Development of a biocatalytic cascade for synthesis of 2-oxo-4-(hydroxymethylphosphinyl) butyric acid in one pot

Article abstract of DOI:10.1080/10242422.2020.1797697

2-Oxo-4-(hydroxymethylphosphinyl) butyric acid (PPO) is an important precursor compound for the broad-spectrum herbicide l-glufosinate (L-PPT). In this study, the gene of d-amino acid oxidase (DAAO) was cloned and expressed in Escherichia coli. By coupling exogenous catalase (CAT), a biocatalytic cascade was constructed for synthesis of PPO in one pot. The bioprocess was optimized on a 300 mL scale reaction by one factor at a time optimization. The conversion of this biocatalytic cascade achieved 46.8% towards 400 mM DL-PPT within 4 h. These results indicated that DAAO could be applied to the large-scale bioproduction of PPO and provide a promising route for the asymmetric synthesis of L-PPT by bio-enzymatic methods using PPO as the substrate.

Full text of DOI:10.1080/10242422.2020.1797697

Biocatalysis and Biotransformation
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Development of a biocatalytic cascade for
synthesis of 2-oxo-4-(hydroxymethylphosphinyl)
butyric acid in one pot
Jianmiao Xu , Kai Zhang , Huiting Cao , Heng Li , Feng Cheng , Chenghao Cao ,
Ya-Ping Xue & Yu-Guo Zheng
To cite this article: Jianmiao Xu , Kai Zhang , Huiting Cao , Heng Li , Feng Cheng , Chenghao
Cao , Ya-Ping Xue & Yu-Guo Zheng (2020): Development of a biocatalytic cascade for synthesis
of 2-oxo-4-(hydroxymethylphosphinyl) butyric acid in one pot, Biocatalysis and Biotransformation,
DOI: 10.1080/10242422.2020.1797697
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2020.1797697
RESEARCH ARTICLE
Development of a biocatalytic cascade for synthesis of 2-oxo-4-
(hydroxymethylphosphinyl) butyric acid in one pot
Jianmiao Xua,b, Kai Zhanga,b, Huiting Caoa,b, Heng Lia,b , Feng Chenga,b, Chenghao Caoa,b
Ya-Ping Xuea,b and Yu-Guo Zhenga,b
,
aKey Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of
b
Technology, Hangzhou, P. R. China; National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals,
Zhejiang University of Technology, Hangzhou, P. R. China
ABSTRACT
ARTICLE HISTORY
Received 27 April 2020
Revised 22 June 2020
Accepted 27 June 2020
2-Oxo-4-(hydroxymethylphosphinyl) butyric acid (PPO) is an important precursor compound for
the broad-spectrum herbicide L-glufosinate (L-PPT). In this study, the gene of D-amino acid oxi-
dase (DAAO) was cloned and expressed in Escherichia coli. By coupling exogenous catalase
(CAT), a biocatalytic cascade was constructed for synthesis of PPO in one pot. The bioprocess
was optimized on a 300 mL scale reaction by one factor at a time optimization. The conversion
of this biocatalytic cascade achieved 46.8% towards 400mM DL-PPT within 4 h. These results
indicated that DAAO could be applied to the large-scale bioproduction of PPO and provide a
promising route for the asymmetric synthesis of L-PPT by bio-enzymatic methods using PPO as
the substrate.
KEYWORDS
2-oxo-4-(hydroxymethyl-
phosphinyl)butyric acid;
D-amino acid oxidase;
catalase; biocatalysis
1. Introduction
more attention in the synthesis of A-keto acid and
non-natural chiral amino acid, exhibiting favourable
application prospect. Additionally, the tightly bound
cofactor FAD, making additional FAD unnecessary dur-
ing the biotransformation process, also favours its
industrial applications in biotransformation (Hsieh
et al. 2009).
Until now, the most important industrial application
of DAAO is the production of 7-aminocephalosporanic
acid (7-ACA), a key pharmaceutical intermediate for
the synthesis of cephalosporin antibiotics (Pollegioni
et al. 2004). Starting from 7-ACA, about 200 tons per
year of semisynthetic cephalosporins are produced,
demonstrating potent potential of DAAO in keto acid
production. However, there is seldom report about
DAAO in preparation of keto acids containing hetero
atoms, such as 2-oxo-4-(hydroxymethylphosphinyl)
butyric acid (PPO). PPO is a vital keto acid intermedi-
ate in the asymmetric synthesis of optically pure L-glu-
fosinate (a low-toxicity, broad-spectrum, and safe
organophosphorus herbicide) which exhibits twice the
herbicidal activity of racemic glufosinate (Jin et al.
