From waste to value-direct utilization of limonene from orange peel in a biocatalytic cascade reaction towards chiral carvolactone

Article abstract of DOI:10.1039/c6gc01138a

In this proof of concept study we demonstrate direct utilization of limonene containing waste product orange peel as starting material for a biocatalytic cascade reaction. The product of this cascade is chiral carvolactone, a promising building block for thermoplastic polymers. Four different concepts were applied to augment limonene availability based on either water extraction solely, addition of extraction enhancers or biomass dissolution.

Full text of DOI:10.1039/c6gc01138a

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From waste to value - Direct utilization of limonene from orange
peel in a biocatalytic cascade reaction towards chiral carvolactone
N. Oberleitner,†a A. K. Ressmann,†a K. Bica,a P. Gärtner,a M. W. Fraaije,b U. T. Bornscheuer,c
F. Rudroff*a and M. D. Mihovilovica
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
In this proof of concept study we demonstrate direct utilization of allowing toluene-free enzyme production.8
limonene containing waste product orange peel as starting In a one-pot resting cell mixed culture approach (Scheme 1) we
material for a biocatalytic cascade reaction. Product of this connected this selective hydroxylation by CumDO expressed in
cascade is chiral carvolactone, a promising building block for P. putida S12 with our previously established synthetic mini-
thermoplastic polymers. Four different concepts were applied to pathway in Escherichia coli BL21(DE3)9, where carveol can
augment limonene availability either based on water extraction serve as starting material. By this new combination
solely, addition of extraction enhancers or biomass dissolution.
limonene (
carvolactone (
carvone ( ). Carvolactones, interesting building blocks for
1
)
could be directly transformed to chiral
5)
via carveol ( ), carvone ( and dihydro-
2
3)
Depletion of fossil resources and increasing demand for
platform chemicals has given rise to utilization of renewable
biomass as sustainable feedstock. To overcome the food vs.
feed problem, valorisation of food supply chain waste (FSCW)
can offer a sustainable route to cheap starting materials for
4
syntheses of bioactive or natural products, can also serve as
monomers for polymer production as they can be subjected to
ring-opening polymerisation and their olefinic side chains can
be easily functionalized and crosslinked.10,
11
Only recently
2
syntheses of valuable compounds.1, More than 15 million
enzymatic oligomerisation of chiral lactones was achieved,
notably in an aqueous system12 and this may be applicable also
for carvolactones.
tons of orange peel waste accumulates as by-product of citrus
fruit industry annually. R-(+)-Limonene (limonene, 1), main
component of most citrus oils, is industrially isolated from
orange peel by energy intensive steam distillation or cold
expression.3 Recent research opens the possibility for
concerted production of biofuels, pectin and limonene from
citrus peel waste.4, 5 Limonene and its oxygenated derivatives
(menthol, perillyl alcohol, carveol, carvone) have great market
potential as solvents, fine chemicals, flavours, fragrances or
even fuels.1 However achieving regio- and stereospecific
hydroxylation by chemical means is difficult, therefore
biocatalytic transformation of limonene has been studied
extensively since the 1960s.6 Duetz et al. showed regio- and
We considered different concepts to utilize FSCW orange peel
as starting material for our biocatalytic cascade towards
carvolactone (Fig. 1). Most commonly liquid biphasic systems13
(concept I) are applied with hydrophobic substrates such as
limonene14. Unfortunately, this concept is not feasible for
in situ conversion of limonene from orange peel as limonene,
due to its high logP value15, would accumulate in the
hydrophobic solvent. With a reasonable biomass loading (ratio
of orange peel to liquid volume) limonene concentrations in
the aqueous phase required for biotransformations cannot be
attained.
stereospecific hydroxylation of limonene (
grown Rhodococcus opacus PWD4 cells and obtained 97%
(+)-trans-carveol (
).7 The gene cluster coding for the enzyme,
1) by using toluene-
2
potentially responsible for this reaction, cumene dioxygenase
(CumDO) was recently cloned into Pseudomonas putida S12
Scheme 1 Cascade from limonene (1) to carvolactone (5), consisting of cumene
dioxygenase (CumDO), an alcohol dehydrogenase (RR-ADH), an enoate reductase
(XenB) and a Baeyer-Villiger monooxygenase (CHMOAcineto) in a mixed-culture set-up.
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Fig. 1 Different strategies for the direct conversion of limonene (1) present in orange peel to chiral carvolactone (5).
