Genome mining reveals new bacterial type I Baeyer-Villiger monooxygenases with (bio)synthetic potential

Article abstract of DOI:10.1016/j.mcat.2020.110875

Baeyer-Villiger monooxygenases (BVMOs) are oxidorreductases that catalyze the oxidation of ketones in a very selective manner. By genome mining we detected seven putative type I BVMOs in Bradyrhizobium diazoefficiens USDA 110. As we established the phylogenetic relationships among them and with other type I BVMOs, we found out that they belong to different clades of the phylogenetic tree. Thus, we decided to clone and heterologously express five of them. Three of them, each one from a divergent phylogenetic group, were obtained as soluble proteins, allowing us to proceed with their biocatalytic assessment and enzymatic characterization. As to substrate scope and selectivity, we observed a complementary behavior among the three BVMOs. BVMO2 was the more versatile biocatalyst in whole-cell systems while BVMO4 and BVMO5 showed a narrow substrate profile with preference for linear ketones and particular regioselectivity for (±)-cis-bicyclo[3.2.0]hept-2-en-6-one.

Full text of DOI:10.1016/j.mcat.2020.110875

Molecular Catalysis 486 (2020) 110875
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
Genome mining reveals new bacterial type I Baeyer-Villiger
monooxygenases with (bio)synthetic potential
a
b
a,1
a,
Romina D. Ceccoli , Dario A. Bianchi , María Ayelén Carabajal , Daniela V. Rial *
a
Área Biología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Consejo
Nacional de Investigaciones Científicas y Técnicas (CONICET), Suipacha 531, S2002LRK, Rosario, Argentina
b
Instituto de Química Rosario (IQUIR, CONICET-UNR), Departamento de Química Orgánica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de
Rosario (UNR), Suipacha 531, S2002LRK, Rosario, Argentina
A R T I C L E I N F O
A B S T R A C T
Keywords:
Baeyer-Villiger monooxygenases (BVMOs) are oxidorreductases that catalyze the oxidation of ketones in a very
selective manner. By genome mining we detected seven putative type I BVMOs in Bradyrhizobium diazoefficiens
USDA 110. As we established the phylogenetic relationships among them and with other type I BVMOs, we found
out that they belong to different clades of the phylogenetic tree. Thus, we decided to clone and heterologously
express five of them. Three of them, each one from a divergent phylogenetic group, were obtained as soluble
proteins, allowing us to proceed with their biocatalytic assessment and enzymatic characterization. As to substrate
scope and selectivity, we observed a complementary behavior among the three BVMOs. BVMO2 was the more
versatile biocatalyst in whole-cell systems while BVMO4 and BVMO5 showed a narrow substrate profile with
preference for linear ketones and particular regioselectivity for ( ± )-cis-bicyclo[3.2.0]hept-2-en-6-one.
Baeyer-Villiger monooxygenases
Flavoenzymes
Ketone biooxidation
Biocatalysis
Biotransformations
1
. Introduction
The inclusion of enzymes in synthetic routes represents a green al-
Many type I BVMOs from bacteria and some enzymes from fungi
have been cloned and studied in the last decades [1,3,4]. Indeed, sev-
eral of them have been cloned, heterologously expressed and biocata-
lytically characterized in the last five years in a raising interest for these
enzymes [5–14]. The accepted enzymatic mechanism of type I BVMOs
proposes the formation of a C4a-peroxyflavin via a covalent bond be-
tween an atom of oxygen and the C4a of the isoalloxazine ring of re-
duced FAD. This intermediate performs a nucleophilic attack on the
carbonyl group of the substrate generating a Criegee intermediate that
rearranges spontaneously to the corresponding ester or lactone. This
mechanism has been reviewed in [1,4,15]. In addition to the enzymatic
characterization of this class of enzymes, the knowledge gained on their
biocatalytic performance has allowed their use for synthetic purposes
and their application on redox cascades. A couple of type I BVMOs were
applied in the synthetic route toward showdomycin, trans-kumausyne
and goniofufurone analogs [16] as well as (+) and (-) non-natural
carba-C-nucleosides [17,18]. Recently, two BVMOs were applied to the
chemo-enzymatic synthesis of the Taniguchi lactone in whole-cells
systems [19]. Besides, BVMO-mediated biooxidations have been com-
bined in multi-enzymatic cascade reactions toward fine chemicals or
ternative to traditional organic synthesis and offers outstanding ad-
vantages regarding selectivity, scalability, efficiency and environmental
impact. Among the strategies toward lactones and esters, the oxidation
of ketones by Baeyer-Villiger monooxygenases (BVMOs) has gained a
high level of acceptance at laboratory scale due, in part, to the avail-
ability of many expression systems and the ability of BVMOs to oxidize
a plethora of structurally diverse substrates [1]. Type I BVMOs contain
non-covalently bound flavin adenine dinucleotide (FAD) as coenzyme
and depend on reduced nicotinamide adenine dinucleotide phosphate
(
NADPH). The importance of BVMOs as oxidizing agents relies on their
ability to replace the use of peroxides, peracids or peroxyacids under
vigorous conditions to achieve ketone oxidation in a reaction known as
Baeyer-Villiger oxidation. BVMOs catalyze the insertion of one atom of
oxygen from molecular oxygen into the substrate while the other one is
reduced to water. In addition to carbonyl compounds, type I BVMOs
can also oxidize organic sulfides, amines and boron- or selenium-con-
taining compounds [1–3].
⁎
Corresponding author.
E-mail addresses: ceccoli@inv.rosario-conicet.gov.ar (R.D. Ceccoli), bianchi@iquir-conicet.gov.ar (D.A. Bianchi), carabajal@ibr-conicet.gov.ar (M.A. Carabajal),
rial@inv.rosario-conicet.gov.ar, drial@fbioyf.unr.edu.ar (D.V. Rial).
1
Present address: Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Ocampo y Esmeralda s/n, Predio CCT‐CONICET‐Rosario, 2000
Rosario, Argentina.
https://doi.org/10.1016/j.mcat.2020.110875
Received 8 August 2019; Received in revised form 21 February 2020; Accepted 3 March 2020
2468-8231/ © 2020 Elsevier B.V. All rights reserved.

R.D. Ceccoli, et al.
Molecular Catalysis 486 (2020) 110875
building blocks [20–25].
amplify them were designed with restriction recognition sites as listed
in Table S2. The PCR amplifications from genomic DNA were per-
formed with Pfu DNA polymerase (Promega Corp, Madison, WI, USA)
according to the manufacturer protocol and supplemented with 5 % (v/
v) DMSO. In the case of blr3857, Taq DNA polymerase (Invitrogen,
Carlsbad, CA, USA) was used to add a 3´ A-tail to the PCR fragment in
order to clone it into pGEM-T Easy (Promega Corp, Madison, WI, USA)
following the manufacturer’s instructions. After digestion with the
corresponding restriction enzymes, the fragments were ligated into
compatible sites of pET-TEV vector to produce pHBd02, pHBd03,
pHBd04, pHBd05 and pHBd06 plasmids (Table S2). The pET-TEV
vector (kindly provided by Dr. Rodolfo M. Rasia, Instituto de Biología
Molecular y Celular de Rosario, CONICET-UNR, Rosario, Argentina) is a
modified pET-28a plasmid in which the thrombin cleavage site was
replaced with the TEV cleavage site [34]. All DNA fragments were
purified using the Wizard® SV Gel and PCR Clean-Up System (Promega
Corp, Madison, WI, USA). The recombinant plasmids were isolated
using Wizard Plus SV Miniprep DNA Purification System (Promega
Corp, Madison, WI, USA) and their sequences were confirmed by DNA
sequencing. E. coli strains were chemically transformed with the plas-
mids by standard procedures [35].
