Download Monooxygenases in the Butane and Cyclohexane Degradation

Monooxygenases in the Butane and
Cyclohexane Degradation Pathways
Authors: [1: Dr. N. Strunk1]
[2: M. Sc. D. Salamanca1]
[3: Prof. Dr. K.-H. Engesser1]
1
Universität Stuttgart, ISWA
Bandtäle 2
70569 Stuttgart, Germany
Monooxygenases are important enzymes in bacterial degradation pathways. In this project we evaluate the possible use of these
monooxygenases for the industrial production of chemicals. We enriched new bacterial strains which use butane or cyclohexane as
a sole source of carbon and energy. The genomes are screened to reveal the sequences of the degradative monooxygenases.
These enzymes will be cloned into acceptable host-strains for further improvement of their catalytic capacity.
Cyclohexane degradation pathway
Butane degradation pathway
Butane
1-Butanole
Butyraldehyde
Cyclohexane
Butyric
acid
Cyclohexanole
Cyclohexanone
OH
O
O2
H2O
NAD(P)H NAD(P)
Butane
monoxygenase
O
NAD(P) NAD(P)H
NAD(P) NAD(P)H
Alcohol
dehydrogenase
O
O
O2 H2O
H2O
OH
Caprolactone
O2 H2O
O
O
Aldehyde
dehydrogenase
There are reports on three different butane monooxygenases.
Two membrane bound types (AMO, pBMO) and one soluble
butane monooxygenase (sBMO), in which our interest lies.
Tab. 1: Our enriched butane degrading bacterial strains.
Strain Gram Taxonomy
Group
BUT1
neg. Stenotrophomonas maltophilia ssp.
γ
BUT2
neg. Haematobacter sp.
α
BUT3
neg. Azoarcus sp.
α
BUT4
neg. Variovorax sp.
β
BUT5
pos. Mycobacterium sp.
BUT6
pos. Mycobacterium sp.
BUT8
pos. Rhodococcus sp.
BDO3 neg. Pseudomonas putida ssp.
γ
NAD(P)H NAD(P)
NAD(P) NAD(P)H
NAD(P)H NAD(P)
Cyclohexane
monooxygenase
Alcohol
dehyd.
Baeyer-Villiger
Monooxygenase
No sequence of a cyclohexane monooxygenase is published
until now. Studies indicated that this enzyme could be related
to the soluble butane monooxygenase.
The cyclohexane pathway contains a second monooxygenase,
type: Baeyer-Villiger monooxygenase (BVO). The sequences
of many of these enzymes are published, their sequences are
diverse. The enrichment of cyclohexane
degrading strains failed several
times. Success was brought by
the use of a biotricklingfilter as
enrichment system.
Fig. 3: Biotrickling filter (right side) and
formation of biomass on the
packing material (bottom)
Fig. 1: sBMO genes (Sluis et. al. 2002)
The sBMO is a three subunit enzyme (bmoX, bmoY, bmoZ).
The gene cassette contains additional genes with codes for an
oxido-reductase (bmoC), a regulatory protein (bmoC) and a
short chain putative protein (bmoD).
• 2 Sequences of the sBMO are
published.
• Primers from Sluis et al. 2002
resulted in unspecific bands.
• Multiple sequence alignment
gave us a primer pair
(red arrows in Fig. 1) for the
bmoX – gene, 993 bp length
according to published
reference sequence.
• Single amplificates appeared in
BUT1, BUT3, BUT8
• Sequencing of the amplificates
revealed in all cases parts
of the bmoX gene.
• The other butane degrading
strains contain no soluble
butane monooxygenase.
Fig 2: Amplification of a
bmoX - fragment
Tab. 2: Our enriched cyclohexane degrading bacterial strains.
Strain Gram Taxonomy
CHX1 neg. Pseudoxanthomonas sp.
CHX2 neg. Stenotrophomonas sp.
CHX4 neg. Sphingomonas sp.
Group
γ
γ
α
• Multiple sequence alignments Fig 4: Amplification of a
gave us several primer pairs.
• One primer pair worked
on CHX1 and CHX4.
• The use of the primers for
bmoX – gene of the butane
degradation pathway
delivered no specific result.
• New primer pairs are to
be developed to help finding
the monooxygenases.
• Transposon mutagenesis
is applied to the CHX
strains to identify the
degradative genes.
BVO - gene fragment