Another possibility is the application of the SFPR (substrate concentrations in that range would be concepts II and III
feed product removal) approach16, taking advantage of the where orange peel itself serves as substrate reservoir. Thus,
orange peel itself as substrate reservoir, constantly feeding the the overall substrate concentration would be below any
reaction with low amounts of water insoluble limonene. toxicity level20 for both microbial hosts and in a suitable
Therefore, mixing orange peel with the resting cells in aqueous concentration range for our biocatalytic cascade.
buffer would be the most facile approach (concept II). Here, Besides water (concept II), two hydrophilic 1-ethyl-3-methyl-
in situ conversion could be enhanced by variation of the imidazolium-based ILs and two biocompatible choline ([chol])
reaction solvent or rather the addition of water miscible ILs21, 22 were chosen additives for possible limonene extraction
solvents. Due to intolerance of microbial expression hosts to enhancement (concept III). 1-Ethyl-3-methyl-imidazolium
organic solvents we opted for the use of hydrophilic ionic acetate [C2mim]OAc was investigated as it is known for its
liquids (ILs) as additives in concept III, as the limited solubility excellent extraction ability of limonene from orange peel.19
of many organic compounds in water could be enhanced in 1-Ethyl-3-methyl-imidazolium chloride [C2mim]Cl as well as
well-defined aqueous IL solutions. Moreover, their ability to [chol]OAc were reported to have no growth inhibitory effect
pre-treat lignocellulosic biomass even in mixtures with water17 on E. coli23 and were therefore included in our study. Choline
make ILs promising additives that were already applied in formate [chol]fom was tested as it previously showed superior
several whole-cell bio-transformations.18 Partial or complete biomass extraction performance.24, 25
dissolution of biomass in pure ILs should enable enhanced First we evaluated the influence of ILs on viability of both
extraction efficiency of limonene from orange peel, as it was bacterial strains based on growth rates, shown in Table 1.
previously shown by Bica et al.19 (concept IV). In contrast to
Table 1 Bacterial growth in presence of ILs. Values given in percentage related to
growth without addition of ILs.
in situ concepts I-III, the latter requires additional dilution of
the dissolved biomass with resting cells after the initial
IL [mM]
50
100
50
100
extraction.
Entry – IL
1 – [C2mim]Cl
E. coli BL21(DE3) growth [%] P. putida S12 growth [%]
For the set-up of
a multi-component system potential
bottlenecks should be ruled out upfront. We investigated
influencing parameters such as (i) performance of limonene
hydroxylation, (ii) compatibility of extraction additives with
both whole-cell biocatalysts, and (iii) compatibility of the two
microbial hosts among themselves.
63±5
83±5
35±7
9±3
87±2
2±1
77±2
1±1
2 – [C2mim]OAc
3 – [chol]fom
4 – [chol]OAc
Concentration of starting material 1 is a relevant parameter for
the biocatalytic cascade, especially for the hydroxylation step.
We investigated different concentrations of limonene in the
first hydroxylation reaction and could improve the yield from
81±4
99±6
60±6
96±6
95±3
86±3
40% at 4 mM limonene (1) to almost 80% at 0.5 mM 1 (ESI,
Fig. S2). The latter concentration seemed to be very low and
unfeasible for further biotechnological applications, but having
a closer look at the total amount of limonene per gram
biomass, only 2-6% of limonene (see ESI, Fig. S1; reference4)
0
0
are available.
A suitable method to obtain limonene
2 | Green Chem., 2016, 00, 1-3
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a
Due to economic reasons, IL concentrations of 50-100 mM
were tested. Growing E. coli BL21(DE3) and P. putida S12
responded differently towards addition of ILs as can be
retrieved from the data in Table 1. Pronounced influence of
the concentration of IL can be seen in case of E. coli BL21(DE3)
where 50 mM [C2mim]OAc were well tolerated but 100 mM
[C2mim]OAc strongly impaired growth (Table 1, entry 2).
P. putida S12 is known to be sensitive to higher acetate
concentrations if not adapted to it.26 This was also observed
here as growth was inhibited by addition of [C2mim]OAc and
[chol]OAc, but not with [C2mim]Cl and [chol]fom. [Chol]fom
had the least effect on viability of both bacterial strains, at
either 50 or 100 mM concentration, and was consequently
elected as the best candidate for subsequent whole-cell
biocatalysis.
