Laboratory evolution of P450 BM-3 for mediated electron transfer.

International University Bremen, Campus Ring 8, 28759 Bremen, Germany.
ChemBioChem (Impact Factor: 3.74). 05/2006; 7(4):638-44. DOI: 10.1002/cbic.200500436
Source: PubMed

ABSTRACT Preparative synthesis with P450 monooxygenases is hampered in cell-free systems by the requirement for cofactors such as NAD(P)H as reduction equivalents. A validated medium-throughput screening system was designed for improving P450 monooxygenases by mediated electron transfer with zinc/cobalt(III)sepulchrate (Zn/Co(III)sep) as an alternative and cost-effective cofactor system. The monooxygenase P450 BM-3 F87A was used as a model system for developing the screening system in a 96-well format. A coefficient of variation of less than 10% was achieved under optimized screening conditions. The mediator evolution screen was validated by comparing the activity of P450 BM-3 to P450 BM-3 F87A and by screening a saturation mutagenesis library at amino acid position R47. For mediated electron transfer, two double mutants P450 BM-3(F87A R47F) and P450 BM-3 (F87A R47Y) were identified with a two-threefold increased catalytic efficiency (up to 32 microM(-1) min(-1) for P450 BM-3(F87A R47F) and 34 microM(-1) min(-1) for P450 BM-3 (F87A R47Y)) compared to P450 BM-3 F87A. The kinetic constants of the double mutants are, in contrast to those of P450 BM-3 F87A, dependent on Co(III)sep concentration in the presence of NADPH. kcat increases from 145 min(-1) (0.25 mM Co(III)sep) to 197 min(-1) (0.5 mM Co(III)sep), and Km decreases simultaneously from 7.0 microM to 3.7 microM, for P450 BM-3 (F87A R47F). For P450 BM-3 (F87A R47Y), kcat increases from 138 min(-1) (0.25 mM Co(III)sep) up to 187 min(-1) (0.5 mM Co(III)sep), and Km decreases from 8.2 microM to 4.2 microM. Due to lower Km values, the catalytic efficiencies were improved six times for P450 BM-3 (F87A R47F) and three times for P450 BM-3 (F87A R47Y), when comparing catalytic efficiencies of the mediated electron-transfer system to the natural reduction equivalent NADPH.

  • [Show abstract] [Hide abstract]
    ABSTRACT: P450(BM3) (CYP102A1), a fatty acid hydroxylase from Bacillus megaterium, has been extensively studied over a period of almost forty years. The enzyme has been redesigned to catalyse the oxidation of non-natural substrates as diverse as pharmaceuticals, terpenes and gaseous alkanes using a variety of engineering strategies. Crystal structures have provided a basis for several of the catalytic effects brought about by mutagenesis, while changes to reduction potentials, inter-domain electron transfer rates and catalytic parameters have yielded functional insights. Areas of active research interest include drug metabolite production, the development of process-scale techniques, unravelling general mechanistic aspects of P450 chemistry, methane oxidation, and improving selectivity control to allow the synthesis of fine chemicals. This review draws together the disparate research themes and places them in a historical context with the aim of creating a resource that can be used as a gateway to the field.
    Chemical Society Reviews 02/2012; 41(3):1218-60. · 24.89 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: : Increasing needs of green energy and concerns of climate change are motivating intensive R&D efforts toward the low-cost production of electricity and bioenergy, such as hydrogen, alcohols, and jet fuel, from renewable sugars. Cell-free biosystems cell-free biosystems for biomanufacturing biomanufacturing (CFB2) have been suggested as an emerging platform to replace mainstream microbial fermentation for the cost-effective production of some biocommodities. As compared to whole-cell factories, cell-free biosystems cell-free biosystems comprised of synthetic enzymatic pathways have numerous advantages, such as high product yield, fast reaction rate, broad reaction condition, easy process control and regulation, tolerance of toxic compound/product, and an unmatched capability of performing unnatural reactions. However, issues pertaining to high costs and low stabilities of enzymes and cofactors as well as compromised optimal conditions for different source enzymes need to be solved before cell-free biosystems cell-free biosystems are scaled up for biomanufacturing biomanufacturing . Here, we review the current status of cell-free technology, update recent advances, and focus on its applications in the production of electricity and bioenergy.
    Advances in biochemical engineering/biotechnology 06/2013; · 1.64 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: : Although cell-free biosystems have been used as a tool for investigating fundamental aspects of biological systems for more than 100 years, they are becoming an emerging biomanufacturing platform in the production of low-value biocommodities (e.g., H(2), ethanol, and isobutanol), fine chemicals, and high-value protein and carbohydrate drugs and their precursors. Here we would like to define the cell-free biosystems containing more than three catalytic components in a single reaction vessel, which although different from one-, two-, or three-enzyme biocatalysis can be regarded as a straightforward extension of multienzymatic biocatalysis. In this chapter, we compare the advantages and disadvantages of cell-free biosystems versus living organisms, briefly review the history of cell-free biosystems, highlight a few examples, analyze any remaining obstacles to the scale-up of cell-free biosystems, and suggest potential solutions. Cell-free biosystems could become a disruptive technology to microbial fermentation, especially in the production of high-impact low-value biocommodities mainly due to the very high product yields and potentially low production costs.
    Advances in biochemical engineering/biotechnology 10/2012; · 1.64 Impact Factor