Dataset Information

Immobilization of Cofactor Self-Sufficient Recombinant Escherichia coli for Enantioselective Biosynthesis of (R)-1-Phenyl-1,2-Ethanediol.

ABSTRACT: (R)-1-phenyl-1,2-ethanediol is an important synthon for the preparation of ?-adrenergic blocking agents. This study identified a (2R,3R)-butanediol dehydrogenase (KgBDH) from Kurthia gibsonii SC0312, which showed high enantioselectivity for production of (R)-1-phenyl-1,2-ethanediol by reduction of 2-hydroxyacetophenone. KgBDH was expressed in a recombinant engineered strain, purified, and characterized. It showed good catalytic activity at pH 6-8 and better stability in alkaline (pH 7.5-8) than an acidic environment (pH 6.0-7.0), providing approximately 73 and 88% of residual activity after 96 h at pH 7.5 and 8.0, respectively. The maximum catalytic activity was obtained at 45°C; nevertheless, poor thermal stability was observed at >30°C. Additionally, the examined metal ions did not activate the catalytic activity of KgBDH. A recombinant Escherichia coli strain coexpressing KgBDH and glucose dehydrogenase (GHD) was constructed and immobilized via entrapment with a mixture of activated carbon and calcium alginate via entrapment. The immobilized cells had 1.8-fold higher catalytic activity than that of cells immobilized by calcium alginate alone. The maximum catalytic activity of the immobilized cells was achieved at pH 7.5, and favorable pH stability was observed at pH 6.0-9.0. Moreover, the immobilized cells showed favorable thermal stability at 25-30°C and better operational stability than free cells, retaining approximately 55% of the initial catalytic activity after four cycles. Finally, 81% yields (195 mM product) and >99% enantiomeric excess (ee) of (R)-1-phenyl-1,2-ethanediol were produced within 12 h through a fed-batch strategy with the immobilized cells (25 mg/ml wet cells) at 35°C and 180 rpm, with a productivity of approximately 54 g/L per day.

SUBMITTER: Peng F

PROVIDER: S-EPMC7046757 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Immobilization of Cofactor Self-Sufficient Recombinant Escherichia coli for Enantioselective Biosynthesis of (R)-1-Phenyl-1,2-Ethanediol.

Peng Fei F Su Hui-Hui HH Ou Xiao-Yang XY Ni Zi-Fu ZF Zong Min-Hua MH Lou Wen-Yong WY

Frontiers in bioengineering and biotechnology 20200221

(R)-1-phenyl-1,2-ethanediol is an important synthon for the preparation of β-adrenergic blocking agents. This study identified a (2R,3R)-butanediol dehydrogenase (KgBDH) from Kurthia gibsonii SC0312, which showed high enantioselectivity for production of (R)-1-phenyl-1,2-ethanediol by reduction of 2-hydroxyacetophenone. KgBDH was expressed in a recombinant engineered strain, purified, and characterized. It showed good catalytic activity at pH 6-8 and bet ...[more]

