Asymmetric bioreduction of γ-and δ-keto acids by native carbonyl reductases from Saccharomyces cerevisiae

Chunlei Ren,Tao Wang,Xiaoyan Zhang,Jiang Pan,Jianhe Xu,2,,Yunpeng Bai,2,

1State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai200237,China

2Shanghai Collaborative Innovation Centre for Biomanufacturing,East China University of Science and Technology,Shanghai200237,China

Keywords:Keto acids/esters(R)-γ-/δ-Decalactones Carbonyl reductase Asymmetric reduction Saccharomyces cerevisiae

ABSTRACT Optically pure(R)-γ-and(R)-δ-lactones can be prepared by intramolecular cyclization of chiral hydroxy acids/esters reduced asymmetrically from γ-and δ-keto acids/esters using Saccharomyces cerevisiae(S.cerevisiae)as a whole-cell biocatalyst.However,some of the enzymes catalyzing these reactions in S.cerevisiae are still unknown up to date.In this report,two carbonyl reductases,OdCR1 and OdCR2,were successfully discovered,and cloned from S.cerevisiae using a genome-mining approach,and overexpressed in Escherichia coli(E.coli).Compared with OdCR1,OdCR2 can reduce 4-oxodecanoic acid and 5-oxodecanoic acid asymmetrically with higher stereoselectivity,generating(R)-γ-decalactone(99%ee)and(R)-δ-decalactone(98%ee)in 85%and 92%yields,respectively.This is the first report of native enzymes from S.cerevisiae for the enzymatic synthesis of chiral γ-and δ-lactones which is of wide uses in food and cosmetic industries.

1.Introduction

Chiral aliphatic γ-and δ-hydroxy acids are key structural components for the production of many fine chemicals and materials[1–8].Currently,the enantioselective reduction of prochiral keto acids is an atom-economic route to these molecules.A variety of methods have been developed to perform such reactions [9–15].For example,Lin et al.reported the asymmetric reduction of aliphatic γ-keto esters in water using a chiral surfactant-type catalyst[11].Arai et al.designed a new catalyst to control the enantioselectivity of chiral lactones and diols by altering the reaction conditions[14].However,although these noble metal complexes can catalyze the asymmetric reduction of keto acids,the reaction conditions are harsh,and the majority of processes are costly and eco-unfriendly.In particular,the enantioselectivity for aliphatic hydroxy acids/esters is still not fully satisfactory.

Compared to chemical catalysis,the bio-catalyzed asymmetric reduction of prochiral carbonyl groups is environmentally benign and highly enantioselective [16–22].The whole cell of S.cerevisiae was first used to convert γ-and δ-keto acids into (R)-hydroxy acids[23,24].Later,optically pure (R)-configuration γ-and δ-lactones,which are used widely as flavors and fragrances in food and cosmetic industries,were produced by S.cerevisiae[25–29].However,until today,no gene sequences have been identified to encode for these carbonyl reductases.Although Kaluzna et al.had investigated systematically 18 key reductases from S.cerevisiae for the bioreduction of α-and β-keto esters [30],none of these enzymes showed measurable activity or high stereoselectivity towards the desired γ-and δ-keto esters(data not shown).Therefore,the exact carbonyl reductase of S.cerevisiae responsible for the asymmetric reduction of γ-and δ-keto esters is still unknown.Recently,we discovered a new carbonyl reductase from Serratia marcescens,SmCR,which showed high enantioselectivity towards a variety of aliphatic γ-and δ-keto acids or esters [31].Using directed evolution technology,its specific activity was enhanced by 86-fold compared with the native enzyme,and the space–time yield of(R)-γ-decalactone reached the unprecedented 1175 g·L?1·d?1so far [32].Based on this work,we hypothesized that SmCR might be used as a template of gene sequences to discover the responsible enzyme(s)for the specific γ-and δ-carbonyl bioreduction by S.cerevisiae.

