Converging conversion - using promiscuous biocatalysts for the cell-free synthesis of chemicals from heterogeneous biomass

Article abstract of DOI:10.1039/d0gc04288a

Production of chemicals from lignocellulosic biomass has been proposed as a suitable replacement to petrochemicals. However, one inherent challenge of biomass utilization is the heterogeneity of the substrate resulting in the presence of mixed sugars after hydrolysis. Fermentation of mixed sugars often leads to poor yield and generation of multiple by-products, thus complicating the subsequent downstream processing. System biocatalysis has thus been developed in recent years to address this challenge. In this work, several novel enzymes with broad substrate promiscuity were identified using a sequence-based discovery approach as suitable biocatalysts in a conversion ofd-xylose andl-arabinose, two major constituents of hemicellulose found in plant biomass. These promiscuous enzymes enabled simultaneous biotransformation ofd-xylose andl-arabinose to yield 1,4-butanediol (BDO) with a maximum production rate of 3 g L^?1h^?1and a yield of >95%. This model system was further adapted toward the production of α-ketoglutarate (2-KG) from the pentoses using O₂as a cosubstrate for cofactor recycling reaching a maximum production rate of 4.2 g L^?1h^?1and a yield of 99%. To verify the potential applicability of our system, we attempted to scale up the BDO and 2-KG production fromd-xylose andl-arabinose. Simple optimization and reaction engineering allowed us to obtain BDO and 2-KG titers of 18 g L^?1and 42 g L^?1, with theoretical yields of >75% and >99%, respectively. One of the promiscuous enzymes identified together with auxiliary promiscuous enzymes was also suitable for stereoconvergent synthesis from a mixture ofd-glucose andd-galactose, predominant sugars found in food waste streams and microalgae biomass.

Full text of DOI:10.1039/d0gc04288a

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Converging conversion – using promiscuous
biocatalysts for the cell-free synthesis of
chemicals from heterogeneous biomass†
a,b,d,e
Cite this: DOI: 10.1039/d0gc04288a
Samuel Sutiono,a,b André Pickc and Volker Sieber
*
Production of chemicals from lignocellulosic biomass has been proposed as a suitable replacement to
petrochemicals. However, one inherent challenge of biomass utilization is the heterogeneity of the sub-
strate resulting in the presence of mixed sugars after hydrolysis. Fermentation of mixed sugars often leads
to poor yield and generation of multiple by-products, thus complicating the subsequent downstream
processing. System biocatalysis has thus been developed in recent years to address this challenge. In this
work, several novel enzymes with broad substrate promiscuity were identified using a sequence-based
discovery approach as suitable biocatalysts in a conversion of D-xylose and L-arabinose, two major con-
stituents of hemicellulose found in plant biomass. These promiscuous enzymes enabled simultaneous
biotransformation of D-xylose and L-arabinose to yield 1,4-butanediol (BDO) with a maximum production
rate of 3 g L−1 h−1 and a yield of >95%. This model system was further adapted toward the production of
α-ketoglutarate (2-KG) from the pentoses using O2 as a cosubstrate for cofactor recycling reaching a
maximum production rate of 4.2 g L−1 h−1 and a yield of 99%. To verify the potential applicability of our
system, we attempted to scale up the BDO and 2-KG production from D-xylose and L-arabinose. Simple
optimization and reaction engineering allowed us to obtain BDO and 2-KG titers of 18 g L−1 and 42 g L−1
,
Received 18th December 2020,
Accepted 7th April 2021
with theoretical yields of >75% and >99%, respectively. One of the promiscuous enzymes identified
together with auxiliary promiscuous enzymes was also suitable for stereoconvergent synthesis from a
mixture of D-glucose and D-galactose, predominant sugars found in food waste streams and microalgae
biomass.
