Chemoenzymatic Synthesis of a Novel Borneol-Based Polyester

Article abstract of DOI:10.1002/cssc.201701146

Terpenes are a class of natural compounds that have recently moved into the focus as a bio-based resource for chemical production, owing to their abundance, their mostly cyclic structures, and the presence of olefin or single hydroxy groups. To apply this raw material in new industrial fields, a second hydroxy group is inserted into borneol by cytochrome P450cam (CYP101) enzymes in a whole-cell catalytic biotransformation with Pseudomonas putida KT2440. Next, a semi-continuous batch system was developed to produce 5-exo-hydroxyborneol with a final concentration of 0.54 g L^?1. The bifunctional terpene was then used for the synthesis of a bio-based polyester by a solvent-free polycondensation reaction. The resulting polymer showed a glass transition temperature of around 70 °C and a molecular weight in the range of 2000–4000 g mol^?1 (M_w). These results show that whole-cell catalytic biotransformation of terpenes could lead to bio-based, higher-functionalized monomers, which might be basic raw materials for different fields of application, such as biopolymers.

Full text of DOI:10.1002/cssc.201701146

Accepted Article
Title: Chemo-enzymatic synthesis of a novel borneol-based polyester
Authors: Steffen Roth, Irina Funk, Michael Hofer, and Volker Sieber
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemSusChem 10.1002/cssc.201701146
Link to VoR: http://dx.doi.org/10.1002/cssc.201701146
A Journal of
www.chemsuschem.org

10.1002/cssc.201701146
ChemSusChem
FULL PAPER
Chemo-enzymatic synthesis of a novel borneol-based polyester
S. Roth,[a] I. Funk,[a] M. Hofer,[b] V. Sieber[a,b]
Abstract: Terpenes are a class of natural compounds that have
recently moved into the focus as bio-based resource for chemicals
productions due to their abundance, their mostly cyclic structure and
the presence of olefin or single hydroxyl groups. In order to apply
this raw material in new industrial fields, a second hydroxyl group is
inserted into borneol by cytochrome P450cam (CYP101) enzymes in
a whole cell catalytic biotransformation with Pseudomonas putida
KT2440. Next, a semi-continuous batch system was developed to
Dihydromyrcenol, for example, has been used for the production
of an amphiphilic biopolymer with emulsifying properties by first
introducing a thiol group at the double bond.[14]
In contrast, the modification of non-activated C-H bonds of
terpenes is more challenging, but could be achieved by
enzymes such as P450 enzymes.[15-19] It has also been shown
that recombinant Pseudomonas putida is able to transform
different terpenes likes limonene or α-pinene to products like
produce 5-exo-hydroxyborneol with a final concentration of 0.54 g L-1. perillyl alcohol or verbenol respectively, which have a higher
Afterwards, bifunctional terpene was used for the synthesis of an all
bio-based polyesters by a solvent free polycondensation reaction.
The resulting polymer showed a glass transition temperature around
70 °C and a molecular weight in the range of 2,000 – 4,000 g mol-1
(Mw). These results show that whole cell catalytic biotransformation
commercial value for the industry than the starting material.[20-22]
P. putida is a ubiquitous, aerobic, Gram-negative bacterium with
a great metabolic versatility. In the last decades, it has become
a bacterial workhorse for several applications in the field of
biotechnology, especially for biotransformation processes.[23-24]
of terpenes could lead to bio-based, higher-functionalized monomers, Today recombinant P. putida is used as a whole cell catalyst to
which might be basic raw materials for different fields of application,
such as biopolymers.
convert various substrates such as styrene,[25] octane[26] or
toluene.[27]
Herein we report that parts of the P450cam operon, P450cam
(camC, monooxygenase), PdX (camB, putidaredoxin), and PdR
(camA, putidaredoxin reductase) can be used to modify borneol
in a biotransformation with Pseudomonas putida. The terpenoid
borneol consists of a bornane scaffold containing one hydroxyl
group at C2 and exists in two stereo configurations. As
constituent of essential oils of many plants and waste residues,
e.g. in spruce needles, borneol has already been known for
many years for its wide occurrence.[28] Even more important, (-)-
borneol is also available in high amounts at a cost range of ca.
5 USD/kg from turpentine oil of the cellulose industry via
established chemical transformation, especially as an
intermediate in the synthesis of camphor (production route via
bornyl chloride) or derived from synthetic camphor (production
route via isobornyl acetate).[29-31] Hence, it is an interesting raw
material for bio-based polymers. To achieve this, a second
functional group has to be introduced. We show the
incorporation of a hydroxyl group at C5 of (-)-borneol by whole
cell catalysis of recombinant P. putida harboring P450cam
enzymes to create a novel monomer that can be used in a
Introduction
Terpenes are a wide class of natural hydrocarbons derived from
isoprene units, which are abundant in nature.[1] Terpenes have a
broad range of application possibilities. Some monoterpenes
have antibacterial[2] and antifungal[3] properties whereas higher
terpenes such as carotenoids are essential for plants, where
they act as energy transfer molecules to the chlorophylls,[4] and
are used as coloring agents in the food industry.[5] Some
terpenes are also used in the pharma industry for example as an
ingredient in transdermal drug delivery systems, where terpenes
enhance the skin permeation of drugs.[6]
Recently terpenes have moved into the focus for the production
of bio-based polymers. There are several reports on α- and β-
pinene[7-10] and limonene[11] being used for polymerization
resulting in homopolymers or copolymers.[12] These examples
make use of the olefinic bonds available in many terpenes,
which could be used for cationic or radical polymerizations[13] or
further converted to epoxides and lactones to open them for ring
opening polymerizations. However, monomers to be used in
polycondensations require two functional groups (i.e. carboxyl or
hydroxyl), yet typical monoterpenes do not fulfill this criterion.
