A biocatalytic cascade for the conversion of fatty acids to fatty amines

Article abstract of DOI:10.1039/c9gc02260k

Fatty amine synthesis from renewable sources is an energetically-demanding process involving toxic metal catalysts and harsh reaction conditions as well as selectivity problems. Herein we present a mild, biocatalytic alternative to the conventional amination of fatty acids through a one-pot tandem cascade performed by a carboxylic acid reductase (CAR) and a transaminase (ω-TA). Saturated and unsaturated fatty acids, with carbon chain lengths ranging from C₆ to C₁₈, were successfully aminated obtaining conversions of up to 96%.

Full text of DOI:10.1039/c9gc02260k

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DOI: 10.1039/C9GC02260K
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A biocatalytic cascade for the conversion of fatty acids to fatty
amines
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
b
a
Joan Citoler, Sasha R. Derrington, James L. Galman, Han Bevinakatti and Nicholas J. Turner *
DOI: 10.1039/x0xx00000x
Fatty amine synthesis from renewable sources is an energetically- lack of selectivity of the catalysts. Mixtures of primary,
demanding process involving toxic metal catalysts and harsh secondary and tertiary amines are commonly obtained during
reaction conditions as well as selectivity problems. Herein we the reaction adding extra complexity to downstream
1
6
present a mild, biocatalytic alternative to the conventional processing.
amination of fatty acids through a one-pot tandem cascade
performed by
transaminase (ω-TA). Saturated and unsaturated fatty acids, with bulk chemicals. The inherent mild reaction conditions together
carbon chain lengths ranging from C to C18, were successfully with attendant chemo- and regio-selectivity make enzymes
a
carboxylic acid reductase (CAR) and
a
Biocatalysis is an attractive alternative for the production of
6
aminated obtaining conversions of up to 96%.
suitable candidates for sustainable and environmentally-
friendly manufacturing processes. Amination of fatty alcohols
1
7
Amines serve as important and versatile intermediates for a
wide range of chemicals synthesised by the pharmaceutical,
agricultural and fine chemical industry. In particular fatty
has previously been achieved in vitro and in vivo by combining
18
ω-transaminases (ω-TA) with alcohol oxidases (AOx) and
1
19–21
alcohol dehydrogenases (ADH)
respectively. Enzyme-
8
amines, with carbon chain lengths ranging from C to C22, are
catalysed in situ aldehyde generation is followed by a
transamination step to yield the amine using an amino donor
such as alanine. Despite the efficiency of these cascades, low
enzyme activities towards non-natural substrates together
with poor substrate solubility in the aqueous phase means that
the synthesis of fatty amines with carbon chain lengths of C14
and above still remains a challenge.
utilised because of their surface active properties, which make
them ideal commodity chemicals for a broad range of
applications such as fabric softening, coating and corrosion
inhibition, emulsification, flotation, anti-caking and crop
2
protection. The fatty amines market is estimated to be the
fastest-growing amine category with an anticipated marked
3
value of USD 2.9 billion by 2025.
Conventional chemistry
From an industrial point of view, fatty amines derived from
"
Nitrile route"
renewable feedstocks are synthesised from fatty acids via the
O
4
–8
[ZnO]
[Ni]
so-called “nitrile route”. Fatty alcohols are also used as
substrates by reacting them with ammonia or alkyl amines,
although this process is less commercially employed and
mainly focussed on secondary and tertiary fatty amine
R
R
CN
R
n
OH
NH3
n
H2
n
NH2
250 °C
200 °C, 35 Bar
Biocatalytic approaches
9
–14
synthesis (Scheme 1).
Both processes are characterised by
Previous work
the use of heterogeneous toxic metal catalysts (metal oxides,
R
ADH/AOx
NAD /O2
R
-TA
Ala
30 °C
R
6
,7
n
OH
+
n
O
n
NH2
nickel and copper)
and harsh energetically-demanding
30 °C
5
reaction conditions (high pressure and temperature). In
addition, the usage of molecular hydrogen in nitrile and imine
reduction steps requires additional safety measures to be
implemented.15 Another drawback of these processes is the
This work
O
R
CAR
NADPH/ATP