100
80
60
40
20
0
R-(+)-limonene (1)
(1R,5S)-carveol (2)
7
6
5
4
3
2
1
0
b
First test biotransformations in the presence of growing cells
and pure limonene (1) led to a massive loss of material due to
the immiscibility and high volatility of
1
(data not
shown).Therefore, we changed from growing to resting cells
and explored the influence of aqueous buffer (concept II) and
aqueous buffer + ILs (concept III) on the biotransformation
performance. Hence, we investigated the hydroxylation of
limonene by CumDO expressing resting cells of P. putida S12 in
the presence of 50 mM and 100 mM IL. In this pre-experiment
the 50 mM showed no interference whereas 100 mM IL
strongly impaired the reaction performance (ESI, Fig. S3).
Consequently 0.5 mM limonene were subjected to
hydroxylation in CumDO expressing resting cells of P. putida
S12 with and without the addition of 50 mM IL. Interestingly
[C2mim]OAc, which was not compatible with growing cells of
P. putida S12, showed nearly no interference with the
biotransformation in resting cells as can be seen in Fig. 2a.
Also [C2mim]Cl had hardly any impact on limonene
hydroxylation whereas both choline ILs reduced the
conversion to carveol significantly. Nevertheless the best
results could be obtained with resting cells in aqueous buffer
without additives (Fig. 2a, concept II).
F
ig. 2 Transformation of a) 0.5 mM R-(+)-limonene (
orange peel (limonene [c] = 13.8 mg ± 4.0 mg / g biomass) to (1R,5S)-carveol (
by CumDO in P. putida S12 resting cells in presence of ILs (50 mM) within 12 h
reaction time. Results are GC yields and deviations and material loss are due to
limonene volatility.
1) b) approx. 3 % (w/v)
2
)
Finally we dissolved the biomass in pure ILs, as proposed in
concept IV, and fed the extract to resting cells expressed
CumDO to 50 mM final concentration of ILs. This required not
only an additional handling step, but also reproducibility was
lowered and did not result in sufficient amounts of product
(data not shown).
To extend concept II
, we combined P. putida S12 cells
expressing CumDO with E. coli BL21(DE3) cells expressing RR-
ADH, XenB and CHMOAcineto in a mixed culture approach
(Scheme 1) in the presence of 0.5 mM limonene. Simultaneous
combination of the bacterial strains in one pot, despite
In order to investigate direct utilization of the waste product
by in situ conversion of limonene, we used orange peel instead
of pure limonene in the presence of aqueous buffer
(concept II) and aqueous buffer + ILs (concept III) (50 mM,
concept III) (Fig. 2a). Orange peel, from a batch with 13.8 mg ±
4.0 mg limonene per g biomass (based on classical EtOAc
extraction of triplicates) was added to CumDO expressing
resting cells of P. putida S12 with a biomass loading of
3% (w/v), which should result in an acceptable concentration
of limonene in the aqueous phase. As limonene contents in
orange peel may vary, we settled on representation of our
results in mg product per g orange peel. As can be seen in
moderate material loss, yielded about 47% of carvolactone (
5)
after 20 h (Fig. 3, stagnation of product formation after 10 h).
100
R-(+)-limonene (1)
(1R,5S)-carveol (2)
80
60
40
20
0
carvolactone (5)
Fig. 2b the conversion of limonene (1) from orange peel to
carveol (2) performed best in the aqueous system without
additives with 4.8 mg carveol per g orange peel to be detected
(GC yield). The addition of ILs led to lower yields of carveol,
where only [C2mim]Cl gave acceptable results as it showed just
minor inhibition of the reaction. Based on those results a clear
preference for concept II, the simple use of orange peel in
water, was gained.
simultaneous
sequential
Fig. 3 Production of carvolactone
simultaneous and 1 mM
BL21(DE3) resting cells after 20 h reaction time.
(
5
)
from 0.5 mM limonene
(
1
)
with
1
with sequential addition of P. putida S12 and E. coli
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However,
a
sequential approach was devised, where limonene from orange peel. We monitored the impact of the
to reach the same ILs on the growth of our bacterial expression hosts as well as
hydroxylation of 1 mM limonene
–
concentration of final product after dilution – by CumDO was on biotransformation activity. Although [C2mim]Cl showed
performed first and E. coli BL21(DE3) resting cells were only promising results as it hardly interfered with the bio-
added to the reaction vessel after 10 h. This enabled nearly full transformation, product formation was not improved by the
conversion to carvolactone (5) in 20 h (Fig. 3). Inspired by addition of ILs.
