Biochemical and Metabolic Insights into Hyoscyamine Dehydrogenase

Fei Qiu, Yijun Yan, Junlan Zeng, Jian-Ping Huang, Lingjiang Zeng, Wei Zhong, Xiaozhong Lan,

Research Article

Biochemical and Metabolic Insights into Hyoscyamine

Dehydrogenase

⊥

Min Chen, Sheng-Xiong Huang,* and Zhihua Liao*

Cite This: ACS Catal. 2021, 11, 2912 −2924

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sı Supporting Information

ABSTRACT: Hyoscyamine, a member of the class of compounds known as tropane alkaloids (TAs), is clinically used as an

anticholinergic drug. Previous research has predicted that hyoscyamine is produced via the reduction of hyoscyamine aldehyde. In

this study, we identiﬁed a root-expressed gene from Atropa belladonna, named AbHDH, which encodes a hyoscyamine

dehydrogenase involved in the formation of hyoscyamine. Enzymatic assays indicated that AbHDH was able to not only reduce

hyoscyamine aldehyde to produce hyoscyamine but also oxidize hyoscyamine to form hyoscyamine aldehyde under diﬀerent

conditions. To elucidate its catalytic mechanism, the crystal structure of AbHDH at a 2.4-Å resolution was also determined.

Overexpression of AbHDH signiﬁcantly enhanced the biosynthesis of hyoscyamine and the production of anisodamine and

scopolamine in root cultures of A. belladonna. However, suppression of AbHDH using RNAi technology did not reduce the

production of hyoscyamine, anisodamine, or scopolamine, though it did increase the accumulation of hyoscyamine aldehyde. In

summary, this study provided timely biochemical and metabolic insights into hyoscyamine dehydrogenase, pointing to an alternative,

promising way to produce pharmaceutical TAs via metabolic engineering in planta.

KEYWORDS: biosynthesis, crystal structure, hyoscyamine, hyoscyamine dehydrogenase, metabolic engineering

INTRODUCTION

ing and synthetic biology oﬀer new hope for engineering the

production of hyoscyamine.

■

Hyoscyamine is a tropane alkaloid (TA) that displays

anticholinergic activity and is used clinically as a drug to

treat various health disorders such as arrhythmias, organo-

Although the biosynthesis of TAs constitutes a century-old

unresolved problem, substantial progress on this front has

been made in recent years (Figure 1). Putrescine, produced

from an ornithine-decarboxylase-catalyzed decarboxylation of

phosphate poisoning, and Parkinson’s symptoms. However,

medicinal plants of the Solanaceae family, such as Atropa

belladonna, Duboisia hybrid, and Datura spp., among others,

are the only naturally occurring materials currently available for

ornithine, is a precursor for the biosynthesis of polyamines,

nicotine, and TAs. In these biosynthesis, ﬁrst putrescine N-

methyltransferase (PMT) catalyzes the N-methylation of

−4

the commercial production of hyoscyamine.

Moreover, the

putrescine, to form N-methylputrescine; next, N-methylpu-

levels of hyoscyamine in these plants are generally very low,

strongly limiting its plant-based production for use in clinical

trescine oxidase (MPO) catalyzes the oxidative deamination of

N-methylputrescine to produce 4-methylaminobutanal, which

applications. Two Nobel laureates, Professor Richard Will-

undergoes spontaneous cyclization to form the N-methyl-Δ -

stat

ter (1872−1942) and Sir Robert Robinson (1886−1975),

pyrrolinium cation. The type III polyketide synthase (PYKS)

made great contributions toward the chemical synthesis of this

alkaloid. Chemical synthesis of hyoscyamine is undoubtedly a

Received: October 27, 2020

Revised: February 6, 2021

Published: February 18, 2021

great success of science, but it fails in the marketplace because

this process is too costly. Plant breeders used genetic, radiant,

and polyploidy breeding methods to attempt to develop new

plant varieties distinguished by high yields of hyoscyamine, but

all of these eﬀorts failed. However now that the TA

biosynthetic pathway has been elucidated, metabolic engineer-

2021 American Chemical Society

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Figure 1. Biosynthetic pathway of tropane alkaloids in Solanaceae. PMT, putrescine N-methyltransferase. MPO, N-methylputrescine oxidase. PYKS,

type III polyketide synthase. CYP82M3, tropinone synthase. TRI, tropine-forming reductase. UGT1, phenyllactate UDP-glycosyltransferase.

ArAT4, aromatic amino acid aminotransferase. PPAR, phenylpyruvic acid reductase. LS, littorine synthase. CYP80F1, littorine mutase. HDH,

hyoscyamine dehydrogenase. H6H, hyoscyamine 6β-hydroxylase.

converts this cation to 4-(1-methyl-2-pyrrolidinyl)-3-oxobuta-

noic acid, which is subsequently converted by CYP83M3 to

milestone in the ﬁeld of synthetic biology. However, the

biochemistry of HDH remains poorly understood, and its role

in TA biosynthesis is also unknown in planta; furthermore, the

value of HDH for engineering hyoscyamine production has not

been studied in TA-producing plants. To address these gaps in

knowledge, the HDH gene of A. belladonna (hereon AbHDH)

was in the current work studied at the molecular, biochemical,

and structural levels. Here, AbHDH was overexpressed to

estimate its potential contribution to engineering the

production of hyoscyamine, anisodamine, and scopolamine

in root cultures of A. belladonna. Also, in planta, AbHDH was

suppressed using RNAi technology to investigate its functional

role in hyoscyamine biosynthesis. Our suite of experiments has

yielded a sounder understanding of HDH at the biochemical

level, and the results have provided an alternative and

competitive way to metabolically engineer the production of

hyoscyamine, anisodamine, and scopolamine in plant materials.

Finally, we anticipate that our ﬁndings will also spur scientists

to think more about the complexity of the process by which

hyoscyamine is formed in planta.

2,13

form tropinone.

Tropine-forming reductase (TRI) reduces

tropinone to tropine, which is used as an acyl acceptor for the

formation of littorine. Aromatic amino acid aminotransferase

ArAT4) then catalyzes the formation of phenylpyruvic acid,

(

by using phenylalanine as a substrate. Phenylpyruvic acid

reductase (PPAR) catalyzes the reduction of phenylpyruvic

acid to phenyllactic acid, which is further converted by UDP-

glycosyltransferase (UGT1) to phenyllactylglucose as the acyl

donor for littorine formation. Then, littorine synthase (LS)

catalyzes the esteriﬁcation of tropine (the acyl acceptor) with

phenyllactylglucose (the acyl donor) to produce littorine.

