Characteristics of l-threonine transaldolase for asymmetric synthesis of β-hydroxy-α-amino acids

Article abstract of DOI:10.1039/c9cy01608b

l-Threonine transaldolase (LTTA) is a putative serine hydroxymethyltransferase (SHMT) that can catalyze the trans-aldehyde reaction of l-threonine and aldehyde to produce l-threo-β-hydroxy-α-amino acids with excellent stereoselectivity. In the present study, an l-threonine transaldolase from Pseudomonas sp. (PsLTTA) was mined and expressed in Escherichia coli BL21 (DE3). A substrate spectrum assay indicated that PsLTTA only consumed l-threonine as the donor substrate and could accept a wide range of aromatic aldehydes as acceptor substrates. Among these substrates, PsLTTA could catalyze p-methylsulfonyl benzaldehyde and l-threonine to produce l-threo-p-methylsulfonylphenylserine with a high conversion rate (74.4%) and a high de value (79.9%). The conversion and stereoselectivity of PsLTTA were found to be dramatically influenced by the concentration of the whole cell, the co-solvent and the reaction temperature. Through conditional optimization, l-threo-p-methylsulfonylphenylserine was obtained with 67.1% conversion and a near-perfect de value (94.5%), the highest stereoselectivity for an l-threo-β-hydroxy-α-amino acid so far reported by enzymatic synthesis. Finally, synthesis of l-threo-p-methylsulfonylphenylserine at a 100 mL scale by whole-cell biocatalysis was conducted. This is the first systematic report of l-threonine transaldolase as a robust biocatalyst for preparation of β-hydroxy-α-amino acids, which can provide new insights for β-hydroxy-α-amino acids synthesis.

Full text of DOI:10.1039/c9cy01608b

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Characteristic of L-threonine transaldolase for asymmetric synthesDisOIo: 1f0.1039/C9CY01608B
β-hydroxy-α-amino acids
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Lian Xu 1, 2, #, Li-Chao Wang 1, 2, #, Xin-Qi Xu 2, Juan Lin 1, 2 *
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1 College of Chemical Engineering, Fuzhou University, Fuzhou 350116, China.
2 College of Biological Science and Engineering, Fuzhou University, Fuzhou 350116,
China.
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# These authors contribute equally to this study.
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* To whom correspondence should be addressed: ljuan@fzu.edu.cn (Juan Lin).
Running title: L-threonine transaldolase for synthesis of β-hydroxy-α-amino acids

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Abstract
L-threonine transaldoase (LTTA) was a putative serine hydroxymethyltransferase
(SHMT) that could catalyze the trans-aldehyde reaction of L-threonine and aldehyde
to produce L-threo-β-hydroxy-α-amino acids with excellent stereoselectivity. In
present study, a L-threonine transaldolase from Pseudomonas sp. (PsLTTA) was
mined and expressed in Escherichia coli BL21 (DE3). Substrate spectrum assay
indicated that PsLTTA only consumed L-threonine as donor substrate and could
accept a wide range of aromatic aldehydes as acceptor substrates. Among those
substrates, PsLTTA could catalyze p-methylsulfonyl benzaldehyde and L-threonine to
produce L-threo-p-methylsulfonylphenylserine with high conversion (74.4%) and
high de value (79.9%). It was found that the conversion and stereoselectivity of
PsLTTA were dramatically influenced by concentration of whole-cell, co-solvent and
reaction
temperature.
By
conditional
optimization,
L-threo-p-methylsulfonylphenylserine was obtained with 67.1% conversion and near
perfect de value (94.5%), the highest stereoselectivity of L-threo-β-hydroxy-α-amino
acids reported by enzymatic synthesis so far. Finally, synthesis of
L-threo-p-methylsulfonylphenylserine in a 100 mL scare by a whole-cell biocatalyst
was conducted. This was for the first time systemically reported that L-threonine
transaldolase as a robust biocatalyst for preparation of β-hydroxy-α-amino acids
which could provide new insights for β-hydroxy-α-amino acids synthesis.
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1. Introduction
β-hydroxy-α-amino acids composed of numerous important building blocks which
were essential for the synthesis of agriculturally or medicinally bioactive products.1-3
For
example,
some
β-hydroxy-α-amino
acids
such
as
L-threo-p-methylsulfonylphenylserine and L-threo-p-nitrophenylserine were known
as key intermediates of thiamphenicol and chloramphenicol, respectively.4,5
L-threo-phenylserine could be found in cyclomarin A, a marine cyclopeptide with
anti-tuberculosis
and
anti-malaria
activities.6
In
addition,
L-threo-3,4-dihydroxyphenylserine (Droxidopa) itself served as a crucial drug for the
treatment of Parkinson’s disease. 7 As a result, β-hydroxy-α-amino acids had attracted
extensive attentions because of their important role in chemical synthesis and
pharmaceutical manufacturing.
β-hydroxy-α-amino contained two-chiral centers and four isomers (L-threo, D-threo,
L-erythro and D-erythro) were produced simultaneously during the chemical
8,9
processes.
This made optically pure of β-hydroxy-α-amino acid difficult to
synthesize. Many approaches including Sharpless asymmetric dihydroxylation,
epoxidation or enzymatic aldol addition had been developed for the synthesis of
β-hydroxy-α-amino acids.10-12 Among them, enzymatic approach was most promising
as it could establish two new chiral-centers in a single reaction with inexpensive
12
substrates as well as under mild and unprotected conditions. Threonine aldolases
(TAs) were such a kind of enzymes that catalyzed the aldol addition of glycine and
various aldehyde to produce β-hydroxy-α-amino acids. TA was a pyridoxal

