One-Pot Cascade Biotransformation for Efficient Synthesis of Benzyl Alcohol and Its Analogs

Article abstract of DOI:10.1002/asia.201901680

Benzyl alcohol is a naturally occurring aromatic alcohol and has been widely used in the cosmetics and flavor/fragrance industries. The whole-cell biotransformation for synthesis of benzyl alcohol directly from bio-based L-phenylalanine (L-Phe) was herein explored using an artificial enzyme cascade in Escherichia coli. Benzaldehyde was first produced from L-Phe via four heterologous enzymatic steps that comprises L-amino acid deaminase (LAAD), hydroxymandelate synthase (HmaS), (S)-mandelate dehydrogenase (SMDH) and benzoylformate decarboxylase (BFD). The subsequent reduction of benzaldehyde to benzyl alcohol was achieved by a broad substrate specificity phenylacetaldehyde reductase (PAR) from Solanum lycopersicum. We found the designed enzyme cascade could efficiently convert L-Phe into benzyl alcohol with conversion above 99%. In addition, we also examined L-tyrosine (L-Tyr) and m-fluoro-phenylalanine (m-f-Phe) as substrates, the cascade biotransformation could also efficiently produce p-hydroxybenzyl alcohol and m-fluoro-benzyl alcohol. In summary, the developed biocatalytic pathway has great potential to produce various high-valued fine chemicals.

Full text of DOI:10.1002/asia.201901680

CHEMISTRY
AN ASIAN JOURNAL
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Accepted Article
Title: One-Pot Cascade Biotransformation for Efficient Synthesis of
Benzyl Alcohol and Its Analogs
Authors: Lijun Liu, Yuling Zhu, Yufen Chen, Huiyu Chen, Cong Fan,
Qiwen Mo, and Jifeng Yuan
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10.1002/asia.201901680
Chemistry - An Asian Journal
One-Pot Cascade Biotransformation for Efficient Synthesis of
Benzyl Alcohol and Its Analogs
Lijun Liu,[a] Yuling Zhu,[a] Yufen Chen,[a] Huiyu Chen,[a] Cong Fan,[a] Qiwen Mo,[a] and Jifeng Yuan*[a]
Abstract: Benzyl alcohol is a naturally occurring aromatic alcohol and
has been widely used in the cosmetics and flavor/fragrance industries.
The whole-cell biotransformation for synthesis of benzyl alcohol
directly from bio-based L-phenylalanine (L-Phe) was herein explored
using an artificial enzyme cascade in Escherichia coli. Benzaldehyde
was first produced from L-Phe via four heterologous enzymatic steps
that comprises L-amino acid deaminase (LAAD), hydroxymandelate
synthase (HmaS), (S)-mandelate dehydrogenase (SMDH) and
benzoylformate decarboxylase (BFD). The subsequent reduction of
benzaldehyde to benzyl alcohol was achieved by a broad substrate
specificity phenylacetaldehyde reductase (PAR) from Solanum
lycopersicum. We found the designed enzyme cascade could
efficiently convert L-Phe into benzyl alcohol with conversion above
99%. In addition, we also examined L-tyrosine (L-Tyr) and m-fluoro-
phenylalanine (m-f-Phe) as substrates, the cascade biotransformation
could also efficiently produce p-hydroxybenzyl alcohol and m-fluoro-
benzyl alcohol. In summary, the developed biocatalytic pathway has
great potential to produce various high-valued fine chemicals.
In this study, we sought to engineer Escherichia coli as a
microbial cell factory[11] for the production of benzyl alcohol and
its analogs by development of an efficient enzyme cascade
(Scheme 1). In particular, L-phenylalanine (L-Phe) is converted to
phenylpyruvate (PPA) via L-amino acid deaminase (LAAD)
mediated deamination process, which is further converted to
benzaldehyde by cascade reactions of hydroxymandelate
synthase (HmaS), (S)-mandelatedehydrogenase (SMDH) and
benzoylformate decarboxylase (BFD). In the last step,
benzaldehyde is reduced by phenylacetaldehyde reductase
(PAR) to benzyl alcohol. From the literature, benzyl alcohol was
synthesized from glucose by a non-natural pathway (HmaS-
SMDH-BFD) that utilizes the endogenous PPA precursor[12]
.
However, the native transaminase encoded by TyrB[13] competes
the PPA precursor for L-Phe biosynthesis and eventually limits
the final product yield. In addition, de novo synthesis of benzyl
alcohol might be limited by the product toxicity to the host.
Therefore, there is a pressing need to develop a whole cell
biotransformation approach for efficient synthesis of benzyl
alcohol.
Benzyl alcohol is generally regarded as a safe and effective
solvent[1] and has been widely used in the production of industrial
chemicals including inks, paints, glues, and hardening products[2].
Besides, it is also commonly used as perfume fixative[3] and
preservative in the food, cosmetic and pharmaceutical
industries[4] because of its floral scent[5] and antimicrobial activity[6].
