Efficient one-step production of (S)-1-phenyl-1,2-ethanediol from (R)-enantiomer plus NAD+–NADPH in-situ regeneration using engineered Escherichia coli
© Zhang et al.; licensee BioMed Central Ltd. 2012
Received: 26 October 2012
Accepted: 19 December 2012
Published: 29 December 2012
Candida parapsilosis CCTCC M203011 catalyzes the stereoinversion of (R)-1-phenyl-1,2-ethanediol (PED) through oxidation and reduction. Its NAD+-linked (R)-carbonyl reductase (RCR) catalyzes the oxidization of (R)-PED to 2-hydroxyacetophenone (HAP), and its NADPH-dependent (S)-carbonyl reductase (SCR) catalyzes the reduction of HAP to (S)-PED. The reactions require NAD+ and NADPH as cofactors. However, even if NAD+ and NADPH are added, the biotransformation of (S)-PED from the (R)-enantiomer by an Escherichia coli strain co-expressing RCR and SCR is slow and gives low yields, probably as a result of insufficient or imbalanced redox cofactors. To prepare (S)-PED from the (R)-enantiomer in one-step efficiently, plus redox cofactor regeneration, we introduced pyridine nucleotide transhydrogenases (PNTs) from E. coli to the metabolic pathway of (S)-PED.
The PNTs were successfully introduced into the E. coli strain RSAB. Most of the PNT activities occurred in the cell membrane of E. coli. The introduction of PNTs increased intracellular NAD+ and NADH concentrations and decreased the NADPH pool without affecting the total nucleotide concentration and cell growth properties. The presence of PNTs increased the NADH/NAD+ ratio slightly and reduced the NADPH/NADP+ ratio about two-fold; the ratio of NADPH/NADP+ to NADH/NAD+ was reduced from 36 to 17. So, the PNTs rebalanced the cofactor pathways: the rate of RCR was increased, while the rate of SCR was decreased. When the ratio of NAD+/NADPH was 3.0 or higher, the RSAB strain produced (S)-PED with the highest optical purity, 97.4%, and a yield of 95.2% at 6 h. The introduction of PNTs stimulated increases of 51.5% and 80.6%, respectively, in optical purity and yield, and simultaneously reduced the reaction time seven-fold.
In this work, PNTs were introduced into E. coli to rebalance the cofactor pools within the engineered (S)-PED pathways. The efficient one-step production of (S)-PED plus NAD+–NADPH in-situ regeneration was realized. This work provided new insights into cofactor rebalancing pathways, using metabolic engineering methods, for efficient chiral alcohol production.
KeywordsRedox cofactor regeneration Chiral alcohol Alcohol dehydrogenases/reductases Metabolic pathway One-step stereoinversion
Alcohol dehydrogenases can catalyze a great variety of reduction–oxidation (redox) reactions during the production of chiral compounds [1–4]. However, their practical applications can be quite challenging since they require expensive cofactors, such as nicotinamide adenine dinucleotide [NAD+ and NADH] and nicotinamide adenine dinucleotide phosphate [NADP+ and NADPH] [5–7]. To address redox reaction limitations, enzyme-mediated cofactor recycling is preferred for industrial processes because of its high selectivity and efficiency [8, 9]. For instance, Verho et al. improved pentose fermentation in Saccharomyces cerevisiae by engineering redox cofactor regeneration through the corresponding fungal pathways .
In general, either the cofactor couple NADH/NAD+ or NADPH/NADP+ is required in a specific biochemical reaction [9, 10]. However, if both NAD(H) (NAD+ and NADH) and NADP(H) (NADP+ and NADPH) are required in a particular cofactor-dependent pathway, the flux is controlled not only by the availability of enzymes, but also by the cofactor amount and the ratio of the reduced forms to the oxidized forms of the cofactor [5, 7, 11]. The different cofactors have to be regenerated in separate processes and their intracellular redox forms need to be balanced to achieve high yields [5, 12]. Several groups achieved the simultaneous regeneration of NAD(H) and NADP(H) in redox reaction systems by the introduction of pyridine nucleotide transhydrogenases (PNTs) [13, 14]. There are two transhydrodenases in Escherichia coli, one soluble, the udhA which is mainly for regeneration of NADH and NADP; a membrane bound, the PNT is used for the regeneration of NAD and NADPH . The PNT enzymes are composed of α and β subunits encoded by the PntA and PntB genes, respectively. Most enzymes are located in the cell membrane and they catalyze the reduction of NADP+ to NADPH via oxidation of NADH to NAD+: NADPH + NAD+ ⇌ NADP+ + NADH[16, 17]. Anderlund et al. studied the physiological effects of interconversion between NAD(H) and NADP(H) in S. cerevisiae expressing membrane-bound PNTs from E. coli during anaerobic glucose fermentation . Boonstra et al. successfully regenerated NAD+ and NADPH in a cell-free system to gain high yields of hydromorphone using the soluble PNT from Pseudomonas fluorescens.