DOI: 10.1039/d0gc04288a
rsc.li/greenchem
lignocellulose) has been proposed.4,5 Degradation of ligno-
cellulose yields D-glucose and, coming from the hemicellulose
Introduction
Development of bio-based chemical production is an approach part, a mixture of sugars such as uronic acids and pentoses.
for alleviating our dependency on fossil resources. Most of Hemicelluloses derived from wheat straw or soft wood, for
these bio-based chemicals are obtained from biotransform- example, largely consists of arabinoxylans and other polysac-
ation of D-glucose, which is mainly derived from starch. This charides that contain either L-arabinose or D-xylose, always
approach, however, possesses the risk of competition with leading to a mixture of these two carbohydrates.6,7 Efficient
food production.1–3 Consequently, second-generation chemical utilization of residual biomass therefore necessitates either a
production based on non-edible plant biomass (i.e. mainly good separation (one sugar one chemical approach), which
can be difficult with carbohydrates, or, preferred convergent
synthesis with several substrates leading to a single product.
Most of the work on the conversion of pentoses has largely
focused on the first approach, with D-xylose being predomi-
aChair of Chemistry of Biogenic Resources, Campus Straubing for Biotechnology and
Sustainability, Technical University of Munich, Schulgasse 16, 94315 Straubing,
Germany. E-mail: sieber@tum.de
nantly utilized for the production of several chemicals, such as
ethylene glycol (EG), glyoxylic acid (GA), glycolic acid (GCA),
1,4-butanediol (BDO), (R/S)-1,2,4-butanetriol (BTO), (R/S)-
3,4-dihydroxybutyric acid (DHB), and mesaconic acid.8,9
Adaptations of the preferred second approach have been
reported only in a few studies. A notable example is the
introduction of fungal D-xylose and L-arabinose pathways
into Saccharomyces cerevisiae for ethanol production. The
bSynBioFoundry@TUM, Technical University of Munich, Schulgasse 22,
94315 Straubing, Germany
cCASCAT GmbH, Europaring 4, 94315 Straubing, Germany
dCatalytic Research Center, Technical University of Munich,
Ernst-Otto-Fischer-Straße 1, 85748 Garching, Germany
eSchool of Chemistry and Molecular Biosciences, The University of Queensland,
68 Copper Road, St. Lucia 4072, Australia
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0gc04288a
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S. cerevisiae strains developed in these works can co-utilize In these pathways, both pentoses are transformed to an achiral
these two sugars, but the generation of by-products (arabitol intermediate, α-ketoglutarate semialdehyde (KGSA), prior to
and xylitol) is prevalent.10–12 Another example is the metabolic subsequent modifications to BDO.30 Our converging conver-
engineering of Bacillus subtilis to co-ferment the two pentoses sion approach was started by identifying and characterizing
for the production of acetoin reaching 60% theoretical yield.13
suitable promiscuous enzymes for the cascade reactions from
In all these works, however, D-glucose is still used as a co- both pentoses to KGSA. Application of a sequence-based bioca-
substrate. In the presence of D-glucose, pentose assimilation is talyst discovery revealed three promiscuous enzymes catalyzing
often repressed, a phenomenon known to be caused by the the first three steps of the modified pathway, thus allowing us
presence of carbon catabolite repression.8,14–16 Hence, low pro- to convert both pentoses to KGSA by using only five enzymes
ductivity and generation of by-products are typically (the original pathways will require eight enzymes). Addition of
observed.17,18 Furthermore, extensive knowledge of chassis two final enzymes enabled simultaneous biotransformation of
microorganisms is often required, as introduction of multiple both pentoses to BDO. To our knowledge, this result has never
functional proteins including sugar transporters is not always been demonstrated by either fermentation or cell-free
straightforward. Another strategy is to apply metabolic engin- approaches before.
