Alkyl Formate Ester Synthesis by a Fungal Baeyer–Villiger Monooxygenase

Article abstract of DOI:10.1002/cbic.201600684

We investigated Baeyer–Villiger monooxygenase (BVMO)-mediated synthesis of alkyl formate esters, which are important flavor and fragrance products. A recombinant fungal BVMO from Aspergillus flavus was found to transform a selection of aliphatic aldehydes into alkyl formates with high regioselectivity. Near complete conversion of 10 mm octanal was achieved within 8 h with a regiomeric excess of ～80 %. Substrate concentration was found to affect specific activity and regioselectivity of the BVMO, as well as the rate of product autohydrolysis to the primary alcohol. More than 80 % conversion of 50 mm octanal was reached after 72 h (TTN nearly 20 000). Biotransformation on a 200 mL scale under unoptimized conditions gave a space-time yield (STY) of 4.2 g L^?1 d^?1 (3.4 g L^?1 d^?1 extracted product).

Full text of DOI:10.1002/cbic.201600684

Accepted Article
Title: Alkyl formate ester synthesis by a fungal Baeyer-Villiger
monooxygenase
Authors: Felix Martin Ferroni, Carmien Tolmie, Martha Sophia Smit,
and Diederik Johannes Opperman
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10.1002/cbic.201600684
ChemBioChem
COMMUNICATION
Alkyl formate ester synthesis by a fungal Baeyer-Villiger
monooxygenase
Felix M. Ferroni[a], Carmien Tolmie[a], Martha S. Smit[a] and Diederik J. Opperman*[a]
Abstract: Baeyer-Villiger monooxygenase (BVMO) mediated
synthesis of alkyl formate esters, important flavour and fragrance
products, was investigated. A recombinant fungal BVMO from
Aspergillus flavus was found to transform a selection of aliphatic
aldehydes to alkyl formates with high regioselectivity. Near complete
conversion of 10 mM octanal was achieved within 8 h with a
regiomeric excess of ~80%. Substrate concentration was found to
affect specific activity and regioselectivity of the BVMO as well as the
rate of product autohydrolysis to the primary alcohol. More than 80%
conversion of 50 mM octanal was reached after 72 h, achieving a TTN
hydrogen migration to form either the acid or the ester products
respectively. Phenylacetaldehyde is converted with low selectivity
for the acid product by CHMO and BVMO4 from Dietzia sp.
D5[14,15], whereas BVMO4 converted 3-phenylpropionaldehyde
exclusively to the acid product[15]. Substitutions on either the
benzene ring or alkyl chain do however affect the
regioselectivity[16,18,19]. It has recently been demonstrated through
mutational studies on CHMO that regioselectivity is mediated via
substrate positioning in the active site due to steric constraints[20]
.
However, to our knowledge, all BVMOs tested to date with
aliphatic aldehydes yielded the corresponding carboxylic
acids[14,15]. We therefore decided to test our library of recombinant
fungal BVMOs from Aspergillus flavus expressed in E. coli (Figure
S2, Supporting Information) against aliphatic aldehydes using
octanal as test substrate (Figure S3, Supporting Information).
Whole-cell (WC) and cell-free extract (CFE) biotransformations of
aldehydes are complicated due to the high degree of background
activity with endogenous E. coli host enzymes. Aldehydes such
of nearly 20 000.
A 200 mL-scale biotransformation under
unoptimized conditions gave a space-time yield (STY) of 4.2 g.L-1.d-1
(3.4 g.L-1.d-1 extracted product).
Alkyl formate esters are considered valuable compounds for the
flavour and fragrance industry with alkyl formates such as heptyl
formate and octyl formate possessing fruity flavours[1]. More
recently they have also attracted attention for their use as the
organic phase in two-liquid phase systems in oxidoreductase
biocatalysis[2,3] as they can dually serve as substrates for co-factor
regeneration by formate dehydrogenase[4]. Alkyl formates are
typically chemically synthesized through the esterification of
formic acid and primary alcohols or through reacting alkyl halides
with formamide[1]. Lipases have also been used to perform similar
transesterification[5] - specifically of ethyl formate with 1-octanol to
produce the corresponding formic acid ester[3]. Due to the
equilibrium constant of the transesterification reaction, yields of
less than 50% octyl formate were obtained. The yield could be
improved to 90% (relative to the alkanol), although only through
the use of excess ethyl formate.
as
octanal
are
rapidly
oxidized
by
aldehyde
dehydrogenases/oxidases to their corresponding fatty acids.
Nevertheless, CFE screening of the recombinant fungal BVMOs
revealed five BVMOs able to produce heptyl formate from octanal.
In aqueous medium, the alkyl formates undergo spontaneous
decarbonylation to an alcohol through hydrolysis by a water
molecule that adds to the carbonyl group of the ester, with small
amounts of heptan-1-ol therefore detected. Of the four BVMOs,
BVMOAFL838 gave the highest concentration of heptan-1-ol.
