File(s) under permanent embargo

Reason: Under embargo until January 2020. After this date a copy can be supplied under Section 51 (2) of the Australian Copyright Act 1968 by submitting a document delivery request through your library, or by emailing

Bacterial biotransformation of 1,8-cineole

posted on 08.06.2020, 23:45 by Birgit Unterweger
The monoterpenoid 1,8-cineole is a major component of Eucalyptus oil and an abundant renewable resource. Whilst 1,8-cineole has many uses in the pharmaceutical, flavour and fragrance industries, its application range is somewhat limited as 1,8-cineole is relatively inert. The utility of 1,8-cineole can be broadened by functionalising its carbon-skeleton with hydroxyl-groups. Hydroxyl-groups provide starting points for further modification making oxygenated 1,8-cineole derivatives interesting synthons with potential applications as antimicrobials, fragrance compounds and bio-derived monomeric building blocks for polymers.
   Oxygenation of 1,8-cineole can be achieved by chemical or biological means. Because of superior environmental-friendliness and regio- and stereoselectivity, biological approaches to oxygenation are preferable. The enzymes catalysing such oxygenations are collectively known as cytochrome P450 monooxygenases (P450s) that are often part of multicomponent enzyme systems and require the support of redox partner proteins to transfer the electrons required for catalysis.
   P450 systems are found in all kingdoms of life. In the area of monoterpenoid oxygenation, the potential of bacterial P450 systems has been widely recognised. However, only a handful of bacterial enzymes catalysing 1,8-cineole oxygenation are known. To expand the enzyme toolbox, the genome sequence of the 1,8-cineole-hydroxylating Sphingobium yanoikuyae B2 was determined. Two 1,8-cineole-binding P450s were purified from cell-free extracts prepared from S. yanoikuyae B2 cultivated in the presence of 1,8-cineole. Partial amino acid sequencing of these proteins enabled identification of full-length gene sequences encoding CYP101J2 and CYP101J3. A third protein, CYP101J4, was identified based on high amino acid sequence similarity to CYP101J2 and CYP101J3. The three P450s from S. yanoikuyae B2 were recombinantly expressed in Escherichia coli and showed the spectroscopic properties typical of P450s. The crystal structure of substrate-free CYP101J2 was solved and comparison to other P450s showed typical structural features and it was found to be similar to CYP101A1 and CYP176A1, which are also known to hydroxylate 1,8-cineole. 1,8-Cineole induced significant type I shifts in all three CYP101J enzymes from S. yanoikuyae indicating that this compound is a likely substrate. Functionality of the P450s was confirmed in E.coli and the main reaction product was hydroxy-1,8-cineole.
   To increase hydroxy-1,8-cineole yields, CYP101J2 was chosen for the development of a scalable production process. Using the enzymatic background from E. coli, low levels of hydroxylated 1,8-cineole were produced and to increase product yield genes encoding potential redox partners were identified in the S. yanoikuyae B2 genome. Combinations of redox partners were screened in E. coli resulting in the discovery of a range of native redox partners that resulted in increased product yields. One particular class I system comprised of CYP101J2, yanoikuyaeredoxin 2 (Yax2) and yanoikuyaeredoxin reductase 3 (YaR3) was shown to be an efficient combination by in vitro reconstitution. The three component system was characterised by high coupling of hydroxy-1,8-cineole formation with NADH consumption. This confirmed the suitability of the system for use in a scalable bioprocess. Thus, an E. coli high-cell density fed-batch process was developed using CYP101J2-Yax2-YaR3 which resulted in the production of ca. 20 g L-1 crude (1S)-2α-hydroxy-1,8-cineole in ca. 10 h.


Principal supervisor

Dena Lyras

Additional supervisor 1

Priscilla Johanesen

Additional supervisor 2

Geoffrey Dumsday

Year of Award


Department, School or Centre



Faculty of Medicine Nursing and Health Sciences