eering to wild type strains that are already able to co-utilize
By simple exchange of a couple of enzymes (recently termed
both sugars. However, it is often hampered by the limited a “plug-and-play” strategy),23,24 we could adapt our cell-free
availability of genetic tools.19–21 To alleviate the problems and approach of BDO production to yield α-ketoglutarate (2-KG)
challenges of fermentation, system biocatalysis based on a from the mixed sugars (Scheme 1), another valuable building
cell-free approach to produce commodities has been developed block that has been used extensively in the food and pharma-
in recent years as an alternative approach; with the concept ceuticals industries.31–33 By reaction design we optimized and
also being termed “synthetic biochemistry”, “synthetic cascade scaled-up our system to reach a BDO titer of 18 g L−1. A similar
manufacturing”, “cell-free synthetic biology” or “cell-free meta- strategy allowed the production of 2-KG to reach 42 g L−1
bolic engineering”.22–25 This approach promises to obtain
We have also attempted the converging conversion of
.
higher production rates and yields, as well as minimize by- D-glucose and D-galactose. These two sugars are predominant
product formation. Being more robust to production para- in microalgae.34,35 Production of chemicals from microalgae
meters (pH, temperature, and substrate or product inhibition) biomass is considered as
a third-generation biorefinery
and simpler to optimize are other advantages of this emerging approach.34,36 Application of different sets of promiscuous bio-
technology.22–24 The major challenge of these approaches is to catalysts allowed the production of ethanol from both hexoses.
keep the number of enzymes small, as they contribute most to Considering that most of the enzymes used in the cascades
the process costs. Similar to the fermentation processes, cell- developed in this work are wild type enzymes, these synthetic
free biotransformation of pentoses has mainly been aimed at cascades hold a promising prospect of further optimization
converting D-xylose. Several compounds that have been pro- toward industrial applications in the near future.
duced from D-xylose in vitro are BTO, EG, GCA, and
α-ketoglutarate (2-KG).26–28 Although higher theoretical yields
were achieved, the final titers obtained were lower than those
of the above-mentioned fermentation processes and still far
below industrial-relevant concentrations.
Results and discussion
Cell-free production of 1,4-butanediol: cascade design and
discovery of promiscuous biocatalysts
Thus, to address the challenges in the co-utilization of
D-xylose and L-arabinose and simplify the design, we set out to The production of BDO from either D-xylose or L-arabinose in
develop a converging conversion approach based on a cell-free E. coli was achieved in a previous study by modifying the oxi-
system. Stereo-convergent synthesis of chemicals is a concept dative Weimberg pathway of each pentose (Scheme 1).30,37 In
to convert two or more diastereomers (here D-xylose and this modified pathway, D-xylose or L-arabinose is converted to
L-arabinose) into one product. Accordingly, for our approach a an achiral intermediate, α-ketoglutarate semialdehyde (KGSA),
product is required that has lost the relevant stereo centers. by respective enzymes of the Weimberg pathways. KGSA is
Accordingly, as the first target compound 1,4-butandiol (BDO) then decarboxylated and reduced to yield BDO. Thus, the first
was chosen, fulfilling this prerequisite and being an important challenge in this work was on how to obtain KGSA from both
commodity chemical with 4.3 million tons annual production pentoses using one enzyme for each reaction.
and an annual growth forecasted to be 8%.8,29 However, due to
To answer this question, we went backward from KGSA to
their typically high selectivity, enzyme based stereo-convergent both sugars (retrosynthesis approach) to identify the promiscu-
synthesis also implies that for every diastereomer or enantio- ous enzymes (Scheme 1). KGSA is obtained from dehydration
mer as a substrate a single enzyme is required, which stands of 2-keto-3-deoxy-D-xylonate (D-KDP) and 2-keto-3-deoxy-L-arabi-
in contrast with the desire to minimize the total amount of nonate (L-KDP). Two different enzyme classes (D-KdpD and
enzymes. Therefore, an essential part of the converging conver- L-KdpD) participated in these reactions. Despite the relatively
sion approach is to be able to utilize promiscuous enzymes.
high activities of both enzymes toward their natural stereoi-
Biotransformation of D-xylose and L-arabinose to BDO was somers, we found no promiscuity of either dehydratase toward
achieved by modifying the Weimberg pathway of each pentose. the other non-preferred stereoisomer, requiring us to still use
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Scheme 1 Cell-free synthetic cascade reaction of biotransformation of D-xylose and L-arabinose to 1,4-butanediol (blue) and α-ketoglutarate (red).
