Totally atom-economical synthesis of lactic acid from formaldehyde: combined bio-carboligation and chemo-rearrangement without the isolation of intermediates

Article abstract of DOI:10.1039/d0gc02433c

Non-fermentative chemoenzymatic transformations have attracted great interest from both academia and industry. Here, we report a green chemoenzymatic cascade reaction that converts the C1 compound formaldehyde into lactic acid using a newly identified formolase variant and NaOH as catalysts with 100% atom economy and 82.9% overall yield under near-ambient conditions. The engineered formolase variant in this study exhibits a 19-fold substantially improved activity and improved formaldehyde resistance (up to 500 mM) and alters the main product from two-carbon glycolaldehyde (GA) to three-carbon dihydroxyacetone (DHA). The crystal structures of the parent formolase and identified variants were resolved to elucidate the molecular reason for the obtained improvement. Molecular dynamics simulation and molecular mechanics/generalized born surface area (MM/GBSA) analysis suggested that the identified amino acid substitutions allow more stable TPP-GA complexes in the active center of the dimeric formolase which is beneficial for the subsequent DHA generation. This journal is

Full text of DOI:10.1039/d0gc02433c

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DOI: 10.1039/D0GC02433C
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Totally atom-economical synthesis of lactic acid from
formaldehyde: combined bio-carboligation and chemo-
rearrangement without the isolation of intermediate
Received 00th January 20xx,
Accepted 00th January 20xx
Tianzhen Li,‡a,b Zijing Tang,‡b Hongli Wei,‡b Zijian Tan,b Pi Liu,b Jinlong Li,b Yingying Zheng,b
Jianping Lin,b Weidong Liu,b Huifeng Jiang,b Haifeng Liu,*c Leilei Zhu,*b and Yanhe Mab
DOI: 10.1039/x0xx00000x
Non-fermentative chemoenzymatic transformations have methanol,5 hydrogenation of CO2,6 partial hydrogenation of CO
attracted great interest of both academia and industry. Here, we and oxidation of methane.7
report a green chemoenzymatic cascade reaction that converts the
C1 compound formaldehyde into lactic acid using a newly identified
a) Microbial production of lactic acid (Fermentation)
OH
formolase variant and NaOH as catalysts with 100% atom economy
OH
OH
OH
O
O
and 82.9% overall yield under near ambient condition. The
engineered formolase variant in this study exhibits substantially
improved activity by 19-fold, improved formaldehyde resistance
(up to 500 mM) and altered the main product from two-carbon
glycolaldehyde (GA) to three-carbon dihydroxyacetone (DHA). The
crystal structures of the parent formolase and identified variant
were resolved to elucidate the molecular reason of the obtained
improvement. Molecular dynamics simulation and molecular
mechanics/generalized born surface area (MM/GBSA) analysis
suggested that the identified amino acid substitutions allow more
stable TPP-GA complex in the active center of the dimeric
formolase which is beneficial for the subsequent DHA generation.
To achieve the goal of reducing our dependence on fossil
resources and turning to sustainable biorefinery, it is essential
to find alternative feed stocks which can sustain large-scale
production of chemicals and fuels.1 C1 molecules including CO2,
CO, CH4, HCOOH, CH3OH and HCHO represent attractive long-
term feedstocks which can potentially sustain production of
fuels and fine chemicals at hundreds of millions of tons per
year.2 Among the cited C1 molecules, formaldehyde is a
particularly versatile and reactive compound which offers great
promise for the production of value-added chemicals.3 More
than 20 million tons of formaldehyde are produced annually
from various available sources,4 e.g. partial oxidation of
OH
Arabinose
OH
OH
OH
OH
OH
OH
Glucose
2-
OPO3
HO
OH
O
O
OH
OH
OH
Xylose
Retro-aldol cleavage
OH
OH
2-
2-
OPO3
OPO3
HO
O
ref 8a
O
OH
O
2-
OPO3
OH
OH
Retro-aldol cleavage
OH
O
O
O
2-O3PO
2-
OPO3
OH
OH
OH
b) Chemical conversion of glucose to lactic acid in water
(Al(III)-Sn(II) cations in H2O under 3 MPa N2 at 180°C for 2 h)
OH
OH
O
OH
O
HO
OH
OH
OH
OH
OH
OH
Glucose
Retro-aldol cleavage
OH
HO
OH ref 8b
O
OH
H2
O
- H2
O
O
O
OH
O
O
OH
OH
OH
O
O
OH
c) Chemical conversion of glycerol to lactic acid in organic solvent-water
(Ru-MACHO + NaOH in H2O/NMP at 140°C for 24 h)
Oxidation
ref 8c
HO
OH
OH
HO
OH
O
O
OH
OH
OH
O
Glycerol
- H2
O
O
O
O
OH
O
OH
d) This work (1. enzymatic carboligation in pH 7.4 KPi buffer at 30°C for 1 h;
2. chemical rearrangement in NaOH/H2O at RT for 19 h)
a. University of Chinese Academy of Sciences, Beijing 100049, China.
b. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin
300308, China. E-mail: zhu_ll@tib.cas.cn.
