Structure-Guided Tuning of a Hydroxynitrile Lyase to Accept Rigid Pharmaco Aldehydes

Article abstract of DOI:10.1021/acscatal.0c01103

The chiral vicinal C-O/C-N bifunctional groups generated from enzymatic hydrocyanation represents a useful methodology. However, construction of the pharmacophore of β₂-adrenoreceptor agonists with this method remains a great challenge because of complete racemization of the benzylic alcohol during deprotection of the acetal groups. In this study, structure-guided redesign of a hydroxynitrile lyase originating from Prunus communis (PcHNL5) enables a highly enantioselective hydrocyanation of rigid benzo-ketal aldehyde which was proved to be resistant against racemization during the deprotection step, with dramatically improved productivity (>95% conversion vs <1%). X-ray structure analysis and kinetic study revealed the side chain of L331 tunes the substrate adaptability of bulky and rigid benzo-ketal aldehyde, thereby facilitating the formation of a series of valuable unnatural cyanohydrins in high enantiopurities and good yields. Furthermore, the HNL variant L331A was successfully applied for a gram-scale chemo-enzymatic synthesis of (R)-salmeterol, a long-term β₂-adrenoreceptor agonist, in an optically pure form (>99% ee) with an overall yield of 54%, which is the highest value reported.

Full text of DOI:10.1021/acscatal.0c01103

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Letter
Structure-Guided Tuning of a Hydroxynitrile Lyase to Accept Rigid
Pharmaco Aldehydes
Yu-Cong Zheng, Fu-Long Li, Zuming Lin, Guo-Qiang Lin, Ran Hong,* Hui-Lei Yu,* and Jian-He Xu*
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ABSTRACT: The chiral vicinal C−O/C−N bifunctional groups
generated from enzymatic hydrocyanation represents a useful
methodology. However, construction of the pharmacophore of β2-
adrenoreceptor agonists with this method remains a great
challenge because of complete racemization of the benzylic alcohol
during deprotection of the acetal groups. In this study, structure-
guided redesign of a hydroxynitrile lyase originating from Prunus
communis (PcHNL5) enables a highly enantioselective hydro-
cyanation of rigid benzo-ketal aldehyde which was proved to be
resistant against racemization during the deprotection step, with
dramatically improved productivity (>95% conversion vs <1%). X-
ray structure analysis and kinetic study revealed the side chain of
L331 tunes the substrate adaptability of bulky and rigid benzo-
ketal aldehyde, thereby facilitating the formation of a series of valuable unnatural cyanohydrins in high enantiopurities and good
yields. Furthermore, the HNL variant L331A was successfully applied for a gram-scale chemo-enzymatic synthesis of (R)-Salmeterol,
a long-term β2-adrenoreceptor agonist, in an optically pure form (>99% ee) with an overall yield of 54%, which is the highest value
reported.
KEYWORDS: cyanohydrin, hydrocyanation, hydroxynitrile lyase, protein engineering, β2-adrenoreceptor agonist
nantioselective C−C bond formation is of the utmost
synthetic values. We noticed a series of β2-adrenergic receptor
agonists are used widely to treat asthma and other pulmonary
disorders.8 Formation of the common 1,2-amino alcohol
moiety of classical β2-adrenergic receptor agonists (e.g.,
salmeterol, salbutamol, vilanterol, and salmefamol) (Scheme
1) has attracted several innovative chemical or biochemical
transformations. These methods involve chemical9 or micro-
bial reduction of ketones,10 metal-11 or chiral auxiliary12
mediated hydrogen transfer, oxy-Michael addition of an nitro-
olefin,13 and the Henry reaction.14 However, these approaches
generally required multistep syntheses of designated substances
and/or high loading of sophisticated catalysts. An early
example of enzymatic hydrocyanation of the 1,3-oxane ring
fused benzaldehydes 1b and 1c applied in the synthesis of
salbutamol failed because of complete racemization and partial
decomposition of the product during the final acetal-
deprotection step.15 The easy removal of the isopropylidene
moiety without appreciable racemization16 of the benzylic
E