Finally, littorine mutase (CYP80F1) catalyzes the rearrange-

ment of littorine into hyoscyamine aldehyde (HYA). It was

postulated a while ago that an alcohol dehydrogenase,

unidentiﬁed at that point, might be responsible for catalyzing

8,19

the formation hyoscyamine via the reduction of HYA.

recent studies reported on a hyoscyamine-producing alcohol

Two

dehydrogenase, named HYA reductase (HAR) in our patent

or hyoscyamine dehydrogenase (HDH), identiﬁed from two

hyoscyamine-producing plants, namely A. belladonna and

Datura stramonium, respectively. With the discovery of HDH

RESULTS AND DISCUSSION

■

Gene Cloning and Expression Analysis. HYA provided

(

or HAR), all of the functional genes involved in hyoscyamine

by CYP80F1 was postulated to be the direct precursor for

biosynthesis have now been identiﬁed. Hyoscyamine 6β-

hydroxylase (H6H) catalyzes the 6β-hydroxylation of hyoscy-

amine, to generate anisodamine, and this same enzyme

subsequently also catalyzes the epoxidation of anisodamine

hyoscyamine formation, leading several scientists to propose

that alcohol dehydrogenase might catalyze the reduction of

19,23

HYA to form hyoscyamine.

The alcohol dehydrogenase

(ADH) conserved domain (PF08240) was used to search the

A . belladonna transcriptomes, and 31 unigenes were identiﬁed

(Table S1). TAs are synthesized in secondary roots, in which

21,22

to form scopolamine.

HDH, as the last identiﬁed gene in TA biosynthesis, has

been used to manufacture hyoscyamine and scopolamine in

the known TA biosynthesis genes are speciﬁcally, or highly,

7,12,15,16,24

yeast by implementing synthetic biology technologies. This

expressed,

and such a root-expressed pattern leads to

biosynthesis of pharmaceutical TAs in yeast represents a great

postulate the candidate alcohol dehydrogenase responsible for

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Figure 2. Bioinformatics and tissue expression analysis of HDH. (a) Digital gene expression patterns of alcohol dehydrogenase and TA biosynthesis

genes from transcriptomes of A. belladonna in its plant tissue types. (b) The constructed phylogenetic tree of the ADH proteins. (c) Expression

levels of HDH in secondary root (SR), primary root (PR), stem (S), and leaf (L) tissues. The bars on the columns show the respective standard

deviations (n = 3). Tissues labeled with diﬀerent letters (a−c) above the columns showed signiﬁcant diﬀerences in HDH expression levels (P <

0.05, as determined by Duncan’s test).

hyoscyamine formation might be speciﬁcally or highly

expressed in secondary roots. Of the 31 ADH genes, just

one (aba_locus_4635) showed a tissue expression proﬁle

similar to that of the characterized TA biosynthesis genes

reaction (qPCR; Figure 2c) and indicated that AbHDH was

highly expressed in the A. belladonna secondary roots,

moderately expressed in the primary roots, but not expressed

in the stems or leaves.

(

Figure 2a). Hence, aba_locus_4635 was considered to be the

Hyoscyamine Dehydrogenase Catalyzes the Recip-

rocal Conversion between HYA and Hyoscyamine. The

His-tagged HDH protein was produced in engineered

only candidate gene for hyoscyamine dehydrogenase. The

cDNAs of AbHDH were cloned and deposited into GenBank

(

under accession number MT981110). A phylogenetic analysis

Escherichia coli and puriﬁed using Ni -resin columns. SDS-

PAGE showed AbHDH at the apparent molecular weight of

42.9 kDa (Figure S1 ), a value consistent with its calculated

molecular weight. Due to the very low stability of HYA and, as

a result, its high susceptibility to quickly undergoing the retro-

Claisen condensation reaction, it is neither commercially

available nor at all easy to produce in the laboratory.

Therefore, here, successive reactions were used to identify

the reduction activity of AbHDH. First, the upstream

CYP80F1 gene of A. belladonna was ectopically expressed in

the yeast strain WAT11, which has been successfully used to

indicated that plant and microbial or algae alcohol dehydro-

genases could be divided into diﬀerent clades (Figure 2b).

Three HDHs (AbHDH, DsHDH and DiHDH) were grouped

to form a subclade separated from plant alcohol dehydro-

genases (Figure 2b), suggesting an evolutionary and functional

divergence between HDHs and plant alcohol dehydrogenases.

AbHDH and DsHDH/DiHDH were found to display 93.7%/

3.2% amino acid sequence identity. These results showed the

three HDHs sharing a common ancestor. Digital gene

expression analysis revealed a tissue proﬁle for AbHDH similar

to the tissue proﬁles of known TA biosynthesis genes. The

expression levels of AbHDH in four representative tissues,

namely secondary roots, primary roots, stems, and leaves, were

determined using the real-time quantitative polymerase chain

25−27

identify catalytic functions of cytochrome P450s.

When

littorine was fed to microsomal proteins with CYP80F1, HYA

(m/z 288.1586; Figure S2) was detected and identiﬁed (Figure

3a). These results were consistent with those reported by Li et

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Figure 3. HDH enzymatic assays. (a) Extracted-ion chromatogram (EIC) trace of the CYP80F1-mediated littorine carbon rearrangement. (b) EIC

traces of the AbHDH-mediated reduction reaction system. Here, dark yellow and orange lines represent authentic samples of hyoscyamine and

littorine, respectively; the violet line indicates the detected metabolites in the reaction system containing littorine, CYP80F1, and puriﬁed HDH

protein; and the dark cyan line shows the detected metabolites in the reaction system containing littorine, CYP80F1 and boiled HDH protein, as a

negative control. (c) EIC traces of the HDH-mediated oxidation reaction system. Here, the red line indicates the detected metabolites in a negative

control using boiled HDH; and the blue line shows the detected metabolites in the HDH-mediated hyoscyamine oxidation reaction. Black arrows

indicate HYA, red arrows represent hyoscyamine, and gray arrows show littorine. The X-axis represents retention time, and the Y-axis represents the

relative abundance.