2019).Asymmetric amination of PPO, using transamin-
ase or glutamate dehydrogenase as biocatalyst, to
D-Amino acid oxidase (DAAO, EC 1.4.3.3) catalyses the
oxidative deamination of a wide range of D-amino
acids to their corresponding imino acids with absolute
stereospecificity, which immediately undergo non-
enzymatic hydrolysis to the respective A-keto acids
and ammonia (Pollegioni and Molla 2011; Han et al.
2015; Xue et al. 2018). With the presence of molecular
oxygen, co-enzyme flavin adenine dinucleotide (FAD)
is re-oxidized, while the electron-acceptor oxygen is
reduced to hydrogen peroxide (H2O2) during the reac-
tion (Scheme 1; Trost and Fischer 2002). The enzyme
is widespread in nature, from microorganisms
(Shimekake et al. 2019) to mammals (Murtas et al.
2019). To date, only a few DAAO have been obtained
as a homogeneous preparation, such as PkDAAO
(Bakke and Kajiyama 2004) from pig kidney and
RgDAAO (Pollegioni et al. 2002) from yeast. The
enzymes from microorganisms appear to be far more
suitable catalysts for bioconversion (Zhu et al. 2011;
Conti et al. 2015). RgDAAO appears to be a suitable
catalyst for industrial application due to its broader
pH activity and a higher activity at saturating oxygen
concentrations. Moreover, it has attracted more and
CONTACT Ya-Ping Xue
xyp@zjut.edu.cn
College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, P.
R. China
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
J. XU ET AL.
2. Materials and methods
2.1. Chemicals
DL-Glufosinate substrate (purity, >82.5%) and PPO
(purity, >99%) were provided by Dezhou Lvba Fine
Chemical Co., Ltd. (Dezhou, China). Plasmid Miniprep
kits and DNA gel extraction kits were purchased from
Axygen Scientific Inc (Hangzhou, China). Phanta Max
Super-Fidelity DNA Polymerase used for mutagenesis
was purchased from Vazyme (Nanjing, China). SDS-
PAGE, Bis-Tris buffer and BCA protein assay kit were
purchased from GenScript (Nanjing, China). Hydrogen
peroxide (H2O2) assay kit was purchased from
Solarbio. Unless otherwise stated, all other chemicals
were of analytical grade and commercially available.
Scheme 1 Preparation of PPO from DL-PPT by DAAO
and catalase
generate L-PPT has the advantage of 100% maximum
theoretical yield, and become a promising strategy for
industrial production of L-PPT. Currently, the synthesis
of PPO in industry is mainly through chemical meth-
ods, and there are no biological methods reported in
related literature. In 1991, using 3-(methylethoxyphos-
phoryl)-propionic acid methyl ester as a starting
material, Hoechst condensed it with diethyl oxalate,
following by the hydrolysis, finally obtaining the target
compound by the decrystallization with 65% total
yield (Zeiss 1991). Serious environmental pollution,
complex process and low yield are always critical
problems in chemosynthesis of PPO (Baillie et al. 1983;
Zeiss 1991). Considering strict stereoselectivity, mild
reaction conditions, high recovery and simple separ-
ation and purification processes, deamination of
racemic glufosinate by DAAO would be an ideal alter-
native due to its strict D-isomer enantioselectivity,
retaining L-isomer with herbicidal activity.
