min for 4E10. These represent lower-bound values since a certain fraction of product is continuously lost by evaporation and/or removed from the solution by the bubbling gases. 2-Propanol
accounts for 90% of the total product, similar to that observed for catalysis in vitro. These data demonstrate that interesting initial activity values can be obtained in the biohydroxylation of propane using engineered P450BM3 mutants. These values are comparable and superior to those reported by other groups using naturally occurring alkane monooxygenases, e.g. 20-25 U/g cdw for nonene epoxidation with AlkB-expressing E. coli (Witholt, 2000) and 2-5 U/g cdw for octane oxidation using AH enzymatic system from Gordonia strain (Kato, 2004). These results also support a good correlation between the activities measured in vitro and in vivo. In addition to the use of even more efficient engineered P450 biocatalysts, other factors can be exploited in order to achieve higher activities in the biotransformation: increasing the biocatalyst content in the cell (currently it is 1.5% but can be raised to 15%) and overcoming limitations through feeding systems. Optimization of oxygen/alkane ratio
has not yet been pursued. [There are no patents resulting from this research to report.]
Impacts
The overall goal of this project is to develop a biological route to converting waste materials via methane to methanol, a valuable liquid fuel and gasoline alternative. In the early phases of the project we are using the direct oxidation of the small alkane propane to produce propanol to study the utility of laboratory-engineered enzymes for oxidation of small alkanes in general. This work could lead to a completely new route to renewable liquid fuels.
Publications
- No publications reported this period
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