importance in organic synthesis.1 Despite the develop-
ment of numerous chemical methods,2 enzymatic C−C bond
formation has emerged as a greener process and an attractive
complementary approach that often affords high enantiose-
lectivity.3 Currently, lyases (e.g., EC 4.1.x.x) are used for
enzymatic C−C bond formation,3a,4 among which only
hydroxynitrile lyase (HNL) catalyzes both C−C and C−X
(X = O or N) bond formation in hydrocyanation or nitro-aldol
reactions.5 The vicinal C−O or C−O/C−N functional groups
generated from the method provide an indispensable tool in
the construction of key structural motifs with pharmaceutical
significance.6 Distinct from purely chemical systems, synthetic
methodologies involving HNLs usually suffer from inherent
conflicts between the harsh synthetic requirements and a
limited availability of suitable biocatalysts, except a few famous
examples through protein engineering.7 In addition, sponta-
neous nonselective HCN addtion/cyanohydrin racemization
happens when pH > 4.5, thus ideal enzymatic hydrocyanations
prefer to be performed under a very acidic environment, which
raises challenges for both the stability and activity of the
enzyme.5a,b In general, HNLs have been reported to be highly
specific toward simple aldehydes (like benzaldehyde or flexible
chain aldehydes); however, further development of this
classical biocatalytic tool remains a great challenge toward
sterically rigid aldehyde substrates in spite of their highly
Received: March 6, 2020
Revised: April 19, 2020
Published: April 28, 2020
https://dx.doi.org/10.1021/acscatal.0c01103
© XXXX American Chemical Society
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Letter
Scheme 1. Strategy to Redesign a Biocatalyst to Facilitate
Enantioselective Hydrocyanation of Pharmaceutically
a
Demanding but Sterically Rigid Aldehydes
a
1a, benzaldehyde, highlighted in gray; 1b, R1 = R2 = H; 1c, R1 = H
or Me, R2 = Me or H; 1d, R1 = R2 = Me.
Figure 1. (A) Structural insights into the binding pose of substrates
1a in the active pocket of crystal complex of wild-type PcHNL5 with
1a (PDB ID 6LQY). The key interactions of the substrate carbonyl
group with residues His498 and Tyr458 or the positions chosen for
mutation (B) are indicated. (B) The candidate residues that might
affect the reactivity of substrate 1d in the PcHNL5 active pocket. The
pale blue shadow represents substrate access calculated by the Caver
3.0 algorithm. Residues surrounding substrate access are shown as
dark blue stick models. Impact of the L331 mutation on substrate
specificity toward bulky benzaldehyde derivates. (C) Asymmetric
hydrocyanation activity of wild-type (WT) PcHNL5 (blue) and its
variant L331A (yellow) toward substrates 1a−1d. (D) The
performance of the PcHNL5 variant L331A versus WT for
hydrocyanation of 1d (1 mL of biphasic reaction mixture was
composed of 0.2 mg of purified HNL, 0.3 mL of citrate buffer (100
mM, pH 3.5), 0.7 mL of HCN-MTBE (2 M), and 0.2 mmol of 1d).
alcohol encouraged us to execute asymmetric hydrocyanation
of 2,2-dimethyl-4H-benzo[d][1,3]dioxine-6-carbaldehyde (1d)
by using PcHNL5 (from Prunus communis), which was recently
identified in our lab, with a good activity and high stability
under an acid environment (Figure S3).17
Although PcHNL5 exhibited a broad substrate tolerance for
many substituted benzaldehydes assayed, a dramatic decrease
in hydrocyanation activity was observed when a rigid 1,3-
dioxane ring was introduced with further decreases for
substrates bearing extra methyl groups on the methylene site.
For example, PcHNL5 showed the highest activity toward its
natural substrate, benzaldehyde (1a) (85 U mg−1 protein),
whereas the activity for aldehyde 1b bearing a 1,3-dioxane ring
was moderate (1.5 U mg−1 protein). The enzyme activity
toward aldehyde 1d proved to be extremely sluggish with the
activity decreasing to less than 0.005% when compared with
that of 1a. Moreover, the optical purity of the enzyme-
catalyzed cyanohydrin product 2d was poor because the
enzymatic activity could not override the competing non-
enzymatic HCN addition to the aldehyde during the reaction.