al. Hyoscyamine (m/z 290.1742; Figure S3) was produced

when the puriﬁed His-tagged HDH protein was added into the

reaction system for the purpose of producing HYA (Figure

the zinc coordinated by one histidine (His74), one glutamate

(Glu75), and two cysteines (Cys52 and Cys168) and together

showing approximate tetrahedral symmetry (Figure 4b). Atop

the active site cavity, three nonpolar residuesMet124,

Leu282, and Ala305together formed a substrate-binding

pocket that, much like three ﬁngers, would appear to control

the HYA substrate binding via shape-dependent van der Waals

interactions (Figure 4c). Furthermore, deep in the pocket, the

two polar residues Ser54 and Cys100 may have formed a

critical hydrogen bond with the carbonyl oxygen of aldehyde

b), and hyoscyamine was not detected in a boiled AbHDH

control (Figure 3b). These results demonstrated the ability of

AbHDH to reduce HYA, to form hyoscyamine, in a cell-free

reaction system producing HYA. When hyoscyamine was used

instead as the substrate, the formation of HYA (m/z 288.1585;

Figure S4) was detected (Figure 3c). Biochemical analysis

revealed AbHDH functioning as an oxidoreductase, i.e., not

only reducing HYA to hyoscyamine, but also oxidizing

hyoscyamine to HYA.

Structural Features of AbHDH and Its Binding of

Substrate. To determine the structural basis for the catalytic

mechanism of AbHDH, its crystal structure was determined to

a resolution of 2.4 Å (Protein Data Bank [PDB] ID: 7CGU;

Table S2). An amino acid sequence alignment indicated the

structure of AbHDH to be similar to that of heteroyohimbine

(

Figure 4c).

Because of the diﬃculty in obtaining ternary complex

crystals for AbHDH, a computational docking program,

namely AutoDock, was used to probe the structural basis

of substrate binding in AbHDH. Judging by the inferred

conformations and binding aﬃnities, the most favorable

orientation of the HYA as well as that of phenylacetaldehyde,

each in the AbHDH active site, was determined as shown in

Figures 4d and S6a respectively. The docking results showed

the binding proﬁle of the benzene ring and aldehyde group of

phenylacetaldehyde to be very similar to the binding proﬁle of

the corresponding groups of HYA (Figure S6b ). In the docked

conformation of HYA, the distances from the HYA oxygen to

alkaloid synthase (PDB ID: 5H81 and 5FI3) and cinnamyl

or sinapyl alcohol dehydrogenase (PDB ID: 2CF5, 5VKT, and

YQD;

9−31

Figure S5 ). Inspection of the crystal structure

showed the AbHDH monomer consisting of two domains,

with one of them being a nucleotide-binding domain

consisting of a classic Rossman fold and the other domain

being a substrate-binding domain. Figure 4a shows the overall

structure of AbHDH, with its active sites deﬁned by

NADP(H) cofactor, a catalytic Zn , and a deep cavity formed

between the two domains. Like those of many members of the

zinc-dependent CAD or SAD family, the active sites cavity of

AbHDH was observed to contain a catalytic zinc sphere, with

the catalytic Zn , the hydroxyl side chain of Ser54, and the

sulfhydryl side chain of Cys100 were measured to be 3.7, 2.8,

and 3.3 Å, respectively (Figure 4e). In addition, the carbonyl

carbon of the HYA was measured to be situated 3.5 Å away

from the C4 atom of the nicotinamide ring of the bound

NADP(H) molecule, which showed the pro-R hydrogen in an

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Figure 4. Structure of AbHDH. (a) Cartoon representation of the AbHDH monomer (colored teal), with NADPH bound to its nucleotide-binding

domain. The location of the active site cavity is indicated by a yellow rectangle. The AbHDH monomer was observed to coordinate one structural

and one catalytic Zn ion, each depicted here as a green sphere with small yellow spheres indicating the coordination bonds. (b) Cartoon and stick

representation of the AbHDH active site. The catalytic Zn ion, observed to be tetrahedrally coordinated by Cys52, His74, Glu75, and Cys168, is

depicted here as a green sphere. Small yellow spheres indicate the coordination bonds formed between the Zn ion and the residues. A stick model

of NADPH is shown. The site of hydride transfer is labeled with an asterisk. (c) Surface representation of the substrate-binding pocket. (d) Surface

representation of the binding pocket docked with HYA. (e) Cartoon and stick representation of the AbHDH active site cavity docked with HYA.

Six residues constituting the substrate-binding pocket are indicated. The docking suggested formation of a hydrogen bond between the carbonyl

group of HYA and the Ser54 residue, postulated to be the key catalytic residue of AbHDH.

appropriate position for ensuring the hydride transfer (Figure

e).

Enzyme Kinetics and Reaction Mechanism. Unlike the

HYA mimics, such as phenylacetaldehyde (Figure S7).

Therefore, enzyme kinetics assays with the wild type and

mutants were carried out in the presence of phenyl-

acetaldehyde for the reduction reaction, and likewise with

hyoscyamine for the reverse reaction. The optimized pH level

and temperature for the AbHDH-catalyzed reduction were,

respectively, 6.4 and 40 °C (Figure S8 ). Under these optimal

conditions, the Km value of the wild-type AbHDH for

reduction reactions catalyzed by some zinc-dependent CAD or

SAD family protein members, the reduction reaction catalyzed

by AbHDH is reversible. Here we relied on hyoscyamine to

derive the enzyme kinetics of this reverse reaction.

Furthermore, we found that AbHDH can reduce various

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Table 1. Enzymatic Kinetics Parameters of Wild-Type AbHDH and Five Mutants

kinetic parameters

wild-type AbHDH

S54A

H74A

C100G

C100Y

C100F

phenylacetaldehyde

K_m(μM)

44.46 ± 10.73

228.4 ± 21.39

5.14

n.d.

199.8 ± 45.77

7.14 ± 0.89

0.036

357.3 ± 83.33

2.83 ± 0.40

0.008

492.4 ± 178.6

3.79 ± 0.91

0.008

k_c_a_t(min⁻¹)

k /K (min⁻μM⁻¹

)

cat

hyoscyamine

K_m(μM)

33.36 ± 4.69

7.49 ± 0.51

0.22

285.9 ± 73.36

0.62 ± 0.04

0.002

n.d.