O
O
O
O
- H2
OH
O
Carboligation
H
OH
HO
O
O
H
OH
O
OH
Formaldehyde
c. Institute of Chemistry, University of Graz, Heinrichstrasse 28, Graz 8010, Austria.
E-mail: haifeng.liu@uni-graz.at
Scheme 1. Represented examples of lactate synthesis from
renewable sources.8 DHA is highlighted in red.
Lactic acid is an important chemical with wide applications in
food, pharmaceutical, cosmetic, detergent and dairy
†Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
‡These authors contributed equally.
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industries,9 as well as polymer synthesis, such as biodegradable rearrangement of DHA into lactic acid (2, Scheme 2) where DHA
View Article Online
polylactic acid, propylene glycol and acrylic polymers.10 was firstly dehydrated to pyruvic aldehyde and then isomerized
DOI: 10.1039/D0GC02433C
Currently, lactic acid are mainly produced through microbial to lactate via intramolecular Cannizzaro reaction under alkaline
fermentation of different carbohydrates, e.g. glucose, fructose condition.
and cellulose (Scheme 1a).8a Chemical transformation of For the initial step of this cascade,
a formolase (BFD-M3:
glycerol and sugars into lactic acid (Scheme 1b-c) have been W86R/N87T/L109G/L110E/A460M) was engineered through
reported and attracted much attention in recent years.8b,8c High iterative rounds of directed evolution to efficiently catalyze the
temperature and expensive metal catalyst are usually required carboligation of formaldehyde into DHA. The parent BFD-M3
for chemical synthesis of lactic acid. As shown in Scheme 1a-c, primarily converts formaldehyde into GA and shows a rather low
DHA is the key intermediate for lactate synthesis in both activity for DHA production.12b Due to limited structure-function
fermentative and chemical synthesis of lactic acid. In microbial relationship of formolase, directed evolution in microtiter plate
fermentation, DHA phosphate is generated through a series of format was performed to obtain improved formolase variants. A
enzymatic step using C5 or C6 sugar as carbon source.8a In coupled screening assay (Scheme S1) was developed to screen epPCR
chemical synthesis, DHA is produced either by retro-aldol libraries. The catalytic activity of formolase variants for DHA
fragmentation of hexose/pentose under harsh reaction generation was determined as following: the generated DHA is
condition8b or selective oxidation of glycerol with Au/CuO and oxidized by galactose oxidase (GalOD) and hydrogen peroxide (H2O2)
Pd catalyst. 11
is produced;13 in the presence of horseradish peroxidase (HRP), 2,2’-
Formolases catalyze the building of three-carbon DHA and two- azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) is oxidized by
carbon GA using formaldehyde as the unique substrate, which H2O2 to ABTS cation radical (ABTS•＋);14 the absorbance of ABTS•＋at
provides an alternative approach for DHA synthesis under mild 410 nm was recorded to quantify produced DHA by different
conditions. Formolase was computationally designed using variants. The two key performance parameters for identifying
benzaldehyde lyase (BAL, from Pseudomonas fluorescens biovar improved variants in microtiter-plate-based screening systems are
I) and benzoylformate decarboxylases (BFD, from Pseudomonas the linear detection range of the employed assay and the true
putida) as starting points, respectively.12 Formolase should be standard variation. A linear detection of DHA ranged up to 7.5 mM
further improved in terms of activity and resistance towards was achieved (Figure S1a). The optimized DHA detection assay in
formaldehyde for efficient biotransformation. Protein microtiter plate screen has a true coefficient of variation of 11.6%
engineering can address these issues and enable formolases to (Figure S1b).
better catalyze formaldehyde into desired products.
Table 1. Apparent kinetic constants of purified parent M3, variants
M5 and M6.a
kcat/Khalf
Formolase
kcat (s-1)
Khalf (M)
n
variants
M3
(s-1·M -1
)
0.018
0.105
0.171
3.7
±0.003
±0.012
±0.004
±0.7
0.048
0.061
0.787
11.2
±0.8
M5
±0.006
±0.020
±0.117
Scheme 2. Two-step chemoenzymatic synthesis of lactate from
formaldehyde through formolase-catalyzed carboligation and NaOH-
promoted rearrangement.