Shared common steps and enzymes for both chemicals are labelled black. Auxiliary enzymes (CbFDH and LpNOX) are drawn to illustrate the choice
of a cofactor balancing system. Three key promiscuous enzymes are highlighted orange.
both enzymes.38 Next, D- and L-KDP as the products of the de- convert KGSA to BDO. An isoenzyme of keto acid decarboxylase
hydration of D-xylonate and L-arabinonate, respectively, were from Lactococcus lactis (LlKdcA) was used for the penultimate
catalyzed by [Fe–S]-dehydratases. In this work, we characterized reaction. This enzyme had been engineered to withstand high
an [Fe–S]-dehydratase from Paralcaligenes ureilyticus (PuDHT) in temperatures and high concentrations of organic miscible sol-
more detail (process parameters and substrate promiscuity of vents (isobutanol).41 Adh Z3 from E. coli (EcAdh Z3) was
PuDHT). To our knowledge, this enzyme has the highest activity chosen to catalyze the last reduction step.42,43 Finally, formate
and total turnover number (TTN) for D-gluconate and D-glycerate dehydrogenase from Candida boidinii (CbFDH) was employed
of dehydratases that have been reported so far.39 PuDHT to achieve a system with balanced cofactor. This regeneration
showed broad substrate acceptance, including high activity on system was chosen because it does not yield by-products,
D-xylonate and L-arabinonate (Table S1†).
unlike the D-glucose to D-gluconate system, and even more due
Both sugar acids are the hydrolysis products of pentonolac- to the possibility to regenerate formates externally using
tones, which are obtained after oxidation of D-xylose and “green” hydrogen, making the whole process following the
L-arabinose. Sugar lactone hydrolysis is often reported to be principles of sustainable chemistry (Scheme 1).44,45
spontaneous. Thus, we focused directly on the oxidation step.
After combining all the enzymes, substrates, and cofactors,
Indeed, the only promiscuous sugar dehydrogenase reported we followed the reaction over time. Both sugars were con-
is glucose dehydrogenase from Saccharolobus solfataricus sumed and 1,4-BDO was formed. However, to our surprise, the
(SsGDH).40 Despite its high activity and promiscuity at elevated reaction progressed relatively slowly (1 mM h−1) (Fig. S4†) in
temperatures, this enzyme is practically inactive at 30 °C (data comparison with a fermentation system using E. coli that
not shown), the temperature chosen for this study. It was still reached BDO production rates of 4.4 mM h−1 from D-xylose
not possible to perform the cascade reaction at significantly and 2.5 mM h−1 from L-arabinose.30 We then analyzed the
higher temperatures because one key enzyme, D-KdpD from HPLC chromatogram of the samples to determine a possible
Pseudomonas putida (PpD-KdpD), is not thermostable.38 Thus, bottleneck. In the chromatogram, only one peak from 1,4-BDO
to allow the simultaneous oxidation of both sugars, we and one peak from sugar acid (either of D-xylonate or of
attempted to find new enzymes.
L-arabinonate) were observed (data not shown).
