identification of additional genes in the cyclohexanol...
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Identification of Additional Genes in the Cyclohexanol Degradation Pathway in
Rhodococcus maris HI-31
Halla Bakheit
A Thesis
In
The Department
Of
Microbiology and Immunology
Presented in -Partial Fulfillment of the Requirements
for the Degree of Master of Microbiology and Immunology at
McGill University
Montréal, Québec, Canada
August 2005
©
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Identification of Additional Genes in the Cyclohexanol Degradation Pathway in
Rhodococcus maris HI-3I
Halla Bakheit
A Thesis
In
The Department
Of
Microbiology and Immunology
Presented in Partial Fulfillment of the Requirements
for the Degree of Master of Microbiology and Immunology at
McGill University
Montréal, Québec, Canada
August 2005
©
II
Abstract
Many bacteria have the ability to degrade cyclohexanol through a five-step
biochemical pathway that leads to the production of adipic acid. Adipic acid is then
processed by ~-oxidation to succinyl-CoA and acetyl-CoA. The original goal of this
study was to locate and characterize the gene that is responsible for the first
dehydrogenation step of the cyclohexanol (chn) degradation pathway in the Gram
positive bacterium Rhodococcus maris strain HI-3I in order to obtain the full gene
complement forcyclohexanol degradation. Instead, three new open reading frames
(ORFs) or genes were obtained as the result of cloning and sequencing of a 5-kb Sad
fragment upstream of the known chn gene cluster in strain HI-3I. Two of the ORFs,
designated chnB2 and chnC2, were found to be homologs of the chnB and chnC
encoding a cyclohexanone monooxygenase (CHMO) and hydrolase, respectively.
This implies possible gene duplication of this portion of the pathway. A third ORF is
a potential long-chain fatty acid Co-A ligase. Characteristics of the three ORFs are
described. In addition, attempts were made to sub-clone the 5-kb Sad fragment into
pT7-6 expression vector for activity assays, etc.
III
/~,
Résumé:
Plusieurs bactéries ont la capacité de dégrader le cyclohexanole par une voie
biochimique à cinq étapes, qui mène à la production d'acide adipique. L'acide
adipique est ensuite converti par beta-oxidation en succinyl-CoA et en acétyl-CoA.
L'objectif initial de ce projet était de localiser et de caractériser le gène responsable
de la première étape de déshydrogenation dans la voie de dégradation du
cyclohexanol (chn), dans la bactérie Gram-positive Rhodococcus maris (souche HI-
31). Trois regions codantes ou gènes ont été obtenus par clonage et séquençage d'un
fragment Sad de 5 kb, localisé en amont du groupement de gènes chn dans la souche
HI-3I. Deux des régions codantes, nommées chnB2 et chnC2, semblent être
homologues à chnB et chnC, qui codent respectivement pour la monooxygénase de la
cyclohexanone (CHMO) et pour l'hydrolase de la cyclohexanone. Ce résultat
suggère une duplication dans cette portion de la voie biochimique. La troisième
région codante est potentiellement une ligase d'acides gras CoA à longues chaînes.
Les caractéristiques des trois régions codantes sont décrites. De plus, nous avons
tenté de sous-cloné le fragment Sad de 5 kb dans un vecteur d'expression pT7-6 pour
faire des essaies d'activité, etc.
IV
Acknowledgement
1 would like to thank my supervisor, Dr. Peter C. K. Lau, for his friendship, excellent
advice and guidance. 1 am also grateful for the opportunity to work in his lab on this
project. His patience is a profound gift for which 1 will always be thankful. He
provided a unique liberating environment. And 1 would like to thank also the
members of the lab especially Hélène Bergeron for providing technical guidance. The
administrative assistance of Jeniffer Dimassimo of the Microbiology and
Immunology Department at McGill University is gratefully acknowledged.
For financial support, 1 would like to thank BioCAP Canada, and the Microbiology
and Immunology Department of McGill for the Harrison Award.
1 want to extend my thanks to my family, mother and father, for supporting my
aspirations and for the years of encouragement.
v
Table of Contents
Page
Title page ......................................................................................... 1
Short Title ........................................................................................ II
Abstract ........................................................................................... .III
Résumé ..................... , ..................................................................... .IV
Acknowledgement. .............................................................................. V
Table of Contents .............................................................................. VI
List of Tables ................................................................................... .IX
List of Figures ................................................................................ , ... X
List of Abbreviations ....................... , .................................................... XI
1. Introduction ................................................................................... 1
2. Literature Review . ............................................................................ 3
2.1. The Chemical Industry ............................................................. 3
2.2. Adipic Acid Production ....................... '" ................................. .4
2.3. Cyclohexanol Degradation Pathway ............................................. 5
2.4. Baeyer and Villiger Reaction ..................................................... 9
2.5. Baeyer-Villiger Monooxygenases .............................................. 10
2.6. Potential Commercial BVMO Biotransformation Process .................. 13
2.7. Rhodococcus Genus ............................................................... 14
2.8. Rhodococcus maris strain HI-31 ............................................... 16
2.9. Objectives .......................................................................... 18
VI
Page
3. Materials and Methods .................................................................... 19
3.1. Bacterial Culture .................................................................. 19
3.2. Bacterial Strains and Plasmids ................................................... 19
3.3. Restriction Enzymes and Agarose Gel Electrophoresis ..................... 21
3.4 .. Genomic DNA Preparation ...................................................... .21
3.5. Southem Blot ....................................................................... 22
3.6. Cloning ofpCMR500 ........................................................... 25
3.7. Colony Hybridization ............................................................. 27
3.8. Sequencing ofthe 5-kb Sad Fragment ........................................ .27
3.9. Sub-cloning of 5-kb Sad Fragment in pT7-6 Expression Vector for
chnC2 Protein Expression ................................................... .30
3.10. Sub-cloning of chnB2 Gene for Protein Expression ....................... .31
4. Results ..... .................................................................................. 33
4.1. Identification of a new DNA Fragment for Cloning ....................... .33
4.1.1. Cloning of Sad 5-kb Fragment.. ................................... 37
4.1.2. Identification ofthe Clone by DNA Sequencing ................. .37
4.2. Sequencing of the 5-kb insert in pCMR500 ................................. .40
4.2.1. Open Reading Frames Analysis .................................... .40
4.3. Sub-cloning of5-kb Sad Fragment in pT7-6 Expression Vector .......... 63
4.4. Sub-cloning of Rhodococcus maris CHM02 for Protein Expression ..... 63
4.4.1. PCR for Rhodococcus maris chnB2 Amplification ............... 63
VII
Page
5. Discussion . ................................................................................... 64
5.1. Rhodococcus maris HI -31 Cyclohexanol Degradation Pathway ........... 64
5.2. Conclusion ........................................................................ 72
6. References .................... ................................................................ 74
VIII
List of Tables
Page
Table 1. Strains and Plasmids used in this Study ................................... 20
Table 2. Primers used in DNA Sequencing Reactions .............................. 29
Table 3. Homology of the Rhodococcus maris HI-3I ORFs .................... .46
Table 4. Percentage Identity of ChnB 1 and ChnB2 Amino Acid Sequences of Rhodococcus maris HI-31 and Brevibacterium sp. HCU ............... 49
Table 5. Substrate Specificity of Rhodococcus maris HI-31 CHMOI and CHM02 ........................................................................ 66
IX
List of Figures
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
Fig. 8.
Fig. 9.
Fig. 10.
Fig. 11.
Fig. 12.
Fig. 13.
Fig. 14.
Fig. 15.
Page
Cyclohexanol Degradation Pathway .......................................... 8
Gene Organization of the Cyclohexanol Degradation Pathway in Rhodococcus maris and Acinetobacter sp. strain 9871 .................. 17
Physical Map of the Cyclohexanol Gene Cluster in Rhodococcus maris HI-31 .................................................................... 35
Southem Blot Analysis of Rhodococcus maris Genomic DNA ........ 36
Screening with Restriction Digestion of the 13 Positive Clones Obtained from Colony Hybridization Experiment. ....................... 38
Electrophoresis Gel Analysis for a Non-digested B-l clone ........... .39
Nucleotide Sequence of the 5-kb Sac l Fragment ....................... .45
Sequence Alignment of the CHMOI and CHM02 from Rhodococcus maris ............................................................................ 48
Sequences of Rhodococcus maris HI-3I ChnB2 Aligned with other Cyclohexanone Monooxygenases of other Bacteria ..................... 52
Phylogenetic Tree of the CHMOs of the Rhodococcus maris HI-31 .. 54
Rhodococcus maris ChnC2 Sequence Aligned with the Hydrolase Sequences of other Bacteria .................................................. 56
Phylogenetic Tree of the Hydrolases of the Rhodococcus maris HI-31 ............................................................................... 58
Rhodococcus maris LCF A CoA ligase Sequence Aligned with Sequences of LCF A CoA ligase of other Bacteria ....................... 60
Phylogenetic Tree of the LCFA Co-A Ligase of the Rhodococcus maris HI-3I .................................................................... 62
Comparison of the Gene Organization of Cyclohexanol Degradation Pathway in Rhodococcus maris HI-31, B. epidermidis HCU, and Acinetobacter sp. strain SE 19 ............................................... 72
x
Abbreviations
AP
BCIP
BSA
BLAST
CHMO
dNTP
DNA
ee
FAD
Kb
KDa
Ilg
ilL
LB
NBT
NAD
NADP
ORF
OD
PCR
List of Abbreviations
Description
Ampicillin
5-bromo-4-chloro-3-indolyl-phosphate
Bovine serum albumin
Basic local alignment search tool
Cyclohexanone Monooxygenase
Deoxy nucleoside triphosphate
Deoxyribonucleic acid
Enantiomeric excess
Flavin Adenine Dinucleotide
Kilo-basepairs
Kilo-Dalton
Microgram
Microlitre
Luri a-Bertani
Nitro blue tetrazolium
Nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide phosphate
Open reading frame
Optical density
Polymerase chain reaction
XI
SDS
TAE
UV
X-gal
Sodium dodecyl sulfate
Tris acetate-ethylene diamine tetra-acetic
Ultraviolet
5-bromo-4-chloro-3-indolyl-~-D-galactoside
XII
1. Introduction
Recently, microbes have been used as environmentally-benign synthetic
catalysts in many applications (Denis-Larose et al., 1997; Morii et al., 1999;
Kostichka et al., 2001), inc1uding the synthesis of adipic acid. Adipic acid is
produced and manufactured by using a petroleum-based product, benzene, as starting
material (Anastas et al., 1994). Due to the fact that benzene is a highly toxic
compound, the commercial production of adipic acid has been associated with health
hazards and environmental concems (Hrelia et al., 2004). Therefore, the use of a
more environmentally friendly production method for adipic acid is desirable.
Many microorganisms have the ability to degrade cyclohexanol to adipic acid
through a five-step reaction (Iwaki et al., 1999; Cheng et al., 2000; Brzostowicz et al.,
2000). The most commonly used bacterium for the study of cyc1ohexanol degradation
pathway isAcinetobacter sp. NCIMB 9871 (Donoghue et al., 1975; Chen et al., 1988;
Iwaki et al., 1999). In this Gram-negative species, ail the five genes involved in the
cyclohexanol degradation pathway have been identified, as weIl as a transcriptional
regulator (Iwaki et al., 1999; Iwaki et al., 2003).
Rhodococcus maris HI-31 is a newly isolated Gram-positive bacterium that
has the ability to use cyclohexanol and cyclohexanone as sole carbon source fo~
growth (Iwaki, Ph.D. Thesis, 2001). Previously, four genes needed for cyc1ohexanol
degradation were identified, chnB, chnC, chnD and chnE, as weIl as a transcriptional
regulator, chnR (Lau laboratory, unpublished data). The chnB, chnC, chnD and chnE,
code for cyclohexanone monooxygenase, ë-caprolactone hydrolase, 6-
hydroxyhexanoic acid dehydrogenase, and 6-oxohexanoic acid dehydrogenase,
respectively.
1
The goal of this study was to locate and characterize chnA, the gene that
produces the first enzyme responsible for the dehydrogenation of cyc1ohexanol to
cyc1ohexanone. Furthermore, it was intended to discover additional genes or, open
reading frames in association with the cyc1ohexanol degradation pathway.
As a result, a 5-kb Sad fragment was c10ned from R. maris DNA in E. coli
and then sequenced. Sequence analysis revealed three ORFs. Two were designated,
chnB2 and chnC2, since they were found to be homologs of the chnB and, chnC
encoding a cyc1ohexanone monooxygenase and hydrolase, respectively, from the
same organism. The third ORF encodes a putative long-chain fatty acid Co-A ligase.
The predicted amino acid sequence of ChnB2 was 61 % identical to ChnB 1 of R.
maris, and 48% identical to the cyc1ohexanone monooxygenase (CHMO) of
Acinetobacter sp. NCIMB 9871. As expected of a flavoprotein, ChnB2 contains a
F AD binding motif and a NADPH cofactor motif (Stehr et al., 1998), and a Baeyer-
Villiger monooxygenase (BVMO) fingerprint motif. In ChnC2, a fingerprint
GlyXSerXGly motif found in other hydrolases can be located. In the case of LCF A
Co-A ligase, a conserved region similar to the ATP/AMP signature motif typically
found in this group of proteins was predicted.
A phylogenetic tree of each of the three sequences was generated to show the
relatedness of the individual family of genes.
Despite the identification of three new ORFs, and three other potential genes
in another fragment of R. maris DNA (Iwaki; Lau, Laboratory, unpublished data), the
chnA gene that encodes the first enzyme in the cyc1ohexanol degradation pathway has
not been located.
2
2. Literature Review
2.1. The Chemical Industry
The chemical industry, often referred to as a keystone industry, serves almost
every sector of the manufacturing economy. Chemical products are needed in aIl
walks of life, including agriculture, medicine, telecommunication, everyday life, etc.
Increased ecological awareness and increasing restrictions are forcing the chemical
industry worldwide to redesign sorne production lines in order to comply with the
protection of the environment. Industrial organic chemistry in particular often makes
use of multi-step synthetic methods, in which, the amount of useful products are
largely out-weighed by the amount of by-products that need disposaI. Persistent
organic pollutants that are produced industrially and are found ubiquitously in the
environment, having a different degree of toxic, mutagenic, and carcinogenic
activities, include: aromatic solvents (e.g. benzene, toluene, xylene isomers);
chlorinated compounds, nitrate, pesticides from agricultural runoffs, and polycyclic
aromatic hydrocarbons (e.g. naphthalene, fluoranthene, pyrene). Organic solvents
also pose a particular concern to the chemical industry, because of the large volume
used in synthe sis, processing, and separations. Many are classified as volatile organic
compounds (VOCs) or hazardous air pollutants and are flammable, toxic, or
carcinogenic. Therefore, the design of environmentally benign solvents and
solventless systems has been one of the most active areas of green chemistry over the
past 10 years (Anastas et al., 2002). Green chemistry can be defined as the utilization
of a set of principles that reduces or eliminates the use or generation of hazardous
3
substances in the design, manufacture, and application of chemical products (Anastas
et al., 2000; Poliakoff et al., 2002).
2.2. Adipic Acid Production
Adipic acid is one of the chemicals of great interest worldwide. Adipic acid
(l,4-butanedicarboxylic acid) is a white, crystalline compound of C6 straight-chain
dicarboxylic acid, which is soluble in alcohol and acetone, but slightly soluble in
water. More than 1.9 billion kgs of adipic acid are needed every year for the
production of nylon 6,6 (Draths et al., 1994). Nylon 6,6 is generated through a step
growth of adipic acid and hexamethylenediamine (Chenier et al., 1992). Nylon 6,6,
having a protein-like structure, is used in the manufacture of carpet fibers, upholstery,
tire reinforcements, auto parts, apparel, etc. There is also a food grade adipic acid,
which is used in relishes, dairy product analogs, frozen dairy desserts, puddings, fats
and oils, gravies, meat products, snack foods, and as a gelling aid. Adipic acid is also
used in the synthesis of other products, such as, resins, foams, plastics, flavoring
agents, pesticides, dyes, adhesives, textile treatment agents, as weIl as fungicides.
Recent studies have presented the use of adipic acid in the self-assembly of gold
nanorods (Orendorff et al., 2005).
To produce adipic acid commerciaIly, benzene, a volatile organic compound
(VOC), is used as starting material at high pressure (800 psi) and high temperature
(250°C). OveraIl, this incurs considerable energy costs. First, benzene is
hydrogenated over a nickel or palladium catalyst to form cyclohexane (Anastas et al.,
1994). Cyclohexane is then air-oxidized over a catalyst to produce both cyclohexanol
and cyclohexanone. Then, cyclohexanol and cyclohexanone are subjected to catalytic
oxidation in the presence of nitric acid to form adipic acid. There are several
4
disadvantages in the use of this method. The use of nitric acid in the terminal
oxidation step, with the release of nitrous oxide into the atmosphere leads to an
increase in the ozone depletion and contributes to global warming. 10% of the nitrous
oxide concentration in the atmosphere is due to nylon production (Thiemens et al.,
1991). In addition to that, benzene cornes primarily from erude oi!, a non-renewable
resource. ContinuaI exposure to benzene can cause leukemia and other forms of
cancer (Lan et al., 2004).
Recently, advances have been made in the production of adipic acid that
renders the process more environmentally friendly and less costly (Hugl et al., 2001).
