Bioproduction of Enantiopure (R)- and (S)-2-Phenylglycinols from Styrenes and Renewable Feedstocks

Article abstract of DOI:10.1002/adsc.202001322

Enantiopure (R)- and (S)-2-phenylglycinols are important chiral building blocks for pharmaceutical manufacturing. Several chemical and enzymatic methods for their synthesis were reported, either involving multi-step synthesis or starting from a relatively complex chemical. Here, we developed one-pot simple syntheses of enantiopure (R)- and (S)-2-phenylglycinols from cheap starting materials and renewable feedstocks. Enzyme cascades consisting of epoxidation-hydrolysis-oxidation-transamination were developed to convert styrene 2 a to (R)- and (S)-2-phenylglycinol 1 a, with butanediol dehydrogenase for alcohol oxidation as well as BmTA and NfTA for (R)- and (S)-enantioselective transamination, respectively. The engineered E. coli strains expressing the cascades produced 1015 mg/L (R)-1 a in >99% ee and 315 mg/L (S)-1 a in 91% ee, respectively, from styrene 2 a. The same cascade also converted substituted styrenes 2 b–k and indene 2 l into substituted (R)-phenylglycinols 1 b–k and (1R, 2R)-1-amino-2-indanol 1 l in 95–>99% ee. To transform bio-based L-phenylalanine 6 to (R)-1 a and (S)-1 a, (R)- and (S)-enantioselective enzyme cascades for deamination-decarboxylation-epoxidation-hydrolysis-oxidation-transamination were developed. The engineered E. coli strains produced (R)-1 a and (S)-1 a in high ee at 576 mg/L and 356 mg/L, respectively, from L-phenylalanine 6, as the first synthesis of these compounds from a bio-based chemical. Finally, L-phenylalanine biosynthesis pathway was combined with (R)- or (S)-enantioselective cascade in one strain or coupled strains, to achieve the first synthesis of (R)-1 a and (S)-1 a from a renewable feedstock. The coupled strain approach enhanced the production, affording 274 and 384 mg/L (R)-1 a and 274 and 301 mg/L (S)-1 a, from glucose and glycerol, respectively. The developed methods could be potentially useful to produce these high-value chemicals from cheap starting materials and renewable feedstocks in a green and sustainable manner. (Figure presented.).

Full text of DOI:10.1002/adsc.202001322

Title: Bioproduction of Enantiopure (<i>R</i>)- and (<i>S</i>)-2-
Phenylglycinols from Styrenes and Renewable Feedstocks
Authors: Balaji Sundara Sekar, Jiwei Mao, Benedict Ryan Lukito,
Zilong Wang, and Zhi Li
This manuscript has been accepted after peer review and appears as an
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content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.202001322
Link to VoR: https://doi.org/10.1002/adsc.202001322

10.1002/adsc.202001322
Advanced Synthesis & Catalysis
Very Important Publication
FULL PAPER
DOI: 10.1002/adsc.202######
Bioproduction of Enantiopure (R)- and (S)-2-Phenylglycinols
from Styrenes and Renewable Feedstocks
Balaji Sundara Sekar a, b †, Jiwei Mao a, b †, Benedict Ryan Lukito a, Zilong Wang a, b,
and Zhi Li a, b
*
a
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4,
Singapore 117585 * E-mail: chelz@nus.edu.sg; Ph: +65-65168416
b
Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of
Singapore, 28 Medical Drive, Singapore 117456
†
These authors contributed equally to this work.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.
Abstract. Enantiopure (R)- and (S)-2-phenylglycinols are enantioselective enzyme cascades for decarboxylation-
important chiral building blocks for pharmaceutical deamination-epoxidation-hydrolysis-oxidation-
manufacturing. Several chemical and enzymatic methods for transamination were developed. The engineered E. coli
their synthesis were reported, either involving multi-step strains produced (R)-1a and (S)-1a in high ee at 576 mg/L
synthesis or starting from a relatively complex chemical. and 356 mg/L, respectively, from L-phenylalanine 6, as the
Here, we developed one-pot simple syntheses of enantiopure first synthesis of these compounds from a bio-based
(R)- and (S)-2-phenylglycinols from cheap starting materials chemical. Finally, L-phenylalanine biosynthesis pathway
and renewable feedstocks. Enzyme cascades consisting of was combined with (R)- or (S)-enantioselective cascade in
epoxidation-hydrolysis-oxidation-transamination
were one strain or coupled strains, to achieve the first synthesis
developed to convert styrene 2a to (R)- and (S)-2- of (R)-1a and (S)-1a from a renewable feedstock. The
phenylglycinol 1a, with butanediol dehydrogenase for coupled strain approach enhanced the production, affording
alcohol oxidation as well as BmTA and NfTA for (R)- and 274 and 384 mg/L (R)-1a and 274 and 301 mg/L (S)-1a,
(S)-enantioselective transamination, respectively. The from glucose and glycerol, respectively. The developed
engineered E. coli strains expressing the cascades produced methods could be potentially useful to produce these high-
1015 mg/L (R)-1a in >99% ee and 315 mg/L (S)-1a in 91% value chemicals from cheap starting materials and
ee, respectively, from styrene 2a. The same cascade also renewable feedstocks in a green and sustainable manner.
converted substituted styrenes 2b-k and indene 2l into
substituted (R)-phenylglycinols 1b-k and (1R, 2R)-1-amino-
2-indanol 1l in 95->99% ee. To transform bio-based L-
phenylalanine 6 to (R)-1a and (S)-1a, (R)- and (S)-
Keywords: Amino alcohols; Biocatalysis;
Enantioselectivity; Enzyme catalysis; One-pot reaction;
Renewable resources
triplex metallohelices[1b] and as chiral ligands for
asymmetric annulation or cyclization. Both (R)- and
(S)-2-phenylglycinol 1a are high value compounds
with the price of $40-$100 per kg. Moreover, the
substituted (R)-2-phenylglycinols 1b-lk (Figure 1a)
are also useful pharmaceutical intermediates, for
instance, (R)-2-amino-2-(4-methoxyphenyl)ethanol
1k and (1R,2R)-1-amino-2-indanol 1l are used in the
synthesis of agonists and Alk5 inhibitors to treat
cancers.[4]
Several chemical methods were established for the
synthesis of (R)- and (S)-1a; however, these methods
have one or more disadvantages. For instance,
methods for the reduction of D- or L-phenylglycine to
(R)- or (S)-1a require expensive enantiopure
substrates and utilizes hazardous chemicals.[5]
Introduction
Enantiopure β-amino alcohols are important high
value chemicals used in the preparation of
pharmaceutics[1] such as randolazine,[2] nebivolol and
metoprolol,[3] as a ligand for chiral catalysts in
asymmetric synthesis, and as a chiral building block
in the synthesis of other chiral compounds.