2.2. Fed batch fermentation production of DAAO
in 5 L fermenter
DAAO gene was synthesized and cloned into the first
ORF frame of pCDFduet-1 vector. The recombinant
strain containing RgDAAO was cultured in
Luria–Bertani (LB) medium containing streptomycin
(50 lg/mL) at 37 ꢀC for 12 hours. A 1% (v/v) cell culture
was then inoculated into a 500 mL shake flask contain-
ing Terrific Broth (TB) medium (100 mL). The cells were
cultivated at 37 ꢀC and 180 rpm for 6 h as a seed
liquid to inoculate the 3 L batch medium (yeast extract
12 g/L, peptone 15 g/L, NaCl 10 g/L, glycerine 12 g/L,
(NH4)2SO4 5 g/L, K2HPO4 1.36 g/L, KH2PO4.3H2O
2.28 g/L, MgSO4.7H2O 0.375 g/L) for fed-batch fermen-
tation in a 5 L fermenter (Winpact thermometer sys-
tem fermenter, New York, USA) cultured under the
following conditions: 37 ꢀC, 500 rpm agitation, 1.6 vvm
aeration, pH 7.0 (kept constant by adding 50% (v/v)
aqueous NH3ꢁH2O and 50% (v/v) aqueous H3PO4).
When the optical density (OD600) value reached 8–10,
lactose (10 g/L) was added, and the culture was
induced at 28 ꢀC for 12 h. Recombinant cells were har-
vested by centrifugation at 8,000 rpm and 4 ꢀC for
10 min. All assays were performed in triplicate.
H2O2, a byproduct of the aforementioned DAAO-
catalysed reaction, is a disadvantageous factor in the
production of keto acid, since the keto acid will be
decarboxylated by H2O2 to form the corresponding
carboxylic acid (Upadhya et al. 2000). The traces of
catalase (E.C. 1.11.1.6) which converts H2O2 to water
and molecular oxygen, can be seen in most DAAO-
ꢀ
ꢀ
preparation processes (Seo et al. 2012; Garcıa-Garcıa
et al. 2018), as catalase is a technical enzyme which is
available in large quantities (Nadler et al. 1986).
Moreover, in most cases, addition of free catalase is
simpler and more economic than chemical methods
to remove H2O2 (Han and Shin 2018).
2.3. Biosynthesis of PPO
Herein, a DAAO from Rhodotorula gracilis (RgDAAO)
was expressed in E. coli. A biocatalytic cascade was
constructed for PPO production by exogenously add-
ing catalase. After optimization of the catalase concen-
tration, aeration flow rates and other reaction
conditions, a maximum PPO yield of 46.8% and an e.e.
value above 99% were obtained. This is the first report
that racemic glufosinate converted to PPO by DAAO/
CAT-combined cascade in one pot.
To develop a more convenient and economical pro-
cess, whole cells were used as catalysts in the reaction.
The collected cells were suspended in deionized
water. The initial reaction mixture exists in two reac-
tion systems: (i) DL-PPT (150 mM) and catalase (193 U/
mL) made up to a final volume of 30 mL in a 50 mL
shake flask. This reaction was used to optimize the
temperature and pH conditions. (ii) DL-PPT (400 mM)

BIOCATALYSIS AND BIOTRANSFORMATION
3
Figure 1. Effect of reaction temperature (a) and pH (b) on the yield of PPO.
and catalase (1926 U/mL) with an aeration flow rate of
0.8 L/min, made up to a final volume of 300 mL in a
1 L bioreactor with 1‰(v/v) antifoam added, condi-
tions such as aeration flow rate and the addition of
catalase were optimized. The wet whole cells contain-
ing DAAO in both the above reactions had concentra-
tions of 20 g/L, and the pH of the initial reaction was
8.0 by titration with NH3ꢁH2O, reactions were per-
formed with magnetic stirring at 500 rpm in a water
bath at 30 ꢀC. The experiments were conducted in
triplicate. Samples were withdrawn from the reaction
mixture at different time points, and 6 M HCl (4 mL)
was added to 200 mL of the sample to terminate the
reaction, followed by the addition of 6 M NaOH (4 mL)
before HPLC analysis.
The concentration of PPO was determined by
Diode Array Detector (UltiMate DAD-3000, 232 nm) of
the same HPLC instrument and column with the same
flow rate. The mobile phase contained 50 mM ammo-
nium dihydrogen phosphate (pH 3.8) buffer, 0.1% tet-
rabutyl ammonium bromide and 12% acetonitrile. The
column temperature was maintained at 40 ꢀC. The
retention time of PPO was 9.4 min.