Although cydohexanol and cydohexanone are also used as starting materials, the by
product, nitrous oxide, is decomposed into nitrogen and oxygen by a two-phase
reductionloxidation process. The energy consumption of the process is reduced by
50%.
However, there has been a novel example of the use of biocatalysts in the
manufacturing of adipic acid. This was studied by Draths and Frost (1994), who used
a genetically altered E. coli to carry out the metabolism of glucose to produce adipic
acid. Glucose is a non-toxic and renewable resource, and through an intermediate,
dehydroshikimic acid (DHS), is converted into protocatechic acid (PCA). PCA is
then converted into catechol, and finally into cis, cis-muconic acid, which is then
hydrogenated to form adipic acid (Draths and Frost, 1994).
2.3. The Cyclohexanol Degradation Pathway
In biological systems, cydohexanol is metabolized through a series of
sequential oxidation reactions. Many micro-organisms are found to have the ability to
5
oxidize cyclohexanol and cyclohexanone to adipic acid through the same pathway
(Murray et al., 1974; Donoghue and Trudgill, 1975): cyclohexanol ~ cyclohexanone
~ epsilon-caprolactone ~ 6-hydroxyhexanoic acid ~ 6-oxohexanoic acid ~ adipic
acid (Cheng et al., 2002). Cyclohexanol is oxidized to cyclohexanone by the NAD
dependent dehydrogenase. The second step is catalyzed by a Baeyer-Villiger
monooxygenase, a flavin NADPH-dependent protein that introduces an oxygen atom
to the ring to yield caprolactone, which is subsequently hydrolyzed into
hydroxycaproate by a lactone hydrolase. The hydroxyl group is then oxidized to a
carboxylic group to yield adipic acid by two NAD-dependent dehydrogenases
(Donoghue et al., 1975). This pathway has been shown to be regulated with chnR, in
Acinetobacter sp. NCIMB 9871, a transcriptional regulator of the AraClXylS family
(Iwaki et al., 1999). chnR is necessary for the induction of CHMO activity by
cyclohexanone (Iwaki et al., 1999).
In the prototypical Acinetobacter sp. NCIMB 9871 (Fig. 1), the genes
encoding the enzymes that are responsible for the cyclohexanol degradation pathway
are named chnA, chnB, chnC, chnD, and chnE, respectively.
The complete set of genes needed for the cyclohexanol degradation pathway
have been determined in two species of Acinetobacter, strain SE19 isolated from an
industrial waste-water bioreactor (Cheng et al., 2002), and the prototypic
Acinetobacter strain NCIMB 9871 (Iwaki et al., 2003).
In bothAcinetobacter sp. strain SE19 and NCIMB 9871, the genes needed for
cyclohexanol oxidation are found in a single stretch of DNA in two groups, chnB,
chnE, and chnR in one group, and the other group transcribed in the opposite
direction, chnA, chnD, and chnC (Fig. 1). It has been suggested that the inversion of
6
the two gene clusters may be due to the presence of certain ORFs between the two
groups. For example, the presence of orD, a pilin inverting-like sequence, and a
putative phage integrase (orf 19) in Acinetobacter sp. strain NCIMB 9871 (Iwaki et
al., 2003). In Acinetobacter sp. strain SE19 these similar genes, designated chnZ and
chnY, are also found (Cheng et al., 2000). The presence of these sequences, are
indicative of recombination events in the two chromosomes.
In Acinetobacter sp. strain NCIMB 9871 and strain SEI9, the sequence of
chnR, a transcriptional regulator, showed homology to the AraC-XyIS family of
transcriptional regulator (Iwaki et al., 1999; Cheng et al., 2000). In R. maris HI-31,
the putative regulator chnR, showed homology to the NtrC-family of transcriptional
regulator (Lau laboratory, unpublished data), which is similar to the Brevibacterum
epidermidis HCU chnRl (Brzostowicz et al., 2002).
Adipic acid metabolism has been proposed to proceed via p-oxidation to
produce p-oxoadipyl-CoA, which is then cleaved to acetyl-CoA and succinyl-CoA
(Chapman and Duggleby, 1967). In Acintobacter sp. NCIMB 9871 and strain SEI9,
the putative-genes that are involved in the p-oxidation of adipic acid to acetyl-CoA
and succinyI-CoA have been determined (Cheng et al., 2000; Iwaki et al., 2003).
7
Cyclorexanol Cyclorexanore E-Caprolactore
chnA chnB chnC chnD
6·0wlBcanate AdipicAcid
1
chnE
l Suc cinyJ-C oA +Acety~CoA
-c:::= 1 o ~
Q.: a .... c :=
Fig. 1: Cyclohexanol degradation pathway by Acinetobacter sp. strain NCIMB 9871. chnA, encodes a cyclohexanol dehydrogenase; chnB, encodes a NADPH-linked cyclohexanone monooxygenase (CHMO); chnC, encodes an epsilbn-caprolactone hydrolase; chnD, encodes a 6-hydroxyhexanoic acid dehydrogenase; and chnE, encodes a 6-oxohexanoic acid dehydrogenase. Further oxidations of adipate to acetyl coenzyme A and succinyl coenzyme A proceed via ~-oxidation (Donoghue et al., 1975; Iwaki et al., 1999).
8
2.4. Baeyer and Villiger Reaction
More than 100 years ago, a chemical reaction discovered by Baeyer and
Villiger showed that the treatment of alicyclic ketones with monoperoxysulfuric acid
results in their conversion to lactones (Baeyer and Villiger, 1899). This reaction
attracted interest because of the broad spectrum of applications including the
synthesis of steroids, antibiotics, pheromones, synthesis of monomers for
polymerization, etc. (Stewart, 1998). For example, bicyclic and polycyclic gamma
lactones have found considerable interest as antitumor compounds, cardiac
sarcoplasmic reticulum Ca2+ -pumping ATPase activators, and as useful intermediates
in the synthesis of drugs for the treatment of glaucoma and hypertension.
Hydroxylated delta-Iactones are other significant structures that were found in
interesting compounds, such as, antihypercholesteremic mevinic acids and the
immunosuppressant discodermolide.
The mechanism of Baeyer and Villiger oxidation was first studied by Criegee
(Criegee, 1948). He showed that there are two steps involved in this reaction. In the
early nucleophilic attack of peroxy acid at the carbonyl carbon, an intermediate
species, called Criegee adduct, is formed. This unstable species goes through
migration of one of the alkyl groups onto the peroxygen, and the concomitant release
of the carboxylate anion yields ester and acid (Strukul, 1998; Sheng et al., 2001).
A big disadvantage of the use of Baeyer and Villiger oxidation is the use of
peracids as oxidants, for example, chloroperoxybenzoic acid or peroxotrifluoroacetic
acid. The shock-sensitivity and the explosive character of peracids, increases the risk
for accidents when performing large-scale reactions. Moreover, peracids are powerful
oxidative agents. Aiso because of many by-product formations, difficult protection
9
and deprotection steps are needed in the synthesis (Kamerbeek et al., 2003). Recent
studies have shown that, the oxidation of thiocolchicone by peracids will lead to the
synthesis, conformation and inhibition of microtubule assembly (Berg et al., 2004).
To avoid the use of peracids, transition metal catalysts and organocatalytic
compounds have been developed (Strukul et al., 1998).
Corma et al. (2001) found that the catalysis reaction of Baeyer and Villiger
oxidation can be generally stereoselective, and it can result in more products than
waste when solid tin catalysts that are water stable, and hydrogen peroxide as the
oxidant are used.
Recently, another environmentally friendly way of the Baeyer and Villiger
oxidation was proposed by Bolm et al. (2002). These authors used compressed CO2 as
a solvent, oxygen as primary oxidant, and benzaldehyde or pivalaldehyde as a co
reductant. By using this technique, they showed that the oxidation of both the cyclic
and acyclic alkanes to the corresponding esters or lactones could be efficiently carried
out.
2.5. Baeyer-Villiger monooxygenases (BVMOs)
There exists a biological form of the Baeyer and Villiger reactions, which was
first discovered by Turfitt in 1948 during the biotransformation of steroids by fungi
(Turfitt, 1948). This biocatalyst is termed Baeyer-Villiger monooxygenase (BVMO).
BVMOs in nature have mainly been found in bacteria such as Acinetobacter,
Pseudomonas, Xanthobacter, Rhodococcus and Nocardia. In these bacteria, BVMOs
generally catalyze the second step of the cyclohexanol degradative pathway that
allows these cells to utilize specific hydrocarbons and/or alcohols as sources of
10
carbon and energy. BVMOs have also been found in fungi like, Dreschlera and
Exophilia, where they appear to play a role in the switch from primary metabolism to
a secondary one (Willetts, 1997).
There are two groups of BVMOs (Willetts, 1997): Type1 BVMOs contain the
cofactor flavin adenine dinuc1eotide (F AD). They consist of identical subunits and
use NADPH as source for electrons. Type2 BVMOs are composed of a2~ trimers,
they contain flavin mononuc1eotide (FMN) as a cofactor and use NADH as an
electron donor.
The cyc1ohexanone monooxygenase (CHMO) from Acintobacter sp.
NCIMB 9871 that oxidizes cyc1ohexanone to caprolactone is a c1assical BVMO
(Donoghue et al., 1976). This CHMO has been shown to form a hydroxyperoxyflavin
intermediate, which is involved in substrate oxidation. This enzyme has been the
reference to study the mechanism, structure and applications of the family of
BVMOs.
The first c10ned BVMOs was that of CHMO, a 60.9-KDa monomeric
flavoprotein, from Acinetobacter sp. NCIMB 9871 (Chen et al., 1988). The potential
utility of this enzyme as an enantioselective and lor chemoselective oxidant allowed
this enzyme to be used for synthetic applications like synthesis of interesting
compounds, such as, bicyc1ic lactones, different sulfates, and thiosulfinates (Stewart
et al., 1998). The c10ning ofthis gene made it possible to produce the enzyme in large
quantities in E. coli. Over-expression of c10ned CHMO allowed the sequence
determination by mass spectrometry (Kneller et al., 2001) that confirmed the DNA
predicted sequence reported by Iwaki et al, (Iwaki et al., 2003). The first sequence
11
determined by Walsh et al. (Walsh et al., 1988) was found to contain several errors
although it consists of the same number of residues.
Several other CHMOs from different bacteria have been cloned and
sequenced. One of the earlier ones was the steroid monooxygenase from
Rhodococcus rhodochrous, that is involved in the oxidation of progesterone to
produce testosterone acetate (Morii et al., 1999). More recently, the CHMO of
Acinetobacter sp. SE19 (Cheng et al., 2000), which is almost identical to the CHMO
of Acintobacter sp. NCIMB 9871 has also been studied. Brzostowicz et al. (2000)
reported the presence of two related CHMOs from Brevibacterium. The specificity of
one is higher for cyclohexanone whereas the other one is better towards
cyclopentanone. Comamonas (previously, Pseudomonas) sp. NCIMB 9872 is another
classical strain capable of BVMO oxidation (Griffin et al., 1976). !ts specificity is
towards cyclopentanone. The complete gene cluster of the cyclopentanone
degradation pathway has been cloned and characterized by Iwaki et al. (Iwaki et al.,
2002). Another notable BVMO is cyclododecanone monooxygenase from
Rhodococcus ruber CD4, which is known to catalyze long-chain cyclic ketones
oxidation (Cll-CI5) (Schumacher et al., 1999). The gene sequence ofthis pathway,
except the first dehydrogenase, has been determined from a related Rhodococcus sp.
SCI (Kostichka et al., 2001). The 4-hydroxyacetophenone monooxygenase of
Pseudomonas jluorescens ABC catalyzes the oxidation of acetophenones to phenyl
acetates (Higson et al., 1990; Kamerbeek et al., 2001). Studies with site-directed
mutants of 4-hydroxyacetophenone monooxygenase from Pseudomonas jluorescens
ABC identified an essential BVMO sequence motif: FXGXXXHXXXW(PID)
(Fraaije et al., 2002). It has been suggested that BVMO fingerprint sequences are
12
r-... mainly involved in catalysis (Fraaije et al., 2002). Replacing the strictly conserved
histidine in hydroxyacetophenone monooxygenase by an alanine resulted in an
inactive protein (Fraaije et al., 2002), while mutagenesis of the conserved histidine
residue with glutamine in cyclohexanone monooxygenase of strain NCIMB 9871
caused a 10-fold reduction in the enzyme activity (Cheesman et al., 2003). The
discovery of the BVMO sequence motif can facilitate the identification of BVMOs in
the microbial genome sequence databank.
2.6. Potential Commercial BVMO Biotransformation Process
Chiral lactones made by using BVMOs are important synthons for the
production of prostaglandins (Banerjee, 2000). However, up to now there has not
been any commercial process for the BVMO technology.
The possibility of the first commercial-based process of Baeyer-Villiger
monooxygenases has been recently reviewed by Alphand et al. (Alphand et al., 2003).
They showed the great progress that has been made in the scale-up of BVMOs
reaction system. However, there are problems with regards to the substrates and
products inhibitions. Aiso the NADPH cofactor, that is used in the production are
expensive and subject to degradation, cell death, etc.
In general, the c10ned BVMOs have the possibility to pro duce a number of
alkyl-substituted E:-caprolactones in homochiral form, widely used as building blocks
in organic and polymer synthe sis, by performing either a single enzymatic oxidation
or a combination of chemoenzymatic methods. Recent laboratory scale results
indicated a spectrum of substituted E:-caprolactones that can be produced in 2::98% ee
(Ky te et al., 2004).
13
A more powerful, productive, and efficient process to scale up the BVMOs
reactions that overcome the inhibition, cell toxicity, and substrate solubility problems
was recently developed by Hilker et al. (2004). They used a technique named
substrate feeding and product removal (SFPR). Whole cell and an adsorbent resin
were used to perform 'a two in one' method to combine in situ substrate feeding and
product removal. More than 98% ee of the corresponding lactones produced from this
reaction was obtained with high yield.
By using directed evolution methods, production of a very highly
enantioselective oxidation reaction of prochiral thioether using CHMO was obtained
(Reetz et al., 2004). Directed evolution is a set of techniques for a desirable
production, evaluation and selection of variants of a biological sequence, usually a
protein or nucleic acid. These techniques, have allowed the generation of enzymes
with greatly enhanced characteristics (Gibbs et al., 2001). An enantioselective mutant
of CHMO was shown to have the ability to control the direction and degree of
enantioselectivity in the CHMO-catalyzed air-oxidation of prochiral thioethers. The
directed evolution method also can provide biocatalysts for the efficient kinetic
resolution of racemic sulfoxides.
2.7. The Rhodococcus Genus
Rhodococci are gram-positive bacteria found in many parts of the
environment like soils, seawaters, and plants. Rhodococcus, is also known as a taxon
of a genetically poorly characterized bacteria with the ability to transform a wide
range of xenobiotic compounds (Finnerty, 1992 [aD. They are also a useful system
14
for studying gene transfer, recombination, plasmid replication, etc. (Larkin et al.,
1998).
The genetic instability and plasticity of the Rhodococcus genome have
appeared to play a significant role in its adaptation to a wide variety of substrates in
the environment. Several rhodococci are used for industrial applications, e.g., the
production of acrylamide and nicotinamide (Kobayashi and Shimizu, 1998). Others
have the ability to convert nitriles into amides (Bunch, 1998). Rhodococcus sp. strain
IGTS8 has been a model for studying the genes responsible for sulfur oxidation.
These studies are aimed at finding biocatalysts for the removal of organic sulfur from
coal and petroleum products, since combustion of the se compounds emits noxious
oxides of sulfur that contribute to acid rain (Finnerty, 1992 [b]; Kilbane, 1990; Denis-
Larose et al., 1997).
Rhodococcus sp. NCIMB 9784 has the ability to degrade bicyclic
monoterpene camphor (Roberts et al., 2004). Rhodococcus sp. RHAI plays an
important role in bioremediation since it is a strong de grader of polychlorinated
biphenyls (PCB) (Fukuda et al., 1998).
Recently, Rhodococcus opacus ISO-5 was shown to exhibit the ability to
utilize taurine (2-aminoethanesulfonate) as a sole source of carbon, nitrogen, or suIf ur
for growth (Denger et al., 2004). Taurine is a phylogenetically ancient compound that
has been known to be a sulfur source for the growth of aerobic microorganisms
(Huxtable, 1992). Taurine also belongs to sulphur-containing amino acids. It has been
used in the treatment of different human diseases, such as epilepsy, Alzheimer's
disease, hepatic disorders, cystic fibrosis and hypercholesterolemia (Souza et al.,
2005).
15
Three Rhodococcus strains, strains NCIMBI2038, P200, and P400, were
shown to be involved in naphthalene catabolism (Kulakov et al., 2005). Rhodococcus
wratislaviensis is another diverse strain, that could utilize 4-nitrpcatechol, 3-
nitrophenol and 5-nitroguaiacol as sole carbon and energy sources (Navratilova et al.,
2005).
Another notable Rhodococcus is strain RRl, that has the ability to de grade N
nitrosodimethylamine (NDMA), a water contaminant found in the environment due
to the release of rocket fuel (Sharp et al., 2005). NDMA, is a possible carcinogenic
compound.