Bacterial genomes are a rich but largely untapped source of BVMOs
[
26,27]; therefore, mining for genes encoding them offers a golden
opportunity to discover novel enzymes. In a previous work, we iden-
tified a BVMO coding sequence in the genome of Leptospira biflexa and
characterized this enzyme thoroughly [8]. By in silico mining the as-
sembled metagenomic dataset obtained from polar and subpolar coastal
sediments [28], new BVMO sequences with novel features were pre-
dicted [29]. In the present work, we identified seven genes of type I
BVMOs by sequence similarity searching in the genome of Bradyrhizo-
bium diazoefficiens USDA 110 and established their phylogenetic re-
lationships with other BVMOs. B. diazoefficiens USDA 110 is an im-
portant nitrogen-fixing soil bacterium that is able to live in a symbiotic
relationship with soybean plants. We cloned and expressed in Escher-
ichia coli five of the identified sequences, and we characterized the
biocatalytic performance of three soluble enzymes both in whole-cell
systems and in pure forms. These newly identified BVMOs showed
differences in their substrate profile and enzyme properties both be-
tween them and with other known BVMOs, making them suitable
candidates for synthetic applications.
2
. Experimental
2.5. Protein expression and purification
2.1. General
Pre-cultures of E. coli BL21(DE3) cells harboring recombinant
All chemicals, enzymes and other reagents for Molecular Biology
plasmids were grown overnight in LB-kanamycin medium. Then, each
culture was prepared inoculating fresh LB-kanamycin medium with 2 %
were purchased from commercial sources (Promega Corp., Madison,
WI, USA; Invitrogen Corp., Carlsbad, CA, USA; Sigma-Aldrich Corp., St.
Louis, MO, USA; Merck KGaA, Darmstadt, Germany; Genbiotech S.R.L.,
CABA, Argentina; BD (Becton, Dickinson and Company), Franklin
Lakes, NJ, USA; Cicarelli Laboratorios, San Lorenzo, Argentina; Bio
Basic Inc., Markham, ON, Canada; MP Biomedicals, Santa Ana, CA,
USA, Alfa Aesar, Tewksbury, MA; USA, QIAGEN GmbH, Hilden,
Germany).
(
v/v) of the overnight pre-culture and incubated at 37 °C until the
bacterial density reached OD600 = 0.4-0.6. IPTG was added to a final
concentration of 0.3 mM and the culture was transferred to 24 °C at
2
00 rpm for 16 h. Then, E. coli cells were harvested by centrifugation
and resuspended in buffer A (50 mM Tris-HCl pH 8.0 and 150 mM NaCl)
containing 0.05 mg/mL lysozyme, 0.1 mM benzamidine and 0.5 % (v/
v) Triton X-100.
To purify the BVMOs, resuspended cells expressing the His-tagged
proteins were lyzed by sonication and, subsequently, centrifuged at
2
.2. Sequence searching and analysis
1
2000 g for 45 min at 4 °C. The cleared cell extract was incubated with
Sequences of putative BVMOs were identified by NCBI tblastn
Ni-NTA Agarose (QIAGEN GmbH, Germany) which had been equili-
brated with buffer A, for 1 h at 4 °C. Then, the resin was packed in an
empty column and washed with fifteen-resin volumes of buffer A sup-
plemented with 10 or 20 mM imidazole to remove nonspecifically-
bound proteins. The BVMO protein was eluted with two-resin volumes
of buffer A supplemented with 200 mM imidazole. Eluted fractions
containing FAD-bound protein were pooled and dialyzed against buffer
A. The purified protein was stored at −80 °C.
analysis against the B. diazoefficiens USDA 110 genome (www.ncbi.nlm.
nih.gov). The retrieved sequences and the sequences from already
cloned BVMOs (Table S1) were aligned with MAFFT version 7 [30].
Phylogenetic trees were generated using the LG substitution model in
PhyML 3.0 [31]. Branch support was calculated using the approximate
likelihood ratio test (aLRT) with a Shimodaira-Hasegawa-like (SH-like)
procedure. Phylogenetic trees were visualized using FigTree v1.3.1
(
http://tree.bio.ed.ac.uk/software/figtree/, [32]).
The presence of each BVMO in the soluble and insoluble fractions,
and the integrity and purity of the proteins were analyzed by SDS-PAGE
on a 12 % polyacrylamide gel followed by Coomassie blue staining.
2
.3. Bacterial strains and culture conditions
B. diazoefficiens USDA 110 was provided by Prof. Dr. Anibal Lodeiro
2
.6. Spectrophotometric analysis
from the Instituto de Bioquímica
y Biología Molecular (IBBM),
Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, Centro Científico Tecnológico
CONICET La Plata, La Plata, Argentina. This strain was grown at 30 °C
Purified protein concentration was determined based on FAD con-
tent. The molar absorption coefficient of protein-bound FAD for each
BVMO was determined by comparing the UV–vis spectrum of the native
in liquid Yeast Extract-Mannitol broth (YEM, 0.5 g/L K
2
HPO , 0.2 g/L
4
−
1
-1
protein with the spectrum of free FAD (ε450 = 11300 M cm ) ob-
tained after treatment of each BVMO with 0.2 % (w/v) sodium dodecyl
sulphate.
MgSO , 0.1 g/L NaCl, 10 g/L mannitol and 0.4 g/L yeast extract, pH
4
6
.8) at 150 rpm for 5–7 days. E. coli strains were grown at 37 °C in
Lysogeny Broth (LB, 5 g/L yeast extract, 10 g/L peptone, and 10 g/L
NaCl) or LB-agar medium (20 g/L agar). If necessary, media were
supplemented with 50 μg/mL kanamycin (LB-kanamycin).
2.7. Flavin analysis
2
.4. Genomic DNA extraction, cloning and gene expression
The flavin was released from the BVMO holoprotein by heat treat-
ment at 70 °C for 10 min. Protein precipitate was removed by cen-
trifugation and the supernatant was analyzed by thin-layer chromato-
graphy (TLC) on silica gel plates (0.25 mm, Merck, Darmstadt,
Germany) as described in [36].
Total genomic DNA was isolated as described in [33]. Five type I
BVMO genes from B. diazoefficiens USDA 110 (blr3857, blr2938,
bll3180, blr0964, blr6984) were selected and the primers used to
2

R.D. Ceccoli, et al.
