Direct terminal alkylamino-functionalization via multistep biocatalysis in one recombinant whole-cell catalyst

Article abstract of DOI:10.1002/adsc.201200958

Direct and regiospecific amino-functionalization of non-activated carbon could be achieved using one recombinant microbial catalyst. The presented proof of concept shows that heterologous pathway engineering allowed the construction of a whole-cell biocatalyst catalyzing the terminal amino-functionalization of fatty acid methyl esters (e.g., dodecanoic acid methyl ester) and alkanes (e.g., octane). By coupling oxygenase and transaminase catalysis in vivo, both substrates are converted with absolute regiospecificity to the terminal amine via two sequential oxidation reactions followed by an amination step. Such demanding chemical three-step reactions achieved with a single catalyst demonstrate the tremendous potential of whole-cell biocatalysts for the production of industrially relevant building blocks. Copyright

Full text of DOI:10.1002/adsc.201200958

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DOI: 10.1002/adsc.201200958
Direct Terminal Alkylamino-Functionalization via Multistep
Biocatalysis in One Recombinant Whole-Cell Catalyst
Manfred Schrewe,a Nadine Ladkau,a Bruno Bꢀhler,a,* and Andreas Schmida
a
Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund
University, Emil-Figge-Str. 66, 44227 Dortmund, Germany
Fax : (+49)-231-755-7382; phone: (+49)-231-755-7384; e-mail: bruno.buehler@bci.tu-dortmund.de
Received: October 31, 2012; Published online: February 22, 2013
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201200958.
Abstract: Direct and regiospecific amino-function-
alization of non-activated carbon could be achieved
using one recombinant microbial catalyst. The pre-
sented proof of concept shows that heterologous
pathway engineering allowed the construction of
a whole-cell biocatalyst catalyzing the terminal
amino-functionalization of fatty acid methyl esters
(e.g., dodecanoic acid methyl ester) and alkanes
(e.g., octane). By coupling oxygenase and transami-
nase catalysis in vivo, both substrates are converted
with absolute regiospecificity to the terminal amine
via two sequential oxidation reactions followed by
an amination step. Such demanding chemical three-
step reactions achieved with a single catalyst dem-
onstrate the tremendous potential of whole-cell bio-
catalysts for the production of industrially relevant
building blocks.
terminal functionalities, e.g., alcohols or amines.[8] As
an alternative, oxygenases can be applied for the
direct and regioselective oxyfunctionalization of sp3
[9]
À
coordinated C H bonds. Besides alcohol formation,
further oxidation is described for various oxygenases
resulting in the accumulation of aldehydes and/or
acids.[10,11,12] Terminal amines can be obtained from al-
dehydes for example, via w-transaminase (w-TA) cat-
alysis. Omega-transaminases catalyze the reversible
amino group transfer from a donor to an acceptor,
whereby one of the two compounds can be a non-acti-
vated aldehyde, ketone, or amine. Transaminases are
also versatile enzymes for the synthesis of chiral
amines.[13] Here, combining oxygenase and transami-
nase catalysis, we report the development of a biocata-
lyst catalyzing directly the specific amino-functionali-
À
zation of an unactivated, terminal C H bond. The re-
newable educt dodecanoic acid methyl ester (1) was
chosen as model substrate giving 12-aminododecanoic
acid methyl ester (4) as product (Figure 1). As a bi-
functional molecule, 4 constitutes a building block for
polymer production.[14] The synthesis of 4 was realized
via a consecutive three-step reaction in one recombi-
À
Keywords: amination; C H activation; multicom-
ponent reactions; terminal functionalization; whole-
cell biocatalysis
Direct and selective functionalization of saturated hy-
drocarbons remains a major challenge in organic
3
chemistry.[1,2] Direct activation of sp C H bonds can
À
be achieved with solid metal catalysts and non-heme
iron complexes.[3,4] However, the application of such
catalysts usually suffers from low efficiency and poor
selectivity.[5] Despite the general inertness of unacti-
À
vated C H bonds in alkanes, the reactivity even
varies among primary, secondary, and tertiary sites.
