Bioprocess and Biosystems Engineering

https://doi.org/10.1007/s00449-020-02325-5

RESEARCH PAPER

Characterization of two new aromatic amino acid lyases

from actinomycetes for highly efficient production of p‑coumaric acid
Peiwu Cui1,2,3 · Weihong Zhong1 · Yong Qin4 · Fuping Tao4 · Wei Wang2 · Jixun Zhan2,3

Received: 20 December 2019 / Accepted: 4 March 2020

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
p-Coumaric acid (p-CA) is a bioactive natural product and an important industrial material for pharmaceuticals and nutraceu-
ticals. It can be synthesized from deamination of l-tyrosine by tyrosine ammonia lyase (TAL). In this work, we discovered
two aromatic amino acid lyase genes, Sas-tal and Sts-tal, from Saccharothrix sp. NRRL B-16348 and Streptomyces sp. NRRL
F-4489, respectively, and expressed them in Escherichia coli BL21(DE3). The two enzymes were functionally characterized
as TAL. The optimum reaction temperature for Sas-TAL and Sts-TAL is 55 °C and 50 °C, respectively; while, the optimum
pH for both TALs is 11. Sas-TAL had a kcat/Km value of 6.2 μM−1 min−1, while Sts-TAL had a much higher efficiency with
a kcat/Km value of 78.3 μM−1 min−1. Both Sts-TAL and Sas-TAL can also take l-phenylalanine as the substrate to yield
trans-cinnamic acid, and Sas-TAL showed much higher phenylalanine ammonia lyase activity than Sts-TAL. Using E. coli/
Sts-TAL as a whole-cell biocatalyst, the productivity of p-CA reached 2.88 ± 0.12 g (L h)−1, which represents the highest
efficiency for microbial production of p-CA. Therefore, this work not only reports the identification of two new TALs from
actinomycetes, but also provides an efficient way to produce the industrially valuable material p-CA.

Keywords Actinomycetes · p-Coumaric acid · Escherichia coli · Phenylalanine ammonia lyase · Tyrosine ammonia lyase

Introduction [1, 2]. However, many of these bioactive natural products

Phenylpropanoids, one of the largest categories of plant
secondary metabolites, have received considerable atten-
tion due to their antioxidant, anticancer, anti-atherosclerosis,
anti-inflammatory and antiviral properties and wide utili-
zation in the fields of pharmaceuticals and nutraceuticals
* Wei Wang
wangwei402@hotmail.com
* Jixun Zhan
jixun.zhan@usu.edu
1
College of Biotechnology and Bioengineering, Zhejiang
University of Technology, Hangzhou 310030, Zhejiang,
China
2
TCM and Ethnomedicine Innovation and Development
Laboratory, School of Pharmacy, Hunan University
of Chinese Medicine, Changsha 410208, Hunan, China
3
Department of Biological Engineering, Utah State
University, 4105 Old Main Hill, Logan, UT 84322‑4105,
USA
4
Hangzhou Viablife Biotech Co., Ltd., 1 Jingyi Road, Yuhang
District, Hangzhou 311113, Zhejiang, China
are present in plants at low concentrations, which makes
extraction of these compounds from plants inefficient and
cost-ineffective [3]. Chemical synthesis of these compounds
often involves toxic chemicals and harsh conditions, which
is, thus, not environment-friendly and sustainable. With the
development of the biosynthetic technology, microbial pro-
duction of useful phenylpropanoids has become an attractive
approach to producing these valuable products.
As one of the most important intermediate metabolites in
phenylpropanoid biosynthesis, p-coumaric acid (p-CA) is a
highly significant chemical building block for the produc-
tion of a variety of valuable substances such as flavonoids,
polyphenols, coumarins, stilbenes, lignans, and polymeric
materials [4–9]. Besides, p-CA itself is also a bioactive agent
exhibiting protective effects against carcinogenesis, athero-
sclerosis, oxidative cardiac damage, neuronal injury and
anti-inflammatory activities [10, 11]. p-CA can be formed
directly from non-oxidative deamination of l-tyrosine cat-
alyzed by tyrosine ammonia lyase (TAL, Fig. 1) or from
deamination of l-phenylalanine by phenylalanine ammonia
lyase (PAL, Fig. 1) followed by the hydroxylation catalyzed
by cinnamate-4-hydroxylse (C4H) [5]. However, expression