Molecular Catalysis 486 (2020) 110875
2
.8. Enzyme activity
reaction was stopped after 24 h of incubation at 24 °C and 200 rpm.
Time-course biotransformation assays of ( ± )-cis-bicyclo[3.2.0]
hept-2-en-6-one were performed in 1 mL reactions containing 0.1 g wet
weight of E. coli BL21(DE3) cells expressing BVMOs, 200 mM Tris-HCl
pH 8.0, 200 mM glucose and 100 mM glycerol. Substrate was added to a
final concentration of 0.5 mg/mL and reactions were stopped at 0.5, 1,
2, 4, 8 or 24 h of incubation at 24 °C.
BVMO activity was determined spectrophotometrically by mon-
itoring the absorbance decrease of NADPH at 340 nm (ε340 = 6220
−1
−1
M
cm ) after addition of the enzyme. Reaction mixtures (1.0 mL)
typically contained 50 mM Tris-HCl buffer pH 8.5 or 100 mM sodium
phosphate buffer pH 7.5, 100 μM NADPH, 1–4 mM ketone substrate and
0
.075 to 0.3 μM enzyme. Steady state kinetic measurements were car-
Analytical samples (0.7 mL) were extracted with ethyl acetate or
dichloromethane (0.7 mL) supplemented with 0.25 mg/mL 1,3,5-tri-
methylbenzene as internal standard. The organic phase was removed,
ried out by varying either the cofactor NADPH (5–150 μM) or the
substrate (1-phenylpropan-2-one (0.02–10 mM) or heptan-3-one
(
0.01–40 mM)) concentration at 25 °C. UV–vis absorption spectra and
dried with anhydrous Na
2
SO , and analyzed by gas chromatography-
4
all measurements were performed using a Shimadzu UV-2450 spec-
trophotometer (Shimadzu, Kyoto, Japan) and quartz cuvettes of 1 cm
path length. One unit of BVMO activity is defined as the amount of
protein that oxidizes 1 μmol NADPH per minute. The kinetic parameters
mass spectrometry (GC–MS; Shimadzu GCMS-QP2010 Plus from
Shimadzu Corporation, Kyoto, Japan) using achiral or chiral capillary
column (SPB-1 Capillary GC Column or β-DEX 325, respectively, 30 m x
0.25 mm ID, 0.25 μm film, from Supelco, Bellefonte, PA, USA) (Table
S3). Biotransformations were performed as triplicates. Selectivity va-
(
K and kcat) of the BVMOs were determined by fitting the data with the
m
Michaelis-Menten equation under steady-state conditions.
lues (E) were calculated according to Sih’s equation: E= [ln(1-ee
S
)-ln
The effect of pH or temperature on the activity of the BVMOs was
determined by performing the reaction at different pH values (6.0–9.0)
or temperatures (20–45 °C). In order to determine the effect of pH on
enzyme stability, each BVMO was incubated at pH 7.0, 7.5, 8.0, 8.5 or
(1+ee
S
/ee
P
)]/[ln(1+ee
S
)-ln(1+ee
S
/ee
P
)] where ee
S
and ee
P
are the
substrate and product enantiomeric excesses in kinetic resolution re-
actions. The optical rotation signs of products were assigned on the
basis of published reference biotransformations. Reaction products
were predicted by GC–MS or by comparison with reference bio-
transformations, when possible.
9
.0, at 4 °C for 2 h, and then the remaining activity was assayed in the
corresponding optimal buffer (50 mM Tris-HCl pH 8.5 or 100 mM so-
dium phosphate pH 7.5) at 25 °C. Similarly, thermal stability was stu-
died by incubating each BVMO at 25, 28, 35 or 40 °C in its optimal
buffer and measuring the activity of periodically-withdrawn samples
with 1-phenylpropan-2-one as substrate. The residual activity was de-
termined against the control assay without treatment. ThermoFAD
analysis was carried out in order to measure the melting temperature
3
. Results and discussion
3.1. Sequence analysis of predicted BVMOs
In order to investigate the presence of sequences coding for BVMOs
(
T ) of the three BVMOs as described in [37]. Samples of 20 μL con-
m
in B. diazoefficiens USDA 110, we used the protein sequences of cyclo-
hexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus
NCIMB 9871, phenylacetone monooxygenase (PAMO) from
Thermobifida fusca and cyclopentanone monooxygenase (CPMO) from
Comamonas sp. NCIMB 9872 as queries for tblastn searches against the
genome of this strain. Seven putative BVMO sequences were retrieved.
Upon sequence analysis we determined that all of them contained two
Rossmann fold motifs GxGxx[G/A] as well as the type I BVMO con-
sensus sequences [A/G]GxWxxxx[F/Y]P[G/M]xxxD and FxGxxxHxxxW
tained 0.5 or 1 mg/mL flavoenzyme in 50 mM Tris-HCl pH 8.5 or
1
00 mM sodium phosphate pH 7.5 and 150 mM NaCl. A gradient tem-
perature from 20 °C to 90 °C, increasing 0.5 °C every 10 s, was applied in
a real-time PCR system (MJ Mini Gradient Thermal Cycler and Mini-
Opticon real-time PCR system, Bio-Rad Laboratories, Hercules, CA,
USA). The fluorescence measurements were performed at an excitation
wavelength range between 470 and 500 nm and with a SYBR Green
emission filter (523−543 nm). All experiments were performed at least
three times and the T
m
was determined as the maximum of the deri-
[
P/D] (Table 1). It is worth mentioning that, within the latter finger-
vative of the sigmoid curve.
print, the P/D position is occupied by a threonine in BVMO1 and by a
serine in BVMO4, while the typically-conserved histidine is substituted
for a phenylalanine in BVMO7 (Table 1). Although some BVMOs tol-
erate other amino acids in this position [38–41], it has been proposed
that this histidine is important for catalysis due to the observation that
its replacement by alanine affected the performance of 4-hydro-
xyacetophenone monooxygenase (HAPMO) from Pseudomonas fluor-
escens ACB (His296Ala mutant) [42] and a His163Gln mutation im-
paired the enzymatic activity of CHMO from A. calcoaceticus [43].
Accordingly, an improved biocatalytic performance was observed when
the Tyr160 of the BVMO from Physcomitrella patens was mutated to His
(Tyr160His mutant) [39].
2
.9. Whole-cell biotransformations
Under growing conditions, biotransformations were carried out in
5
mL cultures of E. coli BL21(DE3) cells expressing BVMOs. Each culture
was prepared inoculating fresh LB-kanamycin medium with 2 % (v/v)
of an overnight grown pre-culture of E. coli BL21(DE3) recombinant
cells and incubated at 37 °C and 200 rpm until the bacterial density
reached OD600 = 0.4-0.6. Then, IPTG was added to a final concentra-
tion of 0.3 mM and the culture was incubated at 24 °C and 200 rpm for
3
0 min. Each ketone tested as substrate was added as an ethanol solu-
tion (0.1 or 0.5 mg/mL final concentration) and the biotransformation
In order to study the phylogenetic relationships among the
Table 1
Consensus sequences in identified type I BVMOs.