21.69 ± 3.19

1.47 ± 0.09

0.068

485.5 ± 154.6

0.28 ± 0.04

0.0006

937.4 ± 258.4

0.52 ± 0.09

0.0006

k_c_a_t(min⁻¹)

k /K (min⁻μM⁻¹

cat

n.d., enzyme reaction not detected.

phenylacetaldehyde was 44.46 ± 10.73 μM, and its

corresponding k and k /K values were 228.4 ± 21.39

results in the catalytic Zn²⁺establishing an electrostatic

interaction with the aldehyde group of substrate HYA; then

the pro-R hydride of NADPH is transferred from the C4 atom

to the C1 atom of the aldehyde group in HYA; at this point,

the hydroxyl group of Ser54 acts as a general acid, donating a

cat

−

−1

min and 5.14 min μM , respectively (Table 1). The

reverse reactions (i.e., oxidation activity) of AbHDH, using

hyoscyamine and phenylethanol as substrates, were also

analyzed. Maximum activity of AbHDH-mediated oxidation

of hyoscyamine was detected at pH 10.4 and 45 °C (Figure

S8 ). Under these conditions, the K_mvalue of AbHDH for

hyoscyamine was 33.36 ± 4.69 μM, for which the k and k /

proton to the Zn -coordinated oxygen of HYA; and ﬁnally,

the corresponding hyoscyamine is produced (Figure 5). In

cat

−1

−

K values were, respectively, 7.49 ± 0.51 min and 0.22 min

−

μM (Table 1); however, there was no activity when

phenylethanol served as the substrate. Under optimal

conditions, the reduction eﬃciency of HDH was 23.36 times

that of its oxidation eﬃciency. Speciﬁcally, the catalytic

eﬃciency of HDH associated with reduction was much higher

than that with oxidation under optimal conditions (Table 1),

and the equilibrium constants of the reactions at pH 6.4, 7.2,

and 10.4 when hyoscyamine was used as the substrate were

Figure 5. Proton shuttling mechanism proposed to operate during the

reduction process in the active site of AbHDH. Solid arrows indicate

the movement of two electrons between functional groups during the

substrate reduction. The likely hydrogen bonds involved are shown as

dashed lines.

323.82, 915.06, and 12.57 respectively. These results

suggested that HDH is more prone to reducing aldehyde to

alcohol rather than oxidizing alcohol to aldehyde under

physiological conditions of plants (around pH 7.2).

To investigate the role of key catalytic residues in the

functioning of the active site pocket of AbHDH, ﬁve mutants

order to determine which hydrogen of NADPH is responsible

for hydride transfer, [4S- H] NADPH (Figure S9a) and

(

S54A, H74A, C100G, C100Y, and C100F) were generated

[

4R- H] (Figure S9b) NADPH were synthesized and used for

through site-directed mutagenesis. The H74A site mutation led

to loss of enzyme activity for each of the two tested substrates

AbHDH-catalyzed reactions. A GC-MS analysis revealed an

incorporation of deuterium into phenylacetaldehyde only in

(

Table 1); enzyme activity proﬁles of C100G showed only

.70% of the wild-type activity for phenylacetaldehyde and

0.90% of the wild-type activity for hyoscyamine, and both

the presence of [4 R - H] NADPH but not with [4S- H]

NADPH (Figure S10 ). Deuterated phenylethanol was

separated by liquid chromatography and further identiﬁed by

EI-HRMS (Figure S11 ). These results were consistent with the

pro-R hydride of NADPH being used for the reduction

reaction.

C100Y and C100F showed only 0.16% and 0.27% of the wild-

type activity for phenylacetaldehyde and hyoscyamine,

respectively (Table 1). By contrast, mutant S54A displayed

no catalytic activity at all when using phenylacetaldehyde as

the substrate; and when hyoscyamine was used as the

substrate, its activity amounted to just 0.91% of that of the

wild type (Table 1), highlighting the key participation of Ser54

for a successful HDH-catalyzed aldehyde reduction. The highly

reduced catalytic activity of HDH for the Ser54 and Cys100

mutations, and their respective 2.8 and 3.3 Å distances to the

carbonyl oxygen of HYA, indicated that Cys100 may form a

hydrogen bond with the aldehyde group to stabilize the

substrate, and that Ser54 may participate in a proton shuttle

system during the reduction of substrate, instead of Ser54

forming polar interactions with a water molecule as reported

HDH Is Valuable for Promoting the Production of

Pharmaceutical TAs in Planta. Since HDH was clearly able

to reduce HYA to form hyoscyamine, we became interested in

studying its relevance for engineering the production of

hyoscyamine, anisodamine, and scopolamine in planta. To do

so, root cultures of A. belladonna, in which HDH was

overexpressed (driven by the CaMV 35S promoter, p35S),

were established. PCR detection showed that the rooting gene

(rolB) and the marker gene (NPTII) were both ampliﬁed from

the positive control pBI121-HDH, control root lines, and

HDH-overexpressing root lines. Fragments of 35S::HDH (a

genomic DNA from p35S to HDH) were speciﬁcally detected

from the pBI121-HDH and HDH -overexpressing root lines yet

not from the control root lines (Figure S12 ). Gene expression

analysis, based on qPCR, indicated that the transcript levels of

HDH were much higher in the HDH-overexpressing root lines

than in the control root lines (Figure 6a). These results

recently by Srinivasan and Smolke.

The docking results and the above site-mutation data

prompted us to posit a simple catalytic mechanism for

AbHDH: as observed in some zinc-dependent CAD or SAD

families, ﬁrst a change in the conformation of AbHDH

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Figure 6. Eﬀects of overexpression of HDH on the biosynthesis of TAs in root cultures of A. belladonna. (a) HDH expression levels in the root

cultures. (b−d) Contents of alkaloids in the root cultures: (b) hyoscyamine, (c) anisodamine, and (d) scopolamine. DW, dry weight. OC, vector

control root lines transformed with pBI121 vector. OHDH-4, 9, 14, 15, 19, 20, 26, 30 denote the representative HDH-overexpressing root cultures.

The bars denote means ± standard deviations (n ≥ 3). *, **, and *** indicate signiﬁcant diﬀerences from control at the levels of P < 0.05, P <

0.01, and P < 0.001, respectively, as determined by t tests.

conﬁrmed that the expression levels of HDH were substantially

elevated in the HDH-overexpressing root lines.

metabolic engineering studies implied that it is worth trying to

deploy some TA biosynthesis genes, such as HDH, in

engineering hyoscyamine production in plants.