2.8. Rhodococcus maris strain HI - 31
In this study we have chosen a strain of Rhodococcus, designated Rhodococcus
maris strain HI-31. This strain was isolated by Dr. Hiroaki Iwaki (Kans ai University
of Osaka, Japan) from a petroleum contaminated soil sample collected from the Kinki
area in Japan. This bacterium is capable of growth on cyclohexanol or cyclohexanone
as sole carbon source.
Prior to this study, equivalents of the Acinetobacter sp. NCIMB 9871 chnB,
chnC, chnD, and chnE genes have been cloned from Rhodococcus maris strain HI-
31. In addition, a putative transcriptional regulator, chnR gene has been located (Lau
laboratory, unpublished data). As shown in Fig. 2, chnC, chnB, and chnE are found in
one orientation where as chnD and chnR are located in the opposite direction. The
chnB, chnC, chnD, chnE, and chnR are located in three clones (pCMRI00,
pCMR200, and pCMR300) in a total length of 8-kb of the R. maris chromos omal
DNA (Fig. 3). However, the chnA gene has not been located.
16
A
Rhodococcus maris HI-31
chnD chnE
cl QQ ~~ ~ 0l:f4 0/:/5 orj6 C'
'r?c ~~",. ~ ~b(\ v: ~ .è~ ~ ~ ~% '\ ~ '-: ~ ::.L ",,-, ~ ~~ ~ ~ ~ ~. ~ 0 (9 0 ~~ ~ ~~ ~ ~ ~. :ô ~ n. ~ ~t~ "L. t;!. ~ ~ •. .L-, ~~ ~ Q t'>d
~~ ~ ~ ~ ~ ~ ?~. \.) Q ~. \ ~ ~ ~ ~ ~~ ~ ~~ t. r> ~. \~ ~ ~ ~ ... ('> ~ t. (9 ~ ~
('> ~ ~ t; (9 ~'U ~l1. ~ ~~ .... ~ <;. ~ % ~ ~ "â ~ 'ô ~
.... ~ ~ ~. ~ ~ -;.
B
Acinetobacter sp. NCIMB 9871
orfI4 orfI3 orfI2 chnB chnE chnR
qqq ..... chnA
~ c~ Fig. 2: Gene organization of the cyclohexanol degradation pathway in Rhodococcus maris HI-31 [A] (Lau laboratory, unpublished data) compared to that of Acinetobacter sp. strain 9871[B] (Iwaki et al., 1999; Iwaki et al., 2003). The orientation of the arrows indicates the direction of gene transcription. Genes involved in the cyclohexanol pathway are indicated by arrows in different colours. Other ORFs are marked by a non-bright yellow arrows. The chnABCDE genes encode, cyclohexanol dehydrogenase, NADPH-linked cyclohexanone monooxygenase (CHMO), epsilon-caprolactone hydrolase, NAD (NADP)-linked, and flavin dinucleotide(F AD) dependent. 6-hydroxyhexanoic acid dehydrogenase and 6-oxohexanoic acid dehydrogenase respectively. ORFs 12, 13, 14 in Acinetobacter sp. strain 9871are genes suggested to be involved in ~-oxidation (Iwaki et al., 2003).
17
2.9. Objectives
In this study, the primary objective was to clone and characterize the chnA
gene that encodes the cyclohexanol dehydrogenase. This enzyme catalyzes the first
step in cyclohexanol degradation in R. maris HI-3I. Another goal was to identify
other genes or open reading frames that may be associated with the cyclohexanol
degradation pathway gene locus.
18
3. Materials and Methods
3.1. Bacterial Culture
Bacteria were grown in Luria-Bertani (LB) medium (Miller, 1972) at either 37°C
or 30°C where indicated, containing 1.4% (w/v) agar. Arnpicillin (100 mg/ml) and 40
/lI of X-gal (20 mg/ml in dimethylformamide) were added when necessary.
3.2. Bacterial Strains and Plasmids
Bacterial strains and plasmids used in this study are listed in (Table 1). E. coli
DH5a was used as a host for cloning, the production of plasmid DNA for sequencing,
and as a host strain for pCMR400 and pCMR500.
19
r--.. Table 1: List ofbacterial strains and plasmids used in this study.
Name Relevant characteristics Reference or source Strains Rhodoeoeeus maris HI- Grows on cyclohexanol Kansai University of 31 Osaka, Japan.
E. coli DH5a Host for recombinant Hanahan, 1983 plasmids. supE44 hsdRl7 reeAI endAl gyrAI thi-l relAI
Plasmids pUC19 Cloning vector ApT Yanisch-Perron et al,.
1985 pGEM-3Zf(+) Cloning vector ApT Promega
laeZ pT7-5 Expression vector ApT Tabor et al,. 1990 pT7-6 Expression vector ApT Tabor et al,. 1990 pCMR400 6-kb BamHI/SphI This study
fragment in pUC 19 pCMR500 5-kb Sad fragment in This study
pGEM-3Zf(+)
20
3.3. Restriction Enzymes and Agarose Gel Electrophoresis
Restriction endonucleases, T4 DNA ligase and the large fragment of DNA
polymerase1 (Klenow fragment) were obtained from New England Biolabs, Inc.
(Mississauga, ON). AlI restriction endonuclease reactions were performed with
buffers provided by the manufacturers.
At the end of the reaction time, a sample of the DNA was loaded directly on
the agarose gel for electrophoresis. Agarose gels were prepared by melting agarose to
a concentration of 0.8% in TAE buffer, 0.04 M Tris acetate, 0.001 M EDTA, and
adding 0.5 ~l/ml of ethidium bromide (l~g/ml) (Sambrook et al., 1989). The 1 Kb
ladder was used as molecular weight marker (New England Biolabs, Inc.
Mississauga, ON).
DNA fragments for cloning and sub-cloning were excised from agarose gel
using the QIAEX II gel extraction kit (Qiagen).
3.4. Genomic DNA Preparation
Rhodococcus maris HI-31 genomic DNA was prepared by using QIAGEN
Genomic-Tip Proto col (Qiagen). Bacteria were grown in 50 ml LB medium,
incubated at 30°C overnight. The O.D at 600 nm was taken, and the celI concentration
was determined to be approximately 1.032x108 c/ml. The culture (about 2.2xlOlO
celIs) was centrifuged at 4°C, 3500 rpm rotor, for 15 minutes. The supernatant was
removed. CelIs were then re-suspended in 3.5 ml of buffer BI (50 mM Tris-Cl, pH
8.0; 50 mM EDTA, pH 8.0; 0.5% Tween-20; 0.5% Triton X-lOO) with RNAse A
enzyme (100 mg/ml), 30 mg/ml of lysozyme, and 100 ~l of the protease enzyme (28
21
mgll.4 ml H20) and incubated at 37°C for 1 hour (bacterial lysis). To denature the
proteins, 1.2 ml of buffer B2 (3 M guanidine HCI; 20% Tween-20) was added,
mixed, and incubated at 50°C for 30 minutes. To clear the lysate, the sample was
centrifuged at 4°C, using a 3500 rpm rotor, for 10 minutes and the supematant was
transferred into a 50 ml sterile tube. In the mean time, the QIAGEN Genomic-Tip
100/G was equilibrated with 4 ml ofbuffer QBT (750 mM NaCI; 50 mM MOPS, pH
7.0; 15% isopropanol, 0.15% Triton X-lOO). The sample was vortexed for 10 seconds
and applied to the equilibrated QIAGEN Genomic-Tip. The QIAGEN Genomic-Tip
was then washed two times with 7.5 ml of buffer QC (1.0 M NaCI; 50 mM MOPS,
pH 7.0; 15% ,isopropanol). Thereafter, the genomic DNA was eluted with 5 ml of a
pre-warmed buffer QF (1.25 M NaCI; 50 mM Tris-HCI, pH 8.5; 15% isopropanol).
3.5 ml of isopropanol was then added to the eluted DNA, mixed and centrifuged at
4°C, 6500 rpm rotor, for 15 minutes to precipitate the DNA. The supematant was
removed aild DNA was air-dried. Finally, DNA was re-suspended with 200 J.tl of
buffer TE (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0) and analyzed on 0.65%
TAE IX agarose gel electrophoresis gel stained with ethidium bromide (Sambrook et
al., 1989).
3.5. Southern Blot
Southem blot analysis was done according to the methods of Sambrook et al
(1989).
pCMR400 plasmid DNA was extracted from E. coli DH5a cells by QIAprep
Spin miniprep. kit (Qiagen). pCMR400 plasmid DNA was then digested with
BglII/BamHI enzymes sequentially to isolate the probe, a 0.919-kb fragment (Fig. 3).
22
For BamHI digestion, 12.5 !lg ofpCMR400 plasmid DNA and 60 units of the BamHI
enzyme in BamHI buffer, and BSA added at final concentration of IX each, were
used. The reaction was incubated at 37°C for 3 hours. Following DNA purification,
the second digestion was carried out with BgllI restriction enzyme. 60 units of BgllI
enzyme, and buffer #3 at final concentration of IX were added and incubated at 37°C
for 3 hours. A 0.919-Kb BamHI/BgllI DNA fragment was then excised and isolated
from an agarose gel. The probe was then labelled by random priming method using
the digoxigenin (DIO) labelling kit (Roche Molecular Biochemicals). 1 !lg of the
probe DNA was diluted with water to 15 !lI, boiled for 10 minutes in a water bath,
and then cooled on ice for 30 minutes. The hexanucIeotide mixture, dNTP labelling
mixture and (20 units) Klenow enzyme were added, mixed, span down briefly and the
reaction was incubated at 37°C for 60 minutes. To estimate the yield of the DIO
labelled probe, the manufacturer's instruction (Roche Molecular Biochemicals) was
followed. The labelled probe concentration was 10 ng/ !lI.
R. maris genomic DNA was digested with the enzymes: KpnI, XhoI, Sad,
SalI, EcoRI, BgllI, and PstI (40 units of each enzyme, 6 !lg of R. maris HI-31
genomic DNA, buffers were added to final concentration IX, BSA was added to final
concentration of IX). Incubation was carried out at 37°C for 4 hours. The digested
samples were then analysed on 0.8% agarose gel electrophoresis in IX TAE buffer.
The gel was stained with ethidium bromide.
DNA was transferred onto nylon membrane (OeneScreen™ ) by Salt Transfer
Protocol. The membrane was first pre-wet with distilled water and equilibrated in
lOX SSC. The gel was agitated in (0.25 N HCL) to facilitate the transfer by partially
nick and depurinate the DNA. The gel was then agitated in (0.4 N NaOH / 0.6 M
23
,"'-'
NaCI) to cleave the DNA at the depurinated sites and separate the strands. This was
followed by neutralization in 1.5 M NaCI / 0.5 M Tris-HCL pH 7.5. The capillary
blot device was then set up to run ovemight. 20X SSC (3 M NaCI, 0.3 M Sodium
citrate dihydrate) was used as a transfer solution. Finally, the DNA was fixed by
cross-linking with UV Stratalinker 1800, Auto Crosslink.
Hybridizations were carried out using standard hybridization buffer at 65°C
for southem blot, and 68°C for colony blots. The membranes were first placed in pre
hybridization buffer (5x SSC, 1.0% (w/v) blocking agent, 0.1 % N-Iauroylsarcosine,
0.02% SDS). Pre-hybridization was done for 1 hour at 65°C (southem blot), and at
68°C (colony blots). During that time, the labelled probe was boiled for 10 minutes to
be denatured, then chilled on ice, and span down. 180 ng of the labelled probe was
diluted with pre-hybridization buffer and used to hybridize southem blot. Incubation
ovemight at 65°C (southem blot), or 68°C (colony blots) was done. Then washing
was performed for southem blot as follows: initial wash with 2X SSC/0.1 % SDS,
then with 0.5X SSC/0.1 % SDS at 65°C. For colony blots, washing was done with 2X
SSC/0.1 % SDS at room temperature then at 68°C, followed by a wash with 0.5X
SSC/0.1 % SDS at 68°C.
Detection of southem and colony blots was carried out us mg alkaline
phosphatase coupled to anti-DIG antibodies, with CSPD (10 mg/ml [4-methoxy-4-(
phosphate-phenyl)-spiro(1 ,2-dioxetane-,2-adamantane) di sodium salt]) as the
chemiluminescent substrate. First, the membranes were washed with (100 mM
Maleic acid, 150 mM NaCI, 0.3% Tween 20 pH 7.5). Membranes were then placed in
blocking buffer (1% blocking reagent w/v dissolved in Maleic acid buffer) and
incubated for 1 hour (southem blot) or for 40 minutes (colony blots) at room
24
temperature with agitation. Next, anti-DIG-AP (Anti-Digoxigenin Fab fragments
conjugated to alkaline phosphatase) diluted in blocking buffer 1: 1 0,000 was added,
incubated at room temperature for 30 minutes. Then washing with washing buffer
(100 mM Maleic acid, 150 mM NaCI, 0.3% Tween 20 pH 7.5) was done.
Equilibration in detection buffer (100 mM Tris-HCL, pH 9.5, 100 mM NaCI) for 5
minutes with agitation was followed. Then, CSPD diluted in detection buffer (1: 1 00)
was added and incubation at 37°C for 15 minutes was performed. The membranes
were then exposed to X-ray film (BioMax-MR) and the films were developed.
3.6. Cloning of pCMR500
pGEM-3Zf(+), a 3.l99-kb vector (Promega, catalog # P227l), was chosen to
clone the 5-kb Sad fragment, which was probed positive, and the plasmid was
transformed in E. coli DH5a competent cells. The vector has a lacZ codon, T7 RNA
Polymerase promoter, and ~-lactamase (Apr) coding region. Initially, pGEM-3Zf(+)
plasmid DNA was prepared by using QIAprep Spin miniprep. Kit (Qiagen). For Sad
enzyme digestion (40 units), 2 /-lg of pGEM plasmid DNA was added to buffer #1
with final concentration of IX, and BSA was added at final concentration of lX. To
avoid re-circularization of the vector, the vector DNA was treated with alkaline
phosphatase (5 units of alkaline phosphatase, the vector DNA, buffer #3 with final
concentration of IX). The reaction was incubated at 37°C for an hour, then run on
0.8% agarose gel and the gel stained with ethidium bromide. The linearized vector
plasmid DNA was then isolated.
To prepare the insert, R. maris genomic DNA was digested with Sad enzyme ~
as follows: 6 /-lg of R. maris genomic DNA, buffer #1 with final concentration of IX,
25
100 units of Sad enzyme, and BSA at final concentration of IX. Incubation was done
at 37°C for 4 hours. The reaction was then ran on 0.8% agarose gel electrophoresis to
separate the fragments. A range of 4.5 to 5.5-kb of DNA was cut from the gel, and
DNA was extracted. For ligation, 400 ng of the vector DNA was added to 1.2 Ilg of
the insert, Ligase buffer at final concentration of IX, 200 units of T4 DNA ligase
enzyme, and 1mM of ATP. The reaction was incubated ovemigh at 16°C.
Transformation of the ligation mix was done in E. coli DH5a competent cells
using the calcium chloride method (Maniatis et al 1982). 2 ml of an ovemight culture
were transferred into 100 ml of LB broth in a 250 ml Erlenmeyer flask and the culture
was grown to an O.D. 550 of 0.5 in a 37°C shaker. The culture was collected in two 50
ml sterile polypropylene centrifuge tubes and chilled on ice for 15 minutes. After
centrifugation at 3000 rpm forI5 minutes at 4°C, the cell pellet was resuspended in
35 ml of buffer consisting of (100 mM RbCI, 50 mM MnClz.4H20, 30 mM
Potassium acetate (pH 7.5), 10 mM CaClz.2H20, and 15% (w/v) Glycerol). Final pH
was 6.8. After incubation on ice for one hour, cells were centrifuged as above. The
supematant fluid was discarded and the cell pellet was resuspended in 8 ml of buffer
consisting of (10 mM MOPS (pH 6.8), 10 mM RbCI, 75 mM CaClz.2H20, and 15%
(w/v) Glycerol) at a final pH 6.8. After incubation on ice for 15 minutes, aliquots
were taken and frozen on a solid C02 /a1cohol bath.
100 III of competent cells were mixed with the ligation mix (in 1.5 ml
Eppendorf microfuge tube) and incubated on ice for 30 minutes, then heat shocked
for 90 seconds in 42°C water bath, and retumed on ice for 3 minutes. 800 III of LB
broth was added and the mixture incubated forI hour at 37°C (phenotypic delay). The
26
~
cells were then plated on LB agar + AP (100 J.tg/ml) + X-gal medium, and incubated
at 37°C over night.
3.7. Colony Hybridization
To search for the right clone that contains the 5-kb Sad fragment, colony
hybridization was performed using a 919-bp BgnIlBamHI labelled probe. Colonies
were transferred from LB plates onto nylon membrane for colony and plaque
hybridization. Plates were first pre-cooled for 30 minutes at 4°C. Then membrane
disks were placed onto the surface of the plates for a minute. N ext, disks were placed
on prepared filter paper soaked with (004 N NaOH, 0.6 M NaCI) for denaturation.
This was followed by neutralization on prepared filter paper soaked with (1.5 M
NaCI, 0.5 M Tris HCL pH 7.5). Finally, the filter paper was soaked with 2X SSC.
The transferred DNA was then fixed by UV -crosslink. Hybridization and detection
were done as described above for the Southem blot.