Specifically, (R)-2-phenylglycinol 1a is used in the
synthesis of novel antibiotic pyrazolopyrimidines,
EGFR inhibitor thienopyrimidines, and 3-
phosphoinositide-dependent protein kinase-1 (PDK1)
inhibitor.[1e] (S)-2-phenylglycinol 1a is used in the
synthesis of a neurotrophic agent, potent histone
deacetylase inhibitors, novel anti-cancer asymmetric
1
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10.1002/adsc.202001322
Advanced Synthesis & Catalysis
Figure 1: Production of enantiopure (R)-2-phenylglycinols 1a-l and (S)-2-phenylglycinol 1a from styrenes 2a-k, indene 2l,
bio-based L-phenylalanine 6 and renewable feedstocks glucose 8 and glycerol 9. (a) Artificial enzyme cascade for the
conversion of styrene 2a-k, indene 2l to (R)-1a-l and (S)-1a via one-pot epoxidation-hydrolysis-oxidation-amination
reaction. (b) Artificial enzyme cascade for the biotransformation of L-phenylalanine 6 to (R)- and (S)-1a via one-pot
deamination-decarboxylation-epoxidation-hydrolysis-oxidation-amination reaction. (c) Conversion of glucose 8 and
glycerol 9 to (R)- and (S)-1a via the combination of natural Shikimate pathway for the synthesis of 6 and the artificial
enzyme cascade for the conversion of 6 to (R)- and (S)-1a. Enzymes involved are styrene monooxygenase (SMO), epoxide
hydrolase (StEH), alcohol dehydrogenase (ADH), transaminase (TA), phenylalanine ammonia lyase (PAL) and
phenylacrylic acid decarboxylase (PAD).
Methods for the kinetic resolution of racemic 1a to
obtain enantiopure (R)- or (S)-1a suffers from low
theoretical yield (50%).[6] Enzyme catalysis is
attractive due to high regio- and stereo-selectivity,
mild reaction conditions, and environmental
friendliness.[7] However, the enzymatic approaches
developed for the synthesis of (R)- and (S)-1a also
have drawbacks. For example, kinetic resolution of
racemic 1a using transaminase suffers from low
theoretical yield (50%).[8] Amination of 2-
hydroxyacetophenone 5a using transaminase to give
(R)-1a requires special substrate.[9] Recently, one-pot
cascade biotransformation of styrene oxide 3a to (R)-
1a[10] and styrene oxides 3a, d, f, h to (S)-1a, d, f,
h[11] were reported, but needs to synthesize the
starting material and solve the inhibition by the
substrates. In this study, we are interested in
developing the enzymatic method for the synthesis of
enantiopure β-amino alcohols such as (R)-1a and (S)-
1a from renewable feedstocks as well as (R)-1a-l and
(S)-1a from cheap and easily available starting
materials.
To reach our goal we are focusing on the
development of one-pot cascade biotransformation
which is an emerging technique for the production of
high-value chemicals from cheap substrates and
renewable feedstocks. This technique uses several
enzymes in one-pot to perform multi-step reactions
under mild reaction conditions, being of
advantageous over traditional multi-step synthesis
2
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10.1002/adsc.202001322
Advanced Synthesis & Catalysis
Figure 2: (a) Production of hydroxyacetophenone 5a from 2 and 10 mM of (R)-phenylethanediol 4a by E. coli-BDHA or
E. coli-CpSADH (10 g cdw/L). (b) Screening of transaminases for the conversion of 5a to (R)- or (S)- 2-phenylglycinol 1a
by biotransformation of 10 mM 5a with E. coli strains expressing transaminases (20 g cdw/L) in phosphate buffer (200
mM, pH 8) containing 0.5% glucose and 200 mM amino donor. Biotransformations were performed in duplicates with
error bars showing ± SD.
without product recovery in each step.[7c, 12] Several
studies were reported on one-pot whole-cell
biotransformation for the production of high-value
chemicals such as enantiopure amino acids, α-
hydroxy acids, amino alcohols and aroma chemicals
from cheap and renewable substrates in an
economical and sustainable manner.[12f, 13] Here we
report the development of artificial enzyme cascades
for the one-pot synthesis of enantiopure (R)-1a-l and
(S)-1a from cheap substrate styrenes 2a-l, further
engineering of the cascade to convert bio-based L-
phenylalanine 6 to (R)- and (S)-1a, and the
combination of natural L-phenylalanine biosynthesis
pathway with artificial enzyme cascade for the
conversion of renewable feedstocks as glucose 8 and
glycerol 9 to (R)- and (S)-1a.
For the oxidation of (R)-phenylethanediol 4a to 2-
hydroxyacetophenone 5a, butanediol dehydrogenase
(BDHA) from Bacillus subtilis BGSC1A1 and
secondary alcohol dehydrogenase (CpSADH) from
Candida parapsilosis were examined. These enzymes
were reported for the efficient production of hydroxy
ketones.[12f, 13d, 15] The genes coding for BDHA and
CpSADH were cloned in individual pET28a plasmid
under the control of T7 promoter, and the two
recombinant plasmids were transformed into separate
E. coli T7 strains to obtain E. coli-BDHA expressing
BDHA and E. coli-CpSADH expressing CpSADH,
respectively. The resting cells of E. coli-BDHA and E.
coli-CpSADH (10 g cdw/L) were evaluated for
oxidation of 2 mM of 4a, giving 95-100% conversion
to 5a for both cases (Figure 2a). The concentration of
4a was increased to 10 mM, and E. coli-BDHA
produced 9.4 mM 5a with 94% conversion within 12
h, whereas E. coli-CpSADH produced only 6.24 mM
of 5a with 62% conversion at the same time.
Therefore, BDHA was chosen as the enzyme for the
oxidation of 4a in the cascade biotransformations.
