Biocascade Synthesis of L-Tyrosine Derivatives by Coupling a Thermophilic Tyrosine Phenol-Lyase and L-Lactate Oxidase

Article abstract of DOI:10.1002/ejoc.202000061

A one-pot biocascade of two enzymatic steps catalyzed by an l-lactate oxidase and a tyrosine phenol-lyase has been successfully developed in the present study. The reaction provides an efficient method for the synthesis of l-tyrosine derivatives, which exhibits readily available starting materials and excellent yields. In the first step, an in situ generation of pyruvate from readily available bio-based l-lactate catalyzed by a highly active l-lactate oxidase from Aerococcus viridans (AvLOX) was developed (using oxygen as oxidant and catalase as hydrogen peroxide removing reagent). Pyruvate thus produced underwent C–C coupling with phenol derivatives as acceptor substrate using specially designed thermophilic tyrosine phenol-lyase mutants from Symbiobacterium toebii (TTPL). Overall, this cascade avoids the high cost and easy decomposition of pyruvate and offered an efficient and environmentally friendly procedure for l-tyrosine derivatives synthesis.

Full text of DOI:10.1002/ejoc.202000061

DOI: 10.1002/ejoc.202000061
Full Paper
Enzyme Catalysis
Biocascade Synthesis of L-Tyrosine Derivatives by Coupling a
Thermophilic Tyrosine Phenol-Lyase and L-Lactate Oxidase
Guosi Li,[a] Jiazhang Lian,[a] Hailong Xue,[a] Yiqi Jiang,[a] Shuyun Ju,[a] Mianbin Wu,[a]
Jianping Lin,*[a] and Lirong Yang[a]
Abstract: A one-pot biocascade of two enzymatic steps cata- was developed (using oxygen as oxidant and catalase as
lyzed by an
been successfully developed in the present study. The reaction underwent C–C coupling with phenol derivatives as acceptor
provides an efficient method for the synthesis of -tyrosine de- substrate using specially designed thermophilic tyrosine
L-lactate oxidase and a tyrosine phenol-lyase has hydrogen peroxide removing reagent). Pyruvate thus produced
L
rivatives, which exhibits readily available starting materials and phenol-lyase mutants from Symbiobacterium toebii (TTPL). Over-
excellent yields. In the first step, an in situ generation of pyru- all, this cascade avoids the high cost and easy decomposition of
vate from readily available bio-based
L-lactate catalyzed by a pyruvate and offered an efficient and environmentally friendly
highly active -lactate oxidase from Aerococcus viridans (AvLOX) procedure for
L
L-tyrosine derivatives synthesis.
Introduction
still many problems to be resolved before industrial production,
such as (i) the purification of pyruvate from complex fermenta-
tion broth is generally difficult and expensive, (ii) the recombi-
nant strains are not stable enough, and (iii) the yield and
productivity of pyruvate is still low. The direct oxidative de-
hydrogenation (ODH) of lactate is regarded as another green
alternative for pyruvate production, since lactate, either in its
chiral or racemic form, is commercially available at much
cheaper price than pyruvate and can be produced at industrial
scale by microbial fermentation.[8] Various chemical catalysts in-
cluding Pd/Pt,[9] metal oxides (Mo, Ti, Zr, W and Sn)[10] and bi-
nary oxides (TeO2-MoO3 and SnO2-MoO3)[11] were reported for
the ODH of lactate, but often resulted in C–C scission and ac-
cordingly the formation of acetaldehyde and CO2. Biocatalysis
is then regarded as a preferred strategy due to its moderate
reaction conditions. It is reported that lactate dehydroge-
nases,[12] NAD-independent lactate dehydrogenase,[13] glycolate
oxidase (GOX),[14] and lactate oxidase (LOX)[15] have great po-
Proteinogenic and nonproteinogenic α-amino acids are among
the top-five families of natural compounds used for the synthe-
sis of pharmaceuticals, materials, and nutrients.[1] Among them,
enantiomerically pure L-tyrosine derivatives represent key pre-
cursors for biochemical markers and anticancer drugs and are
important noncanonical amino acids for synthetic biology.[2]
The classical chemical synthesis of these tyrosine derivatives
usually comprises several steps including protection and depro-
tection of the functional amino acid groups and leads to race-
mic material, which requires late-stage racemate resolution.[3]
In contrast, PLP-dependent tyrosine-phenol lyases (TPL, EC
4.1.99.2) can catalyze the regio- and stereoselective reversible
formation of tyrosine derivatives from donor substrate pyruvate
and acceptor substrate phenol or derivatives in a single and
green reaction.[4] Among various TPLs, thermophilic TPL from
Symbiobacterium toebii (TTPL) exhibited markedly high stability
against phenols and have been employed as biocatalysts for
tential to be used for the production of pyruvate from
Among them, GOX and LOX, both of which belong to flavin
mononucleotide (FMN)-containing short-chain -2-hydroxy acid
oxidase (L-2-HAO, EC 1.1.3.15), can catalyze the ODH of -lactate
L-lactate.