3.8. Sequencing of the 5-kb SacI Fragment
DNA sequencing was performed using the dideoxy chain termination method
(Sanger et al., 1977) on both strands using the ABI PRISM ™ 377 DNA Automated
Sequencer. Confirmation of the positive clone was done by a sequencing reaction
using the universal and reverse primers to sequence a clone named B-I (Fig. 5). This
clone was chosen randomly. Interestingly, B-1 was found to be the right clone. This
was named pCMR500. Following the clone authentication, the entire 5-kb insert was
sequenced. The oligonucleotide synthesis (Table 2) was done by HUKABEL
SCIENTIFIC L TD. The Sequenase enzyme kit (PE APPLIED BIOSYSTEMS) was
27
used as described by the manufacturer. 500 ng of pCMR500 plasmid DNA was added
to 3.2 pmole of each primer, then 8 ~L of Big dye version 2 mix [Big dye (TM)
terminator RR mix (PE APPLIED BIOSYSTEMS # 430315512218)] was added. The
reaction was diluted to 20 ~l with sterile H20. Each reaction was mixed, paraffin oil
was added and then amplified in a PCR thermal cycler. The PCR condition for each
reaction was performed in Perkin-ELMER Cetus thermal cycler under the condition
96°C for 30 seconds, melting temperature for 5 seconds according to the melting
temperature of the primer (Table 2), and 60°C for 4 minutes, and 25 cycles.
Following PCR, each reaction was purified from the non-incorporated dNTP by
Centri-sep column protocol (PRINCETON SEPARATIONS). Sequence reactions
were performed by Hélène Bergeron at the Biotechnology Research Institute (BRI) of
NRC.
The DNA sequencing gels were 0.2 mm thick. The gels contained 4.25%
acrylamide with a ratio of acrylamide to bis-acrylamide of 20:1 (w/w) , made in IX
TBE gel solution containing 6M urea. The samples were resuspended in 2.5~L (5:1
deionized formamide, 25 mM EDTA with 50 mg/mL blue dextran) and denatured 2-3
min at 95°C and kept on ice before loading. For a 36 cm plate length, run time was 7
hours, gel temperature 50°C, voltage 1.68 kV, current 50 mA, and power 150 W. The
sequence was assembled using a commercial Omega software.
28
Table 2: Primers design used in DNA sequencing reactions.
NO Primer's Primer sequence Size Melting name Temperature
1 Universal 5'TGTAAAACGACGGCCAGT '3 18 54 oC 2 Reverse 5' TTACGCCAAGCTATTTAG'3 18 50 0 C 3 Cmr 10 5' GGCTCGGATACAAGACGT'3 18 52 oC 4 Cmr Il 5' AAGGTTCGATGGTGCTCT '3 18 54 oC 5 Cmr 12 5' GCCACGTCGATCTACTC'3 17 54 oC 6 Cmr13 5' TCTCGAGGTGAATGTGCT'3 18 54 oC 7 Cmr 14 5'GACCACTATCGCAGCCG'3 17 56 oC 8 Cmr 15 5'GTGGTTGACGAGGACAG'3 17 54 oC 9 Cmr 17 5' AACCGTCAACTCGCGCA'3 17 54 oC 10 Crnr18 5'GTGTCCTCGGCCATGTC'3 17 56 oC 11 Cmr 19 5'TATTGGCGAAGCTCCTC'3 17 52 oC 12 Cmr21 5' GACCCCGGCTTGGATTT'3 17 54 oC
29
3.9. Sub-cloning of 5-kb SacI Fragment in pT7-6 Expression Vector for chnC2
Protein Expression
Due to the fact that the 5-kb Sad fragment in pCMR500 was cloned in the
opposite direction to the vector promoter, the 5-kb Sad fragment from pCMR500
was sub-cloned in pT7-6 expression vector in an attempt to test the activity of
ChnC2. pT7-6 is a 2.2-kb plasmid, that contains a T7 RNA polymerase and ApT
coding region (Tabor et al., 1990). This plasmid is inducible by heat.
pT7-6 plasmid was extracted by using QIA Spin miniprep. Kit (Qiagen). The
plasmid (4 flg ofpT7-6) was digested with 60 units of Sad enzyme, (buffer #1 with
final concentration IX, and BSA at final concentration IX). The reaction was
incubated at 37°C for 3 hours, then vector plasmid DNA was purified. To avoid re
circulation of the vector, alkaline phosphatase reaction was performed as mentioned
previously.
To prepare the insert, pCMR500 was digested with Sad to liberate the 5-kb
DNA insert (5 flg ofpCMR500 plasmid DNA, buffer #1 with final concentration IX,
60 units of Sad restriction enzyme, BSA with final concentration IX). Incubation at
37°C for 3 hours was done. The reaction was run on a 0.8% agarose gel, and the 5-kb
fragment was cut and the DNA extracted. The DNA insert was then ligated over night
at16°C with the linearized pT7-6 vector (1.5 flg of the insert, 500 ng of the vector,
200 units T4 DNA ligase enzyme, ligase buffer with final concentration IX).
Transformation of the ligation reaction was done in E. coli DH5a competent cells as
mentioned before. After cultivation, 100 colonies from AP resistant cells were chosen
and screened for the presence of7.2-kb sized clones.
30
3.10. Sub-c1oning of chnB2 Gene for Protein Expression
chnB2 gene was found to be contained in two separate clones, pCMR400 and
pCMR500. Therefore, PCR was performed in R. maris total genomic DNA to sub
clone chnB2 for prote in expression. DNA fragment carrying chnB2 gene was
amplified by using PCR primers CHMO-A: 5'TCACACGATTGCGGGCC 3',
CHMO-B:5'GAGGTAGAACAGCACGC 3'. The reaction contains (200 ng of R.
maris genomic DNA, 10 mM of each dNTP mix, 50 pmole of CHMO-A primer, 50
pmole of CHMO-B primer, 5 units of rTaq DNA polymerase (Pharmacia Biotech),
Taq buffer to final concentration IX, H20), and paraffin oil. PCR conditions were
performed 96°C for 1 minute, 54°C for 1 minute, and 72°C for 2 minutes, for 25
cycles. The reaction was then ron on 0.8% TAE IX agarose gel electrophoresis gel
. stained with ethidium bromide, and the amplified DNA 1.9-kb fragment was cut and
purified. Subsequent Cial digestion (2 !!g of 1.9-kb PCR DNA fragment, buffer # 4
with final concentration of IX, BSA at final concentration of IX, 30 units Cial
enzyme) at 37°C for 3 hours of the amplified fragment revealed the presence of a
1.73-kb fragment that was to be cloned in a linearized pT7-5 expression vector.
pT7-5 expression Cial vector was prepared with QIA Spin miniprep kit and
then digested with Cial enzyme (2 !!g of pT7-5 plasmid DNA, buffer # 4 with final
concentration of IX, BSA at final concentration of IX, 30 units Cial enzyme, H20) at
37°C for 3 hours. The DNA was purified from an agarose gel. After that, alkaline
phosphatase reaction was performed as mentioned before. Finally, ligation of the
vector and the insert was performed (400 ng of the vector DNA was added to 1.2 !!g
of the insert, the Ligase buffer with final concentration of 1 X, and A TP with final
concentration 1 mM). The ligation reaction was mixed, incubated at 16°C ovemight.
31
After that, transformation in E. coli DH5a competent cells was done as described
previously.
32
4. Results
4.1. Identification of a new DNA Fragment for Cloning
Figure 3 shows a summary of the physical and genetic map of the
cyclohexanol pathway in R. maris HI-3I. The chnRDCIBIE gene cluster in
pCMR200 and pCMR300 was cloned previously as weIl as the pCMR400 containing
orf4, orf5 and orf6 (Lau laboratory, unpublished data). The predicted sequence of
ChnD (369 amino acids) shows homology to a 6-hydroxyhexanoate dehydrogenase
from Rhodococcus sp. TK6 (AAR27822), with 86% identity and 86% similarity.
ChnE (460 amino acids) is 96% identical to a 6-oxohexanoate dehydrogenase of
Rhodococcus sp. TK6 (AAR27825). ChnCl (310 amino acids) shows 98% sequence
identity to a caprolactone hydrolase of Rhodococcus sp. TK6 (AAR27823). ChnR
(608 amino acids) is 37% identical to the NtrC family transcriptional regulator of
Thermoanaerobacter tengcongensis MB4 (NP6223 75). The sequence of ChnB 1 (540
amino acids) is 90% identical to the cyclohexanone monooxygenase of Rhodococcus
sp. TK6 (AAR27824). The numbers above in parentheses refer to the GenBank
accession numbers.
There are three additional ORFs identified as 3-hydroxyisobutyrate
dehydrogenase (orf4), y-carboxymuconolactone decarboxylase (orf5), and a putative
benzoate transporter (orf6), respectively. These ORFs are contained in a 6-kb
BamHIISphI fragment cloned in plasmid pCMR400 (Fig. 3). Since there was no
homolog of the chnA gene in this fragment, the upstream region of orf4 was targeted.
Southern blot of R. maris genomic DNA digested with the enzymes KpnI,
XhoI, Sad, SalI, EcoRI, BglII, and PstI showed the following positive hybridization ,.,.---, 1
signaIs when probed with a 0.919-kb BamHIIBglII labeIled DNA fragment: KpnI, 4-
33
kb; SacI, 5-kb; EcoRI, 7-kb; and PstI, 5.2-kb (Fig. 4). BglII, and SalI did not give a
positive signal. And in the case of XhoI, the probe hybridized to a more than one
large fragment probably due to incomplete digestion of the genomic DNA.
34
')
-1 kb probe
Bai HI pCMR400 Spr 1 Bam, HI pCMR100 Ban; HI
Sac 1 pCMR500 Sac 1 Psi 1 pCMR200 Psi 1 , , 1· ,
P~/I pCMR300 Pi/
i i i ii:r (.) lE :: t,) IE~:::::::: b (.) .t: lE _ t,) lE lE b lE:: l; ~ 8!. l; ~l; 8!. 8!. 8!.8!. l; l; : ~ 8!. l; ~~ l; ~8!. 1 " Il'' 1 " 1 1 1 1 l , , i 1 o 5 10 15 kb
Orf3 (LCFA)
.... ... " -----"
orf2 (ChnC2)
---- >-Orf1 (ChnB2)
orf4 C>C:> t .. ~ -
orf5 orf6 chnR chnD
... 10.. .......
" " ,.
chnCl chnB 1 chnE
)
Fig. 3: Physical and genetic map of the cyclohexanol gene cluster in R. maris, and sub-clones of the 19-kb region. The orientation of the arrows indicates the direction of gene transcription. The black arrows are genes involved in cyclohexanol degradation. chnR is a transcriptional regulator gene. Other ORFs marked by open arrows. Orf4 encodes a 3-hydroxyisobutrate dehydrogenase, orf5 encodes a y -carboxymuconolactone decarboxylase, and orf6 encodes a putative benzoate transporter (Dr. Lau, unpublished data).
35
1 2 3 4 5 6 7
-
kb 10 8 -6
.. 5
4
3 2.5
2 1.5
1
Fig. 4: Southem blot analysis of R. maris genomic DNA digested with various restriction enzymes. Hybridization was performed with DlG-labelled probe, a O.919-kb BamHI/BglII DNA fragment. The 5-kb Sad fragment (circled) was chosen for cloning.
36
4.1.1. Cloning of SacI-5 kb Fragment
From the result of Southem blot experiment, the 5-kb Sad fragment that gave
a positive hybridization signal was chosen to be cloned into the pGEM-3Zf(+) vector
and transformed in E. coli DH5a competent cells.
To search for colonies that contained the right clone, a colony hybridization
experiment was performed using the O.919-Kb BarnHIIBglII labelled probe. As a
result, 13 colonies gave a positive hybridization signal. Plasmid DNA isolated from
these colonies were screened with Sad digestion, and the electrophoresis gel analysis
revealed a 3.2-kb fragment that was expected of the vector, and a 5-kb fragment that
corresponded to the cloned insert (Fig. 5).
4.1.2. Identification of the Clone by DNA Sequencing
It was of interest to establish by sequencing, using a reverse and universal
primers, the expected clone among the 13 colonies that showed positive signaIs in
addition to the analysis of the positive hybridization data shown in FigA. One clone,
designated B-l, was selected for plasmid DNA extraction and sequencing. As a
result, the sequence of one end of the B-l clone was found to match the partial 3' -end
sequence of chnB found in pCMR400. The B-l plasmid derivative was re-named
pCMR500 (Fig. 3).
37
A
B
KB
10 6 4
2.5
1.5
1
0.5
KB
10 6
B-1--"
2.5 2
1.5
1
0.5
1 2 3 4 5 6 7 8 9 10 11 12 13 14
~ 5.0KB +- 3.2 KB
~ 5.0KB +- 3.2 KB
Fig. 5: Electrophoresis gel analysis for the 13 positive clones screened with Sacl restriction digestion. Odd-numbered lanes are non-digested DNAs. Even-numbered lanes are Sac 1 digestions of the clones. The upper part (A) of the picture screened seven clones whereas the lower part (B) screened six clones. The leftmost lane in both panels shows the molecular size marker in kb.
38
KB
16.2 10.1
B-1 8.0 \
6.0 5.0 3.9 2.9 2.1
Fig. 6: Electrophoresis gel analysis for a non-digested B~l clone: Left lane shows a supercoiled DNA marker, right lane shows the 8.2-kb B-I clone.
39
4.2. Sequencing of the 5-kb insert in pCMR500
FoUowing the authentication of the clone by sequencing, the rest of the DNA
insert was sequenced by using primers that were designed from the new sequence. As
a result, the totallength of the Sac! 5-kb insert was established to be 50 II-bp (Fig. 7).
This sequence has not been submitted to the GenBank database but will be prior to
manuscript submission.
4.2.1. Open Reading Frames Analysis
Sequence analysis of the 5-kb Sac! was carried out using the BLAST
programs of the NCBI from http://www.ncbi.nlm.nih.gov/. The translation of
nucleotides into amino acids was done using GENETYX-MAC program. As a result,
the presence ofthree ORFs (Table 3), aU encoded in the same direction was predicted
(Fig. 3 and Fig. 7). The G+C content of this DNA was found to be a 65%, which is
typical of rhodococcal genes (http://www.bcgsc.ca/gc/rhodococcus; Karlson et al.,
1993). The G+C content was calculated usmg a calculator m
http://www.cnr.berkeley.edul~zimmer/oligoTMcalc.html.
ORF1, 1680-bp in length between nucleotides (3877-5557), was predicted to
encode a cyclohexanone monooxygenase. A 'suggested Shine-Dalgamo sequence,
GGA, was located 8-bp before the start codon of ORFI (Fig. 7). The highest score
given by the BLASTX search was the CHMO of Rhodococcus sp. Phil with 61 %
amino acid identity and 75% similarity (AAN37494.1). The second highest score was
found with the CHMO of Acinetobacter sp. NCIMB 9871 (BAB61738.1) showing
50% identity and 68% similarity (Table 3). However, comparison with our
unpublished chnB sequence from R. maris HI-31 gene showed 80% identity at the
40
nucleotide level and 61 % identity at the amino acid level. Accordingly, ORF1 was
designated chnB2 and we re-assigned chnB as chnBJ.