3. Results and discussion
3.1. Effect of temperature and pH on DAAO-
mediated chiral resolution of racemic glufosinate
Reaction temperature has
a profound effect on
enzyme activity (Thakur et al. 2019). Therefore, the
influence of temperature on reaction was studied
between 20 and 50 ꢀC. As shown in Figure 1(a), the
DAAO exhibits absolute enantioselectivity (>99% e.e.)
under all reaction temperature. In the range of
25–40 ꢀC, the yield of PPO reached 43.1%, indicating
that the enzyme had excellent temperature adaptabil-
ity. Above and below this range, a decreased PPO
yield was observed. A sharp decline in PPO yield was
observed above 50 ꢀC. Therefore, 30 ꢀC was selected as
the optimal reaction temperature for further study.
The effect of pH on the enzymatic reaction was
also investigated in the pH range of 5.0–9.0 using two
different buffers, K2HPO4-KH2PO4 (pH 6.0–8.0) and Tris-
HCl (pH 8.5–9.0), with the same concentrations of
150 mM. As shown in Figure 1(b), the PPO yield was
greater than 42% in the pH range of 7.5–8.5. The
highest PPO yield was obtained at pH 8.0. When the
pH was lower than 6.5 or higher than 8.5, the PPO
yield decreased, indicating that these pH conditions
are harmful to the enzyme stability and activity. It is
2.4. Analytical methods
DL-PPT was derivatized at 30 ꢀC for 5 minutes with
derivative reagent [2.01 g o-phthalaldehyde and 2.43 g
N-acetyl-L-cysteine dissolved in 1 L boric acid buffer
(100 mM, pH 9.8)]. Then the concentration and excess
(e.e.) of DL-PPT were determined by fluorescence
detector (UltiMate FLD-3100, Ex 340 nm and Em
450 nm) of high performance liquid chromatography
(HPLC, ThermoFisher UltiMate 3000) on a C18 column
(Unitary C18, 5 lm, 100 A, 4.6 mm ꢂ 250 mm) at a flow
rate of 1.0 mL/min. The column temperature was
maintained at 35 ꢀC. Each sample (10 lL) was eluted
with 50 mM ammonium acetate (pH 5.7) , containing
10% methanol as mobile phase. The retention time of
D-PPT was 13.6 min (Lv et al. 2019). The calculation
CLꢃPPT ꢃCDꢃPPT
formula of e.e. value is e:e: ¼
ꢂ 100%,
CLꢃPPT þCDꢃPPT
CL-PPT and CD-PPT are measured by the peak area.

4
J. XU ET AL.
Figure 2. Time courses of the catalytic reaction under different conditions. Effect of DL-PPT on the yield of PPO in 50 mL (a) and
1 L reactor (b); Effect of flow rate of air (c) and pure oxygen (d) on the yield of PPO.
reported that the stereoselectivity of DAAO is strict
and is not related to the reaction pH. Furthermore,
when the pH was adjusted to 8.0 with aqueous
ammonia, the same yield was obtained as in the phos-
phate buffer (pH 8.0). Combining the above results,
and considering the subsequent separation and purifi-
cation, and cascade reaction with transaminase or glu-
tamate dehydrogenase to synthesize L-PPT, ammonia
was used in subsequent experiments to adjust the
reaction system to pH 8.0.
reactor (Toftgaard Pedersen et al. 2015). Firstly, the
oxidative reaction was performed in the 50 mL shake
flask, When the reaction substrate was only 150 mM,
the aeration had little effect on the reaction, and the
conversion could be completely achieved without
addition of air (Figure 2(a)). The practical concentra-
tion of O2 (aq) is quiet low (data not shown), indicat-
ing the effect of O2 on the reaction process is not
being exhibited since the O2 mass transfer was lim-
ited. Previous study has demonstrated that the mass
transfer capability of O2 is limited by selected equip-
ment and the maximum oxygen transfer rate in an
industrial scale bioreactor is typically around
100 mmol/L/h (Charles 1985; Toftgaard Pedersen et al.
2015; Chapman et al. 2018). Subsequently, the reac-
tion was performed in the 1 L stirred-tank reactor since
3.2. Effect of oxygen for DAAO
catalysed oxidation
Oxidases require oxygen, which in biotechnology trad-
itionally is supplied by bubbling air through the

BIOCATALYSIS AND BIOTRANSFORMATION
5
Figure 3. Effect of catalase concentration on the yield of PPO.
