Complementary selectivity to (S)-1-phenyl-1,2-ethanediol-forming Candida parapsilosis by expressing its carbonyl reductase in Escherichia coli for (R)-specific reduction of 2-hydroxyacetophenone

An (R)-specific carbonyl reductase from Candida parapsilosis CCTCCM203011 (CprCR) was shown to catalyze the asymmetric reduction of 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol (PED), which is a critical chiral building block in organic synthesis. The gene (rcr) encoding CprCR was cloned based on the amino acid sequences of tryptic fragments of the enzyme. Sequence analysis revealed that rcr is comprised of 1008 nucleotides encoding a 35 977 Da polypeptide, and shares similarity to proteins of the medium-chain dehydrogenase/reductase (MDR) superfamily. Recombinant rcr expressed in Escherichia coli showed a specific 2-hydroxyacetophenone-reducing activity. Using rcr expressing cells, (R)-PED was obtained by asymmetric reduction, which is complementary in enantiomeric configuration to (S)-PED obtained by using whole cells of C. parapsilosis. After optimization of reaction conditions, (R)-PED was produced at 95.5% enantiomeric excess with a yield of 92.6% when isopropanol was used for cofactor regeneration.     

Complementary selectivity to (S)-1-phenyl-1,2-ethanediol-forming Candida parapsilosis by expressing its carbonyl reductase in Escherichia coli for (R)-specific reduction of 2-hydroxyacetophenone

An (R)-specific carbonyl reductase from Candida parapsilosis CCTCCM203011 (CprCR) was shown to catalyze the asymmetric reduction of 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol (PED), which is a critical chiral building block in organic synthesis. The gene (rcr) encoding CprCR was cloned based on the amino acid sequences of tryptic fragments of the enzyme. Sequence analysis revealed that rcr is comprised of 1008 nucleotides encoding a 35 977 Da polypeptide, and shares similarity to proteins of the medium-chain dehydrogenase/reductase (MDR) superfamily. Recombinant rcr expressed in Escherichia coli showed a specific 2-hydroxyacetophenone-reducing activity. Using rcr expressing cells, (R)-PED was obtained by asymmetric reduction, which is complementary in enantiomeric configuration to (S)-PED obtained by using whole cells of C. parapsilosis. After optimization of reaction conditions, (R)-PED was produced at 95.5% enantiomeric excess with a yield of 92.6% when isopropanol was used for cofactor regeneration.     

Cloning and expression of the gene encoding (R)-specific carbonyl reductase from Candida parapsilosis CCTCC M203011

The gene which encodes (R)-specific carbonyl reductase (rCR) from Candida parapsilosis CCTCC M203011 was cloned, sequenced and compared with genes from the GenBank. The results indicated that rCR gene was 1011 bp, encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa, and its nucleotide sequence showed 99% similarity to those of other members of the alcohol dehydrogenase superfamily. The rCR gene could express in recombinant strain Escherichia coli JM 109, and the expression plasmid could produce (R)-1-pheny-1,2-ethanediol (100% e.e., 80.14% yield) fromβ-hydroxyacetophenone without any additive to regenerate NAD+ from NADH.     

一种新型(S)-羰基还原酶的克隆及其功能表达

【目的】从近平滑假丝酵母(Candida parapsilosis CCTCC M203011)基因组中钓取新型(S)-羰基还原酶基因(scrⅡ),对其生物转化手性醇的功能进行了验证。【方法】采用PCR的方法,从C.parapsilosis基因组中扩增出一段可能的羰基还原酶基因scrⅡ。以构建的重组菌Escherichia coli BL21/pET28a-scrⅡ为生物催化剂,2-羟基苯乙酮为底物进行催化反应,经HPLC分析,计算终产物的光学纯度和产率,确定了转化反应的最适温度和pH值。【结果】scrⅡ基因全长为840bp,编码279个氨基酸,与已报道的(S)-羰基还原酶基因scr的一致性为85%。氨基酸序列分析表明SCRⅡ具有典型短链醇脱氢酶的功能域:辅酶结合区域Thr40-Gly41-(X)3-Gly45-X-Gly47和催化三联体结构Ser172-(X)n-Tyr187-(X)3-Lys191。在30℃,0.1mmol/LIPTG的诱导下,(S)-羰基还原酶(SCRⅡ)在E.coli中过量表达。以10%(w/v)的重组菌为催化剂,高浓度(6g/L)2-羟基苯乙酮为底物,在最适反应温度35℃和pH5.5的条件下,转化产物(S)-苯基乙二醇的光学纯度高达99.1%e.e.,产率为89.6%。与(S)-羰基还原酶SCR相比较,底物浓度提高了一倍,产物的光学纯度和产率分别提高了10%和28%。【结论】采用分子克隆技术分离出新型羰基还原酶SCRⅡ的编码基因,该酶的发现为手性醇的高效制备奠定了坚实的研究基础。     