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5'-phosphate (PLP)-dependent enzyme and could be classified into LTA and DTA
based on the stereospecificity at the Cα of its products.13-15 In most cases, TA was
highly stereoselective (>99% enantiomeric excess, ee) at the Cα while showed
moderate stereoselectivity at the Cβ (about 10-45% diastereomeric excess, de).16 As a
result, both threo and erythro isomers were produced in a TA-catalyzed reaction. This
“Cβ problem” had greatly hampered and limited the synthetic application of TA. One
possible reason for the poor stereoselectivity at Cβ was that two active histidine
residues in TA could both interact with the hydroxyl group on Cβ from the opposite
direction.16 It was noteworthy that up to now, attempts to improve the Cβ
stereoselectivity of TA by protein engineering had ended in failure mainly due to the
complicated interactions in the enzyme active center participating in the aldol addition
reactions.14,17,18 DTA was reported could obtain a high stereoselectivity by keeping
the reactions under kinetically control (usually in conditions of low temperature and
low amount of enzyme).19,20 Unfortunately, almost all of active intermediates were
L-threo isomers and only produced by LTA4-7. Recently, Chen et al. had developed a
high-throughput screening method and improved the stereoselectivity of LTA.
However, the de value of mutant enzyme was up to about 74% which was still too
low for the industrial application.18 As a result, the mining and characterization of
novel L-threonine aldolase-like enzyme with high stereospecificity was requisite to
meet the needs of industrial production as well as to expand toolkit for asymmetric
synthesis of β-hydroxy-α-amino acids.

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In recent years, L-threonine transaldoase (LTTA) was reported as a meDmObI: e10r.10o3f9/C9CY01608B
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antibiotic gene cluster and participated in the biosynthesis of antibiotics.
The
characteristic of LTTA was that it could catalyze the formation of β-hydroxy-α-amino
acids with high stereoselectivity at Cβ using L-threonine and aldehyde as substrates. 22
Phylogenetic analysis indicated that LTTA, LTA and serine hydroxymethyl
transferase (SHMT) shared a common evolutionary origin and thus might have a
similar catalytic mechanism.22 In present study, we characterized a LTTA form
Pseudomonas sp. with excellent stereoselectivity in β-hydroxy-α-amino acids
synthesis. It showed that PsLTTA only consumed L-threonine as donor substrate and
could accept a wide range of aromatic aldehydes as acceptor substrates. The
conversion and de value of L-threo-p-methylsulfonylphenylserine by PsLTTA were
up to 67.1% and 94.5% respectively after reaction conditions optimization. Finally,
synthesis of L-threo-p-methylsulfonylphenylserine in a 100 mL scare by a whole-cell
biocatalyst was conducted. Taken together, our results systematic evaluated the
catalytic property of PsLTTA and suggested that PsLTTA could serve as a promising
biocatalyst for preparation of L-threo-β-hydroxy-α-amino acids.
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2. Results and discussion
2.1 Sequence analysis and expressing of PsLTTA
PsLTTA was identified as a SHMT-like enzyme as it showed about 26-33%
identity in amino acid sequence with SHMTs (Table S1).
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Multiple sequences alignment demonstrated that the PLP-binding residues were quite
conservative among PsLTTA and SHMTs, suggesting that they might have a similar
catalytic mechanism (Fig S1). This was consistent with the previous report that LTTA
and SHMT shared a common evolutionary origin.22
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PsLTTA was inducing expressed under the optimum conditions (0.2 mM IPTG,
28℃, 16h) and further purified with His-tag affinity chromatography. The purified
PsLTTA revealed a pink color in solution (Fig S2A). SDS-PAGE indicated that most
of PsLTTA was soluble protein and its monomer molecular weight was about 48 kDa,
which was in accordance with the predicted molecular size (Fig S2B). PsLTTA could
catalyzed the trans-aldehyde reaction of L-threonine and aldehyde to produce
β-hydroxy-α-amino acids and acetaldehyde (Fig S3). Transaldolase activity assay
using p-methylsulfonyl benzaldehyde and L-threonine as substrates demonstrated that
the cell-free extracts and purified enzyme had specific activities of 0.87 U mg−1 and
1.32 U mg−1, respectively.
2.2 Enzymatic properties
Thermostability profile indicated that PsLTTA was quite stable in the range of
4-30℃. The enzymatic activity of PsLTTA was rapidly decreased when the
temperature was above 37℃ and was complete inactivation at 60℃ (Fig 1A). The