enzymatic synthesis of L
-tyrosine and L-DOPA.[5] However, the
cost of the donor substrate accounts for a considerable propor-
tion in these processes. Today, pyruvate is still made through
the classic dehydrative decarboxylation of tartaric acid at high
temperature (300 °C).[6] The excess amount of KHSO4 and the
high temperature make this process unsustainable.
Metabolic engineering and synthetic biology have enabled
the fermentation of lignocellulose-derived sugars to produce
pyruvate at high titers in recent decades.[7] However, there are
L
L
in the presence of oxygen without requiring expensive cofac-
tors (NAD+ or NADP+).[15a,16] The tightly bound cofactor favors
the industrial application of L-2-HAO, since it is not necessary
to add FMN during biotransformation processed. In order to
develop an efficient and sustainable process for the synthesis
of L-tyrosine derivatives, a one-pot, two step biocascade was
designed by combining activity of a short chain L-2-HAO and
TTPL (Scheme 1).
[a] Key Laboratory of Biomass Chemical Engineering of Ministry of Education,
College of Chemical and Biological Engineering, Zhejiang University,
Hangzhou, 310027, China
Results and Discussion
Our first goal was to produce the α-ketoacid pyruvate from the
E-mail: linjp@zju.edu.cn
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejoc.202000061.
naturally occurring α-hydroxy acid
1050
L-lactate using an appropri-
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Scheme 1. One-pot, two-step biocascade for the transformation of L-lactate
1 and phenols 3a–g into the corresponding enantiomerically pure tyrosine
derivatives L-4a–g. L-2-HAO = L-2-hydroxy acid oxidases; CAT = catalase;
TTPL = thermophilic tyrosine phenol lyases; PLP = pyridoxal phosphate.
ate short-chain L-2-HAO. The most efficient ODH of L-lactate
reported was catalyzed by recombinant GOX from Spinach oler-
acea (SoGOX, XP_021866980.1) under 40 psig of oxygen,[14,17]
unfortunately this enzyme suffered from low activity under
atmospheric conditions. Therefore, five other short-chain L-2-
HAOs including a GOX from Arabidopsis thaliana (AtGOX, OA-
O97698.1) and four LOXs from Enterococcus faecium (EfLOX,
WP_002296473.1), Nostoc sp. (NsLOX, WP_044522577.1), Aero-
coccus viridans (AvLOX, WP_003142047.1), Geotrichum candidum
(GcLOX, CDO53315.1) were selected as potential catalytic candi-
dates based on the sequence features of reported L-2-HAOs
and their homologous proteins in the Pfam database. The six
short-chain L-2-HAOs were heterologously expressed in E. coli
and compared for their efficiency in pyruvate synthesis. Among
them, AvLOX was demonstrated to exhibit a volume activity
(per volume of fermentation broth) of 13.5 U mL–1, which were
much higher than SoGOX and other five L-2-HAOs (Figure 1a).
Figure 1. Oxidative dehydrogenation of
volume activity of six short-chain L-2-HAOs from different species. (b) Pyru-
vate synthesis with different amount of -lactate using AvLOX. Reaction con-
ditions: -lactate 1 (50–300 m ), EDTA (1.0 m ), AvLOX (0.4 mg/mL, 10.0 U/
mL), CAT (0.3 mg/mL, 3000 U/mL), pKi (50 m , pH 7.0). Experiments were
performed with a final volume of 10 mL with 200 rpm shaking at 37 °C.
L-lactate using AvLOX. (a) Specific
L
L
M
M
M
AvLOX was therefore selected for the subsequent studies. amount of substrate with optimal enzyme activity ratio were
AvLOX has been extensively studied for the applications in carried out. As shown in Figure 1b, the conversion of -lactate
-lactate as the substrate,
-lactate greater than 200 m
L
quantifying the concentration of lactate in the blood or other reached > 99 % with 50–200 m
fluids,[18] but has never been investigated to produce pyruvate while significantly decreased with
M L
L
M
from
L
-lactate, especially at the preparative scale.
even with extended reaction time. It seemed that the ODH step
was inhibited by high concentration of substrate or product.
