Stereoselective synthesis of (R)-phenylephrine using recombinant Escherichia coli cells expressing a novel short-chain dehydrogenase/reductase gene from Serratia marcescens BCRC 10948

Peng, Guan-Jhih; Kuan, Yi-Chia; Chou, Hsiao-Yi; Fu, Tze-Kai; Lin, Jia-Shin; Hsu, Wen-Hwei; Yang, Ming-Te

doi:10.1016/j.jbiotec.2013.11.011

Cited by 18 publications

(9 citation statements)

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“…In this case, the metal chelator EDTA did not impact the activity, and most of the tested metal ions did not significantly improved Bg ADH3 activity, revealing that Bg ADH3 is typical of non-metal SDR. Several SCRs have been described capable of oxidizing both NADH and NADPH in the asymmetric reduction [ 26 , 27 ]. In this study, cofactor preference of Bg ADH3 was evaluated using COBE as substrate, indicating that Bg ADH3 was not completely specific to NADH or NADPH as well.…”

Section: Discussionmentioning

confidence: 99%

Efficient biosynthesis of ethyl (R)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli

et al. 2016

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BackgroundEthyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE) is a versatile chiral precursor for many pharmaceuticals. Although several biosynthesis strategies have been documented to convert ethyl 4-chloro-3-oxobutanoate (COBE) to (R)-CHBE, the catalytic efficiency and stereoselectivity are still too low to be scaled up for industrial applications. Due to the increasing demand of (R)-CHBE, it is essential to explore more robust biocatalyst capable of preparing (R)-CHBE efficiently.ResultsA stereoselective carbonyl reductase toolbox was constructed and employed into the asymmetric reduction of COBE to (R)-CHBE. A robust enzyme designed as BgADH3 from Burkholderia gladioli CCTCC M 2012379 exhibited excellent activity and enantioselectivity, and was further characterized and investigated in the asymmetric synthesis of (R)-CHBE. An economical and satisfactory enzyme-coupled cofactor recycling system was created using recombinant Escherichia coli cells co-expressing BgADH3 and glucose dehydrogenase genes to regenerate NADPH in situ. In an aqueous/octanol biphasic system, as much as 1200 mmol COBE was completely converted by using substrate fed-batch strategy to afford (R)-CHBE with 99.9 % ee at a space-time yield per gram of biomass of 4.47 mmol∙L−1∙h−1∙g DCW−1.ConclusionsThese data demonstrate the promising of BgADH3 in practical synthesis of (R)-CHBE as a valuable chiral synthon. This study allows for the further application of BgADH3 in the biosynthesis of chiral alcohols, and establishes a preparative scale process for producing (R)-CHBE with excellent enantiopurity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0301-x) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Efficient biosynthesis of ethyl (R)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli

et al. 2016

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“…Chromosomal S . marcescens DNA was isolated as described previously 21 and used as the template. The amplified product was inserted into the pET30a vector (Novagen, Inc., USA) to generate pET30a· smsdr plasmid, followed by transformation using E .…”

Section: Methodsmentioning

confidence: 99%

“…A few schemes for the asymmetric synthesis of ( R )-PE have been developed 17 – 20 . However, such chemical processes demand the use of high pressure and temperature, as well as environmentally damaging organic solvents 21 .…”

Section: Introductionmentioning

confidence: 99%

“…Sm SDR is a 3-oxoacyl-acyl-carrier-protein (OACP) reductase of the classical SDR subfamily that catalyzes the reduction of its authentic substrate, OACP, to (3 R )-3-hydroxy-ACP, participating in the fourth step of 16-carbon palmate biosynthesis 22 , 23 . Hence, expectedly, the whole-cell conversion process to synthesize ( R )-PE using Sm SDR has a relatively low conversion rate 21 , 24 , 25 . To engineer Sm SDR with higher activity and improve this biotransformation process, a detailed structure-function analysis of Sm SDR and Sm SDR-substrate interactions is necessary.…”

Section: Introductionmentioning

confidence: 99%

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Structure-guided design of Serratia marcescens short-chain dehydrogenase/reductase for stereoselective synthesis of (R)-phenylephrine

