Biosynthesis of Phenylglyoxylic Acid by LhDMDH, a Novel d -Mandelate Dehydrogenase with High Catalytic Activity

Article abstract of DOI:10.1021/acs.jafc.7b05835

d-Mandelate dehydrogenase (DMDH) has the potential to convert d-mandelic acid to phenylglyoxylic acid (PGA), which is a key building block in the field of chemical synthesis and is widely used to synthesize pharmaceutical intermediates or food additives.

Full text of DOI:10.1021/acs.jafc.7b05835

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Article
Biosynthesis of Phenylglyoxylic Acid by LhDMDH, a Novel
D-Mandelate Dehydrogenase with High Catalytic Activity
Cunduo Tang, HongLing Shi, Jian-He Xu, Zhu-Jin Jiao, Fei Liu,
Peng-Ju Ding, Hong-Fei Shi, Lun-Guang Yao, and Yunchao Kan
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05835 • Publication Date (Web): 20 Feb 2018
Downloaded from http://pubs.acs.org on February 22, 2018
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Biosynthesis of Phenylglyoxylic Acid by LhDMDH, a Novel
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D-Mandelate Dehydrogenase with High Catalytic Activity
†
§#*
†#
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†
†
†
CunꢀDuo Tang , HongꢀLing Shi , JianꢀHe Xu , ZhuꢀJin Jiao , Fei Liu , PengꢀJu Ding ,
†
*†
*†
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HongꢀFei Shi , LunꢀGuang Yao and YunꢀChao Kan
†
Henan Provincial Engineering Laboratory of Insect Bioꢀreactor and Henan Key Laboratory
of Ecological Security for Water Source Region of Midꢀline of SouthꢀtoꢀNorth, Nanyang
Normal University, 1638 Wolong Road, Nanyang, Henan 473061, People’s Republic of
China
§
State Key Laboratory of Bioreactor Engineering, East China University of Science and
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Technology, Shanghai 200237, People’s Republic of China
#
These authors contribute equally to this work
*Corresponding Author: Dr. CunꢀDuo Tang, Prof. LunꢀGuang Yao and YunꢀChao Kan
Corresponding Address:
Henan Provincial Engineering Laboratory of Insect Bioꢀreactor, Nanyang Normal
University, 1638 Wolong Road, Nanyang, Henan 473061, People’s Republic of China
Phone: +86ꢀ377ꢀ63525087, Fax: 86ꢀ377ꢀ63525087, Eꢀmail: tcd530@126.com;
kanyunchao@163.com; lunguangyao@163.com
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ABSTRACT
Dꢀmandelate dehydrogenase (DMDH) has the potential of converting Dꢀmandelic acid to
phenylglyoxylic acid (PGA), which is a key building block in the field of chemical synthesis
and is widely used to synthesize pharmaceutical intermediates or food additives. A novel
+
NAD ꢀdependent Dꢀmandelate dehydrogenase was cloned from Lactobacillus harbinensi
(LhDMDH) by genome mining and expressed in Escherichia coli BL21. After being
purified to homogeneity, the oxidation activity of LhDMDH toward Dꢀmandelic acid was
ꢀ1
approximately 1200 U·mg , which was close to four times the activity of the probe.
ꢀ1
ꢀ1
m
Meanwhile, the kcat/K value of LhDMDH was 28.80 S ·mM , which was distinctly higher
than the probe. By coꢀculturing two E. coli strains respectively expressing LhDMDH and
LcLDH, we developed a system for the efficient synthesis of PGA, achieving a 60%
theoretical yield and 99% purity without adding coenzyme or coꢀsubstrate. Our data
supports the implementation of a promising strategy for the chiral resolution of racemic
mandelic acid and the biosynthesis of PGA.
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KEYWORDS: mandelate dehydrogenase, biosynthesis, phenylglyoxylic acid, whole cell
catalysis, green chemistry
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INTRODUCTION
Phenylglyoxylic acid (PGA) plays an important role as a building block in the field of
chemical synthesis and is widely used to synthesize pharmaceutical intermediates or food
1
, 2
additives. Compared with chemical preparation via dehydrogenation of mandelic acid,
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, 3
hydrolysis of benzoyl cyanide, or oxidation of styrene, the microbial synthesis of PGA
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, 4
from mandelic acid provides an ecoꢀfriendly approach to industrialꢀscale production,
which addresses the concerns about the environmental aspects of chemical manufacturing.