those results, we finally explored the direct valorisation of The most facile and economic approach (concept II), making
waste product orange peel to chiral carvolactone in the mixed- use of orange peel as substrate reservoir in a SFPR manner in
culture system applying concept II. From a biomass loading of aqueous buffer, emerged in promising results. With a biomass
about 3% (w/v), which yielded in 4.8 mg carveol per g orange loading of 1.5% (w/v) we detected the production of 6.3 mg
peel (limonene [c] = 13.8 mg ± 4.0 mg / g biomass) through carvolactone per g orange peel (29% yield over 4 steps) in a
hydroxylation with CumDO in P. putida S12 (Fig. 2b), 3.2 mg one-pot sequential biocatalyst addition approach. This direct
carvolactone per g orange peel (limonene [c] = 17.9 mg ± utilization of waste product orange peel is not only avoiding
3.7 mg / g biomass) could be produced. To acertain no orange tedious limonene extraction and purification, but also limits
peel overloading or to avoid a toxic effect limiting the reaction, volatility problems with the starting material. Acting as
a lower biomass loading of 1.5% (w/v) orange peel (limonene substrate reservoir, orange peel constantly releases limonene
[c] = 17.9 mg ± 3.7 mg / g biomass) was tested with concept II
.
to the aqueous phase where it can be directly converted in the
In a simultaneous addition approach only low amounts of multi-step biotransformation within
carvolactone could be detected. However, combination of the concept.
a principal proof-of-
mixed-culture sequential combination set-up, which proved Studies on improvement of parameters for the set-up of the
feasible with limonene as starting material, and the lower multi-component system will be part of future research.
orange peel loading, yielded 6.3 mg carvolactone per g orange Bacterial strains could be engineered for tolerance to
peel (limonene [c] = 17.9 mg ± 3.7 mg / g biomass) as can be increased IL concentrations as already shown for E. coli.27
seen in Fig. 4. This promising result, 29% carvolactone from Higher orange peel loadings, resulting in higher limonene
limonene over 4 biocatalytic steps (73% per step), is thus only concentration, could be handled by adaptation of P. putida
relying on orange peel as substrate reservoir in aqueous buffer S1226 or introduction of the hydroxylation reaction in a
without additives, consequently avoiding any additional constitutive solvent tolerant bacterial host28.
parameters increasing complexity of the overall process.
Through assembly of a biocatalytic cascade in vivo we
demonstrated the valorisation of waste product orange peel to
chiral carvolactone, a promising chiral polymer building block.
This direct multi-step conversion was performed in a one-pot
whole cell biotransformation cascade in aqueous buffer
without the need of any additives and underlines the power of
cascade biocatalysis.
Conclusions
We successfully combined two established biotransformation
9
pathways8, gaining access to a novel direct conversion of
natural product limonene (1) to chiral carvolactone (5). This
was realized in a one-pot sequential biocatalyst addition
approach where almost full conversion of limonene
concentrations in the mM range could be achieved.
Acknowledgements
In advanced investigations we explored different concepts for We thank FWF (grant no. I723-N17) and the DFG (grant no.
the valorisation of FSCW orange peel. Several ILs were Bo1862/6-1) for financial support.
considered as additives to enhance in situ conversion of
Notes and references
12
11
10
9
R-(+)-limonene (1)
(1R,5S)-carveol (2)
carvolactone (5)
1
2
3
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/ g biomass) in different approaches and with altered biomass loadings.
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TOC
From waste to value - Direct utilization of limonene from orange peel in a biocatalytic cascade reaction
towards chiral carvolactone
Nikolin Oberleitner,†a Anna K. Ressmann,†a Katharina Bica,a Peter Gärtner,a Marco W. Fraaije,b Uwe T.
Bornscheuerb, Florian Rudroff*a and Marko D. Mihovilovica
From waste to value: We investigated the valorisation of limonene containing waste product orange
peel, and performed a biocatalytic cascade for the production of chiral carvolactone, which can serve as
building block for thermoplastic polymers. Overall, we were able to produce carvolactone starting from
small pieces of orange peel in a 4-step biocatalytic cascade in 30% yield, based on continued extraction
solely with water.
a.Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9/163, 1060 Wien,
Austria, E-mail: florian.rudroff@tuwien.ac.at
b.Molecular Enzymology Group, University of Groningen, Nijenborgh 4, 9747 AG Groningen,
The Netherlands
c. Institute of Biochemistry, Dept. of Biotechnology
&
Enzyme Catalysis,
Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
† These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available: Experimental work and additional
results are described in the ESI. See DOI: 10.1039/x0xx00000x