Thus, the initial ODH rate within 5 min at different L-lactate and
pyruvate concentration was investigated (Figure S2). The initial
ODH rate decreased obviously with the increase of both sub-
strate and product, indicating the existence of both substrate
and product inhibition, which can be addressed if pyruvate was
Notably, the AvLOX catalyzed reaction leads to the formation
of hydrogen peroxide. This reactive species is disadvantageous
for the ODH process due to the chemical decarboxylation of
pyruvate to acetate as well as the inactivation of AvLOX.[19] To
remove the generated H2O2 during biotransformation immedi-
ately, a highly active and stable catalase from Ureibacillus ther-
mosphaericus (CAT)[20] was introduced to construct a two en-
zyme coupled cascade that hydrogen peroxide was in situ de-
composed by catalase yielding only water and oxygen as the
constantly consumed by TTPL and
uous or fed-batch mode.
L-lactate was fed in a contin-
In the second step, TTPL from thermophilic microorganism
products. To obtain the optimal enzyme activity ratio of AvLOX/ S. toebii[5b,21] was selected for the C–C coupling reaction, since
CAT, the ODH of
-lactate was carried out with the supplemen- it is much more stable than TPLs from Citrobacter freundii[22]
tation of different amounts of CAT (Figure S1). Although AvLOX and Erwinia herbicola.[23] However, we found the enzyme has a
could convert -lactate into pyruvate without the supplementa- very narrow substrate scope, which failed to catalyze the con-
L
L
tion of external catalase, its yield was only 5.8 % due to the version of phenol derivatives of interest such as o-chlorophenol
insufficient activity of the native catalase. With 300-fold excess 3d, o-bromophenol 3e, o-methylphenol 3f, and guaiacol 3g
of catalase activity in relation to AvLOX activity, the
L-lactate (Table 1). Therefore, protein engineering was required to
(50 m ) conversion was increased up to almost 100 %, which change the substrate specificity of this C–C bond-forming en-
M
was 17.2-fold higher than that in the absence of catalase zyme. The crystal structure of the TPL from C. freundii, sharing
(5.8 %). These results indicated that the optimal enzyme activity a high sequence identity of 69 % with TTPL, was chosen for
ratio of CAT:AvLOX should be 300:1 in this cascade biocatalytic homology modeling and structure-guided, site-directed muta-
system. Then, conversions in the presence of an increased genesis. Docking simulations were performed using the Discov-
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ery Studio, and
L-tyrosine was successfully docked in the
homology model (Figure 2a). The chemical structure of
L-tyro-
sine differs from its derivatives by only a substituent group at
the o-position of the phenolic group (Figure 2b). However, the
catalytic activity of TTPL towards o-phenols was very low (3b,
3c) or even below the detection limit (3d–g) (Table 1). It was
speculated that the o-phenolic group of L-tyrosine derivatives
cannot enter the active site pocket because of steric hindrance.
Based on this analysis, the enlargement of the pocket in TTPL
should improve o-phenols binding and accordingly its catalytic
activity. Thus, nine of the residues (Phe37, Phe124, Thr129,
Met187, Met380, Arg382, Leu447, F449 and Phe450) forming
the active-site pocket (Figure 2b) were mutated to the smaller
residue valine, which showed a more stereoselective preference
than alanine. In addition, Thr126 was substituted by serine in
order to preserve the hydrogen bonding interaction with
Glu104 (Figure 2b).
Table 1. Specific activity of wild type TTPL and engineered variants toward
various o-phenols.[a]
Substrate
R
Specific activity [U/mg]
WT F37V
T126S
F380V
F37VM380V
1
2
3
4
5
6
7
H (3a)
1.25 0.03 1.08 0.04 1.02 0.05 1.13 0.05 0.98 0.06
OH (3b) 0.55 0.01 N.A. 0.12 0.01 N.A. N.A.
0.48 0.01 0.56 0.03 0.37 0.03 0.64 0.01 0.58 0.02
F (3c)
Cl (3d)
Br (3e)
CH3 (3f) N.A.
OMe (3g) N.A.
N.A.
N.A.