Enantioselective conversion of Dꢀmandelic acid to PGA has been reported in Alcaligenes
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,
bronchisepticus, Pseudomonas polycolor, Gibberella fujikuroi, and Pseudomonas putida.
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Dꢀmandelate dehydrogenase (DMDH), a member of the ketopantoate reductase family, has
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, 6, 7
much potential for use in the conversion Dꢀmandelic acid to PGA.
It also has
tremendous potential in the biosynthesis of enantiopure mandelic acids, which are also
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, 9
important intermediates for synthesis of pharmaceutical and natural products.
The
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heterologous expression of DMDH genes from Lactobacillus brevis , Pseudomonas putida
3
and Pseudomonas aeruginosa in E. coli was capable of oxidizing mandelic acid into PGA.
However, the poor activity of these recombinant mandelate dehydrogenases impedes their
industrial application. Genome mining in microbial sequences could be an economical
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strategy to identify novel mandelate dehydrogenases with higher catalytic activity.
In
addition, recombinant DNA techniques can be used to overproduce a desired enzyme in
microorganisms.
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Based on the technoꢀeconomic impact analysis, NAD has to be regenerated from
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NADH for oxidation reactions with dehydrogenases. To address this problem, the systems
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alcohol dehydrogenase (ADH)/ acetone or acetaldehyde,
lactate dehydrogenase
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(LDH)/pyruvate, and NADH oxidase (NOX)/O
are now used universally. Theoretically,
such a system could also be applied to the biosynthesis of PGA by coupled enzymatic
catalysis, as shown in Scheme 1. However, large amounts of these coꢀsubstrates are needed
in the coupled enzymatic catalysis, which can impede their industrial application and make
these methods inefficient and expensive. In addition, complex processes for enzyme
separation can be omitted by utilizing microbial whole cells as a biocatalyst, in addition to
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the supplementary addition of cofactor regeneration enzymes.
Therefore, wholeꢀcell
catalysis is recognized as a most promising approach for the applied biosynthesis of PGA.
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In this work, we identified a novel NAD ꢀdependent Dꢀmandelate dehydrogenase
(LhDMDH) from L. harbinensis and successfully expressed it in E. coli. The purified
recombinant LhDMDH exhibits higher catalytic activity and better kinetic properties than
the probe. Furthermore, we developed a coꢀculture system consisting of two recombinant E.
coli strains, which can efficiently biosynthesize PGA without adding coenzyme or
coꢀsubstrate. Taken together, our results provide an alternative strategy for the chiral
resolution of racemic mandelic acid and the biosynthesis of PGA.
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MATERIALS AND METHODS
Reagents and Kits
®
PrimeSTAR HS (Premix), DNA Marker Restriction enzymes and DNA Ligation Kit
Ver.2.1 were purchased from TaKaRa (Dalian, China). BamH I and Xho I were purchased
from New England Biolabs, Inc. (Ipswich, MA, England). Dꢀmandelic acid, Lꢀmandelic
acid, Dꢀ2ꢀchloromandelic acid, Lꢀ2ꢀchloromandelic acid, Lꢀlactic acid and PGA were
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purchased from Aladdin (Shanghai, China). NAD , NADP , NADH and NADPH were
purchased from Bontacꢀbio (Shenzhen, China). BCAꢀ200 protein assay kit, IPTG and
®
protein markers were purchased from Sangon (Shanghai, China). Axygen AxyPrep™ PCR
®
®
CleanꢀUp Kit, Axygen AxyPrep™ DNA Gel Extraction Kit and Axygen AxyPrep™
Plasmid Miniprep Kit were purchased from Corning (New York, United States). EasyPure
Bacteria Genomic DNA Kit was purchased from TransGen Biotech (Beijing, China). All
other chemicals were of analytical grade and purchased from Solarbio (Beijing China).
Strains, Plasmids and Culture Media
E. coli BL21 and E. coli DH5α/pET28a, preserved by our group, were cultured in LB
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medium containing 10 g·L tryptone, 5 g·L yeast extract and 10 g·L NaCl, pH 7.2.