13
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Fig. 1  Enzymatic synthesis of p-coumaric acid/trans-cinnamic acid
by TAL and PAL. TAL tyrosine ammonia lyase, PAL phenylalanine
ammonia lyase, C4H cinnamate-4-hydroxylse
of C4H, a cytochrome P450 hydroxylase, in a heterologous
prokaryotic system is often not satisfactory and its func-
tion requires an efficient electron transfer system [1, 12].
Therefore, the most effective route for p-CA production is
through deamination of l-tyrosine by TAL. Both TAL and
PAL belong to the aromatic amino acid lyase (AAAL) fam-
ily, which is classified by the substrate specificity as histi-
dine ammonia lyase (HAL, EC 4.3.1.3), TAL (EC 4.3.1.23),
PAL (EC 4.3.1.24) or PAL/TAL (EC 4.3.1.25) [13]. Unlike
other AAALs, TAL is relatively rare, especially in bacteria
[14]. To our knowledge, TAL has only been identified in
Rhodobacter capsulatus (Integrated Genomics accession
number: RRC01844) [15], Rhodobacter sphaeroides (Gen-
Bank accession number: YP_355075) [16], Saccharothrix
espanaensis (GenBank accession number: ABC88669)
[17], Rhodotorula glutinis (GenBank accession number:
M18261.1) [18], Phanerochaete chrysosporium [19], and
Streptomyces sp. (GenBank accession number: JN587500)
[5]. Discovery of more efficient TALs will facilitate engi-
neered production of p-CA in microbial systems.
In this study, two new actinomycete AAALs were discov-
ered from Saccharothrix sp. NRRL B-16348 (Sas-tal; Gen-
Bank accession number: WP_053717807.1) and Streptomy-
ces sp. NRRL F-4489 (Sts-tal; GenBank accession number:
WP_066973014.1), respectively. They were heterologously
expressed in Escherichia coli BL21 (DE3) and functionally
characterized. Their enzymatic properties on tyrosine were
Table 1  Primers for Gene Primers
amplification of the two AAAL
genes from Saccharothrix Sas-tal-F 5′-AACAT​ATG​ACC​GAC​GCC​CCC​GTG​GAC​CT-3′
sp. NRRL B-16348 and
Sas-tal-R 5′-TAAAG​CTT​CTC​GAG​TCA​GTA​CCG​GCC​CCC​GGT​GACCA-3′
Streptomyces sp. NRRL F-4489
Sts-tal-F 5′-AACAT​ATG​CCG​AGT​CTG​GAC​AGCAT-3′
Sts-tal-R 5′-TAAAG​CTT​CTC​GAG​TCA​GGC​GGC​GCC​GGT​CAG​CT-3′
13
Bioprocess and Biosystems Engineering
studied. The engineered E. coli strain harboring the Sts-tal
gene was found to be efficient in producing p-CA, represent-
ing a promising strain for the production of this industrially
valuable compound.
Materials and methods
Materials
Phusion High-Fidelity DNA polymerase, restriction
enzymes and T4 DNA ligase were purchased from New Eng-
land Biolabs. l-tyrosine, l-phenylalanine, trans-cinnamic
acid and p-CA were purchased from Sigma-Aldrich (St.
Louis, MO, USA). His Pur™ Ni-NTA resin and Luria–Ber-
tani (LB) medium were purchased from Thermo Fisher Sci-
entific (Rockford, IL, USA). Bradford assay solution was
purchased from TCI America (St. Portland, OR, USA).
Except high-performance liquid chromatography (HPLC)
solvents, all other chemicals used were of analytical grade
and were purchased from Fisher Scientific (Rockford, IL,
USA).
Vectors and strains
The vectors pJET1.2 (Thermo Fisher Scientific, USA) and
pET28a (Millipore Sigma, USA) were, respectively, used
for general cloning and gene expression. E. coli XL-1 Blue
and E. coli BL21(DE3) were routinely grown in LB medium
at 37 °C and used for plasmid amplification and enzyme
expression, respectively. Saccharothrix sp. NRRL B-16348
and Streptomyces sp. NRRL F-4489 were obtained from the
Agricultural Research Service Culture Collection of USDA,
and were grown in YM medium at 28 °C.
Amplification of the two putative aromatic amino
acid lyase genes
The primers designed for cloning of Sas-tal (1485 bp) and
Sts-tal (1479 bp) were synthesized by Thermo Fisher Sci-
entific and are shown in Table 1. The genomic DNA was
extracted from Saccharothrix sp. NRRL B-16348 and Strep-
tomyces sp. NRRL F-4489 using the Quick-DNA™ Fungal/
Bacterial Microprep Kit (Zymo Research, USA). To amplify
the AAAL genes, the primers (1  μM), genomic DNA
Restriction site
NdeI
HindIII
NdeI
HindIII
Bioprocess and Biosystems Engineering
(0.2 μL), dNTP mix (200 μM), 5 × buffer (4 μL), DMSO
(0.4 μL), Phusion High-Fidelity DNA Polymerase (0.2 μL at
2 U μL−1) and nuclease-free water were mixed to bring the
volume to 20 μL. The PCR conditions are as follows: initial
denaturation at 98 °C for 5 min, 30 cycles at 98 °C for 40 s,
56 °C for 40 s, and 72 °C for 3.5 min, and finally, 72 °C for
10 min of elongation.
Plasmid construction
After recovery from a 0.8% agarose gel with a GeneJET
Gel Extraction Kit (Thermo Fisher Scientific), the target