Results and discussion
Construction of recombinant E. coli harboring RCR, SCR, PntA, and PntB
In any system based on carbonyl reductase catalysis of the bioconversion of a chiral alcohol, insufficient cofactors or their unbalanced ratios results in low biotransfomation efficiency [5, 7, 11]. Heterogeneous expressions of PNTs (PntA and PntB) were expected to increase the overall intracellular NAD+ and NADPH pool, or to rebalance them, thus improving the flux of NAD+- and/or NADPH-dependent pathways .
It has been reported that the NAD+-linked RCR from C. parapsilosis reduces (R)-PED to HAP, and the NADPH-dependent SCR catalyzes HAP to (S)-PED. To prepare (S)-PED from the (R)-enantiomer efficiently in one-step, PNTs from E. coli were introduced into the pathway catalyzed by RCR and SCR through a “multi-enzyme approach”. Two compatible plasmids pETDuet™-1 and pACYCDuet™-1 (Novagen, Darmstadt, Germany) were used, each of which contained two multiple cloning sites. The expression plasmids pET-RS and pACYC-AB were constructed and then transformed in competent cells of E. coli using standard techniques, as shown in Additional file 1: Figure S1 in the supplemental materials. The recombinant RS, AB, and RSAB strains were shaped after being verified by DNA sequencing.
Co-expression of recombinant enzymes
The recombinant strains were induced with 0.1 mM isopropyl-β-thiogalactopyranoside (IPTG) at 37°C. SDS–PAGE analysis (Additional file 1: Figure S2 in the supplemental materials) showed that two obvious bands (about 37 kDa and 31 kDa) corresponding to the sizes of RCR and SCR  were observed in cell-free extracts of RS and RSAB. The other two bands (about 50 kDa and 47 kDa), corresponding to the sizes of PntA and PntB, were apparent in cell extracts (without centrifugation) of AB and RSAB [6, 7, 13, 14]. So, in the cells of RSAB, four proteins were all expressed.
Enzyme activities and stereoconversions of ( R )-PED a to ( S )-enantiomer by recombinant E. coli strains
Specific activities (U/mg)
Optical purity (%e.e.)
Rebalancing of intracellular nucleotides by introduction of PNTs
Intracellular concentrations of NAD + , NADH, NADP + , and NADPH in recombinant cells during exponential growth
Intracellular concentrations (μmol/g [dry wt] of biomass) of:
Distribution of intracellular nucleotides had little effect on cell growth
One-step stereoconversion of (R)-PED to (S)-enantiomer plus NAD+–NADPH in-situ regeneration using the RSAB strain
Bioconversions of ( R )-PED to ( S )-isomer with RSAB cells for different NAD + /NADPH ratios
NAD+ : NADPH
1 : 1
1 : 2
1 : 3
1 : 4
2 : 1
3 : 1
4 : 1
Optical purity (% e.e.)
Improving stereoconversion efficiency by optimizing ratio of cofactors
To improve the stereoconversion efficiency, the whole-cell biotransformation was investigated in the presence of various ratios of NAD+ (1.0–2.0 mM) and NADPH (1.0–2.0 mM), with both NAD+ and NADPH, and with neither. The optical purity and yield of (S)-PED was measured when the substrate (R)-PED concentration was 100 mM and the incubation of the reaction mixture lasted for 6 hours. As shown in Table 3, even if the initial addition of NAD+ and/or NADPH was omitted, the reaction proceeded as a result of the natural existence of cofactors in E. coli cells. However, the biotransformation efficiency was very low. If neither of the cofactors was added, the optical purity and yield of (S)-PED were at least three times lower than when both cofactors were present. In the case of NADPH, the biotransformation was less than that with NAD+, which suggested that improving the metabolic flux of NAD+-mediated RCR would be more beneficial for the biotransformation efficiency than strengthening the branch of NADPH-dependent SCR. (S)-PED was efficiently produced if both NAD+ and NADPH were present in the reaction mixture, but different NADH/NADP+ ratios resulted in different biotransformation efficiencies. When the NAD+/NADPH ratio was lower than 0.5, the optical purities and yields were not satisfactory. When the ratio was 3.0 or higher, the optical purities and yields of (S)-PED reached the highest levels, i.e., about 97% and 95%, respectively. Compared with the RS strain, RSAB expressing PNTs stimulated an increase of 51.5% and 80.6% in the optical purity and yield of (S)-PED, while simultaneously reducing the substrate-use time seven-fold.