The TA contents were determined in the root cultures of A.

belladonna, by using LC-MS. The contents of hyoscyamine,

anisodamine, and scopolamine were respectively 1.35 mg/g

dry weight (DW), 0.69 mg/g DW, and 0.43 mg/g DW in the

control root lines (Figure 6b−d). Hyoscyamine production

was 1.83 to 4.03 mg/g DW in the HDH-overexpressing root

lines (Figure 6b), suggesting that they could produce 35.56%−

In this study, hyoscyamine production was signiﬁcantly

increased when HDH was overexpressed. Due to the resulting

greater availability of hyoscyamine for H6H, the production of

anisodamine and that of scopolamine were promoted as well.

In contrast to the relatively low level of productivity in titers of

hyoscyamine (around 80 μg/L) and scopolamine (around 30

μg/L) achieved by engineered yeast cells based on synthetic

98.52% more hyoscyamine than could the control root lines.

The anisodamine levels ranged from 1.16 to 1.73 mg/g DW in

the HDH-overexpressing root lines (Figure 6c), while their

scopolamine contents were slightly lower, at 0.62 to 1.28 mg/g

DW (Figure 6d). These levels of anisodamine and scopolamine

were also signiﬁcantly higher in the HDH-overexpressing root

lines than in the control root lines.

biology, the productivity levels of hyoscyamine and scopol-

amine in engineered root cultures of A. belladonna were,

respectively, 1.83−4.03 and 0.62−1.28 mg/g DW. For

scopolamine in particular, its levels reached 1% DW in leaves

of HnH6H-overexpressing A. belladonna plants. Due to these

results, and because commercial production of pharmaceutical

TAs in engineered yeast needs their titer levels to be higher

A few TA biosynthesis genes, namely PMT, CYP80F1, and

H6H, have been used to engineer TA production in root

cultures or plants. Overexpression of PMT markedly increased

N-methylputrescine levels in A. belladonna and a Duboisia

hybrid, but did not promote the biosynthesis of either

than 5 g/L, we are conﬁdent that metabolic engineering of TA

production in planta oﬀers a promising way forward that can

compete against synthetic-biology-based manufacturing of TAs

in yeast cell factories. In fact, artemisinin is a prime example of

this potential. Engineered yeast cells can produce artemisinin

acid at a very high level (of about 25 g/L) and have been used

33,34

hyoscyamine or scopolamine,

attributed to TA biosyn-

thesis being limited downstream rather than at the PMT-

catalyzed step. Overexpression of H6H from Hyoscyamus

niger markedly increased the scopolamine production in root

cultures of A. belladonna, H. niger, and other TA-producing

plants, and impressively, hyoscyamine was almost completely

converted into scopolamine in leaves of HnH6H-overexpress-

to synthesize artemisinin at a relatively low cost. The

pharmaceutical company Sanoﬁ S.A. had ambitiously declared

a plan to launch the large-scale production of artemisinin using

synthetic biology, back in 2013. However, the marketplace

did not cooperate with Sanoﬁ’s aspirations. The artemisinin

generated via synthetic biology met market resistance because

,35−37

ing A. belladonna plants.

Overexpression of CYP80F1,

whose product catalyzes the conversion of littorine to HYA,

did not alter the overall production of hyoscyamine. These

it still cost more than Artemisia annua plants cultivated by

farmers,

such that today commercial production of

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Research Article

Figure 7. Eﬀects of suppressing HDH on the biosynthesis of TAs in root cultures of A. belladonna. (a) HDH expression levels in the root cultures.

(

b−f) Contents of alkaloids in the root cultures: (b) hyoscyamine, (c) anisodamine, (d) scopolamine, (e) hyoscyamine aldehyde relative to the

amount of hyoscyamine, and (f) littorine. DW, dry weight. IC, control root lines transformed with pBin19 vector. IHDH-2, 3, 6, 7, 8, 10, 12, and 18

denote the representative HDH-suppressing root cultures. The bars denote means ± standard deviations (n ≥ 3). *, **, and *** indicate signiﬁcant

diﬀerences from the control at the levels of P < 0.05, P < 0.01, and P < 0.001, respectively, as determined by t tests.

,12,15,16,41

artemisinin still depends on A. annua plants. Although

microbial synthetic biology still represents a promising

technology for manufacturing active pharmaceutical ingre-

dients (APIs) with complicated structures, plant metabolic

engineering might be considered at the present time to be a

more promising way to produce some APIs.

biosynthesis genes were suppressed.

For example,

when ArAT4 was suppressed, the levels of both phenylpyruvic

acid (the direct product of the reaction catalyzed by ArAT4)

and phenylalanine (the direct substrate for ArAT4) went

unchanged, while the production of metabolites downstream of

phenylpyruvic acid, including phenyllactic acid, hyoscyamine,

and scopolamine, were each considerably reduced. This result

was explained by the ﬁve other isozymes catalyzing the same

reaction as ArAT4, and probably cell-speciﬁc expression of

ArAT4, leading to functional redundancy with respect to the

HDH Is Replaceable for Hyoscyamine Formation in

Planta. To further understand the role of HDH in

hyoscyamine biosynthesis in planta, we established root

cultures of A. belladonna, in which HDH was suppressed

using RNAi technology. PCR detection conﬁrmed the T-DNA

harboring the HDH-RNAi fragment (driven by the 35S

promoter) had been integrated into the genome of A.

belladonna (Figure S12). The corresponding gene expression

analysis showed that HDH transcript levels were markedly

decreased in HDH-RNAi root lines (Figure 7a), indicating that

HDH was substantially suppressed in transgenic root cultures

of A. belladonna. Metabolite detection revealed, however, that

the hyoscyamine (Figure 7b), anisodamine (Figure 7c), and

scopolamine (Figure 7d) levels were not signiﬁcantly altered

when HDH was suppressed, demonstrating that suppression of

HDH was not able to disrupt the formation of hyoscyamine.

HYA was detected at relatively high levels in the HDH-RNAi

root lines, but only so at trace levels in control root lines

15,16

formation of phenylpyruvic acid.

