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Apr 25, 2012 - Abstract The strain Achromobacter sp. JA81, which pro- duced enoate reductase, was applied in the asymmetric reduction of activated alkenes.
Appl Microbiol Biotechnol (2012) 95:635–645 DOI 10.1007/s00253-012-4064-6

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Asymmetric bioreduction of activated alkenes by a novel isolate of Achromobacter species producing enoate reductase Yan-Jie Liu & Xiao-Qiong Pei & Hui Lin & Ping Gai & Yu-Chang Liu & Zhong-Liu Wu

Received: 6 January 2012 / Revised: 27 March 2012 / Accepted: 28 March 2012 / Published online: 25 April 2012 # Springer-Verlag 2012

Abstract The strain Achromobacter sp. JA81, which produced enoate reductase, was applied in the asymmetric reduction of activated alkenes. The strain could catalyze the bioreduction of alkenes to form enantiopure (R)-β-arylβ-cyano-propanoic acids, a precursor of (R)-γ-amino butyric acids, including the pharmaceutically active enantiomer of the chiral drug (R)-baclofen with excellent enantioselectivity. It could catalyze as well the stereoselective bioreduction of other activated alkenes such as cyclic imides, β-nitro acrylates, and nitro-alkenes with up to >99 % ee and >99 % conversion. The draft genome sequencing of JA81 revealed six putative old yellow enzyme homologies, and the transcription of one of them, Achr-OYE3, was detected using reverse transcription polymerase chain reaction. The recombinant Escherichia coli expressing Achr-OYE3 displayed enoate reductase activity toward (Z)-3-cyano-3-phenyl-propenoic acid (2a). Keywords Asymmetric reduction . Old yellow enzyme . Enoate reductase . Activated alkene . Achromobacter sp. . Baclofen

Yan-Jie Liu and Xiao-Qiong Pei contributed equally to this study. Electronic supplementary material The online version of this article (doi:10.1007/s00253-012-4064-6) contains supplementary material, which is available to authorized users. Y.-J. Liu : X.-Q. Pei : H. Lin : P. Gai : Y.-C. Liu : Z.-L. Wu (*) Chengdu Institute of Biology, Chinese Academy of Sciences, P.O. Box 416, Chengdu 610041, China e-mail: [email protected] Y.-J. Liu : X.-Q. Pei : H. Lin : P. Gai : Y.-C. Liu Graduate University of the Chinese Academy of Sciences, Beijing 100049, China

Introduction The asymmetric reduction of alkenes to the corresponding enantiopure alkanes can generate up to two chiral centers and is thus one of the most widely used strategies for the production of chiral compounds (Rendler and Oestreich 2007; Stuermer et al. 2007). The biocatalytic approach is catalyzed by enoate reductases [EC1.3.1.31], members of the “old yellow enzyme” (OYE) family (Stuermer et al. 2007; Williams and Bruce 2002), which were first isolated from Saccharomyces carlsbergensis (Warburg and Christian 1932) and later found to be able to catalyze the asymmetric reduction of α,β-unsaturated ketones (Leuenberger et al. 1976) and other activated alkenes (Ohta et al. 1989). These enzymes are ubiquitous and widely distributed in biological sources including higher plants, bacteria, and lower fungi. In the last few decades, both whole cell preparations and isolated enoate reductases have been applied in chemical synthesis for asymmetric C0C reductions (Brenna et al. 2012; Toogood et al. 2010). The substrate spectrum of the OYE family as a whole covers a variety of activated alkenes and alkynes, such as conjugated enals, enones, and ynones, nitro-alkenes, α,βunsaturated nitriles, β-nitro acrylates, and α,β-unsaturated carboxylic acids and their derivatives (Luo et al. 2011; Stueckler et al. 2011; Toogood et al. 2010). Recently, Fryszkowska et al. have reported the asymmetric bioreduction of a new group of alkenes, β-aryl-β-cyanoα,β-unsaturated carboxylic acid (Fryszkowska et al. 2010). The reduction products can be easily transformed into enantiopure γ-amino butyric acids, which serves as the most important inhibitory amino acid transmitter in the mammalian central nervous system (Ong and Kerr 2000). A representative member of this group is 3-(p-chlorophenyl)-γamino butyric acid (baclofen), an antispastic agent on the market under the trade name of Lioresal for treating spasm