This extremely low reactivity of PcHNL5 toward bulky
substrate 1d motivated us to undertake the redesign of the
HNL structure through protein engineering which has
emerged as a powerful tool in tailoring the enzyme for desired
properties.18 We recently determined the crystal structures of
deglycosylated PcHNL5 (PDB ID 6JBY) as well as the enzyme
in complex with 1a (PDB ID 6JQY) and its corresponding
cyanohydrin (R)-2a (PDB ID 7BWP). The geometry of the
active pocket of wild-type (WT) PcHNL5 comprises a winding
substrate tunnel and a deeply buried, but relatively large,
hydrophobic pocket (Figure S120). Similar to other FAD-
dependent HNLs,19 the FAD in PcHNL5 does not directly
participate in proton transfer, instead the cyanohydrin
cleavage/formation proceeds via the general acid/base catalysis
through residue His498 (Figure S122). The substrate pose is
stabilized by hydrogen bonding of the substrate carbonyl group
with His498 and Tyr460, while the aromatic ring is stabilized
in the large hydrophobic pocket (Figure 1A). Because of the
essential position of Phe72, Leu331, Leu343, and His358 in
the substrate tunnel as revealed by Caver 3.020 as well as their
presumed roles in accommodating the dioxane ring and the
hydrophobic ketal group, these four residues were chosen for
valine scanning (i.e., the four targeted residues were replaced
by the relatively smaller and moderately hydrophobic valine to
explore if steric hindrance or hydrophobicity will affect the
activity significantly).21
Pronounced increases were observed with all of the residues
considered (Table S5). The most substantial increases in
specific activity toward 1d were achieved upon mutation of
Leu331. Thus, this residue was further replaced by six
preselected amino acids (Trp, Phe, Thr, Ser, Ala, and Gly)
to reshape the entrance to the substrate pocket by varying the
extent of steric hindrance and the degree of hydrophobicity
(Figure 1B). The single site mutant L331A showed remarkable
enhancements in activity toward 1,3-oxane ring-fused
benzaldehydes (Figure 1C). In particular, the activity of
variant L331A toward 1d (2.4 U mg−1 protein) was 545-fold
higher than that of the WT PcHNL5. These results encouraged
us to evaluate the catalytic performance of structurally tailored
PcHNL5 in hydrocyanation reactions, with a biphasic system
containing 200 mM 1d and an equal amount of the purified
enzyme (Figure 1D). For variant L331A, the reaction was
completed in 16 h, and product 2d was yielded with 96%
conversion and >99.8% ee. In contrast, only a trace conversion
(<1%) and a low optical purity (55% ee) of the product were
obtained with WT PcHNL5, even if the reaction time was
prolonged to 24 h.
To understand how the mutagenesis causes a significant
increase of enzyme activity, the crystal structure of L331A was
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solved (PDB ID 6LR8). Further insights were obtained by
docking simulation of 1d with the wild type and the mutant
crystal structures since attempts to obtain the crystal
complexes with 1d were unsuccessful. A distinct difference in
the substrate binding pose was observed (Figure 2): replacing
Table 1. Saturation Kinetic Data of PcHNL5WT and Selected
Variants
a
Km
kcat
Kcat/Km
b
aldehyde enzyme
[mM]
[s−1
]
[s−1 mM−1
]
folds
1.0
1a
1b
1c
1d
WT
1.8
5.4
28
137
133
247
76
25
L331V
L331A
L331G
WT
L331V
L331A
L331G
WT
L331V
L331A
L331G
WT
0.33
0.12
<0.018
1.0
8.8
c
d
e
f
>30
>40
12
n.a.
37
6.9
7.6
13
70
11
4.0
24
36
14
2.2
2.2
0.32
3.8
3.4
0.18
0.014
0.72
4.2
26
26
2.3
1.0
8.2
17
13
11
0.57
1.0
53
297
39
13
0.55
0.026
0.87
4.1
1.4
7.2 × 10−4
0.063
1.8
1.0
87
L331V
L331A
L331G
Figure 2. Docking simulations of 1d with PcHNL5 wild-type (A) and
L331A mutant (B). Side chain of position 331 is presented as a red
stick, with the dashed line highlighting the contact with the substrate
tunnel. Shadow represents the substrate tunnels of the WT enzyme
(pale blue) and L331A (pale green).