polymerization reaction for
(Scheme 1).
a
new bio-based polyester
Results and Discussion
Engineering of
biotransformation of different borneols
a P. putida whole cell catalyst for
[a]
[b]
S. Roth, I. Funk, Prof. Dr. V. Sieber
Technical University of Munich
Chair of Chemistry of Biogenic Resources
Schulgasse 16, 94315 Straubing, Germany
E-mail: sieber@tum.de
The cam operon[32] is responsible for camphor degradation in P.
putida ATC17453, where in the first step camphor is
hydroxylated at C5 by the enzyme P450cam and then further
metabolized.[33] For this hydroxylation two more proteins, PdR
and PdX, are necessary as they act as an electron transporter
system towards the P450cam.[33] In order to obtain a P. putida
strain capable of borneol hydroxylation without further
Dr. M. Hofer
Fraunhofer Institute for Interfacial Engineering and Biotechnology
Bio, Electro and Chemocatalysis BioCat, Straubing branch
Schulgasse 11a, 94315 Straubing, Germany
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/cssc.201701146
ChemSusChem
FULL PAPER
Pseudomonas
putida KT2440
Scheme 1. Biotransformation of (-)-borneol to 5-exo-hydroxyborneol by engineered Pseudomonas putida KT2440. The bio-based monomer was subsequently
used for polymerization to produce a terpene-based biopolymer.
degradation, parts of the cam operon were cloned into the broad
host vector pBBR122. It is known that the camD gene codes for
a dehydrogenase transforming the 5-hydroxyl group into a
ketone[34-35] and the camR repressor negatively regulates the
operon,[34, 36] which would both result in a reduced yield of the
hydroxylated borneols. Hence, a synthetic operon was designed,
which does not contain these two regions. After successful
cloning and sequencing, the plasmid was transferred to P.
putida KT2440 to yield a whole cell catalytic system. Afterwards,
this strain was used to convert (-)-borneol to the corresponding
peracids,[40] so pH optimum for each conversion is also
dependent on the substrate. Interestingly, in S/P and Tris-HCl
buffers the selectivity increased at alkaline pH values, whereas it
remains nearly constant in MOPS. The selectivity is also slightly
influenced by the buffer substance as high activity was obtaiend
in Tris-HCl and phosphate buffer by contrast to the MOPS
buffered system. As mentioned above, the purified enzymes
showed a lower activity in phosphate buffers.[39] Using whole
cells for the conversion protect the enzymes, as the yields are
similar in Tris-HCl and phosphate buffered systems at pH 7 and
8 (Table 1). Based on these results the preferred buffer system
for the conversion of (-)-borneol is S/P buffer at pH 6.
hydroxyborneol in
a biotransformation. To determine the
stereochemistry of the product and to clarify if isomers are
produced, NMR and GC-MS analysis of the purified product and
substrates were performed (see Supplement). As there is no 5-
endo-hydroxyborneol available, (-)-borneol and its isomer (-)-
isoborneol were analyzed by GC-MS and could be differentiated
as two separated signals were obtained (Supplement Figure S8).
NMR analysis indicated that (-)-borneol is hydroxylated in only
one orientation as well (Supplement Figure S1, S7).
Furthermore it is reported that hydroxylation of camphor by
P450cam enzymes is highly site-specific[37] and solely leads to
5-exo-hydroxycamphor,[37-38]. Therefore the resulting product of
the biotransformation should be enantiomeric pure 5-exo-
hydroxyborneol.
Table 1. Substrate consumption and product formation in different buffer
systems after 6 h at 30 °C. Selectivity is calculated based on conversion and
yield.
Yield 5-exo-
hydroxyborneol
[%]
Conversion (-)-
borneol [%]
Selectivity
[%]
S/P buffer pH 6
100 ± 0.0
76.3 ± 2.0
63.0 ± 1.5
100 ± 0.0
74.9 ± 5.4
100 ± 0.0
100 ± 0.0
76.6 ± 1.9
78.6 ± 11.1
69.8 ± 3.8
63.6 ± 1.6
70.2 ± 2.8
70.7 ± 3.7
64.8 ± 1.7
67.1 ± 0.9
51.9 ± 4.0
78.6
92.6
100
S/P buffer pH 7
S/P buffer pH 8
Optimization of the biotransformation of (-)-borneol
Tris-HCl buffer pH 7
Tris-HCl buffer pH 8
MOPS buffer pH 6
MOPS buffer pH 7
MOPS buffer pH 8
70.2
94.4
64.8
67.1
67.8
For an optimal production of hydroxylated (-)-borneol the
substrate conversion was analyzed in dependence on different
temperatures as well as different buffers and buffer systems, as
the activity of P450cam enzymes is influence by both. Michizoe
et al. tested different buffers at different pH values for the
hydroxylation of camphor by the P450cam monooxygenase and
obtained an optimum at pH 7.4.[39] For the conversion with the
purified enzymes it could be shown that the buffer substance
itself had an influence on the stability of PdX, which was more
stable in Tris-HCl buffer than phosphate buffered systems.[39]
Hence, the buffered systems itself, using Tris-HCl, MOPS and
sodium-potassium-phosphate (S/P) buffers, as well as the pH
values were varied (pH 6.0 – 8.0) to identify the optimal
parameters for the conversion of (-)-borneol. Conversion rates of
(-)-borneol and yields of 5-exo-hydroxyborneol decreased in all
buffers at alkaline pH values (Table 1). Contrary to the pH
optimum at pH 7.4 as mentioned above, the optimum for the
conversion of (-)-borneol was observed at pH 6. This differences
may occur because whole cells are used as catalysts.