0.343
0.218
1.573
1.1
M6
±0.059
±0.043
±0.392
±0.2
Among the reported reaction mechanisms of converting DHA to
lactic acid, dehydration and the subsequent rearrangement of
DHA in aqueous alkaline solution (Scheme 1c) attracted our
attention. The integration of chemical reactions with
biotransformation can contribute to shifting the raw material
supply from a petrochemical to a biorenewable basis. Here, we
report the first chemoenzymatic synthesis of lactic acid from
formaldehyde. The cascades start with the formolase-catalyzed
carboligation of formaldehyde into DHA (1, Scheme 2) with GA
as intermediate, subsequently followed by NaOH-promoted
a The initial-velocity data were fitted to allosteric sigmoidal equation
(v=Vmax×Sn/(Khalfn+Sn), where v is the initial enzyme velocity, S is
substrate concentration, Vmax is the maximum enzyme velocity, Khalf
is the ligand concentration at which half of the active sites are
occupied (concentration of half saturation). The experiments were
performed in presence of 0.5 mM TPP.
2 | J. Name., 2012, 00, 1-3
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After screening of ~8000 clones within three rounds of directed
evolution, three most beneficial substitutions including Ser26Phe,
Gly109Ser and His281Tyr were identified and the respective
variants were named M4 (W86R/N87T/L109G/L110E/A460M
/H281Y), M5 (W86R/N87T/L109G/L110E/A460M/H281Y/S26F)
and M6 (W86R/N87T/L110E/A460M/H281Y/S26F/G109S). The
parent BFD-M3, variants M5 and M6 were characterized in
detail for their kinetic parameters at 30℃ (Figure S2). As shown
in Table 1, the best variant M6 improves the kcat value by 19.0-
fold (0.343 s-1) compared to M3 (0.018 s-1). Furthermore, variant
M6 also show an obvious shift in the product distribution. The
DHA/GA ratio increased from 1:3.1 of the parent M3 to 6.2:1 of
the variant M6 (Figure 1). DHA becoming the main product is
very beneficial for its subsequent utilization.
View Article Online
DOI: 10.1039/D0GC02433C
Figure 2. Thermal inactivation profiles of parent M3, variants M5 and
M6. Residual activities of M3 ( ▲ ), variants M5 (●), M6 ( ◆ ) were
measured after heat treatment (4 h) at varied temperature (36℃-
65℃).
As shown in Figure 3, the DHA yield of M3 and M6 was also
investigated with varied concentration of formaldehyde (200 mM
and 500 mM) at different time points. When the formaldehyde
concentration is 200 mM and 500 mM, M3 generated DHA mainly in
the first 2 h and maintained steady afterwards, while M6 was able to
continue the DHA synthesis until 10 h. M6 possesses higher tolerance
towards substrate formaldehyde which is a great advantage for its
application in formaldehyde transformation. To the best of our
knowledge, M6 presents highest activity for DHA generation at
formaldehyde concentration higher than 200 mM compared to other
reported formolases.15 Formolase was reported as one of the
biocatalysts in cascade reactions to produce sugars (e.g.
dendroketose, L-erythrulose and L-sorbose) with DHA as an
intermediate.15b,16 Up to 40 mM formaldehyde was used as substrate
due to the reason that conversion rate significantly decreased when
formaldehyde was > 60 mM. Further improvement of M6 for the
resistance towards higher concentration of formaldehyde is
beneficial for its potential application.
Figure 1. The amount of GA (white bar) and DHA (black bar) produced
by the parent M3, variants M4, M5 and M6. The reaction was carried
out with 0.027 mM enzyme and 100 mM formaldehyde at 30℃ for 3
h.
Remarkably, identified variants also showed improved thermal
resistance, as shown by residual activity measurement after heat
treatment at varied temperatures for 4 h (Figure 2). The half-
inactivation temperatures (Tm) were found to be 46.5℃ (M3), 52.3℃
(M5), 56.0℃ (M6), respectively. The most thermally resistant variant
M6 had a 9.5℃ higher Tm value than the parent M3. Unfortunately,
there are no Tm values reported for other purified formolase for
comparison. To determine the secondary structural integrity, CD
spectra of each variant was recorded after heat incubation at varied
temperatures between 36℃ to 55℃ (Table S1). With increased
temperature, the absorbance of the CD spectra (from 190 to 240 nm)
was reduced, thus indicating at least a partial unfolding of α-helical
structures (Figure S3). The α-helical content of variants at 36℃ (23.8-
24.2%) and at 55℃ (3.0-9.4%) different significantly. Overall, M6 lost
less α-helical content than M3 at elevated temperature, indicating
that M6 is more thermal stable than the parent M3 which supports
the thermal inactivation profile in Figure 2.