Lactococcus lactis CICC 20209 and Leuconostoc citreum CICC 23234, purchased from
China Center of Industrial Culture Collection (Beijing, China), were used for genomic DNA
extraction. Lactococcus lactis CICC 20209 was cultured in skim milk medium containing
ꢀ1
120 g·L skim milk powder with natural pH. Leuconostoc citreum CICC 23234 was
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cultured in MRS medium containing 10 g·L tryptone, 10 g·L beef extract, 5 g·L yeast
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extract, 5 g·L glucose, 5 g·L sodium acetate, 2 g·L ammonium citrate, 1 g·L Tween 80,
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2 g·L K HPO , 0.2 g·L MgSO ·7H O, 0.05 g·L MnSO ·H O, 20 g·L CaCO , pH 6.8.
Klebsiella oxytoca (gifted by Prof. JianꢀPing Wu) and Staphylococcus aureus (gifted by the
group of HongꢀFei Shi) were cultured in LB medium and used for genomic DNA extraction.
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Genome Mining for Putative D-mandelate Dehydrogenases
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LbDMDH, a NAD ꢀdependent Dꢀmandelate dehydrogenase with high activity encoded by
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Lactobacillus brevis (GenBank accession no: WP_011668914.1), was used as probe for the
BLAST search in the NCBI database (http://www.ncbi.nlm.nih.gov) for 2ꢀdehydropantoate
2ꢀreductase and potential novel Dꢀmandelate dehydrogenases, which was mainly from a
bacterial genome and an uncharacterized sequence. The phylogenetic tree of the potential
novel Dꢀmandelate dehydrogenases was constructed using both the ClustalX2 program and
MEGA 6.0 software. Based on the phylogenetic analysis, ten potential novel Dꢀmandelate
dehydrogenases with 40–70% identical amino acid sequences to LbDMDH were selected.
Gene Cloning and Expression of Selected D-mandelate Dehydrogenases
Based on the results of genome mining, a series of paired primers shown in Table S1 were
designed and synthesized by Synbio Technologies Genes for Life (Suzhou, Jiangsu, China),
and used to amplify the gene fragments of potential novel Dꢀmandelate dehydrogenases.
The other potential Dꢀmandelate dehydrogenase genes from unobtainable or pathogenic
microorganisms were codon optimized and artificially synthesized by Synbio Technologies
Genes for Life. The original strains of potential Dꢀmandelate dehydrogenases were
cultivated in the abovementioned media at 37°C for 14 h on a rotary incubator at 220 rpm
and harvested through high speed centrifugation. The collected microbial cells were
thoroughly washed with sterile deionized water. The genomic DNA was extracted from the
collected microbial cells using the EasyPure Bacteria Genomic DNA Kit according to the
manufacturer’s instructions. Using these genomic DNA as template, the gene fragments of
potential Dꢀmandelate dehydrogenases were amplified with the above primers. The
amplified and artificially synthesized genes were cloned into pET28a, and transformed into
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E. coli BL21. The recombinant transformants were confirmed by DNA sequencing and
selected for culturing.
The selected recombinant transformants were cultured for 14 h in tubes containing 4 mL
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LB medium with 50 ꢁg mL kanamycin at 37°C and 200 rpm, and then the cultures were
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transferred into flasks containing 100 mL fresh LB medium containing 50 ꢁg mL
kanamycin. After cultivation at 37°C for 2.5 h, IPTG was added to a final concentration of
0.1 mM, and induction was allowed to proceed for 20 h at 16°C. The induced cells were
harvested by centrifugation and lysed by ultrasonication, and the cell lysate was centrifuged
at 10000 ×g and 4°C for 30 min. The supernatant was then purified to homogeneity by
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Niꢀchelating affinity chromatography.
Enzyme Activity and Protein Assays
The dehydrogenase activity of Dꢀmandelate dehydrogenase on Dꢀmandelic acid was
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assayed by coupling the increase of NADH.
The reaction mixture contained 100 mM
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glycineꢀNaOH buffer, pH 9.5, 1.0 mM NAD and 6.0 mM substrate. The oxidation activity
◦
was assayed spectrophotometrically at 30 C by monitoring the increase of NADH
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absorbance at 340 nm. The molar absorption coefficient of NADH at 340 nm is 6.22 × 10
ꢀ1
ꢀ1 6, 18
M cm .