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of skeletal muscles, muscle clonus, rigidity, and pain caused by disorders such as multiple sclerosis. Baclofen is currently been used as a racemic mixture, but only (R)-(−)-baclofen is well acknowledged as the pharmaceutically active element (Olpe et al. 1978). The asymmetric bioreduction would afford a promising way to achieve the enantiopure key intermediate. However, despite the efforts to obtain the (R)-enantiomer, Fryszkowska group only obtained the (S)-enantiomer with high conversion and excellent stereoselectivity (89 % to 96 % ee) at a substrate concentration of ~1 g/l using the crude extracts from Clostridium sporogenes, Acetobacterium woodii, and Ruminococcus productus containing enoate reductases (Fryszkowska et al. 2010), and the cultivation and handling of those anaerobic microorganisms required great effort and expertise. Here, we report the isolation of an aerobic strain of Achromobacter sp. producing enoate reductase for the production of (R)-β-phenyl-β-cyano-propanoic acid with excellent stereoselectivity, which could be developed into a biocatalytic approach for the production of precursors of (R)-γ-amino butyric acids, such as (R)-baclofen. The strain can catalyze the stereoselective bioreduction of other activated alkenes as well.

Materials and methods Chemicals Maleimide 1a, 2-methyl-N-phenylmaleimide 4a, [(1E)-2nitro-1-propenyl] benzene 7a and (2E)-2-cyano-3-phenyl2-propenoic acid 8a were purchased from Sigma–Aldrich. Substrates 2a, 3a, and 6a were synthesized according to the literatures (Dean and Blum 1993; Fryszkowska et al. 2008). The substrate (Z)-ethyl 3-nitro-2-phenylacrylate 5a was synthesized from ethyl benzoylformate through Henry reaction (Martin et al. 2008). Racemic 1b and 4b were obtained by Pd/C-catalyzed hydrogenation of 1a and 4a, respectively. Racemic 2b and 3b were synthesized from phenylacetonitrile and 4-chlorophenylacetonitrile, respectively, following literature method (Makosza and Marcinowicz 2001), and the corresponding methyl esters were synthesized from 2b and 3b using Steglich esterification (Neises and Steglich 1978). The reduction of 5a and 6a by sodium borohydride yielded racemic 5b and 6b, respectively. Racemic products 7b and 8b were obtained from 7a and 8a through the reduction with magnesium (Profitt et al. 1975). All other chemicals used were of analytical grade. Isolation and cultivation of microorganisms One gram of soil samples (collected from the Longquan orchard in the suburb of Chengdu of China) was added into

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a 250 ml flask containing 50 ml screening medium (0.1 % (w/v) KH2PO4, 0.2 % (w/v) Na2HPO4, 0.04 % (w/v) MgCl2, 0.04 % (w/v) NH4Cl, pH 7.0) supplemented with 1.5 g citral/l and incubated at 30°C with shaking for 7 days at 180 rpm. Then, 1 ml of the culture was transferred into 50 ml fresh screening medium supplemented with 1.5 g citral/l and incubated at 30°C with shaking at 180 rpm. After 7 days, appropriately diluted broth was plated onto agar plates containing the screening medium supplemented with 2 g citral/l and 15 g agar/l and then incubated at 30°C for 48 h. Each of the resulting single colonies was inoculated into 10 ml fermentation medium (1 % (w/v) peptone, 0.5 % (w/v) yeast extract, 1 % (w/v) NaCl, pH 7.0) and incubated at 30°C for 24 h with shaking at 230 rpm. Then, 2 ml of the culture was transferred into 200 ml fresh fermentation medium and held at 30°C for 36 h with shaking at 230 rpm. Cells were harvested by centrifugation (8,600×g for 8 min at 4°C), washed twice with aqueous NaCl (0.8 %, w/v), and stored at 4°C for further use. 16S rDNA sequence determination Chromosomal DNA of strain JA81 was extracted according to the procedure reported by Wilson (1997). A segment of 16S rDNA was amplified by polymerase chain reaction (PCR) with Taq polymerase using two universal primers: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 5′-GGTT ACCTTGTTACGACTT-3′, cloned into the pMD19-T vector (Takara, Dalian, China), and sequenced at the Invitrogen Life Technologies (Shanghai, China). The sequence was deposited at GenBank under accession no. JN836430. Highly homologous strains were obtained by comparing this sequence with the GenBank database by using nucleotide BLAST. The strain JA81 was deposited at China Center for Type Culture Collection (Wuhan, China) under the acquisition no. CCTCC M 2011369. General procedure with whole cells and analysis of products The whole procedure was performed under aerobic conditions. For microbial screening, biotransformation was carried out at 30°C in 10 ml potassium phosphate buffer (0.1 M, pH 7.0) containing 1 g/l substrate 1a or 2a (dissolved in 100 μl dimethyl sulfoxide), 20 g cell dry weight (CDW)/l, 5 % (w/v) glucose and 5 % (v/v) 2-propanol. After 48 h at 230 rpm, the reaction was terminated by extraction with ethyl acetate three times. The combined organic layers were dried with anhydrous sodium sulfate, concentrated under reduced pressure, and then analyzed. Control experiments were performed in the same reaction system in the absence of microorganisms. Reaction parameters such as co-substrate for coenzyme regeneration, and pH were