Figure 4. Effect of biocatalyst concentration on the yield
of PPO.
the propeller caused more stronger force for
O2(g)!O2(aq). The corresponding results are shown in
Figure 2(c), when the air flow rate was 0.8 L/min, the
maximum substrate concentration was increased from
150 mM to 400 mM gradually, representing a 1.67-fold
increase compared with that of the 50 mL reaction sys-
tem (Figure 2(b)). At the maximum substrate concen-
tration, the yield of PPO improved from 30% to 43.1%
after 12 h gradually, with the flow rate of air increased
from 0 L/min to 0.8 L/min (Figure 2(c)). In addition, the
catalytic rate was slightly accelerated at the beginning
of the reaction (from 12.2 mM/h to 22.4 mM/h).
To further improve the catalytic efficiency, or in
other words, relieve the limit of O2, pure oxygen with
different flow rate was introduced to the reactor. The
catalytic time was shortened and the maximum PPO
yield of 46.8% was achieved at 4 h (Figure 2(d)).
Compared with 0.8 L/min air, 0.6 L/min pure oxygen
increases the maximum yield of PPO from 43.1% to
46.8%. However, from an applied point of view, it is
optional to introduce 0.8 L/min of air into the catalytic
system for the reaction.
being degraded and protecting the activity of the
enzyme. As shown in Figure 3, D-PPT was completely
converted with 193 U/mL catalase. Hydrogen peroxide
generated in the reaction was not effectively removed
and PPO was oxidized to 3-(hydroxymethylphosphinyl)
propionic acid (MPP), resulting in a low PPO yield.
Interestingly, the PPO yield achieved 43.1% with
1926 U/mL catalase adding, but 50% yield could not
be achieved with addition of more catalase.
Furthermore, we found the concentration of H2O2 in
the reaction mixture is quite low (about 0.5 mM), while
the concentration in the whole cells of the reaction
solution is very high (about 5.8 mM). We concluded
that much more catalase was not helpful for removing
H2O2 thoroughly and some PPO was oxidized in the
whole cells, leading to the loss of total yield. Thus,
1926 U/mL catalase was selected as the optimal add-
ing amount.
3.4. Effect of biocatalyst concentration on
catalytic reaction
Insufficient addition of biocatalyst will cause slow con-
version of substrate, while excessive biocatalyst con-
centration might affect gas transmission. To further
improve the catalytic performance of DAAO, the whole
cell catalyst concentration was optimized. As shown in
the Figure 4, biocatalyst concentration below 20 g/L
could not convert substrate completely, and the prod-
uct yield increased with increasing whole cells concen-
tration. But when the concentration of the biocatalyst
was higher than 20 g/L, the PPO yield remained basic-
ally unchanged. Therefore, the optimal DAAO whole
cells concentration was selected at 20 g/L.
3.3. Effect of H2O2 and protection by catalase in
the reaction
H2O2 is a byproduct of the DAAO-catalysed oxidative
deamination of D-amino acids. Previous studies have
demonstrated that it not only decarboxylates oxidized
keto acids to form a carboxylic acid with one carbon
less, but also inactivates DAAO by oxidizing cysteine
ꢀ
and serine residues of the enzyme (Fernandez-
Lafuente et al. 1999; Trost and Fischer 2002).
Removing H2O2 by catalase would generate more dis-
solved oxygen to facilitate reaction, preventing PPO

6
J. XU ET AL.
which is the preferred temperature in industry. Mild
pH profiles may be a favourable characterization for
industrial application.
Oxidation reactions are notorious types of reaction
where O2 is always a bottleneck (Umhau et al. 2000;
Pollegioni et al. 2007; Rosini et al. 2011). In the 50 mL
flask shake, the initial rate was significantly limited by
the depletion of oxygen. When the reaction was
scaled up to 1 L stirred-tank reactor, initial rate and
catalytic performance were improved with addition of
air, while the feeding of pure oxygen further verified
the effect of O2. We found the maximum PPO yield
was obtained at 4 h when pure oxygen was intro-
duced into the reactor. The shortened reaction time
also contributed to an increase in yield, as only a small
part of H2O2 was generated and less PPO was con-
sumed. The higher oxygen transfer rate and dissolved
oxygen concentration (decomposition of H2O2) poten-
tially increased both the catalytic efficiency and bioca-
talyst yield. Nevertheless, the benefit from applying
pure oxygen has to be weighed against the product
value and added cost from purifying.