BVMO
Gene
Rossmann
fold motif
GxGxx[G/A]
Type I BVMO
Type I BVMO consensus
FxGxxxHxxxW[P/D]
Rossmann fold motif
GxGxx[G/A]
consensus
[A/G]GxWxxxx[F/Y]P[G/M]xxxD
1
2
3
4
5
6
7
bll5942
blr3857
blr2938
bll3180
blr0964
blr6984
blr6465
GAGLSG
GGGFGG
GAGFAG
GAGVCA
GAGISG
GAGFSG
GAGFAG
GGTWDLFRYPGIRSD
GGTWYWNRYPGAACD
GGTWYWNRYPGARCD
GGTWWINRYPGCGVD
GGTWSTHRYPGIRSD
GGTWYWNRYPGARCD
GGTWYWNRYPGARCD
FAGRIVHPQKWT
FGGHSFHTSRWD
FKGPVYHTGNWP
FKGTILHSALWS
FKGRIVHPQTWP
FKGKCYHTSQWP
FKGEVYFTGRWP
GSGATA
GTGATA
GTGSSG
GTGATA
GSGATA
GTGATA
GTGSSG
3

R.D. Ceccoli, et al.
Molecular Catalysis 486 (2020) 110875
Fig. 1. Phylogenetic relationships of seven BVMOs from B. diazoefficiens. A maximum-likelihood phylogenetic tree was inferred based on recombinant BVMOs. The
BVMO groups I (pink), II (orange), III (green), IV (purple), V (blue), VI (cyan), VII (yellow) and VIII (red) are indicated as previously defined [5,44]. BVMOs from B.
diazoefficiens are indicated in bold. The scale bar indicates the number of substitutions per site. The color of the node indicates the aLRT value ranging from 0
(
orange) to 1 (purple). Protein sequences with their corresponding accession numbers are listed in Table S1. (For interpretation of references to color in this figure
legend, the reader is referred to the web version of this article.).
predicted BVMOs from B. diazoefficiens USDA 110 and the re-
combinantly available type I BVMOs (Fig. 1; Table S1), a maximum-
likelihood tree was inferred. The topology of the unrooted BVMO tree
showed the clades previously defined as groups I-VII [5,44], with the
rhizobial representatives locating within four of these clades (Fig. 1).
The seven BVMOs sequences are highly divergent and the identity be-
tween sequence pairs ranges from 26 % to 52 % (Table S4). BVMO3,
BVMO6 and BVMO7 locate in group II and share 54 %, 50 % and 48 %
sequence identity with PAMO from T. fusca, respectively (Table S5).
BVMO1 and BVMO5 cluster in group V and share 44 % and 39 % se-
quence identity with the BVMO3 from Dietzia sp. D5, respectively
A. calcoaceticus and CPMO from Comamonas sp.
The length of the identified proteins ranges from 488 to 636 amino
acids, except for BVMO7 that contains 896 residues. The sequence of
BVMO4 includes an N-terminal extension of ∼120 residues typically
found in characterized HAPMOs [45–47] while BVMO5 presents 24 N-
terminal residues that are absent in BVMO sequences from highly si-
milar Bradyrhizobium species and in closely related BVMOs (group V,
Fig. 1). Furthermore, by sequence similarity search, we detected that
the retrieved full-length BVMO7 sequence includes a putative esterase
in its C-terminal domain. A similar fusion protein had been reported in
Rhodococcus rhodochrous DSM 11097 [41], and a fusion between a
BVMO and a polynucleotide synthase was found in Aspergillus fumigatus
Af293 [38].
(
Table S5). BVMO1 is 79 % identical to the recently reported BVMO
from Bradyrhizobium oligotrophicum [12] (Table S5). BVMO2 shares 50
%
sequence identity and groups together with cyclododecanone
monooxygenase (CDMO) from Rhodococcus ruber SC1 in a well-sup-
ported clade that we designated as “group VIII”. BVMO4 locates in
group IV and shares 35 % sequence identity with HAPMO from P.
fluorescens DSM50106 (Table S5). Table S5 also shows the pairwise
identity between each of studied BVMO with the classical CHMO from
3.2. Heterologous expression, purification and spectral properties of BVMOs
Taking into account sequence information, the level of protein si-
milarity and the inferred phylogenetic relationships, we cloned 5 out of
the 7 genes coding for the putative BVMOs that we named BVMO2,
4

R.D. Ceccoli, et al.
Molecular Catalysis 486 (2020) 110875
Fig. 2. Purification and spectral properties of BVMO2, BVMO4 and BVMO5 from B. diazoefficiens. A) Soluble fractions of protein extracts of E. coli BL21(DE3)
expressing the recombinant BVMOs and, the three pure His-tagged BVMOs (BVMO2, BVMO4 and BVMO5) were subjected to 12 % SDS-PAGE followed by Coomassie
blue staining. M, molecular mass marker, S, soluble protein extract, P, purified His-tagged BVMO. B) TLC analysis of flavin content of purified BVMOs. FMN and FAD
were loaded as controls, flavin extracted from purified BVMO2, BVMO4 and BVMO5 were loaded onto adjacent lanes of the silica-coated plate. C) Absorbance spectra
of purified BVMO2, BVMO4 and BVMO5 before (solid lines) and after (dashed lines) treatment with 0.2 % (w/v) SDS. (For interpretation of references to color in this
figure legend, the reader is referred to the web version of this article.).
BVMO3, BVMO4, BVMO5 and BVMO6 (Table 1). The selected genes
were amplified by PCR using genomic DNA from B. diazoefficiens as
template and cloned into the pET-TEV plasmid. In the particular case of
BVMO5, a 1509 pb DNA fragment lacking the region coding for 24 N-
terminal residues of the full-length sequence was cloned. Gene ex-
pression was induced in transformed E. coli BL21(DE3) cells to obtain
N-terminally His-tagged fusion proteins. BVMO2, BVMO4 and BVMO5
were observed in the soluble fractions as proteins of about 71.6, 74.1
and 59.2 kDa, respectively (Fig. 2A). On the other hand, the re-
combinant BVMO3 and BVMO6 were undetectable as soluble proteins
under the assay conditions; in fact, they were obtained in the insoluble
fractions with masses of approximately 63.5 and 64.6 kDa, respectively
8.0 to 9.0 [9], and the BVMO from B. oligotrophicum, which exhibited
optimum activity at pH 9.0 [12]. However, BVMOs, such as the BVMO4
from Dietzia sp. D5, CHMO from Amycolaptosis thermoflava and acetone
monooxygenase from Gordonia sp. strain TY‑5 (ACMO) showed max-
imal activity at pH 7.5 [6,51,52]. We also observed that, even at pH 7.5,
the specific activity of BVMO5 in sodium phosphate was double the
value obtained in Tris-HCl buffer (Fig. 3A). A similar effect has been
observed for BVMO4 from Dietzia sp. D5 previously [6]. The three
rhizobial enzymes showed the highest specific activity at 40 °C, under
the given assay conditions (Fig. 3B).