Herbaspirillum seropedicae was the first microorganism we
Accumulation of D-xylonate has previously been reported as
investigated. It was reported to have multiple [Fe–S]-dependent one of the major challenges in cell-based and cell-free bio-
dehydratases, as well as a functional D-KdpD and L-KdpD,38,39 transformation of D-xylose, significantly affecting the pro-
indicating that this microorganism has two working Weimberg ductivity and yield. It was argued that the slow turnover of [Fe–
pathways, one from D-xylose and the other from L-arabinose. S]-dependent sugar acid dehydratases was the main bottleneck
This could imply the presence of promiscuous sugar dehydro- for the utilization of the Weimberg pathway in vitro.26 The
genases catalyzing the first step of the Weimberg pathways. poor loading of the [Fe–S]-cluster of the dehydratase was likely
Exploring its genome using the sequence of xylose dehydro- the reason of D-xylonate accumulation contributing to its tox-
genase from Caulobacter crescentus (CcXylDH) indicated two icity in in vivo approaches.46,47 However, we ruled out these
putative dehydrogenases that belong to the Short-chain possibilities in our study, as PuDHT was demonstrated to have
Dehydrogenase/Reductase (SDR) family. These two enzymes high activity for both sugar acids (Table S1†). We then hypoth-
were cloned, expressed, and partially characterized. One esized that the observed sugar acid peak in our HPLC was actu-
enzyme (HsXylDH2) showed the highest promiscuity in com- ally attributed to the lactones of each corresponding sugar
parison with CcXylDH and HsXylDH1 (Fig. S3†) and was acid, which were hydrolyzed to their sugar acids during the
further used to oxidize D-xylose and L-arabinose.
HPLC measurement (65 °C, pH 4).
After having identified all enzymes for simultaneous con-
To test this hypothesis and also to overcome this limitation,
version of D-xylose and L-arabinose to achiral KGSA, we we embarked on the discovery of a promiscuous lactonase
selected two last enzymes, decarboxylase and reductase, to using a sequence-based discovery approach to successfully
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production from the mixed pentoses we chose to adapt a
nutating compartment system as previously employed in the
production of pyruvate from glycerol39
Most enzymes used for 2-KG production in this study
favoured slightly alkaline pH. As it is generally known that the
opening of sugar acid lactones is greatly favoured at higher
pHs, we first attempted to perform the reaction without
NmLac2. Using a nutating compartment system (pH 7.5,
25 °C), oxidation of both sugars (total of 50 mM) was com-
pleted in the first hour, and 5 mM 2-KG was observed
(Fig. S8†). With 5 mM initial NAD+ used, this result showed
that LpNOX was able to oxidize 50 mM NADH produced per
hour. This validated the suitability of the nutating compart-
ment system for LpNOX. Performing the reaction at a slightly
higher pH and temperature (pH 8.2, 30 °C) to promote spon-
taneous lactones hydrolysis allowed us to complete 2-KG pro-
duction after 8 h.
Fig. 1 Biotransformation of 1,4-butanediol (BDO) from D-xylose and
L-arabinose at pH 7.0, 30 °C, 700 rpm with NmLac2. D-XylA is D-xylonate
and L-AraA is L-arabinonate. Error bars are standard deviations from
three independent replications. When errors are too small they are
covered by the markers and thus not displayed.
To further maximize the production rate of 2-KG pro-
duction, NmLac2 was added. Complete production of 2-KG
(>99% theoretical yield) was achieved in less than 2 h, corres-
ponding to an overall rate of 28.7 mM h−1 (4.2 g L−1 h−1
)
(Fig. 2). At these optimized conditions, the activity of LpNOX
was able to reach 104 mM h−1 in the first 15 min (Fig. S9†).
The activity gradually decreased to reach an average of 60 mM
h−1. In a previous study in which the LpNOX concentration
reached 98 mM h−1 using a bubble reactor, LpNOX had to be
added every 20 min due to enzyme deactivation by the air–
liquid interfacial area.49 In the nutating compartment system,
however, no additional NOX was necessary.
identify
a lactonase from Noviherbaspirillum massiliense
(NmLac2) (Discovery of novel promiscuous lactonases in the
ESI†). This lactonase also showed broad promiscuity toward
different sugar acid lactones (Fig. S7†). Addition of this
enzyme then allowed BDO production to reach 33 mM h−1 (3 g
L−1 h−1) with >95% theoretical yield (Fig. 1). In this improved
cascade reaction, L-arabinose was consumed faster than
D-xylose. The main reason of this phenomenon is the higher
affinity of HsXylDH2 to L-arabinose than D-xylose (Table S2†).