Although numerous efforts were made, the yield of
PPO was still unable to reach 50%. The byproduct of
oxidation of D-PPT, H2O2, probably decomposed PPO
to MPP, and the chemical structure of DAAO was pos-
sibly affected (Grey et al. 2007). Hence, catalase is
highly desirable to keep the concentration of hydro-
gen peroxide, formed as a byproduct, at a minimum
to avoid enzyme deactivation and product degrad-
ation. In this work, we dramatically enhanced the yield
of PPO through feeding of catalase, however, the max-
imum yield was reached up to 46.8% with 49.9% con-
version, indicating the formation of byproduct MPP
under the interference by H2O2 in this reaction was
inevitable, and the H2O2 could not be completely
removed. How to effectively remove H2O2 is a major
challenge in the DAAO-catalysed keto acid production
(Volpato et al. 2010). Finding a catalase with lower Km
value and co-expression with DAAO in single cell
would be worthwhile.
Figure 5. Time course of recombinant cell catalytic synthesis
of PPO.
3.5. Large-scale preparation of PPO under
optimal conditions
To further evaluate the feasibility of the bioprocess for
production of PPO for practical application, a 3 L scale
reaction system for the oxidization of DL-PPT under
the above-mentioned conditions was performed in a
5 L stirred-tank reactor. The initial reaction mixture
contained DL-PPT (400 mM), recombinant wet whole
cells harbouring DAAO (20 g/L), and catalase (1926 U/
mL), with aeration of 0.8 L/min. The final volume was
made up to 3 L in a 5 L bioreactor. The reaction mix-
ture was agitated at a speed of 500 rpm and a tem-
perature of 30 ꢀC. The reaction profile is shown in
Figure 5, indicating that the total yield and efficiency
were not affected by scaling up. As expected, a con-
version of >49.9% was reached, and a product yield
of 43.1% was obtained after 12 h. To date, using
racemic glufosinate as a substrate to produce PPO by
bioenzymatic method is the method with the highest
yield, indicating that this biocatalytic method is the
method of choice for industrial production of PPO.
3.6. Discussion
From an applied point of view, exorbitant biocata-
lysts concentration will cause additional cost of solid–-
liquid separation and superfluous waste. 20 g/L wet
cell was enough to achieve maximum yield.
Furthermore, whole cells are easy to operate, do not
need to go through steps such as crushing and purifi-
cation, and can be immobilized to recycle easily.
After optimization of pH, temperature, biocatalyst
concentration, oxygen, and catalase, the reaction was
further scaled up to 3 L with 400 mM substrate. 43.1%
yield was obtained after 12 h which is the same with
PPO is a vital keto acid containing phosphorus atom,
but biological or chemical strategies for synthesis of
PPO have seldom been reported. In this work, we suc-
cessfully constructed a recombinant E. coli harbouring
RgDAAO and used it to produce PPO.
Firstly, we investigated pH and temperature profiles
of RgDAAO. To our surprise, the RgDAAO exhibited
excellent stability in range of pH 7.5–8.5 and 25–40 ꢀC,
extending the great applied potential in PPO synthe-
sis. The enzyme was probably denatured at 50 ꢀC

BIOCATALYSIS AND BIOTRANSFORMATION
7
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4. Conclusions
In summary, we have successfully constructed recom-
binant E. coli strains expressing DAAO for the produc-
tion of PPO. The ventilation flow rate and amount of
exogenous catalase addition were the rate-determin-
ing factors. Introducing air was more cost-effective,
although the effect of introducing pure oxygen was
better. Consequently, a certain flow rate of air must
be introduced into the reaction system and an appro-
priate amount of catalase must be added to promote
the reaction process. More importantly, this is the
highest reported yield of D, L-PPT produced by bioca-
talytic production of PPO, laying the foundation for
industrial applications.
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Disclosure statement
No potential conflict of interest was reported by the authors.
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pathways and enhancement of reactivity. FEBS J. 278:
482–492.
Funding
This work was funded by National Natural Science
Foundation of China [No. 21978268 and No. 31970046].
ORCID
Heng Li
http://orcid.org/0000-0002-6406-7269
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