In order to analyze the effect of pH on enzyme stability, pre-in-
cubation assays were carried out according to the procedures described
in Experimental. As shown in Fig. 3C, BVMO2 and BVMO4 from B.
diazoefficiens retained almost full activity both in Tris-HCl and sodium
phosphate buffers after incubation at different pH values in the range
from 7.0 to 9.0. Besides, BVMO5 from B. diazoefficiens retained full
activity in sodium phosphate buffer both at pH 7.5 and 8.0, but it lost
about 20 % of activity after incubation at pH 7.0. Moreover, the in-
cubation of BVMO5 in Tris-HCl buffer at pH 7.5, 8.0, 8.5 or 9.0 was
detrimental to enzyme stability since a notorious decrease in BVMO5
activity was observed after the treatments, suggesting a drastic effect of
buffer composition on enzyme stability (Fig. 3C). Thermal stability of
the three BVMOs was evaluated at different temperatures and as a
function of the incubation period (Fig. 3D). After 30 min at 25 °C,
BVMO2, BVMO4 and BVMO5 retained 80.4 %, 79.2 % and 91.9 % of
their initial activity, respectively. We observed that the three BVMOs
were less stable at 40 °C. Indeed, after a 5-min treatment at this tem-
perature, BVMO2 retained only 36.7 % of its initial activity while the
residual activities of BVMO4 and BVMO5 were 69.1 % and 76.9 %,
respectively (Fig. 3D). Additionally, the ThermoFAD assay was used to
(
Fig. S1). Thus, we continued working with the three BVMOs that could
be obtained in soluble form, i.e. BVMO2, BVMO4 and BVMO5. The
three recombinant His-tagged BVMOs were purified to homogeneity by
affinity chromatography followed by dialysis of selected (yellow)
fractions and SDS-PAGE analysis. They migrated as single bands with
estimated molecular masses in good agreement with their calculated
masses (Fig. 2A). By TLC analysis of heat-treated enzymes, we con-
firmed that FAD is the cofactor of each of the three BVMOs (Fig. 2B).
UV–vis spectra of the pure enzymes revealed two maxima corre-
sponding to enzyme-bound FAD (BVMO2: 370 and 442 nm, BVMO4:
3
83 and 443 nm; BVMO5: 382 and 445 nm) that correlate with the
typical maxima values for flavoenzymes (Fig. 2C, solid lines). Upon
unfolding of the enzymes by SDS-treatment, free FAD showed maxima
at 370 and 448 nm (Fig. 2C, dashed lines). From the difference in ab-
sorbance between free FAD and protein-bound FAD, we calculated a
molar
absorption
coefficient
at
442 nm
(ε442
)
of
−
1
−1
−1
1
3.89 ± 0.15 mM cm
for BVMO2, ε443 = 13.80 ± 0.43 mM
−1 −1
−1
cm
for BVMO4 and ε445= 13.94 ± 0.10 mM cm
for BVMO5.
determine the apparent T
fluorescence upon protein unfolding. The measured T
BVMO2, BVMO4 and BVMO5 were 33.8 ± 0.7 °C, 35.8 ± 0.6 °C and
m
of these flavoenzymes by measuring FAD
m
values for
3.3. Enzyme stability and activity
3
5.5 ± 0.5 °C, respectively. These values are within the normal range
We determined the specific activity of BVMO2, BVMO4 and BVMO5
for enzymes from a mesophile such as B. diazoefficiens. Similar protein
unfolding temperatures have been reported for CHMO from A. calcoa-
from B. diazoefficiens USDA 110 in the pH range from 6.0 to 9.0 and at
temperatures ranging between 20 and 45 °C, with 1-phenylpropan-2-
one (phenylacetone) as substrate (Fig. 3A and B). We found out that the
most favorable buffer for the activity of BVMO2 and BVMO4 was Tris-
HCl pH 8.5 while BVMO5 was optimally active in sodium phosphate
buffer at pH 7.5 (Fig. 3A). Several BVMOs display optimum pH values
for activity between 8.0 and 9.5, such as the CHMO from A. calcoace-
ticus NCIMB 9871 with a pH optimum at 8.6 [48], cyclopentadecanone
monooxygenase (CPDMO) from Pseudomonas sp. HI-70, which showed
optimum enzyme activity at pH 9.0 [49], MekA from Pseudomonas
veronii MEK700 with optimum activity between pH 9.0 and 10.0 [50],
the polycyclic ketone monooxygenase from Thermothelomyces thermo-
phila (PockeMO), which showed maximal activity in the pH range from
ceticus NCIMB 9871 (T
sp. HI-31 (T
5 °C as the reaction temperature for further assays.
m
= 37 °C) [53] and for CHMO from Rhodococcus
m
= 36 °C) [54]. Taken these results together, we confirmed
2
The steady-state kinetic parameters of the recombinant BVMO2,
BVMO4 and BVMO5 from B. diazoefficiens were determined for either 1-
phenylpropan-2-one (phenylacetone) or heptan-3-one (3-heptanone),
under the conditions described in Experimental, by following the con-
sumption of NADPH at 340 nm (Table 2, Fig. S2). BVMO2 from B.
diazoefficiens oxidized 1-phenylpropan-2-one and heptan-3-one at rea-
sonable rates under the given assay conditions and showed relatively
high
K
m
values for each substrate (1.579 mM and 1.242 mM,
5

R.D. Ceccoli, et al.
Molecular Catalysis 486 (2020) 110875
Fig. 3. Enzyme activity of B. diazoefficiens BVMOs with 1-phenylpropan-2-one as substrate. A) Influence of pH on the activity of BVMO2 (red), BVMO4 (purple) and
BVMO5 (blue). Activity measurements were performed in 100 mM sodium phosphate (triangles) or 50 mM Tris-HCl (circles) buffer. B) Influence of temperature on
the activity of BVMO2 (red), BVMO4 (purple) and BVMO5 (blue). Activity measurements were performed in 100 mM sodium phosphate pH 7.5 (triangles) or 50 mM
Tris-HCl pH 8.5 (circles) buffer, as required by each enzyme. C) pH stability of BVMO2 (red), BVMO4 (purple) and BVMO5 (blue). Enzymes were incubated at
different pHs in sodium phosphate (grey bars) or Tris-HCl (colored bars) buffers. For each enzyme, the activity without treatment was defined as 100 %. Error bars
represent the standard deviation of three measurements. D) Thermal stability of BVMO2 (red), BVMO4 (purple) and BVMO5 (blue). Enzymes were incubated at
different temperatures in sodium phosphate or Tris-HCl buffer, as required by each enzyme. Residual activity after defined time intervals was determined under
standard assay conditions. For each enzyme, the activity without treatment was defined as 100 %. Error bars represent the standard deviation of three measurements.
