Cascade Biocatalysis for Sustainable Asymmetric Synthesis: From Biobased l-Phenylalanine to High-Value Chiral Chemicals

Article abstract of DOI:10.1002/anie.201606235

Sustainable synthesis of useful and valuable chiral fine chemicals from renewable feedstocks is highly desirable but remains challenging. Reported herein is a designed and engineered set of unique non-natural biocatalytic cascades to achieve the asymmetric synthesis of chiral epoxide, diols, hydroxy acid, and amino acid in high yield and with excellent ee values from the easily available biobased l-phenylalanine. Each of the cascades was efficiently performed in one pot by using the cells of a single recombinant strain over-expressing 4–10 different enzymes. The cascade biocatalysis approach is promising for upgrading biobased bulk chemicals to high-value chiral chemicals. In addition, combining the non-natural enzyme cascades with the natural metabolic pathway of the host strain enabled the fermentative production of the chiral fine chemicals from glucose.

Full text of DOI:10.1002/anie.201606235

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201606235
German Edition: DOI: 10.1002/ange.201606235
Biotransformations
Cascade Biocatalysis for Sustainable Asymmetric Synthesis: From
Biobased l-Phenylalanine to High-Value Chiral Chemicals
+
+
Yi Zhou , Shuke Wu , and Zhi Li*
Abstract: Sustainable synthesis of useful and valuable chiral
fine chemicals from renewable feedstocks is highly desirable
but remains challenging. Reported herein is a designed and
engineered set of unique non-natural biocatalytic cascades to
achieve the asymmetric synthesis of chiral epoxide, diols,
hydroxy acid, and amino acid in high yield and with excellent
ee values from the easily available biobased l-phenylalanine.
Each of the cascades was efficiently performed in one pot by
using the cells of a single recombinant strain over-expressing 4–
Scheme 1. General concept of sustainable synthesis of high-value non-
natural chiral chemicals from biobased chemicals or biomass by using
non-natural enzyme cascades.
1
0 different enzymes. The cascade biocatalysis approach is
promising for upgrading biobased bulk chemicals to high-
value chiral chemicals. In addition, combining the non-natural
enzyme cascades with the natural metabolic pathway of the
host strain enabled the fermentative production of the chiral
fine chemicals from glucose.
Amino acids are attractive biobased chemicals for sus-
tainable synthesis of chiral molecules since they are currently
produced by fermentation in large amounts and at low cost.
[6]
One-pot chemical transformations of amino acids into chiral
chemicals are still rare. In contrast, cascade biocatalysis, an
emerging green tool for asymmetric synthesis with high
[7,8]
S
ustainable manufacturing of chemicals from renewable
selectivity and no intermediate separation,
could provide
feedstocks is attracting increasing attention because of the oil
new asymmetric synthesis of chiral chemicals from amino
acids. For instance, l-amino acids were converted into the
corresponding chiral a-hydroxy acids by replacing an amino
group with a hydroxy group via a two-step biocascade, which
[
1]
depletion and global climate change. Significant progress
has been made in chemical or enzymatic conversion of
biomass into bulk chemicals.
[
2]
[3,4]
The advances of metabolic
[
9]
engineering and synthetic biology have enabled the fermen-
tation of (hemi)cellulose-derived sugars to produce a variety
is advantageous over the chemical method. Herein we
report new types of cascade biocatalysis consisting of 3–8
enzymatic steps for the enantioselective conversion of l-
phenylalanine [(S)-1] into chiral epoxide, diols, hydroxy acid,
and amino acid (Scheme 2), for which there are no chemical
counterparts; and the one-pot synthesis of these chiral
compounds, which are useful and valuable synthons and
pharmaceutical intermediates (see Table S1 in the Supporting
Information), from l-phenylalanine or glucose by using the
developed cascades.
[5]
of biobased bulk chemicals. However, the fermentative
production of non-natural high-value fine chemicals still faces
many challenges, including the lack of efficient pathways
towards the non-natural chemicals. We envision a feasible and
potentially general approach for green, efficient, and sustain-
able production of non-natural chiral fine chemicals by
developing novel types of one-pot non-natural biocatalytic
cascades to convert a selected easily available biobased bulk
chemical into the target fine chemicals (Scheme 1). The
selected bulk chemical could be a primary cell metabolite,
thus the designed enzyme cascades to convert this metabolite
can be integrated with the native metabolic pathway of the
host microorganism to achieve the fermentative production of
high-value chiral chemicals directly from sugar.