Herein,we reported two carbonyl reductases identified from S.cerevisiae,OdCR1 and OdCR2,using a genome-mining approach.We found both the enzymes can reduce γ-and δ-carbonyl groups asymmetrically in keto acids/esters,whereas OdCR2 showed high stereoselectivity towards both 4-/5-oxodecanoic acids and their methyl esters,generating γ-/δ-decalactones with excellent enantioselectivity(up to 99%ee).In particular,the substrate preference of OdCR2 is different from that of SmCR,indicating it has a unique catalytic mechanism.OdCR2 is the first enzyme from S.cerevisiae that can be used to synthesize(R)-γ-and(R)-δ-decalactones from γ-and δ-keto acids.It can be further engineered to improve the bioproduction efficiency of chiral aliphatic lactones in the future.

Table 1 Comparison of various carbonyl reductases for 4-oxodecanoic acid reduction

2.Materials and Methods

2.1.Materials

4-Oxodecanoic acid,methyl 4-oxodecanoate,5-oxodecanoic acid and methyl 5-oxodecanoate were obtained from Bestally Biotech (Xiamen,China).Racemic γdecalactone and δ-decalactone were purchased from TCI(Shanghai,China).All chemicals and reagents were purchased from Sigma-Aldrich.E.coli DH5α,E.coli BL21(DE3)and plasmid pET-28a(+)were used for overexpressing enzymes.

2.2.Discovering the genes encoding OdCR1 and OdCR2

The conserved amino acid(aa)sequences of 10 carbonyl reductases were identified by multiple alignment using Clustal X.The conserved aa sequences,e.g.VTGASRGIG,IVNNAGIT and VAPGFI,and the key word“L-3-hydroxyacyl CoA dehydrogenase”were used as the probes to launch WU-Blast2 search in the S.cerevisiae genome database(SGD).In total,nine genes of putative carbonyl reductases were obtained and cloned in the following experiments.

2.3.Cloning,expression,and purification of OdCR1 and OdCR2

Genes of OdCR1 and OdCR2 were amplified by polymerase chain reaction(PCR)using S.cerevisiae genomic DNA as the template.The primers used were listed in Table S2.The PCR products were digested with EcoRI and XhoI and ligated to vector pET-28a(+)digested with the same enzymes to construct pET28a-OdCR1 and pET28a-OdCR2.These plasmids were transformed into E.coli BL21(DE3)cells.The cells were cultured in 100 ml LB broth with 50 mg·L?1Kanamycin at 37°C.Subsequently,isopropyl β-D-1-thiogalactopyranoside(IPTG)of 0.2 mmol·L?1was added to induce the protein expression at OD600of 0.6.The cultures were grown for another 24 h at 16°C and 200 r·min?1.The cells were harvested by centrifugation,sonicated in 100 mmol·L?1phosphate buffer,and centrifuged at 12000g at 4 °C for 20 min.The supernatant was collected and purified using 2 ml of Ni-NTA resin at 4°C.After washing with buffer A(100 mmol·L?1PBS,pH 7.0,plus 0.5 mol·L?1NaCl,20 mmol·L?1imidazole,and 1%Tween 80),the enzyme(OdCR1)was eluted using buffer B(100 mmol·L?1PBS,pH 7.0,200 mmol·L?1imidazole).The purified enzyme was concentrated by ultrafiltration and used for enzyme characterization.The protein concentration was quantified by the Bradford method.The catalytic activity of the putative carbonyl reductases was measured with 4-oxodecanoic acid as substrate at 30°C using the following enzyme assays.

2.4.Enzymatic characterization

The enzyme activity was measured by recording the decrease of NADPH absorbance at 340 nm in a 96-well plate at 30°C.Each well contained a solution with 0.1 mmol·L?1NADPH,2 mmol·L?1substrate,100 mmol·L?1sodium phosphate buffer(pH 7.0)and pure enzyme(OdCR1).For OdCR2,the supernatant of the cell-free extract containing 27.0 mg·ml?1crud enzymes was used to characterize the catalytic properties of OdCR2.The pH-activity profile of OdCR1 and OdCR2 was investigated in 1 ml pH 4–11 buffers containing 2 mmol·L?14-oxodecanoic acid and 0.1 mmol·L?1NADPH at 30 °C.The temperature profile was determined at 20–60°C with the same solution at pH 7.0.Thermostability was determined at 30°C,40°C,and 50°C at pH 7.0.Solvent tolerance was tested by mixing 450 μl enzyme solution with 50 μl organic solvents.After 0.5 h incubation at room temperature,the residual activity was measured in PBS at pH 7.0 and 30°C.A PowerWave XS2 spectrophotometer(BioTek,USA)was used to take the readings.The amount of enzyme that catalyzes the oxidation of 1 μmol NADPH per minute under the above conditions was defined as one unit.