Cloning and expression of the gene encoding (R)-specific carbonyl reductase from Candida parapsilosis CCTCC M203011

The gene which encodes (R)-specific carbonyl reductase (rCR) from Candida parapsilosis CCTCC M203011 was cloned, sequenced and compared with genes from the GenBank. The results indicated that rCR gene was 1011 bp, encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa, and its nucleotide sequence showed 99% similarity to those of other members of the alcohol dehydrogenase superfamily. The rCR gene could express in recombinant strain Escherichia coli JM109, and the expression plasmid could produce (R)-1-pheny-1,2-ethanediol (100% e.e., 80.14% yield) from β-hydroxyacetophenone without any additive to regenerate NAD⁺ from NADH. __________ Translated from Microbiology, 2006, 33(4): 112–118 [译自: 微生物学通报]     

（R）与（S）-羰基还原酶偶联一步法制备（S）-苯乙二醇

【目的】通过 (R) - 和(S) -羰基还原酶在大肠杆菌中偶联,实现了一步法制备（S）-苯乙二醇的生物转化过程。【方法】将来源于近平滑假丝酵母(Candida parapsilosis CCTCC M203011)的(R)- 羰基还原酶基因（rcr）和(S) -羰基还原酶基因（scr）串联于共表达载体pETDuetTM-1上。重组质粒pETDuet-rcr-scr转化稀有密码子优化型菌株Escherichia coli Rosetta,获得酶偶联重组菌株E. coli Rosetta / pETDuet-rcr-scr。当重组菌体培养至OD600 0.6-0.8时,添加终浓度1 mmol/L IPTG,30℃诱导蛋白表达10 h。【结果】SDS-PAGE结果表明(R)- 和(S) -羰基还原酶均明显表达,它们的相对分子质量分别为37 kDa和30 kDa。重组菌生物转化结果表明：在pH7.0的磷酸缓冲液中,添加5 mmol/L Zn2+时,获得产物(S)-苯乙二醇,产物光学纯度为91.3% e.e.,产率为75.9%。【讨论】采用分子重组技术成功整合了两种氧化还原酶的催化功能,实现了(S)- 苯乙二醇的一步法转化,为简化手性醇制备途径提供了一条崭新的思路。     

Purification and characterization of the NADH-dependent (S)-specific 3-oxobutyryl-CoA reductase from Clostridium tyrobutyricum

An NADH-dependent (S)-specific 3-oxobutyryl-CoA reductase from Clostridium tyrobutyricum was purified 15-fold with a yield of 46%. It was homogeneous by gel electrophoresis after three chromatographic steps. The apparent molecular mass was estimated by column chromatography to be 240 kDa. SDS-gel electrophoresis revealed the presence of 33 kDa subunits. Substrates of the enzyme were ethyl and methyl 3-oxobutyrate, 3-oxobutyryl-N-acetylcysteamine thioester, and 3-oxobutyryl coenzyme A. The specific activities were 340 and 10 U (mg protein)^-1 for the reduction of 3-oxobutyryl coenzyme A and ethyl 3-oxobutyrate, respectively; the Michaelis constants were 300 M and 300 mM, respectively. The identity of 12 N-terminal amino acid residues was determined. The ezmyme was used in a preparative reduction of substrate, yielding ethyl (S)-3-hydroxybutyrate (>99% enantiomeric excess).     