In the present study, since suppressing HDH did not

decrease the production of hyoscyamine, anisodamine or

scopolamine, it may be concluded that HDH is not essential

for hyoscyamine to be formed in planta, and it is therefore

reasonable to postulate that some other yet-to-be identiﬁed

enzymes might complement the activity of HDH in planta. In

fact, although hyoscyamine was produced, HYA was not

detected in littorine-fed tobacco root cultures that expressed

CYP80F1, which normally catalyzes littorine to form HYA.

Here we also used HDH to search tobacco transcriptomes and

genomes, but could not ﬁnd any HDH genes in tobacco (data

not shown). Taken together, we may therefore deduce that

some other enzymes in tobacco catalyze the reduction of HYA

into hyoscyamine. Since the oxidoreductase family is large and

composed of hundreds of members, others besides HDH

might also be capable of reducing HYA to form hyoscyamine

in planta. These yet-to-be identiﬁed enzymes that are able to

reduce HYA to hyoscyamine await discovery and testing by

scientists, to better understand and appreciate the complexity

of the hyoscyamine biosynthesis process in planta.

(

Figure 7e); hence, suppression of HDH led to the

accumulation of HYA. Littorine (Figure 7f) levels were not

signiﬁcantly increased when HDH was suppressed.

Generally, metabolite biosynthesis is disrupted when one or

more key biosynthesis genes are suppressed or knocked out.

For hyoscyamine, its production has been shown to be

dramatically decreased, and in most cases the levels of

candidate substrates signiﬁcantly elevated, when certain TA

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Research Article

CONCLUSIONS

Transformants were selected on a synthetic dropout medium

deﬁcient in uracil with 2% glucose added. The microsomal

protein of CYP80F1 was induced and isolated as previously

■

Hyoscyamine dehydrogenase was identiﬁed from A. belladon-

na, and named AbHDH. Under physiological conditions in

plant cells, AbHDH was concluded to mainly catalyze the

reduction of hyoscyamine aldehyde to form hyoscyamine. The

in vitro characterization and crystal structure of AbHDH to

relatively high resolution revealed its catalytic mechanism and

high aﬃnity for substrates, which may allow for the syntheses

of useful alcohols for the purpose of preparing complex natural

products and pharmaceutical ingredients in future research.

AbHDH was found to be a zinc-containing long-chain

dehydrogenase, transferring the pro-R hydrogen from the 4-

position of the NADPH cofactor. In addition, AbHDH was

shown to be of value in engineering the production of

pharmaceutical TAs in planta through an overexpression

strategy.

17,44

described.

The HDH CDS was inserted into pET28a with

EcoRI and XhoI, whose resulting vector was introduced into

the Escherichia coli strain BL21 (DE3). The His-tagged HDH

16,45

was induced and puriﬁed as described elsewhere.

For the

HDH reduction activity assays, a reaction mixture (500 μL)

containing 600 μg of microsomal protein, 5 mM NADPH, and

mM (R)-(−)-littorine in 100 mM potassium phosphate (pH

.5) was made; the reaction was initiated by adding

microsomal and HDH proteins (100 μg) and run at 30 °C

for 12 h. Boiled HDH served as a negative control. For the

HDH oxidation activity assays, a reaction mixture (500 μL)

containing 2 mM NADP and 1 mM L-hyoscyamine in a 100

mM glycine buﬀer (pH 8.0) was made, and to this mixture was

added the HDH protein (100 μg) to oxidize hyoscyamine, at

0 °C for 2 h (with boiled HDH used as a negative control).

EXPERIMENTAL SECTION

■

The oxidation and reduction reactions were each terminated

by adding enough ammonium hydroxide to bring the ﬁnal pH

to a value of 10. The reaction mixtures were then added to

Extrelut NT and incubated at room temperature for 30 min,

after which the alkaloids were eluted with 20 mL of

dichloromethane. A nitrogen stream was applied to remove

the solvent, and the resulting residue was dissolved in 10 mL of

methanol and immediately analyzed using LC-HRMS.

Catalytic products were separated and detected using a

Thermo Scientiﬁc Q Exactive mass spectrometer equipped

with a Hypersil GOLD C18 column (100 × 2.1 mm, 1.9 μm;

Thermo Scientiﬁc). The ﬂow rate was set to 0.4 mL/min and

the oven temperature was 35 °C. The samples were separated

using a binary gradient elution with 0.1% formic acid in water

(A) and acetonitrile (B). The corresponding elution

procedures are described in Table S4. The injection volume

was 2 μL. All measurements were taken using electron spray

ionization (ESI) in its positive ion mode and full MS mode.

Regarding the instrument parameters, sheath gas pressure was

set at 35 psi, aux gas ﬂow rate at 20 L/h, spray voltage at 3.00

kV, capillary temperature at 350 °C, S-lens RF level at 50, and

aux gas heater temperature at 350 °C. The R-(−)-littorine

standards were purchased from Toronto Research Chemicals

(ON, Canada). The L-hyoscyamine standards were purchased

from MCE (MedChemExpress, SHH, China).

Crystallization and Structure Determination of

AbHDH. Crystals of AbHDH were grown using the hanging-

drop vapor diﬀusion method at 293 K. The drops contained

each a 1:1 mixture of 10 mg·ml protein and a crystallization

buﬀer (0.2 M ammonium sulfate, 0.1 M Tris-HCl [pH 8.0],

30% PEG 3350). Diﬀraction data were collected using X-rays

at a wavelength of 0.9795 Å and a temperature of 100 K at

beamline 18U of the Shanghai Synchrotron Radiation Facility

(SSRF), and then processed using HKL3000 software. The

structure was determined by carrying out the molecular

replacement method in Molrep based on using 1YQD as the

search model. An initial model was built using PHENIX.-

Gene Screen and Bioinformatics Analysis. The

reduction of HYA to hyoscyamine is a typical reduction of

an aldehyde, and many such reactions occur in biological

settings and are catalyzed by alcohol dehydrogenase. There-

fore, we speculated that alcohol dehydrogenase might catalyze

HYA to produce hyoscyamine. To mine the members of the

alcohol dehydrogenase family in an A. belladonna tran-

scriptome database based on the Hidden Markov Model

(

“

PF08240), a clustering analysis was performed using the

pheatmap” package (to derive correlations between the

expressions of various genes) in ‘R’ software. The correspond-

ing phylogenetic tree was generated using the neighbor-joining

method, in MEGA7 software.