Scaling up the biosynthesis of 1,4-butanediol and
α-ketoglutarate: simple optimization and reaction engineering
Production of α-ketoglutarate: application of a “plug-and-play”
strategy
BDO is an important building block that is still largely pro-
duced using petrochemicals. An impressive work on high-titer
One of the main benefits of a cell-free approach is the possi-
bility of using different substrates or of obtaining different pro-
ducts by replacing, eliminating, or adding one to a few
enzymes only. This easy adaptability is often called “plug-and-
play”.23,24 In this work, we applied this strategy to produce
2-KG. In order to adapt our cell-free system of BDO production
to now produce 2-KG, we replaced the last three enzymes
(colored blue in Scheme 1) with two enzymes (colored red in
Scheme 1).
For regeneration of NAD+, we opted to use an NADH
oxidase from Lactobacillus pentosus (LpNOX), which was
reported to be suitable for the oxidation of NADH to NAD+
using molecular oxygen to form only water.48 Although high
activity of LpNOX has been reported, utilization of this enzyme
in a cell-free system is not straightforward. It is partly due to
the limitation of oxygen availability in aqueous solutions.
Quiescent solution has been shown to be unsuitable for a reac-
tion catalyzed by NOX. Several strategies have thus been devel-
oped to address this challenge, including stirred or agitated
and sparged bubble reactors. However, all these strategies had
come at the expense of enzyme stability due to rapid protein
Fig. 2 Biotransformation of α-ketoglutarate (2-KG) from D-xylose and
L-arabinose at pH 7.5, 30 °C with NmLac2. The reaction was performed
in a nutating compartment nutated at 50 rpm. D-XylA is D-xylonate and
L-AraA is L-arabinonate. Error bars are standard deviations from three
independent replications. When errors are too small they are covered by
deactivation on air–liquid interfacial area.49,50 Thus, for 2-KG the markers and thus not displayed.
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bioproduction of BDO from D-glucose has been performed
through years of integrative efforts.51 We now attempted to
increase the BDO titer of our cell-free converging conversion
approach from both pentoses by increasing the substrate load
(D-xylose and L-arabinose) five-fold (40 g L−1 or 266 mM).
In the first optimization step, we varied the enzyme concen-
trations to determine the importance of each enzyme in the
cascade. This simple approach allowed for a reduction in the
enzyme load by 30% to achieve the same yield as in the experi-
ment shown in Fig. 1 (Fig. S11†). After determining the opti-
mized enzyme combination, we perform the reaction in the
batch mode. After 20 h biotransformation, the BDO yield
reached only 55% of the theoretical value (Fig. S12†). Although
this result was already significantly higher than those for pre-
vious fermentation approaches, we attempted to further
increase the yield.30,52 Addition of each enzyme after 20 h bio-
transformation did not increase the yield further (data not
shown). Previous studies showed that several different enzyme
variants of the last two steps, i.e. decarboxylation of KGSA and
reduction of SA, influenced BDO yields. LlKivD, an isoenzyme
of LlKdcA, was shown to be the most suitable enzyme; thus, we
did not attempt to replace LlKdcA. Substituting AdhZ3 (LND)
with AdhP from E. coli, however, allowed us to improve the
BDO yield from 55% to 75% (Fig. S12†).
In a time-course experiment using AdhP, accumulation of
sugar acids was observed up to 9 h and gradually decreased
(Fig. 3A). This indicated slower dehydration reaction of the
sugar acids catalyzed by PuDHT in comparison with a reaction
using lower substrate load (Fig. 1). Measuring the activity of
PuDHT in the presence of the substrates (D-xylose, L-arabinose)
and co-substrate (formate) revealed that PuDHT was inhibited
by the latter with an I50 (concentration of an inhibitor to give
50% reduced initial activity) of 150 mM (Fig. S13A†). In
addition, formation of BDO seemed to be slower in the begin-
ning and was expedited when L-arabinose had been consumed.
This was likely due to an inhibitory effect of L-arabinose at
high concentrations to HsXylDH2 (Ki = 130 mM) (Table S2†).
D-Xylose, on the other hand, did not inhibit HsXylDH2
(Table S2†).