2526
888
0.64
a
The kinetic parameters were determined using substrate concen-
trations of 0.25−30 mM for 1a, 0.5−30 mM for 1b, 0.5−20 mM for
1c, and 0.5−15 mM for 1d. See the Supporting Information for
b
of the side chain of Leu331 into alanine leads to a dramatically
enlarged substrate tunnel and an extra space for accommoda-
tion of the rigid dioxane ring and two methyl groups. Besides,
the attacking distances between the aldehyde group of 1d and
residues His498 and Tyr460 in L331A become much shorter
(∼3 Å vs >5 Å) than the wild type, due to a 99.3° deflection of
the benzo-ring plane (Figure S120) from the original hard-to-
attack conformation, indicating that a sufficiently strong
hydrogen bonding network with 1d is successfully rebuilt by
mutation for facilitating the hydrocyanation reaction.
details. The folds of kcat/Km change for the specified variant over the
c
WT enzyme. The Km value was beyond the maximal concentration
due to limited solubility. Thus, it was calculated by an extrapolation
d
e
method. Calculated from the highest reaction rate determined. Not
f
available in this case. Calculated from the highest kcat/Km detected.
2f),22 anticonvulsants (2g),23 β-blockers (2h, 2j, 2k),24 β1-
adrenergic receptor agonists (2l),25 and ACE-inhibitors
(2p).26 Each of these aldehydes was used successfully as a
substrate for at least one of the L331 variants with much higher
activity than the WT PcHNL5. Substitution of Leu 331 into
other hydrophobic residues with a smaller side chain, such as
alanine or valine, gave up to 2 orders of magnitude higher
activity toward the majority of 3,4-substituted ring-fused
aldehydes (1b−1j) and the more complicated 7-ethyl-
benzofuran-2-carbaldehyde (1k). Similar phenomena were
observed when substrates bearing an alkyl linker with an
aromatic ring (1g, 1p) were assayed with the L331 variants,
albeit the top hit for 1g was L331G, which may be due to the
larger size of the piperonyl ring when compared with that of
1p. In contrast, different substitutions at other positions of the
benzene ring, such as the 3,5-substituted (1o) or 2,3-
substituted aryl aldehyde (1i), were also accepted by a few
L331 variants with a bulky side chain like phenylalanine
(L331F) or tryptophan (L331W). Notably, when L331
variants with either a hydrophobic or hydrophilic residue
were used to catalyze 4-hydroxy benzaldehyde (1l) bearing an
electronically withdrawing but less sterically hindered group,
only a slight increase in activity was observed. In contrast,
L331 variants with smaller hydrophobic residues (e.g., L331 V
or L331A) showed a clear increase in enzyme activity toward
the bulky 4-phenyl-benzaldehyde (1m). These results suggest
that the steric effect of the side chain of L331 or its variants
interacting with the 2,3,4-substituted benzene ring of
substrates, rather than the hydrophobicity, is mainly
responsible for the observed difference in enzymatic hydro-
cyanation reactions.
We subsequently determined the saturation kinetic con-
stants of WT PcHNL5 and several representative variants
(L331A, L331V, L331G) with substrates 1a−1d (Table 1).
Substitution of Leu331 to the smaller-sized hydrophobic
residue alanine caused a remarkable decrease in Km (from 36
to 2.2 mM) and a large increase in turnover rate (kcat from 2.6
× 10−2 to 4.1 s−1) for substrate 1d. However, further reduction
in size by mutation to glycine did not affect the affinity with 1d
(i.e., no further decrease in Km beyond that of L331A). Instead,
variant L331G showed a clear decrease in kcat from 4.1 to 1.4
s−1, which is consistent with the initially assayed specific
activities (Table S5). The best variant for 1d (L331A) also
exerts strong activity toward 1c, with a 297-fold higher catalytic
efficiency (kcat/Km) than the WT enzyme. However, for
aldehyde 1b (with a relatively smaller substituent), the best
Leu331 variant was L331 V, with the lowest Km (6.9 mM)
observed. Moreover, larger Km values were observed toward
substrate 1a when Leu331 was replaced by smaller side chain
amino acids Val, Ala, and Gly. Because Leu331 is located at the
bottleneck of the substrate tunnel, these results suggest that the
side chain shape and/or size of residue 331 can regulate the
substrate adaption of the enzyme for bulky aryl aldehydes.