Since the steps in the reactions catalyzed by RCR or SCR occurred in a specific order (i.e., the free enzyme was first combined with the coenzyme and then with the substrate) , the addition of cofactors could seriously affect the enzymatic efficiency. Furthermore, the SCR enzyme had a much higher expression level and a much stronger enzymatic function than those of RCR in E. coli. The moderate increase in NAD(H) could strengthen the RCR function, and improve the balance between NAD+-dependent RCR and NADP(H)-linked SCR in the enzyme-coupled system. These results suggested that the NAD(H)-mediated or NADP(H)-dependent fluxes are partly determined by their cofactor availability, and can be improved by rebalancing the metabolic branches , i.e., strengthening or weakening their cofactor regeneration systems [29, 30].
Using a multi-coenzyme approach, PNT enzymes, involved in cofactor regeneration, were introduced into the metabolic pathway, driving it in the direction of (S)-PED production, in the RSAB strain. The introduction of PNT enzymes resulted in slightly higher intracellular NAD(H) concentrations and a much lower NADPH pool, with cell growth and total nucleotides unaffected. The cofactor balancing of (S)-PED pathways caused a sharply reduced NADPH/NADP+ ratio but maintained the NADH/NAD+ ratio at a constant level during the exponential growth phase. The efficient one-step production of (S)-PED from the (R)-enantiomer, plus NAD+–NADPH in situ regeneration, was achieved by the introduction of PNTs. When the ratio of NAD+/NADPH was three or higher, PNT introduction resulted in significant increases in the optical purity and yield of (S)-PED. The results demonstrate that in certain microbial systems the cofactor-dependent (S)-PED pathway is not only controlled by the availability of target enzymes (RCR and SCR), but is also determined by the amount of cofactor and the ratio of reduced forms to oxidized forms [12, 29]. This work provides a new strategy for preparing (S)-PED efficiently, using cofactor rebalancing to engineer chiral alcohol pathways by the introduction of PNT enzymes.
Microorganisms and chemicals
C. parapsilosis CCTCC M203011, obtained from the American Type Culture Collection (ATCC, USA), was used as the DNA donor of the SCR gene (scr). The organisms were cultivated as described previously [18, 19]. E. coli K12 was used as the DNA donor of pyridine nucleotide transhydrogenase A and B genes (PntA and PntB). E. coli BL21 (DE3) and JM109 were used as host cells for gene cloning and expression experiments. E. coli cells were cultured at 37°C in Luria–Bertani (LB) medium, supplemented with ampicillin (100 μg/mL) and/or chloramphenicol (34 μg/mL) when necessary. After starting the cultivation, IPTG (0.1 mM) was added to the medium at 5 h. The cultures were shaken for 8 h at 37°C and harvested by centrifugation after addition of IPTG.
The enzymes and cofactors were purchased from the Sigma-Aldrich Chemical Co., Inc. The restricted enzymes, vectors, and marker DNA used for cloning and the expression experiments were purchased from Qiagen (Germany), Takara-Bio (Kyoto, Japan), Novagen (Germany), and New England Biolabs (USA). All other chemicals were of the highest grade that could be obtained commercially.
Plasmids, strains and primers used
Plasmids, strains, primers
2.7 kb, Ampr
3.7 kb, pMD18-T containing rcr, Ampr
3.5 kb, pMD18-T containing scr, Ampr
4.2 kb, pMD18-T containing PntA, Ampr
4.1 kb, pMD18-T containing PntB, Ampr
5.4 kb, contains two multiple cloning sites, Ampr
4.0 kb, contains two multiple cloning sites, Cm r
7.4 kb, pETDuet™-1 containing rcr and scr, Ampr
6.8 kb, pACYCDuet™-1 containing PntA and PntB, Cm r
C. parapsilosis CCTCC M203011
DNA donors of rcr and scr genes
E. coli K12
DNA donors of PntA and PntB genes
E. coli JM109
recA 1 supE 44 endA 1 hsdR 17 gyrA 96 relA 1 thi△(Lac-proAB)F’
E. coli BL21(DE3)
F-ompT hsdS B (r B -m B -) gal dcm (DE3)
E. coli BL21 bearing pETDuet™-1 and pACYCDuet™-1
E. coli BL21 bearing pET-RS
E. coli BL21 bearing pACYC-AB
E. coli BL21 bearing pET-RS and pACYC-AB
5′ → 3′
TGACTCTCGAG CTATGGATTAAAAACAACACGACC(Xho I)
ATCGAATTC GATGGGCGAAATCGAATCTTATTG(Eco RI)
TGACTGCGGCCGC CTATGGACACGTGTATCCACCGTC(Not I)
CGCGGATCCATGCGAATTGGCATACCAAG (Bam HI)
CCCAAGCTT TTAATTTTTGCGGAACATTTTC (Hin dIII)
CGCGATATC ATGTCTGGAGGATTAGTTAC (Eco RV)
CCCCTCGAG TTACAGAGCTTTCAGGATTG (Xho I)
Construction of co-expression systems
To co-express rcr, scr, PntA, and PntB in E. coli BL21 (DE3), two compatible plasmids, pETDuet™-1 and pACYCDuet™-1 (Novagen), were used. The plasmids pMD-RCR and pMD-SCR were digested with Nde I/Xho I and Eco R I/Not I, respectively, and then ligated into pETDuet™-1 in sequence to construct pET-RS. The fragments of PntA (Bam H I/Hin d III) and PntB (Eco R V/Xho I) were successively inserted into the corresponding sites of pACYCDuet™-1 to obtain pACYC-AB. The construction of pET-RS and pACYC-AB is shown in Additional file 1: Figure S1 in the supplemental materials. They were then introduced into E. coli BL21 cells individually or simultaneously. The positive strains RS, AB, and RSAB were obtained. The plasmids and strains containing different antibiotic resistances and the primers used in this study are listed in Table 4.