Figure 3. The yield of DHA generated by variant M6 under 200 mM
(●), 500 mM (■) formaldehyde and parent M3 under 200 mM (○), 500
mM (□) formaldehyde at varied reaction time. The reaction was
carried out with 0.027 mM of the tested proteins.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Inspired by previous report on lactic acid generation through d YLF is the yield of lactic acid from formaldehyde.
View Article Online
DOI: 10.1039/D0GC02433C
chemical catalysis,17 we decided to use hydroxides as catalyst for the
conversion of DHA into lactic acid. Investigation was firstly
performed for the effect of the different alkaline metal hydroxides
(NaOH, KOH) and alkaline-earth metal hydroxides (Ba(OH)2, Ca(OH)2)
on the yield of lactic acid from commercial DHA. NaOH and KOH were
dissolved in aqueous solution, while Ba(OH)2 and Ca(OH)2 were used
as suspension due to their poor solubility. Both NaOH and KOH
showed higher activity for lactic acid formation at room temperature
(Figure 4).
e
Atom economy = (molecular mass of desired product / molecular
mass of all reactants) × 100. Atom economy of lactic acid from
formaldehyde = (molecular mass of lactic acid) / (3×molecular mass
of formaldehyde) × 100.
With the engineered formolase variant M6 and NaOH as catalysts, a
chemoenzymatic synthesis of lactic acid from one-carbon
formaldehyde was successfully constructed. 300 mM formaldehyde
was used as initial substrate. As shown in Table 2 and Figure S5, M6
effectively catalyzed the polymerization of formaldehyde with a DHA
yield of 91.7%. In the subsequent chemical rearrangement step
catalyzed by NaOH, the enzyme-free solution containing the
generated DHA was added through a dispensing funnel to NaOH
aqueous solution at room temperature. DHA was transformed to
lactic acid and the final yield of lactic acid from formaldehyde was
82.9%. The atom economy of this chemoenzymatic system was 100%.
The excellent atom efficiency (100%) and final yield (82.9%) verify
that this chemoenzymatic approach is a promising approach for lactic
acid production.
To investigate the mechanistic principles underlying the improved
activity and the shift in GA/DHA ratio of the identified variant M6,
crystal structures of the parent M3 (PDB ID: 6M2Z) and variant M6
(PDB ID: 6M2Y) were solved with the cofactor TPP. As the active
center of BFD is located in the monomer-monomer interface,20 the
structural analysis in this study were performed using the active
dimer. The three beneficial substitutions Ser26Phe, Gly109Ser and
His281Tyr (Figure 5b) are all located in the monomer-monomer
interface and in the proximity of active center. The MM/GBSA
analysis was performed after molecular docking of TPP-GA complex
and molecular dynamics simulation. 1.5-fold higher binding free
energy of the monomer-monomer interaction was observed for M6
compared to the parent M3, indicating an improved monomer-
monomer interaction in M6 (Table S2) which can stabilize the
functional dimer structure. Structure analysis revealed that
substitutions Ser26Phe and His281Tyr tend to form a new π-π
stacking interaction between chain A and chain B. The enhanced
interaction between Ser26Phe and His281Tyr can stabilize the
monomer-monomer interaction which is beneficial for the
architecture of the active center. Furthermore, substitution
Ser26Phe also leads to increased interaction with the hydrophobic
patch (Leu461, Phe464, Val467 and Leu468) of the neighboring
monomer, which can further stabilize the active dimer structure. All
the above-mentioned interface interactions must give rise to the
stable existence of the functional dimer structure and make variant
M6 more active and stable compared the parent M3. The MM/GBSA
analysis21 was also applied to calculate the binding free energy
(ΔGbind) of TPP-GA complex to the active sites of parent M3 and
variant M6 (Table S2). The ΔGbind value of TPP-GA binding to the
dimeric M6 is 1.2-fold higher than the parent M3 (-165.3 kcal/mol vs
-133.2 kcal/mol). The higher binding affinity of TPP-GA to M6
suggests more stable TPP-GA complex in the active site, which
facilitates the subsequent DHA generation. This observation explains
the mechanistic principle underlying the shift in GA/DHA ratio of M6.
Figure 4. The yield of lactic acid from commercial DHA (50 mM)
catalyzed by NaOH, KOH, Ba(OH)2 and Ca(OH)2 at room temperature
for 24 h.