(
For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.).
respectively) (Table 2). BVMO Sle_62070 from Streptomyces leeu-
wenhoekii C34 and BVMOAFL838 from Aspergillus flavus NRRL3357 also
Both 1-phenylpropan-2-one and heptan-3-one were very good sub-
strates for BVMO5 from B. diazoefficiens. BVMO5 showed high affinity
exhibited K
m
values for 1-phenylpropan-2-one in the milimolar range
for 1-phenylpropan-2-one (K = 0.099 mM) and for heptan-3-one
m
under the experimental conditions reported [11,55]. Thus, the catalytic
efficiency of BVMO2, estimated by the ratio kcat/K , was similar for
both substrates. BVMO4 from B. diazoefficiens performed very well on
-phenylpropan-2-one under the established assay conditions, since we
determined a K in the micromolar range (0.081 mM) and a reasonable
cat value, which explain the good kcat/K value obtained (Table 2).
However, heptan-3-one was a poor substrate for BVMO4, as evidenced
by a K in the milimolar range and a meager kcat/K value (Table 2).
(K
m
= 0.022 mM) as well as relatively high activities toward both
m
substrates that led to significantly high kcat/K
m
values (Table 2). The
affinity of BVMO4 and BVMO5 from B. diazoefficiens for 1-phenyl-
propan-2-one seemed to be in the same range as the affinity previously
informed for the BVMO from Cyanidioschyzon merolae and for PAMO
from T. fusca, under the respective assay conditions [39,56,57]. Up to
our knowledge, this work is the first report of Michaelis-Menten para-
meters for the oxidation of heptan-3-one by BVMOs. Information about
the kinetic parameters toward other linear 3-ketones of length similar
to C7 (i.e. C6, C8) is available for few BVMOs only. Indeed, the
1
m
k
m
m
m
Therefore, the kcat/K
m
values for 1-phenylpropan-2-one and heptan-3-
one clearly favored 1-phenylpropan-2-one as substrate for BVMO4.
Table 2
Kinetic parameters of the recombinant BVMOs.
Substrate
Enzyme
K
m
(mM)
k
cat (s−1)
kcat/K
m
(mM−1s−1
)
1
-phenylpropan-2-one
BVMO2
BVMO4
BVMO5
BVMO2
BVMO4
BVMO5
BVMO2
BVMO4
BVMO5
1.579 ± 0.125
0.081 ± 0.006
0.099 ± 0.010
1.242 ± 0.106
5.38 ± 1.19
2.902 ± 0.066
5.30 ± 0.09
9.39 ± 0.18
2.206 ± 0.044
0.534 ± 0.031
9.62 ± 0.20
1.31 ± 0.05
5.27 ± 0.14
8.67 ± 0.12
1.838 ± 0.152
65.51 ± 5.31
95.07 ± 9.34
1.776 ± 0.155
0.099 ± 0.023
447.3 ± 57.03
1773 ± 1115
1491 ± 214
heptan-3-one
NADPH
0.022 ± 0.003
0.0007 ± 0.0005
0.0035 ± 0.0005
0.0056 ± 0.0003
1554 ± 95
6

R.D. Ceccoli, et al.
Molecular Catalysis 486 (2020) 110875
BVMOAFL838 from A. flavus exhibited relatively low affinity for octan-3-
one and the BVMO from Rhodococcus pyridinivorans showed low affinity
for either hexan-3-one or octan-3-one [55,58]. In order to further in-
vestigate our new enzymes, we evaluated their cofactor dependent-
activity. No activity was detected when NADH instead of NADPH was
tested as hydride donor, in line with the typical requirements of type I
BVMOs [1,3,15]. The affinity of the three BVMOs for NADPH was very
previous reports, the biotransformation of ketone 7 by whole-cells ex-
pressing BVMO2, BVMO4 or BVMO5 from B. diazoefficiens resulted in
the production of heptyl acetate exclusively (Table 3). Regarding the
conversion of nonan-4-one (8), each of these three BVMOs showed a
different behavior. BVMO2 produced propyl hexanoate mainly (80 %),
BVMO4 generated pentyl butyrate preferentially (65 %) and BVMO5
showed no clear regioselectivity for the oxygenation of this substrate
(Table 3). Our group has previously described nonan-4-one as a sub-
strate for the BVMO from L. biflexa in whole-cell biotransformations,
resulting in the selective production of pentyl butyrate [8]. Hence,
BVMO2 and BVMO from L. biflexa expand our toolbox toward the re-
gioisomeric esters produced by oxidation of nonan-4-one. Table 3
shows that most of the short linear- and branched-chain ketones with a
carbonyl functionality at C2 (1, 2, 4, 6 and 7) were regioselectively
converted to the corresponding alkyl acetates, except for pentan-2-one
(2). Pentan-2-one was oxidized by BVMO5 to propyl acetate mainly, but
produced measurable amounts of methyl butyrate (rr 96:4) (Table 3).
Although the ability of several BVMOs to oxidize acetophenone (9) and
1-phenylpropan-2-one (10) has been studied before, the Baeyer-Villiger
oxidation of 1-(p-tolyl)propan-2-one (11) was first reported in 2017 by
our group [8]. Acetophenone (9) was no substrate for whole-cells ex-
pressing the BVMO2, BVMO4 or BVMO5 from B. diazoefficiens; how-
ever, 1-phenylpropan-2-one (10) and 1-(p-tolyl)propan-2-one (11) were
converted to benzyl acetate and 4-methylbenzyl acetate, respectively,
by the three BMVOs in an outstanding way (Table 3). The results on
these three ketones were similar to the ones we had reported for the
BVMO from L. biflexa [8], with the insertion of the oxygen atom oc-
curring to the more substituted carbon in ketones 10 and 11.
high, as K values in the micromolar range were determined (Table 2).
m
These estimated affinities for NADPH are comparable with those in-
formed for other BVMOs [6,59,60]. However, it is worth noting the
particularly low K
m
of BVMO2 for NADPH, which indicates that the
affinity of BVMO2 toward NADPH is remarkably high during catalysis.
3.4. Whole-cell biotransformations
In order to assess the substrate profile of BVMO2, BVMO4 and
BVMO5 from B. diazoefficiens USDA 110, biotransformations of several
ketones with variable structure were performed in whole-cell systems
under growing conditions to provide NADPH in vivo. When possible,
we compared the performance of these three biocatalysts among them,
with closely related BVMOs or with other reported BVMOs. The ketones
chosen as candidate substrates allowed us to explore spatial and che-
mical constraints for activity, which may have an influence on substrate
preference and selectivity of the biocatalysts. In addition, most of the
products obtained are aroma compounds, solvents or flavors of
common use. The enzymatic alternative we proposed represents a new
perspective and a great advantage over the chemical procedures used to
obtain these esters.
