Engineered Alkane-Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties

Department of Chemistry and Chemical Engineering, California Institute of Technology, 1200 California Blvd. MC 210-41, Pasadena, CA 91125, USA.
Angewandte Chemie International Edition (Impact Factor: 11.26). 02/2007; 46(44):8414-8. DOI: 10.1002/anie.200702616
Source: PubMed


(Figure Presented) Divide, evolve, and conquer: A domain-based strategy (see scheme) was used to engineer high catalytic and coupling efficiency for propane hydroxylation in a multidomain cytochrome P450 enzyme. The engineered enzymes exhibit high total activities in whole-cell bioconversions of propane to propanol under mild conditions, using air as oxidant.

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    • "Six mutants of the uncharged Ser72 and Ala74 residues that contained a negatively charged amino acid at these respective positions were selected. The Ser72 and Ala74 residues are located around the substrate binding channel (Li et al., 2001b; Otey et al., 2006; Dietrich et al., 2009) of the protein and have been shown to influence regioselectivity (Otey et al., 2006; Dietrich et al., 2009) and metabolic efficiency (Li et al., 2000, 2001b; Fasan et al., 2007) in previous studies. Two M01 mutants that contained mutations at the Ser72 position, MT43 (S72D) and MT44 (S72E), and one M01 mutant that contained a mutation at the Ala74 position (MT41; A74E) were selected. "
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    ABSTRACT: In the present study, the diversity of a library of drug-metabolizing bacterial cytochrome P450 (P450) BM3 mutants was evaluated by a liquid chromatography-mass spectrometry (LC-MS)-based screening method. A strategy was designed to identify a minimal set of BM3 mutants that displays differences in regio- and stereoselectivities and is suitable to metabolize a large fraction of drug chemistry space. We first screened the activities of six structurally diverse BM3 mutants toward a library of 43 marketed drugs (encompassing a wide range of human P450 phenotypes, cLogP values, charges, and molecular weights) using a rapid LC-MS method with an automated method development and data-processing system. Significant differences in metabolic activity were found for the mutants tested and based on this drug library screen; nine structurally diverse probe drugs were selected that were subsequently used to study the metabolism of a library of 14 BM3 mutants in more detail. Using this alternative screening strategy, we were able to select a minimal set of BM3 mutants with high metabolic activities and diversity with respect to substrate specificity and regiospecificity that could produce both human relevant and BM3 unique drug metabolites. This panel of four mutants (M02, MT35, MT38, and MT43) was capable of producing P450-mediated metabolites for 41 of the 43 drugs tested while metabolizing 77% of the drugs by more than 20%. We observed this as the first step in our approach to use of bacterial P450 enzymes as general reagents for lead diversification in the drug development process and the biosynthesis of drug(-like) metabolites.
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    • "It is difficult to accurately measure the "in-cell conversion rate", but apparently the AMO/CPR-oxidation capability needs to be improved to fully convert the amorphadiene to artemisinic acid. Improvement of the kinetic properties of P450s is likely to be challenging, though such improvements have been demonstrated in a microbial system [28]. Nonetheless, genes/enzymes in plant secondary metabolism are evolutionarily recent, and hence there may be room for further improvement of their catalytic efficiencies by in vitro evolution or site-directed mutagenesis. "
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    ABSTRACT: Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required. Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 microg mL(-1) in shake-flask cultures and 1 g L(-1) in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast. The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast.
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