After 24 h, DHA in NaOH and KOH solution were completely
consumed and the major product was lactic acid (Figure S4a). The
yield of lactic acid from DHA with NaOH as catalyst was highest
(99.7%) compared to KOH (83.2%), Ba(OH)2 (30.7%) and Ca(OH)2
(14.7%). NaOH led to fewer by-products compared to KOH, Ba(OH)2
and Ca(OH)2 (Figure S4). According to previous reports, the by-
products included glyceric acid, glycolic acid, formic acid, acetic
acid.17a The yield of lactic acid from DHA catalyzed by NaOH in this
study is also higher than several reported metal salts catalysts (AlCl3,
ZnSO4)18 and heterogeneous catalysts (Carbon−Silica catalysts,
Zeolite catalysts H-USY and Sn-Beta),19 which required high reaction
temperature. Therefore, NaOH is the optimal hydroxide catalyst for
lactic acid formation from DHA and it was used as the catalyst in the
cascade reaction.
Table 2. The yield of DHA and lactic acid from formaldehyde.a
Atom
Formaldehyde
(mM)
YDF
YLD
YLF
economy
(%) b
(%) c
(%) d
(%) e
300
91.7±3.7 90.4±1.0
82.9±4.3
100
a
The generation of DHA from formaldehyde (first step) was
performed with 300 mM formaldehyde as substrate at 30℃ for 1h.
The yield of lactic acid from DHA produced by the first step was
performed under 2.5
temperature for 19 h.
M NaOH aqueous solution at room
b YDF is the yield of DHA from formaldehyde.
c YLD is the yield of lactic acid from DHA.
4 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/D0GC02433C
COMMUNICATION
Author contribution
Z. Tang, T. L. and H. W. contributed equally to this work. Z. Tang
and T. L. identified improved formolase variants and performed
characterization. T. L. and H. L. integrated chemical catalysis
with enzymatic carboligation and set up chemoenzymatic
approach. P. L., J. Li and Z. Tan performed molecular dynamic
simulation and molecular mechanics/generalized born surface
area (MM/GBSA) analysis. H. W., Y. Z. and W. L. solved the
crystal structure of formolase variants. L. Z. and H. L. conceived
and designed the overall experiments. The draft of manuscript
was mainly written by T. L., H. L. and L. Z., Y. M., J. Lin and H. J.
revised the manuscript. L. Z. supervised the project and finally
approved the current version.
Figure 5. Structural alignment of the parent BFD-M3 (PDB code:
6M2Z) and variant M6 (PDB code: 6M2Y). Chain A of M3 and M6 are
in green and pale green, respectively; chains B of M3 and M6 are in
cyan and light teal, respectively. The cofactor TPP is shown in ball
(red). Image (a) shows the overall dimeric structures of M3 and M6.
The substitutions Ser26Phe, Gly109Ser and His281Tyr as well as
residues Leu461, Phe464, Val467 and Leu468 are located in the
circles. (b) to (d) represent the detailed view of the circled area of
image (a). Image (b) shows the three substitutions of Ser26Phe,
Gly109Ser and His281Tyr in M6 located in the monomer-monomer
interface. Image (c) shows hydrophobic interaction between
substitution of Ser26Phe of Chain B and residue Leu461, Phe464,
Val467 and Leu468 of chain A. Image (d) emphasizes the π-π stacking
interaction between Ser26Phe of Chain B and H281Y of Chain A. The
figure was rendered using PyMOL (TM) Molecular Graphics System
(version 2.3.0).
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by grants of National Key Research and
Development Program of China (2018YFA0901500) and the Key
Research Program of the Chinese Academy of Sciences (ZDRW-ZS-
2016). Y. Z and W. L. thank the final support of Youth Innovation
Promotion Association CAS. We thank Shanghai Synchrotron
Radiation Facility (SSRF) and National Facility for Protein Science in
Shanghai (NFPS) for beam time allocation and data collection
assistance.
In summary, a two-step chemoenzymatic approach for converting
formaldehyde into lactic acid was established using an engineered
formolase and NaOH as catalysts at temperature ≤ 30℃ with perfect
atom-economy efficiency and overall yield. The chemoenzymatic
synthesis of lactic acid will open a new route for the lactic acid
synthesis from C1 compound. The engineered formolase variant M6
displays highly improved efficiency in the carboligation of
formaldehyde, benefiting from its increased activity and resistance
towards formaldehyde as well as shifted product spectrum. Crystal
structure analysis, molecular dynamics simulation and MM/GBSA
analysis provided a molecular explanation for the improvement of
engineered formolase variant M6, demonstrating that the beneficial
amino acid substitutions lead to stabilized dimerization of M6 and
improved binding of TPP-GA complex to the active center which
promotes the DHA generation.
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