Engineering cytochrome P450 BM3 of Bacillus megaterium for terminal oxidation of palmitic acid.
Directed evolution via iterative cycles of random and targeted mutagenesis was applied to the P450 domain of the subterminal fatty acid hydroxylase CYP102A1 of Bacillus megaterium to shift its regioselectivity towards the terminal position of palmitic acid. A powerful and versatile high throughput assay based on LC-MS allowed the simultaneous detection of primary and secondary oxidation products, which was instrumental for identifying variants with a strong preference for the terminal oxidation of palmitic acid. The best variants identified acquired up to 11 amino acid alterations. Substitutions at F87, I263, and A328, relatively close to the bound substrate based on available crystallographic information contributed significantly to the altered regioselectivity. However, non-obvious residues much more distant from the bound substrate showed surprising strong contributions to the increased selectivity for the terminal position of palmitic acid.
Firmenich SA, Corporate R&D, Route des Jeunes 1, CH-1211, Geneva 8, Switzerland. Electronic address: fredi.bruhlmann@firmenich.com.
Fredi
F
Brühlmann
Protéus, Parc Georges Besse, 70 allée Graham Bell, 30035 Nîmes cedex 1, France; Total, New Energies, R&D Biotechnology, 24 cours Michelet, la Défense 10, Paris La Défense cedex 92069, France.
Laurent
L
Fourage
Protéus, Parc Georges Besse, 70 allée Graham Bell, 30035 Nîmes cedex 1, France.
Christophe
C
Ullmann
Firmenich SA, Corporate R&D, Route des Jeunes 1, CH-1211, Geneva 8, Switzerland.
Olivier P
OP
Haefliger
Firmenich SA, Corporate R&D, Route des Jeunes 1, CH-1211, Geneva 8, Switzerland.
Nicolas
N
Jeckelmann
Firmenich SA, Corporate R&D, Route des Jeunes 1, CH-1211, Geneva 8, Switzerland.
Cédric
C
Dubois
Protéus, Parc Georges Besse, 70 allée Graham Bell, 30035 Nîmes cedex 1, France; SEPPIC, 127 chemin de la Poudrerie, Castres cedex 81105, France.
Denis
D
Wahler
D019943:Amino Acid Substitution / Q000235:genetics; D001410:Bacillus megaterium / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution; D005818:Genetic Engineering; D057166:High-Throughput Screening Assays; D016296:Mutagenesis / Q000235:genetics*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D010084:Oxidation-Reduction; D019308:Palmitic Acid / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D019308:Palmitic Acid; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases
Bacillus megaterium; Cytochrome P450; Directed evolution; Palmitic acid; Terminal oxidation