The reduction activity of Lꢀlactate dehydrogenase on sodium pyruvate was
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assayed by coupling the decrease of NADH. One unit of enzyme activity was defined as
the amount of enzyme reducing or oxidizing 1 ꢁmol NADH per minute under the above
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reaction conditions. The protein concentration was measured with the BCAꢀ200 Protein
Assay Kit (Pierce, Rockford, IL), using bovine serum albumin as the standard. Sodium
dodecyl sulfateꢀpolyacrylamide gel electrophoresis (SDSꢀPAGE) was carried out on a
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12.5% gel using the reported method, and the isolated protein bands were visualized by
staining with Coomassie Brilliant Blue Rꢀ250 (Sigma, St. Louis, MO, USA). The apparent
molecular weights were estimated using Quantity One software based on the standard
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marker proteins.
Biochemical Characterization of Purified Recombinant Enzymes
The temperature optima of these recombinant Dꢀmandelate dehydrogenases were
determined under the standard assay conditions described above, except for temperatures
ranging from 20 to 60°C. To estimate their thermostability, these recombinant Dꢀmandelate
dehydrogenases were incubated at pH 9.0 and various temperatures (20–50°C) for 1.0 h, and
then, the residual enzyme activity was measured under the optimal reaction temperatures.
Here, thermostability was defined as a temperature, at or below which the residual activity
retained more than 85% of its original activity. The pH optima of these recombinant
Dꢀmandelate dehydrogenases were assayed by the standard activity assay method as stated
above with 100 mM glycineꢀNaOH buffer over the pH range of 7.0−11.0. To estimate the
pH stability, aliquots of these recombinant DMDHs were preincubated at 0°C for 1.0 h in
varied pH values from 7.0 to 11.0, and the residual activities were assayed under the optimal
reaction temperatures and pH values. The pH stability, in this work, was defined as the pH
range over which the residual activities were more than 85% of the original activity.
The kinetic parameters of the purified Dꢀmandelate dehydrogenases were determined
by assaying (in triplicate) the activities on various substrate concentrations (1–12 mM) at a
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fixed NAD concentration of 2 mM. On the other hand, the kinetic parameters of the
purified dehydrogenase were determined by assaying (in triplicate) their activities on
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various NAD concentrations (0.2–1.2 mM) at a fixed substrate concentration of 10 mM.
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The correlation curve of reaction rate to substrate or NAD concentration was plotted and
analyzed using nonlinear fitting by the Origin 9.0 software.
The substrate specificities of the purified Dꢀmandelate dehydrogenases were
determined by measuring the enzyme activities, as described above, toward different
substrates, including D, Lꢀmandelic acid, Lꢀmandelic acid, Dꢀ2ꢀchloromandelic acid,
Lꢀ2ꢀchloromandelic acid and Lꢀlactic acid.
Analysis of PGA
PGA was dissolved in deionized water at concentrations ranging from 0.5 to 2.5 mM, and
determined using an HPLC system equipped with a Thermo Hypersil C18 column (250 mm
× 4.6 mm, 5 ꢁm, ThermoFisher Scientific), and a UV detector at 215 nm. The mobile phase
−1
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contained 20% methanol and 80% ddH O, and the flow rate was 1.0 mL min . Retention
time of PGA was 3.1 min. The correlation curve of peak area to PGA concentration was
plotted and analyzed using Origin 9.0, which provided one linear regression equation.
Meanwhile, PGA was dissolved in deuterium dimethylsulfoxide at a concentration of 1 mM
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and fully characterized by H NMR spectroscopy at 400 MHz.
‘One3 pot’ Bioconversion of D-mandelic acid by Recombinant E. coli Co-culture
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To enhance the capacity of NAD regeneration, another recombinant E. coli strain
containing the gene for LcLDH, an efficient Lꢀlactate dehydrogenase (GenBank accession
21
no. OJF74586) from Lactobacillus casei, was constructed and named as E. coli
BL21/pET28aꢀLcLDH. These recombinant E. coli strains were coꢀcultured and induced as
described above. Then, Dꢀmandelic acid was added to a final concentration of 50 mM, and
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transformed for 48 h at 30°C. During this period, 100 ꢁL samples were removed for HPLC
analysis every 4 h. The E. coli BL21/pET28a and E. coli BL21/pET28aꢀLhDMDH were
used as control respectively.
