Enantioselective cascade biocatalysis for deracemization of 2-hydroxy acids using a three-enzyme system

Background Enantiopure 2-hydroxy acids are key intermediates for the synthesis of pharmaceuticals and fine chemicals. We present an enantioselective cascade biocatalysis using recombinant microbial cells for deracemization of racemic 2-hydroxy acids that allows for efficient production of enantiopure 2-hydroxy acids. Results The method was realized by a single recombinant Escherichia coli strain coexpressing three enzymes: (S)-2-hydroxy acid dehydrogenase, (R)-2-keto acid reductase and glucose dehydrogenase. One enantiomer [(S)-2-hydroxy acid] is firstly oxidized to the keto acid with (S)-2-hydroxy acid dehydrogenase, while the other enantiomer [(R)-2-hydroxy acid] remains unchanged. Then, the keto acid obtained reduced to the opposite enantiomer with (R)-2-keto acid reductase plus cofactor regeneration enzyme glucose dehydrogenase subsequently. The recombinant E. coli strain coexpressing the three enzymes was proven to be a promising biocatalyst for the cascade bioconversion of a structurally diverse range of racemic 2-hydroxy acids, giving the corresponding (R)-2-hydroxy acids in up to 98.5 % conversion and >99 % enantiomeric excess. Conclusions In summary, a cascade biocatalysis was successfully developed to prepare valuable (R)-2-hydroxy acids with an efficient three-enzyme system. The developed elegant cascade biocatalysis possesses high atom efficiency and represents a promising strategy for production of highly valued (R)-2-hydroxy acids. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0560-1) contains supplementary material, which is available to authorized users.


Background
Enantiopure 2-hydroxy acids are among the most important building blocks for synthesizing pharmaceuticals and fine chemicals [1]. For example, (R)-(-)-mandelic acid is widely used as an intermediate for the preparation of antibiotics, antiobesity drugs, and antitumor agents [2]. (R)-o-Chloromandelic acid is the most preferred chiral building block for the industrial synthesis of anti-thrombotic agent, a best-selling cardiovascular drug [3]. (R)-2-Hydroxy-4-phenylbutyric acid is an intermediate in the manufacture of angiotensin converting enzyme inhibitors [4]. (R)-3-Phenyllactic acid is used as a precursor for the synthesis of englitazone which has excellent hypoglycemic effect [5]. Due to their importance, many enantioselective routes for their synthesis have been developed and a great progress has been achieved in recent years. Traditionally, their industrial production mainly relies on the chemical approaches such as chemical kinetic resolution with chiral agent. However, it does not always satisfactorily work because of expensive agent, unsatisfied enantiomeric excess (ee) of product or low yield (e.g., <50 %).
Biocatalysis is increasingly being used to develop efficient and green processes for chiral 2-hydroxy acids synthesis [6]. Several enzymatic approaches have been reported for synthesizing chiral 2-hydroxy acids in the literature [1,7,8], including reduction of 2-keto acids with stereoselective 2-keto acid reductase [9], enantioselective oxidation of racemic 2-hydroxy acids with 2-hydroxy acid
In this work, we aim to develop an enantioselective cascade biocatalysis for deracemization of racemic 2-hydroxy acids to (R)-2-hydroxy acids via an oxidation-reduction sequence using a recombinant Escherichia coli expressing three enzymes (Scheme 2). One enantiomer [(S)-2-hydroxy acid] is firstly oxidized to the keto acid with enantioselective (S)-2-hydroxy acid dehydrogenase [(S)-2-HADH] while the other enantiomer [(R)-2-hydroxy acid] remains unchanged. The keto acid obtained is then bioreduced to the opposite enantiomer with stereoselective (R)-2-keto acid reductase [(R)-2-KAR] plus cofactor regeneration enzyme glucose dehydrogenase (GDH) subsequently. The recombinant E. coli strain coexpressing (S)-2-HADH, (R)-2-KAR and GDH was proven to be a promising biocatalyst. A wide range of 2-hydroxy acids can be deracemized to (R)-2-hydroxy acids with near-perfect stereo purity and high conversion. This method by cascade enantioselective oxidation and asymmetric reduction with a single recombinant strain represents a cheap, easy and environmental approach for synthesizing (R)-2-hydroxy acids from their racemates.

