CYP101J2, CYP101J3, and CYP101J4, 1,8-cineolehydroxylating cytochrome P450 monooxygenases from Sphingobium yanoikuyae strain B2

Birgit Unterweger, Dieter M. Bulach, Judith Scoble, David J. Midgley, Paul Greenfield, Dena Lyras, Priscilla Johanesen, Geoffrey J. Dumsday

Research output: Contribution to journalArticleResearchpeer-review

Abstract

We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8- cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida. Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications.

Original languageEnglish
Pages (from-to)6507-6517
Number of pages11
JournalApplied and Environmental Microbiology
Volume82
Issue number22
DOIs
Publication statusPublished - Nov 2016

Cite this

@article{0bbff0c5e2a64deab8287aedf8ad49bc,
title = "CYP101J2, CYP101J3, and CYP101J4, 1,8-cineolehydroxylating cytochrome P450 monooxygenases from Sphingobium yanoikuyae strain B2",
abstract = "We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8- cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47{\%} and 44{\%} identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida. Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30{\%} amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications.",
author = "Birgit Unterweger and Bulach, {Dieter M.} and Judith Scoble and Midgley, {David J.} and Paul Greenfield and Dena Lyras and Priscilla Johanesen and Dumsday, {Geoffrey J.}",
year = "2016",
month = "11",
doi = "10.1128/AEM.02067-16",
language = "English",
volume = "82",
pages = "6507--6517",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "Am Soc Microbiol",
number = "22",

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CYP101J2, CYP101J3, and CYP101J4, 1,8-cineolehydroxylating cytochrome P450 monooxygenases from Sphingobium yanoikuyae strain B2. / Unterweger, Birgit; Bulach, Dieter M.; Scoble, Judith; Midgley, David J.; Greenfield, Paul; Lyras, Dena; Johanesen, Priscilla; Dumsday, Geoffrey J.

In: Applied and Environmental Microbiology, Vol. 82, No. 22, 11.2016, p. 6507-6517.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - CYP101J2, CYP101J3, and CYP101J4, 1,8-cineolehydroxylating cytochrome P450 monooxygenases from Sphingobium yanoikuyae strain B2

AU - Unterweger, Birgit

AU - Bulach, Dieter M.

AU - Scoble, Judith

AU - Midgley, David J.

AU - Greenfield, Paul

AU - Lyras, Dena

AU - Johanesen, Priscilla

AU - Dumsday, Geoffrey J.

PY - 2016/11

Y1 - 2016/11

N2 - We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8- cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida. Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications.

AB - We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8- cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida. Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications.

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U2 - 10.1128/AEM.02067-16

DO - 10.1128/AEM.02067-16

M3 - Article

VL - 82

SP - 6507

EP - 6517

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 22

ER -