Directed evolution of a carbonyl reductase LsCR for the enantioselective synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl) ethanol

Liu, Hua-Tao; Weng, Chun-Yue; Xu, Shenyuan; Li, Shufang; Wang, Ya‐Jun; Zheng, Yu‐Guo

doi:10.1016/j.bioorg.2022.105991

Cited by 10 publications

(6 citation statements)

References 37 publications

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“…The mutant Ls CR M3 constructed in the previous study (Liu et al, 2022) still suffered from modest activity (18.7 U/mg) and short t 1/2 (only 4.1 h at 40°C). In this study, to search for residues that can affect the activity and thermostability of the enzyme, Ala with higher conformational potential and Pro with higher rigidity were introduced at the organization near the active site (Jones et al, 2017; Figure S1a,b).…”

Section: Resultsmentioning

confidence: 88%

“…Therefore, it is inferred whether the α‐helix composed of these four amino acids will have a stronger positive and negative kinetic correlation with this position. From the 10 sequences used in the previous study (Liu et al, 2022), five sequences (CR1, CR2, CR3, CR4, and CR5) were randomly selected for homology modeling and their 3D structures were built. All the five CRs were modified to have the short α‐helix consisting of the motif I‐A‐G‐L through protein engineering (Figure S6a).…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, it is inferred whether the α-helix composed of these four amino acids will have a stronger positive and negative kinetic correlation with this position. From the 10 sequences used in the previous study (Liu et al, 2022), five sequences (CR1, CR2, CR3, CR4, and CR5)…”

Section: Mechanism Elucidation Of Thermostability Enhancementmentioning

confidence: 99%

“…The insolubility of the substrate can be effectively solved by introducing an organic phase to achieve a high substrate loading. For example, the isopropanol‐water reaction system was developed for redox reactions (Liu et al, 2022), in which isopropanol acted as the substrate and the solvent. More and more green media are developed, such as deep eutectic solvents (Gorke et al, 2008), ionic liquids (Singh & Savoy, 2020), and supercritical fluids (Manjare & Dhingra, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…In the previous study, we cloned a carbonyl reductase (EC 1.1.1.184) from Levilactobacillus suantsaii , and it belongs to an NADPH‐dependent short‐chain dehydrogenase/reductase. Its mutant Ls CR M3 was developed by protein engineering, which had much‐improved activity but poor thermostability (Liu et al, 2022). Herein, we achieved the coevolution of stability and activity of Ls CR M3 through only a single point mutation E145A, yielding a robust mutant Ls CR M4 .…”

Section: Introductionmentioning

confidence: 99%

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Coevolving stability and activity of LsCR by a single point mutation and constructing neat substrate bioreaction system

Liu

Weng

Zhou

et al. 2023

Biotech & Bioengineering

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Carbonyl reductase (CR)-catalyzed bioreduction in the organic phase and the neat substrate reaction system is a lasting challenge, placing higher requirements on the performance of enzymes. Protein engineering is an effective method to enhance the properties of enzymes for industrial applications. In the present work, a single point mutation E145A on our previously constructed CR mutant LsCR M3 , coevolved thermostability, and activity. Compared with LsCR M3 , the catalytic efficiency k cat /K M of LsCR M3 -E145A (LsCR M4 ) was increased from 6.6 to 21.9 s −1 mM −1 . Moreover, E145A prolonged the half-life t 1/2 at 40°C from 4.1 to 117 h, T m was increased by 5°C, T 50 30 was increased by 14.6°C, and T opt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing LsCR M4 completely reduced up to 600 g/L 2-chloro-1-(3,4-difluorophenyl)ethanone (CFPO) within 13 h at 45°C, yielding the corresponding (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL) in 99.5% ee P , with a space-time yield of 1.0 kg/L d, the substrate to catalyst ratios (S/C) of 600 g/g.Compared with LsCR M3 , the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCR WT , the substrate loading was increased by 6.5 times. In contrast, LsCR M4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system.

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Section: Resultsmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Mechanism Elucidation Of Thermostability Enhancementmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Coevolving stability and activity of LsCR by a single point mutation and constructing neat substrate bioreaction system

Liu

Weng

Zhou

et al. 2023

Biotech & Bioengineering

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Stairway to Stereoisomers: Engineering Short‐ and Medium‐Chain Ketoreductases To Produce Chiral Alcohols

Shanbhag

2023

ChemBioChem

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The short-and medium-chain dehydrogenase/reductase superfamilies are responsible for most chiral alcohol production in laboratories and industries. In nature, they participate in diverse roles such as detoxification, housekeeping, secondary metabolite production, and catalysis of several chemicals with commercial and environmental significance. As a result, they are used in industries to create biopolymers, active pharmaceutical intermediates (APIs), and are also used as components of modular enzymes like polyketide synthases for fabricating bioactive molecules. Consequently, random, semi-rational and rational engineering have helped transform these enzymes into product-oriented efficient catalysts. The rise of newer synthetic chemicals and their enantiopure counterparts has proved challenging, and engineering them has been the subject of numerous studies. However, they are frequently limited to the synthesis of a single chiral alcohol. The study attempts to defragment and describe hotspots of engineering short-and medium-chain dehydrogenases/reductases for the production of chiral synthons.

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Enhancing the Activity of an Alcohol Dehydrogenase by Using “Aromatic Residue Scanning” at Potential Plasticity Sites

Xie

Liu

et al. 2023

Chemistry A European J

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An alcohol dehydrogenase LkADH was successfully engineered to exhibit improved activity and substrate tolerance for the production of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol, an important precursor of ticagrelor. Five potential hotspots were identified for enzyme mutagenesis by using natural residue abundance as an indicator to evaluate their potential plasticity. A semi-rational strategy named "aromatic residue scanning" was applied to randomly mutate these five sites simultaneously by using tyrosine, tryptophan, and phenylalanine as "exploratory residues" to introduce steric hindrance or potential π-π interactions. The best variant Lk-S96Y/L199W identified with 17.2-fold improvement in catalytic efficiency could completely reduce up to 600 g/L (3.1 M) 2-chloro-1-(3,4-difluorophenyl)ethenone in 12 h with > 99.5 % ee, giving the highest space-time yield ever reported. This study, therefore, offers a strategy for mutating alcohol dehydrogenase to reduce aromatic substrates and provides an efficient variant for the efficient synthesis of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol.

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Directed evolution of a carbonyl reductase LsCR for the enantioselective synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl) ethanol

Cited by 10 publications

References 37 publications

Coevolving stability and activity of LsCR by a single point mutation and constructing neat substrate bioreaction system

Coevolving stability and activity of LsCR by a single point mutation and constructing neat substrate bioreaction system

Stairway to Stereoisomers: Engineering Short‐ and Medium‐Chain Ketoreductases To Produce Chiral Alcohols

Enhancing the Activity of an Alcohol Dehydrogenase by Using “Aromatic Residue Scanning” at Potential Plasticity Sites

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