For the conversion of 5a to (R)-1a, databases were
searched for (S)-enantioselective transaminases with
substrate preference similar to 5a and simple amino
donors such as glutamate, L-alanine and
Results and Discussion
Design of cascade biotransformation for the
production of (R)- and (S)-2-phenylglycinol 1a
from styrene 2a
To convert styrene 2a to (R)- and (S)- 2-
phenylglycinol 1a, we designed artificial cascade
with four enzymes for the epoxidation-hydrolysis-
oxidation-transamination reactions (Figure 1a).
Conversion of styrene 2a to (R)-phenylethanediol 4a
by epoxidation-hydrolysis reaction were achieved by
styrene monoxygenase (SMO) from Pseudomonas sp.
isopropylamine.
Eight
(S)-enantioselective
transaminases such as HnTA, DpgTA, EeaTA, VfTA,
CvTA, mVfTA[9], BmTA and MmTA were identified
with substrate preference similar to 5a. The genes
coding for eight (S)-enantioselective transaminases
were cloned in individual pRSFDuet-1 plasmid and
transformed into E. coli T7 strains, each expressing
one of eight different transaminases. 10 g cdw/L of
resting cells of E. coli strains expressing eight
transaminases were used for the biotransformation of
10 mM 5a in KP buffer containing 0.5% glucose and
200 mM amino donor (D-, L-alanine, glutamate) for
24 h.
and epoxide hydrolase (StEH) from Solanum
14]
tuberosum.[13f,
Conversion of 4a to 2-
hydroxyacetophenone 5a by oxidation reaction could
be achieved by the use of alcohol dehydrogenase.
Amination of 5a to (R)- and (S)-2-phenylglycinol 1a,
respectively, could be achieved by using (R) and (S)-
enantioselective transaminases.
3
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10.1002/adsc.202001322
Advanced Synthesis & Catalysis
Figure 3: Cascade biotransformation of styrene 2a to (R)- and (S)-1a using E. coli strains expressing the artificial enzyme
cascade shown in Figure 1a. (a) E. coli-SSBB-1-6 harboring three expression plasmids with different copy numbers for the
expression of SMO, StEH, BDHA, AlaDH and BmTA at various expression levels for producing (R)-1a. (b) SDS-PAGE
analysis of crude extracts of E. coli-SSBB-1-6 showing the expression of enzymes for producing (R)-1a. Lane M, protein
ladder; Lane 1-6, E. coli-SSBB-1-6. (c) Synthesis of (R)-1a from 10 mM 2a by E. coli-SSBB-1-6 (20 g cdw/L) at 30°C for
24 h. (d) E. coli-SSBN-1-6 harboring three expression plasmids with different copy numbers to express SMO, StEH,
BDHA, AlaDH and NfTA at various expression levels for producing (S)-1a. (e) SDS-PAGE analysis of crude extracts of E.
coli-SSBN-1-6 showing the expression of enzymes for producing (S)-1a. Lane M, protein ladder; Lane 7-12, E. coli-
SSBN-1-6. (f) Synthesis of (S)-1a from 5 mM 2a by E. coli-SSBN-1-6 (20 g cdw/L) at 30°C for 48 h. Reaction conditions:
Two-phase system of 2 mL phosphate buffer (200 mM, pH 8) and 2 mL n-hexadecane containing 2a. Biotransformations
were performed in duplicates with error bars showing ± SD.
As shown in Figure 2b, several transaminases were
able to convert 5a to (R)-1a and the best result was
obtained from E. coli-BmTA expressing the
transaminase from Bacillus megaterium. BmTA was
able to convert 10 mM 5a to (R)-1a with 82%
efficiency and >99% ee with L-alanine as amino
donor. BmTA was thus chosen for the artificial
cascade for the production of (R)-1a. Similarly, (R)-
enantioselective transaminases were searched in
database and three transaminase candidates such as
NfTA, ArRTA and AtTA were identified for the
examination on the conversion of 5a to (S)-1a. The
selected transaminases were cloned in individual
pRSFDuet-1 plasmid, and transformed into three
different E. coli T7 strains. 10 g cdw/L of the resting
cells of E. coli strains expressing three (R)-
enantioselective transaminases were used for the
biotransformation of 10 mM 5a in KP buffer
containing 0.5% glucose and 200 mM amino donor
(L-alanine, isopropylamine) for 24 h. As shown in
figure 2a, only E. coli-NfTA expressing transaminase
from Neosartora fischeri was able to convert 5a to
(S)-1a with relatively high efficiency. NfTA was able
to convert 10 mM 5a to (S)-1a in 24% and 91% ee
with L-alanine as amino donor. Therefore, NfTA was
chosen for the artificial cascade for the production of
(S)-1a.
Engineering of E. coli co-expressing 5 enzymes of
the artificial cascade for the conversion of styrene
2a to (R)- and (S)-2-phenylglycinol 1a
For the efficient whole cell biotransformation of 2a to
(R)- and (S)-1a with a single strain, combinatorial
approach was attempted for the expression of
enzymes in the artificial cascade. Combinatorial
approach facilitates to obtain maximum conversion
efficiency by optimizing the expression level of
multiple enzymes in the cascade. This could be
achieved by expressing the enzymes of the artificial
cascade from three plasmids (pCDFDuet-1,
pETDuet-1 and pRSFDuet-1) with varying copy
numbers of 10 to >100.[16] Different combinations of
these plasmids could result in different expression
levels of the enzymes.[14a, 17] Thus, SMO and StEH for
the conversion of 2a to 4a were cloned together in
4
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10.1002/adsc.202001322
Advanced Synthesis & Catalysis
Table 1: Cascade biotransformation of styrenes 2a-2k and indene 2l to (R)-2-phenylglycinols-1a-1k and (1R,2R)-1-amino-
2-indanol 1l with resting cells of E. coli-SSBB-1
Substrate conc. b)
Cell conc.