2.5.Preparative synthesis of chiral lactones

The reaction mixture contained the substrate (50 mmol·L?1),D-glucose (100 mmol·L?1),NADP+(0.2 mmol·L?1),lyophilized Escherichia.coli (E.coli)/BmGDH (4 mg·ml?1),wet cells of E.coli/OdCR2(50 or 60 mg·ml?1)and 50 mL PBS(100 mmol·L?1,pH 6.0).The reaction was run at 30°C and pH 6.0 with tittering of 1.0 mol·L?1NaOH solution.After reaction,the pH of the reaction solution was decreased to 2.0 with 20%H2SO4,and the mixture was then heated at 90°C for 2 h.The product was extracted with ethyl acetate,dried over anhydrous Na2SO4,and concentrated under reduced pressure.The product was further purified by column chromatography with a mixture of petroleum ether and ethyl acetate(10∶1,v/v)as the eluent.The enantiopurity of product was analyzed by chiral gas chromatography using a Shimadzu GC-2014 equipped with a CP-Chirasil-Dex CB column(25 m×0.25 mm×0.39 mm,Varian)and a flame ionization detector(FID).Nitrogen was used as the carrier gas.Temperatures of the inlet and detector were set to 280°C.For γdecalactone,the column temperature started at 110°C,increased to 140°C at the rate of 2°C·min?1and hold at this temperature for 10 min.For δ-decalactone,the column temperature hold at 80°C for 10 min,raised to 100°C by 1°C·min?1and hold for 30 min;then raised to 130°C by 0.5°C·min?1and hold for 10 min;then raised to 180°C by 10°C·min?1and hold for 10 min.

3.Results and Discussion

3.1.Discovery of OdCR1 and OdCR2

Recently,we reported a novel carbonyl reductase(SmCR)which can reduce 4-oxodecanoic acid with high enantioselectivity[31].During the screening of SmCR,we constructed a small library of 10 carbonyl reductases from different bacteria with excellent stereoselectivity(>99%ee)that have different sequence identities(40%–70%)and activities as compared with the template enzyme PpCR(Table 1).

Although all of these enzymes were discovered from bacteria,they bear the conserved aa sequences which can also be owned by similar enzymes in S.cerevisiae.Therefore,we used ClustalX to perform a multiple alignment of 10 carbonyl reductases,and found three highly conserved sequences,VTGASRGIG,LVNNAGIT and VAPGFI(Fig.1),which were used as templates to search similar reductases in S.cerevisiae.In addition to conserved sequences,L-3-hydroxyacyl CoA dehydrogenase is a common annotation which was also used to perform the Blast search.In total,9 genes encoding putative carbonyl reductases were found and cloned in E.coli(Table S1,Supplementary Material).Among the 9 yeast enzymes,YKL071W and YIL124W were found to be catalytically active towards 4-oxodecanoic acid,which were used and renamed OdCR1 and OdCR2,respectively.

Fig.1.Multiple alignment of the amino acid sequences of 10 microbial carbonyl reductases with high stereoselectivity towards 4-oxodecanoic acid.The conserved sequences are labeled.

Table 2 Specific activities of OdCR1 and OdCR2 towards different substrates

The genomic annotation indicates that OdCR1 has not been characterized,and OdCR2 is an NADPH-dependent 1-acyl dihydroxyacetonephosphate reductase which has not been investigated for the asymmetric reduction of carbonyls.Both OdCR1 and OdCR2 could be well expressed in E.coli,and the more active OdCR1 was further purified for characterization (Fig.S1).OdCR2 was found to be located in mitochondria of S.cerevisiae according to the genomic annotation in SGD,which is in agreement with the previous report that the responsible enzyme was confirmed to exist in mitochondria [24].However,it was hard to be purified compared to OdCR1 so we had to use the crude enzyme for the preliminary examination of its catalytic properties.

Table 3 Kinetic parameters of OdCR1 towards different substrates

3.2.Substrate specificity of OdCR1 and OdCR2

Fig.2.Enzymatic characterization of OdCR1 for the catalysis of 4-oxodecanoic acid.(a) pH profile,(b)temperature profile,(c) solvent tolerance,and(d)half-times at different temperatures.