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optimized using the same procedure using substrate 2a with a concentration of 1 g/l for 18 h. Biotransformation of other activated alkenes using the whole cells of JA81 was performed in the same reaction system except that glucose was added at a concentration of 1 % (w/v). Preparative biotransformation was carried out under the same conditions except that a higher cell concentration of 20–50 g cell dry weight/l was applied to achieve a complete conversion of each substrate. The final product was purified with column chromatography and subjected to nuclear magnetic resonance (NMR) and optical rotation analysis to confirm the structure and purity. The conversion of 1a to 1b was determined on a Fuli 9790 II gas chromatography (GC) system connected to a flame ionization detector using column 30QC2/AC5 (30 m × 0.22 mm ID, 0.25 μm film thickness; SGE Analytical Science, Australia). Products from 2a and 3a were converted to the corresponding methyl esters to determine the concentrations and enantiomeric excesses. High-performance liquid chromatography (HPLC) analysis was performed on a Shimadzu Prominence LC20 AD system connected to a PDA-detector. The conversion rates of 2a-8a to 2b-8b were analyzed using Inertsil SIL-100A column (4.6×250 mm, GL Sciences Inc., Japan) with n-hexane/2-propanol (70:30v/v) at 0.8 ml/min. Enantiomeric excess were analyzed using Chiralcel OD-H (2b-4b) or OJ-H (5b-8b) columns (4.6×250 mm, Daicel, Japan) with n-hexane/2-propanol (90:10v/v) at 0.8 ml/min. The absolute configurations of the products were established by comparison with the optical rotation reported in the literature. Spectral data for biotransformation products Pyrrolidine-2,5-dione (1b): white solid; 1 H NMR (600 MHz, DMSO): δ 2.55 (t, 4 H, CH2CH2, J01.7 Hz), 3.34 (s, 1 H, NH). (R)-3-Cyano-3-phenyl-propanoicacid (2b): yellow oil; 1 H NMR (600 MHz, CDCl3)—δ ppm 2.93 (dd, 1 H, CH2, J0 6.5, 17.1 Hz), 3.10 (dd, 1 H, CH2, J08.2, 17.1 Hz ), 4.28 (dd, 1 H, CH, J06.6, 8.2 Hz), 7.36–7.48 (m, 5 H, Ar-H); (R)-3-cyano-3- (phenyl)-propanoic acid methyl ester: light yellow oil; 1 H NMR (600 MHz, CDCl3)—δ ppm 2.86 (dd, 1 H, CH2, J06.6, 16.6 Hz), 3.03 (dd, 1 H, CH2, J08.3, 16.6 Hz), 3.72 (s, 3 H, CH3), 4.30 (t, 1 H, CH, J07.4 Hz), 7.34–7.40 (m, 5 H, Ar-H); [α]D25 0+16.3 (c00.4, MeOH) for 98 % ee {lit. (Fryszkowska et al. 2010) [α]D29 0−15.3 (c01.15, MeOH) for 94 % ee, (S)}; retention times—tR (R) 11.5 min, tR (S) 14.8 min. (R)-3-(4-Chloro-phenyl)-3-cyano-propanoic acid (3b): yellow solid; 1 H NMR (600 MHz, CDCl3)—δ ppm 2.88 (dd, 1 H, CH2, J06.8, 17.2 Hz), 3.04 (dd, 1 H, CH2, J08.0, 17.2 Hz), 4.25 (t, 1 H, CH, J07.3 Hz), 7.32 (d, 2 H, Ar-H,