The primary site is least reactive making specific ter-
minal functionalization most demanding.[2,4,6] Transi-
tion metal-boryl complexes catalyze the functionaliza-
tion of alkanes specifically at the terminal position.[7]
In an additional reaction step, the resulting borane
Figure 1. Terminal amino-functionalization of dodecanoic
acid methyl ester (1) via 12-hydroxydodecanoic acid methyl
ester (2) and 12-oxododecanoic acid methyl ester (3) to 12-
aminododecanoic acid methyl ester (4) with E. coli BL21
(DE3) (pBT10, pTA) containing alkane monooxygenase
AlkBGT and w-transaminase CV2025. See the Supporting
products can be converted to products with different Information for the detailed reaction scheme.
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nant microbe expressing the genes for alkane mono-
oxygenase AlkBGT from Pseudomonas putida GPo1
and w-TA CV2025 from Chromobacterium violaceum.
AlkBGT catalyzes the NADH-dependent terminal
oxyfunctionalization of medium-chain length alkanes
and fatty acids[15,16] and was recently shown to form
terminal alcohols and the corresponding aldehydes
from fatty acid methyl esters.[17] The w-TA CV2025
accepts a wide range of amino donors and accept-
ors[18] with pyridoxal 5’-phosphate (PLP) as the typical
coenzyme of TAs enabling reversible amino group
transfer.[19]
In a single whole-cell catalyst based on Escherichia
coli BL21 (DE3), the oxygenase and transaminase re-
actions are coupled via heterologous pathway engi-
neering. Thereby, 1 is converted by AlkBGT in a two-
step reaction via 12-hydroxydodecanoic acid methyl
ester (2) to 12-oxododecanoic acid methyl ester (3),
which is further converted to 4 by CV2025 with l-ala-
nine serving as the amino donor. Additionally, the
same concept for terminal amino-functionalization is
applied for the synthesis of octylamine from octane.
In a recent study, recombinant E. coli W3110 con-
taining the alkBGT expression vector pBT10 was
shown to produce 2 and 3 from 1.[17] Here, E. coli
Figure 2. Oxyfunctionalization of 1 with E. coli BL21 (DE3)
(pBT10) in KPi buffer (pH 7.4) containing 1% (w/v) glu-
cose; biomass conc.: 1.5 gCDW LÀ1. Half diamonds: 1, trian-
gles: alcohol, diamonds: aldehyde, squares: acid, asterisks:
sum of educt and products. Alcohol and acid refer to the
sums of 12-hydroxydodecanoic acid methyl ester (2) and 12-
hydroxydodecanoic acid and of dodecanedioic acid mono-
AHCTUNGTREGmNNNU ethyl ester (5) and dodecanedioic acid, respectively. See
the Supporting Information for details on ester hydrolysis.
Aldehyde is 12-oxododecanoic acid methyl ester (3).
BL21 (DE3) was transformed with the same plasmid (DE3) carrying the cv2025 expression vector pTA,
enabling an initial hydroxylation activity of but lacking AlkBGT (Figure 3, a) and in control ex-
À1
2.2 UgCDW (1 U=1 mmol product formed per min, periments with E. coli BL21 (DE3) (Figure 3, b), the
CDW=cell dry weight) (Table 1), which is in the alkane monooxygenase can be considered as the main
same range as observed for E. coli W3110 (pBT10) cause for aldehyde oxidation. Overoxidation of the
(1.9 UgCDWÀ1).[17] Besides the expected products 2 and substrate is a characteristic often observed for mono-
3, accumulation of dodecanedioic acid monomethyl oxygenases[10] and three subsequent oxidation steps
ester (5) was observed after 15 min (Figure 2) which catalyzed by one enzyme are also described for
was not seen in the short-term assays performed for xylene monooxygenase XylM from P. putida mt-2
5 min.[17] After 60 min, the acid concentration exceed- showing 25% amino acid sequence homology with
ed alcohol and aldehyde concentrations. Since acid AlkB.[11,20] Aiming at an intracellular coupling of oxy-
formation from 3 was almost absent with E. coli BL21 genase and transaminase catalysis allowing production
Table 1. Maximal specific net oxygenation and transamination rates achieved with E. coli BL21 (DE3) containing alkane
monooxygenase AlkBGT and/or w-transaminase CV2025.