‘One3 pot’ Bioconversion of D,L-mandelic acid
To evaluate the application potential of LhDMDH in the chiral resolution of racemic
mandelic acid, these recombinant E. coli strains were coꢀcultured and induced as described
above. Then, D,Lꢀmandelic acid was added to a final concentration of 50 mM, and
transformed for 48 h at 30°C. During this period, 100 ꢁL samples were removed for HPLC
analysis every 4 h.
Purification and Identification of Biotransformation Products
After the catalytic reaction was terminated, the resulting solution was centrifuged at 8228
×g for 10 min to remove the cells, and the supernatant was extracted three times by
isovolumetric acetate. The extracts were combined and vacuum evaporated to obtain the
crude products. The crude products were purified to homogeneity by silica gel filtration
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chromatography, and the yield was determined by HPLC analysis as described above. The
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product was fully characterized by H NMR spectroscopy at 400 MHz.
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Gene Cloning and Expression of D-mandelate Dehydrogenases
To search for an efficient Dꢀmandelate dehydrogenase for the biosynthesis of PGA, a
genome mining strategy was adopted. Four gene fragments of potentially novel
Dꢀmandelate dehydrogenases were amplified, as shown in Figure S1, and four gene
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fragments were artificially synthesized. Eight potential NAD ꢀdependent mandelate
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dehydrogenases were cloned and heterogeneously overꢀexpressed in E. coli BL21 and
SDSꢀPAGE analysis of four representative recombinant dehydrogenases are shown in
Figure 1. Interestingly, several of them displayed oxidation activity toward Dꢀmandelic acid.
Among these, a putative 2ꢀdehydropantoate 2ꢀreductase (GenBank accession no:
WP_027828400.1) from L. harbinensis, herein designated LhDMDH, was confirmed with
ꢀ1
the highest oxidation activity (230 U·mL fermentation liquor on Dꢀmandelic acid), which
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was close to ten times that of the probe. Sequence alignment revealed a relatively low
(<55%) amino acid sequence identity with the probe (Figure S2), indicating that it is a
novel Dꢀmandelate dehydrogenase. The highest sequence identity (39.9%) found using
BLASTp in the protein database of NCBI was with a putative 2ꢀdehydropantoate
2ꢀreductase (PDB accession no. 2EW2) from Enterococcus faecalis V583, belonging to the
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ketopantoate reductase family.
To characterize their enzymatic properties, the recombinant Dꢀmandelate
dehydrogenases were purified through NiꢀNTA affinity chromatography. As shown in
Figure 1, they were purified to homogeneity, seen as a single band on an SDSꢀPAGE gel,
corresponding to an apparent molecular weight of 38.0 kDa. The specific oxidation activity
−1
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of purified LhDMDH was 1200 U·mg , which was close to four times that of the probe
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, 6, 22
and significantly higher than the other reported Dꢀmandelate dehydrogenases.
The
specific oxidation activity of LcDMDH, a Dꢀmandelate dehydrogenase cloned from
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Leuconostoc citreum, was 40.6 U·mg . On the other hand, the specific oxidation activity of
LlDMDHꢀ2, a Dꢀmandelate dehydrogenase cloned from Lactococcus lactis, was 11.7
−
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U·mg . Among the purified, recombinant Dꢀmandelate dehydrogenases, no activity was
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detected with NADP as cofactor, indicating that LhDMDH is an NAD ꢀdependent
Dꢀmandelate dehydrogenase.
Comparative Enzymatic Properties of D-mandelate Dehydrogenases
The enzymatic properties of purified recombinant Dꢀmandelate dehydrogenases were
investigated using Dꢀmandelic acid as the model substrate. The temperature properties of
these purified Dꢀmandelate dehydrogenases are shown in Figure 2, which shows higher
catalytic activity at a temperature range of 25ꢀ30°C. The temperature optimum of LhDMDH
and probe was 30°C, while the other was 25°C (Figure 2a). All of the purified Dꢀmandelate
dehydrogenases were highly stable at 20°C after being incubated for 1 h, and they retained
levels of residual activities at 80% or above (Figure 2b). Unfortunately, the residual activity
of LhDMDH was less than 50% after being incubated at 30°C for 1 h, and its low
thermostability would be impeded during biosynthesis of PGA. Hence, whole cell catalysis
is a preferred option to eliminate the influence of low thermostability, in the biosynthesis of
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PGA.
It should be noted that with the emergence of various bioinformatics software, in
silico design is playing a more significant role in molecular modification of thermostability.