Specifically, the novel biocatalytic cascades to synthesize
five chiral molecules from l-phenylalanine [(S)-1; Scheme 2]
were designed as follows. Cascade 1: (S)-1 undergoes deam-
ination/decarboxylation to styrene (3) by using phenylalanine
[
10]
ammonia lyase (PAL)
and phenylacrylic acid decarbox-
[11]
ylase (PAD), followed by S-selective epoxidation of 3 to
give (S)-styrene oxide [(S)-4] with styrene monooxygenase
[
+]
[+]
[12]
[
*] Dr. Y. Zhou, Dr. S. Wu, Prof. Dr. Z. Li
(SMO).
Cascades 2 and 3: combining cascade 1 and
Synthetic Biology for Clinical and Technological Innovation (SynCTI)
Life Sciences Institute, National University of Singapore
regioselective hydrolysis of (S)-4 with epoxide hydrolase
from potato (StEH) or from Sphingomonas HXN-200
2
8 Medical Drive, Singapore 117456 (Singapore)
[13]
(
SpEH) gives rise to (R)- or (S)-1-phenylethane-1,2-diol
[
+]
Dr. S. Wu, Prof. Dr. Z. Li
[
(R)-5 or (S)-5]. Cascade 4: extension of cascade 3 by
regioselective double oxidation of (S)-5 using alcohol dehy-
drogenase (AlkJ) and aldehyde dehydrogenase
EcALDH) produces (S)-mandelic acid [(S)-7]. Cascade 5:
addition of oxidation–transamination to cascade 4 by using
hydroxymandelate oxidase (HMO), a-transaminase
EcaTA), glutamate dehydrogenase (GluDH), and catalase
Department of Chemical and Biomolecular Engineering
National University of Singapore
4
Engineering Drive 4, Singapore 117585 (Singapore)
[14]
(
E-mail: chelz@nus.edu.sg
+
[
] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
(
[14]
1002/anie.201606235.
(CAT), forms (S)-phenylglycine [(S)-9].
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1
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Scheme 2. New types of cascade biocatalysis for one-pot transformation of biobased l-phenylalanine [(S)-1] into (S)-styrene oxide [(S)-4], (S)- and
(R)-1-phenylethane-1,2-diol [(S)-5 and (R)-5], (S)-mandelic acid [(S)-7], and (S)-phenylglycine [(S)-9].
To engineer cascade 1, AtPAL2 from Arabidopsis thali-
[
10]
ana was selected for the deamination of (S)-1 (see Fig-
[
11]
ure S1), and AnPAD (fdc1 & pad1 ) from Aspergillus niger
were chosen for the decarboxylation of cinnamic acid (2; see
Figure S2). Both AtPAL2 and AnPAD were then cloned
together as PAL-PAD on compatible plasmids (see Fig-
[12]
ure S3). Combinations of PAL-PAD and SMO
plasmids
gave E. coli strains (LZ01-LZ12; Table S2) for cascade 1 (see
Figure S4). Similarly, combining PAL-PAD with either SMO-
[13]
[14]
StEH
or SMO-SpEH
plasmids gave E. coli strains
(
LZ13-LZ24; Table S2) for cascade 2 (see Figure S5) or E.
coli strains (LZ25-LZ36; Table S2) for cascade 3 (see Fig-
ure S6). For cascade 4, E.coli strains (LZ37-LZ60; Table S2)
[
14]
containing PAL-PAD, SMO-SpEH, and AlkJ-EcALDH
plasmids were constructed (see Figure S7). For cascade 5, E.
coli strains (LZ61-LZ84; Table S2) containing PAL-PAD,
SMO-SpEH, AlkJ-EcALDH, and HMO-EcaTA-GluDH-
Figure 1. E. coli (LZ76) containing four recombinant plasmids and
over-expressing 10 enzymes for converting (S)-1 into (S)-9.
[14]
CAT plasmids were engineered (see Figure S8). The best
strains for the cascade biotransformations 1–5 are given in
Table 1. Among them, E. coli (LZ76) containing four
plasmids with 12 genes for cascade 5 catalyzes 8 reaction
steps (Figure 1).
The E. coli (pRSF-PAL-PAD) and the best five strains for
each cascade grew well in M9 medium, and the resting cells
harvested at late exponential phase were used for the
corresponding biotransformation in a two-phase system
containing KP buffer and n-hexadecane (1:1). The time
courses of the biotransformations are shown in Figure 2. E.
conversion at 10 hours (Figure 2b). Bioconversion of 120 mm
of (S)-1 with E. coli (LZ20) and E. coli (LZ26) for 10 hours
À1
gave 109 mm (15 gL ) of (R)-5 in 96% ee and 91%
À1
conversion (Figure 2c) and 107 mm (15 gL ) of (S)-5 in
97% ee and 89% conversion, respectively (Figure 2d).
