Parallel anti-sense two-step cascade for alcohol amination leading to ω-amino fatty acids and α,ω-diamines

Article abstract of DOI:10.1039/c8gc02122h

Running two two-step cascades in parallel anti-sense to transform an alcohol to an amine allowed the conversion of ω-hydroxy fatty acids (ω-HFAs) and α,ω-diols to the corresponding ω-amino fatty acids (ω-AmFAs) and α,ω-diamines, respectively. The network required only two enzymes namely an aldehyde reductase (AHR) and a transaminase (TA). Benzylamine served on the one hand as amine donor and on the other hand after deamination to benzaldehyde also as oxidant. All ω-HFAs tested were efficiently transformed to their corresponding ω-AmFAs using purified enzymes as well as a whole-cell system, separately expressing both the enzymes, with conversions ranging from 80-95%. Additionally, a single-cell co-expressing all enzymes successfully produced the ω-AmFAs as well as the α,ω-diamines with >90% yield. This system was extended by employing a lactonase, enabling the transformation of ?-caprolactone to its corresponding ω-AmFA with >80% conversion.

Full text of DOI:10.1039/c8gc02122h

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Parallel Anti-Sense Two-Step Cascade for Alcohol Amination
leading to ω-Amino Fatty Acids and α,ω-Diamines
Sihyong Sung#a, Hyunwoo Jeon#a, Sharad Saraka, Md Murshidul Ahsanb, Mahesh D. Patila,
Wolfgang Kroutilc, Byung-Gee Kimd, Hyungdon Yun*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Running two two-step cascades in parallel anti-sense to transform cascade reactions can be classified into four different designs
an alcohol to an amine allowed the conversion of ω-hydroxy fatty such as linear, orthogonal, cyclic and parallel reactions.[6b] In
acids (ω-HFAs) and α,ω-diols to the corresponding ω-amino fatty contrast to the other designs, the orthogonal reaction is
acids (ω-AmFAs) and α,ω-diamines, respectively. The network mostly refereeing to the recycling of
a
cofactor.[6b]
required only two enzymes namely an aldehyde reductase (AHR) Nevertheless, several ‘parallel inter-connected approaches’,
and a transaminase (TA). Benzylamine served on the one hand as have been reported to produce enantiomerically enriched
amine donor and on the other hand after deamination to alcohols by both parallel single-step reactions.[6c,d] While in the
benzaldehyde also as oxidant. All ω-HFAs tested were efficiently parallel reactions, the redox equivalent is provided by another
transformed to their corresponding ω-AmFAs using purified molecule not part of the cascade, in redox self-sufficient linear
enzymes as well as a whole-cell system, separately expressing cascades, the redox equivalents originate from a redox step
both the enzymes, with conversions ranging from 80-95%. within the linear sequence.[6b] For instance, various hydrogen
Additionally, a single-cell co-expressing all enzymes successfully borrowing cascades have been reported for the amination of
produced the ω-AmFAs as well as the α,ω-diamines with >90% alcohols.[6e, 7] In the present study, we developed an parallel
yield. This system was extended by employing a lactonase, anti-sense cascade reaction for the synthesis of ω-amino fatty
enabling the transformation of ε-caprolactone to its acids (ω-AmFAs) and α,ω-diamines from their corresponding
corresponding ω-AmFA with >80% conversion.
ω-Hydroxy fatty acids (ω-HFAs) and α,ω-diols, respectively.