Conv. c)
(%)
74
ee c)
(%)
>99
>99
>99
99
>99
>99
95
>99
>99
>99
>99
>99
Substrate a)
R
Product
(mM)
10
10
10
10
10
5
5
5
10
5
5
(g cdw L-1)
H
o-F
m-F
20
20
20
20
20
25
25
25
20
25
25
25
2a
2b
2c
2d
2e
2f
2g
2h
2i
1a
1b
1c
1d
1e
1f
1g
1h
1i
57
69
73
56
53
34
22
67
p-F
m-Cl
p-Cl
m-Br
p-Br
m-Me
p-Me
p-OMe
-
57
61
24
2j
2k
2l
1j
1k
1l
5
a)
Substrates 2a-2l were converted to 1a-1l by E. coli SSBB-1 (20 g cdw/L) at 30°C for 48 h in a two-phase system of 2
mL phosphate buffer (200 mM, pH 8) and 0.4 mL n-hexadecane containing 2a-2l. b) normalized to aqueous phase volume.
c)
Concentration and enantiomeric excess of 1a-1l were analysed using reverse phase HPLC with SB-C18 and Crownpak
CR (+) chiral column, respectively. Biotransformations were performed in duplicates and the mean values were presented.
pCDFDuet-1, pETDuet-1 and pRSFDuet-1. BDHA
for the conversion of 4a to 5a and alanine
dehydrogenase[13f] for recycling amino donor (L-
alanine) were cloned together in pCDFDuet-1,
pETDuet-1 and pRSFDuet-1. BmTA for (R)-1a or
NfTA for (S)-1a production from 5a was cloned in
pCDFDuet-1, pETDuet-1 and pRSFDuet-1. The
23%. As NfTA was not efficient like BmTA,
relatively low production of (S)-1a was observed in E.
coli-SSBN-1-6. Evolution of NfTA and search for
new
R-selective
transaminase
for
further
improvement of (S)-1a production is under progress.
To further improve the conversion of 2a to (R)-1a by
E. coli-SSBB-1, the reaction conditions were
optimized (Table S2). Firstly, the biotransformation
of 2a to (R)-1a was performed with E. coli-SSBB-1
(20 g cdw/L) with 0- or 100-mM supplementation of
L-alanine. E. coli naturally produces L-alanine and E.
coli-SSBB-1 express alanine dehydrogenase for
recycling L-alanine using NH3/NH4Cl, therefore the
supplementation of L-alanine might not be necessary.
In fact, same conversion efficiency (68-69%) was
obtained with or without L-alanine supplementation.
Secondly, the organic phase n-hexadecane was
replaced by ethyl oleate and the synthesis of (R)-1a
from 2a was evaluated. As n-hexadecane is obtained
from petroleum, a greener and biocompatible organic
phase such as ethyl oleate which can be obtained
from biodiesel was tested. No significant difference
was observed in the conversion (68-69%) suggesting
that ethyl oleate could be used as a greener alternative.
Further, the volume of organic phase (n-hexadecane
or ethyl oleate) was reduced by changing the ratio of
organic phase: phosphate buffer ratio from 1:1 to
0.2:1. Improved conversion of 2a to (R)-1a was
observed with both n-hexadecane: phosphate buffer
and ethyl oleate: phosphate buffer in 0.2:1 ratio,
respectively. 74% conversion of 2a to (R)-1a was
achieved in biotransformation with n-hexadecane:
phosphate buffer in 0.2:1 ratio. Reduced volume of
organic phase usage increased the conversion
efficiency and could also contribute to reducing
operation cost in large-scale production.
recombinant
pCDFDuet-1,
pETDuet-1
and
pRSFDuet-1 plasmids were transformed into twelve
different E. coli strains so that all five enzymes are
present in compatible plasmids in each E. coli strain
(Figure 3a, 3d and Table S1). The E. coli strains
containing the five-enzyme cascade for the
conversion of 2a to (R)-1a and (S)-1a, were named E.
coli-SSBB-1-6 and E. coli-SSBN-1-6, respectively.
SDS-PAGE analysis of E. coli-SSBB-1-6 and E. coli-
SSBN-1-6 were performed, which confirmed the
expression of all five enzymes in the artificial
cascade (Figure 3b, 3e). The expression of BmTA
was relatively higher in E. coli-SSBB-1 and E. coli-
SSBB-2 compared to other strains.
One-pot biotransformation of styrene 2a to (R)-
and (S)-2-phenylglycinol 1a with E. coli co-
expressing 5 enzymes of artificial cascade
One-pot biotransformation of 10 mM 2a to (R)-1a
was performed with resting cells of E. coli-SSBB-1-6
(20 g cdw/L) at 30°C for 24 h. As shown in Figure 3c,
difference in copy numbers of expression vectors
showed huge variation in the synthesis of (R)-1a by E.
coli-SSBB-1-6. E. coli-SSBB-1 (pCDF-SMO-StEH,
pET-BDHA- AlaDH, pRSF-BmTA) was the best
strain to produce 6.83 mM of (R)-1a (>99% ee and
68% conversion) from 2a (Figure 3c). One-pot
biotransformation of 5 mM 2a to (S)-1a was
performed with resting cells of E. coli-SSBN-1-6 (20
g cdw/L) at 30°C for 48 h. As shown in Figure 3f, E.
coli-SSBN-2 (pCDF-SMO-StEH, pET-NFTA, pRSF-
BDHA- AlaDH) showed the highest conversion of
5
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10.1002/adsc.202001322
Advanced Synthesis & Catalysis
Figure 4: Cascade biotransformation of L-phenylalanine 6 to (R)- and (S)-1a using E. coli strains expressing the artificial
enzyme cascade shown in Figure 1b. (a) E. coli-PPSSBB and E. coli-PPSSBN harboring four expression plasmids for the
expression of PAL, PAD, SMO, StEH, BDHA, AlaDH and BmTA or NfTA for the production of (R)- or (S)-1a,
respectively. (b) SDS-PAGE analysis of crude extracts of E. coli-PPSSBB and E. coli-PPSSBN showing the expression of
enzymes for producing (R)- or (S)-1a. Lane M, protein ladder; Lane C, E. coli T7 (negative control); Lane 13, E. coli-
PPSSBB; Lane 14, E. coli-PPSSBN. (c) Synthesis of (R)- and (S)-1a from 10 mM 6 by E. coli-PPSSBB and E. coli-
PPSSBN (15 g cdw/L), respectively in a two-phase system of 5 mL phosphate buffer (200 mM, pH 8) containing 6 and 1
mL n-hexadecane at 30°C for 24 h. Biotransformations were performed in duplicates with error bars showing ± SD.