Reaction of 120 mm of (S)-1 with E. coli (LZ37) afforded
À1
100 mm of (S)-7 at 10 hours and 110 mm (17 gL ) of (S)-7 at
24 hours in 99% ee (92% conversion; Figure 2e). In all these
biotransformations, nearly no intermediates or byproducts
remained in the final reaction mixtures.
coli (pRSF-PAL-PAD) efficiently converted 150 mm of 1 into
À1
1
39 mm (14 gL ) 3 in 92% conversion at 5 hours, with no
Bioconversion of 40 mm of (S)-1 with E. coli (LZ76) gave
À1
accumulation of the intermediate or byproducts (Figure 2a).
34 mm (5.1 gL ) of (S)-9 in greater than 99% ee and 85%
In comparison, fermentative production of 3 gave a much
lower concentration, even if designed micelles were used.
conversion at 24 hours. Thus, the very challenging eight-step
biocatalytic cascade was successfully achieved with a single
recombinant strain coexpressing 10 enzymes. During the
bioconversion, significant amounts of (S)-7 accumulated
before the addition of new portions
[15]
Biotransformation of 140 mm of (S)-1 with E. coli (LZ03)
À1
produced 125 mm (15 gL ) of (S)-4 in 99% ee and 89%
Table 1: Engineered recombinant E. coli strains for the designed cascade biotransformations.
of NH /NH Cl and glucose at
3 4
[
a]
[b]
10 hours. Thus, the HMO-EcaTA-
GluDH-CAT part of the cascade is
less efficient than the PAL-PAD-
SMO-SpEH-AlkJ-EcALDH part.
Choosing other more active
enzymes for the cascade from (S)-
7 to (S)-9 could further improve the
efficiency and productivity of cas-
cade 5.
Strain
Recombinant plasmids
Reactions
Conv. [%]
E. coli (LZ03)
E. coli (LZ20)
E. coli (LZ26)
E. coli (LZ37)
E. coli (LZ76)
pACYC-PAL-PAD, pRSF-SMO
pET-PAL-PAD, pCDF-SMO-StEH
pACYC-PAL-PAD, pET-SMO-SpEH
pACYC-PAL-PAD, pCDF-SMO-SpEH, pET-AlkJ-EcALDH
pET-PAL-PAD, pCDF-SMO-SpEH, pRSF-AlkJ-EcALDH,
pACYC-HMO-EcaTA-GluDH-CAT
(S)-1 to (S)-4
(S)-1 to (R)-5
(S)-1 to (S)-5
(S)-1 to (S)-7
(S)-1 to (S)-9
89
91
89
92
85
[
a] E. coli T7 expression strains harboring the recombinant plasmids. [b] Plasmids pACYC, pCDF, pET,
and pRSF were constructed on empty pACYCDuet-1, pCDFDuet-1, pETDuet-1, and pRSFDuet-1,
respectively.
2
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Figure 2. Time curves of cascade biotransformation of l-phenylalanine into various chemicals. a) (S)-1 to 3 with E. coli (pRSF-PAL-PAD).
b) (S)-1 to (S)-4 with E. coli (LZ03). c) (S)-1 to (R)-5 with E. coli (LZ20). d) (S)-1 to (S)-5 with E. coli (LZ26). e) (S)-1 to (S)-7 with E. coli (LZ37).
À1
f) (S)-1 to (S)-9 with E. coli (LZ76). All biotransformations were performed with resting cells (15 gcdwL ) in a two-phase system containing KP
buffer (200 mm, pH 8, 0.5–2% glucose) and n-hexadecane (1:1) at 250 rpm and 308C. In the biotransformation shown in (f), 200 mm NH /NH Cl
3
4
and 2% glucose was added at 10 h. Concentrations are normalized to the volume of aqueous solution. Error bars represent the standard
deviation of triplicated biotransformations.
Table 2: Preparation of (S)-4, (R)-5, (S)-5, (S)-7, and (S)-9 by cascade
biotransformations of l-phenylalanine [(S)-1]
To further enhance the sustainability of the bioprocesses,
the petro-based n-hexadecane was replaced by ethyl oleate
[
16]
[a]
[b]
[c]
(
EO), which could be produced from biobased oleic acid
Catalyst
Subs. conc.
mm]
Prod.