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J08.4 Hz), 7.38 (d, 2 H, Ar-H, J08.4 Hz); (R)-3-(4-chlorophenyl)-3-cyano-propanoicacid methyl ester: white solid; 1 H NMR (600 MHz, CDCl3)—δ ppm 2.85 (dd, 1 H, CH2, J0 7.0, 16.7 Hz), 3.03 (dd, 1 H, CH2, J07.8, 16.7 Hz), 3.73 (s, 3 H, CH3), 4.30 (t, 1 H, CH, J07.4 Hz), 7.32 (d, 2 H, Ar-H, J08.4 Hz), 7.38 (d, 2 H, Ar-H, J08.4 Hz); [α]D25 0+8.2 (c0 0.6, MeOH) for 94 % ee {lit. (Fryszkowska et al. 2010) [α]D29 0−7.5 (c02.15, MeOH) for 91 % ee, (S)}; retention times—tR (R) 13.7 min, tR (S) 15.5 min. (R)-N-Phenyl-2-methylsuccinimide (4b): white solid; 1 H NMR (600 MHz, CDCl3)—δ 1.45 (d, 3 H, CH3, J07.2 Hz), 2.50 (dd, 1 H, CH, J04.5, 17.8 Hz), 3.00–3.09 (m, 1 H, CH2), 3.10 (dd, 1 H, CH2, J09.2, 17.8 Hz ), 7.25–7.50 (m, 5 H, Ar-H); [α]D25 0+7.4 (c02.0, CHCl3) for >99 % ee {lit. (Shimoda et al. 2004) [α]D25 0+7.3 (c00.5, CHCl3) for 100 % ee, (R)}; retention times—tR (R) 19.8 min, tR (S) 21.6 min. (R)-Ethyl-3-nitro-2-phenylpropanoate (5b): light yellow oil; 1 H NMR (600 MHz, CDCl3)—δ ppm 1.22 (t, 3 H, CH3, J07.1 Hz), 4.13–4.25 (m, 2 H, OCH2CH3), 4.42 (dd, 1 H, ArCHCO, J05.2, 10.0 Hz), 4.45 (dd, 1 H, CH2NO2, J05.2, 14.6 Hz), 5.10 (dd, 1 H, CH2NO2, J010.0, 14.6 Hz), 7.34– 7.50 (m, 5 H, Ar-H); [α]D25 0+70 (c00.4, CHCl3) for 45 % ee {lit. (Martin et al. 2008) [α]D25 0−126.2 (c02.8, CHCl3) for 94 % ee, (S)}; retention times—tR(R) 24.2 min, tR (S) 32.3 min. (S)-1-Nitro-2-phenylpropane (6b): light yellow oil; 1 H NMR (600 MHz, CDCl3)—δ ppm 1.39 (d, 3 H, CH3, J0 6.9 Hz), 3.58–3.68 (m, 1 H, CH), 4.45–4.56 (m, 2 H, CH2NO2), 7.20–7.36 (m, 5 H, Ar-H); [α]D25 0−40.0 (c0 1.0, CHCl3) for 97 % ee {lit. (Ohta et al. 1989) [α]D27 0+44.3 (c03.4, CHCl3) for 98 % ee, (R)}; retention times—tR (S) 14.3 min, tR (R) 15.6 min. 1-Phenyl-2-nitropropane (7b): light yellow oil; 1 H NMR (600 MHz, CDCl3)—δ ppm 1.55 (d, 3 H, CH3, J06.6 Hz), 3.02 (dd, 1 H, CH, J06.8, 14.0 Hz), 3.34 (dd, 1 H, CH2, J0 7.4, 14.0 Hz), 4.78 (m, 1 H, CHNO2), 7.16–7.33 (m, 5 H, Ar-H). 2-Cyano-3-phenylpropanoic acid (8b): light yellow oil; 1 H NMR (600 MHz, CDCl3)—δ ppm 3.20 (dd, 1 H, CH2, J0 8.6, 13.9 Hz), 3.28 (dd, 1 H, CH2, J05.4, 13.9 Hz), 3.78 (dd, 1 H, CH, J05.4, 8.6 Hz), 7.24-7.35 (m, 5 H, Ar-H). Draft genome sequencing of Achromobacter sp. JA81 and gene annotation Genomic DNA of Achromobacter sp. JA81 was extracted following previously reported procedure (Wilson 1997). And 10μg DNA was subjected to BGI–Shenzhen (Shenzhen, China) to construct the PCR-free library. Short-insert (350 bp) genomic DNA library was constructed and paired-end sequenced on a Genome Analyzer II (Illumina). The sequence data from library were verified and

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low-quality sequences, base-calling duplicates, and adaptors removed. Paired-end sequence data from the genomic DNA libraries were assembled using SOAPdenovo (Li et al. 2010). All paired-end reads were aligned to contigs to construct scaffolds. Each nucleotide position in the final assembly was assessed for accuracy by aligning all filtered reads to the scaffolds using SOAPaligner (http://soap.genomics.org.cn/ soapdenovo.html). The open reading frame of each gene was predicted and was annotated by KEGG/COG/SwissProt databases. The sequences of the six putative OYE homologies were verified by direct cloning from the genomic DNA and subsequent sequencing. Reverse transcription polymerase chain reaction The total RNA was isolated from freshly harvested cells of Achromobacter sp. JA81 using the RNAiso Plus kit (Takara Shuzo Co., Ltd, Dalian, China) and was used to synthesize cDNA with PrimeScript reverse transcription polymerase chain reaction (RT-PCR) kit following the supplier’s instruction (Takara Shuzo Co., Ltd, Dalian, China). The cDNA was used as the template to amplify target genes using PCR with sequence-specific primers (Table 1). The amplified PCR product was ligated into pMD19-T vector (Takara Shuzo Co., Ltd, Dalian, China), and the sequence was verified by DNA sequencing at Invitrogen Life Technologies (Shanghai, China). Heterologous expression of Achr-OYE3 and biotransformation The Achr-OYE3 gene was amplified with PCR using primers 5′-GGATCC ATG AAG ATC GTT TGC ATC GGC G-3′ and 5′-AAGCTT TCATCG GCC GGC CGC GAT-3′, ligated into pMD19-T vector, digested with EcoR I and Hind III restriction enzymes, and ligated into pET32a(+) plasmid digested with the same enzymes. The resulting plasmid encoding AchrOYE3 was transformed into Escherichia coli Rosetta-gami 2 (DE3) (Novagen, Madison, WI, USA) competent cells and cultivated overnight at 37°C in Luria-Bertani (LB) medium. Then, the starter culture was transferred into fresh LB medium. After incubation at 37°C for 3 h, 0.5 mM IPTG was added, and incubation was continued at 30°C for 3 h with shaking at 230 rpm. The recombinant E. coli cells were harvested by centrifugation and washed with potassium phosphate buffer (0.1 M, pH 7.0). And 0.5 g DCW was applied in the biotransformation in 10 ml potassium phosphate buffer (0.1 M, pH 7.0) containing 1 g/l substrate 2a (dissolved in 100 μl dimethyl sulfoxide), 1 % (w/v) glucose and 5 % (v/v) 2-propanol. The reaction was carried out at 30°C for 48 h with shaking at 230 rpm. The product was extracted and