PCR products were ligated into the pJET1.2 vector to yield
pJET1.2-Sas-tal and pJET1.2-Sts-tal, respectively. After the
plasmids were confirmed by digestion check and sequencing,
the two genes were excised from the corresponding plasmids
using NdeI and HindIII and ligated into pET28a between the
same sites to construct pET28a-Sas-tal and pET28a-Sts-tal.
The ligation products were transferred into E. coli XL1-Blue
competent cells through chemical transformation, and the
transformants were selected on LB agar plate containing
50 μg mL−1 kanamycin. The correct plasmids were con-
firmed by digestion check with NdeI and HindIII.
Expression and purification of recombinant
enzymes
The two expression plasmids were, respectively, trans-
ferred into E. coli BL21(DE3) competent cells. The result-
ant strains were subsequently cultivated in LB broth sup-
plemented with 50  μg  mL −1 kanamycin. The cultures
were shaken at 250 rpm and 37 °C until the O ­ D600 reached
0.4–0.6. The cultivation temperature was then decreased to
28 °C and the cells were induced with 200 μM isopropyl-β-
d-1-thiogalactopyranoside (IPTG) for 12 h. The cells were
harvested by centrifugation at 3000×g and 4 °C for 10 min,
and subsequently washed twice with distilled water and
resuspended in 20 mM pH 7.9 Tris–HCl buffer containing
0.5 M NaCl. The harvested cells were disrupted by sonica-
tion on ice. The cell lysates were centrifuged at 10,000×g
and 4 °C for 10 min to collect the soluble fraction contain-
ing target protein. The supernatants were subsequently sub-
jected to 15% SDS-PAGE analysis using a premixed protein
marker (EZ-Run™ Pre-Stained Rec Protein Ladder, molecu-
lar range: 10–170 kDa, Fisher BioReagents) as the reference.
Gels were stained with Coomassie Brilliant Blue R250.
To purify the enzymes, the supernatants were loaded
onto a HisPur™ Ni-NTA affinity column (Thermo Sci-
entific, Rockford, USA) according to the manufacturer’s
protocols. After eluting the column with cool (4 °C) wash-
ing buffer (50 mM Tris–HCl, 2 mM EDTA, 10 mM imida-
zole, pH 7.9), the recombinant Sas-TAL and Sts-TAL were
finally eluted with elution buffer (50 mM Tris–HCl, 2 mM
EDTA, pH 7.9) containing 100 mM and 50 mM imidazole,
respectively.
In vivo functional characterization of Sas‑TAL
and Sts‑TAL
The engineered E. coli cells harboring the Sas-tal or Sts-
tal gene were grown and induced as described above. After
induction for 12 h, cells were harvested by centrifugation of
the broth at 3000×g and 4 °C for 10 min and subsequently
washed twice with distilled water. Whole-cell biotransfor-
mation was performed using a 10-mL system, which con-
sisted of the harvested E. coli cells ­(OD600 1.0) and 3 mM
l-tyrosine in 10 mM sodium phosphate buffer (pH 8.0). The
reaction mixture was incubated at 28 °C and 250 rpm for
6 h. 3 M HCl (2.0 mL) was then added to terminate the reac-
tion. The reaction mixture was centrifuged at 13,000×g for
15 min, and 50 μL of the supernatant was analyzed by HPLC
on an Agilent 1200 HPLC system (Agilent, USA) equipped
with an Agilent Eclipse Plus ­C18 column (4.6 mm × 250 mm,
5 μm) and a diode array detector. The HPLC was eluted with
15% (v/v) acetonitrile–water at 30 °C and the product was
detected at 310 nm. The ESI–MS spectrum was collected
on an Agilent 6130 single quadruple mass spectrometer. A
standard curve was established using commercial p-CA as
the standard to quantify the production of p-CA.
For in vivo PAL activity assay, the experiment was per-
formed in a similar way except that 3 mM l-phenylalanine
was added as the substrate. HPLC analysis for PAL activ-
ity was programmed with a gradient of methanol–water
containing 0.1% formic acid (0–5 min, 5:95; 5–14 min,
5:95–20:80; 14–18 min, 20:80–80:20; 18–25 min, 80:20;
v/v) over 25 min at 278 nm.
In vitro functional characterization of recombinant
enzymes
Enzymatic assays were performed in a 200-μL system
containing 10 mM glycine–NaOH buffer (pH 11.0), 3 mM
l-tyrosine, 6 μM enzyme for purified Sas-TAL and 3 μM
enzyme for purified Sts-TAL. The mixture was incubated at
50 °C for 1 h before the reaction was terminated by boiling at
100 °C for another 10 min. Then, the denatured protein was
removed by centrifugation at 13,000×g for 15 min, and 50
μL of the supernatant was analyzed by HPLC for the forma-
tion of p-CA with the methods mentioned above. Similarly,
the PAL activity of Sas-TAL and Sts-TAL was determined
using l-phenylalanine as the substrate. A standard curve was
established using commercial trans-cinnamic acid to meas-
ure the production of trans-cinnamic acid.
13