PMID: 32154222

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Project description:Candida parapsilosis CCTCC M203011 catalyzes the stereoinversion of (R)-1-phenyl-1,2-ethanediol (PED) through oxidation and reduction. Its NAD(+)-linked (R)-carbonyl reductase (RCR) catalyzes the oxidization of (R)-PED to 2-hydroxyacetophenone (HAP), and its NADPH-dependent (S)-carbonyl reductase (SCR) catalyzes the reduction of HAP to (S)-PED. The reactions require NAD(+) and NADPH as cofactors. However, even if NAD(+) and NADPH are added, the biotransformation of (S)-PED from the (R)-enantiomer by an Escherichia coli strain co-expressing RCR and SCR is slow and gives low yields, probably as a result of insufficient or imbalanced redox cofactors. To prepare (S)-PED from the (R)-enantiomer in one-step efficiently, plus redox cofactor regeneration, we introduced pyridine nucleotide transhydrogenases (PNTs) from E. coli to the metabolic pathway of (S)-PED.The PNTs were successfully introduced into the E. coli strain RSAB. Most of the PNT activities occurred in the cell membrane of E. coli. The introduction of PNTs increased intracellular NAD(+) and NADH concentrations and decreased the NADPH pool without affecting the total nucleotide concentration and cell growth properties. The presence of PNTs increased the NADH/NAD(+) ratio slightly and reduced the NADPH/NADP(+) ratio about two-fold; the ratio of NADPH/NADP(+) to NADH/NAD(+) was reduced from 36 to 17. So, the PNTs rebalanced the cofactor pathways: the rate of RCR was increased, while the rate of SCR was decreased. When the ratio of NAD(+)/NADPH was 3.0 or higher, the RSAB strain produced (S)-PED with the highest optical purity, 97.4%, and a yield of 95.2% at 6 h. The introduction of PNTs stimulated increases of 51.5% and 80.6%, respectively, in optical purity and yield, and simultaneously reduced the reaction time seven-fold.In this work, PNTs were introduced into E. coli to rebalance the cofactor pools within the engineered (S)-PED pathways. The efficient one-step production of (S)-PED plus NAD(+)-NADPH in-situ regeneration was realized. This work provided new insights into cofactor rebalancing pathways, using metabolic engineering methods, for efficient chiral alcohol production.

Project description:Candida parapsilosis (R)-carbonyl reductase (RCR) and (S)-carbonyl reductase (SCR) are involved in the stereoconversion of racemic (R,S)-1-phenyl-1,2-ethanediol (PED) to its (S)-isomer. RCR catalyzes (R)-PED to 2-hydroxyacetophenone (2-HAP), and SCR catalyzes 2-HAP to (S)-PED. However, the stereoconversion efficiency of racemic mixture to (S)-PED is not high because of an activity imbalance between RCR and SCR, with RCR performing at a lower rate than SCR. To realize the efficient preparation of racemic mixture to (S)-PED, an in situ expression of RCR and a two-stage control strategy were introduced to rebalance the RCR- and SCR-mediated pathways.An in situ expression plasmid pCP was designed and RCR was successfully expressed in C. parapsilosis. With respect to wild-type, recombinant C. parapsilosis/pCP-RCR exhibited over four-fold higher activity for catalyzing racemic (R,S)-PED to 2-HAP, while maintained the activity for catalyzing 2-HAP to (S)-PED. The ratio of k cat /K M for SCR catalyzing (R)-PED and RCR catalyzing 2-HAP was about 1.0, showing the good balance between the functions of SCR and RCR. Based on pH and temperature preferences of RCR and SCR, a two-stage control strategy was devised, where pH and temperature were initially set at 5.0 and 30 °C for RCR rapidly catalyzing racemic PED to 2-HAP, and then adjusted to 4.5 and 35 °C for SCR transforming 2-HAP to (S)-PED. Using these strategies, the recombinant C. parapsilosis/pCP-RCR catalyzed racemic PED to its (S)-isomer with an optical purity of 98.8 % and a yield of 48.4 %. Most notably, the biotransformation duration was reduced from 48 to 13 h.We established an in situ expression system for RCR in C. parapsilosis to rebalance the functions between RCR and SCR. Then we designed a two-stage control strategy based on pH and temperature preferences of RCR and SCR, better rebalancing RCR and SCR-mediated chiral biosynthesis pathways. This work demonstrates a method to improve chiral biosyntheses via in situ expression of rate-limiting enzyme and a multi-stage control strategy to rebalance asymmetric pathways.

Dataset Information

Immobilization of Cofactor Self-Sufficient Recombinant Escherichia coli for Enantioselective Biosynthesis of (R)-1-Phenyl-1,2-Ethanediol.

Publications

Immobilization of Cofactor Self-Sufficient Recombinant <i>Escherichia coli</i> for Enantioselective Biosynthesis of (<i>R</i>)-1-Phenyl-1,2-Ethanediol.

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