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analyzed following the same procedure as using the Achromobacter sp. JA81 cells. Nucleotide sequences These nucleotide sequences of Achr-OYE1 to 6 (Achromobacter sp. JA81) were deposited in the GenBank database with accession no. JQ765594, JQ765595, JQ765596, JQ765597, JQ765598, and JQ765599, respectively.

Results Screening of microorganisms for (R)-selective reduction of β-phenyl-β-cyano-α,β-unsaturated carboxylic acid Forty-six strains were isolated from soil samples enriched using citral as their sole carbon and energy source, a condition suitable for the survival of microbial strains producing enoate reductases. Those strains were first tested for their ability to catalyze the reduction of maleimide (1a) to pyrrolidine-2,5-dione (1b) (Fig. 1a), and 16 microbial strains that showed reductive capabilities toward 1a were then subjected to the enantioselective reduction of (Z)-3cyano-3-phenyl-propenoic acid 2a (Fig. 1b). Seven strains were found to catalyze the formation of (R)-3-cyano-3-phenylpropanoic acid (2b) (Table 2) with 35–92 % ee. The partial 16S rDNA sequences were then determined for these strains. The strain JA81 shared the highest identity of 99 % with several strains from the genus of Achromobacter and other six strains belonged to the genus of Pseudomonas. Unlike those of the Pseudomonas species, strains of Achromobacter have not been reported in the asymmetric reduction of carbon–carbon double bonds. Moreover, the strain JA81 afforded the highest conversion of 70 % with Table 1 Sequences of oligonucleotides used in RT-PCR analysis Predicted OYEs

Oligonucleotide sequences

Achr-OYE1

5′-ATGAACCCCCTGTTTGAACCGCTGAAAG-3′ 5′-TCAGGCGGACAGCGCCGGGTAG-3′

Achr-OYE2

5′-ATGAGTCACCTGTTCAGCACCAC-3′ 5′-CTATCGCTGGCCGAAGCGC-3′ 5′-ATGAAGATCGTTTGCATCGGCG-3′ 5′-TCATCGGCCGGCCGCGAT-3′ 5′-ATGAACACACCCGATCCCCTGTTTG-3′ 5′-TCAGGCGCGGTCCGGAAC-3′ 5′-ATGAGCCAGTACTCCGCG-3′ 5′-CTACCGATTGGACGTCTGAG-3′ 5′-ATGAACACGTTCACCGGTCGC-3′ 5′-TCACGCCAGCTGAACCTGC-3′

Achr-OYE3 Achr-OYE4 Achr-OYE5 Achr-OYE6

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Fig. 1 Reactions applied in the screening of microbial strains producing enoate reductases

good enantioselectivity (90 % ee) (Table 2). Therefore, JA81, designated as Achromobacter sp. JA81, was chosen for further study, and its 16S rDNA sequence was deposited in GenBank with accession no. JN836430. The strain Achromobacter sp. JA81 was able to produce the enoate reductase responsible for the reduction of 2a in the absence of any inducer. Although citral was used to enrich the strains producing enoate reductase(s), the addition of citral in the fermentation medium was found to negatively affect both cell growth and the activity (data not shown).