A cascade for the amination of prim-alcohols was designed
where two two-step sequences run parallel in anti-sense
direction interconnected (Scheme 1). In the upper sequence,
from left to right an aldehyde reductase (AHR) converts ω-
HFAs to oxo-fatty acids (OFAs), which subsequently get
aminated by a ω-transaminase (ω-TA) using a benzylamine as
an amine donor. In the bottom sequence, the deaminated
amine donor (i.e. benzaldehyde) obtained in the second step
of the upper sequence is converted to its corresponding
alcohol by the AHR, thereby minimizing the inhibition of the ω-
TA by deaminated product[8,9] and effectively regenerating the
cofactor[10b] (Scheme 1)
The NAD(P)+-dependent AHR (slr1192) from Synechocystis
species[10b] exhibits a good activity towards a broad range of
aliphatic and aromatic alcohols.[10] To start with, we examined
the reactivity of AHR in presence of ω-TA from S. pomeroyi
(PMTA) by using 20 mM benzylamine as amino donor and 5
Amine derived bioplastics extend the conventional access to
petroleum-based products by minimizing harmful greenhouse
effects. These compounds have gained tremendous popularity
due to their unique functionality and efficient
biodegradability.[1,2] Owing to their numerous advantages, the
biocatalytic methods are maturing and proving to be valuable
alternatives.[3] Scientific findings have evidenced various
optimized protocols to produce lower-molecular weight
monomers (e.g. adipic acid, lactic acid). However, protocols for
medium- to long-chain bioplastic monomers (e.g. ω-amino
dodecanoic acid, α,ω-diamines.) remain a challenging task.[4,5]
For instance, till date only <5.0 mM ω-amino dodecanoic acid
(ω-AmDDA) was produced by using whole-cell reaction.[4]
A multi-enzyme cascade reaction is notably appealing since
biocatalysts are highly specific and most importantly
compatible with each other.[2b,
6a]
In general, biocatalytic
mM ω-hydroxy dodecanoic acid (ω-HDDA) as
substrate.
a model
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Scheme 1. Design of two interconnected two-step sequences, parallel anti-sense cascade, to produce ω-AmFAs.
This reaction resulted in the formation of 2.5 mM ω-AmDDA. AmDDA was obtained (Supporting Information Fig. S7).
As the analysis of OFAs is comparatively difficult,[4a] we Furthermore, the reaction was carried out with different
measured the amine products of these reactions. In order to amount of whole-cell catalysts, ranging from
3 to 27
examine the applicability of AHR in regenerating NAD(P)+ mgDCW/mL, wherein 27 mgDCW/mL of both the cells yielded 94
cofactor via ‘parallel anti-sense’ cascade network, purified AHR mM of ω-AmDDA (Supporting Information Fig. S8).
(0.05 mg/mL) was reacted with 30 mM benzaldehyde in the
Enzymatic reaction systems comprising of two or more
presence of 10% ethanol. This reaction yielded 29 mM benzyl cells, each expressing different protein, may exhibit limitation
alcohol, implying the high efficiency of AHR in regenerating of mass transfer between two whole-cell catalysts.[4,11] In order
NAD(P)+ cofactor (Supporting Information Fig. S1)
to address these issues, we developed a single-cell system
Once the AHR system was instituted, we performed the expressing both the enzymes. Among the three different
screening of ω-TAs from our ‘in-house’ library (Supporting vector combinations developed ( : pQE-80L and pET-24ma
information Table S1). Ten purified ω-TAs (0.1 mg/mL each) pET-24ma and pET-Duet1 : pET-Duet1 and pET-24ma for AHR
were reacted with 5 mM ω-HDDA and 20 mM of benzylamine and PMTA, respectively), combination best expressed both
A
B:
C
C
in the presence of AHR (0.5 mg/mL). Among the tested ω-TAs, the proteins resulting into the >99 mM conversion to ω-
four enzymes showed good reactivity with >75% conversions AmDDA (Supporting Information Fig. S9 and S10). These
(Supporting Information Fig. S2). The optimum pH for all the results indicated that endogenous NAD(P)H is sufficient for the
four ω-TAs was measured at pH 8.0. Therefore, all the further recycling of cofactor. Thus, all the further reactions were
reactions were carried out at pH 8.0 (Supporting Information performed using vector combination
C.
Fig. S3). In addition, we compared the reactivity of Having the single-cell expression system in hand, we
benzylamine with that of one of the most widely used amino performed further experiments with various ω-HFA substrates
donor (S)-α-methyl benzylamine (S-α-MBA). The results (20 mM and 100 mM each) in order of increasing carbon chain
suggested that the benzylamine also can act as a good amino length between C6-C16 (Supporting information Table S2 and
donor for all the tested ω-TAs (Supporting Information Fig. S4). Table 1) The conversions for all the tested substrates was
Following the successful biotransformations using purified excellent (>90 mM). It is worth mentioning that the highest
enzyme system, we decided to move forward to a whole-cell conversion of 96.7 mM was achieved in the case of ω-amino
system. Whole cell systems offer advantages such as simpler hexanoic acid (ω-AmHA) (1b). Slightly lowered conversions
handling leading to cost-optimized bioconversions compared (84.7 and 69%) were obtained in the cases of 6b and 7b,
to their purified enzymes counterparts.[4,11] Initially, respectively and the unreacted substrates were observed in
recombinant E. coli (BW25113, ∆fadD) cells, separately the process. This might be related with the poor solubility of
expressing AHR and ω-TAs, were added to the reaction 6a and 7a compared to 1a (Table 1).