One-pot biotransformation of styrenes 2a-2k and
indene 2l to (R)-phenylglycinols 1a-1k and
(1R,2R)-1-amino-2-indanol 1l with E. coli co-
expressing 5 enzymes of artificial cascade
bio-based L-phenylalanine 6 to (R)- and (S)-2-
phenylglycinol 1a
For the conversion of bio-based L-phenylalanine 6 to
enantiopure (R)- and (S)-1a, artificial enzyme cascade
Substituted (R)-phenylglycinols 1a-1k and (1R,2R)-1-
amino-2-indanol 1l are also important building blocks
in the synthesis of pharmaceutics. Therefore, E. coli-
SSBB-1 cells were examined for the conversion of
substituted styrenes 2a-k and indene 2l to (R)-1a-1k
consisting
of
decarboxylation-deamination-
epoxidation-hydrolysis-oxidation-transamination,
Phenylalanine ammonia lyase (PAL) of Arabidopsis
thaliana and phenylacrylic acid decarboxylase (PAD)
of Saccharomyces cerevisiae were expressed together
in the cascade for 2a to (R)- and (S)-1a (Figure 1b).
We previously proved that PAL and PAD efficiently
produces 2a from 6.[13a, 13c, 14a, 18] Thus, PAL and PAD
enzymes cloned in pACYCDuet-1 (pACYC-PAL-
PAD)[13c] were co-expressed in E. coli-SSBB-1
(pCDF-SMO-StEH, pET-BDHA-AlaDH, pRSF-
BmTA) and E. coli-SSBN-2 (pCDF-SMO-StEH,
and
(1R,2R)-1-amino-2-indanol
1l.
One-pot
biotransformation was performed in the two-phase
system with n-hexadecane and phosphate buffer
(0.2:1) with 5-10 mM 2a-2l, E. coli-SSBB-1 (20 g
cdw/L) and the conversion to 1a-1l were listed in
Table 1. All the tested substrates (2a-2k) were
converted to their respective (R)-phenylglycinols (1a-
1k) and (1R,2R)-1-amino-2-indanol 1l in 95->99% ee. pET-NFTA, pRSF-BDHA-AlaDH) and the resulted
Among the eleven substituted substrates, eight
substrates were converted to their respective (R)-
phenylglycinols with >50% conversion. Specifically,
2c, 2d, 2i, 2k showed >60% conversion. Thus, the
developed artificial cascade is useful for the
production of various (R)-2-phenylglycinols 1a-1k
and (1R,2R)-1-amino-2-indanol 1l.
One-pot biotransformation of styrenes 2a, 2d, 2e, 2j,
2k to (R)-2-phenylglycinols 1a, 1d, 1e, 1j, 1k were
scaled up. Preparative biotransformation was
performed with resting cells of E. coli-SSBB-1 (25 g
cdw/L) in 100 mL phosphate buffer with 20 mL of n-
hexadecane containing 2a, 2d, 2e, 2j, 2k (50 mM,
normalized to aqueous phase volume). After reaction
for 48 h at 30°C, (R)- 1a, 1d, 1e, 1j and 1k were
isolated from the aqueous phase in high purity with
59%, 50%, 37%, 32%, and 44% yield, respectively.
recombinant strains were named as E. coli-PPSSBB
and E. coli-PPSSBN, respectively (Figure 4a). SDS-
PAGE analysis of the crude cell lysate of E. coli-
PPSSBB and E. coli-PPSSBN was performed (Figure
4b), showing the expression of all seven enzymes of
the artificial cascade in E. coli-PPSSBB and E. coli-
PPSSBN.
Cascade biotransformation of 10 mM 6 to (R)- and
(S)-1a was performed with the resting cells (20 g
cdw/L) of E. coli-PPSSBB and E. coli-PPSSBN,
respectively, in the two-phase system with n-
hexadecane and phosphate buffer (0.2:1). After 24 h
of reaction at 30°C, 576 mg/L (R)-1a and 356 mg/L
(S)-1a was produced by E. coli-PPSSBB and E. coli-
PPSSBN, respectively (Figure 4c). This is the first
report on the synthesis of (R)- and (S)-1a from bio-
based substrate 6. Since the accumulation of
intermediates such as 4a and 5a was observed, further
enhancing the productivity might be achieved by
improving the activity of the alcohol dehydrogenase
and transaminase.
Design of cascade biotransformations and
development of resting-cells bioprocess to convert
6
This article is protected by copyright. All rights reserved.

10.1002/adsc.202001322
Advanced Synthesis & Catalysis
Figure 5: Fermentation of glucose 8 or glycerol 9 to (R)- and (S)-1a using E. coli NST74(DE3) strains expressing the
artificial enzyme cascade shown in Figure 1c. (a) E. coli NST-PPSSBB and E. coli NST-PPSSBN harboring engineered
Shikimate pathway and four expression plasmids for the expression of PAL, PAD, SMO, StEH, BDHA, AlaDH and
BmTA or NfTA for the production of (R)- or (S)-1a, respectively. (b) SDS-PAGE analysis of crude extracts of E. coli
NST-PPSSBB and E. coli NST-PPSSBN showing the expression of enzymes for the conversion of 6 to (R)- and (S)-1a.
Lane M, protein ladder; Lane C, E. coli NST74(DE3); Lane 15, E. coli NST-PPSSBB; Lane 16, E. coli NST-PPSSBN. (c)
Synthesis of (R)- and (S)-1a from 20 g/L 8 or 9 by E. coli NST-PPSSBB and E. coli NST-PPSSBN, respectively via
fermentation with 50 mL M9 medium containing 8 or 9 and 10 mL n-hexadecane at 25°C for 24 h. Experiment was
performed in duplicates with error bars showing ± SD.
Design of cascade biotransformations and
development of growing-cells bioprocess to
convert renewable feedstocks glucose 8 and
glycerol 9 to (R)- and (S)-2-phenylglycinol 1a
bioproduction of (R)- and (S)-1a from renewable
feedstocks such as 8 and 9.
Production of (R)- and (S)-2-phenylglycinol 1a
from renewable feedstocks glucose 8 and glycerol
9
by coupling two strains expressing L-
E. coli naturally produces L-phenylalanine 6 by
fermentation of renewable feedstocks such as glucose
8 and glycerol 9 through Shikimate pathway.[13b, 19]
The natural L-phenylalanine biosynthesis pathway
was combined with the above developed artificial
enzyme cascade of converting 6 to (R)- or (S)-1a, to
transform 8 and 9 to (R)- and (S)-1a, respectively. As
the biosynthesis of 6 is strictly regulated in E. coli by
feedback regulation[20], we engineered E. coli NST74
phenylalanine biosynthesis pathway and the
artificial cascade
The fermentative synthesis of (R)- and (S)-1a from
sugars 8, 9 could burden the cells due to
overexpression of several enzymes in a single strain.
In addition, the supplementation of organic phase to
growing cells and the accumulation of reaction
intermediates could inhibit the cell growth thus
reducing the fermentative synthesis of (R)- and (S)-1a.