Determination of the optimal conditions for Sas‑TAL
and Sts‑TAL and their kinetic parameters
The effects of temperature on the TAL activity of Sas-
TAL and Sts-TAL were determined by measuring the
enzyme activity at various temperatures ranging from 30
to 70 °C under the standard assay conditions in 10 mM
glycine–NaOH buffer; while, the effects of pH on the TAL
activity were measured at varying pH values ranging from
7.0 to 12.0 using 10 mM sodium phosphate buffer (pH 7.0
and 8.0), 10 mM glycine–NaOH buffer (pH 9.0, 10.0, 10.5,
11.0 and 11.5) and 10 mM potassium chloride-NaOH (pH
12.0) at 50 °C.
For the kinetic studies, the initial reaction rates of the
purified TAL were evaluated by reacting the enzyme with
different concentrations of l-tyrosine ranging from 100
to 3 mM for both Sas-TAL and Sts-TAL, in 10 mM gly-
cine–NaOH buffer (pH 11.0) at 50  °C for 30  min. The
respective kinetic parameters, including Km and Vmax, were
calculated using the Lineweaver–Burk plots [20]. The value
of the turnover number (kcat) was calculated from the equa-
tion: kcat = Vmax/[E], where [E] is the enzyme concentration
in the reaction system and Vmax is the maximum velocity.
Furthermore, the effects of metal ions on the activity of
TAL were also analyzed by testing the purified enzymes
with 3  mM salt solutions of NaCl, KCl, F ­ eSO4, ­FeCl3,
­ZnCl2, ­CaCl2, ­CuCl2, ­MgCl2 and M ­ nCl2. The TAL activity
was studied under standard reaction conditions with 10 mM
glycine–NaOH buffer (pH 11.0), 3 mM l-tyrosine, 6 μM
enzyme for purified Sas-TAL and 3 μM enzyme for purified
Sts-TAL at 50 °C.
Production of p‑CA from l‑tyrosine using
engineered E. coli cells as the whole‑cell biocatalysts
The effects of temperature and pH on the efficiency of
whole-cell biotransformation were also studied with a simi-
lar method mentioned above. The tested temperatures ranged
from 30 to 70 °C and the pH values from 7.0 to 12.0.
Two different cell concentrations and substrate concentra-
tions were also tested. The cells (­ OD600 1.0 or 10.0) were
reacted with l-tyrosine (3 mM or 30 mM) in 10 mM gly-
cine–NaOH buffer (pH 11.0) at 50 °C for 1 h, with a total
volume of 10.0 mL. The bioconversion was terminated by
addition of 2.0 mL of 3 M HCl into the system. The reaction
mixtures were centrifuged at 13,000×g for 15 min and the
supernatants were subjected to HPLC analysis.
Statistical analysis
All experiments were carried out in triplicate and the
mean values were calculated. The standard deviation for
13
Bioprocess and Biosystems Engineering
each test was calculated using SPSS 20.0 and indicated
as error bars.
Results and discussion
Sequence analysis to two putative aromatic amino
acid lyases from actinomycetes
p-CA is a well-known antioxidant from nature. It is also
a common chemical precursor to synthesize other com-
pounds. TAL-catalyzed deamination of the l -tyrosine
is a convenient biological process for the production of
p-CA. Therefore, discovery of efficient TALs is critical
for developing cost-effective production processes of this
compound. TALs have been previously found from a few
sources such as R. capsulatus [15] and S. espanaensis [17].
They are often involved in the biosynthesis of proteins
and secondary metabolites. For example, a TAL from R.
capsulatus was found to be involved in the biosynthesis
of a photoactive yellow protein [15]. Sam8 is a TAL from
S. espanaensis that is involved in the biosynthesis of caf-
feic acid, a part of the aglycon of saccharomicins A and
B [17]. Discovery and heterologous expression of these
TALs makes it possible to produce p-CA in engineered
microorganisms such as bacteria and yeast [13].
Genome mining of potential TALs led to the discovery
of two putative AAAL genes from two actinomycetes.
The 1485 bp Sas-tal gene was found in Saccharothrix sp.
NRRL B-16348 and the 1479 bp Sts-tal gene was found
from Streptomyces sp. NRRL F-4489. BLAST analysis of
the protein sequences of these two proteins revealed that
they are homologous to a number of TALs and unchar-
acterized AAALs. Phylogenetic analysis of Sas-TAL and
Sts-TAL with 11 other proteins including TALs, PALs and
TALs/PALs from different organisms (Fig. 2a) showed
that Sas-TAL and Sts-TAL belong to two different but
closely related clades. Both of them showed closer rela-
tionship to reported TALs rather than PALs. Multiple pro-
tein sequence alignment (Fig. 2b) revealed the presence
of the conserved Ala-Ser-Gly motif that forms the unique
modified amino acid cofactor 3,5-dihyro-5-methylidene-
4H-imidazol-4-one (MIO) [14]. It was reported that there
is a substrate selectivity switch region (boxed in Fig. 2b)
in AAALs. In general, PALs have FL (phenylalanine and
leucine) in this region, TALs have HL (histidine and leu-
cine), and PALs/TALs have HQ (histidine and glutamine),
respectively [21]. A previous work reported that replac-
ing the active site His residue of TAL with Phe switched
its substrate specify from l-tyrosine to l-phenylalanine.
Similarly, mutation of P ­ he 144 with His in Arabidopsis
thaliana PAL1 converted it into a selective TAL [14].
Bioprocess and Biosystems Engineering
Fig. 2  Phylogenetic and sequence analysis of Sas-TAL and Sts-TAL.
a Phylogenetic analysis of Sas-TAL, Sts-TAL and 11 other aromatic
amino acid lyases (AAALs). b Partial amino acid sequence align-
ment of Sas-TAL, Sts-TAL and 11 other AAALs. The enzymes and
GenBank accession numbers are: 1, Sas-TAL from Saccharothrix sp.
NRRL B-16348 (WP_053717807.1); 2, Sts-TAL from Streptomyces
sp. NRRL F-4489 (WP_066973014.1); 3, Rc-TAL from Rhodobacter
capsulatus (WP_013066811.1) [15]; 4, Rs-TAL from Rhodobacter
sphaeroides (WP_011339422.1) [16]; 5, Se-TAL from Saccharothrix
espanaensis (WP_015103237.1) [11, 17]; 6, Pc-PAL from Petroseli-
num crispum Nym (CAA57056.1) [24]; 7, Jr-PAL from Juglans regia
(JX069977.1) [25]; 8, Jc-PAL from Jatropha curcas L. (ABI33979.1)
Therefore, the active site His residue plays a critical role
in the TAL activity and substrate selectivity. Both Sas-
TAL and Sts-TAL have HL in this region, suggesting that
they are TALs. Based on the results of the phylogenetic
[26]; 9, Tp-PAL from Trifolium pretense (AAZ29733.1) [27]; 10,
Ib-PAL from Inonotus baumii (OCB91043.1) [28]; 11, Rg-PAL
from Rhodotorula glutinis (AHB63479.1) [29]; 12, Zm-PAL from
Zea mays L. cv Corso (AAL40137.1) [30]; 13, Tc-PAL from Tri-
chosporon cutaneum (ABA69898.1) [21]. Sas: Saccharothrix sp.;
Sts: Streptomyces sp.; Rc: Rhodobacter capsulatus; Rs: Rhodobac-
ter sphaeroides; Se: Saccharothrix espanaensis; Pc: Petroselinum
crispum; Jr: Juglans regia; Jc: Jatropha curcas; Tp: Trifolium pre-
tense; Ib: Inonotus baumii; Rg: Rhodotorula glutinis; Zm: Zea mays;
Tc: Trichosporon cutaneum. The substrate selectivity switch is boxed
and the MIO-forming Ala-Ser-Gly motif is asterisked
analysis and protein sequence alignment, Sas-TAL and
Sts-TAL were predicted to be TALs that catalyze deami-
nation of l-tyrosine to yield p-CA.
13