mixture containing 20 mM ω-HDDA (Supporting Information
Nevertheless, the robustness of the established cascade
Fig. S6). Cells expressing AHR showed the best conversion (18
was further tested at an elevated scale of 200 mM using ω-
mM) in the presence of ω-TA from S. pomeroyi (PMTA ). To
HDDA as a model substrate. This reaction, performed in the
further check the applicability of PMTA at higher scale, 100
presence of 20% DMSO as a co-solvent, also achieved an
mM ω-HDDA was used, wherein 91 mM of corresponding ω-
excellent conversion (85%) with the formation of 170 mM 5b
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(Supporting Information Fig. S12). In addition, to demonstrate µM).[13b] Nevertheless, the present studies, employing the
the scope of this enzymatic reaction, we utilized a methyl ester novel parallel anti-sense cascade, successfully minimized all
derivative of ω-HDDA (7a) as model compound (Table 1, Entry these limitations and excellent conversion of ω-HFAs to their
7), which yielded 69 mM of the ω-amino dodecanoic acid corresponding ω-AmFAs was achieved. Similar to ω-AmFAs,
methyl ester (ω-AmDDME) (7b) (Supporting Information Fig. α,ω-diamines are also widely used as a valuable feedstock
15]
S13 and S14). However, in this case 6.4 mM of by-product ω- material to produce industrially important polymers.[11a,
AmDDA was formed, plausibly due to the hydrolysis of an ester Recently, the access for transformation of α, ω-diols into their
group into carboxylic acid. However, it is unclear that first corresponding α,ω-diamines by employing three enzymes
hydrolysis of 7a occurs followed by amination or vice-versa.
(ADH, ω-TA and AlaDH) with utilizing alanine as amino donor
has been reported.[16]
Table 1. Amination of ω-HFA through a novel ‘parallel anti-
sense’ cascade reaction employing ω-TA and AHR [a][b]
After successful transformation into ω-AmFAs, the broad
substrate specificities of AHR and ω-TA encouraged us to
examine the feasibility of this system to produce α,ω-diamines
from their corresponding α, ω-diols (Scheme 2) (Supporting
Information Fig. S15).
No
Substrate
n
R
Amine product
[mM]
1
2
3
4
5
6
7
1a
2a
4
6
H
H
1b
2b
3b
4b
5b
6b
7b
96.7
95.5
95.2
94.9
91.5
84.7
69.0
Table 2. Comparative biosynthesis of α, ω-diamines using α,ω-
diols[a][b]
3a
8
H
4a
9
H
No Substra
te
n
Conversion Diamine
Aminol
[mM]
[%][c]
[mM]
5a
10
14
10
H
6a
7a[c]
H
1
2
3
4
5
1c
2c
3c
4c
5c
6
99.2
1d 98.7 1e
2d 96.2 2e
0.5
1.2
CH3
8
97.4
67.9
40.7
23.8
[a] Reaction condition:100 mM substrate, 200 mM benzyl-
amine, 36 mgCDW/mL AHR-PMTA Cells, 37°C, 100 mM Tris-HCl
buffer (pH 8.0), 10% (v/v) DMSO, 24 h.
10
12
14
3d 56.8 3e 11.1
4d 38.9 4e
5d 21.4 5e
1.8
2.4
[b] The information regarding reagents and products is shown
in Table S3
[c] 6.4 mM of 12-AmDDA was produced as hydrolysis product.
[a] Reaction condition: 100 mM substrate, 400 mM
benzylamine, 36 mgCDW/mL of AHR/PMTA cell, 37°C, 200 mM
Tris-HCl buffer (pH 8.0), 10 % (v/v) DMSO, 24 h.
[b] The information regarding reagents and products is shown
in Table S3.