To better understand the influence of residue 331 on
substrate adaption and to explore the synthetic potential of the
already engineered enzyme variants, a series of structurally
diverse aromatic aldehydes were subjected to hydrocyanation
with the PcHNL5 variants (Table S5, for details see the
Supporting Information). The corresponding cyanohydrins of
these aldehydes tested are found in scaffolds of pharmaco- or
natural product compounds (Figure S115): tranquilizers (2e,
Performance of the WT enzyme with the best-performing
variants in a biphasic hydrocyanation reaction was sub-
sequently compared (Table 2). Most aldehydes were loaded
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addition,27 the most effective method to mitigate the problem
is to employ an enzyme with higher activity or dosage to
override the competing reaction.28 Thus, the L331 variants of
PcHNL5 are a set of attractive tools that provide a biocatalytic
solution for substrates (e.g., 1g, 1p) suffering from severe
reactivity of nonenzymatic addition, where both the optical
purity and productivity of the cyanohydrin products were
clearly improved. Notably, because a high cell-density
fermentation of the recombinant Pichia pastoris could produce
over 1 g L−1 of secreted PcHNL5 (Table S6), the direct use of
a simple acidized fermentation broth (the supernatant portion)
and subsequent acetylation afforded all of the O-protected
unnatural cyanohydrins (Table 2), including the poorest
substrate 1-naphthaldehyde (1i), with moderate to satisfying
yields (41%−97%) and ideal enantiopurity (96% to >99% ee)
within 12 h.
Table 2. Asymmetric Hydrocyanation of Structurally
Diverse Aryl Aldehydes by PcHNL5WT and Its Variants
a
Finally, to demonstrate the practical feasibility of the tailored
PcHNL5 variants, we devised the chemo-enzymatic synthesis
of (R)-salmeterol (Scheme 2), which is a popular long-term β2-
Scheme 2. Chemo-Enzymatic Gram-Scale Synthesis of (R)-
a
Salmeterol
a
A biphasic reaction mixture (1 mL) was composed of 0.1 mg of
purified HNL, 0.3 mL citrate buffer (100 mM, pH 3.5), 0.7 mL of
HCN-MTBE (2 M), and varying amounts of aldehydes 1a−1p (1b,
1c, 1f, 1g, and 1h, 0.5 mmol; 1e, 1j, 1o, and 1p, 1 mmol; 1f, 0.3
mmol; 1d, 1i, 1m, and 1k, 0.2 mmol; 1l, 0.1 mmol; 1n, 0.05 mmol).
Reactions were carried out at 10 °C and 1500 rpm for 12 h. For
experimental details of TOF determination, see Supporting
a
Reaction and conditions: (a) PcHNL5L331A (0.5 wt %), HCN (∼1 M
in MTBE, 4 equiv), citrate buffer (100 mM, pH 3.5, 50% v/v), 10 °C,
16 h, 93%; (b) LiAlH4 (1.8 equiv), THF, 0 °C to RT, 8 h. 67%
(recrystallization); (c) bromide 5, DMF, RT, 70 h, 86%; (d) HOAc/
water (3/1, v/v), 50 °C, 4 h, quant. MTBE = methyl tert-butyl ether;
THF = tetrahydrofuran; DMF = N,N-dimethylformamide.
b
c
Information. 0.2 mg of HNL was used. 0.3 mg of HNL was used.
d
e
0.02 mg of HNL was used. The preparative scale reactions were
performed using fermentation broths (supernatant portion) of
PcHNL5-L331X variants after high cell-density fermentation in a 5-
L bioreactor. Isolated yields and ee values were calculated based on O-
acetylated cyanohydrins obtained after a two-step transformation. See
the Supporting Information for details.
agonist used in the clinic.29 In a 100 mL biphasic reaction
system, 5 g of substrate 1d was subjected to hydrocyanation by
employing only 0.5 wt % of PcHNL5-L331A. The reaction
proceeded smoothly to give (R)-2d in 93% isolated yield and
>99% ee within 16 h. A reduction with lithium aluminum
hydride in THF gave the corresponding 2-amino-1-alcohol 4
without deterioration of enantiopurity. The subsequent N′-
alkylation with bromide 5 and deprotection of the
isopropylidene group in an aqueous solution of acetic acid
afforded (R)-salmeterol acetate in an optically pure form
(>99% ee). The overall transformation from the readily
available aldehyde (1d) to the target molecule was realized in
four steps with 54% overall yield. This chemo-enzymatic route
represents the highest overall yield and optical purity reported
for (R)-salmeterol. Importantly, these results demonstrate the
over a final concentration range of 0.1−1.0 M with a minimum
enzyme dosage of 0.015 wt %. Mutagenesis did not affect the
good stability of the L331 variants under acidic conditions,
even with increased acid tolerance (Figure S3). This allows the
enzymatic hydrocyanation to be performed at low pH to
suppress the nonselective chemical HCN addition reaction.