Construction of recombinant E. coli strain expressing (S)-2-HADH
We recently established a high-throughput screening method to screen stereoselective (S)-2-HADH producing  [35]. Pseudomonas aeruginosa CCTCC M 2011394 harboring a flavine mononucleotice (FMN)-dependent (S)-2-HADH, which specifically oxidizes the (S)-isomer of 2-hydroxy acids to 2-keto acids [36], was isolated from the soil samples. Thus, (S)-2-HADH may be used as the biocatalyst for the oxidation step in the designed enantioselective cascade biocatalysis. The gene of (S)-2-HADH from P. aeruginosa CCTCC M 2011394 (Gen-Bank accession number: KU612124) was cloned and expressed in E. coli BL21(DE3). The recombinant E. coli was cultured in LB medium at 37 °C to reach an OD 600 of 0.6 and then induced by the addition of isopropyl β-D-1thiogalactopyranoside (IPTG) at 0.1 mM. The cells were continually grown at 28 °C, 150 rpm for 12 h. The resting cells of the recombinant E. coli strain (E. coli BL21(DE3)/ pET28b-HADH) were used as biocatalysts for the enantioselective oxidation of racemic 2-hydroxy acids with rac-1a as model substrate. The result showed that the activity was lower than 5.0 U/g dry cell weight (DCW) and conversion of 2-keto acid 2a was only 5.1 % after 2 h reaction ( Table 1, entry 1). To achieve economic feasibility and competitiveness for the enantioselective oxidation of 2-hydroxy acids, it is necessary to find an promising (S)-2-HADH showing high activity and enantioselectivity (E). We adopted a genome mining strategy to search for more efficient (S)-2-HADHs. A pBLAST search was conducted by using (S)-2-HADH from P. aeruginosa CCTCCM 2011394 as the template in the NCBI database. Four representative (S)-2-HADHs from Burkholderia xenovorans LB400 (ABE35802.1), P. putida ATCC 12633 (AAC15503.1), P. aeruginosa NUST (AGM49308.1), P.
fluorescens strain EBC191(AAW79575.1) were selected (Additional file 1: Figure S1). After being synthesized in vitro and cloned into pET28b, the four (S)-2-HADH genes were then expressed in E. coli BL21(DE3), respectively. Rac-1a was used as the model substrate to evaluate their activity and enantioselectivity. The results indicated that they all displayed oxidation activities. The resting cells of recombinant E. coli BL21(DE3) expressing the (S)-2-HADH from the B. xenovorans LB400 and P. aeruginosa NUST showed relatively higher activity (>90 U/g DCW) with excellent enantioselectivity (E > 200). After 2 h reaction, the conversions of keto acid 2a reach 49.0 and 48.9 %, respectively, which were near theoretical conversions. However, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that (S)-2-HADH from B. xenovorans LB400 has been expressed in partially soluble state. In the forthcoming experiments, the (S)-2-HADH from P. aeruginosa NSUT was selected for further studies. The requirement of coenzyme in the stereoselective oxidation catalyzed by (S)-2-HADH from P. aeruginosa NSUT was investigated. The activity of (S)-2-HADH was almost lost upon flavin removal. The activity of the apoenzyme was partly reactivated by the addition of FMN. These results confirmed that the (S)-2-HADH from P. aeruginosa NUST is a flavoprotein with FMN as cofactor. The reaction that oxidizes (S)-2-hydroxy acids to 2-keto acids consists of the steps involved in substrate oxidation and FMN reduction [37]. The FMN is then reoxidized by electron transfer to the oxidant. The FMN-dependent (S)-2-HADH family can be divided into three subgroups based on the different oxidants including oxygen, flavocytochrome b2s and ubiquinone utilized in the second oxidative half-reaction [37,38]. Operation parameters, including optimum temperature and pH of the dehydrogenation by the recombinant E. coli BL21(DE3)/pET28b-HADH were investigated. The result showed that the resting cells of recombinant E. coli BL21(DE3)/pET28b-HADH showed high activity at 35-55 °C and pH 7.5-8.5 (Additional file 1: Figure S2). The wide ranges of optimum temperature and pH are very beneficial for the cascade biocatalysis.