41
1: 61: 121: 181:
GAGCTCGCCGCAGACCAGGAGCACTCGCTGGCAATGTTCGGTTCGCTCGGCTTCACCGGC GAGGCGCTGCTGCGCGACCATATCCGTGACCGCGAGGGCAACCTGCGCGACCTGGTCATG CTCGCTCACTTCGTCGACGCCACCTGGTCGTCCATCGACACCATCGGGGTCAGTGCCGAA CTCGGCCTGGAGGACAACACCCCGTGACCGGAACGACGCAACGAGGCCACACGGCGCCGG
241: GCCGCCTGCTCTATTTCACCGACCCCGCACGCGCGGCCGTCGACTATGCGCTCCTGGCCT 301: ACTCTGCACCGCTGCTGGCCGCACTTCCTCGCGGCGACAAACACCCCGTCCTCGTGCT CC 361: CCGGACTGAACACGAGCGATGCCTCCACCTACACCCTGCGGACCGTGCTCAAAGGGCTCG 421: GATACAAGACGTACGGGTGGCAGCTCG~CCGCAACATCGGCCCCACGAGCAAGGCGGTGC
481: ACGGCACGCAGGCGCGGCTCGACTACTTGACCAACCGCTACCAGCAGCCGGTCACGCTCA 541: TCGGGTGGAGCCTCGGCGGCATTTTCGCGCGCAAACTCGCCCGCCGCACCCCGTCGGCGG 601: TTCGACAGGTCATCACCCTGGGCAGCCCGATCCGGCTCGCCCGGCACGAACAGAGCCGCG 661: CCAACCGCCTGTTCCATCGCAACTCCCATGAGCACATCGAGCCGTTGGATCTGCCGCT CG 721: AACGCGGCGCAGGGCCGCTTCCGGTTCCCGCCACGTCGATCTACTCCAAGCTCGACGGGA 781: TCCTCGCCTGGCGGGCCTGCCTGGACGAGCCCTCGCCACGTGCCGAGAACATCGCCGTGC 841: 901: 961:
TTGCCAGTCACTTCGGCATCACCGGTAACCCGGCGACACTTTGGGCGGTCGCGGACCGGC TCGCCCAGCCTCCGGACCGATGGGCCCCGTTCCGACCGCCCGCTCTCCTCCGAATGGCCT CCCAGCACCC~gCCGATCATGACCAACCTCGCCCACATCCTGACCGAGTCCGCGCGC
ORF3:M T N L A H 1 L TES A R 1021: CGTCATCCCGACCGGCCGGCGATCCGTCTGGACGATCTCGTCATCACGTATGCCGAACTC
R H P 0 R PAl R L D D LVI T Y A E L 1081: GACGATCTGACCGCACGCGCGGCAGGCTGGCTGCAGGCCCGCGGTATCCGGCCGGGCGAC
DOL T A R A A G W L Q A R G 1 R P G D 1141: CGGGTCGGGATCGCGCTGCCGAACATCGTGCCGTTCCCGGTCTTCTACTACGGTGTGTTG
R V GIA L P N 1 V P F P V F Y Y G V L 1201: CGCGCCGGCGCGACGGTCGTGCCGATGAATCCGCTCCTCAAGGCCCGCGAGATCGAGCAC
R A GAT V V P M N P L L K ARE 1 E H 1261: GCCCTTCGCGATTCCGGTGCCGCTCTGGCGCTGGTGGGGACCACTATCGCAGCCGAGGCG
A L R D S G A A L A L V G T T 1 A A E A 1321: CAGGCCGCGGCCGCAGCGACCGGCACCGACATCGTGGTCATCGACGACGACACCCTGGCC
Q A A A A A T G TOI V V 1 0 0 D T L A 1381: GGCGCTGCCCAATGGCCGCGCCTGCCGGAGGTGACCGCCCGGGCCGACGACGACACGGCG
G A A Q W P R L P E V T A RAD DDT A 1441: GTCCTCCTCTACACCTCGGGCACGACCGGCGCGCCGAAGGGCGCACAGCTCACCCACTCG
V L L Y T S G T T GAP K G A Q L T H S 1501: AACATGTACCGCAACGCCACCACCTTCGTCGCCATGCTCGACATCCGTAAGGAGGATGTG
N M Y RNA T T F V A M L D 1 R K E D V 1561: GTGCTGGGTTGCCTCCCGTTCTTCCACGCGTTCGGGCAGAGCAACGCGCTGAACGCCTCC
V L G C L P F F H A F G Q S N A L N A S 1621: CTCGCGGCCGGCGCGTGTGTGTCGCTGGTGCCGCGATTCGAAGCCGTAGCGGTCGTGCGG
L A A G A C V S L V P R F E A V A V V R 1681: CTCATCGAACGCCACCGGGTGACCGTCTTCGAGGGCGTCCCCACGATGTACGTTTCCCTG
LIE R H R V T V F E G V P T M Y V S L 1741: CTCCACGCCGACTTGTCGGAGGCGGACACCTCCAGTCTGCGGATCTGCATCTCGGGTGGC
L H A D L S E A D T S S L R 1 C 1 S G G 1801: GCGGCGTTGCCGATCGAGGTCCTGAACGGATTCCAGGGAGCCTTCGGCGCACCGATTCTC
A A L PIE V L N G F Q G A F GAP 1 L 1861: GAGGGCTACGGCCTGTCCGAGACATCGCCGACCGCCACGTTCAACCGGATCGGGAAATCC
E G Y G L SET S P T A T F N R 1 G K S 1921: AAGCCGGGGTCGATCGGACTGCCGATCGACGGTGTCGAACTGAAGCTGGTCGCTCGGGAC
K P G SIG L P 1 D G V E L K L V A R D 1981: GGCACCGAAACCCTGCCCGGCGAGGTGGGCGAGATCGTCATCCGCGGCCACAACGTGATG
G TET L P G E V GEl V 1 R G H N V M 2041: AAGGGCTACTGGAAGCGGCCCGACGCGACTGCCGCCGCGATAGTCGACGGCTGGTTCCAT
K G Y W K R P 0 A T A A A l V 0 G W F H 2101: ACGGGAGACATGGCCACTCGCGACGAGGACGGCTTCTACTTCATCGTCGACCGCAAGAAG
T G D MAT ROE 0 G F Y F 1 V D R K K 2161: GACATCATCATCCGGGGCGGCTACAACGTCTACCCCCGCGAAATCGAAGAAGTACTGTAC
D 1 1 1 R G G Y N V Y PRE 1 E E V L Y 2221: GAACACCCCGCGGTGCGCGAAGTCGCCGTCATCGGATTGCCTCATCCCACCTACGGCGAA
E H P A V R E V A V 1 G L P H P T Y G E 2281: GAGGTTGCCGCCGCGATCACCTTGCGACCCGGTGCGGAGGCAACTCCCGAGGAGCTTCGC
E V A A AIT L R P G A E A T PEE L R 2341: CAATACGTCAAGAGCCGGGTCGCCGCGTACAAGTACCCGCGCCACGTCTGGCTTGCCGAC
42
Q y V K S R V A A Y K Y P R H V W LAD 2401: AAGCTGCCCAAGGGTGCCACCGGCAAGATCCTCAAACGCGAAATCGGCATCCCCGCCCAC
K L P K GAT G KIL K REl G l P A H 2461: CTTCTCGAACAGCCGCCGGCGTGACCGGCCCGGCCGTCGTACCGACCCGGT~~JlACAC
L L E Q P P A * 2521: CCATGGCTGATCGGACCCGACGGGTGGCCGTCGGCGAGCTGGAACTGGCCTACGAGACCT
ORE'2: MAD R T R R V A V GEL ELA Y E T 2581: TCGGGGACGCCGGCAACTCACCCCTGCTGCTCATCTCGGGGCTCGCCACCCAGATGCTCG
F G D A G N S P L L LIS G LAT Q M L 2641: GCTGGGACGAACGGTTCTGCGACCAACTCGCCGACCACGGCTTCCATGTGATCCGGTT CG
G W DER F C D Q LAD H G F H V l R F 2701: ACAACCGCGACATCGGGTTGTCCACCCACCTTCACGAGGGCGGGATTCCGAATCTGGGGG
D N R D l G L S T H L H E G G l P N L G 2761: CCCTGCTTCGCGGGGAGGCCGCGCCGGCGCCGCCGTACACGCTGGCGGACATGGCCGAGG
A L L R G E A A P A P P y T LAD M A E 2821: ACACCGCTGGGCTGCTCGACGCGCTGGGTCTCGAGTCCGCGCACATCGTCGGCGCCTCGA
D T A G L L D A L G LES A H l V GAS 2881: TGGGTGGCATGATCGCGCAGCAGCTCGCGCTTCACCACGGACACCGGGTCCGGAGTCTGA
M G G M l A Q Q L A L H H G H R V R S L 2941: CCTCCATCATGTCCACTCCCTCGCGCCAGGTG~GACAGGCGCGGCCGGAGGCGCAGGCGG
T SIM S T P S R Q V G Q ARP E A Q A 3001: TGTTGTTCCTGCCGCCGCCGACCGACCCGGACACCGCCGCCGAACGCTCCCTGACGGTGT
V L F L PPP T D P D T A A ERS L T V 3061: ATCGGGTGATCGGATCACCCGGGTACCCGCTCGACGAGCCTCGCGTCGCCGAGGTCGCGC
y R V l G S P GYP L D E P R V A E V A 3121: GACGCGCCTTCGCGCGAGGCAACAACCCAGCCGGCGTGGCGCGGCAGTACGCGGCGATTG
RRA FAR G N N P A G V A R Q y A A l 3181: TGGTCTCCCCGGACCGCACGCCGGGACTGCGCGAGTTGACGGTTCCGACGCTGGTGATCC
V V S P D R T P G L REL T V P T LVI 3241: ACGGCGAGGACGATCCCCTGGTTCAGGTCGAAGGCGGTCTGGCGACGGCCGACGCGGTCC
H G E D D PLV Q V E G G LAT A D A V 3301: CGGGCGCCCGTCTGGTCGTGGTGCCGGGCATGGGCCACAACCTGCCCCAGCCCCTGTGGC
P G A R L V V V P G M G H N L P Q P L W 3361: CGCAGGTGATCGACGAGATCGTCGCGCACGCCCGGGCCGCCGAGGTCGGCCCCGCGGCTC
P Q V IDE l V A H A R A A E V G P A A 3421: ACGCCTGACGACCTGCGGGCGGAACCGCACGATCACACGATTGCGGGCCGCCGAGCCGCC
H A * 3481: GAGGCGCTCGAGATCGAATCAGTGTGCACGAAAAATCCTCCATCAGAACACTTCTCGATC 3541: GATAGGCCCGTACGTTCCGGGTCGTCGTCGGCCCGGCAGCAGAGCACTGCGCCGGGCCGA 3601: CTCATGCCGCTCGAAGCGGGGTGCTCACAGGCTGTCCTCGTCAACCACGCCGTCCCTACC 3661: GACGCCGACGGGGCCGTCCGGAATCCCGAGCCCGTGAACCGCCCCGTTCCCTGCCGCCCC 3721: CATGCCGTCGATTCGCCCGTTTCGGGAGTGTGTCGAGCCCCGAGTTCCGGAGTGTTTCAT 3781: ATTGGAACGCTCGGACGGCAGTCGCGCATCTCACCATTGTGGACAGAACATGAACCACGC 3841: CACATGCGGGTTCGCACCATCGANGGGATACAGGCCATGGATGGACTCATCCACGACCAG
ORF'l:M D G L l H D Q 3901: ATCCGCGATCTCGACGTTCTTGTCGTGGGAGCCGGGTTCGGCGGAATCTACACGCTGCAC
l R D L D V L V V GAG F G G l Y T L H 3961: AAGCTGCGCAACGAGCAGGGACTCGACGTCGTCGCCATCGACAAGGCGGGCGGGGTGGGG
K L R N E Q G L D V V A l D K A G G V G 4021: GGTACCTGGTACTGGAACAAGTACCCGGGTGCGTTGTCGGATTCGCAGTCCTTCGTCTAC
G T W y W N K Y P GAL S D S Q S F V Y 4081: CAGTACTCGTTCGACCGCGACCTGTACACGAACAACACGTGGACCCACCGGTTCATCAAG
Q y S F D R D L Y T N N T W T H R F l K 4141: GGGCCCGAGGTCCTGGCCTACCTCAACAAGGTGGTGGACCGTTTCGGGCTGCGCGAGCAC
G P E V L A Y L N K V V D R F G L R E H 4201: ATTCACCTCGAGACCGGCATGACCGAGGCCGTGTGGGACGAACTCTCCGGTACCTGGACC
l H LET GMT E A V W DEL S G T W T 4261: GTCCGCACCGACCGCGGCATCACCTACCGGGCCCGCTTCCTCGTCACCGGGCTGGGCATT
V R T D R G l TYR A R F L V T G L G l 4321: CTGTCGGCGACCAACACCCCCGAGATCCACGGCATCGAGCATTTCGAGGGCCGGGTCGTG
L S A TNT PEI H G l E H F E G R V V 4381: CACAGCGGGGCCTGGCCGGAAGAACTGGACCTGACGGGCAAGCGGGTCGGGGTGATCGGT
H S G A W PEE L D L T G K R V G V l G
43
4441: AACGGCTCGACCGGCAATCAGATCATCACGGCCACGGCTCCGATTGCCGGGCATCTGATC N G S T G N Q lIT A T API A G H L l
4501: TCGTTCCAGCGGTCCCCGCAGTACAGCGTGCCGGTCGGCAACCGGGAGGTGAGCCCCGAG S F Q R S P Q y SVP V G N R E V S P E
4561: CAGTTGCGGGCCGACCACGACAACTTCGACGCCACCTGGGAGCAGGTGCGCAATTCCAGT Q L RAD H D N F D A T W E Q V R N S S
4621: GTGGCCATGGGTTTCGAGGAGAGCACCATCGAGACCTTCAGTGTGTCCGCGGAGGAGCGC V A M G FEE S T lET F S V S A E E R
4681: GAACGCATCTTCCAGGAGGCCTGGGACAAGGGCGGGGGGTTCCAGTTCATGTTCGGGACC E RIF Q E A W D K G G G F Q F M F G T
4741: TTCTGCGACATCGCCACCGACGAGGCGGCCAACGAGGAGGCGGCGAAGTTCATCCGCCGC F. C DIA T DEA A NEE A A K F l R R
4801: AAGATCGGCGAGATCGTGCA~eG9S3Aq,?}ÇCGqe:èGêl~G~llCA~~êC1l~~~'!'~ K IGE l V Q D PET A R K L T P T D M
4861: T17;:,J;.GçjG:CGÇ.ê$'G'êq$n~1ïIIIl~~gz€Jih.'r~g,[G99~.J y ARR P L CDS G FYE A F N R P N V
4921: [~hrî@~j~1ll~~~&.q~fi~~($1f"ç~Gi:i1r~iq~"Wg~§@œ,1'ç:G'Ï';~Aç~ S L V N V KEN PlV R M T R K G l V T
4981: ~i~~~ii~Îç,,~~t;9J~~[J;'çg~1\i!W;;'Fç(lç,çAç;Ç!g.@T~T'C1~q~ltIm E D G TEH E L D V L V FAT G F D A V
5041: GA~G~.ê.~W~ô~t1.~a:~~g~!F~~~~ÂœifJ\G'TçMêA<;l D G N Y M R V D L K G R· D G K LIS Q H
T··.;:;", ... ·".'·" ... ·;.,.·.·.·;;::4(c ... '.'G':G· .. '.'G· ·C··C'··Gm'·'··C'· ...... '''r<;i;l.'WNil:ijcitli#o··:::-;..''''.>'''T·;'<;4·''''C···A·C···C·.··A····G·C· .. G:.·.·G·· G'T'·.·T'·C·C· Cv',."·.~,,M .. l.·B·:·l·f"#.'ii.~~ .• · .. ,,· 5101 : "'S'f?;!""''''<:!\ ••. M,,,.,:.,, .. ,,.~ •.. ,~,,,,;I..J::1"'.~~19"'''''''''' . "'''''''" . ... "»' ."" ~4;-t"J;'NI&lF_ W N D G PTS y L G V A T S G F P N M F
5161: A\liraTGèfèGGlif~}~'fIfi!'§':l:GT.~C'i!ffi,..riÊ.~G~i!f{~_el.l'rtG~GGê élGG'=G ,', ,~;8f.:?- ". "'d,, "~, ~~~JMb>:,'I\ft.'rl ~"d~H\ ":,.",,<l~~.A~t* .' I~~.Ad"o% "'11:<N' , .. <'.'.M ,.,;C ,.,~,', ,>~,.\A'8é MIL G P N G P F T N L P P T l E A Q V
5221: ~~ftt~~~liî:œG'~~~~€!f'J;'.~~G'ltI»G'ltI:ti:iG<;~OOCACGGÂG~êJ::iG~9ll:.GGA'~&.~(ij E FIT D TIR K V A A TET G R -1 D L
5281: AGGçaCG1J;~~c~~lÇ~~:ç~Âê,..G~.AèèGÂ~;;.çÇ!:t~€i!:«C~~~~~~!§éGp~~~1l~l<i R P E A E A D W TET CRE V A A A T V
5341: ~TC~~~;t~~GGA!t!lJ.:rm:~G'Il~~G~èAT'L:CCÇGGç;~ç.G.Ç~f5A~CGWG F G K V D S W 1 F GAN l P G K K R S V
5401: _'l?Tl!!tJir;.ce'l'eGG,~r&(iiCC!1'Eil9\i!~~AG'I'Aé:g;G:~~lML~~t~,.~~'f!iV~f~~ L F Y L G G L G E Y R K 1 V A A E V A A
5461: GG1rT~~~t5'ç?i~il:'tg:G:r~~êA[~q~l!:~~g~c~CCGAeT~«GGÇ9~e~eATCCt;;~ GYP S F V T K S P P L P L G A A H P K
5521: C«~Cfa;p,Ct~CA~~~.~tF'iFCi:S31~i~C'Ç)AC(jCMel~I~~~~A~Ir~jj~qGÂT
5581: 5641: 5701:
R T S Q P F R T A T Q H *
ORF4: M T S T 5761: ~'±\~Ç~9CGM;®î~A~ .. S9.m~~~l!!;~~TGTGGGTCGTATCCCTCGCC
1 T T D Q E ARR K R T V M W V V S L A 5821: ACCGTCGGCATGATCTTCGATGGTTACGACCTCGTTGTATACGGCGCGGTGCTGTCGACC
T V G MlF D G Y D L V V Y G A V L S T 5881: TTCCTGAACGACCCGAGCCATCTCGGGGAAGTTACGCCCGCGGTCGCGGGGACCCTGGGC
FLN D P S H L G E V T P A V A G T L G 5941: AGCTACGCCCTGCTGGGAATGATGTTCGGTGCTCTGCTCGCCGGAGCCTTCGGTGACATC
S y A L L G M M F GAL L A G A F G D 1 6001: CTCGGCCGCCGCAAGGTCATGCTGCTCTCCTACGCGTGGTTCTCCATCGGCATGTTCGTC
L G R R K V M L L S y A W F SIG M F V 6061: ACCGCGCTGATGAGCAGTACCACGACGTTCGGCATCATGCGGTTCCTCACCGGCCTCGGT
T A L MSS T T T F G 1 M R F L T G L G 6121: ATCGGCGCGCTGATCGCCACCACCGGCACCTTGGTCACCGAATTCGCCCCGCCGGGAAAG
1 GAL lAT T G T L V T E F .A P P G K 6181: AAGAACCTCTGCAGTGCCATCAGCTACTCCGGCGTGCCGCTGGGGAGCCTGCTGGGTGCC
K N L C SAI S y S G V P L G S L L G A 6241: CTGCTTGCCATCCTTCTGTTGGAGTCCCTCGGTTGGCGCGGACTGTTCATGATCGGGGCG
L LAI L L LES L G W R G L FMI G A 6301: TTGCCGATCGTGACCCTGTTGCCGTTGGCGTTTCTGAAGATGCCCGAGTCGGTGCCGTGG
L P 1 V T L L PLA F L K M P E SVP W 6361: TTGGTCGCCCGGGGCCAGATCGACAAGGCCCGGGCGATCTCCGAGCGCACCGGTGTGCCG
44
L V A R G Q l 0 K A RAI S E R TGV P 6421: ATGCCCGAGAGCGTCCCGGAATCGGAGAAGGCCGAACGGGTGGGCTTCGCCGGCCTGATC
M P E SVP E S E K A E R V G F A G L l 6481: AGCCGCGGCTTCCTGCTCTCGACGGTGCTGGTGGGATTGATGAGCGCCCTCGCCCAGTTC
S R G F L L S T V L V G L M S A L A Q F 6841: CTCAACTACTACCTCAACACCTGGTTGCCGGTGCTGATGGAACAAGTGGGGTTCAATACC
L N Y Y L N T W L P V L M E Q V G F N T 6901: AAAGGCTCGCTCGCCTTCCTTCTCGTGTTGAGCGGAGGCGCAATCCTCGGAGCGCTGGGC
K G S L A F L L V L S G GAI L GAL G 6961: GGCTCCCGGTTCGCCGACCGGTTCGGTCCCAAA
G S RFA D R F G P K
Fig. 7: Nucleotide sequence of the 5-kb SacI fragment (clone pCMR500) plus part of pCMR400 sequence that contains the 3'-end of chnB2 and the complete orf4. The bold faced sequences are the various restriction sites shown in (Fig. 3). Deduced ORFs are labelled and the position (orf3: 981-2481. orf2: 2522-3426. orfl: 3876-5556), transcription direction is indicated. The deduced ShineDalgamo sequences were highlighted with yellow colour. Asterisks indicate stop codons. The sequence of the probe used in hybridization was highlighted with grey colour. The sequence of oligonucleotides primers used to sub-clone chnB2 was highlighted with bold red colour. CIal restriction site was highlighted with bold blue colour. SacI restriction site was highlighted with bold brown colour
45
Table 3: Homology of the Rhodococcus maris HI-3I ORFs.