-TA
IPA
R
R
n
OH
NH2
n
O
n
3
0 °C
30 °C
a. School of Chemistry, University of Manchester, Manchester Institute of
Biotechnology, 131 Princess Street, Manchester, M1 7DN, United Kingdom.
AkzoNobel Surface Chemistry, 10 Finderne Ave, Bridgewater, NJ 08807, USA
Scheme 1 Chemical and enzymatic routes towards the synthesis of fatty
amines from renewable vegetable oils and animal fats.
b.
†Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Journal Name
Carboxylic acid reductases (CARs) have increasingly been further experiments. Sp-TA is known for having a broad
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2
2–
DOI: 10.1039/C9GC02260K
40
highlighted as attractive biocatalysts for chemical synthesis.
substrate scope which includes aliphatic substrates. McCAR
2
9
25
Through a complex 3-domain structure, CARs are capable of has previously been identified, although no biochemical
successfully catalysing the reduction of a broad range of characterisation has been carried out. Generating structural
carboxylic acids, yielding the corresponding aldehydes, at the data could provide insight into the properties of the enzyme
expense of ATP and NADPH. The reported activity towards scaffold that make McCAR better performing biocatalyst than
fatty acids2
3,25,30–32
prompted us to consider CARs as suitable the rest of screened CAR enzymes.
enzymes to access fatty amines in combination with other After initial screening, subsequent reaction intensification was
enzymes. The only previous report of using CARs to access
Table 1. Biocatalytic amination of fatty acidsa
fatty amines is contained in a patent wherein fatty amine
Conversion to
amine (%)
synthesis is carried out through a engineered microbial
metabolic pathway with carbon sources as starting materials.
However, the substrate scope as reported is limited to
dodecylamine and a lack of quantitative data is provided.
Substrate
Structure
n
3
3
1
a
3
5
7
94
96
93
92
44
9
1b
1c
O
1
1
1
1
1
d
e
f
g
h
9
OH
n
11
13
15
2
4
6
In order to identify potentially suitable combinations of
2
2
enzymes we selected four CARs (from Nocardia iowensis,
1
2
3
27
Mycobactarium marinum,
Segniliparus rugosus
and
O
89
58
6
Mycolicibacterium chlorophenolicum), shown to be active
towards fatty acids, and coupled them with six in-house ω-TAs
1i
1j
OH
n
5
3
7
21
O
(
from Arthrobacter sp, Vibrio fluvialis, Pseudomonas putida
1
k
5
5
35
63
3
8
18
OH
OH
NBRC 14161, Chromobacterium violaceum, Agrobactarium
tumefaciens39 and Silicibacter pomeroyi ). For each pair we
examined the conversion of dodecanoic acid 1d to
doceylamine 3d as the model reaction. Due to the poor
substrate solubility under aqueous conditions, n-heptane
containing 5 mM carboxylic acid was overlayed on the
aqueous phase as a reservoir together with 5% (v/v) dimethyl
sulfoxide (DMSO) to enable a gradual interphase substrate
transfer. NADPH was recycled in situ by addition of glucose/
glucose dehydrogenase (GDH – Codexis CDX 901), whereas
ATP was recycled by polyphosphate-AMP phosphotransferase
n
n
40
O
1
l
a
-1
-1
Reaction conditions: 5 mM carboxylic acid, 31.25 mM IPA, 2 mg mL
McCAR lysate, 2 mg mL Sp-TA lysate, 2 mg mL PAP lysate, 2 mg mL
-1
-1
-1
Adk lysate, 0.2 mg mL CDX-901 GDH, 0.5 mM NADP+, 0.5 mM PLP, 4 mg
mL PolyP, 25 mM D-glucose, 5.5 mM MgCl , 5% (v/v) DMSO, 100 mM
2
pH 8 Tris buffer, 500 µL reaction volume, 500 µL n-heptane. 30 °C, 180
rpm, 20 h.
-1
carried out by varying several reaction parameters including
2
, glucose and IPA as well
as recycling enzymes and substrate loading (ESI Fig. S4-S9).
Conversion increased to >90% upon reduction of MgCl
concentration to near-stoichiometric amounts (1.1
equivalents) suggesting potential enzyme inhibition. In our
previous work, CAR and NADPH/ATP recycling enzymes have
been demonstrated to successfully perform carboxylic acid
the concentration of PolyP, ATP, MgCl
(PAP) and adenylate kinase (Adk) from Acinetobacter johnsonii
employing polyphosphate (PolyP) as the phosphate group
donor.41 Isopropylamine (IPA) was used in excess (50 equiv.) as
amino donor for the transaminase-catalysed reaction in the
presence of pyridoxal 5′-phosphate (PLP) as cofactor (Scheme
2
2
).
4
2
O
2
reduction with MgCl concentrations up to 100 mM,
CAR
-TA
R
R
suggesting that ω-TA could be the enzyme which is inhibited.
In an attempt to facilitate enzyme preparation and reduce
costs of purification, we focussed our attention on using cell
free extracts (CFE) instead of purified enzyme. To our surprise
there was no overall negative impact on the percentage
conversion when using CFEs.
R
NH2
OH