Fig. 3  SDS-PAGE analysis of the expression and purification of Sas-
TAL and Sts-TAL. a Expression and purification of Sas-TAL from
E. coli BL21(DE3)/pET28a-Sas-tal. b Expression and purification
of Sas-TAL from E. coli BL21(DE3)/pET28a-Sts-tal. M: protein
ladder; 1: lysate of E. coli BL21(DE3)/pET28a; 2. lysate of E. coli
BL21(DE3)/pET28a-Sas-tal (a) or -Sts-tal (b); 3: purified Sas-TAL
(a) or Sts-TAL (b)
Expression and purification of Sas‑TAL and Sts‑TAL
The two genes were amplified from the genomic DNA
of corresponding hosts, and ligated to pET28a to yield
the corresponding expression plasmids, pET28a-Sas-tal
Fig. 4  In vivo functional characterization of Sas-TAL and Sts-TAL. a
HPLC analysis of conversion of l-tyrosine into p-coumaric acid by
Sas-TAL and Sts-TAL in E. coli at 310  nm. (i) l-tyrosine + E. coli
BL21(DE3)/pET28a; (ii) l-tyrosine + E. coli BL21(DE3)/pET28a-
13
Bioprocess and Biosystems Engineering
and pET28a-Sts-tal. The two plasmids were expressed in
E. coli BL21(DE3) with IPTG induction. The cells were
lysed by sonication for SDS-PAGE analysis. As shown in
Fig. 3a, compared to the vector control (E. coli BL21(DE3)/
pET28a, lane 1), a new ~ 55 kb protein band showed up in
the lysate of E. coli BL21(DE3)/pET28a-Sas-tal (lane 2),
suggesting that SAS-TAL has been successfully expressed.
Similarly, Sts-TAL was also expressed in E. coli BL21(DE3)
(Fig. 3b). These two enzymes were purified by Ni-NTA
chromatography to homogeneity (line 3 of Fig. 3a, b). The
yields of Sas-TAL and Sts-TAL were 92.4 ± 0.8 mg L−1 and
202.2 ± 5.7 mg L−1, respectively.
In vivo and in vitro functional characterization
of Sas‑TAL and Sts‑TAL
To verify the function of recombinant TALs, 3 mM l-tyros-
ine was, respectively, reacted with IPTG-induced E. coli
BL21(DE3)/pET28a-Sas-tal, E. coli BL21(DE3)/pET28a-
Sts-tal and E. coli BL21(DE3)/pET28a in 10 mM sodium
phosphate buffer (pH 8.0). HPLC analysis showed that the
vector control did not convert the substrate into any detect-
able product (trace i, Fig. 4a); while, both the engineered
Sas-tal; (iii) l-tyrosine + E. coli BL21(DE3)/pET28a-Sts-tal; (iv)
standard of p-coumaric acid. b UV spectrum of the bioconversion
product at 15.0  min. c ESI–MS (−) spectrum of the bioconversion
product at 15.0 min
Bioprocess and Biosystems Engineering
strains yielded a less polar product at 15 min (traces ii and
iii, Fig. 4a). This product has the same retention time as the
commercial standard of p-CA (trace iv, Fig. 4a). Further-
more, the product showed the same UV spectrum (Fig. 4b)
as that reported for p-CA [22]. Finally, the ESI–MS spec-
trum (Fig. 4c) of this compound showed a [M−H]− ion peak
at m/z 163.1, indicating that its molecular weight is 164 Da,
which is consistent with that of p-CA. Therefore, this prod-
uct was characterized as p-CA, and both Sas-TAL and Sts-
TAL can catalyze the deamination of l-tyrosine to yield p-
CA when they were expressed in E. coli BL21(DE3). The
purified enzymes were also reacted with l-tyrosine in vitro
and showed the same product, further confirming that these
two enzymes are TALs.
Determination of optimum reaction temperature
and pH for Sas‑TAL and Sts‑TAL
To determine the kinetic parameters of Sas-TAL and Sts-
TAL, we first measured how the reaction temperature and
pH affect the enzymatic activity of these two TALs. A total
of seven temperatures, including 30, 40, 45, 50, 55, 60 and
70 °C, were tested. We established the standard curves of
p-CA (Fig. 5a) and cinnamic acid (Fig. 5b) using authen-
tic samples. As shown in Fig. 6a, the activity of Sas-TAL
increased from 55.50 ± 0.14 to 181.81 ± 8.86 µM p-CA
­mg−1 min−1 when the reaction temperature was increased
from 30 to 55 °C. However, further increase in the tem-
perature resulted in decreased enzymatic activity, which
might be due to the inactivation of the enzyme at high tem-
peratures. Therefore, 55 °C is the optimum temperature
for Sas-TAL for in vitro enzymatic reactions. Similarly,
the optimum temperature for Sts-TAL was determined to
Fig. 5  The standard curves of p-CA (a) and trans-cinnamic acid (b) for quantification of the product formation
be 50 °C. These are higher than the optimum temperatures
of several reported TALs that are typically in the range of
25–40 °C (Table 2).
We next tested the effect of pH on the activity of Sas-
TAL and Sts-TAL. Eight pH values, ranging from 7 to
12, were examined for the reaction systems of these two
enzymes. As shown in Fig.  6b, the enzymatic activ-
ity of Sts-TAL increased when the pH was increased
from 7 to 11, and the maximum activity of Sts-TAL
(330.57 ± 3.05  µM p-CA ­m g −1  min −1) was achieved at
pH 11. Apparent drop in the activity of Sts-TAL was
observed when the pH was further increased to 11.5 and
12. As such, the optimum pH value for Sts-TAL was
determined to be 11. For Sas-TAL, its enzymatic activity
increased from 11.94 ± 0.97 to 147.99 ± 1.59 µM p-CA
­mg−1 min−1 when the pH was increased from 7 to 10.5.
Similar enzymatic activity was observed for Sas-TAL 10.5,
11.0 and 11.5, and a significant decrease in the activity
was observed when the reaction pH was increased to 12.
Thus, we chose pH 11 for both enzymes for the following
studies. Alkaline pH is generally preferred by TALs to
catalyze the deamination reaction of l-tyrosine. The opti-
mum pH for Sas-TAL and Sts-TAL is much higher than the
optimum pH values for several previously reported TALs
(Table 2), such as the TAL from R. glutinis (pH 8.5) and
Sam8 from S. espanaensis (pH 8.8). This indicated that
Sas-TAL and Sts-TAL might be more stable and perform
more efficiently at pH 11 than others. We also calculated
the theoretical isoelectric point (pI) values of related TALs
using the ExPASy-ProtParam tool. As shown in Table 2,
both Sas-TAL and Sts-TAL have a much lower pI value
than Rg-TAL, Rc-TAL and Rs-TAL. By contrast, Sam8 has
a similar pI value with Sas-TAL and Sts-TAL.
13
 Bioprocess and Biosystems Engineering