To overcome these issues, we coupled fermentation-
biotransformation,[13a, 13b, 21] to fermentatively produce
6 from 8 or 9 and sequentially biotransform 6 to (R)-
and (S)-1a, respectively.
(DE3)
containing
natural
L-phenylalanine
biosynthesis pathway for the overproduction of 6
[13b, 18]
from 8 or 9 with relaxed feedback regulation.
This strain was used as the host. The plasmids
pACYC-PAL-PAD, pCDF-SMO-StEH, pET-BDHA-
AlaDH, pRSF-BmTA and pACYC-PAL-PAD,
pCDF-SMO-StEH, pET-BDHA- AlaDH, pRSF-
NfTA were transformed into E. coli NST74 (DE3) to
produce E. coli NST-PPSSBB and E. coli NST-
PPSSBN, respectively (Figure 5a). SDS-PAGE
Fermentative production of L-phenylalanine 6 from
sugars 8, 9 was performed by E. coli NST-Phe to
reach high concentrations of 6, followed by the one-
pot biotransformation of 6 to (R)- and (S)-1a by the
analysis was performed with the crude cell lysate of E. resting cells of E. coli-PPSSBB and E. coli-PPSSBN,
coli NST-PPSSBB and E. coli NST-PPSSBN (Figure
5b). The expression of all seven enzymes in the
artificial cascade were confirmed in E. coli NST-
PPSSBB and E. coli NST-PPSSBN.
The fermentation of 8 or 9 to (R)- and (S)-1a was
performed with E. coli NST-PPSSBB and E. coli
NST-PPSSBN, respectively, in 50 mL M9 media
containing 10 g/L 8 or 9 and 10 mL n-hexadecane at
25°C for 24 h. E. coli NST-PPSSBB produced 123
and 178 mg/L (R)-1a from glucose 8 and glycerol 9,
respectively, while E. coli NST-PPSSBN produced
110 and 151 mg/L of (S)-1a from 8 and 9,
respectively (Figure 5c). This is the first report on the
respectively (Figure 6a). E. coli NST-Phe
overexpressing five key enzymes of Shikimate
pathway (AroG*, AroK, YdiB, PheA*, and TyrB)
13b]
was employed to produce 67-80 mM 7.[13a,
Resting cells (20 g cdw/L) of E. coli-PPSSBB and E.
coli-PPSSBN were added to the fermentation broth
with biosynthesized 6, to perform biotransformation
of 6 to (R)- and (S)-1a, respectively. By coupled
fermentation-biotransformation approach, 274 and
384 mg/L of (R)-1a were obtained from glucose 8
and glycerol 9, respectively. For the synthesis of (S)-
1a, the product titer of 274 and 301 mg/L were
achieved from glucose 8 and glycerol 9, respectively
(Figure 6b).
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.202001322
Advanced Synthesis & Catalysis
Figure 6: Coupled fermentation and biotransformation for the conversion of glucose 8 and glycerol 9 to (R)- and (S)-1a
from. (a) E. coli NST-Phe harboring expression plasmid for the expression of key enzymes of Shikimate pathway for
improved production of L-phenylalanine 6 was coupled with E. coli-PPSSBB and E. coli-PPSSBN harboring four
expression plasmids for the expression of PAL, PAD, SMO, StEH, BDHA, AlaDH and BmTA or NfTA for the production
of (R)- or (S)-1a, respectively from 8 and 9. (b) Synthesis of (R)- and (S)-1a by E. coli-PPSSBB and E. coli-PPSSBN,
respectively from biosynthesized 6 produced by the fermentation of 8 and 9 by E. coli NST-Phe. Reaction procedure:
Fermentation was performed using growing cells of E. coli NST-Phe at 37°C for 24-28 h for the production of 67-80 mM
6 from 8 or 9. Biotransformation was performed by E. coli-PPSSBB or E. coli-PPSSBN (20 g cdw/L) at 30°C for 24 h in a
two-phase system containing 5 mL fermented mixture containing biosynthesized 6 (10 mM), phosphate buffer (200 mM,
pH 8) and 5 mL n-hexadecane. Experiment was performed in duplicates with error bars showing ± SD.
engineered E. coli cells expressing the L-
phenylalanine biosynthesis pathway and the artificial
Conclusion
cascade afforded (R)-1a at 123 and 178 mg/L and (S)-
1a at 110 and 151 mg/L from glucose and glycerol,
respectively. These results represent the first
examples of synthesis of (R)-1a and (S)-1a from
renewable feedstocks. Coupling of E. coli strain
expressing L-phenylalanine biosynthesis pathway
with E. coli strain expressing the artificial cascade
enhanced the synthesis of (R)-1a and (S)-1a from
glucose and glycerol, respectively, giving 274 and
384 mg/L (R)-1a and 274 and 301 mg/L (S)-1a,
respectively. The developed strategies are potentially
useful for producing high-value chemicals from
cheap and renewable feedstocks.
We successfully designed and developed novel
artificial enzyme cascades consisting of epoxidation-
hydrolysis-oxidation-transamination for the one-pot
conversion of styrene 2a to enantiopure (R)- and (S)-
2-phenylglycinol 1a, respectively. The engineered E.
coli strains expressing the developed enzyme cascade
converted styrene 2a to (R)-1a in >99% ee at 1015
mg/L and (S)-1a in 91% ee at 315 mg/L, respectively.
The enzyme cascade was successfully applied for the
biotransformation of substituted styrenes 2b-k and
indene 2l to produce the corresponding (R)-
phenylglycinols 1b-k (95->99% ee) and (1R,2R)-1-
amino-2-indanol 1l (>99% ee), respectively, with
>60% conversion of four substituted styrenes. While
the syntheses of (R)-1a and (S)-1a from styrene 2a
were achieved for the first time via the developed
cascade, the conversion of 2b-l to (R)-1b-l represents
the first cascade transformation to produce these
chemicals.