Fig.3.Enzymatic characterization of OdCR2 for the catalysis of 4-oxodecanoic acid.(a)pH profile,(b)temperature profile,(c)solvent tolerance,and(d)thermostability at different temperatures.

We are interested in the capability of OdCR1 and OdCR2 for reducing aliphatic γ-and δ-keto acids/esters.Therefore,the specific activity and enantioselectivity of OdCR1 and OdCR2 towards γ-and δ-keto acids/esters were explored(Table 2).As a result,OdCR1 shows a much higher activity(1.05 U·(mg protein)?1towards methyl 4-oxodecanoate(1b)than 4-oxodecanoic acid (1a) (0.02 U·(mg protein)?1).The kinetic parameters confirmed this observation,as OdCR1 shows a smaller Kmand a higher kcattowards 1b,leading to a higher catalytic efficiency(Table 3).However,the enantioselectivity of OdCR1 towards 1a(99%)is higher than 1b(81%),which is similar to the property of SmCR that the stereoselectivity is higher but the activity is lower towards keto acids.In addition,the activity and enantioselectivity of OdCR1 towards δ-keto ester(1d)decreased compared to γ-keto ester 1b respectively,and the enantioselectivity of OdCR1 towards 1c decreased to 6%(S)although its activity increased to 21.0 U·(mg protein)?1,indicating that OdCR1 has a substrate preference for γ-acids/esters.

Notably,the catalytic activity of OdCR2 towards 1a-d is quite different from that of OdCR1(Table 2).OdCR1 displayed lower activity and higher enantioselectivity towards keto acid than keto esters.However,OdCR2 displayed both significantly higher activity(34.8 for 1a vs 1.98 for 1b;31.7 for 1c vs 0.91 for 1d)and higher enantioselectivity(99%,98% for 1a and 1c vs 97%,93% for 1b and 1d,respectively) towards keto acids,which indicates that the catalytic mechanism of OdCR2 from eukaryotic cells is different from SmCR from bacteria.For keto reduction,ketoreductases usually bind the substrate with a large pocket and a small pocket,which determines the direction of the hydride attack and thus stereoselectivity[33,34].The difference of the activity and selectivity of OdCR1 and OdCR2 towards keto acids and keto esters should be caused by the different substrate binding modes,which is studied in our laboratory.

3.3.Characterization of OdCR1

The reduction of 4-oxodecanoic acid by OdCR1 was studied systematically.The pH profile showed that the highest activity was observed at pH 7.0 in a Tirs-HCl buffer(Fig.2a).The optimal temperature is 35°C,at which it was 20%faster than at 25°C(Fig.2b).OdCR1 showed higher tolerance towards organic solvents such as DMSO,DMF and methanol(Fig.2c).The enzymatic activity was studied as a function of time,and half-lives of 91 h,50 h and 0.67 h at 30 °C,40°C and 50°C were recorded,respectively(Fig.2d).This result indicates OdCR1 has a moderate thermostability for industrial applications.

3.4.Characterization of OdCR2

To characterize OdCR2,cells were cultured for 24 h after IPTG induction and then lysed.After centrifuge,the supernatant that contained the crude enzyme(27.0 mg·ml?1)was collected and used for enzyme assay without further purification.OdCR2 showed the highest activity towards 4-oxodecanoic acid at pH 6.5 and 40 °C in PBS buffer (Fig.3a and b).When OdCR2 was incubated with different organic solvents,only DMSO improves its activity by 20%while other solvents decreased its activity significantly(Fig.3c),indicating that DMSO can be used as a co-solvent to increase both the substrate solubility and the enzyme activity.As shown in Fig.3d,the stability of OdCR2 was measured at different temperatures,and its half-lives were 4.3 h,1.5 h and 0.1 h at 30°C,40°C and 50°C,respectively.Compared with OdCR1,OdCR2 showed a much lower thermostability which explained why it was hard to be purified.To apply this enzyme for industrial biocatalysis,the thermostability of OdCR2 needs to be improved significantly in the future.