Nevertheless Spolitak et al. reported similar activities even with
purified P450cam at pH 6.2, 7.4 and 8.0 for the reaction with
Substrate conversion by P450cam enzymes is also influenced
by the temperature as shown for whole cell biotransformation of
camphor with recombinant E. coli.[41] Furthermore, P. putida
tolerates a wide range of temperatures[42] and so influence of
different temperatures on the biotransformation was investigated
(Figure 1). Conversion rates were nearly similar in a range of
30 – 40 °C and decreased at lower temperatures. In contrast,
product yields decreased at higher temperatures reaching their
maximum at 30 °C. Mouri et al. figured out that lower
temperatures for the hydroxylation of camphor by P450cam
enzymes in recombinant E. coli resulted in
a
faster
conversion.[41] The authors used inducible cells for the protein
expression, which were harvested, concentrated and then used
This article is protected by copyright. All rights reserved.

10.1002/cssc.201701146
ChemSusChem
FULL PAPER
for the hydroxylation of 2 mM camphor.[41] Following this
procedure an optimal temperature of 20 °C was obtained.[41] In
contrast, herein hydroxylation of (-)-borneol was performed
using a constitutive expression of the enzymes with growing
cells. Therefore 30 °C seemed to be the optimal temperature for
the conversion, as it also is the optimal growth temperature of P.
putida.[42]
Schewe et al. investigated the biotransformation of α-pinene
using recombinant E. coli as a whole cell catalytic system.[49]
They make use of the P450BM-3 monooxygenase from Bacillus
megaterium for the conversion of α-pinene to α-pinene oxide,
trans-verbenol and myrtenol respectively.[49] As a main product
-1
20 mg gcdw α-pinene oxide were obtained in 1.5 h reaction
time.[49] They obtained constant conversion rates for the first
30 min, which then decreased. The authors suggested that the
main problem was the toxicity of the substrate α-pinene.[49] To
determine any toxicity of (-)-borneol or 5-exo-hydroxyborneol to
P. putida, which would affect conversion rates, a toxicity test
was performed using 2 mM of substrate or product respectively.
Bacterial growth was analyzed over 24 h, but neither (-)-borneol
nor 5-exo-hydroxyborneol showed any toxic effect on the cells.
All these results indicate that the best parameters for the
conversion of (-)-borneol are a low oxygen level in the media
(low amount of side-products), S/P buffer pH 6 as the medium
(high product yields) and 30 °C as the conversion temperature
(fastest conversion with the highest yield).
Conversion (-)-borneol
Yield 5-exo-hydroxyborneol
100
80
60
Development of a semi-continuous batch system for the
production of 5-exo-hydroxyborneol
For the production of larger quantities of 5-exo-hydroxyborneol a
semi-continuous batch system was developed, which allowed
the reuse of the cells as a catalyst (Figure 2).
40
23
30
T / °C
37
40
Figure 1. Conversion of (-)-borneol and obtained yield of 5-exo-
hydroxyborneol at different temperatures after 5 h reaction time using the
P450cam enzymes in recombinant P. putida KT2440 as a whole cell catalytic
system.
Both experiments showed the formation of 5-oxocamphor as a
side-product at lower pH values and higher temperatures
respectively. Side product formation is attributed to different
effects: (i) products are further converted by P450cam enzymes
due to unspecific oxidation as shown for camphor, which is
oxidized to 5-oxo-camphor,[43] (ii) a high oxygen level in the
media is also responsible for further oxidation of products by
P450cam enzymes.[44] To prevent further product oxidation the
biotransformation was performed in a two phase system, where
the product should be protected by dissolving in the organic
phase. Moreover, high volatility as well as insolubility of (-)-
borneol in aqueous media as two major challenges could be
circumvented by the two phase system as well. As
Pseudomonas species are known to have a high tolerance to
different organic solvents,[45-46] they are excellent candidates for
biotransformations in presence of organic solvents. n-Hexane
and n-dodecane, both of which are reported to minimize toxic
effects of substrate or product,[47] containing (-)-borneol were
used and space-time yields (STY) were calculated accordingly.
STYs for both solvents (n-hexane and n-dodecane) were similar
(STY ~ 32 mg L-1 h-1) and also comparable to the conversion
without an organic solvent (STYP-media ~ 34 mg L-1 h-1). Similar
yields were reported for α- and β-pinene (STY = 22 and 41 mg L-
1 h-1), which were converted by resting cells of Pseudomonas
strain PIN.[48] The major product of the metabolism of this strain
was p-cymene, which was further metabolized to a broad
spectrum of terpenes (limonene, α-terpinolene, α-terpineol).[48]
Figure 2. Flow chart of the biotransformation process of (-)-borneol.
Biotransformation is performed in a 1.5 L air-flushed stirred tank reactor (1).
After complete conversion the product is separated from the cells via cross
flow filtration (2) and subsequently extracted with an organic solvent (3), while
the cells are returned to the reactor and used for further biotransformations
after addition of fresh media and substrate.
After complete substrate conversion was observed, the product
was separated from the cells by cross flow filtration and
extracted with ethyl acetate. Consequently fresh substrate was
added to the cells, which could be reused up to three times
(Figure 3).
Substrate conversion was high in every batch process (> 98 %;
Table 2), which confirmed that the cells are reusable as a
catalyst for the conversion of (-)-borneol. The time to reach full
conversion increased from 8 to 40 h, but was even shorter
compared to other hydroxylation reactions by P450cam
enzymes (e. g. α- and β-ionone whole cell biotransformation with
recombinant E. coli).[50] Product yields varied slightly with the
This article is protected by copyright. All rights reserved.