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optimum reaction temperature of PsLTTA was determined at 30℃ (Fig 1B). PsLTTA
was active at pH 6.0-8.0 as more than 80% activity could be detected (Fig 1C). The
maximum activity of PsLTTA was observed at 100 mM potassium phosphate buffer
with a pH value of 7.0 (Fig 1D). Those results suggested that PsLTTA was a pH and
temperature sensitive enzyme. The effects of metal ions on the activity of PsLTTA
were further investigated at concentrations of 1 mM and 10 mM. As shown in Fig 1E,
the transaldolase activity of PsLTTA was slightly promoted by Mg2+ and Li+ when
compared to the control group. Ni2+, Cu2+, Fe2+ and Zn2+ could inhibit PsLTTA both
in 1 mM and 10 mM. Other metal ions such as Cs2+, Ba2+, Ca2+ and Mn2+, showed
little effect on PsLTTA in 1 mM but a moderate inhibition in 10 mM. PLP
concentration assay suggested that PLP was required for the catalysis of PsLTTA
since a PLP-free group showed 65.8% activity compared to the control group (250
μM PLP) (Fig 1F). This result was similar to the previous report that LTTA was a
PLP-dependent enzyme.24 The partial activity in PLP-free group was likely on
account that the enzyme itself combined with a portion of PLP during the process of
folding intracellularly.
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2.3 Substrate specificity of PsLTTA
The donor substrate specificity of PsLTTA was evaluated with p-methylsulfonyl
benzaldehyde as the acceptor. The Km and kcat of PsLTTA using L-threonine as donor
substrate was 9.85 mM and 63.4 min−1 while PsLTTA was not active toward
L-allo-threonine, D-threonine, D-allo-threonine, L-serine and glycine (Table S2 and
Fig S4). Similar to the enzyme assay results, HPLC analysis indicated that PsLTTA

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could catalyze p-methylsulfonyl benzaldehyde and L-threonine to form
L-threo-p-methylsulfonylphenylserine with high stereoselectivity (80.1% de) (Fig 2A).
However, no products were detected when using L-allo-threonine, D-threonine,
D-allo-threonine, and glycine as donor substrates (Fig 2C-2F). PsLTTA could also
catalyzed the trans-aldehyde action of p-methylsulfonyl benzaldehyde and L-serine,
however, the transaldolase activity sharply declined and the major product was
L-erythro-p-methylsulfonylphenylserine (-43.8% de) (Fig 2B). Those data suggested
that PsLTTA was high selective in donor substrate. Coincidentally, the configuration
of L-threonine was L-threo form which was the same as the major product, this
finding let us speculated that the strict donor specificity might contribute to the high
stereoselectivity of PsLTTA.
The acceptor substrate specificity was further assessed using L-threonine as the
donor. We evaluated the catalytic activity of PsLTTA on aliphatic aldehydes such as
formaldehyde or acetaldehyde. The results showed that PsLTTA could not consume
those two aldehydes mainly due to their oxidability. It demonstrated that PsLTTA
could utilize a wide range of aromatic aldehydes as acceptor substrate to produce
β-hydroxy-α-amino acids. As expected, all the major products were L-threo forms
(Table 1). This was quite important since the active intermediates of thiamphenicol,
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chloramphenicol and Droxidopa were L-threo isomers.
We found the substituent
type on the phenyl ring of aromatic aldehydes could affect the activity of PsLTTA.
For example, PsLTTA could catalyze aromatic aldehydes with electron-withdrawing
substituents such as -CH3SO2, -NO2, -F, -Cl and -Br, while electron-donating

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substituents (-CH3, -OCH3 and -OH) could not serve as the substrates of PsLTTA. For
-Cl and -Br substituted benzaldehydes, it showed that PsLTTA only catalyzed
o-substituted benzaldehydes, suggesting that the position of substituent also had an
effect on PsLTTA. Those results were quite similar when using DTA to catalyze
aromatic aldehydes and glycine. Chen et al. reported that the type of substituting as
well as the position on the phenyl ring played an essential role in the catalytic
performance of DTA by affecting the interaction between the β-OH-group of the
substrate and the manganese ion during the process of substrate recognition.20 This
conclusion seemed to be useful for PsLTTA and could be the basis for protein
engineering of PsLTTA.
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It was worth mentioning that PsLTTA could catalyze p-methylsulfonyl
benzaldehyde with a high conversion (74.4%) and high stereoselectivity (79.9% de).
The product L-threo-p-methylsulfonylphenylserine was the main intermediate of
thiamphenicol and florfenicol. L-threonine aldolase (LTA) also reported that could
catalyze the formation of p-methylsulfonylphenylserine using p-methylsulfonyl
25
benzaldehyde and glycine as substrates.
We assessed the catalytic activity and
stereoselectivity of PsLTTA and a LTA from Clavibacter michiganensis (ClLTA).
The conversion of p-methylsulfonyl benzaldehyde by PsLTTA and ClLTA were 75.2%
and
85.6%,
respectively.
However,
the
de
value
of
L-threo-p-methylsulfonylphenylserine by PsLTTA was 79.6%, much higher than that
of ClLTA with a de value of 28.6% (Fig S5). This result suggested that PsLTTA was
more stereoselective compared to ClLTA. Furthermore, the reverse activities of