Fig. 2 Effect of glucose concentration on the conversion rate (open circle) and enantioselectivity (filled circle). Bioreduction was carried out with substrate 2a (1 g/l) and 20 g cell dry weight (CDW)/l at 30°C for 18 h in the presence of 5 % (v/v) isopropanol

dropped to 20 % at pH 6.0 from the highest value of 54 % at pH 7.0 but remained at 48 % at pH 8.0. Substrate specificity of Achromobacter sp. JA81

Biotransformation conditions and cofactor dependency of Achromobacter sp. JA81 The biotransformation was initially carried out in the presence of 5 % (w/v) glucose and 5 % (v/v) 2-propanol to facilitate co-enzyme regeneration. Further investigation on the effect of glucose concentration revealed that the conversion and optical purity of product (R)-2b were significantly influenced. The highest enantiomeric excess of 98 % ee was achieved at a glucose concentration of 1 % (w/v) with a 47 % conversion for an 18-h reaction (Fig. 2). The concentration of 2-propanol also affected the activity and selectivity of the reaction, and a concentration of 5 % (v/v) resulted in the best substrate conversion and ee value of product 2b (Fig. 3). The pH of the reaction buffer had little effect on the enantioselectivity of the bioreduction of 2a. However, the conversion rate Table 2 Asymmetric bioreduction of (Z)-3-cyano-3-(phenyl)-propenoic acid 2a Strains

Conv. (%)a

ee (%)

Pseudomonas sp. JB61 Pseudomonas sp. JB62 Pseudomonas sp. CN13 Pseudomonas sp. CN21 Pseudomonas sp. CN11 Achromobacter sp. JA81 Pseudomonas sp. JA91

11 54 4 10 10 70 60

35 80 54 40 50 90 92

a

Conversion was measured using HPLC after 48-h reaction

The bioreductions of a spectrum of typical activated alkenes were investigated to test the substrate tolerance of the strain Achromobacter sp. JA81. After 48-h reaction, 80 % of the screening substrate 2a could be converted to yield (R)-2b in 98 % ee. Substrate 3a, a close analogue of 2a, is a precursor of baclofen. It was reduced to the saturated counterpart (R)-3b with 80 % conversion and excellent enantioselectivity (94 % ee) (Table 3). The optical purity can be easily enhanced to >99 % ee through single crystallization, and the product can be transformed into the pharmaceutically active (R)-enantiomer of the chiral drug baclofen via established chemical transformations without loss of optical purity (Fryszkowska et al. 2010). The substrate 4a turned out to be an excellent substrate, which was readily reduced to the saturated counterparts (R)-4b with quantitative conversion and excellent enantioselectivity (>99 % ee) (Table 3). At an elevated substrate concentration of 54 mM (10 g/l), the reaction remained efficient, yielding the product (R)-4b with 96 % conversion and 97 % ee. The β-substituted nitro-alkane 6a was reduced to the corresponding product (S)-6b with excellent enantioselectivity (97 % ee) and good activity (87 % conversion) (Table 3). Alkenes 5a, 7a, and 8a were also accepted as substrates for the strain Achromobacter sp. JA81, but the resulting products were either having lower enantiopurity or racemic (Table 3). Since the newly formed stereogenic center was adjacent to strong electron withdrawing group(s), the proton at the center would be acidic enough to encounter racemization under the reaction conditions.

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Two activated alkynes, 4-phenyl-3-butyn-2-one and 3phenyl-2-propynoic acid, were also tested for the bioreduction but were not accepted as substrates for the strain Achromobacter sp. JA81. In general, no side reaction or by-product was observed for all the biotransformation reactions, and the unconverted substrates could be recovered from the reaction mixture. Each of the bioreduction reaction of substrates 2a to 8a could reach a conversion rate of >99 % when higher cell concentration of 20–50 g CDW/l was applied, and the increased cell concentration did not affect the enantiopurity of the product. Putative enoate reductases in Achromobacter sp. JA81 Fig. 3 Effect of isopropanol concentration on the conversion rate (open circle) and enantioselectivity (filled circle). Bioreduction was carried out with 2a (1 g/l) and 20 g CDW/l at 30°C for 18 h in the presence of 1 % (w/v) glucose

The draft genome sequence of the chromosome of Achromobacter sp. JA81 had a total assembly size of 6,564,157 bp, with a mean GC content of 66.7 %. This assembly was comprised of 1,404 contigs, which were joined into 43 scaffolds. According to the annotation of genome sequencing