Plasmid
Substrate Alcohol formation
rate[a]
Aldehyde formation
rate[a]
Acid formation
rate[a]
Amine formation
rate[a]
[b]
–
3
3
1
1
16[f]
–
–
0.6 (0)
0.8 (0)
1.9 (0)
1.4 (0)
–
82
–
pTA[c]
17[f]
pBT10[c]
pBT10,
pTA[d]
3.7 (2.2)
2.9 (2.7)
2.2 (0.5)
2.4 (0.8)
1.5 (0.5)
pBT10,
Octane
26
12
2.6 (1.6)
10 (8.6)
pTA[e]
[a]
À1
Formation rates are given as specific activities in UgCDW
Biomass conc.: 0.9 gCDW LÀ1, substrate conc.: 0.5 mM.
Biomass conc.: 1.5 gCDW LÀ1, substrate conc.: 2.5 mM.
biomass conc.: 1.4 gCDW LÀ1, substrate conc.: 2.9 mM.
Biomass conc.: 1.3 gCDW LÀ1, substrate conc.: 1.4 mM.
.
[b]
[c]
[d]
[e]
[f]
Host intrinsic aldehyde reduction rate, in brackets: initial specific activities after 5 min of reaction; CDW: cell dry weight;
U=1 mmolminÀ1
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Direct Terminal Alkylamino-Functionalization via Multistep Biocatalysis
Table 2. In vitro and apparent in vivo kinetic parameters for
the conversion of 3 to 4 with w-TA CV2025.
In vitro[a]
In vivo[b]
À1
À1
Vmax
KM, KS
KI
7.1Æ0.2 Umgw-TA
117Æ14 UgCDW
0.54Æ0.13 mM
7.5Æ2.7 mM
[c]
80Æ10 mM
–
[a]
Based on NADH consumption during coupled spectro-
photometric assay (see the Supporting Information); 6 mg
of purified w-TA were applied.
[b]
[c]
Based on product formation with resting E. coli BL21
(DE3) (pTA); biomass conc.: 0.1 gCDW LÀ1.
KS: substrate uptake constant for whole cells (equivalent
to KM for cell-free enzymes).
the desired reaction. As indicated by the high Vmax
and the low KM (Table 2) 3 can be considered a good
substrate for the w-TA. Substrate inhibition could not
be observed up to the maximum concentration ap-
plied (2.5 mM).
Aiming at the in vivo coupling of AlkBGT and
CV2025, whole-cell aminations of 3 were conducted
with E. coli BL21 (DE3) (pTA). l-Alanine has been
shown to be a preferred amino donor for CV2025.[18]
In order to investigate whether the intracellular ala-
nine synthesis allows efficient formation of 4, the re-
action was performed over 30 min applying different
extracellular l-alanine and NH4Cl concentrations.
Without l-alanine or NH4Cl, only low amine concen-
trations were formed (0.01 mmolgCDWÀ1) (see the Sup-
porting Information). The addition of 187 mM NH4Cl
resulted in a 4-fold increased amine formation rate
(0.04 mmolgCDWÀ1) whereas supplying 50 mM of l-ala-
nine led to an almost 60-fold increased rate of amine
formation (0.59 mmolgCDWÀ1). A further increase in l-
alanine concentration only slightly enhanced amine
formation. During the transamination reaction, ester
hydrolysis could be detected resulting in small
amounts of 12-aminododecanoic acid (ADA) (<10%
of 4).
Figure 3. Whole-cell conversion of 3 with a) E. coli BL21
(DE3) (pTA) containing the transaminase CV2025 (KPi
buffer (pH 7.4) containing 50 mM of l-alanine and 1%
ACHTUNGTRENNUNG
(w/v) glucose; biomass conc.: 1.5 gCDW LÀ1) and b) E. coli
BL21 (DE3) lacking the transaminase (KPi buffer (pH 7.4)
containing 1% (w/v) glucose; biomass conc.: 0.9 gCDW LÀ1).
Circles: amine, triangles: alcohol, diamonds: aldehyde,
squares: acid, asterisks: sum of educt and products. Amine,
alcohol, and acid refer to the sums of 12-aminododecanoic
acid methyl ester (4) and 12-aminododecanoic acid, of 12-
hydroxydodecanoic acid (2) methyl ester and 12-hydroxydo-
decanoic acid, and of dodecanedioic acid monomethyl ester
(5) and dodecanedioic acid, respectively. See the Supporting
Information for details on ester hydrolysis.
of 4, acid formation constitutes a competing reaction.
