Cytochrome P450 Monooxygenases in Biotechnology and Synthetic Biology

Cytochromes P450 are ubiquitous enzymes accepting a tremendous number of substrates and catalyzing a broad range of reactions with potential applications in biotechnology and synthetic biology. P450s were engineered to catalyze abiotic reactions such as carbene or nitrene transfers, opening up completely new perspectives in synthetic chemistry. Lately, P450s have successfully been introduced into artificial multi-enzyme cascades, both in vitro and in vivo, providing alternative routes for retro-synthetic production of high-value oxyfunctionalized compounds. Harnessing the synthetic potential of P450s in chemo-enzymatic processes or as part of reconstituted biosynthetic pathways in microbial hosts provides promising strategies for de novo synthesis of synthons and complex natural products, even though there are still some obstacles to overcome. Cytochromes P450 (P450 or CYP) are heme-containing enzymes that catalyze the introduction of one atom of molecular oxygen into nonactivated C–H bonds, often in a regio- and stereoselective manner. This ability, combined with a tremendous number of accepted substrates, makes P450s powerful biocatalysts. Sixty years after their discovery, P450 systems are recognized as essential bio-bricks in synthetic biology approaches to enable production of high-value complex molecules in recombinant hosts. Recent impressive results in protein engineering led to P450s with tailored properties that are even able to catalyze abiotic reactions. The introduction of P450s in artificial multi-enzymatic cascades reactions and chemo-enzymatic processes offers exciting future perspectives to access novel compounds that cannot be synthesized by nature or by chemical routes. Cytochromes P450 (P450 or CYP) are heme-containing enzymes that catalyze the introduction of one atom of molecular oxygen into nonactivated C–H bonds, often in a regio- and stereoselective manner. This ability, combined with a tremendous number of accepted substrates, makes P450s powerful biocatalysts. Sixty years after their discovery, P450 systems are recognized as essential bio-bricks in synthetic biology approaches to enable production of high-value complex molecules in recombinant hosts. Recent impressive results in protein engineering led to P450s with tailored properties that are even able to catalyze abiotic reactions. The introduction of P450s in artificial multi-enzymatic cascades reactions and chemo-enzymatic processes offers exciting future perspectives to access novel compounds that cannot be synthesized by nature or by chemical routes. a reaction that has (so far) not been described as being catalyzed by any known enzyme. antimicrobial substances that either kill bacteria or inhibit their growth and that are widely used in the treatment and prevention of bacterial infections. a method of protein engineering that mimics the evolutionary process in nature and combines the generation of large mutant libraries with their rapid and sensitive high-throughput screening. chemical substances used to treat diseases or to promote well-being. signaling molecules made by oxidation of arachidonic acid or other polyunsaturated fatty acids. European Medicines Agency; an agency of the European Union responsible for the evaluation and supervision of medicines for human and veterinary use. Food and Drug Administration; an agency of the United States Department of Health and Human Services responsible for protecting and promoting public health through supervision of drugs, vaccines, and biopharmaceuticals. a semi-rational method of protein engineering. Based on a mutant library constructed via first-sphere mutagenesis, this method involves mapping of the active site configurations of P450 variants using a set of semisynthetic chromogenic probes, and prioritization of those variants with potential regio- and/or stereoselectivity towards the target substrate. flavin adenine dinucleotide, a prosthetic group. flavin mononucleotide, a prosthetic group. an iron ion coordinated by four nitrogen atoms of porphyrin, a prosthetic group. enzymes in which the heme prosthetic group is linked to the apoprotein via cysteine thiolate axial ligand. the practice of optimizing genetic and regulatory processes within a cell to increase the cells’ production of a substance of interest. a protein engineering technique that allows the discrimination between beneficial and neutral or deleterious mutations. a computational method to predict the preferred orientation of a substrate in the enzyme active site realized by geometric matching and shape complementarity. a computational method to predict the preferred positioning and state of a substrate in the enzyme active site during catalysis. The method takes into account the conformational space and rigid body transformations (translations and rotations as well as internal changes to the substrate’s structure, for example, torsion angle rotations). nicotinamide adenine dinucleotide, a cofactor consisting of two nucleotides joined through phosphate groups; one nucleotide contains an adenine base and the other nicotinamide. nicotinamide adenine dinucleotide phosphate; differs from NADH in the presence of an additional phosphate group. an organic molecule that is tightly, for instance, covalently bound to the enzyme. mutagenesis of enzymes based on mechanistic and structural data. chemically reactive species containing oxygen (e.g., peroxides, superoxide) formed as natural byproducts of the normal metabolism of oxygen. a protein engineering technique, in which a DNA codon is randomized to produce all possible amino acids in the protein at that position. biologically active organic compounds with a specific core structure composed of 17 carbon atoms bonded in four fused rings. an interdisciplinary branch of biology and engineering that combines and applies various disciplines from these domains with the aim of building artificial biological systems for research, engineering, and medical applications. a part within a molecule that is related to a possible synthetic operation; a retro-synthetic fragmentation structure, often used to mean ‘synthetic building block’. a large and diverse class of secondary metabolites produced mainly by plants. Terpenes consist of different numbers of isoprene units. Terpenes that contain additional functional groups are called terpenoids. organic compounds important to health; an essential nutrient that cannot be produced in the body, but must be procured from food. chemical substances found within an organism that are not naturally produced by the organism. Natural compounds can become xenobiotics if they are taken up by another organism.