Experimental Section
Recombinant strain construction
E. coli strain expressing enzymes for the cascade
conversion of styrene 2a to (R)- and (S)-2-phenylglycinol
1a
We also successfully designed and developed novel
artificial
enzyme
cascade
consisting
of
The genes coding for enzymes in the cascade (SMO, StEH,
BdhA, AlaDH and BmTA or NfTA) were cloned into three
expression plasmids (pETDuet, pCDFDuet and pRSFDuet)
with different copy numbers and antibiotic resistance. The
expression plasmids were transformed into E. coli T7 to
obtain 12 different recombinant strains (E. coli-SSBB-1-6
and E. coli-SSBN-1-6) with varying expression levels of
the enzymes in the artificial cascade (Table S1, Supporting
information).
decarboxylation-deamination-epoxidation-hydrolysis-
oxidation-transamination for the conversion of bio-
based L-phenylalanine 6 to (R)-1a and (S)-1a,
respectively. The engineered E. coli cells expressing
the cascade were able to transform L-phenylalanine 6
to (R)-1a and (S)-1a in high ee at 576 mg/L and 356
mg/L, respectively, demonstrating the first example
of synthesis of (R)-1a and (S)-1a from an easily
available bio-based chemical.
E. coli strain expressing enzymes for the cascade
conversion of L-phenylalanine 6 to (R)- and (S)-2-
phenylglycinol 1a
E. coli-SSBB-1 and E. coli-SSBN-2 were further
engineered for the cascade conversion of L-phenylalanine
Combination of L-phenylalanine biosynthesis
pathway with the developed artificial enzyme cascade
to produce (R)-1a and (S)-1a was successfully
demonstrated. Fermentative biotransformation with
6 to phenylglycinol 1a. pACYCDuet expressing PAL and
[13e]
PAD from our previous study
was transformed into E.
coli-SSBB-1 (pCDF-SMO-StEH, pET-BDHA- AlaDH,
pRSF-NFTA) and E. coli-SSBN-2 (pCDF-SMO-StEH,
8
This article is protected by copyright. All rights reserved.

10.1002/adsc.202001322
Advanced Synthesis & Catalysis
pET-NFTA, pRSF-BDHA- AlaDH) to obtain E. coli-
PPSSBB (produce (R)-1a) and E. coli-PPSSBN (produce
(S)-1a), respectively.
aqueous phase was adjusted to pH>12 with NaOH (10 M),
followed by extraction with ethyl acetate (3×100 ml). The
organic phase was separated and dried over Na2SO4. After
filtration, the organic phase was subjected to evaporation.
The crude phenylglycinols was purified by flash
E. coli strain expressing enzymes for the production of
(R)- and (S)-2-phenylglycinol 1a from glucose 8 and
glycerol 9
chromatography on
a
silica gel column with
For the synthesis of phenylglycinol 1a from glucose 8 and
glycerol 9, E. coli NST74 (DE3) was used as expression
strain which was previously engineered to produce L-
phenylalanine.[18] pCDF-SMO-StEH, pET-BdhA-AlaDH,
CH2Cl2:MeOH:NH3 (28% aqueous solution) of 100:10:1 as
eluent (Rf ≈ 0.3–0.6 for (R)-1a, 1d, 1e, 1j and 1k). The
collected fraction containing the product was dried over
Na2SO4. After filtration, the organic solvent was removed
by evaporation, and the product was dried under vacuum
overnight to obtain pure product (>98% HPLC) (1a, 81 mg,
59% yield; 1d, 78 mg, 50% yield; 1e, 64 mg, 37% yield; 1j,
49 mg, 32% yield; 1k, 73 mg, 44% yield). The isolated
pRSF-BmTA/NfTA
and
pACYC-PAL-PAD
were
transformed into E. coli NST74 (DE3) to obtain E. coli
NST-PPSSBB (produce (R)-1a) and E. coli NST-PPSSBN
(produce (S)-1a), respectively.
1
product was confirmed by HPLC, H-NMR and 13C-NMR.
1
1a: H NMR (400 MHz, CDCl3) δ 7.44 – 7.21 (m, 5H),
General procedure for the cultivation of E. coli strains
4.04 (dd, J = 8.4, 4.3 Hz, 1H), 3.73 (dd, J = 10.9, 4.3 Hz,
1H), 3.56 (dd, J = 10.9, 8.4 Hz, 1H). 13C NMR (101 MHz,
CDCl3) δ 142.57, 128.84, 127.75, 126.70, 68.02, 57.55. 1d:
1H NMR (400 MHz, CDCl3) δ 7.36 – 7.23 (m, 2H), 7.08 –
6.96 (m, 2H), 4.05 (dd, J = 8.2, 4.3 Hz, 1H), 3.70 (dd, J =
10.8, 4.3 Hz, 1H), 3.53 (dd, J = 10.8, 8.3 Hz, 1H). 13C
NMR (101 MHz, CDCl3) δ 162.36 (d, J = 245.8 Hz),
128.33 (d, J = 8.0 Hz), 139.69, 115.66 (d, J = 21.3 Hz),
The glycerol stock of recombinant E. coli was inoculated
in 2 mL of LB medium with appropriate antibiotics and
grown at 37°C and 220 rpm for 6-8 h. 1 mL of the culture
was added to 50 mL of modified M9 medium with
appropriate antibiotics (50 µg/mL kanamycin, 50 µg/mL
chloramphenicol, 50 µg/mL streptomycin, and 100 µg/mL
ampicillin) in 250 mL flask and grown at 37°C and 220
rpm. Antibiotics were reduced to 50% for strains
containing more than two plasmids. At 2 h, 0.5 mM IPTG
was added and the growth temperature was set at 22°C.
After 16 h of growth at 22°C, the cells were harvested by
centrifugation at 2680 × g for 10 min. The cell pellets were
resuspended in appropriate volume of phosphate buffer and
used for biotransformation.
1
68.00, 56.85. 1e: H NMR (400 MHz, CDCl3) δ 7.40 –
7.14 (m, 4H), 4.03 (dd, J = 7.2, 4.0 Hz, 1H), 3.73 (dd, J =
10.6, 3.6 Hz, 1H), 3.53 (dd, J = 10.4, 8.1 Hz, 1H). 13C
NMR (101 MHz, CDCl3) δ 145.03, 134.72, 130.10, 127.86,
1
126.96, 124.94, 68.03, 57.14. 1j: H NMR (400 MHz,
CDCl3) δ 7.18 (dd, J = 23.7, 8.1 Hz, 4H), 4.01 (dd, J = 8.4,
4.3 Hz, 1H), 3.71 (dd, J = 10.9, 4.3 Hz, 1H), 3.54 (dd, J =
10.8, 8.5 Hz, 1H), 2.33 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 139.53, 137.43, 129.51, 126.59, 68.02, 57.25,
1
Cascade conversion of styrene 2a to (R)- and (S)-2-
phenylglycinol 1a
21.24. 1k: H NMR (400 MHz, CDCl3) δ 7.30 – 7.20 (m,
2H), 6.92 – 6.84 (m, 2H), 4.00 (dd, J = 8.0, 4.1 Hz, 1H),
3.79 (s, 3H), 3.69 (dd, J = 10.8, 4.3 Hz, 1H), 3.59 – 3.48
(m, 1H). 13C NMR (101 MHz, CDCl3) δ 159.03, 127.63,
114.05, 100.00, 67.86, 56.72, 55.30.