BVMOAFL838 has previously been shown to catalyze the
conversion of various 2-alkanones with absolute regioselectivity
to the corresponding alkyl acetate products[21,22]
.
To investigate the substrate specificity and selectivity of
BVMOAFL838 towards aldehydes, the enzyme was purified to
homogeneity and biotransformations performed using glucose
dehydrogenase (BmGDH) for co-factor regeneration. Gratifyingly
all the aldehydes tested (Scheme 1), yielded the corresponding
formate esters (Table 1).
Baeyer-Villiger monooxygenases (BVMOs) are flavoproteins
that convert ketones and cyclic ketones to esters and lactones
respectively using molecular oxygen and NAD(P)H under mild
conditions[6–9]. In addition, BVMOs have been shown to catalyze
heteroatom oxidation[10–12]
oxidation of aldehydes[14–16]
,
epoxidation of alkenes[13] and
Conversion of the aromatic
.
aldehydes benzaldehyde and 4-hydroxybenzaldehyde results in
carbon migration to form the corresponding ester with
autohydrolysis to the phenol product[17]. The distance between the
aldehyde group and the benzene ring also affects carbon vs
[a]
Dr. F.M. Ferroni, Miss C. Tolmie, Prof. M.S. Smit, Dr. D.J.
Opperman
Department of Biotechnology
University of the Free State
205 Nelson Mandela Drive, Bloemfontein, 9300 (South Africa)
E-mail: opperdj@ufs.ac.za
Supporting information for this article is given via a link at the end of
the document.
Scheme 1. Biotransformations of aliphatic aldehydes (a) to alkyl formate esters
(b) and their spontaneous hydrolysis to alcohols (c). Detailed reaction scheme
given in Supporting Information (Scheme S1).
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10.1002/cbic.201600684
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COMMUNICATION
Table 1. Conversion of aliphatic aldehydes (a, 10 mM) to alkyl formate esters
(b) and their spontaneous hydrolysis to alcohols (c) using purified BVMOAFL838
(2 µM) at 20°C and BmGDH for co-factor regeneration.
Substrate
Conversions (%)
2h
8h
RE[a]
95%
93%
82%
66%
99%
99%
1a
2a
3a
4a
5a
6a
>99 (11% 1c)
98 (11% 2c)
84 (5% 3c)
69 (1% 4d)
98 (1% 5c)
64 (12% 6c)
>99 (37% 1c)
99 (37% 2c)
97 (15% 3c)
93 (2% 4c)
>99 (4% 5c)
76 (42% 6c)
Figure 1. Conversion (sum of total oxygenated products) of octanal. Reaction
conditions: 1 mL reaction volumes, containing 2 µM BVMO, 1 U BmGDH, 100
mM glucose, 0.3 mM NADP+, 10-100 mM octanal, 1-2% (v/v) methanol as
cosolvent, 100 mM Tris-HCl (pH 8), 20°C, 200 rpm.
[a] Regiomeric excess (RE) in organic phase = (areas of ester product + alcohol
from autohydrolysis – acid product)/(total areas of oxygenated products)
Complete or near complete conversions of valeraldehyde (1a)
and hexanal (2a) and more than 80% conversion of octanal (3a)
were obtained within 2 h. These products were slowly hydrolyzed
to primary alcohols with the shorter chain length alkyl formates
giving more of the hydrolyzed alcohol products. This could be
attributed to the higher solubility of the shorter chain alkyl
formates in the aqueous phase or simply due to the slower
reaction rates of the longer chain aldehydes. Extractions were
performed without acidification, yielding higher effective
regiomeric excesses due to the lower solubility of the fatty acid
anions at high pH in organic solvents (Table 1). The effective
regioselectivity decreased with increased chain length with
decanoic acid constituting approximately 18% of the total
oxygenated products from decanal (4a). Branched chain
aldehydes, citronellal (5a) and 7-hydroxycitronellal (6a), were
converted exclusively to the corresponding formate esters. Here
the autohydrolysis rate of 6a was significantly higher than that of
5a, although the specific activity was lower, supporting the
argument that hydrolysis rate is correlated to water solubility.
The regioselectivity of BVMOAFL838 was also negatively affected
by the increased substrate concentration, with octanoic acid
constituting approximately 20% and 30% of the total extracted
oxygenated products from 50 mM and 100 mM octanal
respectively. Interestingly, the regioselectivity increased during
the time-course of the reaction, as the effective concentration of
substrate decreased (Figure S7, Supporting Information). Bisagni
and coworkers similarly found that both substrate concentration
and enzyme concentration can influence the regioselectivity of
BVMO mediated oxygenation of 2-phenylpropionaldehyde[15]
.
Surprisingly, the rate of authohydrolysis of the heptyl formate
decreased with increasing substrate concentration: whereas
nearly 90% of the heptyl formate was converted to heptan-1-ol
after 72 h during the biotransformation of 10 mM octanal, only
approximately 4% and less than 1% of the heptyl formate in the
50 mM and 100 mM reactions were hydrolyzed to heptan-1-ol
(Figure 2, S5-7, Supporting Information).