Table 3 Asymmetric bioreduction of various activated alkenes catalyzed with Achromobacter sp. JA81

a

Conversion was measured using HPLC after 48-h reaction. And >99 % conversion could be achieved using 20–50 g CDW/l for substrates 2a-8a

b

Data in brackets were measured at a substrate concentration of 10 g/l

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data of Achromobacter sp. JA81, we found six putative OYE family members that were designated as Achr-OYE1, AchrOYE2, Achr-OYE3, Achr-OYE4, Achr-OYE5, and AchrOYE6. All of the six putative OYEs were directly cloned from the genome of JA81, and their sequences were confirmed. Except Achr-OYE3, all putative OYE genes were of typical length compared with other known OYEs, which contains around 350 amino acids. Achr-OYE3 was the largest and had one complete open reading frame coding for 776 amino acid residues (see, Electronic supplementary material for sequence information). Phylogenetic analysis was performed for the six putative OYEs and 29 OYE enzymes that had been reported to Fig. 4 Phylogenetic relationship of six predicted old yellow enzyme (underlined) homologies in Achromobacter sp. JA81 to other enaote reductases with known function. Amino acid sequences were used to generate the distance tree with Clustal W method with default settings. A distance neighbor-joining tree was then created using the Mega (version 4.0). Bootstrap values expressed as percentages of 1,000 replications are showed at the nodes. The names of the proteins are indicated to the right of the tree with the NCBI accession nos. given in parentheses

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reduce activated alkenes (Fig. 4). The results indicated that these predicted OYE enzymes are only distantly related and belong to phylogenetically distant taxa; Achr-OYE1 Achromobacter sp. is most closely related to the xenobiotic reductase XenB from Pseudomonas fluorescens with 69.5 % identity; Achr-OYE4 is located in a branch with the Shewanella Yellow Enzyme SYE1 from Shewanella oneidensis with 64.6 % identity; Achr-OYE2 is most closely related to the chromate reductase CrS from Thermus scotoductus with 49.3 % identity; Achr-OYE3 and Achr-OYE5 are clustered together with 19.7 % identity; Achr-OYE6 is not clustered with other OYE enzymes but shares a 29.5 % identity with PETN reductase from Enterobacter cloacae.

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Identification of the enoate reductase produced in Achromobacter sp. JA81 To identify the enoate reductase(s) produced in Achromobacter sp. JA81, the transcription of the six putative OYE homologies was examined by RT-PCR. Because the strain JA81 typically required a cultivation period of 36 h to achieve a good catalytic activity, three total RNA samples were isolated from cells that were freshly harvested after 16-h, 24-h, and 36-h cultivation. The transcription of Achr-OYE3 was consistently observed from all three RNA samples, while none of the other OYE homologies was detected (Fig. 5) despite the effort to using different specific primers for the amplification, which clearly indicated the expression of Achr-OYE3 in Achromobacter sp. JA81. The biotransformation of substrate 2a using recombinant E. coli expressing Achr-OYE3 resulted in the product (R)2b with 84 % yield and 97 % ee, while under the same conditions, the E. coli cells harboring the empty vector only yielded around 2 % (R)-2b. A high cell concentration of recombinant E. coli was applied for the biotransformation due to the extremely limited expression of active AchrOYE3 in E. coli. Most of the proteins were formed in inclusion bodies, and the amount of holoprotein in soluble form was rather limited despite our effort to enhance its expression in E. coli, which, in addition to possible instability of the protein, resulted in the unsuccessful attempt to obtain homogenous Achr-OYE3 from recombinant E. coli. Nevertheless, current results including those from RT-PCR and the biotransformation have clearly demonstrated the

Fig. 5 Agarose gel electrophoresis of RT-PCR analysis. M1, marker; 1, Achr-OYE1; 2, Achr-OYE2; 3, Achr-OYE3; 4, Achr-OYE4; 5, Achr-OYE5; 6, Achr-OYE6

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expression of Achr-OYE3 in Achromobacter sp. JA81 and its function as an enoate reductase.

Discussion The strains of Achromobacter sp. have been known for their abilities to biodegrade methyl tert-butyl ether (Eixarch and Constanti 2010), p-nitrophenol (Wan et al. 2007), bisphenol (Zhang et al. 2007), endosulfan (Li et al. 2009), and hexavalent chromium (Zhu et al. 2008) and to catalyze the asymmetric acyloin condensation reaction (Guo et al. 2001) but not the reduction of alkenes. Bacteria from other genera such as Pseudomonas, Bacillus, Clostridium, Ruminococcus, Acetobacterium, Zymomonas, and Agrobacterium have been reported to contain enoate reductases (Toogood et al. 2010). By mining the database, we found several protein sequences originated from the genome sequencing data of Achromobacter strains that are predicted to be OYE family members. However, so far, there has been no experimental proof of their functions. In the current work, a newly isolated strain of Achromobacter sp. JA81 was applied to the biocatalytic (R)-selective reduction of β-aryl-β-cyano-α,β-unsaturated carboxylic acid to produce precursors of γ-amino butyric acid including the pharmaceutically active (R)-enantiomer of the antispastic drug baclofen. Over the years, despite efforts to make enantiopure (R)-baclofen through chemical or biocatalytical resolution (Brenna et al. 1997) or enantioselective synthesis (Thakur et al. 2003), none of the process has been commercialized due to disadvantages such as the need for separation of diastereo-isomers and the use of expensive chiral reagents in stoichiometric amounts. Therefore, baclofen remains sold as a racemic mixture. The asymmetric bioreduction of 3a catalyzed with Achromobacter sp. JA81 would afford a promising approach to achieve the optically pure (R)-enantiomer. Moreover, the strain can also catalyze the stereoselective bioreduction of cyclic imides, β-nitro acrylates, and nitroalkenes, and the corresponding products often serve as important chiral building blocks in chemical synthesis. For example, the enantiopure succinimides 4b are building blocks for the synthesis of biologically active natural products, such as, pyrrolizidine and indolizidine alkaloids (Hegazy et al. 2006). Although it has been achieved using other enoate reductases (Hegazy et al. 2006; Sortino and Alicia Zacchino 2010; Sortino et al. 2009), the reported substrate concentration only reached 5 mM (0.94 g/l). On the contrary, the present system can well tolerate a substrate concentration of 54 mM (10 g/l). The draft genome sequence of Achromobacter sp. JA81 has revealed six OYE homologies, but the transcription of only one of them, Achr-OYE3, has been detected.