Fig. 6  Effects of the temperature (a), pH (b), substrate concentration (c) and metal ions (d) on the activity/reaction velocity of purified Sas-TAL
and Sts-TAL

Table 2  Comparison of optimum temperature and pH as well as kinetic parameters of Sas-TAL and Sts-TAL with other reported TALs

Enzyme Source Optimum Optimum pH Theoretical pI Km (μM) kcat ­(s−1) kcat/Km (μM−1 s−1) References
temperature
(°C)

Sas-TAL Saccharothrix sp. NRRL 55 11.0 5.14 1492.2 155.2 0.104 This work
B-16348
Sts-TAL Streptomyces sp. NRRL F-4489 50 11.0 4.95 336.5 439.1 1.3 This work
Rg-TAL Rhodotorula glutinis 40 8.5 6.44 380.0 114 0.3 [1]
Rc-TAL Rhodobacter capsulatus DSMZ 35 8.5 6.50 15.6 27.7 1.77 [15]
1710
Rs-TAL Rhodobacter sphaeroides 25 8.5 7.30 60 0.02 0.333 × 10–3 [16]
Sam8 Saccharothrix espanaensis 30 8.8 5.27 15.5 0.015 0.968 × 10–3 [17]
NRRL-15764

13
Bioprocess and Biosystems Engineering
Determination of the kinetic parameters of Sas‑TAL
and Sts‑TAL
We next measured the kinetic parameters of Sas-TAL and
Sts-TAL at the optimal temperature and pH. Specifically, pH
11 and 55 °C were used for Sas-TAL, and pH 11 and 50 °C
for Sts-TAL. Figure 6c showed the kinetic behaviors of the
two TALs. The two linear double-reciprocal plots obtained
with higher correlation coefficients indicated that both TALs
obeyed Michaelis–Menten kinetics with regard to l-tyrosine.
Based on the nonlinear regression analysis, Sas-TAL showed
Kcat, Km and kcat/Km values of 9.3 × 103 min−1 (or 155.2 s−1),
1492.2 μM and 6.2 μM−1 min−1 (or 0.104 μM−1 s−1), while
these values for Sts-TAL were 2.6 × 104 min−1 (or 439.1 s−1),
336.5 μM and 78.3 μM−1 min−1 (or 1.3 μM−1 s−1), respec-
tively. The turnover numbers of Sas-TAL and Sts-TAL are
higher than those of known TALs, and their Km values are
larger than most reported TALs as well. Both high kcat and
Km values are likely due to the higher reaction temperatures.
Compared with the kinetic parameters of reported microbial
Fig. 7  The PAL activity of Sas-TAL and Sts-TAL. a HPLC analysis
(278  nm) of the formation of cinnamic acid from l-phenylalanine
by Sas-TAL- and Sts-TAL-harboring E. coli. (i) l-tyrosine + E. coli
BL21(DE3)/pET28a; (ii) l-tyrosine + E. coli BL21(DE3)/pET28a-
TALs shown in Table 2, both Sas-TAL and Sts-TAL exhib-
ited a much higher turnover number (Kcat) toward l-tyros-
ine than reported TALs, and Sts-TAL also showed higher
catalytic efficiency (kcat/Km), indicating that these enzymes,
especially Sts-TAL, represent efficient biocatalysts for p-CA
production.
Effect of metal ions on the enzymatic activity
of Sas‑TAL and Sts‑TAL
To analyze the effect of metal ions on the activity of Sas-
TAL and Sts-TAL, 3 mM of different metal ions was added
to the reaction system. As shown in Fig. 6d, addition of
­Na+, ­K+, ­Ca2+ and ­Mg2+ slightly increased the enzymatic
activity of Sas-TAL (less than 10%), while ­Fe2+, ­Fe3+, ­Zn2+,
­Cu2+ and M ­ n2+ showed strong inhibition to this enzyme.
However, there were no ions that increased the activity of
Sts-TAL. All tested groups showed weaker activity than the
control (showing 25.2–92.2% of the original activity), except
Sas-tal; (iii) l-tyrosine + E. coli BL21(DE3)/pET28a-Sts-tal; (iv)
standard of p-coumaric acid. b Comparison of in vitro TAL activity
and PAL activity for Sas-TAL and Sts-TAL
13