O
n
n
n
1a-l
2a-l
3a-l
NADPH
ATP
NADP+
AMP
IPA
Acetone
Gluconolactone
PolyP(n-1)
Glucose
PolyP(n)
PAP/Adk/GDH
CFEs
In order to explore the substrate scope, McCAR and Sp-TA
were screened against a panel of saturated and unsaturated
Scheme 2 Cascade design for the crossed-combination of CARs and ω-
TAs with dodecanoic acid as substrate.
6
fatty acids with chain lengths ranging from C to C18 (Table 1).
To our delight, most reactions resulted in the amination of 1d Some unwanted further reduction of the aldehyde
to 3d with conversions ranging from 17 to 60% (ESI Fig. S3). intermediate 2 was observed (≤6%) in some of the reactions
Conversion of the aldehyde to amine was dependent on the yielding the alcohol as a side by-product. This reaction is
particular ω-TA, whereas CAR activities were generally catalysed by endogenous alcohol dehydrogenases from E. coli
comparable. The best performing pair of enzymes was shown present in the cell lysate. Substrates with fully-saturated
to be ω-transaminase from Silicibacter pomeroyi (Sp-TA) and a carbon chains ranging from C to C12 (1a-d) were successfully
6
novel CAR from Mycolicibacterium chlorophenolicum (McCAR), converted to the corresponding amine in high conversions
and therefore this enzyme combination was selected for (>90%). However, a reduction in conversion was observed for
2
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substrates with saturated chains ranging from C14 to C18, with a to find more suitable biocatalysts which can VaiellwoAwrtichleiOgnhliener
6% and 8% conversion of myristic acid 1e and palmitic acid 1f substrate loadings and a broader substrDaOtIe: 1s0c.1o0p3e9/.CE9vGaCl0u2a2t6io0nK
4
to the amine products respectively. A negligible amount of and optimisation of the co-solvents used in the reaction could
product was obtained with stearic acid 1g. In the case of also play an important role in assuring the maximum solubility
substrates with unsaturated side-chains chains, namely of the substrate and an optimal substrate interphase transfer.
myristoleic 1h, palmitoleic 1i and oleic acid 1j, increased
conversions were observed compared to their saturated chain In summary, we have designed a one-pot multi-enzyme
analogue substrates (89% versus 44%, 58% versus 9% and 6% cascade process for the amination of saturated and
versus 1% respectively). The unsaturated nature of the unsaturated fatty acids with a carbon chain length ranging
6
aliphatic chain increases the substrate solubility under from C to C18. Efficient cofactor recycling enzymes have been
aqueous conditions suggesting that increased conversions may successfully coupled with McCAR and Sp-TA in a biphasic
not only be due to increased enzyme activity but also to system with n-heptane as a solvent overlay and conversions up
increased solubility in the reaction phase. Increasing the to 96% were achieved. Carbon chain length and chain
degree of unsaturation (linoleic 1k and linolenic acid 1l) further unsaturation played a crucial role in substrate solubility, which
improved solubility resulting in conversions of 35% and 63% is directly related to the reaction performance. To the best of
respectively. In order to confirm the reaction dependence on our knowledge, this proof-of-concept approach represents the
solubility and enzyme activity, we performed specific activity first in vitro biocatalytic cascade to access fatty amines from
assays for substrates 1d, 1e and 1h in the aqueous phase and renewable fatty acids and the first biocatalytic cascade to
concentrations under the solubility limit. McCAR showed access fatty amines with a chain length greater than 12
different activity for each of the substrates, thus carbons.
demonstrating that both the solubility and the enzyme activity
play an important role in the reaction performance (ESI Fig.
S11).
Conflicts of interest
1
00
The authors declare no conflicts of interest.
90
80
70
60
50
40
30
20
10
0
Acknowledgements
Amine
Acid
J.C. kindly acknowledge AkzoNobel for funding for a PhD
studentship. J.L.G acknowledges the support of BBSRC-FAPESP
Grant agreement no. BB/P01738X/1. N.J.T. is grateful to the
European Research Council for an Advanced Grant funding
Alcohol*
(grant no. 742987). The authors are also grateful to Dr.
Reynard Spiess for the HRMS data.
0
10
20
30
Time (h)
Notes and references
Figure 1 Reaction time-course for the amination of 1d. *Alcohol could
only be seen in late stages of the reaction, probably because its low
concentration could not be detected by GC-FID.
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