(R)-专一性羰基还原酶与甲酸脱氢酶基因在大肠杆菌中的共表达

【目的】通过研究(R)-专一性羰基还原酶和甲酸脱氢酶基因在大肠杆菌中的共表达,解决较高底物浓度下不对称转化反应的辅酶限制性问题。【方法】分别以近平滑假丝酵母(Candida parapsilosis CCTCC M203011)和博伊丁假丝酵母(Candida boidinii)基因组为模板,采用PCR方法扩增得到(R)-专一性羰基还原酶基因（rcr）和甲酸脱氢酶基因（fdh）,克隆到共表达载体pETDuetTM-1中进行表达。共表达质粒pETDuet-rcr-fdh转化稀有密码子优化型菌株E. coli Rosetta,获得重组菌E. coli Rosetta/pETDuet-rcr-fdh。【结果】在30℃条件下,经1 mmol/L IPTG诱导表达8 h后,SDS-PAGE结果表明(R)-专一性羰基还原酶和甲酸脱氢酶均有明显的表达,其相对分子质量分别为37 kDa和 40 kDa。以高浓度(6 g/L)2-羟基苯乙酮为底物时,0.1 g重组菌细胞催化产生(R)-苯基乙二醇,产物光学纯度为100% e.e.,产率为85.9%。与无甲酸脱氢酶参与辅酶再生循环的重组菌E. coli Rosetta/pETDuet-rcr相比,产物光学纯度和产率分别提高了1.3和2.7倍。【讨论】该重组菌的构建为基因工程法生物合成(R)-苯基乙二醇的工业应用奠定了基础。     

A novel NADPH-dependent aldehyde reductase, catalyzing asymmetric reduction of ß-keto acid esters, from Sporobolomyces salmonicolor: purification and characterization

Abstract NADPH-dependent aldehyde reductase (EC 1.1.1.2) was purified 23-fold with an overall yield of 11% from Sporobolomyces salmonicolor AKU 4429, in 4 steps and, by adding ammonium sulfate, the enzyme was crystallized. The enzyme has a strict requirement for NADPH and irrversibly reduces a number of aldehydes, such as p -nitrobenzaldehyde, pyridine-3-aldehyde and d -glyceraldehyde. Furthermore, it was found that the enzyme catalyses stereospecific reduction of 4-halo-3-oxobutanoate esters to the corresponding ( R )-4-halo-3-hydroxybutanoate esters, which are promising chiral compounds for the chemical synthesis of l -carnitine.     

Carbonyl reductase SCRII from Candida parapsilosis catalyzes anti-Prelog reaction to (S)-1-phenyl-1,2-ethanediol with absolute stereochemical selectivity

An (S)-specific carbonyl reductase (SCRII) was purified to homogeneity from Candida parapsilosis by following an anti-Prelog reducing activity of 2-hydroxyacetophenone. Peptide mass fingerprinting analysis shows SCRII belongs to short-chain dehydrogenase/reductase family. Its coding gene was cloned and overexpressed in Escherichia coli. The recombinant SCRII displays the similar enzymatic characterization and catalytic properties to SCR. It catalyzes the enantioselective reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with excellent optical purity of 100% in higher yield than SCR. Based on the sequence-structure alignment, several single-point mutations inside or adjacent to the substrate-binding loop or active site were designed. With respect to recombinant native SCRII, the A220 and E228 mutations almost lost enantioselectivity towards 2-hydroxyacetophenone reduction. The catalytic efficiencies (k_cat/K_m) for the A220 or E228 variants are <7% that of the unmutated enzyme. This work provides an excellent catalyst for enantiopure alcohol preparation and the lethal mutations of A220 and E228 suggest their importance in substrate-binding and/or catalysis.     

Coexpression of a carbonyl reductase and glucose 6-phosphate dehydrogenase in Pichia pastoris improves the production of (S)-1-phenyl-1,2-ethanediol

Abstract

To develop an efficient biocatalyst to produce optically active (S)-phenyl ethanediol (PED), a carbonyl reductase SCRII and glucose 6-phosphate dehydrogenase were coexpressed intracellularly in Pichia pastoris. The recombinant enzyme PpSCRII was purified with a specific activity of 8.32 U mg^?1, over 36% higher than that of Escherichia coli SCRII. The recombinant cells P. pastoris/SCRIIG catalyzed the reduction of 2-hydroxyacetophenone to give (S)-PED with optical purity of >99% in a yield of 96.3%. The yield was improved by 19.9% and 25.7% over E. coli BL21/SCRII and Candida parapsilosis, respectively, when the reaction duration was shorted from 48 h to 24 h. When using glucose 50 g L^?1 as co-substrate, these P. pastoris/SCRIIG cells could be reused ten times and the optical purity and yield of (S)-PED kept at >99% enantiomeric excess and >85%, respectively.     