ORF Gene Length Size a.a. Hom%gous Source Identity GeneBank Name bp kDa protein Species (%) Accession
Number
Orfl ChnB2 1680 62.2 560 Cyclohexan- Rhodococcus sp.Phil. 61 AAN37494 one monooxyge- Acinetobacter sp. 50 BAB61738 nase NCIMB9871
Orf2 ChnC2 903 33.4 301 Hydrolase Bradyrhizobium 47 NP-769524 japonicum USDA
Clostridium 40 NP-149260 acetobutylicum ATCC
Orf3 LCFA- 1500 55.5 500 Long chain Streptomyces 56 BAC68086 CoA fatty acid- avermitilis ligase CoA ligase MA-4680
Kineococcus 55 ZP-radioto/erans 00226705 SRS30216
46
Comparison of the ChnBl (CHM01) and ChnB2 (CHM02) sequences (Fig.
8). was done using the LALIGN pro gram for alignments between two sequences
(Huang et al., 1991; http://www.ch.embnet.orgD. As expected of a BVMO
flavoprotein, CHM02 contains several conserved sequence regions: a characteristic
F AD-binding fingerprint, with the sequence GxGxxG motif found at amino acids 17-
24 amino acids; a NADP binding motif at position 187-194 amino acids. The
presence of these two GxGxxG motifs were suggested to differentiate these proteins
from another mechanistically related flavoproteins (Eppink et al., 1997). Aiso found
in ChnB2 is the sequence FEGRVVHSGAWP at amino acid positions 163-175. This
has been suggested as a "BVMO identifying sequence motif' (Fraaije et al., 2002).
Another conserved region found among FAD-and NAD(P)H-binding proteins, ATG
motif, was located near the end of the ChnB2 (Fig. 8). This ATG motif, recognized
recently by Vallon (2000), apparently has a role in NAD(P) binding. Furthermore,
another short sequence motif (GG motif), was found in ChnB2. The GG motif has
been identified to be indicative of a Rossman fold domain involved in F AD binding
(Vallon, 2000).
Table 4, shows the pairwise amino acid identity comparison between R. maris
ChnB 1 and ChnB2, with Brevibacterium epidermidis HCU ChnB 1 and ChnB2. The
result showed that both of the ChnB 1 are related to each other with 60% sequence
identity.
~. 1
47
10 20 30 40 50 ~m· CnnSl - •• M'!AQ'1":mT'IlMWX~~nA'lfflKUlHS:U;;:LTT'\I'Gf'OAAOO~YWNJit,{PQAL
" .. ~ ~ ~ ! .. :;:"": 4 ~ i !, ~ :: .... :: :. :: .. ~:: t:: ,.!,. .. 1: :::;: i. :"t~ f : :: :: :: .. : : :- :: ~ Rm~ChnB2 MDOt.1HOQ:t!U')WVl.W_~lYTW~WWA!l)~VOOT\1i'YWmO!!i'OAL
10 20 30 40 50 60
60 70 SO 90 100 110 Rm- ehnBl St1rESUL YRFSF1lru:»:..LORS1'WKTTnl'Qpa!LBYLlilD~t.RRHl"'KFQ'1'lW1'SALY
Rm-C'I'mB2 $œœFVYQYSli'PRDL~RFTKGPEVLAYLNKWORFGt.rmli!lit.~vw 70 au .9() 100 :UO 120
120 130 140 150 160 170 b-C'l'mEl t.DDil:Nu{~~W\KY'V'mAV(JLL$AIl'WPm.~t.PT~~ftl~~.t"~
RIlI-Chna:z oÈLsQ~T~R;~R~l~~Fi~LdiL~~~;RiH~ÎÊ»~i~gcii~t~ELÔ~T 130 140 150 HO 170 180
180 I.D 200 21D 220 230 Rm-ChnEl GaRVGV!~GS~VITSLAPSVEHLTVFVRT?QY8VPVGNRPVNPEOlAErKADYOR!
Rln-ChnB2 Gl('.RVGVI~G$'kGNQ!ITATAFIMHLl$FQn5.t"QYSVi'VœRSVSPSQ~NF1)J\T ll)O 200 :11.0 220 230 240
240 250 260 270 2$0 290 Rm-Chnal ~SAVAmFU:$TLl?AMSV$:eltSl<:NR!FQEAwmtOOCn1i'MOO'!'FGD!Arn~
Rm-Chnl$2 WEQVRNSSVAMGF.a:es'!'IB'!'l"SVSAEERER.!FQEAwtlI<CJOOFQFMFG1'1"CO:tATtlI>l\ANE 250 260 270 280 290 300
300 110 320 330 340 350 Rm-ChnBl AL~:eFrRAKVAEtIEPPBTARKLMPKG~FAKRPLCDSGYY~PWVeAVAXKENP!Rn
Rm-Chn$2 EAAKFI~OBtVODFBTARKLTPTnMYARRPLCDSGFY1lA~~PNVSLVNVKnNPIVR 310 320 330 340 350 360
360 37' 380 390 4DO 410 R:n-Cll.'1Bl V'!'~"VV'!'EOOVt.liELDVl.VFATÇ)JJDJ\'!Jl:lONYRRIETRGROOLll:r~PTSY1AVS
Rm-ChrB;:I, MTRlCOTVTROOTlrH~VFA:ruFt\AVJ:)(l;N'ltMRVDLJ.(CJ:uXlIKL!SOliWNOOPTS'1!l:.GVA 310 l80 '90 400 410 420
420 430 440 450 460 41D Rm-Chnrn TANFPNWl"WJLCPf~Pf'Tl\.'LPPSIETOVRW:tSMICYABlmGVR.AIBPTPs.M:ASW'l'B1'C
! .... ::: ::t.:i::!!:~!::l ... :: .. : .. ::~::!: ~ •. :ltl:~~ .. !~:::::::
I<:m- Chn1l2 'l'SCFPNMFMILGPOOPl"1'NL?PTl EPRWSFITM!GK'VMTE'l"GRID~RPEAE'Al:lW1'BTC 430 HO 450 460 470 4ao
4an 41011) 500 510 520 530 RI1l-ClmBl TEIANATLIi'TKG.oS'WlFGAN.tOOlŒPSVLFYWOU~):YEAVMf\.t::Vlt.J\OOYRGFlWKSAEM
!,,! 1: .. : !: :t~:l~;;:::.:t: !::::$::;;;: .. il .... .: ,.; =.= .: ~:t ~
km-ChnS2 RSVAAA'l'VOOKVDSWIPGJ\N!.PGKDSVLF''iDGGInBYRKIVMt::VMGYPSFVTKSPPL 490 500 510 520 53;) 51Q
S<\I(J
Rm-ChnB1 ~V-----_·_---------
Rm ~ ChrJ>2 PLr',MliPPmTSQPFRTATQli SM 560
Fig. 8: Sequence alignment of the cyclohexanone monooxygenase (CHMO) 1, and 2 from R. maris. The highlighted conserved sequences are described in the text: dot, indicates identity; colon, indicates similarity.
48
Table 4: Comparison of ChnB1 and ChnB2 amino acid identity of Rhodococcus maris Hl-31 and Brevibacterium sp. HCU.
Identity% Brevibacterium Brevibacterium R. maris
ChnBl ChnB2 CbnB2
R. maris ChnBl 60% 32% 61%
R. maris ChnB2 39% 34%
49
At the time of this writing, an updated sequence homology se arch was carried
out. This resulted in the multiple sequence alignment shown in Fig. 9. Additional
homologous sequences are those of Rhodococcus Phi2 (AAN37491), Rhodococcus
sp. TK6 (AY486161), Rhodococcus Phil (AAN37494), Arthrobacter sp. BP2
(AAN37479), and Xanthobacter jlavus (CAD10801), (Fig. 9). Overall, there was
39.4% sequence identity. AIso, F AD and NADP(H) binding motifs, the suggested
Bayer-Villiger monooxygenas-binding motif, ATG motif and GG motif are located.
In the multiple sequence alignment that was done by using ClustalW 1.75
(Thompson et al, 1994) (Fig. 9), most of the sequence motifs that are found in
BVMOs, besides the BVMO motif, could be identified displaying the following
arrangement: GXGXXG-GG-ATG-GXGXXG-A TG, except for the first ATG motif
that was only found in Xanthobacter. This ATG motif was described to be indicative
of F AD and NAD(P) binding domains (Vallon, 2000). AIso, it appears that ChnB2 of
R. maris has an extended C-terminus by about 12-amino acids. On the other hand, the
sequence of Arthrobacter sp. BP2 appears to have an N-terminus extension of 50-
amino acids. This may be due to a miss-identification of the methionine start codon
since there is an internaI methionine at about the same position of the start codon of
the rest of the sequences.
50
,~
r'
Phi2 TK6 Phil BP2 ChnB2 Xanthobacter
Phi2 TK6 Phil BP2 ChnB2 Xanthobacter
Phi2 TK6 Phil BP2 ChnB2 Xanthobacter
Phi2 TK6 Phil BP2 ChnB2 Xanthobacter
Phi2 TK6 Phil BP2 ChnB2 Xanthobacter
Phi2 TK6 Phil BP2 ChnB2 Xanthobacter
Phi2 TK6 Phil BP2 ChnB2 Xanthobacter
Phi2 TK6 Phil BP2 ChnB2 Xanthobacter
Phi2 TK6 Phil BP2 ChnB2 Xanthobacter
--------------------------------------------------lMTAQTIHTV 9 --------------------------------------------------lMTAQTIHTV 9 -------------------------------------------------lMTAQISPTVV 10
1MSTRSWPGGPPSWHRSSTSSRPGTGNNPATLRSHTIHYFVPCIRTTKEFAMTAQNTFQTV 60 -----------------------------------------------lMDGLIHDQIRDL 12 --------------------------------------------lMTMTVEKTRTGGADY 15
10DAVVI IYAVHKLHHELGLTTVGFDKADGPGiTWYWNRYPGALSDTESHLYRFSF 69 10DAVVI IYAVHKLHHELGLTTVGFDKADGPS(;;TWYWNRYPGALSDTESHLYRFSF 69 Il DA VVIQA~Jr,GGIYAVHKLHNEQGLTVVGFDKADGP~<hWYWNRYPGALSDTESHLYRFSF 70 61DAVVI IYAVHKLHNEQGLTVVGFDKADGP~~TWYWNRYPGALSDTESHVYRFSF 120 13DVLVV IYTLHKLRNEQGLDVVAIDKAGGV~GTWYWNKYPGALSDSQSFVYQYSF 72 16DAVV YAVHKLRNEQGMNVKAYDNAADI~~TWFWNRYPGAVSDTESFVYRFSF 75
*.:*:******:*::***::* *: .. *:* ****:**:****:**: :*. :*: :**
70DRDLLQDGTWKNTYVTQPEILEYLEDVVDRFDLRRHFRFGTEVTSAIYLDDENLWEVTTD 129 70DRDLLQESTWKSTYITQPEILEYLEDVVDRFDLRRHFKFGTEVTSALYLDDENLWEVTTD 129 71DRDLLQDGTWKTTYITQPEILEYLESVVDRFDLRRHFRFGTEVTSAIYLEDENLWEVSTD 130
121DKGLLQDGTWKHTYITQPEILEYLEDVVDRFDLRRHFRFGTEVKSATYLEDEGLWEVTTG 180 73DRDLYTNNTWTHRFIKGPEVLAYLNKVVDRFGLREHIHLETGMTEAVWDELSGTWTVRTD 132 76DRELLQRGRWKNRYVTQPEILAYLNEVADHLDLRRSYEFNTKVSAAQFDoIIILWKVTTD 135
*: * * **:* **:.*.*::.** .. * .. * : : * * *
136KGQAVTAKYLITGLGLLSATNLPK * * ::: .. :*:*** * *.: *:: *:*. :*:.***: .*:*:***:**.***
189 189 190 240 192 195
190èQQVITALAPEVEHLTVFVRTPQYSVPVGNRPVTPEQIDAIKADYDRIWEQAKNSAVAFG 249 190GQQVITSLAPEVEHLTVFVRTPQYSVPVGNRPVTPEQIAEIKADYDRIWERAKNSAVAFG 249 1915QQVITALAPEVEHLTVFVRTPQYSVPVGNRPVTKEQIDAIKADYDGIWDSVKKSAVAFG 250 241GQQVITALAPEVEHLTVFVRTPQYSVPVGKRPVTTQQIDEIKADYDNIWAQVKRSGVAFG 300 193GNQIITATAPIAGHLISFQRSPQYSVPVGNREVSPEQLRADHDNFDATWEQVRNSSVAMG 252 196~VQVITATAPIAKHLTVFQRSAQFVVPIGNTPQDAETIARQKATYDDIWKQVKSSAVAFG 255
* *:**: ** ** * *:.*: **:*: :* * .: *.**:*
250FEESTLPAMSVSEEERNRIFQEAWDHGGGFRFMFGTFGDIATDEAANEAAASFIRSKIAE 309 250FEESTLPAMSVSEEERNRIFQEAWDHGGGFRFMFGTFGDIATDEAANEAAASFlRAKVAE 309 251FEESTLPAMSVSEEERNRIFQEAWDHGGGFRFMFGTFGDIATDEAANEAAASFIRSKIAE 310 301FEESTVPAMSVTEEERRQVYEKAWEYGGGFRFMFETFSDIATDEEANETAASFIRNKIVE 360 253FEESTIETFSVSAEERERIFQEAWDKGGGFQFMFGTFCDIATDEAANEEAAKFIRRKIGE 312 256FEESTIPAETASPEERDRVFEAAWQRGGGFYFMFGTFSDIATSQVANDAAADFIKRKLKQ 315
*****: : :.: *** :::: **: **** *** ** ****.: **: **.**: *: :
310IIEDPETARKLMPTGLFAKRPLCDAGYHQVFNRPNVEAVAIKENPIREVTAKGVVTEDGV 369 310IIEDPETARKLMPTGLFAKRPLCDSGYYEVYNRPNVEAVAIKENPIREVTAKGVVTEDGV 369 311IIEDPETARKLMPTGLYAKRPLCDNGYYEVYNRPNVEAVAIKENPIREVTAKGVVTEDGV 370 361TIKDPETARKLTPTGLFARRPLCDDGYFQVFNRPNVEAVAIKENPIREVTAKGVVTEDGV 420 313 IVQDPETARKLTPTDMYARRPLCDSGFYEAFNRPNVSLVNVKENPIVRMTRK GIVTEDGT 372 316IVKDPETARKLTPSDLYAKRPLCGDDYYGVYNRDNVTLADVKADPIAEFTLTGIRLASGA 375
::******** *:. ::*:**** .. : .. :** ** .:* :** * . *: . *.