Compared with the wild type enzyme, the turnover frequency
(TOF or kcat) of the variants improved by 1.6 to 792-fold, even
when the substrate was well accepted by WT PcHNL5 (e.g.,
1b, 1e, 1j, 1p). Because asymmetric biohydrocyanation is a
competitive process against a spontaneous nonselective
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great potential of protein engineering for resolving conflicts
between the demanding synthetic requirements and the
inherent properties of native enzymes in the development of
new chemo-enzymatic methods.
Fu-Long Li − State Key Laboratory of Bioreactor Engineering,
Shanghai Collaborative Innovation Centre for
Biomanufacturing, East China University of Science and
Technology, Shanghai 200237, China; orcid.org/0000-
0002-2460-3266
Zuming Lin − CAS Key Laboratory of Synthetic Chemistry of
Natural Substances, Center for Excellence in Molecular
Synthesis, Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, Shanghai 200032, China
Guo-Qiang Lin − CAS Key Laboratory of Synthetic Chemistry
of Natural Substances, Center for Excellence in Molecular
Synthesis, Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, Shanghai 200032, China; University of
the Chinese Academy of Sciences, Beijing 100049, China
In summary, we have successfully engineered a hydroxyni-
trile lyase that enables asymmetric hydrocyanation of a very
challenging rigid benzo-ketal aldehyde. By utilizing the
rationally redesigned variants, we were able to realize the
first and highly efficient chemo-enzymatic synthesis of an
optically pure long-term β2-agonist via asymmetric C−C bond
formation. Furthermore, exploring the substrate spectrum of
the engineered enzymes allowed a deeper insight into the
crucial role of residue 331 in precisely regulating the substrate
acceptability of this enzyme family, thereby providing guidance
for future bioconversion of various unnatural aldehydes,
especially those with bulky and rigid substitution groups on
the aromatic ring. We anticipate that this work will act as a
valuable example and lay the foundation for overcoming
enzyme limitations in the development of new chemo-
enzymatic approaches to atom-economic and eco-friendly
syntheses.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acscatal.0c01103
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acscatal.0c01103.
■
sı
ACKNOWLEDGMENTS
■
This work was financially supported by the National Key
Research and Development Program of China (Grants
2019YFA09005000 and 2018YFC1706200), the National
Natural Science Foundation of China (Grants 21536004,
21922804, 21776085, and 21871085), the Chinese Academy
of Sciences (Grants XDB20020000 and QYZDY-
SSWSLH026), and the Fundamental Research Funds for the
Central Universities (Grant 22221818014). We thank
Professor Romas J. Kazlauskas (UMN) for his critical reading
and valuable suggestions, Miss Lian Wu (SIOC), and Dr.
Guang Liu (ECUST) for their helpful guidance in the addition
of NAG during the data refinement of the crystal structure, Dr.
Xu-Dong Kong (ECUST), Dr. Fei-Fei Chen (ECUST), and
Dr. Qi Chen (ECUST) for their insightful discussions, and Mr.
Jun Zhu (ECUST) for his experimental assistance. We are also
grateful for the access to beamline BL17U1 and BL19U1 at
Shanghai Synchrotron Radiation Facility and thank the
beamline staff for the technical help.
Materials and chemicals; detailed experimental and
computational protocols; supporting schemes, tables,
and figures for the primers design; expression,
purification, and crystallization of PcHNL5; synthetic
details; GC and HPLC analytic conditions; GC and
HPLC profiles, NMR profiles, and MS profiles; and
fermentation details (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Ran Hong − CAS Key Laboratory of Synthetic Chemistry of
Natural Substances, Center for Excellence in Molecular
Synthesis, Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, Shanghai 200032, China; University of
the Chinese Academy of Sciences, Beijing 100049, China;
orcid.org/0000-0002-7602-539X; Email: rhong@
sioc.ac.cn
Hui-Lei Yu − State Key Laboratory of Bioreactor Engineering,
Shanghai Collaborative Innovation Centre for
Biomanufacturing and Frontiers Science Center for
Materiobiology and Dynamic Chemistry, East China University
of Science and Technology, Shanghai 200237, China;
Email: huileiyu@ecust.edu.cn
Jian-He Xu − State Key Laboratory of Bioreactor Engineering,
Shanghai Collaborative Innovation Centre for
Biomanufacturing and Frontiers Science Center for
Materiobiology and Dynamic Chemistry, East China University
of Science and Technology, Shanghai 200237, China;
orcid.org/0000-0003-0140-3808; Email: jianhexu@
ecust.edu.cn
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