Bacterial biotransformation of 1,8-cineole
Creators: name: Unterweger, Birgit
name: Monash University Theses
Publisher: name: Figshare
Year: 2016
Subjects: Biochemistry

description: The
monoterpenoid 1,8-cineole is a major component of <i>Eucalyptus</i> 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. <br>
   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. <br>
   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 <i>Sphingobium yanoikuyae</i> B2 was
determined. Two 1,8-cineole-binding P450s were purified from cell-free extracts
prepared from <i>S. yanoikuyae</i> 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 <i>S. yanoikuyae</i> 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 <i>S. yanoikuyae</i> indicating that this compound is a likely substrate.
Functionality of the P450s was confirmed in <i>E.coli</i> and the main reaction
product was hydroxy-1,8-cineole. <br>
   To increase hydroxy-1,8-cineole yields, CYP101J2 was chosen
for the development of a scalable production process. Using the enzymatic
background from <i>E. coli</i>, low levels of hydroxylated 1,8-cineole were produced
and to increase product yield genes encoding potential redox partners were
identified in the <i>S. yanoikuyae</i> B2 genome. Combinations of redox partners were
screened in <i>E. coli</i> 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 <i>in vitro</i>
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 <i>E. coli</i>
high-cell density fed-batch process was developed using CYP101J2-Yax2-YaR3
which resulted in the production of ca. 20 g L<sup>-1</sup> crude
(1S)-2α-hydroxy-1,8-cineole in ca. 10 h.