Benzyl alcohol can be directly extracted from edible fruits and tea
leaves, as well as from essential oils of jasmine and hyacinth[7].
However, benzyl alcohol in its natural sources usually suffers low
concentration, which leads to low product yields and high
extraction costs. In addition, conventional chemical synthesis of
benzyl alcohol is mostly achieved from petroleum-derived
feedstocks and this method involves toxic reagents and harsh
conditions[8] which is considered as an unsustainable approach.
In comparison, biotransformation could efficiently catalyze
multistep reactions and convert cheap and renewable feedstocks
into bulk and fine chemicals without the use of toxic reagents[9].
Therefore, biotransformation provides an economic and
environmentally-friendly alternative to traditional chemical
Scheme 1. Artificial biosynthesis pathway of 1a-1c to 6a-6c by enzyme
cascades. LAAD, L-amino acid deaminase; HmaS, hydroxymandelate
synthase; SMDH, (S)-mandelate dehydrogenase; BFD, benzoylformate
decarboxylase; PAR, phenylacetaldehyde reductase.
LAAD is a FAD-containing enzyme, which has broad substrate
specificities to catalyze the deamination of many L-amino acids
into the corresponding α-keto acids[14]. Hou et al found that LAAD
from Proteus mirabilis KCTC 2566 could efficiently catalyze the
deamination of L-Phe into PPA with a conversion ratio of 100%[15]
.
Besides, LAAD has been used for producing 2-phenylethanol[16]
and phenyllactic acid[17] in engineered E. coli. In order to improve
the deamination of L-Phe for benzyl alcohol production, we
decided to introduce LAAD from P. mirabilis (PmLAAD) into our
recombinant E. coli. Although HmaS from Amycolatopsis
orientalis utilizes p-hydroxyphenylpyruvate as its natural
substrate[18], it also displayed great activity towards converting
PPA into (S)-mandelic acid[19]. To examine the effect of PmLAAD
on the deamination step, we compared the yield of (S)-mandelic
acid from strains transformed with plasmid pET-PmLAAD-HmaS
methods[10]
.
[a]
[*]
Lijun Liu, Yuling Zhu, Yufen Chen, Huiyu Chen, Cong Fan, Qiwen
Mo, Jifeng Yuan
State Key Laboratory of Cellular Stress Biology
School of Life Sciences, Xiamen University, Fujian, PR China,
361102
or pET-HmaS. As shown from Figure S1, after 24
h
biotransformation at 30°C, the recombinant E. coli with additional
expression of PmLAAD yielded 40.1 mM (S)-mandelic acid from
50 mM L-Phe, whereas strain harbouring the plasmid pET-HmaS
only gave 4.3 mM product, suggesting that PmLAAD did promote
the deamination of L-Phe thus favor efficient synthesis of PPA
derived (S)-mandelic acid.
Corresponding author: Prof. Jifeng Yuan
State Key Laboratory of Cellular Stress Biology
School of Life Sciences, Xiamen University, Fujian, PR China,
361102. E-mail: jfyuan@xmu.edu.cn
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/asia.201901680
Chemistry - An Asian Journal
biotransformation by PmLAAD-HmaS-SMDH-BFD-PAR enzyme
cascade has great potential for industrial conversion of L-Phe into
benzyl alcohol.
Figure1. Biotransformation of L-Phe into benzyl alcohol. (A) Recombinant
E. coli containing three plasmids for overexpressing five enzymes. PmLAAD
from P. mirabilis; HmaS from A. orientalis; SMDH from P. putida KT2440; BFD
from P. putida KT2440; PAR from S. lycopersicum. (B) Cascade
biotransformation of L-Phe (10 mM) into benzyl alcohol with the resting cells of
E. coli MG1655 RARE (10 g cdw L–1, CDW=cell dry weight) at 30°C for 24 h.
The reactions were conducted in KP buffer (200 mM, pH 8.0, 2% glucose). All
experiments were carried out in triplicate and the data are the mean values with
standard deviation.
Figure 2. Biotransformation of L-Tyr and m-f-Phe into benzyl alcohol
analogs. (A) Cascade biotransformation of L-Tyr (5 mM) into p-hydroxybenzyl
alcohol with the resting cells of E. coli MG1655 RARE (10 g cdw L–1). (B)
Cascade biotransformation of m-f-Phe (10 mM) into m-fluoro-benzyl alcohol
with the resting cells of E. coli MG1655 RARE (10 g cdw L–1). The reactions
were conducted in KP buffer (200 mM, pH 8.0, 2% glucose) at 30°C for 24 h.
All experiments were carried out in triplicate and the data are the mean values
with standard deviation.