Fig. 3 Productions of 1,4-butanediol (1,4-BDO) (A) and α-ketoglutarate
(2-KG) (B) from a mixture of D-xylose and L-arabinose. Error bars rep-
resent standard deviation from three independent replications. The
experiments were performed at 30 °C, pH 7.0 and 700 rpm (A) and pH
8.0 and 50 rpm (B). D-XylA/L-AraA is the total amount of D-xylonate and
L-arabinonate. 2-KGSA is α-ketoglutarate semialdehyde. When errors are
too small they are covered by the markers and thus not displayed.
With this approach, the BDO production reached 18 g L−1
(200 mM) in 16 h (Fig. 3A), significantly faster than in fermen-
tation processes.30,52 A higher yield (>75%) was also achieved
as compared to two previous fermentation attempts that
reached <40% theoretical yield.30,52 At the end of the biotrans-
formation, KGSA was observed, suggesting LlKdcA being inhib-
ited (Fig. 3A). No BTO formation was detected, unlike reported
by a previous work using a modified E. coli strain from
D-xylose.30 This is mainly due to the higher catalytic efficiency
of PpD-KdpD toward D-KDP in comparison with that of LlKdcA
toward D-KDP.30,38
observed (data not shown), suggesting that all the enzymes cat-
alyzing the forward reactions were still active. Because the pro-
duction of 2-KG was based on cofactor recycling, a halted reac-
tion was then reasoned to be caused by the unavailability of
the oxidizing cofactor, NAD+, which was catalyzed by LpNOX.
Addition of LpNOX every 3 h allowed for the production of
2-KG to reach 41.6 g L−1 (>99% of the theoretical yield) after
9 h biotransformation (Fig. 3B) with an overall space–time–
yield of 4.6 g L−1 h−1
.
The same substrate load of 40 g l−1 for each sugar was also
used to produce 2-KG. The reaction was performed in a nutat-
ing compartment system with a breathable sealing to allow for
Promiscuous biocatalysts toward third generation chemical
production
better oxygen transfer. Following the experiment over time Besides D-xylose and L-arabinose rich lignocellulose, there are
showed that the production of 2-KG stagnated after 6 h other mixed sugar waste streams available. For example,
(Fig. S14†). HPLC analysis showed that no intermediates were D-glucose together with D-galactose are the main hexoses
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found in microalgae. These organisms are receiving increased simple kinetic parameters.22–24 Intermediates such as
attention due to their rapid growth in comparison with D-gluconate and D-galactonate were not observed, showing that
plants.34–36 Biomass of microalgae is also often found as waste PuDHT had high reactivity toward both sugar acids as opposed
in the production of high value compounds from these organ- to a previous system using SsDHAD. In the latter system,
isms.53 The approach of chemical production from microalgae accumulation of D-gluconate was observed in the first 10 h.56
biomass is widely known as a third-generation approach.34,36 Identical yields (>90%) were obtained compared to a system
Similar to D-xylose and L-arabinose, it is of practical interest to solely using D-glucose.39
enable the simultaneous conversion of D-glucose and
D-galactose.