Fig.4.Bioconversion time-course of substrates 1a (a) and 1c (b) by OdCR2.Reaction mixtures (10 ml):substrate (10 mmol·L?1),D-glucose (20 mmol·L?1),NADP+ (0.2 mmol·L?1),wet cells(0.5 g),E.coli/BmGDH(0.02 g),PBS buffer(pH 6.0,100 mmol·L?1).

3.5.Time courses of asymmetric reduction of 1a and 1c by OdCR2

Subsequently,the asymmetric reduction of 1a and 1c by E.coli/OdCR2 was carried out at 30°C in a 10 ml reaction solution.To recycle NADPH,E.coli expressing a glucose dehydrogenase from Bacillus megaterium was used to regenerate NADPH by catalyzing the oxidation of glucose.96%1a(10 mmol·L?1)was converted into(R)-4-hydroxydecanoic acid within 6 h,which was then cyclized into(R)-γ-decalactone in 99%ee(Fig.4a).Furthermore,10 mmol·L?11c was transformed into (R)-5-hydroxydecanoic acid in 4 h with 85%conversion(Fig.4b),and(R)-δdecalactone was yielded in 98%ee.The high ee values of both the products and the fast conversion rate indicate that OdCR2 is a good candidate for asymmetric reduction of long-chain keto acids.

3.6.Preparative synthesis of(R)-γ-and(R)-δ-decalactones

To further demonstrate the capability of new carbonyl reductases for asymmetric reduction,we employed OdCR2 for the preparative synthesis of (R)-(+)-γ-and (R)-(+)-δ-decalactones through a one-pot chemo-enzymatic route (Table 4).After reduction,the reaction was terminated with 20%H2SO4,and the mixture was heated for intramolecular cyclization.Typically,50 mmol·L?11a or 1c was converted by the whole-cell catalyst E.coli/OdCR2 in a 100 ml system.(R)-γdecalactone of 99% ee was obtained in 85% yield after 6 h,whereas(R)-δ-decalactone of 98%ee was prepared in 92%yield(Table 4).Previously,the wild-type SmCR can also reduce γ-and δ-keto acids asymmetrically,however,the efficiency is low[31].To convert 50 mmol·L?11a or 1c,150 mg·ml?1lyophilized cell-free extracts of E.coli/SmCR were loaded,and the conversions were 95%and 92%after 16 h,respectively.For(R)-δ-decalactone,the ee was 95%.In contrast,OdCR2 can reduce the γ-and δ-keto acids of high concentration with high stereoselectivity and high efficiency,which is difficult to achieve using metal catalysts due to the toxicity of keto acids.

4.Conclusions

Here,we reported two novel carbonyl reductases OdCR1 and OdCR2 cloned from S.cerevisiae for the asymmetric reduction of aliphatic long-chain γ-and δ-keto acids/esters.OdCR1 displayed only high stereoselectivity towards 4-oxodecanoic acid(1a,99%ee (R))while OdCR2 showed high stereoselectivity towards both 4?/5-oxodecanoic acids and their methyl esters(1a-1d,93%–99%ee(R)).Notably,OdCR2 displayed higher activity towards keto acids than keto esters,which is different from the catalytic preference of SmCR reported previously.However,OdCR1 has a better thermostability(91 h half-life)than OdCR2(4.3 h half-life)at 30°C.In a 100 ml system,4-oxo-and 5-oxodecanoic acids could be reduced to (R)-hydroxy acids by OdCR2,which were easily cyclized into (R)-γdecalactone 99% ee and (R)-δ-decalactone (98% ee) in 85%–92%yields.The reactions were performed under mild conditions with a high conversion of up to 99%.As the first isolated enzymes from S.cerevisiae for asymmetric reduction of γ-and δ-keto acids,OdCR1 and OdCR2 provide new examples to study the differences in the mechanism of asymmetric reduction catalyzed by different enzymes,which will pave the way for future applications.

Table 4 Preparative synthesis of(R)-γ-and(R)-δ-decalactones by OdCR2

Acknowledgements

This work was financially sponsored by the National Key Research and Development Program of China(2016YFA0204300,2019YFA09005000),the National Natural Science Foundation of China(21536004,21776085,21871085),the Natural Science Foundation of Shanghai(18ZR1409900),Key Project of the Shanghai Science and Technology Committee(18DZ1112703)and the Fundamental Research Funds for the Central Universities(WF1714026).

Supplementary Material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cjche.2020.07.014.