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PsLTTA and ClLTA were evaluated using L-threo-p-methylsulfonylphenylserine as
substrate. As shown in Fig S6, ClLTA could total change
L-threo-p-methylsulfonylphenylserine into p-methylsulfonyl benzaldehyde and
glycine within 3 hours. This result was consistence with the conclusion that
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LTA-catalyzed reaction was reversible.
activity of PsLTTA was
To our surprise, the reverse transaldol
undetectable when using
almost
L-threo-p-methylsulfonylphenylserine and acetaldehyde as substrates, suggesting the
LTA-catalyzed reaction might not a reversible reaction. In LTA catalyzed-reaction,
the excess substrate (usually glycine was 10 folds to aldehyde) was required to make
sure that the reaction equilibrium shifted in the direction of producing
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β-hydroxy-α-amino acids, which resulted in a waste of substrates. In PsLTTA, the
use of superfluous substrates could be avoidable as its poor reverse transaldol activity.
Considering the stereoselectivity and substrate availability, it revealed that PsLTTA
was more suitable for industrialized application of producing β-hydroxy-α-amino
acids.
2.4 Effects of reaction conditions on conversion and stereoselectivity of PsLTTA
Although the enzyme activity assay indicated that PsLTTA achieved the
maximum activity at phosphate buffer (Fig 1D), it revealed that the transformation of
β-hydroxy-α-amino acid by whole-cell biocatalyst could be improved more than 30%
when using Tris-HCl buffer (100 mM Tris-HCl, pH 7.0) as reaction buffer. This result
suggested Tris-HCl buffer was more suitable for whole-cell biocatalyst and was used
in the following reactions. 10% acetonitrile (v/v) was selected for co-solvent since

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p-methylsulfonyl benzaldehyde dissolved well in acetonitrile anDdOI: 10.t10h3e9/C9CY01608B
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optimal reaction temperature (30 ℃) was used as initial reaction temperature.
We first evaluated the concentration of wet cells (3 to 50 mg/ml) and substrate (10
to 40 mM) on the conversion and stereoselectivity of PsLTTA using p-methylsulfonyl
benzaldehyde and L-threonine as substrates. As shown in Fig 3, similar trends were
constantly observed that the increase of wet cells led to a decrease of stereoselectivity
in PsLTTA under different substrate concentration. The de values of products were
about 86.1% when the wet cells concentrations were 3.0 and 6.25 mg/ml. Then the de
values declined from 86.1% to 72.0% with the increase of wet cells concentrations
from 6.25 up to 50 mg/ml. Those results strongly indicated that low dose of wet cells
contributed to the formation of L-threo isomer. The 3.0 and 6.25 mg/ml of wet cells
concentrations had little difference in the stereoselectivity of PsLTTA, but greatly
affected its conversion (about 5-22% improvement in conversions), suggesting that
6.25 mg/ml of wet cells was suitable for achieving high stereoselectivity performance.
The concentration of substrates affected the conversion of PsLTTA but not its
stereoselectivity. Under 6.25 mg/ml wet cells concentration, the conversions fall from
90.1% to 50.5% when the substrate concentrations ranged from 10 mM to 40 mM.
We found that the products could reach the maximum yield at 30 mM substrate with a
conversion of 69.5%. Based on those results, 6.25 mg/ml and 30 mM were selected as
the final concentration of wet cells and substrate, respectively.
The effects of co-solvents were further investigated. As shown in Table 2, the none
organic solvents group gave a comparatively low conversion (41.6%) and de value

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(71.3%), suggesting that organic solvents were required for the catalysis of PsLTTA.
It demonstrated that low percentage of organic solvents (10%) greatly promoted the
conversion of PsLTTA while high percentage of organic solvents (30%) leaded to a
decline of conversion. Among the tested organic solvents, high conversion (80.4%)
and de value (90.2%) were obtained when using 10% ethyl acetate as co-solvents. 10%
MeOH and 10% DMSO exerted promotion effects on the conversion but showed little
enhancements on the stereoselectivity of PsLTTA when compared to 10% CH3CN
group. The addition of 10% acetone and 10% EtOH groups both lowered the
conversion and the stereoselectivity of PsLTTA. Thus, 10% ethyl acetate was selected
as an optimal co-solvent for enzymatic reaction.
The trans-aldehyde reaction of PsLTTA was evaluated under different temperature
for 6 hours. As shown in Fig 4, the highest conversion of products (79.8%) were
observed at 30℃, which was consistence with the conclusion that the optimum
reaction temperature of PsLTTA was 30℃. For stereoselectivity, we surprising found
that increasing temperature from 10℃ to 35℃ led to a reduction of stereoselectivity in
PsLTTA. The de values had little change (about 94.5%) but the conversion rates were
rapidly increased from 44.7% to 67.8% at the temperature of 10, 15 and 20 ℃,
respectively. The de values sharply declined (from 94.5% to 87.2%) when the
temperature was 25, 30 and 37℃. Those results indicated that lower temperature
contributed to the formation of L-threo isomer and suggested 20 ℃ as the best
temperature for catalysis reaction of PsLTTA.
Taken together, our results strongly suggested that low concentration of wet cells