Cloning and Expression Analysis of HDH. The total

RNA of A. belladonna secondary root tissue was isolated by

using the RNAsimple Total RNA Kit (Tiangen, Beijing,

China), after which a cDNA library for rapid ampliﬁcation of

cDNA ends (RACE) was constructed using the SMARTer

RACE cDNA Ampliﬁcation Kit according to its operational

instructions (Clontech, CA, U.S.A.). The 3′-RACE and 5′-

RACE of HDH were respectively carried out, according to the

manufacturer’s protocol. The full-length HDH was isolated by

using a primer pair consisting of f-HDH-F and f-HDH-R, with

ampliﬁcation of the HDH gene carried out using HyPerFUsion

DNA polymerase (APExBIO Technology, TX, USA). The

ensuing PCR products were subcloned into a pJET1.2/blunt

vector (Thermo Fisher Scientiﬁc, MA, USA) and sequenced.

Tissue proﬁling of HDH was analyzed by carrying out qPCR,

−

for which PGK served as the internal reference gene.

Secondary roots, primary roots, stems, and leaves of A.

belladonna plants grown in the ﬁeld for 4 months were

harvested. The diﬀerent tissues of A. belladonna were used for

RNA isolation and cDNA synthesis, using kits from Tiangen

Biotech (BJ, China), with SYBR qPCR Mix purchased from

Novoprotein (SHH, China). The qPCR was performed using

an IQ5 machine (Bio-Rad, CA, U.S.A.), which was also used to

assess the expression levels of HDH in suppressed and

overexpressed root cultures. All primers used in this study

are listed in Table S3.

Isolation of the AbCYP80F1 Microsomal Protein and

Determination of the HDH Catalytic Activity. The

CYP80F1 coding sequence (CDS) was cloned into pYES2

with BamHI and XhoI, and the resulting yeast expression

vector was then transformed into the yeast strain WAT11.

autobuild and subjected to manual adjustment using

COOT, with all models reﬁned using PHENIX.reﬁnement

and Refmac5.

Molecular Docking of Substrate to AbHDH. Rigid

molecular docking was performed using Autodock 4.2. The

ligand NADPH was downloaded from the PDB database

(http://www.rcsb.org/) and docked into the cofactor-binding

site of AbHDH. The ligands HYA and phenylacetaldehyde

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https://pubs.acs.org/doi/10.1021/acscatal.0c04667.

Research Article

were generated using ChemBio3D Ultra 12.0, and then energy

minimized with Molecular Mechanics (MM2) until a

minimum root-mean-square (RMS) gradient of 0.100 was

achieved (https://www.chemdraw.com.cn/). When docking,

the key parameters such as grid number and algorithm were set

to default values, but rotatable bonds in the ligand were not set

in this manner in order to allow for ﬂexible docking. Finally,

one hundred independent docking runs were performed, and

the complex structure with the best combination of low

binding energy and favorable orientation was selected. PyMOL

Determination of the Stereospeciﬁcity of the Hydro-

gen Transfer from NADPH. [4S- H] NADPH and [4R- H]

NADPH were synthesized according to the method reported

by Barber but with a slight modiﬁcation. For [4S- H]

NADPH, a reaction system (500 μL) containing 83 mM

phosphate buﬀer (pH 8.0), 14.7 mM D-glucose-1- H, 9.3 mM

NADP , 40% DMSO and 5 units of glucose-6-phosphate

dehydrogenase (Saccharomyces cerevisiae) was made. For

[4R- H] NADPH, a reaction system (500 μL) containing 25

mM Tris buﬀer (pH 9.0), 2.8 mM NADP , 1 M 2-

.7 (http://www.pymol.org) was used for viewing the

propanol- H , and 5 units of alcohol dehydrogenase

molecular interactions and image processing.

(Thermoanaerobium brockii) was made. Each of these reaction

system was incubated at 30 °C for 1 h, and then 20 μL of the

resulting reaction solution was added to an AbHDH reaction

system including potassium phosphate (100 mM, pH 7.0), 1

mM phenylacetaldehyde, and 50 μg AbHDH. The enzymatic

reaction was quenched by adding to it an equal volume of 0.8

M formic acid; the product was extracted twice using 80 μL of

ethyl acetate, and analyzed using GC-MS. In order to further

identify the deuterated phenylethanol, the ampliﬁcation

reaction of AbHDH catalysis was performed in a total volume

of 2 mL. The organic phase was distilled to dryness under

reduced pressure, and then the residues were dissolved in

ethanol. Product of interest was isolated by HPLC and

identiﬁed by EI-HRMS.

GC-MS Identiﬁcation of the Products of Phenyl-

acetaldehyde Reduction Mediated by AbHDH. Phenyl-

acetaldehyde reduction reactions were carried out in 400 μL

mixture containing potassium phosphate (100 mM, pH 7.0), 1

mM phenylacetaldehyde, 2 mM NADPH, and 50 μg of

AbHDH. A similar reaction, but with preboiled AbHDH, was

used as a negative control. These reactions were each initiated

at 30 °C, and then stopped with an equal volume of 0.8 M

formic acid after 3 h, extracted with 80 μL of ethyl acetate

twice, and analyzed using GC-MS (GCMS-QP2010Plus,

SHIMADZU, Kyoto, Japan). The MS analysis was carried

out in electron ionization (EI) mode. Samples were separated

using an Rtx-5 column (30 m × 0.25 mm × 0.25 μm, Restek)

with a temperature gradient of 45 °C for 4 min, 45−185 °C at

Establishment of Hairy Root Cultures and Molecular

Detection. To investigate the functioning of HDH in the

biosynthesis of TAs, overexpression or suppression of HDH

and corresponding control root cultures were established

a rate of 14 °C/min, and 185 °C for 2 min.

Analysis of Enzyme Kinetics Parameters and Equili-

brium Constants. The ﬁve mutant HDH proteins, made with

S54A, H74A, C100G, C100F, and C100Y substitutions,

respectively, were each obtained by carrying out overlapping

PCR. The primers used for this procedure are described in

Table S3, and the corresponding proteins were puriﬁed by

deploying essentially the same procedure used to purify the

wild-type enzyme. The puriﬁed HDH protein (125 μg) was

used in each oxidation/reduction enzymatic assay in 1 mL

reaction buﬀers that contained 0.2 mM NADP-2Na/NADPH-

,37

according to published references.