Yield
[mg]
ee [%]
[
[%]
and ethanol, in the two-phase system. As illustrated in
Table S3, biotransformations of (S)-1 to 3, (S)-4, (R)-5, (S)-
, (S)-7, and (S)-9, afforded similar high conversion (84–96%)
and product ee values (96– > 99%) as those in the two-phase
system using n-hexadecane.
Preparative biotransformations were carried out with the
resting cells of the corresponding strains in KP buffer/EO
10:1) except for the production of (S)-4. The products were
purified by solvent extraction, crystallization, precipitation,
or flash chromatography. As shown in Table 2, (S)-4, (R)-5,
[
d]
E. coli (LZ03)
E. coli (LZ20)
E. coli (LZ26)
E. coli (LZ37)
E. coli (LZ76)
100
100
100
100
40
(S)-4
(R)-5
(S)-5
(S)-7
(S)-9
926
975
1082
1058
376
77
71
78
70
62
99
96
97
99
>99
5
[
a] Reaction was performed in 100 mL KP buffer (200 mm, pH 8, 0.5–2%
À1
glucose) and 10 mL ethyl oleate with resting cells (20 gcdwL ) at
(
2
50 rpm and 308C for 24 h. [b] Yield of isolated product. [c] Determined
by chiral HPLC analysis. [d] 100 mL n-hexadecane was used instead of
0 mL ethyl oleate.
1
(
S)-5, (S)-7, and (S)-9 were isolated in 376–1082 mg (62–78%
yield) with excellent ee values (96– > 99%).
The five recombinant strains contain the natural pathway
from glucose to l-phenylalanine and the engineered non-
natural pathway from l-phenylalanine to the corresponding
chiral chemicals (S)-4, (R)-5, (S)-5, (S)-7, and (S)-9. These
strains grew in M9 medium supplemented with glucose, and
ing the cells using synthetic biology tools. At this moment, the
production of the chiral fine chemicals from the biobased
chemical by cascade biotransformations is much more
practical. In general, this approach is also much simpler and
more flexible for the sustainable production of chiral fine
chemicals with versatile structures and production volumes.
In summary, we designed and constructed novel types of
non-natural biocatalytic cascades for one-pot enantioselective
conversion of biobased l-phenylalanine [(S)-1] into a number
of high-value chiral fine chemicals, with no chemical counter-
parts. The engineering of non-natural biocatalytic cascades
with up to 8 steps of enzymatic reactions, 10 enzymes, and 12
genes was also successfully demonstrated, thus serving as
a showcase of developing highly efficient and complex
(S)-4, (R)-5, (S)-5, (S)-7, and (S)-9 were directly produced in
the culture medium at 0.7–1.6 mm (see Table S4). In compar-
ison, the product concentrations are lower than those from
the cascade biotransformations of (S)-1. Nevertheless, it
opens a new opportunity for sustainable production of the
non-natural chiral fine chemicals by fermentation via inte-
grating the non-natural pathway from a primary metabolite
towards the target non-natural product. We are currently
working on the improvement of the productivity by engineer-
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3
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Angewandte
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engineered whole-cell biocatalysts produced the target chiral
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amino acid (S)-9 in 85–91% conversion, 62–78% yield, and
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Acknowledgments
This work was supported by the Science & Engineering
Research Council of A*STAR, Singapore, through a research
grant (Project No. 1021010026) and the National University
of Singapore through Synthetic Biology for Clinical and
Technological Innovation (SynCTI) program.
Keywords: amino acids · asymmetric synthesis · biocatalysis ·
biomass · enzymes
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Chem. Biomol. Eng. 2013, 4, 259; d) J. Becker, C. Wittmann,
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Received: June 27, 2016
3383; e) J. Nielsen, J. D. Keasling, Cell 2016, 164, 1185.
Published online: && &&, &&&&
4
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Biotransformations
All fine: Asymmetric synthesis of chiral
epoxide, diols, hydroxy acid, and amino
acid in high yield and with excellent
ee value from biobased l-phenylalanine
was achieved with engineered whole-cell
biocatalysts containing designed non-
natural enzyme cascades, respectively.
Fermentative production of the chiral
chemicals from glucose was also dem-
onstrated by combining the non-natural
cascades with the metabolic pathway of
the host.
Y. Zhou, S. Wu, Z. Li*
&&&& — &&&&
Cascade Biocatalysis for Sustainable
Asymmetric Synthesis: From Biobased l-
Phenylalanine to High-Value Chiral
Chemicals
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!