Preparation of cell-free extracts
The cultured E. coli BL21 cells were harvested by centrifugation, suspended in 20 mM Tris–HCl (pH 8.0) and 150 mM NaCl, and then disrupted with an ultrasonic oscillator (Insonater 201 M; Kubota, Japan). After centrifugation (16 000 rpm × 40 min) at 4°C, the cell-free extracts were used for the enzyme assays. Preparation of the cell membranes of E. coli BL21 was performed as described by Clarke et al. .
The enzymatic activities of RCR for oxidation of (R)-PED were measured at 35°C and pH 9.0 by spectrophotometrically recording the rate of change of NADH absorbance at 340 nm. The SCR activities for reduction of HAP were assayed at 340 nm by monitoring the change in NADPH. One unit of enzyme activity is defined as the amount of enzyme catalyzing the reduction/oxidation of 1 μmol of NAD(P)H per minute under the measurement conditions. The specific activity is the number of enzyme units per milligram. The standard assays were performed as described by Nie et al. .
Because one isoform is a membrane-bound protein, the PNT activities were determined in cell extracts without centrifugation . Briefly, the enzyme activity was measured spectrophotometrically for 1 min at 30°C at 375 nm, using 3-acetylpyridine adenine nucleotides, and 10–100 μL crude cell extracts, as described by Rydström et al. . Protein concentrations were measured using the method described by Bradford .
Biotransformations and analytical methods
Using washed cells of RS, AB, and RSAB as the catalysts, the reaction was carried out as described previously . When necessary, 1% toluene was added to the reaction mixture . The (S)-PED product was extracted with ethyl acetate, and the organic layer was used for analysis. The optical purities and yields of (S)-PED were determined using high-performance liquid chromatography on a Chiralcel OB-H column (Daicel Chemical Ind. Ltd., Japan). All plots were shown as the means of three independent experiments.
Determination of intracellular nucleotide concentrations
The extraction of intracellular nucleotides was carried out as previously described by Nissen et al. , with minor modifications. The collected 5.0 mL of recombinant E. coli culture was mixed with 20 mL of 60% methanol (−40°C) within 1 s. A 50 mM KPO4 buffer (pH 5.0) and 50 mM Tris–HCl (pH 9.0) were used for extraction of NAD+/NADP+ and NADH/NADPH, respectively. The nucleotide concentrations were measured immediately after reducing the sample volumes by evaporation under vacuum (30 min, 5°C). The contents of NAD+, NADH, NADP+, and NADPH in the samples, obtained by cold methanol extraction, were determined as described by Hu , using standard curves for each compound. Assays were performed in triplicate.
Pyridine nucleotide transhydrogenase subunit α
- PntA :
Pyridine nucleotide transhydrogenase A gene
Pyridine nucleotide transhydrogenase subunit β
- PntB :
Pyridine nucleotide transhydrogenase B gene
- rcr (R:
)-carbonyl reductase gene
- scr (S:
)-carbonyl reductase gene.
The authors thank Mr. Yimin Zhu and Dr. Helen Mcpherson for help with the English writing. This project was supported by the National Science Foundation of China (NSFC, No. 31070059), the National Key Basic Research and Development Program of China (973 Program No. 2009CB724706 and 2011CB710800), the Fundamental Research Funds for the Central Universities (No. JUSRP21121), and the Program of Introducing Talents of Discipline to Universities (No. 111-2-06), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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