­ a+ and K
N ­ + that had no effect on the activity of Sts-TAL at
3 mM.
Deamination of l‑phenylalanine by Sas‑TAL
and Sts‑TAL
Both TALs and PALs belong to the family of aro-
matic amino acid lyases. There is also one type of PAL/TAL
capable of catalyzing the deamination of both phenylalanine
and tyrosine [13]. We, therefore, investigated whether Sas-
TAL and Sts-TAL can convert l-phenylalanine into trans-
cinnamic acid. HPLC analysis showed that compared to the
control (trace i, Fig. 7a), the whole-cell biotransformation of
l-phenylalanine with E. coli BL21(DE3)/Sas-TAL generated
a less polar product (trace ii, Fig. 7a). Similarly, the same
product was obtained from the reaction of l-phenylalanine
with E. coli BL21(DE3)/Sas-TAL (trace iii, Fig. 7a). This
product was found to be trans-cinnamic acid by a compari-
son with the commercial standard (trace iv, Fig. 7a). These
results indicated that the two actinomycete TALs also have
the PAL activity.
To compare the TAL and PAL activities of Sas-TAL,
in vitro reactions were conducted at pH 11 and 55 °C, and
calculated according to the standard curves (Fig. 5a, b). As
shown in Fig. 7b, under the same reaction conditions, Sas-
TAL showed a PAL activity of 169.24 ± 1.71 µM trans-cin-
namic acid·mg−1·min−1, which is 36% higher than its TAL
activity. By contrast, when TAL and PAL activities of Sts-
TAL were measured at pH 11 and 55 °C, the TAL activity
was found to be 301.05 ± 4.15 µM p-CA ­mg−1 min−1, and
its PAL activity was only 19.67 ± 0.44 µM trans-cinnamic
acid ­mg−1 min−1. The TAL activity of Sts-TAL is 15.3-
fold higher than its PAL activity. This results indicate that
both Sas-TAL and Sts-TAL can catalyze the deamination
of l-tyrosine and l-phenylalanine. Sts-TAL strongly prefers
l-tyrosine as the substrate; whereas, Sas-TAL slightly prefers
l-phenylalanine to l-tyrosine.
Determination of the optimal conditions
for the production of p‑CA using E. coli BL21(DE3)/
Sas‑TAL and E. coli BL21(DE3)/Sts‑TAL cells
Since purification of enzymes from E. coli cells is costly,
whole-cell biocatalysis represents a convenient and cost-
effective way to produce industrially valuable products
[23]. We already showed that the TAL-harboring E. coli
cells can convert l-tyrosine into p-CA in 3.3. To find out
the optimal conditions for the whole-cell biotransforma-
tion process, we next investigated the effects of the reac-
tion temperature and pH on the production of p-CA. To
test the optimal temperature for whole-cell conversion of
l-tyrosine into p-CA, E. coli BL21(DE3)/Sas-TAL and E.
coli BL21(DE3)/Sts-TAL cells ­(OD600 1.0) were incubated
13
Bioprocess and Biosystems Engineering
with 3 mM l-tyrosine in 10 mM glycine–NaOH buffer (pH
11) at different temperatures ranging from 30 to 70 °C for
1 h. As shown in Fig. 8a, when the reaction temperature was
increased, enhanced specific activity of E. coli BL21(DE3)/
Sts-TAL was also obtained. At 50 °C, the specific activity
reached 2291.0 ± 17.2 μM p-CA per ­OD600 unit cells h−1.
Further increase in the reaction temperature to 60 °C and
70 °C led to a decrease in the activity. A similar trend was
observed for E. coli BL21(DE3)/Sas-TAL, except that its
overall efficiency was much lower than E. coli BL21(DE3)/
Sts-TAL. Therefore, our results suggested that the optimum
temperature for the production of p-CA by both strains are
50 °C (Fig. 8a). We also evaluated what pH value is opti-
mal for whole-cell biotransformation of l-tyrosine to p-CA.
Eight different pH values (pH 7, 8, 9, 10, 10.5, 11, 11.5 and
12) were tested, among which pH 11 was the best for both E.
coli BL21(DE3)/Sas-TAL and E. coli BL21(DE3)/Sts-TAL
(Fig. 8b). This is consistent with the optimum pH for the
purified enzymes.
We next compared two different cell concentrations in the
whole-cell biotransformation process. E. coli BL21(DE3)/