Short linear- and branched-chain ketones (1-4) were oxidized with
good to excellent conversion and regioselectivity toward the ester
product formed by the insertion of the oxygen atom on the side of the
longer or branched-alkyl chain (Table 3). E. coli cells expressing BVMO2
or BVMO5 oxidized these ketones moderately, but cells expressing
BVMO4 showed the best performance on this set of short-chain ketones,
a behavior similar to the one we observed for the BVMO from L. biflexa
in whole-cell systems [8]. Biotransformations of linear- and branched-
chain ketones containing 7–9 carbon atoms (5-8) by cells expressing
BVMO2 or BVMO5 proceeded to almost full conversion in 24 h,
whereas BVMO4, as a whole-cell biocatalyst, achieved moderate con-
version of ketones 5, 6 and 8, even at low substrate concentration. It is
worth noting that in the presence of heptan-3-one (5), BVMO2 and
BVMO5 produced butyl propionate preferentially, while BVMO4 gen-
erated 62 % of ethyl pentanoate (Table 3). To the best of our knowl-
edge, only our group has tested heptan-3-one (5) as a BVMO substrate
in whole-cell systems before [8]. In that work, we demonstrated that E.
coli cells expressing the BVMO from L biflexa are able to catalyze the
regioselective oxidation of heptan-3-one to butyl propionate. Therefore,
we focused upon this reaction further by comparing the results shown
in Table 3 with the kinetic parameters obtained with the pure enzymes
Cyclopentanone (12), cyclohexanone (13), cycloheptanone (14)
and cyclododecanone (15) were evaluated as substrates in order to
examine the ring size effect of cyclic ketones on the biocatalytic per-
formance of these BVMOs from B. diazoefficiens USDA 110 (Table 4).
Ketones 12, 13 and 15 were poorly converted by BVMO2, cyclo-
pentanone (12) was hardly accepted by BVMO4 and BVMO5, and cy-
cloheptanone (14) was no substrate for these biocatalysts. The 4-me-
thylcyclohexanone (16) was only moderately converted (43 %) by
BVMO2 with an enantiomeric excess (ee) of 81 % in favor of the
(-)-enantiomer of the lactone (Table 4). We concluded that the mono-
cyclic ketones shown in Table 4 are poor or no substrates for either
BVMO4 or BVMO5, while BVMO2 has the less restrictive acceptance
profile on this kind of ketones.
In this work, we showed that BVMO2, BVMO4 and BVMO5 from B.
diazoefficiens USDA 110 belong to different phylogentic clades. BVMO2
is able to accept linear and cyclic ketones, including cyclododecanone.
This substrate preference is in agreement with its phylogenetic location
(Fig. 1) since BVMO2 from B. diazoefficiens clusters together with
BVMOs that are active on bulky substrates and large cyclic ketones,
such as CPDMO from Pseudomonas sp. HI-70 [49], CDMO from R. ruber
SC1 [62], BVMO4 from Dietzia sp. D5 [6] and PockeMO from T. ther-
mophila [9]. Our phylogenetic analysis presented in Fig. 1 also revealed
that the BVMO4 from B. diazoefficiens is located in group IV, a cluster
shared with two BVMOs from Rhodococcus jostii RHA1 that showed
activity on some aliphatic ketones [40,44], with HAPMO from P. putida
and P. fluorescens, which are active on aromatic compounds and some
aliphatic ketones [45,63] and with a BVMO, named IfnQ, which is
proposed to catalyze a crucial step in the biosynthesis of the iso-
furanonaphthoquinones JBIR-76 and JBIR-77 in Streptomyces sp. RI-77
[64]. Consistently, the biocatalytic performance of BVMO4 conformed
to this profile. Beside, our phylogenetic study assigned BVMO5 from B.
diazoefficiens to group V, which is composed by enzymes particularly
active on linear ketones (Fig. 1). Indeed, this a feature shared by
BVMO5 according to the data presented in Table 3. The BVMO from P.
putida KT2440 [61], the BVMO3 from Dietzia sp. D5 [7] and the re-
cently characterized BVMO from B. oligotrophicum [12] are some re-
presentatives of this group. Therefore, the substrate scope of each of the
three uncovered BVMOs from B. diazoefficiens USDA 110 (Tables 3 and
(
Table 2). The heptan-3-one (5) is a very good substrate for the BVMO5
from B. diazoefficiens according to both the biotransformation results
(
(
Table 3) and the kinetic parameters determined with the pure enzyme
Table 2). On the other hand, the high K and low kcat values measured
m
for the oxidation of heptan-3-one (5) by BVMO4, and thus the meager
k
cat/K
m
obtained, together with the biotransfomation results demon-
strated that heptan-3-one is a poor substrate for this enzyme. Despite
this latter result, biooxidation of heptan-3-one (5) gave us access to
both regioisomeric esters (butyl propionate and ethyl pentanoate) for
the first time in BVMO-mediated reactions (Table 3). The bio-
transformations of nonan-2-one (7) or nonan-4-one (8) were initially
carried out under standard conditions. However, as we observed cel-
lular lysis or growth inhibition, ketones 7 and 8 were consequently used
at a final concentration of 0.1 mg/mL (Table 3). The nonan-2-one (7)
has also been described as a substrate for whole-cells expressing the
BVMO3 from Dietzia sp. D5 [7], the BVMO from Pseudomonas putida
KT2440 [61] and the BVMO from L. biflexa [8]. In line with these
7

R.D. Ceccoli, et al.
Molecular Catalysis 486 (2020) 110875
Table 3
In vivo biotransformations of acyclic ketones.a
rr: regioisomeric ratio of esters (P1:P2 %); nd: not detected; nc: no conversion.
a
b
Results are the means of three independent experiments.
As a whole-cell system in growing conditions.
c
d
Relative conversion of ketone to the corresponding ester determined by GC.
0
.1 mg/mL of starting material.
4
) and the phylogenetic relationships established with other char-
reaction to assay regio- and enantioselectivity of BVMOs-catalyzed re-
actions (Fig. 4A). E. coli cells expressing BVMO2, BVMO4 or BVMO5
from B. diazoefficiens USDA 110 oxidized ( ± )-cis-bicyclo[3.2.0]hept-2-
en-6-one with significantly different regio- and enantioselectivities, but
the activity of either BVMO3 or BVMO6 was negligible or undetectable.
After 24 h of biotransformation, BVMO2 achieved > 99 % conversion
acterized type I BVMOs (Fig. 1) are in accordance with the proposed
link between BVMO sequence and substrate profile [65] and with
previous classifications [5,44].
The ( ± )-cis-bicyclo[3.2.0]hept-2-en-6-one is a well-known, non-
natural substrate of many BVMOs and it is accepted as a benchmark
Table 4
Biooxidation of cyclic ketones.a
N°
Substrate
Product
BVMO2b
BVMO4b
BVMO5b
Conversion (%)c
1
1
1
1
1
2
3
4
5
6
cyclopentanone
tetrahydro-2H-pyran-2-one
oxepan-2-one
32
35
nc
16
8
9
cyclohexanone
nc
nc
nc
nc
nc
nc
nc
nc
cycloheptanone
–
cyclododecanone
4-methylcyclohexanone
oxacyclotridecan-2-one
(S)-5-methyloxepan-2-one
d
43, 81(-)
nc: no conversion (< 5%).
a
b
c
d
Results are the means of three independent experiments.
As a whole-cell system in growing conditions.
Relative conversion of ketone to the corresponding ester determined by GC.
ee: enantiomeric excess of lactones. The sign of specific rotation is indicated in brackets.
8

R.D. Ceccoli, et al.