10.1002/cssc.201701146
ChemSusChem
FULL PAPER
addition of fresh substrate, but were similar to those in the
preliminary tests and also those reported for other hydroxylation
reactions (hydroxylation of limonene by P. putida MTCC 1072
cells).[21]
However, in the semi-continuous process, the oxygen level is
controlled by an external oxygen supply. By varying the level of
oxygen, specific product yields could be increased and best
results were obtained at an airflow of 2.75 L (air) L-1 (culture
volume) min-1. In each cross-flow filtration step, a certain amount
of media remained in the reactor, otherwise cell density would
increase and cells would be damaged by increased shear stress,
and so product yields remained at ~ 80 %. There also could be
some loss of substrate during the biotransformation because of
the volatile nature of the substrate.
(-)-Borneol
5-exo-Hydroxyborneol
4.0
3.0
2.0
1.0
0.0
I
II
III
IV
Polymerization of a bio-based borneol polyester
The melting point of 5-exo-hydroxyborneol obtained from the
biotransformation was determined (m. p. = 130 – 140 °C) and
purity could be increased by recrystallization (> 98 %). To see
whether the purified product can be used for polymerization, a
proof of concept was done by the polycondensation of 5-exo-
hydroxyborneol and succinic acid dimethylester. Succinic acid
could be obtained from renewable resources like carbohydrates
by fermentation.[51] There are several reports for the
biotechnological production of succinic acid by metabolic
engineered bacteria[52] and yeasts,[53] which can produce
succinic acid at high titers (45 g L-1).[53] Using this monomer for
the polycondensation reaction, the resulting biopolymer is a
novel all bio-based polyester. Furthermore, this monomer was
chosen to produce a polymer, which is somewhat similar to
polybutylene terephthalate (PBT), where a C4-alkyl chain and a
ring structure are present as well. In the bio-based polyester, the
aromatic ring is exchanged by the bicyclic alkyl ring of the
terpene monomer and the ester bond now is reversed (Figure 4).
0
20
40
t / h
60
80
Figure 3. Substrate consumption and product formation during the
biotransformation of (-)-borneol with cell recycling. Each dotted vertical line
indicates the addition of new substrate. All in all cells could be used four times
for the conversion of (-)-borneol (Phase I – IV).
Table 2. Parameters obtained during the biotransformation. STYs refer to
wet biomass, for calculation see material and methods.
Phase I
Phase II
Phase III
Phase IV
t [h]
8
13
99.6
75.3
28
21
98.0
81.7
20
40
98.8
83.3
11
Conversion [%]
Yield [%]
99.8
80.9
48
STY [mg L-1 h-1]
STY [mg mg-1 h-1]
Figure 4. Structure of the commercial polybutylene terephthalate (PBT, left)
12
6.0
4.0
2.0
compared to the terpene-based biopolymer (right).
During the first step of the solvent free polymerization, methanol
was released and used as a marker to follow the reaction
progress. First, polymerizations were carried out at lower
reaction temperatures and times, which resulted in viscous,
glutinous reaction mixtures. On increasing reaction temperature
and time, an amber-colored, transparent solid was obtained. To
get more details about the new material, thermal properties and
molecular mass distribution of the biopolymer and an industrial
PBT (as reference) were analyzed using differential scanning
By implementing the semi-continuous process, several
advantages could be achieved compared to a batch process.
The batch process is limited to a substrate concentration of
5 mM, because of the low substrate solubility and after the
conversion the product has to be isolated from the cells. With
the semi-continuous process it is possible to convert more
substrate over time, thereby increasing the total product yield.
Additionally, the amount of product per cell could be increased,
as the cells can be reused. Furthermore, when cells are present
during the extraction with ethyl acetate, an interphase is formed
and so extraction after cell removal by cross-flow filtration was
more efficient.
calorimetry
(DSC)
(Figure 5)
and
gel
permeation
chromatography (GPC) respectively. DSC analysis of the crude
biopolymer showed a glass transition temperature (Tg) in the
range of 35 – 55 °C. After the removal of residual monomers,
the Tg increased to about 70 °C, which is higher than the Tg of
In contrast to the preliminary tests no side-product formation
was observed, which is most likely due to the controlled lower
oxygen level in the media. The preliminary tests were performed
in shake flasks, where the oxygen level is supposed to be high.
PBT (45 – 60 °C). The terpene-based copolymer had
a
molecular weight in the range of Mw = 2,000 – 4,000 g mol-1
(PDI = 1.9), which is much lower compared to the industrial PBT
This article is protected by copyright. All rights reserved.

10.1002/cssc.201701146
ChemSusChem
FULL PAPER
(MW = 55,000 g mol-1). The difference in the molecular weights
might be attributed to the non-optimized polymerization
procedure. Polycondensation of modified terpenoids is a rather
unexplored area in the wide field of terpene-based polymers.