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4
In particular, molecular dynamics simulation has become a more mature tool . Our future
research will focus on the use of in silico design to modify LhDMDH at the molecular level
to improve its thermostability.
The pH properties of the purified Dꢀmandelate dehydrogenases are shown in Figure 3.
As shown in Figure 3a, all of them are alkalophilic dehydrogenases, and the pH optimum of
LhDMDH was 9.5. Except for LlDMDHꢀ2, they were highly stable at pH values ranging
from 9.5 to 10.5 retaining more than 80% of their original activity (Figure 3b). The
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LlDMDHꢀ2 was stable only within a narrow pH range, and its residual activity declined
markedly at pH values less than 8.5 or more than 9.5.
The kinetic parameters of the purified Dꢀmandelate dehydrogenases toward
+
Dꢀmandelic acid and NAD were determined and summarized in Table 1 and Table 2,
respectively. The K value of LhDMDH toward Dꢀmandelic acid was 1.05 mM, which was
m
distinctly lower than that of the probe, indicating that LhDMDH had a higher affi nity for
7
ꢀ1
ꢀ1
m
Dꢀmandelic acid. In addition, the kcat/K value was 28.80 S ·mM , which was distinctly
higher than the one of probe, which further indicates the advantage of using LhDMDH in
the biosynthesis of PGA.
The substrate specificity of the purified Dꢀmandelate dehydrogenases were determined
and summarized in Table 3. They all clearly displayed the greatest oxidation activity toward
Dꢀmandelic acid, and only extremely low activity was detected toward Dꢀ2ꢀchloromandelic
acid. In addition, nearly no activity was detected toward Lꢀmandelic acid,
Lꢀ2ꢀchloromandelic acid and Lꢀlactic acid, indicating that these Dꢀmandelate
dehydrogenases could be applied to chirally resolve racemic mandelic acid during the
biosynthesis of PGA. Furthermore, the absence of activity toward Lꢀlactic acid is beneficial
+
14, 15
to NAD regeneration with the aid of an efficient Lꢀlactate dehydrogenase.
‘One3 pot’ Biosynthesis of PGA by E. coli-E. coli Co3 cultures
Biosynthesis at the industrial scale will benefit from reducing the amount of coenzyme
+
or coꢀsubstrate used and from enhancing the regeneration of NAD . Therefore, we
investigated the feasibility of the biosynthesis of PGA in a ‘oneꢀpot’ fermentation system by
coꢀculturing the two strains E. coli BL21/pET28aꢀLhDMDH and E. coli
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BL21/pET28aꢀLcLDH. In this coꢀculture, E. coli BL21/pET28aꢀ LhDMDH is dedicated to
the conversion of Dꢀmandelic acid into PGA and E. coli BL21/pET28aꢀ LcLDH is dedicated
+
to enhance the regeneration of NAD by utilizing the pyruvic acid generated by itself. To
detect the expression level of the coffi culture, SDSꢀPAGE and enzyme activities analysis
were carried out. As shown in Figure 4, SDSꢀPAGE analysis of the coffi cultured bacteria
found two specific bands with apparent molecular weights of 38.0 and 40 kDa, which
indicates that LhDMDH and LcLDH were successfully expressed in the process of
coffi culturing. In addition, the oxidation activity toward Dꢀmandelic acid of the coffi cultured
ꢀ1
bacteria was 190 U·mL fermentation liquor, which showed a slight decline compared with
single culture. Meanwhile, the coffi cultured bacteria’s reduction activity toward sodium
ꢀ1
pyruvate was 210 U·mL fermentation liquor, which further indicates that LhDMDH and
LcLDH were successfully expressed in activated states.
Dꢀmandelic acid was bioconverted in 48 h by the coffi culture, and the product was
purified to chromatographically pure homogeneity (Figure S3a), which means that purity
was over 99%. As shown in Figure S3, the retention time of the purified product was 3.1
min, which was in accordance with the retention time of PGA (Figure S3b). This result,
1
combined with the H NMR spectra of Dꢀmandelic acid (Figure S4) and the product
(Figure S5), sufficiently demonstrates that the Dꢀmandelic acid had been converted to PGA
by coꢀculturing the two strains E. coli BL21/pET28aꢀLhDMDH and E. coli
BL21/pET28aꢀLcLDH. These results also indicate that the PGA was successfully
synthesized without adding coenzyme or coꢀsubstrate, implying that the synthesis cost of
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PGA can be significantly reduced by this strategy, thus fully demonstrating the advantage of
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whole cell catalysis.
