title: Semi-rational engineering of cytochrome CYP153A from Marinobacter aquaeolei for improved ω-hydroxylation activity towards oleic acid.
abstract: ω-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved ω-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket. The in vitro-reconstituted activity of Variant II with purified Pdx and Pdr was 2.7-fold that of the template, while the whole cell transformation activity was 2.0-fold that of the template. A 96-well format-based screening scheme for CYP153A was also developed, which should be useful for engineering of other P450s with low activity. Kinetic analyses indicated that the activity improvement for CYP153A variants largely resulted from enhanced electron transfer. This further demonstrates the importance of the electron transfer between P450s and the non-native redox partners for the overall performance of hybrid P450 systems.
authors.affiliation: Department of Chemical Engineering, National Engineering Laboratory for Industrial Enzymes, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Duan
authors.affiliation: Department of Chemical Engineering, National Engineering Laboratory for Industrial Enzymes, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China.|Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, 1000 Jinqi Road, Shanghai, 201403, China.
authors.forename: Lina
authors.initials: L
authors.lastname: Ba
authors.affiliation: Department of Chemical Engineering, National Engineering Laboratory for Industrial Enzymes, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Gao
authors.affiliation: Department of Chemical Engineering, National Engineering Laboratory for Industrial Enzymes, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China.
authors.forename: Xianxing
authors.initials: X
authors.lastname: Gao
authors.affiliation: National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin, 300308, China.
authors.forename: Dunming
authors.initials: D
authors.lastname: Zhu
authors.affiliation: DSM Biotechnology Center, Alexander Fleminglaan 1, 2613 AX, Delft, The Netherlands.
authors.forename: René M
authors.initials: RM
authors.lastname: de Jong
authors.affiliation: DSM Ahead R&D B.V.-Innovative Synthesis, Urmonderbaan 22, 6167 RD, Geleen, The Netherlands.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mink
authors.affiliation: DSM Ahead R&D B.V.-Innovative Synthesis, Urmonderbaan 22, 6167 RD, Geleen, The Netherlands. iwona.kaluzna@dsm.com.
authors.forename: Iwona
authors.initials: I
authors.lastname: Kaluzna
authors.affiliation: Department of Chemical Engineering, National Engineering Laboratory for Industrial Enzymes, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China. zhanglinlin@mail.tsinghua.edu.cn.
authors.forename: Zhanglin
authors.initials: Z
authors.lastname: Lin
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004579:Electron Transport; D005288:Ferredoxins / Q000378:metabolism; D006900:Hydroxylation; D053520:Marinobacter / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D019301:Oleic Acid / Q000378:metabolism*; D015202:Protein Engineering
chemical_list: D005288:Ferredoxins; D050505:Mutant Proteins; D019301:Oleic Acid; C100137:putidaredoxin; D003577:Cytochrome P-450 Enzyme System; C435228:cytochrome P-450 CYP153 (Acinetobacter); D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase
keywords: Cytochrome CYP153A; Electron transfer; Iterative saturation mutagenesis; Oleic acid; Site-directed saturation mutagenesis