A novel carbonyl reductase from Pichia stipitis for the production of ethyl (S)-4-chloro-3-hydroxybutanoate

An NADPH-dependent carbonyl reductase (PsCR) gene from Pichia stipitis was cloned. It contains an open reading frame of 849 bp encoding 283 amino acids whose sequence had less than 60% identity to known reductases that produce ethyl (S)-4-chloro-3-hydroxybutanoates (S-CHBE). When expressed in Escherichia coli, the recombinant PsCR exhibited an activity of 27 U/mg using ethyl 4-chloro-3-oxobutanoate (COBE) as a substrate. Reduction of COBE to (S)-CHBE by transformants in an aqueous mono-phase system for 18 h, gave a molar yield of 94% and an optical purity of the (S)-isomer of more than 99% enantiomeric excess.     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。     

Steady-state kinetics of the oxidation of (S)-1-phenyl-1,2-ethanediol catalyzed by alcohol dehydrogenase from Candida parapsilosis CCTCC M203011

Kinetic studies of oxidation reaction of (S)-1-phenyl-1,2-ethanediol (PED) catalyzed by a NAD⁺-dependent alcohol dehydrogenase from Candida parapsilosis CCTCC M203011 obtained from China Center for Type Culture Collection (CPADH) were observed for getting insight into the deracemization redox reaction. The data of initial velocity experiments in the absence of product, product (β-hydroxy-hypnone) inhibition experiments and dead-end (pyrazole) inhibition experiments strongly suggest that the reaction follows Theorell-Chance BiBi mechanism in which the coenzymes bind to the free form of the enzyme firstly. The kinetic parameters of this model were estimated by using non-linear regression analysis software.     

A novel NADH-dependent carbonyl reductase from Kluyveromyces aestuarii and comparison of NADH-regeneration system for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate

To compare NADH-regeneration systems for the synthesis of (S)-4-chloro-3-hydroxybutanoate (ECHB), a novel NADH-dependent carbonyl reductase (KaCR1), which reduced ethyl 4-chloroacetoacetate (ECAA) to form (S)-ECHB, was screened and purified from Kluyveromyces aestuarii and a gene encoding KaCR1 was cloned. Glucose dehydrogenase (GDH) and formate dehydrogenase (FDH) were compared as enzymes for NADH regeneration using Escherichia coli cells coexpressing each enzyme with KaCR1. E. coli cells coexpressing GDH produced 45.6 g/l of (S)-ECHB from 50 g/l of ECAA and E. coli cells coexpressing FDH, alternatively, produced only 19.0 g/l. The low productivity in the case of FDH was suggested to result from the low activity and instability of FDH.     

Purification and characterization of a novel NADH-dependent carbonyl reductase from Pichia stipitis involved in biosynthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate

A novel NADH-dependent dehydrogenases/reductases (SDRs) superfamily reductase (PsCRII) was isolated from Pichia stipitis. It produced ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] in greater than 99% enantiomeric excess. This enzyme was purified to homogeneity by ammonium sulfate precipitation followed by Q-Sepharose chromatography. Compared to similar known reductases producing (S)-CHBE, PsCR II was more suitable for production since the purified PsCRII preferred the inexpensive cofactor NADH to NADPH as the electron donor. Furthermore, the Km of PsCRII for ethyl 4-chloro-3-oxobutanoate (COBE) was 3.3 mM, and the corresponding Vmax was 224 μmol/mg protein/min. The catalytic efficiency is the highest value ever reported for NADH-dependent reductases from yeasts that produce CHBE with high enantioselectivity. In addition, this enzyme exhibited broad substrate specificity for several β-keto esters using NADH as the coenzyme. The properties of PsCRII with those of other carbonyl reductases from yeasts were also compared in this study.     

Purification and some properties of (1R,2S)-1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase from Comamonas testosteroni T-2

Abstract Inducible (1 R ,2 S )-1,2-dihydroxy-3,5-cyclohexadiene-l,4-dicarboxylate (diene-diol) dehydrogenase was found in extracts of Comamonas testosteroni T-2 grown in p -toluate-or terephthalate-salts medium and it was purified using anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is a homodimer with subunit M r 39000. It had a specific activity of 500 mkat/kg of protein and was activated by the addition of Fe²⁺. The dehydrogenase converted 1 mol diene-diol and 1 mol NAD⁺ to 1 mol protocatechuic acid, 1 mol NADH and 1 mol CO₂. Apparent K _m-values of 43 μM (NAD⁺) and about 90 μM (diene-diol) were determined. The hydride ion was transferred to the si face of NAD⁺.     