To fully assess the productivity of E. coliꢀE. coli coꢀcultures in comparison with E. coli
BL21/pET28aꢀLhDMDH or E. coli BL21/pET28a single culture for PGA synthesis, three
different culture modes were performed, and their yields measured. As shown in Figure 5,
after coffi culturing for 48 h, the yield of PGA was over 60%, which was significantly higher
by approximately 20% than the yields of the single cultures. This improvement is attributed
+
to that the enhanced the capacity of NAD regeneration by utilizing the pyruvic acid
14
generated within the coffi cultured E. coli/pET28aꢀLcLDH. Moreover, this result also
+
indicates that the NAD and NADH can be transmitted between cells. Unfortunately, the
yield of PGA by coꢀculture could not be sustained after 48 h under these culture conditions.
+
These conditions may restrict the regeneration or transmission capacity of NAD , thus the
next research priority will be optimizing the culture conditions or coꢀexpressing the
LhDMDH and LcLDH. In addition, the strategy for Lꢀphenylglycine biosynthesis from PGA
by coꢀexpressing leucine dehydrogenase could be used to improve the capacity to regenerate
+
7, 27
NAD and synthesize the derivatives of PGA
.
We also used the coꢀculture system to transform racemic mandelic acid into PGA. As
shown in Figure 6, after coffi culturing for 48 h, the yield of PGA was approximately 40%
toward 50 mM D, Lꢀmandelic acid, which is close to the theoretical yield of 50%. Therefore,
the LhDMDH has tremendous application potential in the chiral resolution of racemic
mandelic acid and the biosynthesis of PGA, which is an important intermediate for the
8, 9
synthesis of pharmaceutical and natural products.
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In conclusion, a novel Dꢀmandelate dehydrogenase (LhDMDH) from L. harbinensis was
successfully mined and expressed in E. coli BL21. The LhDMDH displayed high catalytic
activity and excellent kinetic parameters. Interestingly, the LhDMDH had no activity toward
Lꢀlactic acid and Lꢀmandelic acid which, with the aid of an efficient Lꢀlactate
+
dehydrogenase, is suitable for NAD regeneration and in the chiral resolution of racemic
mandelic acid and the biosynthesis of PGA. We successfully biosynthesized PGA by
coꢀculturing two recombinant E. coli strains without adding coenzyme or coꢀsubstrate. All
of these results establish a solid foundation for the chiral resolution of racemic mandelic
acid and the biosynthesis of PGA.
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31 ASSOCIATED CONTENT
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37
38
39
Supporting Information
Additional experimental results and other data are available free of charge via the Internet at
http://pubs.acs.org. These include: sequences of the primers used for cloning and expression
of novel Dꢀmandelate dehydrogenases (Table S1); the PCR amplification for the four
Dꢀmandelate dehydrogenase genes (Figure S1); multiꢀsequence alignment of the
representative Dꢀmandelate dehydrogenases (Figure S2); the HPLC analysis spectrum of the
1
product (Figure S3); the H NMR analysis spectra of the Dꢀmandelic acid (Figure S4) and
product (Figure S5).
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40 AUTHOR INFORMATION
41
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52
Authors’ Contributions
CunꢀDuo Tang, HongꢀLing Shi and the first author contributed equally to this work.
Notes
The authors declare that they have no competing interests.
ACKNOWLEDGMENTS
We are grateful to Prof. Minchen Wu (Wuxi Medical School, Jiangnan University) and Prof.
Ting Lei (Nanyang Normal University) for providing technical assistance. This work was
supported by the grants from Scientific and Technological Project of China (31372381),
Scientific and Technological Project of Henan Province (162102210116), Scientific research
and service platform fund of Henan Province (2016151), fund of scientific and
technological innovation team of water ecological security for Water Source Region of
Midꢀline of SouthꢀtoꢀNorth Diversion Project of Henan Province (17454), High School Key
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Research Project of Henan Province (15A416008), and the Special Funded Projects of
Nanyang Normal University (17359).
55 REFERENCES
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Scheme and Figure Legends
Scheme 1. Coupled enzymatic synthesis of PGA from D-mandelic acid.