There are a lot of reports, which use other polymerization
techniques for terpene-based polymers. Often double bonds are
used for the polymerization of terpenes, as a straightforward
approach shown for cationic polymerizations reported for the
homopolymerization of β-pinene.[54] With this method Satoh et al.
could obtain poly(β-pinene) with high molecular weights (up to
50,000 MN) and a high transparency.[55] Terpenes without double
bonds often have to be modified before they are suitable for
polymerization. (-)-Menthone for example, carrying a single
carbonyl group, can be converted to lactams[56] or lactones,[57]
which could be used in ring opening polymerization (ROP) to
create polyamides (1.700 – 3.400 g mol-1 Mw) and polyesters (up
to 90,000 g mol-1 MN) respectively. One of the most common
techniques for synthesis of terpene-based polyesters is the
combination of ROP with lactones as monomers. For example
carvone-derived lactones lead to homopolymers with molecular
weights in the range of 700 – 10,500 g mol-1 (MN) by ROP.[58]
Compared to this terpene-based polymers, the novel borneol-
based biopolymer is an interesting starting point for further
the purified product was used for the polymerization with
succinic acid dimethylester. The biopolymer had a Tg of around
70 °C and a Mw in a range of 2,000 – 4,000 g mol-1. To increase
molecular weights the polymerization procedure has still to be
optimized. Therefore
effecting the polymerization like (i) amount and kind of catalyst,
(ii) polymerization temperatures and pressures, (iii)
a screening on different parameters
polymerization time, will be performed next. Further
investigations on the bio-based borneol polyester, for example
determination of impact resistance or ultimate strength, will be
performed, even with the low molecular weight polymers, to
figure out suitable applications for the material.
Experimental Section
GC-MS
Cell samples were mixed with an equimolar volume of ethyl acetate and
centrifuged after vigorous mixing. A sample of the upper organic phase
(150°µL) was mixed with ethyl acetate (350 µL) and GC-MS was
performed using auto-injector AOC-5000, the Shimadzu GC-2010 Plus
and a SGE BPX-column (30 m x 0.25 mm inner diameter). Separation of
the substances on the GC-MS was achieved at an oven-temperature of
50 °C followed by a temperature programming from 50 °C to 120 °C
(15 °C min-1), then to 170 °C (5 °C min-1), finally to 200 °C (20 °C min-1)
and holding this temperature for 10 min under the constant flow of
1.69 mL helium min-1. The resultant chromatograms were analyzed with
the Shimadzu GCMSsolution software.
investigations, especially using polycondensations as
a
polymerization technique. Thus, novel bio-based materials could
be obtained and by optimizing the polymerization procedure,
also the production of high molecular weight borneol-based
biopolymers should be possible.
DSC
0
-0.1
-0.2
-0.3
-0.4
Thermal properties of polymer materials (e. g. glass transition
temperature (Tg)) were recorded with differential scanning calorimetry
using the METTLER Toledo DSC 1 Star system. Samples of solid
polymer (10 – 15 mg) were heated from 50 °C to 300 °C (25 °C min-1),
hold for 2 min and then cooled down to - 20 °C (- 25 °C min-1) and hold
for 5 min. Next, the samples were heated to 100 °C (10 °C min-1), then to
400 °C (30 °C min-1) and finally cooled down to 50 °C (- 30 °C min-1). The
heating program was performed under constant flow of 50 mL nitrogen
min-1. The DSC curves obtained were analyzed with the METTLER
STARe SW 13.00 software and all Tg values were obtained from the
second scan, after removing the thermal history.
0
20
40
T / °C
60
80
100
GPC
Sample preparation was performed by drying (4 h at 80 °C) the solid
polymer samples (5 – 15 mg) under vacuum. Dried samples were
dissolved in appropriate volume of HFIP containing NaTFA (0.05 mol L-1).
For the calibration curve polymer standards of the ReadyCal-Kit
Poly(methyl methacrylate) low were dissolved in HFIP (1.5 mL)
containing NaTFA (0.05 mol L-1). Samples and polymer standards were
stored for two days at RT protected from light. Samples were then filtered
(0.2 µm) and GPC analysis was performed using the SECcurity GPC
System (Inline degaser and oven TCC600 (Polymer Standards Service),
1260 Infinity pump, 1260 Infinity auto-sampler, 1260 Infinity RI-detector
(Agilent Technologies)), a PSS PFG pre-column (50 mm x 8 mm, particle
size 7 µm) and two PSS PFG columns (300 mm x 8 mm, particle size
7 µm, porosity 100 Å and 1000 Å). The GPC curves obtained were
analyzed using PSS WinGPC UniChrom software (Polymer Standards
Service).
Figure 5. DSC analysis of the terpene-based biopolymer after removal of
residual monomers. The second scan of the sample is shown and glass
transition temperature was calculated by integration.
Conclusions
Within this study, it was successfully proven that it is possible to
hydroxylate (-)-borneol to yield a bifunctional terpene-diol by a
whole cell catalytic system. A process was developed, which
allowed the semi-continuous production of 5-exo-hydroxyborneol
with direct product extraction, in which cells could be reused as
catalyst for repeated biotransformations. In a proof of concept
This article is protected by copyright. All rights reserved.

10.1002/cssc.201701146
ChemSusChem
FULL PAPER
NMR
incubated (30 °C, 120 rpm), samples for GC-MS were taken at different
time intervals and space-time yields (STY) were calculated (Equation 1
and 2).
NMR-spectra were measured on a JNM-ECA 400 MHz spectrometer
from JOEL at 25 °C with the application of standard pulse programs. The
chemicals shifts (δ) are reported in ppm with reference to
tetramethylsilane. Coupling constants J are reported in Hertz (Hz). For
determining CH2-signals, the DEPT135°-technique was used. For correct
assignment of the signals, 2D NMR methods, like COSY, HSQC and
HMBC. For more information see SI.
m (obtained product [g])
[Eq. (1)]
[Eq. (2)]
STY [g L-1 h-1]= V (obtained Volume [L]) t (time [h])
c (obtained product [g L-1])
STY [g g-1 h-1] = m (wet biomass [g L-1
]) t (time [h])
For the toxicity tests of substrate, product or the corresponding solvent,
the cells were incubated (30 °C and 120 rpm) with (-)-borneol, 5-exo-
hydroxyborneol (each 2 mM) or ethyl acetate either for 48 h and OD600
values were measured with the Ultraspec 10 cell densitiy meter
(Amersham Biosciences) at different time intervals and compared to a
control.