Biotransformation was performed at 30°C in a two-phase
system with 2 mL suspension of E. coli-SSBB-1-6 and E.
coli-SSBN-1-6 (20 g cdw/L) in phosphate buffer (200 mM,
pH 8) with n-hexadecane containing 2a (final conc., 10
mM for (R)-1a and 5 mM for (S)-1a). 2% glucose and 100
mM amino donor were added to the reaction at 0 h.
Additional glucose (1%) and amino donor (100 mM) were
added together at 12 and 20 h. The reaction was continued
for 24 h and 48 h for the synthesis of (R)- and (S)-1a,
respectively. (R)- and (S)-1a was present only in the
aqueous phase and 50 µL of sample was taken to analyse
its concentration using reverse phase HPLC.
Cascade conversion of L-phenylalanine 6 to (R)- and
(S)-phenylglycinol 1a
Biotransformation of L-phenylalanine 6 using E. coli-
PPSSBB and E. coli-PPSSBN was performed at 30°C for
24 h for the synthesis of (R)- and (S)-1a, respectively.
Reaction was performed in a two-phase system with 5 mL
suspension of E. coli-PPSSBB or E. coli-PPSSBN (20 g
cdw/L) in phosphate buffer (200 mM, pH 8) with 1 mL n-
hexadecane. 10 mM 6, 2% glucose and 100 mM
NH3/NH4Cl were added to the reaction at 0 h. Additional
glucose (1%) and NH3/NH4Cl (100 mM) were added
together at 12 h. At 24 h, 50 µL of the aqueous phase was
collected to quantify the concentration of (R)- or (S)-1a
using reverse phase HPLC.
General procedure for the cascade conversion of
styrenes 2b-l to (R)-2-phenylglycinols 1b-l
Biotransformation was performed at 30°C in a two-phase
system with 2 mL suspension of E. coli-SSBB-1 in
phosphate buffer (200 mM, pH 8) with 0.4 mL n-
hexadecane containing 2b-1 (final conc., 5-10 mM). 2%
glucose and 100 mM amino donor were added to the
reaction at 0 h. Additional glucose (1%) and amino donor
(100 mM) were added together at 12 and 20 h. After 48 h
of biotransformation, (R)-1b-l were present only in the
aqueous phase and their concentration were analysed using
reverse phase HPLC.
Fermentation of glucose 8 and glycerol 9 to produce
(R)- or (S)-2-phenylglycinol 1a
E. coli NST-PPSSBB and E. coli NST-PPSSBN were
inoculated, respectively in 2 mL LB medium containing
appropriate antibiotics and grown at 37oC, 220 rpm for 6-8
h. 1 mL of the culture of E. coli NST-PPSSBB or E. coli
NST-PPSSBN was added to 50 mL of modified M9
medium with 20 g/L 8 or 9, respectively, and appropriate
antibiotics in 250 mL flask. After 2 h of growth at 37°C,
0.1 mM IPTG and 10 mL n-hexadecane was added and the
growth temperature was set at 25°C. At 24 h, 50 µL of the
aqueous phase was collected to quantify the concentration
of (R)- or (S)-1a using reverse phase HPLC.
Preparative biotransformation of styrenes (2a, 2d, 2e,
2j, 2k) to produce (R)-2-phenylglycinols (1a, 1d, 1e, 1j,
1k)
Preparative biotransformation was performed at 30°C in a
two-phase system with 100 mL suspension of E. coli-
SSBB-1 (25 g cdw/L) in phosphate buffer (200 mM, pH 8)
with 20 mL n-hexadecane containing 50 mM of 2a, 2d, 2e,
2j and 2k. 2% glucose and 100 mM NH3/NH4Cl were
added to the reaction at 0, 16 and 24 h. After 48 h of
biotransformation, the aqueous phase was separated by
centrifugation at 4200 × g for 10 min. The aqueous phase
was saturated with NaCl, adjusted to pH<2 with HCl (10
M), and washed with ethyl acetate (2×30 ml) to remove
trace n-hexadecane and other organic impurities. The
Coupled fermentation-biotransformation for the
production of (R)- or (S)-2-phenylglycinol 1a from
glucose 8 or glycerol 9
Fermentative production of L-phenylalanine 6 from 8 or 9
by E. coli NST-Phe was performed as mentioned
[13b]
previously
, to achieve 67-80 mM 6 and the
fermentation culture containing biosynthesized 6 and E.
9
This article is protected by copyright. All rights reserved.

10.1002/adsc.202001322
Advanced Synthesis & Catalysis
coli NST-Phe cells was stored at 4 °C or used directly in
biotransformation. 5 mL of reaction mixture was prepared
with fermentation broth containing biosynthesized 6 (final
conc. 10 mM), phosphate buffer (200 mM, pH 8), 2%
glucose, 100 mM NH3/NH4Cl. Resting cells of E. coli
NST-PPSSBB or E. coli NST-PPSSBN (20 g cdw/L) and 1
mL of n-hexadecane were added and biotransformation
was performed at 30°C, 220 rpm. Additional glucose (1%)
and NH3/NH4Cl (100 mM) were added together at 12 h. At
24 h, 50 µL of the aqueous phase was collected to quantify
the concentration of (R)- or (S)-1a using reverse phase
HPLC.
Catal. 2018, 1, 12-22. e) B. Hauer, ACS Catal. 2020,
10, 8418-8427.
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L. Wei, J.-H. Xu, J. Biotechnol. 2019, 290, 24-32.
[9] A. Nobili, F. Steffen‐ Munsberg, H. Kohls, I. Trentin,
C. Schulzke, M. Höhne, U. T. Bornscheuer,
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Acknowledgements
This research is supported by the National Research Foundation
(NRF) Singapore under its Advanced Manufacturing and
Engineering Individual Research Grant (Project No.
A1783c0014) and administered by the Agency for Science,
Technology and Research.
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