Next to the in vitro kinetics, the apparent whole-
However, the constantly increasing acid and, thus, cell transamination kinetic parameters were deter-
overall product concentration showed that AlkBGT mined. Also in vivo, the reaction followed Michaelis–
was active throughout the reaction (90 min). There- Menten-type kinetics but showed substrate inhibition
fore, terminal amine formation should be feasible if (Table 2, see the Supporting Information) at concen-
the kinetic properties of CV2025 allow an efficient trations of 3 exceeding the maximum concentrations
conversion of 3 to 4. As observed in all experiments formed during oxyfunctionalization of 1 with E. coli
with cells present, 2 and 5 were hydrolyzed to some BL21 (DE3) (pBT10) by a factor of more than 10
extent to 12-hydrododecanoic acid (HDA) and dodec- (Figure 1). Such behaviour was not unexpected, since
anedioic acid (DDA), respectively (for details of ester transaminase catalysis is often impaired by inhibition
hydrolysis, see the Supporting Information).
at high substrate concentrations.[13] The high substrate
Conversions of 3 were carried out with purified uptake constant (KS) observed in vivo as compared to
CV2025 in a spectrophotometric assay (see the Sup- the KM observed in vitro indicates an uptake limita-
porting Information) to investigate kinetic reaction tion for 3 with whole cells. In contrast, the purified
parameters and the suitability of the transaminase for TA showed a high affinity towards 3 indicated by the
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Manfred Schrewe et al.
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low KM. Therefore, intracellular concentrations of 3
accumulating during AlkBGT-catalyzed oxidation of
1 might still be sufficient to enable the desired forma-
tion of 4.
In whole-cell biotransformations, the host intrinsic
enzymatic background has to be considered to learn
about possible unwanted side reactions. With 3 as
substrate (2.5 mM), E. coli BL21 (DE3) (pTA) pro-
duced 1.4 mM terminal amine in 15 min of reaction
(Figure 3, a), from which 5% were hydrolyzed to
ADA. After 90 min, ADA made up 30% of the total
amine product (see the Supporting InformÀa1tion). The
initial transamination activity of 82 UgCDW (Table 1)
was as expected from the apparent kinetic parameters
upon addition of 2.5 mM of
3
(calculated:
76 UgCDWÀ1) (Table 2). As a result of aldehyde reduc-
Figure 4. Amino-functionalization of 1 with E. coli BL21
(DE3) (pBT10, pTA) containing AlkBGT and CV2025; KPi
buffer (pH 7.4) containing 50 mM of l-alanine and 1%
tion, 2 was accumulating as side product (Figure 3 a)
À1
at a maximum rate of 17 UgCDW (Table 1). Approxi-
AHCTUNGTRENNUNG
(w/v) glucose; biomass conc.: 1.4 gCDW LÀ1. Half diamonds:
mately 0.31 mM terminal alcohol was formed in
30 min of which HDA accounted for 10%. Hydrolysis
of 2, 4, and 5 did not occur in abiotic control experi-
ments and thus can be attributed to host intrinsic en-
zymes. Hydrolysis of 1 and 3 was not observed. In
order to simplify downstream processing of the reac-
1, circles: amine, triangles: alcohol, diamonds: aldehyde,
squares: acid, asterisks: sum of educt and products. Amine,
alcohol, and acid refer to the sums of 12-aminododecanoic
acid methyl ester (4) and 12-aminododecanoic acid, of 12-
hydroxydodecanoic acid methyl ester (2) and 12-hydroxydo-
decanoic acid, and of dodecanedioic acid monomethyl ester
tion mixture, ester hydrolysis should be avoided in (5) and dodecanedioic acid, respectively. See the Supporting
Information for details on ester hydrolysis. Aldehyde is 12-
oxododecanoic acid methyl ester (3).