Cloning
The P450cam operon was amplified by PCR with genomic DNA from
Pseudomonas putida ATCC 17453. For cloning the camCAB part of the
operon primer P1 and P2 (P1: 5’-CAATAAGAACGAGGTAATGCATGA
CGACTGAAACCATACAAAG-3’; P2: 5’-TAATGCGGCCGCGTCCCGG
GATTGGCCATTG-3’) were used for amplifying the camC, camA and
camB genes and adding an overlap sequence at the 3’ end of the PCR
product. For amplification of the promotor region primer P3 and P4 (P3:
5’-GTACTGAATTCGTTGCCCGGCTCGATCCGAG-3’; P2: 5’- GTATGG
TTTCAGTCGTCATGCATTACCTCGTTCTTATTG-3’) were used which
added an overlap sequence at the 5’ end. Both PCR products were then
used in an overlap extension PCR yielding camCAB. This PCR product
was then digested with EcoRI and NotI and ligated into the vector
pBBR122 digested with the same enzymes yielding pBBR122_camCAB.
The plasmid was transformed in E. coli DH10B and plated on LB plates
containing kanamycine (30 mg L-1). Single clones were used for plasmid
DNA preparation and sequencing. After the sequencing confirmed the
success of cloning the plasmids were transformed in P. putida KT2440
by electroporation.
Production of the diol compound
For the biotransformation of borneol to the corresponding diol, cells were
produced as described above and (-)-borneol was used as the substrate.
The OD600 of the main culture was set to 1.0 and 1 L of the culture broth
was then transferred to a stirred tank reactor with a total volume 1.5 L.
The broth was incubated (30 °C, 215 rpm, air flow of 2.75 L L-1 min-1) and
(-)-borneol (3 – 5 mM) was added as substrate. Samples withdrawn at
regular time intervals were analyzed by GC-MS. After full conversion of (-
)-borneol, the wet biomass weight was determined and the product was
separated from the cells by cross flow filtration with a Vivaflow 200
membrane (MWCO = 30 kDa, Sartorius). In addition, the reactor was
filled up with fresh media and substrate. This step was repeated three
more times. The aqueous product was extracted with the same amount
of ethyl acetate and the organic solvent was removed by evaporation.
Biotransformation of (-)-borneol
Purification of the diol compound by recrystallization
Single P. putida clones were used for inoculation of P-media (25 mL; 2 g
L-1 KH2PO4, 4 g L-1 Na2HPO4, 1 g L-1 (NH4)2SO4, 2.5 g L-1 glucose, 30 mg
L-1 kanamycine and 0.4 % trace metal solution (TMS)). The TMS
contained 10 g L-1 MgO, 2 g L-1 CaCO3, 5.6 g L-1 FeSO4x7H2O, 1.44 g L-1
ZnSO4x7H2O, 0.85 g L-1 MnSO4xH2O, 0.25 g L-1 CuSO4x5H2O, 0.28 g L-1
CoSO4x7H2O, 0.062 g L-1 H3BO3 and 50 mL L-1 HCl. The cells were
incubated in Erlenmeyer flasks overnight (30 °C, 150 rpm). This pre
culture was then used to inoculate 1 L fresh media with an OD600 of 0.2.
The cells were then further incubated (30 °C, 150 rpm) for 24 h.
Afterwards (-)-borneol (3 – 5 mM) was dissolved in dodecane or ethyl
acetate and added to the cell broth. Cells were further incubated (30 °C,
150 rpm), samples were taken at different time intervals and analyzed by
GC-MS.
To each gram of product, 6.2 g of ethyl acetate were added and heated
to 77 °C. After 2 –3 min the clear reaction mixture was allowed to cool
down to RT, whereby white crystals precipitated. The suspension was
incubated for 24 h at RT, following an incubation of another 24 h at 4 °C.
After an additional incubation for 30 h at – 20 °C the suspension was
vacuum filtered and white crystals were obtained. The resulting filtrate
was purified once more with the same procedure.
Polymerization
For the transesterification of succinic acid dimethyl ester (1.29 g,
8.8 mmol) with 5-exo-hydroxyborneol (1.05 g, 6.2 mmol) in the presence
of 0.5 mol-% tin(II) acetate (7.1 mg, 0.029 mmol, on 5-exo-
hydroxyborneol), a two-necked round bottom flask equipped with a
magnetic glass stirrer and a reflux condenser with a distillation bridge
were used and heated to 105 °C for 16 h. The educts were added under
an atmosphere of nitrogen and transesterification was conducted solvent-
free for 2 h at 190 °C under a nitrogen atmosphere. In the second step
the reaction mixture was heated to 245 °C and under reduced pressure
(p = 90 mbar), the excess of succinic acid dimethyl ester was removed.
After 4 h, the reaction mixture was cooled down to RT.