In previous work, PuDHT had been used successfully to
Challenges and future prospects of the stereo-convergent
synthesis from biomass
produce ethanol from D-glucose.39 In this preliminary study,
the aptitude of PuDHT for this purpose had been tested. We
used a cell-free cascade to produce ethanol as a model
cascade. For this application, we used SsGDH instead of
HsXylDH2 to oxidize both sugars simultaneously due to its
higher thermostability. SsGDH was also used instead of
BsGDH (glucose dehydrogenase from Bacillus subtilis), the
enzyme used in a previous work, due to low promiscuity of the
latter (data not shown).39,40 Furthermore, the promiscuous
2-keto-3-deoxy-D-gluconate/D-galactonate from Picrophilus torri-
dus (PtKdgA) was used to catalyze the simultaneous aldol-clea-
vage of the intermediates from D-glucose and D-galactose
(Scheme S1†).54
Simple optimization showed that PuDHT together with
SsGDH and PtKdgA was able to facilitate ethanol production
from the mixed hexoses, and higher ethanol yield could be
achieved by using higher volumetric activity of either PuDHT
or D-glyceraldehyde dehydrogenase from Thermoplasma acido-
philum (Fig. S10†).55 After finding an optimized enzyme com-
bination, we followed the ethanol production from D-glucose
and D-galactose (Fig. 4). D-Galactose was consumed faster than
D-glucose according to the higher catalytic efficiency of SsGDH
toward D-galactose than D-glucose.40 Together with the result
of the two previous cascades, this shows the simplicity of pre-
dicting and, thus, modelling of cell-free approaches based on
For BDO production from D-xylose and L-arabinose, LlKdcA was
shown to be a bottleneck enzyme, possibly due to by-product
(CO2) inhibition. Further experiments by adding sodium bicar-
bonate in the reaction mixture resulted in lower BDO yield,
supporting this hypothesis (Fig. S12†). LlKdcA was not inhib-
ited by BDO up to 1 M (data not shown). An engineered LlKivD
variant was shown to be able to catalyze the decarboxylation
reaction at a high substrate load, which would be a promising
alternative for further studies.57 PuDHT, the key enzyme,
showed relatively low inhibition by BDO (I50
> 2 M)
(Fig. S13B†), thus being an already suitable biocatalyst for high
titer BDO production.
In this work, we also demonstrated that the nutating com-
partment system was compatible with LpNOX in the pro-
duction of 2-KG. Constant deactivation of LpNOX was likely
caused by its low thermostability, which was further supported
by the formation of protein clumps over time during 2-KG pro-
duction (Fig. 3B and S15†).48 Hence, future work will be
directed toward improving the thermostability of LpNOX or
other water-forming NOXs. Additionally, we began to search
for a new class of KdpD and to modify the existing D- and
L-KdpDs for promiscuity toward both KDPs, further reducing
the number of enzymes used.
In addition to both pentoses, D-glucuronate and
D-galacturonate are two major uronic acids found in plant
biomass.5,58,59 Two previous works have successfully converted
D-glucuronate to 2-KG and D-galacturonate to BDO via
KGSA.30,49 Therefore, to further reduce the cost of producing
chemicals from hemicellulose, future work will be directed
toward incorporating these uronic acids to our cell-free system
to convert simultaneously D-xylose, L-arabinose, D-glucuronate,
and D-galacturonate to BDO or 2-KG.
We have also demonstrated the compatibility of PuDHT to
convert D-glucose and D-galactose to ethanol via pyruvate as a
model system (Fig. 4). This cell free system thus showed a
potential for simultaneous biotransformation of both hexoses
to other chemicals. This will provide additional alternatives for
third-generation chemical production.34,36
Conclusions
D-Xylose and L-arabinose are the two major pentoses constitut-
ing hemicellulose. Utilization of these pentoses is one of the
keys for the success of second-generation chemical production.
In this work, a cell-free system was developed to transform
Fig. 4 Biotransformation of ethanol from D-glucose and D-galactose.
The reaction was performed at pH 7.0, 50 °C, 700 rpm. Error bars are
standard deviations from three independent replications. When errors
are too small they are covered by the markers and thus not displayed.
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both sugars simultaneously to chemicals of interest. 1,4-
Butanediol (BDO) was chosen as the primary target due to its
high market demand and it currently still being majorly pro-
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André Pick is the CEO of CASCAT GmbH, a biotechnology
company pursuing commercialization of bio-based processes.
20 J. D. McMillan and B. L. Boynton, Appl. Biochem.
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The authors would like to acknowledge partial funding from
the German Federal Ministry for Education and Research
through the HotSysAPP project (Grant No. 031L0078F) and
PFIFF project (Grant No. 031B0351C) and the European Union
through Blue Algae project (Interreg Project 41). The authors
are grateful for fruitful discussion with Samed Güner.
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