Extensive research has enabled the production of ω-HFAs.[12-14]
However, their conversion into corresponding aminated
derivatives is still a challenging process. For instance, a
quinone dependent alcohol dehydrogenase (AlkJ)[4] or
NAD(P)H dependent prim-ADH[13b] were employed for the
generation of oxo-fatty acids (OFAs) followed by amination
using ω-TA. Unfortunately, this approach turned out to be
unsuitable due to first, ‘over-oxidation’ of ω-HFAs by AlkJ.[14]
Moreover, the recycling of quinone cofactor is inefficient
process compared to regeneration of NAD(P)H and second, the
exclusive substrate inhibition to prim-ADH (Ki value =98
[c] Overall conversion of α,ω-diols to α ω-diamines and aminol
To check the applicability of our single cell system, expressing
both the proteins (Vector combination C, as described above),
for the synthesis of α,ω-diamines, a reaction was carried out
using various α,ω-diols as substrates (100 mM each), having
varying carbon chain length ranging from C8-C14.
Scheme 2. Enzymatic synthesis of α,ω-diamines using AHR and ω-TA via a novel ‘parallel anti-sense’ cascade reaction
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The results demonstrated that the conversions of α,ω-diols to Finally, we envisioned to combine the system with a lactonase
their corresponding α,ω-diamines decreased with the (Scheme 3). reaction was performed employing cells
A
increasing chain length of the substrates (Table 2). The expressing lactonase and a single-cell system expressing AHR
conversions of the substrates having carbon chain length <C12 and PMTA in a reaction mixture containing 100 mM of ε-
were moderate to excellent (56-98.7%). However, the caprolactone as a substrate. This reaction successfully yielded
conversions steeply dropped to ~39 and 21% in case of the 80 mM of ω-AmHA (Supporting Information Fig. S19).
diols having carbon chain length of C14 and C16, respectively.
(Table 2, Entry 4 and 5). This could be due to two possible
Conclusions
reasons. First, the poor solubility of the diols. Second, the two
times amination of oxo intermediate decreases the overall
In summary, we have successfully developed an efficient
conversion to α,ω-diamines. To address the solubility issues,
biocatalytic cascade concept involving the combination of two
the effect of co-solvents such as DMSO and dimethoxyethane
parallel anti-sense two-step sequences for the production of
(DME) was tested. The increase in DMSO from 10 to 20% (v/v)
ω-AmFAs and α,ω-diamines by employing a single-cell system
resulted in significant improvement in conversions of long-
expressing AHR and PMTA. This reaction gives access to an
chain α,ω-diols (n =10, 12 and 14) to their corresponding
alternative method to produce valuable precursors of
diamines (Supporting information Fig. S16). For instance, the
petroleum-based products and bioplastic polymers.
conversion of 3c into 3d improved from 56.8 to 88 mM with
Furthermore, the results demonstrate a new avenue that
the increase in DMSO from 10 to 20% (v/v). However, the use
lactones can be used as substrates for the newly developed
of DME as a co-solvent did not improve the conversions
parallel anti-sense bis-cascade employing AHR and ω-TA.
(Supporting information Fig. S17).
Ideally, generating a driving force for the forward reaction is a
Conflicts of interest
key episode in the cascade reactions. In the case of ‘parallel
There are no conflicts to declare
anti-sense’ cascade reported herein, the benzylamine acts as a
better amino donor for PMTA than the diamine (Benzylamine
exhibited five-fold higher activity compared to 1,10-
diaminodecane; Supporting information Fig. S18) and
NAD(P)H-dependent AHR preferably acts as a reductant for
aldehydes than as an oxidant,[10b] thereby acting as a major
driving force of overall forward reaction. Furthermore, in case
of ω-AmFAs, the precipitation of final product assists to drive
forward the reaction to higher conversion of ω-AmFAs.
Acknowledgements
This work was supported by the Ministry of Trade, Industry and
Energy of South Korea (MOTIE, Korea) under the industrial
Technology Innovation Program No. 10062550, ‘The production of
nylon 12 monomer, ω-aminolaruic acid from plant oil by
bioconversion and by basic Science Research program through the
National Research Foundation of Korea (NRF) funded by the
Ministry of Science, ICT and future Planning (2016R1A2B2014794).
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A novel ‘parallel Anti-Sense’ cascade, employing aldehyde reductase and ω-transaminase, has been
reported to produce bioplastic monomers with excellent conversion.