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(6.25 mg/ml), co-solvent (10% ethyl acetate) and low temperature (20 ℃) could
dramatically improve the stereoselectivity of PsLTTA with a de value from 71.3% up
to 94.5%. Our conclusion that low concentration of wet cells and low temperature
contributed to the stereoselectivity of PsLTTA was exactly similar to DTA.20 Under
such conditions, the catalysis reaction was under kinetic control but not
thermodynamic control which was benefit for producing L-threo isomer. 27
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In recent years, lots of attentions were put on TAs as they were the only reported
aldolases for production β-hydroxy-α-amino acids using cheap substrates (glycine and
aldehyde) and under mild conditions. However, the poor stereoselectivity at Cβ (about
10-45% de) severely blocked its industrialized application.16 In present study, the
characterized PsLTTA not only displayed TAs-like merits such as inexpensive
substrates and mild conditions, but also excellent stereoselectivity at Cβ (94.5% de)
and poor reverse transaldol activity. Under the optimized conditions, we further
assessed the stereoselectivity of PsLTTA and ClLTA on other aromatic aldehydes
(Table 3 and Fig S7-14). Similar to p-methylsulfonyl benzaldehyde, PsLTTA could
consume multiple aromatic aldehydes to produce β-hydroxy-α-amino acids with
excellent stereoselectivity. Among those products, L-threo-p-nitrophenylserine (92.8%
de), L-threo-phenylserine (89.1% de), L-threo-o-chlorophenylserine (87.1% de) and
L-threo-o-bromophenylserine (86.9% de) were obtained with excellent
stereoselectivity. The de values of those products were much higher than that
catalyzed by PsLTTA before reaction optimization (Table 2) or by ClLTA (Fig
S7-14). Those results demonstrated that the optimized reaction conditions were

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universally applicable for aromatic aldehydes with different substituents. Considering
that L-threo-p-nitrophenylserine was widely reported as an intermediate of
chloramphenicol and L-threo-phenylserine could be found in serval bioactive
4-6
substances,
it strongly suggested that PsLTTA might take TA’ s place as a
promising and robust biocatalyst for production of useful β-hydroxy-α-amino acids.
2.5 Synthesis of L-threo-p-methylsulfonylphenylserine by whole-cell biocatalyst
A 100-mL scale reaction was then carried out under the optimal reaction conditions,
and time courses of transformation of L-threo-p-methylsulfonylphenylserine were
monitored from 0 to 24 h. As expected, L-threo-p-methylsulfonylphenylserine were
produced with excellent stereoselectivity (90.4% de). The conversion rate was 67.1%
and the concentration of p-methylsulfonyl benzaldehyde decreased from 31.0 mM to
10.2 mM within 24 hours. The synthesis of L-threo-p-methylsulfonylphenylserine
reached 13 mM (95.1% de) during the first 2 hours indicated that the beginning
reaction was efficient. The products added up to 21 mM at 12 h and was not increased
in yield from 12 to 24 h. However, the de value at 12 h was 93.1% and decreased to
90.4% at 24 h. In our opinion, L-threo-p-methylsulfonylphenylserine was synthesized
preferentially in the initial reaction stage. As time went by, the reaction tended to
produce more L-erythro-p-methylsulfonylphenylserine which led to a decline of de
value. Those results suggested that short time reaction contributed to the formation of
L-threo-p-methylsulfonylphenylserine.
Considering
that
L-threo-p-methylsulfonylphenylserine was instable, we changed it into
L-threo-p-methylsulfonylphenylserine ethyl ester by esterification. The final purified

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product was 189 mg in white solid, yield 33.0% and further confirmed by HRDMOSI: 10(.F10i3g9/C9CY01608B
S15), 1H NMR (Fig S16) and 13C NMR (Fig S17) analysis. As far as we knew, these
results represent the highest de value reported far for asymmetric catalysis of
L-threo-p-methylsulfonylphenylserine.
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3. Conclusions
A L-threonine transaldolase from Pseudomonas sp. (PsLTTA) was mined and
expressed in Escherichia coli BL21 (DE3). Substrate spectrum assay indicated that
PsLTTA only consumed L-threonine as donor substrate and could accept a wide range
of aromatic aldehydes as acceptor substrates. Through reaction condition optimization,
the reaction was under kinetic control and L-threo-p-methylsulfonylphenylserine was
obtained with more than 60% conversion and near perfect stereoselectivity (94.5% de),
the highest stereoselectivity of L-threo-β-hydroxy-α-amino acids reported by
enzymatic
synthesis
so
far.
Finally,
synthesis
of
L-threo-p-methylsulfonylphenylserine in a 100 mL scare by a whole-cell biocatalyst
was conducted. It was for the first time systematically demonstrated that L-threonine
transaldolase as a powerful biocatalyst for preparation of L-threo-β-hydroxy-α-amino
acids. However, it should be admitted that the production of
L-threo-p-methylsulfonylphenylserine by PsLTTA was comparatively low since the
maximum concentration was only about 20 mM under the optimized conditions. In
our opinion, the moderate production by PsLTTA mainly due to its
less-efficient catalytic ability. Furthermore, the produced acetaldehyde during the