Sas-TAL and E. coli BL21(DE3)/Sts-TAL were tested for
converting l-tyrosine into p-CA at different O ­ D600 values
­(OD600 1.0 and 10.0). The system with O ­ D600 1.0 cells used
3 mM l-tyrosine as the substrate, while 30 mM l-tyrosine
was used for O­ D600 10.0 cells. The reactions were performed
at pH 11 and 50 °C for 1 h. As shown in Fig. 8c, when
the ­OD600 1.0 cells reacted with 3 mM l-tyrosine, E. coli
BL21(DE3)/Sas-TAL achieved 0.135 ± 0.001  mM p-CA
with a 4.5% conversion rate; whereas, E. coli BL21(DE3)/
Sts-TAL achieved 2.538 ± 0.103 mM p-CA with an 84.6%
conversion rate. When the ­OD600 value was increased to 10.0
and l-tyrosine concentration to 30 mM, the final titers of p-
CA for E. coli BL21(DE3)/Sas-TAL and E. coli BL21(DE3)/
Sts-TAL were 1.206 ± 0.019 mM (equivalent to 0.198 g L−1)
and 17.575 ± 0.727 mM (equivalent to 2.88 g L−1), with
the corresponding conversation rates of 4.0% and 58.6%,
respectively.
Consistent with the in vitro enzymatic activities, E. coli
BL21(DE3)/Sts-TAL is much more efficient than E. coli
BL21(DE3)/Sas-TAL. This is expected because Sts-TAL
has better catalytic efficiency and expression level than
Sas-TAL. The productivity of E. coli BL21(DE3)/Sts-TAL
reached 2.88 ± 0.12 g (L h)−1 (equivalent to 1.76 mM p-CA/
unit of OD600 cells h−1). This is four times the productivity
of p-CA (440 μM p-CA/unit of OD600 cells) in E. coli by
the TALs from Herpetosiphon aurantiacus and Flavobac-
terium johnsoniae, which was measured in the induced fer-
mentation broth for more than 24 h [13]. Therefore, E. coli
BL21(DE3)/Sts-TAL outperformed the previously reported
bacterial strains for the production of p-CA, representing a
promising strain for the production of this valuable chemi-
cal. The production efficiency may be further increased by
Bioprocess and Biosystems Engineering
Fig. 8  Determination of optimal conditions for whole-cell conver-
sion of l-tyrosine to p-CA with engineered E. coli cells. a Effect of
the reaction temperature on the specific conversion activity of E.
coli BL21(DE3)/Sas-TAL and E. coli BL21(DE3)/Sts-TAL. b Effect
directed evolution of Sts-TAL and enhanced expression of
this enzyme.
Conclusions
In this work, we identified two AAALs from two different
actinomycetes. Both Sas-TAL and Sts-TAL were found to
have the TAL and PAL activities. While Sas-TAL showed a
better PAL activity than TAL activity, Sts-TAL strongly pre-
fers l-tyrosine as the deamination substrate. The optimum
temperatures of Sas-TAL and Sts-TAL are 55 °C and 50 °C,
while the optimum pH for both TALs is 11.0, respectively.
Kinetic studies on the purified enzymes revealed that Sts-
TAL is a much more efficient TAL than Sas-TAL, with a
kcat/Km value of 78.3 μM−1 min−1. Besides, the engineered E.
of the reaction pH on the specific conversion activity of E. coli
BL21(DE3)/Sas-TAL and E. coli BL21(DE3)/Sts-TAL. c Compari-
son of the specific activity of E. coli BL21(DE3)/Sas-TAL and E. coli
BL21(DE3)/Sts-TAL at different cell and substrate concentrations
coli strain harboring Sts-tal shows a high conversion rate of
1.758 mM p-CA/unit of O ­ D600 cells, representing the high-
est productivity of p-CA in prokaryotic cells. This discovery
will provide a much more effective way for producing the
industrially valuable material p-CA through an environmen-
tal friendly and sustainable process.
Acknowledgements  This research was financially supported by
National Natural Science Foundation of China (81973211) and Fund-
ing of Changsha Science and Technology Bureau (kq1701069).
Compliance with ethical standards 
Conflict of interest  The authors declare no conflict of interest.
Ethical approval  This study does not contain any studies with human
participants or animal performed by any of the authors.
13
 Bioprocess and Biosystems Engineering

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