Upon confirming (S)-mandelic acid production from the
recombinant E. coli, we next sought to introduce a second
enzymatic module that contains SMDH and BFD[20] from
Pseudomonas putida for converting (S)-mandelic acid into
benzaldehyde[21]. Finally, benzaldehyde was further reduced to
benzyl alcohol by a third enzymatic module mediated by PAR
from Solanum lycopersicum (Figure 1A). According to literature,
PAR from S. lycopersicum was previously employed to efficiently
reduce 2-phenylacetaldehyde into 2-phenylethanol[22] and it also
has broad substrate specificities on benzaldehyde and
cinnamaldehyde[23]. In the present study, we chose E. coli
MG1655 (RARE) strain with majority of endogenous alcohol
dehydrogenases (ADHs) and aldo-keto reductases (AKRs) being
deleted[24] for benzyl alcohol productions. As can been seen from
Figure 1B, 10 mM L-Phe was completely converted to benzyl
alcohol after 24 h biotransformation at 30°, suggesting PAR has
sufficient activity towards benzaldehyde substrate. In the process
of biocatalysis, the substrate of L-Phe was completely
deaminated and negligible amounts of byproducts were
accumulated (Figure S2), indicating that the whole cell
To further explore the compatibility of our established
enzymatic cascade for other aromatic alcohol products, we
employed the same enzymatic cascade for synthesizing p-
hydroxybenzyl alcohol, a useful compound with great commercial
values (Table 1). For example, p-hydroxybenzyl alcohol is the
active phenolic ingredient of Gastrodia elata[25] and it also has
various biological functions including anti-oxidant, anti-
inflammatory, anti-tumor and anti-apoptosis activities[26]. As
shown from Figure 2A, biotransformation studies revealed that L-
Tyr was rapidly converted to p-hydroxybenzyl alcohol, reaching a
concentration of 4.68 mM (580.98 mg L–1) p-hydroxybenzyl
alcohol with 93.6% conversion after 24 h reaction. In addition,
HPLC revealed good purity of p-hydroxybenzyl alcohol (Figure
S3). Therefore, the whole cell biotransformation developed in the
present study offers an effective approach for synthesizing p-
hydroxybenzyl alcohol.
Table 1. The summary of bioproduction of benzyl alcohol and its analogs using recombinant E. coli [a]
Product
Structure
Applications
Substrate conc.
(mM) [b]
Product conc.
(mM) [c]
Yield (mg L–1
1114.92
)
Conv. (%) [c]
>99.0
6a
Mainly used as perfume fixative[5], oil solvent
and plasticizer. It also used in the manufacture
10
10.31
of perfume, soap, medicine and dyestuff[12a]
.
6b
6c
An important pharmaceutical intermediate,
which is mainly used in the synthesis of drugs
5
4.68
580.98
633.17
93.6
and spices[26-27]
,
e.g. for synthesis of
Gastrodin[28]
Key intermediate in organic synthesis[29]
.
5
5.02
>99.0
[a] MG1655 RARE strain is a strain derived from MG1655 (DE3) with the majority of alcohol dehydrogenases deleted.
[b] Reaction conditions: 1a–1c (5–10 mM) and E. coli MG1655 RARE cells (10 g cdw L–1) in the KP buffer (200 mM, pH 8.0) containing 2% glucose at 30°C.
[c] Determined by HPLC analysis.
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10.1002/asia.201901680
Chemistry - An Asian Journal
Furthermore, we also explored the versatility of our designed
biocatalytic pathway towards synthesis of non-natural aromatic
alcohol like m-fluoro-benzyl alcohol, which represents an
important intermediate in organic synthesis (Table 1). As shown
in Figure 2B, when the recombinant E. coli was fed with 10 mM of
m-f-DL-Phe, approximately 5.02 mM m-fluoro-benzyl alcohol
could be obtained. As PmLAAD only deaminates the L-form of
substrate, the D-form of m-f-Phe remained unreacted (Figure S4).
Therefore, our results indicated that the artificial enzyme cascade
could effectively revolve the racemic mixture of m-f-DL-Phe and
convert m-f-L-Phe into the corresponding product.
alcohol and p-hydroxybenzyl alcohol directly from simple carbon
source[30]. However, in situ product removal techniques have to
be developed to address the product toxicity issues. Furthermore,
the reported enzyme cascade might be applicable or amendable
for converting other amino acids such as L-tryptophan into
anticancer drug indole-3-carbinol[31]
. In a word, our study
demonstrated the capability of whole cell biotransformation for
efficient synthesis of benzyl alcohol and other fine chemicals in a
green and sustainable manner.
In summary, an efficient enzymatic cascade was constructed
by co-expressing LAAD, HmaS, SMDH, BFD and PAR in E. coli.
The recombinant E. coli could efficiently synthesize benzyl
alcohol and its analogs with high yield (Table 1), which would
greatly reduce the cost for future product separation and
purification. In addition, LAAD coupled HmaS also efficiently
produced (S)-mandelic acid, which is a useful precursor to various
drugs. In the future, the established enzymatic cascade might also
combine with the native aromatic amino acid biosynthetic
pathway of a host microorganism for de novo synthesis of benzyl
Acknowledgements
We acknowledge financial support from Xiamen University under
grant no. 0660-X2123310, 0660/Z0211701 and ZhenSheng
Biotech, China.
Keywords: Biotransformation • benzyl alcohol • aromatic amino
acids • biocatalysis
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