370LHELDVLV FDAVDGNYRRIEIRGRDGLHINDHWDGQPTSYLGVSTANFPNWFMVLG 429 370LHELDVLV. DAVDGNYRRIEIRGRDGLHINDHWDGQPTSYLGVSTANFPNWFMVLG 429 371LHELDVLVFATGFDAVDGNYRRIEIRGRNGLHINDHWDGQPTSYLGVTTANFPNWFMVLG 430 421LHELDVIVFÀ..r~FDAVDGNYRRMEISGRDGVNINDHWDGQPTSYLGVSTAKFPNWFMVLG 480 373EHELDVLV~lIFDAVDGNYMRVDLKGRDGKLISQHWNDGPTSYLGVATSGFPNMFMILG 432 376EHELDVVIFA1~FDAVDGNYTRMDMRGRNGVSLRDMWKEGPLGYLGIMEAEFPNLFMILG 435
*****::************ *::: **:* :: * * ***: : *** **:**
430PNGPFTNLPPSIETQVEWISDTIGYAERNGVRAIEPTPEAEAEWTETCTAIANATLFTKG 489 430PNGPFTNLPPSIETQVEWISDTIGYAERNGVRAIEPTPEAEAEWTETCTEIANATLFTKG 489 431PNGPFTNLPPSIETQVEWISDTVAYAERNElRAIEPTPEAEEEWTQTCTDIANATLFTRG 490 481PNGPFTNLPPSIETQVEWISDTVAYAEENGlRAIEPTPEAEAEWTETCTQIANMTVFTKV 540 433PNGPFTNLPPTIEAQVEFITDTIRKVAATETGRIDLRPEAEADWTETCREVAAATVFGKV 492 436PNGPFTNLPPSIETQVEWIADMVKTMEAKGLKTSEPTAQARDQWVELCRTIANMTLFPKA 495
51
**********:**:***:*:* : .:*. :*.: * :* *:*:
Phi2 490DSWIFGANIPGKTPSVLFYLGGLRNYRAVLAEVATDGYRGFDVKSAEMVTV--------- 540 TK6 490DSWIFGANIPGKKPSVLFYLGGLRNYRAVMAEVAADGYRGFEVKSAEMVTV--------- 540 Phil 491DSWIFGANVPGKKPSVLFYLGGLGNYRNVLAGVVADSYRGFELKSAVPVTA--------- 541 BP2 541DSWIFGANVPGKKPSVLFYLGGLGNYRGVLDDVTANGYRGFELKSEAAVAA--------- 591 ChnB2 493DSWIFGANIPGKKRSVLFYLGGLGEYRKIVAAEVAAGYPSFVTKSPPLPLGAAHPKRTSQ 552 Xanthobacter 496ESWIFGANIPGKKNTVMFYLAGLGNYRKVLSGLSESGYPTIIFDRAVECVA--------- 546
:*******:***. :*:***.** :** .. *
Phi2 --------TK6 --------Phil --------BP2 --------ChnB2 553PFRTATQH 560 Xanthobacter --------
Fig. 9: Sequence of Rhodococcus maris HI-31 ChnB2 aligned with sequences of cyclohexanone monooxygenases of Rhodococcus Phi2 (AAN3749 1), Rhodococcus sp. TK6 (AY486161), Rhodococcus Phil (AAN37494), Arthrobacter sp. BP2 (AAN37479), and Xanthobacter flavus (CAD10801). The multiple alignments were generated by ClustalW 1.75 (Thompson et al, 1994). The F AD binding motif near the N-terminal, and NADP binding motif near the middle of the protein, centered a round nucleotide signature GXGXXG (Vallon, 2000). Baeyer-Villiger monooxygenase sequence motif found the middle of the protein,' centered around nucleotide signature FXGXXXHXXXW(PID) (Fraaije et al., 2002), besides other conserved regions A TG motif and GG motif (Vallon, 2000) were highlighted with yellow colour. The frrst ATG motif was highlighted in green.
52
~ ..
Figure 10 shows the positions of the two BVMOs of Rhodococcus maris in
relation to other sequences in a phylogenetic tree. The tree was generated by using
the GeneBee pro gram from http://www.genebee.msu.su/services/phtree reduced.html
(Brodsky et al., 1992). This analysis indicated that R. maris ChnB2 (R.mar2) shares
less homology to the rest of the CHMOs of Rhodococcus compared to the R. maris
ChnB 1 (R.mar 1) sequence.
53
/'"""'-. 1
,cluster algorithft PHYLOGENETIC TREE Phglograft
R.narl
~~ R.TlC6
1-1 R.Phi2
R.Phii
R.BP2
XSlallus
-l R.NCIHB
R.nar2
Brelli
---i R.erythro
R.rhodoch
Brell2
Fig. 10: Phylogenetic tree of the CHMOs of the Rhodococcus maris HI-3I ChnBl, Rhodococcus maris HI-31ChnB2, Acinetobacter sp. SE19 (AAGl0021), Acinetobacter sp. NCIMB9871 (BAB61738), Brevibacterium sp. HCU CHMOI (AAGOI289), Brevibacterium sp. HCU CHM02 (AAGOI290), Rhodococcus erythropolis (CAC40956), Rhodococcus rhodochrous (BAA24454), Rhodococcus Phi2 (AAN37491), Rhodococcus sp. TK6 (AY486161), Rhodococcus Phil (AAN37494), Arthrobacter sp. BP2 (AAN37479), and Xanthobacter jlavus (CAD10801). The phylogenetic tree was generated by using the GeneBee program from http://www.genebee.msu.su/services/phtreereduced.htmI(Brodsky et al., 1992).
54
Upstream of chnB2 (CHM02) is an ORF of 903-bp in length, between
nucleotides (2522-3425). We designated this chnC2 since it appears to be a
duplication of the R. maris chnC that encodes a lactone hydrolase. 6-bp from the
beginning of the gene, a consensus ribosome-binding sequence, (GAGGA) was found
(Fig. 6). BLASTX search shows high homology of chnC2 to Bradyrhizobium
japonicum USDA hydrolase with 47% identity and 59% similarity. The deduced
amino acid sequence of ChnC2 contains a pentapeptide, Gly-Asp-Ser-Met-Gly at
position 122-126 amino acids, a feature resembling the conserved GlyXSerXGly
motif found in the family of hydrolases where Ser is a catalytic residue (Ollis et al,.
1992). ChnC2 is also homologous to the hydrolase, chnC, of Clostridium
acetobutylicum ATCC with 40% identity and 56% similarity (Table 3).
Figure 11 shows a sequence alignment of ORF2 (chnC2) with three putative
hydrolase sequences from Bradyrhizobium japonicum USDA 110 (BAC48149),
Clostridium acetobutylicum ATC 824 (AAK76842), and Erwinia carotovora sp.
atroseptica SCRIl043 (CAG75135). The alignment revealed the presence of
GlyXSerXGly motifthat is characteristics of hydrolases.
55
r" NP 149260 YP 050327 ChnC2 NP 769524
NP 149260 YP 050327 ChnC2 NP 769524
NP 149260 YP 050327 ChnC2 NP 769524
NP 149260 YP 050327 ChnC2 NP 769524
NP 149260 YP 050327 ChnC2 NP 769524
NP 149260 YP 050327 ChnC2 NP 769524
-----lMAHVNTNGIQIEYEVFGKRTNPTIVLIAGNGAQLNFWESDFCEMLAQHNLQVIR 54 ----lMYGHIRNKGVNIAYESFGDESTEIIILVAGLGSQMISWSDAFCQTVVSRGYRVIR 55 -1MADRTRRVAVGELELAYETFGDAGNSPLLLISGLATQMLGWDERFCDQLADHGFHVIR 58
1MNAHQPPQTVRANGIDICYEIFGNDNAEPLLLIMGLGAQMIHWDDAFCEQLAVHGFRVIR 60 ::: ** ** : : *: * .: *: * **: :. :. : ***
55FDNRDAGLSTKFDAAGIPDMSKIYQAAQEGKPIKTAYTLEDMADDVAGLLDALEIKKAHI 114 56FDHRDVGCSSHFYDSPVPNIGEVNKAISANRQPDVPYTLDDMAGDLIGLMDALSITAAHF 115 59FDNRDIGLSTHLHEGGIPNLGALLRG---EAAPAPPYTLADMAEDTAGLLDALGLESAHI 115 61FDNRDIGKSSHLTGGKRLTPLELLKLRFLRIPVAATYKLIDMARDTVGLMDALGIKSAHL 120
**:** * *::: * * *** * **:***: **:
IAQVFAYRHPLRICSLISIMSSTGNPNNPKISQETLEIVTATPPN---QRGA 171 lAARHPERVLSLSIIMSSSGNPSLPQTAPDVMALMTQPAPNPFLNEPE 175
PSIR-C1VC;OliRE'EP,OAVLFLPPPT---DPDT 171 177
* ******** .. *: ** ***: ... *
172YIEYTVHMWKKLWSKGFPFEEERAIRYTEESYDRCYYPQGAVRQNAALVANGDRRKYLSL 231 176YLNHRLRLAERLAGKIYPFDTERYRSQFKEELRRCFNPEGFTRQMAALVASGDRRKLLAT 235 172AAERSLTVYRVIGSPGYPLDEPRVAEVARRAFARGNNPAGVARQYAAIVVSPDRTPGLRE 231 178 FVVRYGQTWKVLRAGAFPEEEALDPDRAERVFARGLNPAGVGRQLRAVLASGSR KERLHA 237
: * : * * * ** *:: ... * *
232LRVPTLVIHGTADPLIPVEAGKDTARTIPNAKLLLIEGMGHDMPKGTWKCIVEAIVSLVR 291 236IAAPTLVIHGTDDPLFVPSHGEDVAINIPEAEFMLIDGMGHDIPNQLNALVTDAIVRIAQ 295 232LTVPTLVIHGEDDPLVQVEGGLATADAVPGARLVVVPGMGHNLPQPLWPQVIDEIVAHAR 291 238MKTPTLVIHGTVDPLVHPEGGKDTAASIPGAKLLMVEGMGHALPMRFWPEIIGAIDKHAR 297
.. ******* ***
292DTSHSCWR--- 299 296RRHQNSALHTS 306 292AAEVGPAAHA- 301 298RAAAQAA---- 304
* * : * *.:::: **** : * *
Fig. 11: R. maris ChnC2 sequence aligned with the hydrolase sequences of Bradyrhizobiumjaponicum USDA 110 (BAC48149), Clostridium acetobutylicum ATC 824 (AAK76842), and Erwinia carotovora sp. atroseptica SCRI1043 (CAG75135). The multiple alignments were generated by ClustalW 1.75 (Thompson et al, 1994). GlyXSerXGly motifwas highlighted with yellow colour.
56
A phylogenetic tree of the hydrolases was also done for Rhodococcus maris
ChnCl and Rhodococcus maris ChnC2 sequences in relation to others (Fig. 12). The
result indicated that the two R. maris hydrolases, R.marl and R.mar2, are in two
separate clusters. For example, R. maris ChnC2 is more related to the
Bradyrhizobium japonicum USDA 110 hydrolase, whereas R. maris ChnC 1 is more
related to the hydrolase of Xanthobacter flavus. A similar result was found for the
two Brevibacterium sp. HCU hydrolases (Brzostowicz et al., 2002).
57
Cluster algorithft PHYLOGENETIC TREE Phylograft
1 R.l'larl
1 X.flal/us r---
Bre\lÏ1
R.erythro
~ R.l'lar2
B.japonic
y Cl.acetob
E.caroto\l
r- P.aerugin
- Str'eptol'lY
[ Ji Rcinetoba ~I
::;: ... '" rz Bre\li2 # ~--.~--~.-~~--~~--~~~~--~~--.~---.~--~--~----------~ o 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Fig. 12: Phylogenetic tree of the hydrolases of the Rhodococcus maris HI-31 ChnCl, Rhodococcus maris HI-31ChnC2, Xanthobacter flavus (CADI0800), Rhodococcus erythropolis (CAC 17806), Pseudomonas aeruginosa (AAKOI509), Streptomyces sp. (AABI6939), Acinetobacter sp. no. 6 (BAB68338), Bradyrhizobiumjaponicum USDA 110 (BAC48149), Clostridium acetobutylicum ATC 824 (AAK76842), Brevibacterium sp. HCU. ChnC1 (Q93QG9), Brevibacterium sp. HCU. ChnC2 (Q8VW46), and Erwinia carotovora sp. atroseptica SCRI1043 (CAG75135). The phylogenetic tree was generated by using the GeneBee pro gram from http://www.genebee.msu.su/services/phtree reduced.html (Brodsky et al., 1992).
58
Upstream 'of chnC2 is ORF3 between nucleotides (981-2481), a 1500-bp that
is predicted to encode a long chain fatty acid Co-A ligase (LCFA Co-A ligase). 6-bp
upstream of the start codon is a putative Shine-Dalgarno sequence, GAAAG (Fig. 7).
BLASTX search revealed 56% identity and 68% homology to the Streptomyces
avermitilis MA-4680 (BAC68086). The deduced amino acid sequence of LCF A Co
A ligase contains two conserved regions, YTSGTTGAPKGA at amino acid position
158-170 amino acids, and GYGLSE at position 295-301 amino acids. This motif
(YTSGTTGXPKGV----GYGXTE), is predicted to be the ATP/AMP signature that
was identified on the basis of the sequence similarities found in the family of
adenylate forming enzymes inc1uding, fatty acid Co-A ligase enzymes (Wiemar et al.,
2002).
Fig. 13 shows the sequence alignment of LCF A Co-A ligase with
Streptomyces avermitilis MA-4680 (BAC68086), and Kineococcus radiotolerans
SRS30216 (ZP-00226705). The alignment indicated an overall 45% sequence identity
indicated by asterisks.
59
NP 821551.1 ZP 00226705 LCFA
NP 821551.1 ZP 00226705 LCFA
NP 821551.1 ZP 00226705 LCFA
NP 821551.1 ZP-00226705 LCFA
NP 821551.1 ZP 00226705 LCFA
NP 821551.1 ZP 00226705 LCFA
NP 821551.1 ZP 00226705 LCFA
NP 821551.1 ZP 00226705 LCFA
NP 821551.1 ZP 00226705 LCFA
--lMANLAEFLVETARRQPERPALRLGGQVTSYAELDQRSARAAALLRAEGVRPGDRVAL 57 1MSSPTTFAGAVAEHARTRPDGVAVRLDEHVLTWRELDDATARVAALLRAQGVRPGDRVAL 60
--lMTNLAHILTESARRHPDRPAIRLDDLVITYAELDDLTARAAGWLQARGIRPGDRVGI 57 :.:* :.* ** :*: *:** * :: ***: :** * *:*.*:******.:
58MLPNVPEFVVLYYGVLRAGAVVVPMNPLLKTRETEYHLRDSGAQMLFEWHQAPGEGAQGA 117 61VLPNVPQFPVLYHGILRAGAVVVPMNPLLRGREVNHHFADSGAVLALVWHAVADASHAGA 120 58ALPNIVPFPVFYYGVLRAGATVVPMNPLLKAREIEHALRDSGAALALVGTTlAAEAQAAA 117
***: * *:*:*:*****.********: ** :: : **** :
118AAAGVRHMAVEPAAFAGLLAGHAPLPEVSEPDSEDVAVLLY11SGT 121VGTGTRVVVVEPGATAALLAGVDPDPAVHVPDPGDTAVIL 118AATGTDIVVIDDDTLAGAAQWPR-LPEVTARADDDTAVLL
*
177 LSHRNLL 180 LTHSNMY 176
. . : *. : * * * *.**:******** **** *:* ..
178HNTEVNAVDVQRMTAEDVVVGCLPLFHIFGQICAMSVTVRAGASLTLIPRFDPQTVLDAI 237 181SNALTSQERLIRLGGDDVILGALPLFHAFGQTCAMNTAVVAGRTLSLLPRFSAAAALAVV 240 177RNATT-FVAMLDIRKEDVVLGCLPFFHAFGQSNALNASLAAGACVSLVPRFEAVAVVRLI 235
*: . :**::*.**:** *** *: .. :: ** : : *: ***.. . ..
238ARDRATIFEGVPTMYAALLQHP--SEADVSTLRMCISGGASLPVEILHGFERRFGCVVLE 295 241ERDRVTHFAGVPTMYVALLNEPGAEEFDLSSWRSCVSGGASLPVEVLRGFEARFGVQILE 300 236ERHRVTVFEGVPTMYVSLLHAD-LSEADTSSLRICISGGAALPIEVLNGFQGAFGAPILE 294
* * * * ******. :**: * * *: * *:****:**:*:*.**: ** :**
SPVVTFNHPDRPRKAGSIGTPIKDVQVRLLDEKGQDVTPGEIGELAVRGPNVM 355 SPVASFNHPDAVRKPGTIGVPVRGCEMDLRGADGTPVTEGVG-EIVVRGENVM 359 SPTATFNRIGKS-KPGSIGLPIDGVELKLVARDGTETLPGEVGEIVIRGHNVM 353
*:*:***** .. :**: . * *. ** *: . :: * * * *:. :** ***
356KGYWN-RPEETAATIPDGWLRSGDLAHRDEDGYLYIVDRKKDMIIRGGYNVYPREIEEVL 414 360TGYWRNEAATAAAFTADGWFRTGDLATRDEDGYYAIVDRAKDMIDRGGYNVYPREVEEVL 419 354KGYWK-RPDATAAAIVDGWFHTGDMATRDEDGFYFIVDRKKDIIIRGGYNVYPREIEEVL 412
*** :** ***:::**:* *****: **** **:* **********:****
415HEHPAVALAAVVGLADAHLGEEIAAAVVLRPKAQATPDELRQFVKERVAAYKYPREVWLV 474 420HEHPAVEQVAVVGFPDPLVGEEVGAAVVLKEGACATPEELQQHVKDALAAYKYPRRVWIV 479 413YEHPAVREVAVIGLPHPTYGEEVAAAITLRPGAEATPEELRQYVKSRVAAYKYPRHVWLA 472
:***** **:*: ... ***:.**:.*: * ***:**:*.**. :*******.**:.