0.51 0.01 0.31 0.02 0.43 0.02 0.44 0.01
0.32 0.02 0.15 0.02 0.33 0.01 0.30 0.02
0.43 0.02 0.35 0.01 0.54 0.04 0.39 0.04
0.39 0.01 N.A.
0.31 0.03 0.35 0.01
[a] Specific activity was determined using purified proteins by monitoring the
formation of 1 μmol of -tyrosine product per minute at 37 °C in potassium
phosphate buffer (50 m , pH 8.0). All enzymatic assays were performed in
L
M
triplicate, at least, and the averaged values are reported. WT = recombinant
wild-type enzyme; N.A. = no measurable activity.
Ten single mutants (F37V, F124V, T126S, T129V, M187V,
M380V, R382V, L447V, F449V, and F450V) were constructed and
investigated using o-methylphenol 3f as a model substrate. As
shown in Figure 2c, three superior mutants (F37V, M380V, and
T126S) were obtained. These mutants were then purified to ho-
mogeneity (Figure S3) and their activities towards various o-
phenols were evaluated. As shown in Table 1, the variants F37V
and M380V showed a much broader substrate spectrum, ac-
cepting all substrates tested except for 3b. T125S showed a
similar substrate spectrum as F37V and M380V with reduced
activity but failed to convert 3g. Interestingly, the activity for
the natural substrate catechol 3b was reduced (T125S) or elimi-
nated (F37V and M380V). The active-site pocket was further
expanded by constructing the double mutant F37VM380V. Un-
fortunately, the double mutant failed to continue to facilitate
the catalysis of the non-natural substrate o-phenols. Multiple
sequence alignment shows that these active residues are highly
conserved among the TPL family (Figure S4), thus the molecular
engineering method may be also useful to improve the catalytic
activity toward o-phenols in other members of the family.
Figure 2. Rational design of TTPL. (a) Docking of
TTPL. Docking simulations were performed with discovery studio 2.5. (b) Resi-
dues interacting with -tyrosine in the active site pocket of TTPL. The dotted
line represented the hydrogen bond. The o-position of the phenolic group
was marked with a red circle. (c) Specific volume activity of the TTPL mutants
toward o-methyl-phenol 3f.
L-tyrosine into wild type
L
TTPL mutants were selected depending on the phenol deriva-
tives. Previous substrate specificity studies showed that the best
substrates for wild type TTPL were 3a and 3b. For 3c, 3e, and
3f, the reactions were performed using the above designed
TTPL variant M380V. For C–C coupling of 3d and 3g, TTPL vari-
ant F37V was chosen instead. AvLOX, CAT, and TTPL were all
employed as cell free lyophilized lysate providing an “off-the
shelf” catalyst that can be stored for several months. To identify
a suitable pH value for the three-enzyme cascade process, the
pH ranges of the three individual catalysts were compared (Fig-
ure S5): AvLOX, as well as CAT, achieved the optimal activity at
neutral pH values (pH 7.5 for AvLOX and pH 7.0 for CAT),
Next, the two-step cascade reaction catalyzed by AvLOX and whereas TTPL preferred a pH range of 8.0–8.5. As the optimal
TTPL was performed in a one-pot fashion using -lactate 1 as pH values of the three enzymes were not the same, the de-
L
the donor substrate and o-phenols 3a–g as the acceptor sub- pendence of the overall cascade process on the pH value was
strate. According to a previous study,[24] a donor/acceptor ratio investigated using 3f as the acceptor substrate at a concentra-
of 2:1 was used in the present study. For optimal performance, tion of 23 m
1052
M
(Figure S6). The highest conversion (93 %) was
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Table 2. One-pot biocatalytic cascade for the preparation of L-4a–g.
Entry[a]
R
TTPL sources
Reaction time [h]
L-lactate conversion [%]
3a–g conversion [%][b]
ee L-4a–g [%][c]
1
2
3
4
5
6
7
H (3a)
OH (3b)
F (3c)
Cl (3d)
Br (3e)
CH3 (3f)
OMe (3g)
WT
WT
28
36
28
36
36
28
28
> 99
> 99
> 99
> 99
> 99
> 99
> 99
98 (84)
85 (65)
93 (62)
89 (56)
81 (51)
97 (69)
96 (71)
> 99
> 99
> 99
> 99
> 99
> 99
> 99
M380V
F37V
M380V
M380V
F37V
+
[a] Reaction conditions: o-phenol 3a–g (46 m
(0.3 mg/mL, 3000 U/mL), TTPL (2.1 mg/mL), pKi (50 m
for 28 or 36 h. [b] Purification and recovery yield in brackets. [c] Determined by chiral HPLC analysis.