All root cultures were

identiﬁed by PCR using genomic DNAincluding rolB,

NPTII, 35S::HDH, and 35S::HDH RNAiwith qPCR used

to conﬁrm the expression levels of HDH in diﬀerent root

cultures (as described above). For the gene expression and

metabolite analyses, root lines were cultured in liquid

,16

Murashige and Skoog (MS) medium for 28 days.

primers used are listed in Table S3 .

The

Metabolite Analysis of Root Cultures. Determination of

Na. Potassium phosphate buﬀer and glycine buﬀer (pH 4−

littorine, hyoscyamine aldehyde, hyoscyamine, anisodamine,

2) were used to determine the optimal pH of HDH for its

and scopolamine contents was done following Qiu et al.,

oxidation/reduction, and various temperatures (range: 25−50

albeit with slight modiﬁcations. The harvested root cultures

were lyophilized and ground into ﬁne powder; 25 mg of this

material was used for the metabolites’ extraction and added to

°C) were tested to determine the optimum one for this

oxidation/reduction, whose enzyme kinetics were then

analyzed at these optimal pH and temperature conditions.

Various concentrations of hyoscyamine (0.01−10 mM) and

phenylacetaldehyde (0.01−3 mM) were used for the analysis

of enzyme kinetics. The enzymatic activity was calculated

based on NADPH consumption, which was spectrophoto-

metrically followed at a wavelength of 340 nm (Thermo

Scientiﬁc), and the K and V values were calculated from

mL of extraction buﬀer (20% methnol including 0.1% formic

acid). Metabolites were extracted at 10 °C for 3 h and then

immediately used for detection. To quantify the content of

metabolites, chromatography and mass spectrometry were

applied, using the same conditions as described above except

the sampler was set as 4 °C. The injection volume was 5 μL

after a 10-fold dilution. The standard curves of littorine,

hyoscyamine, anisodamine, and scopolamine were built used

authentic samples obtained from Toronto Research Chemicals

and Sigma-Aldrich (MO, U.S.A.).

max

curve ﬁtting of the Michaelis−Menten equation v = (V

max

[

S])/(K + [S]). The k values were calculated according to

m cat

the equation k_c_a_t= V_m_a_x/[E]. The standards for NADP-2Na,

NADPH-4Na, and phenylacetaldehyde were purchased from

Aladdin (SHH, China). Equilibrium constants of the reactions

with hyoscyamine as the substrate were determined under

various pH conditions (6.4, 7.2, and 10.4). Here, in each case,

a reaction mixture containing 0.25 mM hyoscyamine and 0.25

ASSOCIATED CONTENT

■

sı

Supporting Information

The Supporting Information is available free of charge at

mM NADP was made, and then the reaction was monitored

by measuring the (increase in) absorbance at 340 nm using an

extinction coeﬃcient of 6220 L M⁻cm for NADPH. After

−1

Puriﬁed AbHDH, MS data for hyoscyamine and HYA,

amino acid sequences alignment, HDH docked with

phenylacetaldehyde, identiﬁcation of reduction products

of phenylacetaldehyde, optimal conditions for the

equilibrium was reached, the concentration of product

(

hyoscyamine aldehyde) was calculated, to ﬁnally obtain the

equilibrium constant (K_e_q).

921

ACS Catal. 2021, 11, 2912−2924

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Complete contact information is available at:

Research Article

Min Chen − College of Pharmaceutical Sciences, Key

Laboratory of Luminescent and Real-Time Analytical

enzymatic reaction, MS data of synthesized [4- H]

NADPH, MS data for deuterium exchange experiments,

MS data for deuterated phenylethanol, genomic DNA

detection, candidate HDH genes, X-ray crystallography

data collection, primers used in the study, and mobile

phase gradient (PDF )

Chemistry (Ministry of Education), Southwest University,

Chongqing 400715, China

https://pubs.acs.org/10.1021/acscatal.0c04667

Author Contributions

F.Q., Y.Y., and J.Z. contributed equally to this work.

AUTHOR INFORMATION

■

⊥

Corresponding Authors

Notes

Sheng-Xiong Huang − State Key Laboratory of

Phytochemistry and Plant Resources in West China, and CAS

Center for Excellence in Molecular Plant Sciences, Kunming

Institute of Botany, Chinese Academy of Sciences, Kunming

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

50201, China; orcid.org/0000-0002-3616-8556;

This work is ﬁnancially supported by the fundings listed below:

the NSFC projects (U1902212, 31770335 and 32000239), the

National Key R&D Program of China (2018YFA0900600), the

National Transgenic Major Project of China (2019ZX08010-

Email: sxhuang@mail.kib.ac.cn

Zhihua Liao − Chongqing Key Laboratory of Plant Resource

Conservation and Germplasm Innovation, Key Laboratory of

Eco-environments in Three Gorges Reservoir Region (Ministry

of Education), SWU-TAAHC Medicinal Plant Joint R&D

Centre, School of Life Sciences, Southwest University,

Chongqing 400715, China; orcid.org/0000-0002-1283-

004), the Forth National Survey of Traditional Chinese

Medicine Resources, Chinese or Tibet Medicinal Resources

Investigation in Tibet Autonomous Region (State Admin-

istration of Chinese Traditional Medicine 20191217-540124,

649 ; Email: zhliao@swu.edu.cn

20191223-550126, and 20200501-542329), China Postdoc-

toral Science Foundation (2020M683216), Key Research

Program of Frontier Sciences and the Strategic Priority

Research Program, CAS (XDB27020205 and QYZDB-SSW-

SMC051), and foundations from Yunnan province

Authors

Fei Qiu − Chongqing Key Laboratory of Plant Resource

Conservation and Germplasm Innovation, Key Laboratory of

Eco-environments in Three Gorges Reservoir Region (Ministry

of Education), SWU-TAAHC Medicinal Plant Joint R&D

Centre, School of Life Sciences, Southwest University,

Chongqing 400715, China

(

2019FJ007, 2019ZF011-2, and 2019FA034). The study on

the HDH enzyme has been included in an authorized patent

ZL 201910826205.8) and a PCT patent application (PCT/

(

CN2020/113138) made by Southwest University.

Yijun Yan − State Key Laboratory of Phytochemistry and

Plant Resources in West China, and CAS Center for

Excellence in Molecular Plant Sciences, Kunming Institute of

Botany, Chinese Academy of Sciences, Kunming 650201,

China

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