Large‐scale synthesis of tert‐butyl (3R,5S)‐6‐chloro‐3,5‐dihydroxyhexanoate by a stereoselective carbonyl reductase with high substrate concentration and product yield

To biosynthesize the (3R,5S)‐CDHH in an industrial scale, a newly synthesized stereoselective short chain carbonyl reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW, and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient production of (3R,5S)‐CDHH. The scale‐up synthesis of (3R,5S)‐CDHH was performed in 5000 L bioreactor with 400 g/L of (S)‐CHOH at 30°C, resulting in a space‐time yield of 13.7 mM/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)‐CDHH reached 97.5% and 99.5%, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:612–620, 2017     

A novel FAD-protein that allows effective reduction of methyl viologen by NADH (NADH-methyl viologen reductase) from photosynthetic bacterium, Rhodospirillum rubrum: purification and characterization

It was found that the cytoplasm of light-grown cells of Rhodospirillum rubrum could catalyze the reduction of methyl viologen (MV) (Em, 7 = -0.44 V) by NADH and NADPH. In the present study, the enzyme capable of catalyzing MV reduction by NADH (NADH-MV reductase) was purified 1,500-fold from an extract of cells with a yield of 4.4%. The purification procedure comprised (NH4)2SO4 fractionation, and chromatographies on Sepharose CL-6B, DEAE-Sepharose CL-6B, phenyl-Sepharose CL-4B, Blue-Cellulofine, and TSK-Gel G3000SW. Two NADPH-MV reductases were separated during the purification. The NADH-MV reductase obtained was nearly homogeneous, as judged on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The enzyme has a molecular weight of 220,000 and an isoelectric point of 4.8; it is composed of four subunits with a molecular weight of 57,000, and is bound with about 1 mol FAD/mol subunit. The activity is optimum at pH 8. The Km values for NADH and MV are 115 microM and 1.3 mM, respectively, with a molecular activity of 13,000 min-1. The activity was stimulated 2.4-fold in the presence of 20-100 mM ammonium ions. The enzyme also catalyzed the reduction of benzyl viologen, methylene blue and 2,6-dichlorophenol-indophenol (Em, 7 = -0.36, +0.011, and +0.217 V, respectively) at comparable rates. The ratios of the activity with NADH to that with NADPH were 80, 133, 41, and 5.5 with MV, benzyl viologen, methylene blue and 2,6-dichlorophenolindophenol, respectively. The enzyme was significantly stable in the presence of both 5mM 2-mercaptoethanol and 20% (w/v) glycerol. The activity was not appreciably influenced by the presence of 2 M urea, although the reagent caused dissociation to the subunits.     

Enantioconvergent production of (R)-1-phenyl-1,2-ethanediol from styrene oxide by combining the Solanum tuberosum and an evolved Agrobacterium radiobacter AD1 epoxide hydrolases

Soluble epoxide hydrolase (EH) from the potato Solanum tuberosum and an evolved EH of the bacterium Agrobacterium radiobacter AD1, EchA-I219F, were purified for the enantioconvergent hydrolysis of racemic styrene oxide into the single product (R)-1-phenyl-1,2-ethanediol, which is an important intermediate for pharmaceuticals. EchA-I219F has enhanced enantioselectivity (enantiomeric ratio of 91 based on products) for converting (R)-styrene oxide to (R)-1-phenyl-1,2-ethanediol (2.0 +/- 0.2 micromol/min/mg), and the potato EH converts (S)-styrene oxide primarily to the same enantiomer, (R)-1-phenyl-1,2-ethanediol (22 +/- 1 micromol/min/mg), with an enantiomeric ratio of 40 +/- 17 (based on substrates). By mixing these two purified enzymes, inexpensive racemic styrene oxide (5 mM) was converted at 100% yield to 98% enantiomeric excess (R)-1-phenyl-1,2-ethanediol at 4.7 +/- 0.7 micromol/min/mg. Hence, at least 99% of substrate is converted into a single stereospecific product at a rapid rate.