future by identifying and suppressing the responsible
enzyme activity.[21] Alcohol formation from 3 was also
observed in control experiments with E. coli BL21
(DE3) indicating host intrinsic dehydrogenase activity
À1
towards the supplied substrate (Figure 3, b). Alde- rates of 26 UgCDW were in the same range as ob-
hyde reduction rather than oxidation catalyzed by E. served earlier (Table 1).[23] Within 30 min of reaction,
coli dehydrogenases has been reported before.[11,22]
0.22 mM octylamine were formed as the main product
À1
Since the separately investigated reactions showed with a maximum amine formation rate of 10 UgCDW
.
promising results, E. coli BL21 (DE3) was trans- Again, acid formation was observed (Table 1), giving
formed with both expression plasmids. Co-expression 0.07 mM octanoic acid in the same assay after 30 min.
of alkBGT and cv2025 was verified by SDS-PAGE The higher rates achieved with octane as substrate in
analysis (not shown) and 1 (2.9 mM) was successfully comparison to 1 are a result of the enhanced substrate
converted to 4 (up to 0.13 mM) in the whole-cell reac- accessibility due to the less hydrophobic character of
tion (Figure 4). The maximum specific amine forma- octane.[23]
À1
tion rate of only 1.5 UgCDW (Table 1) clearly indicat-
In conclusion, we have successfully coupled oxygen-
ed a severe limitation of CV2025 by the low aldehyde ase and transaminase catalysis in one single recombi-
availability throughout the whole reaction time. It nant whole-cell biocatalyst allowing for the first time
could be shown in a recent study that the hydroxyl- the direct and specific terminal amino-functionaliza-
ation of 1 is limited by poor mass transfer of the hy- tion of the renewable substrate 1 and of octane via
drophobic substrate over the outer cell mem- three sequential reactions. As a proof of concept, this
brane.[17,23] By enhancing the intracellular availability study shows the versatility of microbial catalysts for
of 1 for AlkBGT catalysis, the aldehyde formation the specific functionalization of unactivated carbons,
rates may be increased as well which in turn should which is difficult to achieve by chemical means. The
improve the transamination reaction significantly. constructed catalyst represents an excellent starting
However, the amine was accumulating as main prod- point for the development of an efficient amino-func-
uct during the first 60 min of reaction. In contrast to tionalization process. Approaches in progress include
the decreasing CV2025 activity after 60 min, AlkBGT the improvement of hydrophobic substrate availability
showed a constant activity, which resulted in similar by catalyst engineering[23] and application of the two-
concentrations of amine and acid after 90 min.
liquid phase concept to tackle substrate/product in-
To demonstrate the broad applicability of the bio- hibition, to exploit reaction kinetics for a directed for-
catalyst, the amino-functionalization of octane mation of the amine, and to facilitate product isola-
(1.4 mM) was investigated. Maximal hydroxylation tion.[24] The versatility of AlkBGT accepting aliphatic,
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Direct Terminal Alkylamino-Functionalization via Multistep Biocatalysis
alicyclic, and aromatic hydrocarbons,[25] fatty acids,[16]
and fatty acid methyl esters as substrate, and the
broad substrate spectrum of CV2025[18] augur well for
a broad applicability of the biocatalyst for the direct
amino-functionalization of unactivated terminal C H
bonds.
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À
Experimental Section
Biotransformation Procedure
Oxidation by AlkBGT-containing E. coli cells (1–
1.5 gCDW LÀ1) was performed as described before.[17] Addi-
tionally, quantification of dodecanedioic acid monomethyl
ester (5) and dodecanedioic acid (DDA) was carried out by
high performance liquid chromatography (HPLC, see the
Supporting Information for details). The reaction was
stopped after different reaction times by addition of 0.5 mL
ice-cold acetonitrile to the reaction mixtures (1 mL). Transa-
mination and coupled oxidation-transamination by E. coli
cells containing CV2025 and AlkBGT/CV2025, respectively,
was performed accordingly, except that the reaction buffer
contained l-alanine (50 mM if not stated otherwise) (see the
Supporting Information for further experimental details).
[10] J. B. van Beilen, W. A. Duetz, A. Schmid, B. Witholt,
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Acknowledgements
We thank the German Federal Ministry of Education and Re-
search (BMBF, grant number 0315205) for financial support
and Phytowelt GreenTechnologies for providing the in vitro
transaminase assay procedure.
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