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reaction could also inhibit the synthesis of L-threo-p-methylsulfonylphenylserine
(data were not shown). So further studies such as engineering PsLTTA or establishing
an
acetaldehyde
removal
system
was
under
way
to
improve
L-threo-β-hydroxy-α-amino acids production.
4. Experimental
4.1 Materials
Escherichia coli BL21 (DE3) and plasmid pET28a were kept in our laboratory and
served as host strain and expression vector, respectively. Yeast alcohol dehydrogenase
(ADH), Pyridoxal 5’-phosphate (PLP) and NADH were purchased from Aladdin
(China). O-phthaldialdehyde (OPA) and N-acetyl-cysteine (NAC) were obtained from
Sigma-Aldrich (America). L-threo-p-methylsulfonylphenylserine standard was
purchased from Shanghai yuanye Bio-Technology Co., Ltd (China). All other
chemicals and reagents used in this work were obtained commercially with the
highest purity unless otherwise stated.
4.2 Gene cloning, expression and purification
The DNA sequences of Pseudomonas sp. L-threonine transaldolase (PsLTTA) and
Clavibacter michiganensis low specificity L-threonine aldolase (ClLTA) based on
their protein sequences (GenBank No. WP_065936857 and WP_011931605) were
optimized and synthesized by Wuhan GeneCreate Biological Engineering Co., Ltd.
(China) and cloned into plasmid pET28a. The recombinant plasmids were further

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transformed into E. coli BL21 (DE3).
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500 mL E. coli BL21 (DE3) cells harbouring pET28a-PsLTTA or pET28a-ClLTA
were grown in LB medium containing 50 μg/ mL kanamycin. The expression of
PsLTTA and ClLTA were induced overnight at 28℃ with 0.2 mM
isopropyl-β-D-thiogalactopyranoside (IPTG) after OD600 nm reached 0.5. Cells were
pelleted at 6000 g under 4℃ and were resuspended with Tris-HCl buffer (100 mM
Tris-HCl, 200 mM NaCl, 25 mM imidazole, pH 7.0). Cells were lysed by
ultrasonication and the soluble PsLTTA protein was purified by a Ni-IDA Prepacked
Column (Sangon, China) according to the instruction manual. The purified enzyme
was exchanged into Tris-HCl buffer (100 mM Tris-HCl, 20% glycerol, pH 7.0) by a
PD-10 desalination column (GE Healthcare, America) and further assessed by
SDS-PAGE. Protein concentration was determined using a BCA Protein Assay Kit
(Beyotime, China). For whole cell biocatalyst, wet cells were collected, weighted and
resuspended in Tris-HCl buffer (100 mM Tris-HCl, pH 7.0) with a concentration of
250 mg/ml. The purified enzymes and wet cells were stored at -80℃ until used.
4.3 Enzyme activity assay
Enzyme activities of PsLTTA was determined by an NADH coupled with ADH
assay as previous described.28 The reduction of NADH was detected by monitoring
the decrease in absorbance at 340 nm (ε=6220 M-1cm-1) using Multiskan Go
microplate reader (Thermo Scientific, America) at 25 °C. Reaction mixtures (180 μL)
consisting of 10 mM p-methylsulfonyl benzaldehyde, 30 mM L-threonine, 0.2 mM
PLP, 0.2 mM NADH and 10 U ADH in Tris-HCl buffer (100 mM Tris-HCl, pH 7.0,

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380
10% acetonitrile) were incubated at 25 ℃ for 1 min. Reactions were initDiaOtIe: 1d0.1b03y9/C9CY01608B
introduction of 10 mg PsLTTA enzyme (in 20 μL Tris-HCl buffer) and monitored at
340 nm for 2 min. A boiled enzyme sample was used as a negative control. One unit
(U) of transaldolase activity was defined as the amount of enzyme catalyzing the
conversion of 1 μmol of p-methylsulfonylphenylserine per minute.29 All experiments
were conducted in triplicate.
4.4 Determination of kinetic parameters
The kinetic parameters were determined by measuring the initial rate of enzymatic
reaction at 10 mM p-methylsulfonyl benzaldehyde and varying concentrations of
L-threonine (0.1-200 mM).29 Five independent replicates were performed for each
concentration of L-threonine assay and the data were fitted to the Michaelis-Menten
equation using Origin 8.0 software.
4.5 Effect of temperature and pH on the stability and activity of PsLTTA
The pH stability of PsLTTA was evaluated by investigating the transaldolase
ability after incubation in 4℃ for 12h at various pHs using 100 mM citric acid-citrate
(pH 3.0-6.0), phosphate (pH 6.0-8.0), Tris-HCl (pH 8.0-9.0) and
carbonate-bicarbonate (pH 9.0-11.0) as buffer. The thermostability of enzyme was
assessed after 1h incubation at varied temperatures from 4 to 80℃.
The pH dependence of enzyme activity was studied at various pHs (pH 6.0-8.0) in
100 mM phosphate buffer. The effect of temperature on enzyme activity was assessed
at varied temperatures from 25 to 45 ℃. The enzyme activity was measured as
described above. All experiments were conducted in triplicate.