Liu

Kuan

Tsou

et al. 2018

Sci Rep

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Bioconversion is useful to produce optically pure enantiomers in the pharmaceutical industry, thereby avoiding problems with side reactions during organic synthesis processes. A short-chain dehydrogenase/reductase from Serratia marcescens BCRC 10948 (SmSDR) can stereoselectively convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into (R)-phenylephrine [(R)-PE], which is marketed medically as a nasal decongestant agent. The whole-cell conversion process for the synthesis of (R)-PE using SmSDR was reported to have an unexpectedly low conversion rate. We reported the crystal structure of the SmSDR and designed profitable variants to improve the enzymatic activity by structure-guided approach. Several important residues in the structure were observed to form hydrophobic clusters that stabilize the mobile loops surrounding the pocket. Of these, Phe98 and Phe202 face toward each other and connect the upper curvature from the two arms (i.e., the α7 helix and loopβ4–α4). The mutant structure of the double substitutions (F98YF202Y) exhibited a hydrogen bond between the curvatures that stabilizes the flexible arms. Site-directed mutagenesis characterization revealed that the mutations (F98Y, F98YF202Y, and F98YF202L) of the flexible loops that stabilize the region exhibited a higher transformation activity toward HPMAE. Together, our results suggest a robust structure-guided approach that can be used to generate a valuable engineered variant for pharmaceutical applications.

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“…These comparative models provide the value of domains/proteins belonging to Serratia spp. in the stereoselective synthesis of pharmaceutically important drugs(Peng et al 2014;Bai et al 2015;Liu et al 2018). In conclusion, preliminary molecular modelling analysis represents the basic study on prnF and further detailing of prnF with FMN and NADPH may pave more insight on two component arylamine oxygenase system in PRN production among rhizobacteria.…”

mentioning

confidence: 90%

Pyrrolnitrin Biosynthesis From Rhizospheric Serratia Spp. With Antifungal Activity and Binding Interactions of PrnF With Ligands

Pawar

Chaudhari

2020

Preprint

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Pyrrolnitrin (PRN) from rhizobacteria displays a key role in biocontrol of phytopathogenic fungi in rhizospheric soil. Therefore, different rhizospheric soils were investigated for the prevalence of PRN producer in minimal salt (MS) medium containing tryptophan (0.2 M NaCl; pH 8) using three successive enrichments. Of 12% isolates, only five bacterial strains had shown PRN secretion, screened with Thin Layer Chromatography (Rf 0.8) and antifungal activity (27 mm) against phytopathogen. The phenetic and 16S rRNA sequence revealed the close affiliation of isolates (KMB, M-2, M-11, TW3, and TO2) to Stenotrophomonas rhizophila (KY800458), Enterobacter spp. (KY800455), Brevibacillus parabrevis (KY800454), Serratia marcescens (KY800456) and Serratia nemtodiphila (KY800457). Purified compound from isolates was characterised using UV, IR, HPLC, LCMS and GCMS as PRN. However, BLASTn hit of prn gene sequences from both Serratia species showed 99% similarity with NADPH dependent FMN reductase component (prnF). The homology protein model of prnF was developed from translated sequence of S. marcescens TW3 with chromate reductase of Escherichia coli K-12. Docking with FMN and NADPH was performed. The study demonstrated the possible role of prnF NADPH dependent FMN reductases in prnD for supply of reduced flavin in rhizobacterial strain of Serratia spp. which may pave a way to understand PRN production.

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Stereoselective synthesis of (R)-phenylephrine using recombinant Escherichia coli cells expressing a novel short-chain dehydrogenase/reductase gene from Serratia marcescens BCRC 10948

Cited by 18 publications

References 5 publications

Efficient biosynthesis of ethyl (R)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli

Efficient biosynthesis of ethyl (R)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli

Structure-guided design of Serratia marcescens short-chain dehydrogenase/reductase for stereoselective synthesis of (R)-phenylephrine

Pyrrolnitrin Biosynthesis From Rhizospheric Serratia Spp. With Antifungal Activity and Binding Interactions of PrnF With Ligands

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