Construction of recombinant E. coli strain coexpressing (R)-2-KAR and GDH
Stereoselective (R)-2-KAR could reduce prochiral 2-keto acids to produce corresponding chiral 2-hydroxy acids. The gene of (R)-2-KAR cloned from Leuconostoc mesenteroides CCTCC M 2016063 (GenBank accession number: KU612125) was expressed in E. coli BL21(DE3). After cultivation, the whole cells of recombinant E. coli BL21(DE3)/pET28b-KAR were collected and disrupted by sonication. The (R)-2-KAR with N-terminal histag in the cell free extract was purified to homogeneity by nickel affinity chromatography. The purified (R)-2-KAR migrated as a single band and located at the position of about 32 kDa on SDS-PAGE (Additional file 1: Figure S3), which is in agreement with the molecular mass deduced from its amino acid sequence. The purified enzyme showed little activity with NADPH but full activity with NADH, indicating an NADH-dependence. For the application of reductase, the addition of expensive cofactor often makes the bioreaction less practically feasible from the viewpoint of economic aspects. A coexpression of two enzymes in one E. coli cell seems to be an efficient approach to solve this problem. Thus, we introduced a GDH from Exiguobacterium sibiricum The substituents in substrates and the distance between the hydroxy group and benzene ring are the important factors to affect the catalytic ability of the biocatalyst. Among all the 2-hydroxy acids tested, substrate 2a-2m could be efficiently reduced to (R)-2-1a-1m in >86 % conversion and >99 % ee within 3.5-10 h ( Table 2, entries 1-13). When the OH and OCH 3 were attached to the phenyl ring of the substrates (2n-2q) and the distance between the hydroxy group and benzene ring increased (2r and 2s), the recombinant E. coli exhibited a relatively low activity ( Table 2, entries [14][15][16][17][18][19].

Deracemization of 2-hydroxy acids with the mixtures of recombinant E. coli BL21(DE3)/pET28b-HADH and E. coli BL21(DE3)/pCDFDuet-KAR-GDH
For developing a process for deracemization of racemic 2-hydroxy acids, we coupled the asymmetric oxidation with the opposite stereoselective reduction. Recombinant E. coli BL21(DE3)/pET28b-HADH and E. coli BL21(DE3)/pCDFDuet-KAR-GDH were cultivated, separately, to achieve the resting cells. The cells of the mixed two strains were designed as the catalytic system. The enantioselective cascade biocatalysis for deracemization of 1a-1s was carried out by a one-pot strategy. The conversion, ee of products and reaction time were detected ( Table 3). The result showed that the (R)-isomers of substrates (1a-1m) were obtained in high conversions (>90 %) with >99 % ee. In the case of 1n-1s (  (Table 3, entry 16-17), (R)-2-hydroxy acids were obtained with high enantiomeric excess (>99 %), which indicated that the (S)-isomer of 2-hydroxy acids were completely oxidized. The lower conversion might be due to the accumulation of 2-keto acids. Figure 1 shows a typical deracemization progress for the preparation of (R)-1a from 1a with the mixtures of recombinant E. coli BL21(DE3)/pET28b-HADH and    Figure S5). The constructed three-enzyme system was used for one-pot cascade biocatalysis. The recombinant E. coli BL21(DE3)/pET28b-HADH/pCD-FDuet-KAR-GDH was cultured to achieve the resting cells. The cascade oxidation-reduction reaction catalyzed by the resting cells of the recombinant E. coli were performed at 35 °C and pH 7.5 ( Table 4). The results showed that most of those substrates (1a-1m) can be obtained in up to 98.5 % conversion and >99 % ee in a shorter reaction time as compared to the mixtures of two recombinant E. coli. The reason for this may be that the multienzyme in one recombinant strain avoid the transfer of substrates in different cells. Figure 2 shows a typical time course for production of (R)-1a from racemic 1a with a single recombinant E. coli BL21(DE3)/pET28b-HADH/pCDFDuet-KAR-GDH expressing all the necessary enzymes. After 100 min reaction, (S)-1a was almost completely converted to (R)-1a. The results confirmed that the three-enzyme coexpressing system was more efficient.