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4.6 Effect of metal ions on transaldolase activity of PsLTTA
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401
402
The influences of different metal ions on transaldolase activity of PsLTTA were
studied in the presence of specific metal ions at final concentrations of 1 mM and 10
mM. The activity measured in the absence of metal ion was taken as a negative
control and all experiments were performed in triplicate.
4.7 Chiral derivatization analysis of stereospecificity of PsLTTA
The standard catalysis reaction was performed in 1 ml volume comprising 100 mM
L-threonine, 40 mM p-methylsulfonyl benzaldehyde, 0.2 mM PLP, 1 mM MgCl2 and
2 μM PsLTTA (or 25mg/ml wet cells) in Tris-HCl buffer (100 mM Tris-HCl, pH 7.0).
Reaction was incubated at 30℃ for 4h with constantly shaking and terminated by
addition of 2 ml MeOH. The reaction mixtures were incubated at 4℃ for 12 h and the
supernatant was used for further study.
The determination of conversion and stereospecificity of PsLTTA was performed
by HPLC after derivatization with O-phthaldialdehyde/N-acetyl-cysteine
(OPA/NAC).30 Briefly, the OPA/NAC reagent was obtained by dissolving of 100 mg
of NAC in 20 mL derivatization buffer (0.2 M Boric acid, 0.2 M KCl) and then 25.6
mg of OPA in 5 mL MeOH was added. OPA/NAC reagent was mixed with diluted
reaction solution at a ratio of 4:1 and kept for 10 min in room temperature.
Chromatographic analysis was carried out using Shimadzu LC-20AT HPLC system
(Japan) with an UV detector at 236 and 340 nm. Column: Agilent ZORBAX
reversed-phase columns (250×4.6 mm, 5 μm), mobile phase: 50 mM KH2PO4, pH
8.0/acetonitrile (81/19 or 79/21), flow rate: 1 ml min-1, temperature: 30 ℃.

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4.8 Donor and acceptor substrate specificity
Donor substrate specificity of PsLTTA was evaluated using L-threonine,
L-allo-threonine, D-threonine, D-allo-threonine, L-serine and glycine as donors while
p-methylsulfonyl benzaldehyde as acceptor. For acceptor substrate specificity,
a series of aromatic aldehydes and L-threonine were selected as acceptor and donor,
respectively. Reaction mixtures were detected by enzyme activity assay or by
analytical HPLC after OPA/NAC derivatization.
4.9 Effect of reaction condition on activity and stereospecificity of PsLTTA
Effect of reaction conditions on whole-cell biocatalyst were further determined.
The initial catalysis condition was performed at 30℃ for 3h, in a 1 ml volume
comprising 100 mM L-threonine, 40 mM p-methylsulfonyl benzaldehyde, 0.2 mM
PLP, 1 mM MgCl2 and 25 mg wet cells in Tris-HCl buffer (100 mM Tris-HCl, 10%
CH3CN, pH 7.0). The reaction conditions including amount of enzyme or substrate,
co-solvents and reaction temperature were further evaluated. The conversion and
stereospecificity catalyzed by whole-cell biocatalyst were detected by analytical
HPLC after OPA/NAC derivatization.
4.10 Reverse activity of PsLTTA
The
L-threo-p-methylsulfonylphenylserine and acetaldehyde as substrates. The reaction
was performed in ml volume comprising 100 mM
reverse
activity
of
PsLTTA
was
assessed
using
1
L-threo-p-methylsulfonylphenylserine, 50 mM acetaldehyde, 0.2 mM PLP, 1 mM
MgCl2 and 25mg/ml wet cells in Tris-HCl buffer (100 mM Tris-HCl, pH 7.0).

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Reaction was incubated at 30℃ for different hours with constantly shaking and
terminated by addition of 2 ml MeOH. The conversions were detected by analytical
HPLC at 236 nm. The reaction catalyzed by ClLTA was performed as a positive
control.
426
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430
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432
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439
440
441
442
443
444
445
446
4.11 Synthesis of L-threo-p-methylsulfonylphenylserine in a large scare of 100
mL system by whole-cell biocatalyst
Transformation of L-threo-p-methylsulfonylphenylserine at a 100 mL system by
whole-cell biocatalyst were performed at the optimized reaction conditions. Briefly,
L-threonine (1.2 g, 10 mmol), p-methylsulfonyl benzaldehyde (0.55 g, 3 mmol), PLP
(5 mg, 0.02 mmol), MgCl2 (10 mg, 0.1 mmol) and wet cells (0.625g) were added in
100 ml Tris-HCl buffer (100 mM Tris-HCl, 10% ethyl acetate, pH 7.0 ). The reaction
mixture was constantly shaking at 20 °C for 24 h. Time course of catalysis processing
was minored in triplicate. The conversion and stereoselectivity were detected by
analytical HPLC after OPA/NAC derivatization.
After reaction, the crude reaction supernatant was added 20 mL ethanol and
adjusted to pH 1.0 by concentrated sulfuric acid. For esterification, the mixture was
incubated at 100℃ for 2 hours. The supernatant was extracted with chloroform twice.
The chloroform layer was combined and evaporated to dryness. The residue was
purified by a C18 SPE column (CHCl3: MeOH = 2 :1) to give 189 mg white solid,
yield 33.0%. 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 8.1 Hz, 2H), 7.62 (d, J = 8.1
Hz, 2H), 4.98 (d, J = 4.5 Hz, 1H), 4.19 (q, J = 7.0 Hz, 2H), 3.64 (d, J = 4.7 Hz, 1H),
3.08 (s, 3H), 1.23 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 172.79, 147.58,