Optimization of the biotransformation
P. putida cells containing the pBBR122_camCAB plasmid were
incubated (2 d, 30 °C, 120 rpm) in shake flasks. To check whether
different buffers at different pH affect conversion, cells were harvested by
centrifugation (4,000 xg, 30 min, 4 °C), washed twice with the
corresponding buffer (pH 6.0 – 8.0) and set to an OD600 of 1.0. All buffers
(20 mM) contained equal amounts of glucose (2.5 g L-1) and TMS (0.4 %)
as the P-media. Cells were treated with (-)-borneol (1 mM), further
incubated (30 °C, 120 rpm) and samples for GC-MS were taken after 0 h
and 6 h. For the conversion at different temperatures (T = 23 °C – 40 °C),
cells were produced as above and also incubated (30 °C, 120 rpm) with
(-)-borneol (1 mM) at different temperatures (23, 30, 37, 40 °C). Samples
for GC-MS were taken at regular time intervals. The conversion in a two
phase system was performed using n-hexane and n-dodecane as
solvents (each 20 % (v/v)) containing (-)-borneol (50 mM). Cells were
For the removal of residual monomers polymer samples (200 – 250 mg)
were pulverized and then mixed with methanol (10 mL) for 16 h at RT.
After filtration, followed by a washing step with methanol (5 mL), the
solvent was removed by evaporation and the filter was dried.
This article is protected by copyright. All rights reserved.

10.1002/cssc.201701146
ChemSusChem
FULL PAPER
[24]
[25]
M. R. Mormile, R. M. Atlas, Canadian Journal of
Microbiology 1989, 35, 603-605.
Acknowledgements
G. Bestetti, E. Galli, C. Benigni, F. Orsini, F. Pelizzoni,
Applied Microbiology and Biotechnology 1989, 30, 252-
256.
T. Vallon, M. Glemser, S. H. Malca, D. Scheps, J. Schmid,
M. Siemann-Herzberg, B. Hauer, R. Takors, Chemie
Ingenieur Technik 2013, 85, 841-848.
M.-I. Ramos-González, A. Ben-Bassat, M.-J. Campos, J. L.
Ramos, Applied and Environmental Microbiology 2003, 69,
5120-5127.
The authors are grateful for the financial support by the German
Federal Environmental Foundation (Deutsche Bundesstiftung
Umwelt, DBU) (PhD grant AZ: 20015/382). The authors also
thank Claudia Falcke, Marion Wölbing and Paul N. Stockmann
for scientific conversations and help with DSC, GPC and NMR
analysis.
[26]
[27]
[28]
[29]
[30]
A. Orav, T. Kailas, M. Liiv, Chromatographia 1996, 43,
215-219.
M. Beri, J. Sarin, Journal of Chemical Technology and
Biotechnology 1936, 55, 605-607.
Z.-d. ZHAO, L.-w. BI, Biomass Chemical Engineering
2009, 4, 000.
Keywords: Biocatalysis • Biotransformations • Copolymerization
• Polymers • Terpenoids
References
[31]
[32]
G. Austerweil, Google Patents, 1911.
[1]
[2]
[3]
[4]
[5]
[6]
L. Janssens, H. L. De Pooter, N. M. Schamp, E. J.
Vandamme, Process Biochemistry 1992, 27, 195-215.
M. Friedman, P. R. Henika, R. E. Mandrell, Journal of
Food Protection 2002, 65, 1545-1560.
K. A. Hammer, C. F. Carson, T. V. Riley, Journal of
Applied Microbiology 2003, 95, 853-860.
J. Hirschberg, Current Opinion in Plant Biology 2001, 4,
210-218.
S. Kiokias, C. Proestos, T. Varzakas, Current Research in
Nutrition and Food Science 2016, 4, 25 - 37.
N. Sharma, B. Parashar, S. Sharma, U. Mahajan, Indo
global Journal of pharmaceutical sciences 2012, 2, 262-
278.
H. Aramaki, M. Fujita, Y. Sagara, A. Amemura, T. Horiuchi,
FEMS microbiology letters 1994, 123, 49-54.
H. Leisch, R. Shi, S. Grosse, K. Morley, H. Bergeron, M.
Cygler, H. Iwaki, Y. Hasegawa, P. C. K. Lau, Applied and
Environmental Microbiology 2012, 78, 2200-2212.
M. Fujita, H. Aramaki, T. Horiuchi, A. Amemura, Journal of
bacteriology 1993, 175, 6953-6958.
R. A. Hartline, I. Gunsalus, Journal of bacteriology 1971,
106, 468-478.
H. Aramaki, Y. Sagara, H. Kabata, N. Shimamoto, T.
Horiuchi, Journal of bacteriology 1995, 177, 3120-3127.
M. H. Gelb, D. C. Heimbrook, P. Malkonen, S. G. Sligar,
Biochemistry 1982, 21, 370-377.
P. V. Gould, M. H. Gelb, S. Sligar, Journal of Biological
Chemistry 1981, 256, 6686-6691.
J. Michizoe, H. Ichinose, N. Kamiya, T. Maruyama, M.
Goto, Journal of bioscience and bioengineering 2005, 99,
12-17.
T. Spolitak, J. H. Dawson, D. P. Ballou, Journal of
Biological Chemistry 2005, 280, 20300-20309.
T. Mouri, N. Kamiya, M. Goto, Biotechnology letters 2006,
28, 1509-1513.
P. Fonseca, R. Moreno, F. Rojo, Environmental
Microbiology Reports 2011, 3, 329-339.
O. Sibbesen, J. J. De Voss, P. R. O. de Montellano,
Journal of Biological Chemistry 1996, 271, 22462-22469.
B. Prasad, D. J. Mah, A. R. Lewis, E. Plettner, PLoS ONE
2013, 8, e61897.
Y. Sardessai, S. Bhosle, Research in Microbiology 2002,
153, 263-268.
J.-L. Ramos, M. S. Cuenca, C. Molina-Santiago, A.
Segura, E. Duque, M. R. Gómez-García, Z. Udaondo, A.
Roca, FEMS microbiology reviews 2015, fuv006.