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Preliminary results on the other five homologies showed that they all displayed activity toward substrate 2a, albeit with varied enantioselectivity ranging from 50 % to 97 % ee (data not shown). It is possible that their transcription and expression could be initiated under varied cultivation conditions, and some might be inducible under certain stress or nutritional deficiency conditions. Their characterizations are worthy of further investigation, which is actively pursued in this laboratory. The sequence of Achr-OYE3 contains 773 amino acids, which is around two times longer than typical OYE enzymes, which usually constitutes around 350 amino acids. Although BLAST search returned many OYE homologies with similar length of over 700 amino acids as Achr-OYE3,

those are all predicted enzymes without any experimental information on their characteristics and share a maximal of 83 % identity with Achr-OYE3 (Fig. 6). The amino acid sequence alignment of Achr-OYE3 with two of those predicted enzymes and two known enoate reductases, YqjM from Bacillus subtilis and XenA from Pseudomonas putida are shown in Fig. 6. The N terminus of Achr-OYE3 is not aligned with YqjM and XenA but well aligned with the two predicted enzymes. This region may contain the C-terminal partial sequence of the Rossmannfold NAD(P)+-binding (NADB) domain (boxed in black), lacking ~250 amino acids from the N terminus of NADB. The NADB domain is found in numerous dehydrogenases of metabolic pathways such as glycolysis and many other

Fig. 6 Multiple sequence alignment of OYE family enzymes and putative dehydrogenases. Accession numbers: JQ765596, AchrOYE3, predicted OYE family enzyme in this work; YP004234554, predicted NADPH dehydrogenase from Acidovorax avenae ATCC 19860; CCA87372, putative oxidoreductase from Ralstonia syzygii R24; P54550, known enoate reductase YqjM from B. subtilis; Q9R9V9, known enoate reductase XenA from P. putida). Residues

that are strictly conserved are shaded in dark blue; well-conserved residues are in light red; consensus of at least 50 % identical amino acid residues is shaded in light blue. The predicted partial Rossmannfold NAD(P)+-binding domain is boxed in black. Three functionally conserved amino acids are boxed with red lines. Residues that may be involved in FMN binding are marked with a star on top

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redox enzymes, but not particularly for enoate reductases. The role of this region in the function of Achr-OYE3 is yet to be investigated. The C terminus of Achr-OYE3 is well aligned with two predicted enzymes, and YqjM and XenA. In this region, AchrOYE3 shares identities of 34.8 % and 35.3 % with YqjM and XenA, respectively. Residues His580, His583, and Tyr585 (numbering according to Achr-OYE3) are the active-site residues strictly conserved for ‘classical’ OYEs. Functionally important residues involved in the binding of FMN are also conserved in Achr-OYE3 (Fig.6). Those sequence analysis information, in combination with the fact that recombinant E. coli expressing Achr-OYE3 can catalyze the asymmetric reduction of 2a, has indicated that AchrOYE3 is indeed an FMN-dependent enoate reductase of the OYE family. In conclusion, a novel enoate-reductase-producing strain Achromobacter sp. JA81 was isolated from soil. The strain can successfully reduce prochiral β-aryl-β-cyano-α,βunsaturated carboxylic acid to (R)-β-aryl-β-cyano-propanoic acid with excellent enantioselectivity and catalyze the stereoselective bioreduction of cyclic imides, β-nitro acrylates, and nitro-alkenes. The OYE homology, Achr-OYE3, appeared to be responsible for the enoate reductase activity. Acknowledgment This work was supported by the National Natural Science Foundation of China (20802073 and 21072183), the 100 Talents Program and the West Light Foundation of the Chinese Academy of Sciences.

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