475DTLPTGPSGKILKREISAPTG------- 495 480SELPKGPTGKILKREIHPPLERTSES-- 505 473DKLPKGATGKILKREIGIPAHLLEQPPA 500
**.*.:******** *
Fig. 13: R. maris LCF A CoA ligase sequence aligned with sequences of LCF A CoA ligas~ of Streptomyces avermitilis MA-4680 (NP-821551) and Kineococcus radiotolerans SRS30216 (ZP-00226705). The multiple alignments were generated by ClustalW 1.75 (Thompson et al, 1994). yTSGTTGXPKGV----GyGXTE predicted ATP/AMP signature was highlighted with yellow colour.
60
,r-... A phylogenetic tree of the LCFA Co-A ligases sequences (Fig. 14) indicated
that R. maris LCFA Co-A ligase (R.mar) is most related to the LCFA Co-A ligase of
Streptomyces avermitilis MA-4680.
61
" "." '
Cluste... algorithn PHYLOGENETIC TREE f>hylog ... an
R.na ...
5.ave ... nit
O.iheyens
B.halodu ...
R.ful4
- R.ful7
R.ful6
r p.ae ... ugin
~, ~ E.coli ~ L-__ ~ __ ~~~ ____ ~.-__ ~~ __ -.~ ____ ~~ __ ~~ ____ ~ ____________ ~
o 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Fig. 14: Phylogenetic tree of the LCFA Co-A ligases of the Rhodococcus maris HI-3I, Escherichia coli KI2 (AAC74875), Pseudomonas aeruginosa PAOI (AAG06687), Archaeoglobus fulgidus DSM 4304(fadD-6) (AAB89737), Archaeoglobus fulgidus DSM 4304 (fadD-7)(AAB89478), Archaeoglobus fulgidus (fadD-4) (AAB90399), Bacil/us halodurans C-125 (BAB06822), and Oceanobaci/lus iheyensis HTE83 1 (BAC 13132). The phylogenetic tree was generated by using the GeneBee program from http://www.genebee.msu.su/services/phtree reduced.html (Brodsky et al., 1992).
62
4.3. Sub-cloning of 5-kb SacI fragment in pT7-6 Expression Vector
Due to the fact that the 5-kb fragment in pCMR500 was cloned in the opposite
orientation of the lac promoter, sub-cloning of the 5-kb SacI fragment was carried out
in the pT7-6 expression vector (a 2.2-kb) in an attempt to express the chnC2 gene
product. Following the transformation in E. coli, seven-clones with the expected size
(7 .2-kb) were found. Restriction analysis of the seven-clones indicated that three of
them were in the right orientation after being tested with Pstl digestion. The
diagnostic fragments were 6-kb and 1.2-kb (data not shown). Unfortunately, the
expression work was not completed, due to personal safety reason and the need to use
radioactivity (candidate was pregnant at that time).
4.4. Sub-cloning of Rhodococcus maris CHM02 for Protein Expression
4.4.1. PCR for Rhodococcus maris chnB2 Amplification
R. maris chnB2 gene was located in two separate clones, pCMR400 and
pCMR500. Therefore, PCR amplification on R. maris genomic DNA was performed
to sub-clone R. maris chnB2. As a result, a 1.9-kb PCR fragment was obtained. After
CIal fragment, 1.37-kb fragment that contains the chnB2 gene was liberated and
ligated in the Clal-linearized pT7-5 expression vector. Uhfortunately in this case, the
desired recombinant clone was not obtained after several attempts.
63
5. Discussion
5.1. Rhodococcus maris HI-31 Cyclohexanol Degradation pathway
Prior to this work, the chnB, chnC, chnD, and chnE genes of the cyclohexanol
degradation pathway in R. maris HI-31 have been identified. The chnBl, chnCl, and
chnE are transcribed in one direction whereas chnR and chnD, found upstream of
chnCl, are transcribed in the opposite direction (Lau laboratory, unpublished data)
(Fig. 15-A).
Despite additional cloning and characterization of a 6-kb DNA locus upstream
of the known cyclohexanol gene cluster there was no region of a chnA homolog that
encodes a cyclohexanol dehydrogenase. AIso, several Kb downstream of chnE have
been sequenced (Lau laboratory, unpublished data) but there was no possible chnA
encoding sequence. Instead, in this study, we discovered a second copy of chnB
(designated chnB2) and chnC (designated chnC2) in addition to a putative long chain
fatty acid Co-A ligase. These ORFs are separated from the principle gene cluster by
three other ORFs, tentatively identified as 3-hydroxyisobutyrate dehydrogenase, y
carboxymuconolactone decarboxylase, and a benzoate transporter (Fig. 15-A).
In the cyclododecanol degradation pathway of Rhodococcus ruber SC 1, the
cyclododecanol dehydrogenase-encoding gene has also been reporting missing
despite vigorous analysis of the pathway (Kostichka et al., 2001).
In Brevibacterium epidermidis HCU, the presence of two BVMOs, ChnBl
and ChnB2, have been reported (Brzostowicz et al., 2000). Strain HCU was selected
for growth on cyclohexanol. Pairwise comparison of amino acid identity of R. maris
ChnB 1 and ChnB2 with ChnB 1 and ChnB2 of B. epidermidis HCU indicate that R.
64
maris ChnB 1 and ChnB 1 of B. epidermidis HCU are close relatives, with 60%
sequence identity (Table-4).
Previous studies of B. epidermidis HCU ChnBl and ChnB2 have found a
dramatic difference between the two proteins in the use of cyclopentanone or
cyclohexanone as substrate (Brzostowicz et al., 2000). While ChnB 1 had little
activity for cyclopentanone, ChnB2 oxidizes cyclopentanone very weIl. On the other
hand, the ChnB 1 has a preference for cyclohexanone. These findings suggest that the
two monooxygenases of R. maris preferentially uses some substrates better than the
other. AIso, the presence ofthese two monooxygenases with distinct specificity in the
same strain may suggest that the activity of these enzymes in nature may be toward
natural products that contain cyclohexanone or cyclopentanone asa substructure, for
example, those in steroids or polyketides.
After the completion of this work, the substrate specificity of R. maris ChnB 1
and ChnB2 was determined (Iwaki, unpublished data). CHMOI (ChnBl) was shown
to be good in oxidizing cyclohexanone and cycloheptanone, but was relatively weak
toward cyclopentanone and cyclooctanone (Table 5). On the other hand, R. maris
ChnB2 was also found to have activity towards the various substrates but
considerably lower than those of ChnB 1. There appears to be no preference of
CHM02 (ChnB2) of R. maris towards cyclopentanone, a situation that is different for
ChnB2 of Brevibacterium epidermidis HCU.
65
Table 5: Substrate specificity of Rhodococcus maris HI-31 CHMOI and CHM02. U/mg: one unit of activity is defined as the amount of enzyme required to convert 1 ~mole of substrate in 1 minute (Iwaki; Lau, Laboratory). The BVMO activity was measured by the decrease in absorbance at 340 nm due to the oxidation ofNADPH to NADP (Iwaki et al., 2002).
Substrate
cyc1ohexanone
cyc1opentanone cycloheptanone cyc1ooctanone
R. maris CHMO-l U/mg
3.66 (100%)
1.05 (28.7%) 2.69 (73.5%) 0.21 (5.7%)
CHM02 U/mg
0.092 (100%)
0.046 (50%) 0.064 (69.6%) o
66
Immediately upstream of chnB2 and transcribed in the same direction is
chnC2. This is a potentiallactone hydrolase and could potentially be involved in the
formation of hydrooxyhexanone in the cyclohexanol degradation pathway in R. maris
via the hydrolysis of caprolactone.
Comparison between the R. maris ChnC 1 and ChnC2 sequences indicated that
ChnCl was 30-bp (10 amino acids) longer than ChnC2. Furthermore, their sequence
alignment showed little homology, a situation similar to that obtained from a
comparison of ChnCl and ChnC2 from Brevibacterum epidermidis HCU
(Brzostowicz et al., 2002). B. epidermidis HCU ChnCl is a close homolog of the
ChnC of Acinetobacter sp. strain SEI9, where as ChnC2 belongs to adifferent family
of hydrolases even though both of them have been shown to have the ability to
hydrolyse caprolactone (Brzostowicz et al., 2002). The phylogenetic tree of R.· maris
ChnC 1 and ChnC2, shows that these proteins are not related to each other. ChnC2
was found to be most closely related to the Bradyrhizobium japonicum USDA 110
hydrolase although its substrate is unknown (Fig. 12).
Hydrolases are one of the most extensively used class of enzymes for
biocatalysis, organic transformations, and industrial applications. The discovery of
new hydrolases will contribute to preparative synthesis and large-scale chemical
production of sorne desired compounds (Cheng et al 2002). For example, ChnC of
Acinetobacter strain NCIMB 9871, was found to be capable ofracemic resolution of
lactones, such as, e-decanolactone in addition to caprolactone (Onakunle et al., 1997).
The availability of additional clones of chnC provides new opportunities for the
exploration of their substrate specificities.
67
r-,
~--"
Finding genes with a functional redundancy in the R. maris cyclohexanol
degradation pathway (chnBl and chnB2, chnCl and chnC2) is not a new matter.
Previously, in Nocardia globerula CLl, two distinct cyclohexanone monooxygenases
have been found (Norris et al., 1976). And more recently, in B. epidermidis HCU
(Brzostowicz et al., 2000), two clusters were identified to contain the genes
responsible for cyclohexanol pathway (Fig 15-B). Cluster 1 contains nine ORFs that
includes chnRl, chnCl, chnBl, chnDl, and chnA. Cluster 2 contains chnD2, chnR2,
chnC2, and chnB2 genes. These clusters of genes were found to have a functional
redundancy, but not aIl of them belong to the same families. For example, chnB2
gene product was found to be capable of oxidizing cyclopentanone while chnBl gene
product has a weak activity towards it (Brzostowicz et al., 2002). On the other hand,
the activity of ChnB 1 is higher towards cyclohexanone (Brzostowicz et al., 2000).
AIso, ChnDI of Brevibacterum epidermidis HCU was found to belong to a Zn
dependent long-chain dehydrogenase, where as ChnD2 of B. epidermidis HCU was
found to be a Zn-dependent 6-hydroxyhexanoate dehydrogenase (Brzostowicz et al.,
2002). R. maris HI-31 ChnD was found to be more related to 6-hydroxyhexanoate
dehydrogenase (Dr Lau, unpublished data), a situation similar to the ChnD2 of B.
epidermidis HCU.
During genome evolution, the duplication of existing genes is mainly the most
important process for the generation of new genes. It can occur by duplication of the
entire genome, duplication of a single chromosome or part of a chromosome, or by
duplication of a single gene or group of genes. If a gene segment coding for a
structural domain is duplicated by unequal crossing-over, replication slippage or
other methods lead to duplication of DNA sequences. This will lead to domain
68
!~-"
duplication. As a result, structural domain will be repeated in the protein. This may
be advantageous by making the protein product more stable. If the coding region of
the protein is changed by mutation over time this may produce protein with new
activity (Brown, 2002).
It has been proposed that domain duplication may cause a gene to become
longer (Brown, 2002). The difference in gene size (R. maris chnB2 is 60-bp longer
than chnBl) and activity, may be due to a general consequence of gene evolution.
Further upstream of chnC2 and transcribed in the same direction, is ORF3,
that encodes a potential long chain fatty acid Co-A ligase. LCF A C, which is also
known as fatty acid acyl-CoA synthetase, plays an essential role lipid biosynthesis
and fatty acid degradation such as catalyzing the formation of acyl-CoA from
intermediary metabolism, which are involved in protein transport, enzyme activation,
protein acylation, cell signalling. Besides that they serve as substrates for ~-oxidation
(Weimar et al., 2002).
The function of LCF A Co-A ligase associated with cyc1ohexanol degradation
pathway is yet to be determined.
It is a common feature among catabolic pathways of aromatic metabolism to
find ORFs or genes that appear to have no apparent contribution to the pathway of
interest. For example, in Acinetobacter strain SE19, ORFs like, chnZ (a conserved
hypothetical sequence), chnY (a pilin invertase sequence), and chnX (an unknown
protein sequence) were found (Cheng et al., 2000) (Fig. 15-C). Aiso in Acinetobacter
strain NCIMB 9871, orD (a pilin gene inverting sequence) and orf5 (an unknown
protein sequence) were found (Iwaki et al., 2003). Moreover, in R. maris HI-31 ORFs
that encode 3-hydroxyisobutyrate dehydrogenase, y-carboxymuconolactone
69
decarboxylase, and a putative benzoate transporter respectively, were also found
between the cyclohexanol degradation genes (Fig. 15-A).
A very recent development in BVMO technology has been the first crystal
structure of phenylacetone monooxygenase (PAMO) determined from a thermophilic
organism, Thermobifidafusca (Malito et al., 2004). This study demonstrated the first
three-dimensional structure of a Baeyer-Villiger monooxygenase. PAMO was shown
to exhibit a two-domain architecture resembling that of the disulfide oxidoreductase.
The active site of the protein is located in a cleft at the domain interface. An arginine
residue located above the flavin ring is predicted to exist in two positions, termed the
"IN" and "OUT" positions. The "IN" position is found in the crystal structure and the
"OUT" position allows NADPH to approach the flavin in order to reduce the cofactor.
The position of this amino acid is suited to stabilize the negatively charged flavin
peroxide and Criegee intermediates.
In the crystal structure of PAMO, a conserved histidine-173 residue in the
BVMO fingerprint motif was found to play a role in the domain rotations and
conformational changes that occur during the catalytic cycle (Malito et al., 2004).
In addition to the determination of BVMO crystal structure, the identification
of other BVMOs will allow for a greater understanding of the structure and function
of these proteins. Moreover, these studies will allow a greater possibility of selecting
and engineering BVMOs to function under non-natural conditions.
Although cyclohexanol and its ketones components are not priority pollutants
there are different naturally occurring molecules in the environment that contain these
core or cycloalkanes structures, e.g. steroids and fossil fuel hydrocarbons, or they
may be formed as a result of hydroxylation of cyclohexane. Xanthobacter sp. is one
70
r---... of the few bacteria that has the ability to utilize cyclohexane as a source of carbon,
and able to transform cyclohexane to cyclohexanol (Trower et al., 1985; Trower et
al., 1989). Very recently, a homolog of a butane monooxygenase was found
responsible for the activation of cyclohexane in a beta-proteobacterium,
Brachymonas petroleovorans CHX (Brzostowicz et al., 2005).
71
A
Rhodococcus maris HI-3I chnC2 chnB2 chnR
q qqc> LCFA Co-A Ligase
B
orf4 orf5 orf6
Brevibacterium HCU Clusterl
chnRl
Brevibacterium HCU Cluster2
Ch~~ chn Le ~
c
Acinetobacter SE19
chnBl chnE
~
chnA chnD chnC chnR
cl \=J"\=J" chnZ chnY chnX
Fig. 15: Comparison of the gene organization of cyclohexanol degradation pathway in R. maris HI-3I (A), B. epidermidis HCU (B), and Acinetobacter sp. strain SEl9 (C). The orientation of the arrows indicate the direction of gene transcription. Genes involved in cyclohexanol pathway are indicated by arrows with different colours. Other ORFs are marked by light yellow arrows. The chnABCDE genes encode, cyclohexanol dehydrogenase, NADPH-linked cyclohexanone monooxygenase (CHMO), epsilon-caprolactone hydrolase, NAD (NADP)-linked, and flavin adinine dinucleotide (F AD) dependent, 6-hydroxyhexanoic acid dehydrogenase and 6-oxohexanoic acid dehydrogenase (Brzostowicz et al,. 2002; Cheng et al., 2000). Orf4, orf5, and orf6 are genes encode: 3-hydroxyisobutyrate dehydrogenase, y-carboxymuconolactone de carboxylase, and a putative benzoate transporter respectively. ChnZ is a conserved hypothetical sequence, chnY is a pilin invertase sequence, and chnX is an unknown protein sequence.
72
5.2. Conclusion
In conclusion, the nucleotide sequence of a 5.011-kb gene region that is
associated with the cyclohexanol degradation pathway in R. maris HI-31 was
obtained. Comparison of the deduced ORFs and phylogenetic analysis with the other
proteins in the NCBI database has allowed assignment of possible functions.
This study has led to the discovery of at least three additional genes, two of
them appear to be the result of gene duplication. Further characterization of the gene
products are needed. Importantly, the chnA gene that is responsible for the first step
of cyclohexanol degradation in R. maris remains to be identified.
73
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