F. van Keulen, C. N. Correia, M. M. R. da Fonseca,
Journal of Molecular Catalysis B: Enzymatic 1998, 5, 295-
299.
S. K. Yoo, D. F. Day, Process Biochemistry 2002, 37, 739-
745.
H. Schewe, B.-A. Kaup, J. Schrader, Applied Microbiology
and Biotechnology 2008, 78, 55-65.
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[7]
[8]
[9]
R. P. F. Guiné, J. A. A. M. Castro, Journal of Applied
Polymer Science 2001, 82, 2558-2565.
J. Lu, M. Kamigaito, M. Sawamoto, T. Higashimura, Y.-X.
Deng, Macromolecules 1997, 30, 22-26.
B. Keszler, J. P. Kennedy, in Macromolecules: Synthesis,
Order and Advanced Properties, Springer Berlin
Heidelberg, Berlin, Heidelberg, 1992, pp. 1-9.
M. Claudino, J.-M. Mathevet, M. Jonsson, M. Johansson,
Polymer Chemistry 2014, 5, 3245-3260.
K. Yao, C. Tang, Macromolecules 2013, 46, 1689-1712.
M. R. Thomsett, T. E. Storr, O. R. Monaghan, R. A.
Stockman, S. M. Howdle, Green Materials 2016, 4, 115-
134.
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[10]
[11]
[12]
[13]
[14]
H. Miyaji, K. Satoh, M. Kamigaito, Angewandte Chemie
International Edition 2016, 55, 1372-1376; Angewandte
Chemie 2016, 128, 1394-1398.
M.-H. Alvès, H. Sfeir, J.-F. Tranchant, E. Gombart, G.
Sagorin, S. Caillol, L. Billon, M. Save, Biomacromolecules
2014, 15, 242-251.
[15]
[16]
M. Hofer, H. Strittmatter, V. Sieber, ChemCatChem 2013,
5, 3351-3357.
G. Grogan, G. Roberts, S. Parsons, N. Turner, S. Flitsch,
Applied Microbiology and Biotechnology 2002, 59, 449-
454.
[47]
[48]
[49]
[50]
[51]
[52]
[17]
[18]
C. Funk, A. E. Koepp, R. Croteau, Archives of
Biochemistry and Biophysics 1992, 294, 306-313.
M. H. Gelb, P. Malkonen, S. G. Sligar, Biochemical and
Biophysical Research Communications 1982, 104, 853-
858.
A. Çelik, S. L. Flitsch, N. J. Turner, Organic &
biomolecular chemistry 2005, 3, 2930-2934.
I. Bechthold, K. Bretz, S. Kabasci, R. Kopitzky, A. Springer,
Chemical engineering & technology 2008, 31, 647-654.
S. J. Lee, D.-Y. Lee, T. Y. Kim, B. H. Kim, J. Lee, S. Y.
Lee, Applied and environmental microbiology 2005, 71,
7880-7887.
T. V. Yuzbashev, E. Y. Yuzbasheva, T. I. Sobolevskaya, I.
A. Laptev, T. V. Vybornaya, A. S. Larina, K. Matsui, K.
Fukui, S. P. Sineoky, Biotechnology and bioengineering
2010, 107, 673-682.
[19]
[20]
R. Bernhardt, Journal of Biotechnology 2006, 124, 128-
145.
S. Cornelissen, M. K. Julsing, J. Volmer, O. Riechert, A.
Schmid, B. Bühler, Biotechnology and Bioengineering
2013, 110, 1282-1292.
[21]
[22]
[23]
T. Chatterjee, D. Bhattacharyya, Applied microbiology and
biotechnology 2001, 55, 541-546.
G. Molina, M. R. Pimentel, G. M. Pastore, Applied
Microbiology and Biotechnology 2013, 97, 1851-1864.
D. M. John, G. F. White, Journal of Bacteriology 1998, 180,
4332-4338.
[53]
This article is protected by copyright. All rights reserved.

10.1002/cssc.201701146
ChemSusChem
FULL PAPER
[54]
[55]
A. Llevot, P. K. Dannecker, M. von Czapiewski, L. C. Over,
Z. Söyler, M. A. Meier, Chemistry-A European Journal
2016, 22, 11510-11521.
K. Satoh, A. Nakahara, K. Mukunoki, H. Sugiyama, H.
Saito, M. Kamigaito, Polymer Chemistry 2014, 5, 3222-
3230.
[56]
[57]
[58]
M. Winnacker, A. Tischner, M. Neumeier, B. Rieger, RSC
Advances 2015, 5, 77699-77705.
M. Winnacker, B. Rieger, ChemSusChem 2015, 8, 2455-
2471.
J. R. Lowe, M. T. Martello, W. B. Tolman, M. A. Hillmyer,
Polymer Chemistry 2011, 2, 702-708.
This article is protected by copyright. All rights reserved.

10.1002/cssc.201701146
ChemSusChem
FULL PAPER
Entry for the Table of Contents
FULL PAPER
S. Roth,[a] I. Funk,[a] M. Hofer,[b]
Pseudomonas
putida KT2440
V. Sieber[a,b]
Page No. – Page No.
Title
A new monomer for terpene-based biopolymers was produced by whole cell
biotransformation. Pseudomonas putida KT2440 was used as a host for the
hydroxylation of (-)-borneol by P450cam enzymes. A semi-continuous batch system
was developed to yield 5-exo-hydroxyborneol in a final concentration of 0.54 g L-1.
In a proof of principle, the purified product was then used to create novel terpene-
based polyesters, which were further characterized by DSC and GPC analysis.
This article is protected by copyright. All rights reserved.