M
),
L
-lactate 1 (2 equiv.), NH4 (10 equiv.), PLP (0.1 m
M
), EDTA (1.0 m
M
), AvLOX (0.4 mg/mL, 10.0 U/mL), CAT
M
, pH 8.0). Experiments were performed with a final volume of 50 mL with 200 rpm shaking at 37 °C
achieved at pH 8.0 in the potassium phosphate buffer. Under successfully transformed into their corresponding
optimal conditions, 46 m substrate could be converted to en- rivatives L-4a–e and L-4g with varied conversion (81–98 %)
antiopure L-4f with an efficiency up to 97 % (Figure S7). How- (Table 2), demonstrating the practical utility of the cascade for
ever, when the substrate concentration was increased to 69 or various -tyrosine derivatives synthesis. Then, all the products
92 m , the C–C coupling step could not reach completion un- were purified through a strong cationic exchanger and then
der the same reaction conditions (Figure S7). It turned out that characterized by NMR and MS. Analysis of the optical purity
the ODH step was inhibited as 19 % and 51 % -lactate were by chiral HPLC verified that all the enzymatically synthesized
unconverted at the substrate concentration of 69 and 92 m
L-tyrosine de-
M
L
M
L
M
,
products eluted exclusively in a single peak (> 99 % ee).
In conclusion, o-phenol derivatives were transformed into
respectively. Most likely, the FMN-dependent enzyme was im-
paired by the formation of a charge-transfer complex between
phenols and flavin as previously reported.[25]
the corresponding
selectivity and high yields using an efficient one-pot, two-step
L-tyrosine derivatives with excellent stereo-
To demonstrate the practical utility of the system, the two- cascade containing TTPL and AvLOX. The efficiency of this ap-
step coupled cascade was performed at 50 mL scale and com- proach was based on the high activity of AvLOX, allowing the
pared with one step C-C coupling using pyruvate 2 as the do- in situ generation of pyruvate, the TTPL donor substrate, from
nor substrate. In order to monitor the reactions in more detail, a
L-lactate. In this case, pyruvate was readily converted by TTPL
time-course study was performed (Figure S8). The C-C coupling by minimizing its accumulation and decomposition. This envi-
reaction of the cascade was much slower in the first few hours. ronmentally friendly procedure, not ever reported, provides an
Nevertheless, after 28 h, the yield of L-4f could be increased interesting alternative to the conventional biocatalytic and
up to 98 %, indicating the efficient coupling of the two steps. chemical synthesis of
L-tyrosine derivatives. In addition, TTPL
Interestingly, the conversion with the one-step system was 9 % activities towards the o-phenol derivatives, which are generally
lower than that with the two-step coupled cascade, although poor TTPL substrates, were greatly enhanced via site-directed
the synthesis from 1 was much easier than that using 2 as the mutagenesis based protein engineering. This cascade biocata-
donor substrate. By detecting the final reaction mixture, it was lytic approach is also promising for upgrading bio-based bulk
found that the remaining pyruvate concentration was 15.3 % chemicals (i.e.
lower in the one-step system than that in the two-step coupled
cascade reaction (Figure S8). As high donor/acceptor ratio can
L-lactate) to high-value chiral chemicals.
Acknowledgments
facilitate the C-C coupling due to the low affinity of most TPL
towards pyruvate,[4a,26] the cascade probably enhanced the
yield of L-4f by avoiding the decrease of pyruvate as well as
donor/acceptor ratio. According to previous studies, pyruvate is
not stable and can undergo an aldol-type condensation to form
polymers, especially under alkaline conditions at high concen-
trations.[27]
This work was financially supported by the National Hi-Tech
Research and Development Program of China (2014AA022105).
Keywords: Biocatalysis · Cascade reactions · Enzyme
catalysis · Synthetic methods · Tyrosine derivatives
Finally, the scope of the biocascade was investigated for a
panel of acceptor o-phenols with a substrate concentration of
46 mM and a scale of 50 mL (Table 2). Besides the model sub-
strate 3f, phenol and o-phenols containing o-hydroxy 3b, o-
fluorine 3c, o-chloro 3d, o-bromo 3e, and o-methoxyl 3g were
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Received: January 15, 2020
Eur. J. Org. Chem. 2020, 1050–1054
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