Conclusions
In summary, a cascade biocatalysis was successfully developed to prepare valuable (R)-2-hydroxy acids with an efficient three-enzyme system, which was constructed by coexpressing (S)-2-HADH, (R)-2-KAR and GDH. The recombinant E. coli strain coexpressing the three enzymes was proven to be a promising biocatalyst for the cascade bioconversion of a structurally diverse range of racemic 2-hydroxy acids, giving the corresponding (R)-2-hydroxy acids in up to 98.5 % conversion and >99 % ee. The developed elegant cascade biocatalysis possesses high atom efficiency and represents a promising strategy for production of highly valued (R)-2-hydroxy acids.

Table 4 Deracemization of 2-hydroxy acids with recombinant E. coli coexpressing (S)-2-HADH, (R)-2-KAR and GDH
Reactions were performed in one pot at 35   Nucleotide sequences of (S)-2-HADHs from these strains were synthesized using the polymerase chain reaction assembly method [39]. The coding genes were ligated into pET28b and expressed in E. coli BL21(DE3). For the selection of E. coli BL21(DE3) transformants, 50 μg/mL kanamycin was added to the Luria-Bertani (LB) medium (5 g yeast extract, 10 g tryptone, and 10 g NaCl in 1 L of distilled water). The requirement of coenzyme in the stereoselective oxidation catalyzed by (S)-2-HADH from P. aeruginosa NSUT was investigated according to the method as described previously [40].

Microorganisms and culture conditions
P. aeruginosa CCTCCM 2011394 was cultured at 30 °C in rich medium containing 10 g glucose, 10 g yeast extract, 2.5 g K 2 HPO 4 ·3H 2 O, 2.5 g KH 2 PO 4 , 0.2 g MgSO 4 ·7 H 2 O, 0.03 g FeSO 4 ·7 H 2 O and 1.0 g NaCl in 1 L of distilled water (pH 7.0). L. mesenteroides CCTCC M 2016063 was cultured at 30 °C in complete medium containing 10 g glucose, 10 g yeast extract, 10 g tryptone, 5 g NaCl and 5 g beef extract in 1 L of distilled water (pH 7.0). E. coli BL21(DE3) (Novagen, Darmstadt, Germany) were used for gene expression. The E. coli strains were grown at 37 °C in LB medium. The recombinant E. coli strains were grown at 37 °C in LB medium containing appropriate antibiotics (50 μg/mL streptomycin, or 50 μg/mL kanamycin, or both) to reach an OD 600 of 0.6 and then induced by adding IPTG at 0.1 mM. The strains were cultured continually at 28 °C, 150 rpm for 12 h. The whole cells were collected by centrifugation at 9000×g under 4 °C for 10 min, washed twice with 100 mM phosphate buffer (pH 7.5) for activity test and biotransformation.

Cascade deracemization of 2-hydroxy acids with mixture of the resting cells of recombinant E. coli BL21(DE3)/ pET28b-HADH and E. coli BL21(DE3)/pCDFDuet-KAR-GDH
The freshly prepared whole cells of E. coli BL21(DE3)/ pET28b-HADH and E. coli BL21(DE3)/pCDFDuet-KAR-GDH were suspended in 10 mL of phosphate buffer (100 mM, pH 7.5) to a cell concentration of 4 and 8 g DCW/L, respectively. Racemic 2-hydroxy acids 1a-1s was added to the mixture at a final concentration of 20 mM. The mixtures were shaken at 35 °C and 150 rpm in 50-mL flasks. Samples were taken at regular intervals and the reactions were terminated through centrifugation (12,000×g, 4 °C, 5 min). The conversion and ee of products were determined by chiral high-performance liquid chromatographic (HPLC) method.

Additional file
Additional file 1: Figure S1. Amino acid sequences multiple alignment of 2-HADH from P. aeruginosa CCTCC M 2011394, B. xenovorans LB400, P. putida ATCC 12633, P. aeruginosa NUST, P. fluorescens strain EBC191. Figure  S2. Optimization of biooxidation reaction conditions by resting cells of recombinant E. coli BL21(DE3)/pET28b-HADH. (A) Effect of temperature on the biooxidation. The optimum temperature was determined over the range from 25 and 65 °C. The recombinant E. coli BL21(DE3)/pET28b-HADH showed high activity at 35-55 °C, and at higher temperatures the activity began to decrease significantly. (B) Effect of pH on the biooxidation; The optimum pH on the oxidation reaction was determined over the range from 6.0 and 9.0. When the pH was below 7.5 or over 8.5, the enzyme activity decreased dramatically.