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465
466
467
468
139.91, 127.48, 127.26, 73.41, 61.64, 60.22, 44.53, 14.08. HRMS (m/z) (M+): calcd.
for C12H18NO5S, 288.0897, found 288.0900.
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgements
We thank Dr. Bing-De Zheng (College of Chemistry, Fuzhou University) for the
technical assistance of HRMS and NMR analysis. This study is financially supported
by Industry University Research project of Fuzhou University (No. 2019090501).
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535
Table
Table 1. Acceptor substrate specificity of PsLTTA.
Table 2. Effects of co-solvents on the conversion and stereoselectivity of PsLTTA.
Table 3 Stereoselectivity of PsLTTA and ClLTA.
Figure captions
Fig. 1. Enzymatic properties of PsLTTA. (A) Temperature stability profile of
PsLTTA after incubation of enzyme solution on various temperatures (4-80 °C) for 60
minutes. (B) Detection of the optimal reaction temperature of PsLTTA. (C) pH
stability profile of PsLTTA after incubation of enzyme solution on various pH (3-11)
for 12 hours at 4℃. (D) Detection of the optimal reaction pH of PsLTTA. (E) Effects
of metal ions (1 mM and 10 mM) on the trans-aldehyde activity of PsLTTA. (F)
Effects of PLP concentration on the trans-aldehyde activity of PsLTTA. All assays
were performed three times.
Fig. 2. Donor substrate specificity of PsLTTA detected by HPLC after OPA/NAC
derivatization. (A) L-threonine. (B) L-serine. (C) D-threonine. (D) L-allo-threonine.
(E) D-allo-threonine. (F) glycine. Product L-p-methylsulfonylphenylserine (tL-threo=7.1
min, tL-erythro=8.4 min)
Fig. 3. Concentration effect of wet cells and substrates on the conversion and
stereoselectivity of PsLTTA. The concentrations of p-methylsulfonyl benzaldehyde
were 10 mM (A), 20 mM (B), 30 mM (C) and 40 mM (D), respectively. The reactions
mixture contained 10% (v/v) CH3CN as co-solvent and incubation at 30 ℃ for 4

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hours.
537
538
539
540
541
542
543
544
Fig. 4. Effect of temperature on the conversion and stereoselectivity of PsLTTA. (A)
The conversion and de value of L-threo-p-methylsulfonylphenylserine under various
temperatures. (B) Determination of stereospecificity of PsLTTA by HPLC analysis
after OPA/NAC derivatization. Product L-p-methylsulfonylphenylserine (tL-threo=7.1
min, tL-erythro=8.4 min).
Fig. 5. Time course for synthesis of L-threo-p-methylsulfonylphenylserine in 100 mL
scare.

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Table 1. Acceptor substrate specificity of PsLTTA.
R
H
Conversion (%) de (%)
R
o-Br
Conversion (%) de (%)
51.0
40.5
25.2
59.4
43.5
44.6
ND
77.2
50.2
29.9
76.7
69.2
66.4
ND
43.3
ND
ND
74.4
ND
ND
ND
ND
ND
ND
80.8
ND
ND
79.9
ND
ND
ND
ND
ND
ND
o-NO2
m-NO2
p-NO2
o-F
m-Br
p-Br
p-MeSO2
p-I
m-F
o-OH
m-OH
p-OH
o-CH3
p-O(CH3)
p-F
o-Cl
m-Cl
p-Cl
37.9
ND
80.3
ND
ND
ND
546
547
548
549
550
551
Reaction conditions: 1 mL reaction mixture containing 40 mM aromatic aldehydes,
100 mM L-threonine, 0.2 mM PLP, 1 mM MgCl2 and 25 mg/mL wet cells in
Tris-HCl buffer (100 mM Tris-HCl, 10% CH3CN, pH 7.0) at 30 ℃ for 4 h.
ND, Not detected.

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Table 2. Effects of co-solvents on the conversion and stereoselectivity of PsLTTA.
co-solvents
none
Conversion (%) de (%) co-solvents
Conversion (%) de (%)
41.6
81.1
76.9
44.6
45.1
55.9
17.7
71.3
85.3
86.9
82.6
80.8
81.0
80.2
80.0
85.1
84.5
10% CH3CN
20% CH3CN
30% CH3CN
10% DMSO
20% DMSO
30% DMSO
70.4
45.1
26.2
78.2
69.9
56.0
84.9
82.1
79.0
84.2
85.7
84.6
90.2
89.4
89.4
89.9
10% MeOH
20% MeOH
30% MeOH
10% EtOH
20% EtOH
30% EtOH
10% Ethyl acetate 80.4
20% Ethyl acetate 74.8
30% Ethyl acetate 69.2
40% Ethyl acetate 61.1
10% Acetone 64.5
20% Acetone 46.2
30% Acetone 39.7
553
554
555
Reaction conditions: 1 mL reaction mixture containing 30 mM p-methylsulfonyl
benzaldehyde, 100 mM L-threonine, 0.2 mM PLP, 1 mM MgCl2 and 6.25 mg/mL wet
cells in Tris-HCl buffer (100 mM Tris-HCl, pH 7.0) at 30 ℃ for 4 h.