Steady-state kinetics with octanal revealed maximum reaction
rates less than that observed for 2-octanone and 3-octanone, but
an affinity (Km) between that of 2-octanone and 3-octanone[21]
.
Substrate inhibition was also less pronounced for octanal than 2-
octanone (Figure S4, Supporting Information). To investigate the
usefulness of BVMOAFL838, we performed time-course reactions
with increasing substrate concentrations. Overall, the reaction
rates decreased significantly after 8 h but activity was still
observed after 24 h. Turnover frequencies (TOFs) decreased with
increasing substrate concentration, but approximately 80%
conversion was still achieved from 50 mM octanal after 72 h
(Figure 1), with extracted yields of 20 mM (2.8 g.L-1) heptyl
formate [calculated to represent 24 mM or 3.5 g.L-1 in
biotransformation reaction mixture (BRM)] and a total turnover
number (TTN) exceeding 12 000 w.r.t. total oxygenated products
extracted and nearly 20 000 w.r.t. products in BRM.
Figure 2. Time-resolved distribution of octanal and oxygenated products
extracted during biotransformation of 50 mM octanal.
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COMMUNICATION
Encouraged by these promising results, we performed a 200 mL
scale synthesis of heptyl formate under these unoptimized
conditions. Biocatalyst concentration was increased to 5 µM with
50 mM octanal added neat. Protein precipitation was already
noticeable within the first few hours of the biotransformation.
Nevertheless, the heptyl formate product constituted
approximately 60% of the extracted reaction mixture after 24 h
(Figure S8, Supporting Information) yielding 0.42 g heptyl formate
in BRM with space-time yield (STY) of 4.2 g.L-1.d-1 (3.4 g.L-1.d-1
extracted product).
Acknowledgements
We thank the National Research Foundation (NRF), South Africa
for financial support and Mr. S. Marais for GC analysis.
Keywords: biocatalysis • Baeyer-Villiger monooxygenase •
formate ester • aldehydes • alkyl formate
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In summary, we have for the first time demonstrated BVMO-
mediated synthesis of alkyl formate esters from aldehydes.
Activity, selectivity as well as the autohydrolysis of the product is
dependent on substrate concentration. We are currently
investigating two-phase liquid systems, as we anticipate this will
decrease the effective concentration of the substrate to the
enzyme for improved selectivity, as well as serve as an organic
reservoir for the product to circumvent product hydrolysis.
Alternatively, improved product hydrolysis can serve as a route to
natural uneven chain length primary alcohols, similar to studies
combining BVMOs with esterases to yield primary alcohols from
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Experimental Section
All substrates were obtained from Sigma-Aldrich and used without any
further purification.
BVMOs were expressed from the pET22b(+) vectors (Novagen) in E. coli
BL21-Gold(DE3) (Strategene) and purified as previously described[22]
.
Glucose dehydrogenase from Bacillus megaterium (pET28:BmGDH) was
similarly expressed and purified to homogeneity using Ni-affinity
chromatography and desalted using a PD10 column (GE Healthcare).
Units for BmGDH is defined as the amount of enzyme that converts 1 µmol
of NADP+ in 1 minute at 20°C (100 mM Tris-HCl pH 8, 100 mM glucose).
Biotransformations were performed in 40 mL capped amber glass vials in
total reaction volumes of 1 mL. Biotransformation mixtures consisted of 2
µM (0.13 mg/ml) purified BVMO, 1 U BmGDH, 100 mM glucose, 0.3 mM
NADP+, 10-100 mM octanal, 1-2% (v/v) methanol as cosolvent and 100
mM Tris-HCl (pH 8). Reactions were performed at 20°C where after the
content was extracted using equal volumes of ethyl acetate containing 2
mM 1-undecanol as internal standard. The 200 mL scale synthesis was
performed in a capped 500 mL Schott bottle containing 5 µM (0.32 mg/ml)
purified BVMO, 1 U.mL-1 BmGDH, 100 mM glucose, 0.3 mM NADP+, 50
mM octanal and 100 mM Tris-HCl (pH 8). Reactions were performed for
24 h and extracted twice with 50 mL ethyl acetate containing 2 mM 1-
undecanol as internal stand. The organic phases were combined and the
extracted products were quantified against authentic standards. GC-FID
and GC-MS were carried out on a Finnigan Trace GC ultra (Thermo Fisher
Scientific) on a FactorFour VF-5ms column (60 m x 0.32 mm x 0.25 µm,
Varian).
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ChemBioChem
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Alkyl formate synthesis is performed
using a Baeyer-Villiger
Felix M. Ferroni, Carmien Tolmie,
Martha S. Smit, Diederik J. Opperman*
monooxygenase. Regioselectivity
and rate of autohydrolysis of the
alkyl formates to the corresponding
primary alcohol product is affected
by substrate concentration.
1 – 3
Alkyl formate ester synthesis by a
fungal Baeyer-Villiger
monooxygenase
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