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Figure 1. The SDS-PAGE analysis for the representative recombinant D-mandelate
dehydrogenases. Lane M, PageRuler Prestained Protein Ladder; lane 1, expressed products
of E. coli/pET28a; lane 2, crude LbDMDH; lane 3, purified LbDMDH; lane 4, crude
LhDMDH; lane 5, purified LhDMDH; lane 6, crude LcDMDH; lane 7, purified LcDMDH;
lane 8, crude LlDMDHꢀ2; lane 9, purified LlDMDHꢀ2.
Figure 2. The temperature properties of the representative recombinant D-mandelate
dehydrogenases. (a) temperature optima of the representative recombinant Dꢀmandelate
dehydrogenases. The temperature optima were assayed spectrophotometrically by
monitoring the increase of NADH absorbance at 340 nm at reaction temperatures ranging
from 20 to 60°C. (b) temperature stabilities of the representative recombinant Dꢀmandelate
dehydrogenases. The temperature stabilities were determined by incubating them at various
temperatures (20ꢀ50°C) for 1.0 h, and then the residual enzyme activities were measured as
describe in the text.
Figure 3. The pH properties of the representative recombinant D-mandelate
dehydrogenases. (a) pH optima of the representative recombinant Dꢀmandelate
dehydrogenases. The pH optima were assayed by the standard activity assay method as
described in the text with 100 mM glycineꢀNaOH buffer over the pH range of 7.0−11.0. (b)
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pH stabilities of the representative recombinant Dꢀmandelate dehydrogenases. To estimate
the pH stability, aliquots of these recombinant Dꢀmandelate dehydrogenases solution were
preincubated at 0°C for 1.0 h at various pH values from 7.0 to11.0, and the residual
activities were assayed under the optimal reaction temperatures and pH values.
Figure 4. SDS-PAGE analysis for the expressed products of single culture and
co-culture. Lane M, PageRuler Prestained Protein Ladder; lane 1, expressed products of
single cultured E. coli/pET28aꢀLhDMDH; lane 2, expressed products of coꢀcultured E.
coli/pET28aꢀLhDMDH and E. coli/pET28aꢀLcLDH.
Figure 5. The yield of different culture mode. The reactions were carried out at 30°C
and 200 rpm for 48 h in a 100ꢀmL fermentation liquor containing 50 mM Dꢀmandelic acid
without adding coenzyme or coꢀsubstrate.
Figure 6. The conversion curve of co-culture toward D, L-mandelic acid. The
reactions were carried out at 30°C and 200 rpm for 48 h in a 100ꢀmL fermentation liquor
containing 50 mM D, Lꢀmandelic acid without adding coenzyme or coꢀsubstrate.
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Tables
Table 1 The kinetic parameter of D-mandelate dehydrogenases for D-mandelic acid.
Kinetic parameters for substrate
Enzyme
ꢀ
1
ꢀ1
ꢀ1
K
m
(mM)
k
cat (S )
K
i
(mM)
k
cat/K
m
(S mM )
LbDMDH
LhDMDH
LcDMDH
LlDMDHꢀ2
1.45
1.05
1.75
1.00
8.82
24.37
26.40
24.86
16.25
6.08
30.28
1.56
28.80
0.89
2.29
2.29
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Table 2 The kinetic parameter of D-mandelate dehydrogenases for NAD .
+
Kinetic parameters for NAD
Enzyme
ꢀ
1
ꢀ1
ꢀ1
K
m
(mM)
k
cat (S )
K
i
(mM)
N
m
kcat/K (S mM )
LbDMDH
LhDMDH
LcDMDH
LlDMDHꢀ2
0.78
0.40
0.85
0.42
9.23
11.83
72.00
2.19
29.05
1.86
N
N
1.85
N
4.40
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Table 3 The substrate spectrum of D-mandelate dehydrogenases
Relative activity (%)
Substrate
LbDMDH
LhDMDH
LcDMDH
LlDMDHꢀ2
Dꢀmandelic acid
D, Lꢀmandelic acid
Dꢀ2ꢀchloromandelic acid
Lꢀmandelic acid
100
75
100
82
100
85
2.51
0
100
80
0.05
0
0.59
0.04
0
0.28
0.02
0
Lꢀ2ꢀchloromandelic acid
Lꢀlactic acid
0
0
0
0
0
0
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Scheme 1.
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Figure 1.
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Figure 2a.
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