Molecular Catalysis 486 (2020) 110875
Table 5
a
Biooxidation of ( ± )-cis-bicyclo[3.2.0]hept-2-en-6-one in 24 h.
b
c
d
eeN, eeABN (%)e
Biocatalyst
Conversion (%)
N:ABN (%)
BVMO2
BVMO4
BVMO5
> 99
97
50:50
45:55
3:97
98(-), > 99(-)
81(+), 73(+)
48(-), 38(-)
72
a
b
c
Results are the means of three independent experiments.
As a whole-cell system in growing conditions.
Relative conversion of ketone to the corresponding lactone determined by
N:ABN: ratio of normal:abnormal lactones.
GC.
d
e
N
ee : enantiomeric excess of normal lactone, eeABN: enantiomeric excess of
abnormal lactone. The sign of specific rotation is indicated in brackets.
the (+) enantiomers preferentially with good ee (81(+), 73(+)), and
BVMO5 achieved almost complete regioselectivity toward the abnormal
lactone (3:97) with moderate ee (48(-), 38(-)) and 72 % conversion
(
Table 5), a result similar to that observed for BVMO5 from Myco-
bacterium tuberculosis [66].
Based on these results, we decided to study the kinetic resolution of
this fused ketone through time-course biotransformations in resting E.
coli cells expressing BVMO2, BVMO4 or BVMO5 from B. diazoefficiens.
The results are depicted in Fig. 4B. The outcomes of our experiments
indicated that BVMO2 catalyzed the oxidation of each enantiomer of
the ( ± )-cis-bicyclo[3.2.0]hept-2-en-6-one in fast reactions and at si-
milar rates to the normal or abnormal lactone with excellent ee,
achieving E-values > 200 for both products (Fig. 4B, Table 6). There-
fore, no kinetic resolution was observed for the BVMO2-mediated
biooxidation of this substrate. The BVMO4-based biocatalyst showed a
fast oxidation of the (+)-(1R,5S) ketone to the (+)-abnormal lactone,
and the conversion of the (-)-(1S,5R) enantiomer of the ketone to both
regioisomeric lactones in a slower reaction (Fig. 4B). At 56 % conver-
sion, approximately 80 % of the (-)-(1S,5R) ketone remained unreacted
and the abnormal lactone was produced preferentially with excellent ee
in favor of the (+)-lactone, achieving an EABN-value > 200 (Table 6).
Noteworthy, the kinetic resolution catalyzed by BVMO4 showed a
rarely reported but interesting result. To the best of our knowledge,
BVMO4 is the first prokaryotic BVMO that allows fast and simple access
to the (+)-abnormal lactone since a similar selectivity had only been
observed for the reaction catalyzed by the eukaryotic BVMOAFL619 from
A. flavus NRRL3357 [5]. The BVMO5-mediated biooxidation of
(
± )-cis-bicyclo[3.2.0]hept-2-en-6-one was completely selective since
BVMO5 oxidized the (-)-(1S,5R) ketone to the abnormal lactone in a
fast reaction and the (+)-(1R,5S) ketone was oxidized to the abnomal
lactone in a slower reaction (Fig. 4B). This biocatalyst successfully
achieved the kinetic resolution of the racemic fused ketone giving the
(
-)-(1R,5S) abnormal lactone with 98 % ee (EABN-value > 200) at 51 %
conversion (Table 6). Hence, by BVMO4 and BVMO5-mediated kinetic
resolution of ( ± )-cis-bicyclo[3.2.0]hept-2-en-6-one, we gained access
to both enantiomers of the abnormal lactone in excellent optical purity,
with negligible or no production of the normal lactones.
4
. Conclusions
Fig. 4. Baeyer-Villiger oxidation of ( ± )-cis-bicyclo[3.2.0]hept-2-en-6-one
catalyzed by rhizobial BVMOs. A) Biooxidation of ketone enantiomers (K) to the
normal 2-oxabicyclo[3.3.0]oct-6-en-3-one (N) and abnormal 3-oxabicyclo
In this work, we identified seven putative BVMO sequences from B.
[
(
3.3.0]oct-6-en-2-one (ABN) lactones. B) Time course of the oxidation of
diazoefficiens USDA 110 and established the phylogenetic relationships
among them and with other known type I BVMOs. We biochemically
characterized three BVMOs that belong to different phylogenetic clades,
i.e. BVMO2, BVMO4 and BVMO5, and we assessed their substrate scope
and selectivity. BVMO2, BVMO4 and BVMO5 from B. diazoefficiens
showed different behaviors on selected ketones tested as candidate
substrates. BVMO2 seems to be the most versatile of the three enzymes
as whole-cell biocatalyst, accepting substrates with variable structure.
BVMO4 and BVMO5 showed narrow substrate profiles, but they are
remarkably regioselective in the monooxygenation of ( ± )-cis-bicyclo
±
)-cis-bicyclo[3.2.0]hept-2-en-6-one by E. coli expressing BVMO2 (red),
BVMO4 (purple) or BVMO5 (blue) from B. diazoefficiens. Error bars represent
the standard deviation of three independent measurements. (For interpretation
of references to color in this figure legend, the reader is referred to the web
version of this article.).
and produced comparable levels of normal (N) and abnormal (ABN)
lactones with excellent ee (98(-), > 99(-)), BVMO4 also gave similar
amounts of each regioisomeric lactone, but, unlike BVMO2, it produced
9

R.D. Ceccoli, et al.
Table 6
Molecular Catalysis 486 (2020) 110875
Kinetic resolution of ( ± )-cis-bicyclo[3.2.0]hept-2-en-6-one.
Biocatalysta
Conversion (%)b
N:ABN (%)c
eeN, eeABN (%)d
E
, EABNe
N
BVMO2
BVMO4
BVMO5
67
56
51
58:42
> 99(-), > 99(-)
55(+), 97(+)
na, 98(-)
> 200, > 200
16, > 200
10:90
nd: > 99
na, > 200
nd: not detected; na not applicable.
a
b
c
d
As a whole-cell system in resting conditions.
Relative conversion of ketone to the corresponding lactone determined by chiral phase GC.
N:ABN: ratio of normal:abnormal lactones.
N
ee : enantiomeric excess of normal lactone, eeABN: enantiomeric excess of abnormal lactone. The sign of specific rotation is indicated in
brackets.
e
Selectivity values (E) were calculated according to Sih’s equation as described in Experimental.
[
3.2.0]hept-2-en-6-one allowing access to both antipodal abnormal
References
lactones in excellent optical purity. These enzymes expand our toolbox
toward regioisomeric esters produced by oxidation of certain linear
ketones. BVMO4 and BVMO5 gave us access to both regioisomeric es-
ters (butyl propionate and ethyl pentanoate) in BVMO-mediated oxi-
dations of heptan-3-one. BVMO2-mediated oxidation of nona-4-one
produced propyl hexanoate mainly, an outcome that complements the
selectivity of BVMO from L. biflexa [8]. Thus, the three characterized
BVMOs from B. diazoefficiens enrich the available collection of BVMOs
and provide eco-friendly alternatives for synthetic purposes.
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