Recent advances in biotechnological applications of alcohol dehydrogenases

Zheng, Yu‐Guo; Yin, Huanhuan; Yu, Dao-Fu; Chen, Xiang; Tang, Xiaoling; Zhang, Xiaojian; Xue, Ya‐Ping; Wang, Ya‐Jun; Liu, Zhi‐Qiang

doi:10.1007/s00253-016-8083-6

Cited by 153 publications

(83 citation statements)

References 98 publications

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“…Chiral alcohols with high enantiomeric purity are important intermediates for the synthesis of optically active fine chemicals that are used for example to produce pharmaceuticals and agrochemicals (Ager et al 1996;Liese et al 2006;Breuer et al 2004). Alcohol dehydrogenases (ADHs) are used for the synthesis of chiral alcohols under very mild reaction conditions due to their high catalytic efficiency and selectivity (Hall and Bommarius 2011;Zheng et al 2017). A prominent example is the NADPH-dependent ADH from Lactobacillus brevis (LbADH), which catalyzes the stereoselective reduction of prochiral ketones to the corresponding, mostly (R)-configured secondary alcohols (Hummel 1999;Rodriguez et al 2014).…”

Section: Introductionmentioning

confidence: 99%

NADPH biosensor-based identification of an alcohol dehydrogenase variant with improved catalytic properties caused by a single charge reversal at the protein surface

et al. 2020

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Alcohol dehydrogenases (ADHs) are used in reductive biotransformations for the production of valuable chiral alcohols. In this study, we used a high-throughput screening approach based on the NADPH biosensor pSenSox and fluorescence-activated cell sorting (FACS) to search for variants of the NADPH-dependent ADH of Lactobacillus brevis (LbADH) with improved activity for the reduction of 2,5-hexanedione to (2R,5R)-hexanediol. In a library of approx. 1.4 × 10 6 clones created by random mutagenesis we identified the variant LbADH K71E. Kinetic analysis of the purified enzyme revealed that LbADH K71E had a ~ 16% lowered K M value and a 17% higher V max for 2,5-hexanedione compared to the wild-type LbADH. Higher activities were also observed for the alternative substrates acetophenone, acetylpyridine, 2-hexanone, 4-hydroxy-2-butanone, and methyl acetoacetate. K71 is solvent-exposed on the surface of LbADH and not located within or close to the active site. Therefore, K71 is not an obvious target for rational protein engineering. The study demonstrates that high-throughput screening using the NADPH biosensor pSenSox represents a powerful method to find unexpected beneficial mutations in NADPH-dependent alcohol dehydrogenases that can be favorable in industrial biotransformations.

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Section: Introductionmentioning

confidence: 99%

NADPH biosensor-based identification of an alcohol dehydrogenase variant with improved catalytic properties caused by a single charge reversal at the protein surface

et al. 2020

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“…Asymmetric reduction of ketones is one of the most important, fundamental and practical reactions for chiral alcohols synthesis . Currently, chiral alcohols can be synthesized by using chemical and biological technology . Chemical stereoselective synthesis of (3 R ,5 R )‐ 2 via boride‐catalyzed asymmetric hydrogenation has been developed, however it requires harsh conditions, especially cryogenic treatment (always ≤60°C), generating huge energy consumption and waste disposal problems, along with unsatisfactory product optical purity .…”

Section: Introductionmentioning

confidence: 99%

Enhanced diastereoselective synthesis of t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate by using aldo‐keto reductase and glucose dehydrogenase co‐producing engineered Escherichia coli

Wang

Shen

Luo

et al. 2017

Biotechnology Progress

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t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is a key chiral diol precursor of atorvastatin calcium (Lipitor®). We have constructed a Kluyveromyces lactis aldo-keto reductase mutant KlAKR-Y295W/W296L (KlAKRm) by rational design in previous research, which displayed high activity and excellent diastereoselectivity (de > 99.5%) toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). To realize in situ cofactor regeneration, a robust KlAKRm and Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) co-producer E. coli BL 21(DE3) pETDuet-esgdh (MCS1)/pET-28b (+)-klakrm was constructed in this work. Under the optimized conditions, AKR and GDH activities of E. coli BL 21(DE3) pETDuet-esgdh (MCS1)/pET-28b (+)-klakrm peaked at 249.9 U/g DCW (dry cellular weight) and 29100 U/g DCW, respectively. It completely converted (5R)-1 at substrate loading size of up to 60.0 g/L (5R)-1 in the absence of exogenous NADH, which was one-fifth higher than that of the separately prepared KlAKRm and EsGDH under the same conditions. In this manner, a biocatalytic process for (3R,5R)-2 with productivity of 243.2 kg/m d was developed. Compared with the combination of separate expressed KlAKRm with EsGDH, co-expression of KlAKRm and EsGDH has the advantages of alleviating cell cultivation burden and elevating substrate load. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1235-1242, 2017.

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“…Compared with isolated enzymes, the use of whole-cell catalysts simplify the procedure, reduces the cost and bene ts the enzyme stability. Despite great potentials, the number of characterized alcohol oxidases was very limited in contrast to a large array of alcohol dehydrogenases with various substrate speci cities [7,[13][14][15][16].…”

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confidence: 99%

Efficient Whole-Cell Oxidation of α,β-Unsaturated Alcohols to α,β-Unsaturated Aldehydes through Cascade Biocatalysis of Alcohol Dehydrogenase, NADPH Oxidase and Hemoglobin

Qiao

Wang

Zeng

et al. 2020

Preprint

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Background: α,β-unsaturated aldehydes are widely used in as organic synthesis of fine chemicals such as flavor, fragrances and pharmaceuticals. The selective oxidation of α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes remains challenging to overcome poor selectivity, over-oxidation and low atom efficiency in chemical routes.Results: An E. coli strain co-expressing NADP+-specific alcohol dehydrogenase YsADH and oxygen-dependent NADPH oxidase TkNOX was constructed, which enabled the NADP+ regeneration and catalyzed the oxidation of 100 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal with the yield of 21.3%. The oxygen supply was strengthened by introducing the hemoglobin VsHGB into recombinant E. coli cells and replacing the atmosphere of the reactor with pure oxygen, which increased the yield up to 51.3%. To further improve catalytic performance, the E. coli cells expressing the multifunctional fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB was achieved, which totally converted 250 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal after 8 h of whole-cell oxidation. The reaction conditions of the cascade biocatalysis were optimized, in which the supplement of 0.2 mM FAD and 0.4 NADP+ was essential for maintaining high catalytic activity. Finally, the established whole-cell system could serve as a platform for the synthesis of valuable α,β-unsaturated aldehydes from selective oxidation of various α,β-unsaturated aldehydes.Conclusions: The construction of the strain expressing the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB fulfilled efficient NADP+ regeneration and selective oxidation of various α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes. With the scope of redox enzymes, the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB has become the latest successful example to improve catalytic performance in comparison with separated counterparts.

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Recent advances in biotechnological applications of alcohol dehydrogenases

Cited by 153 publications

References 98 publications

NADPH biosensor-based identification of an alcohol dehydrogenase variant with improved catalytic properties caused by a single charge reversal at the protein surface

NADPH biosensor-based identification of an alcohol dehydrogenase variant with improved catalytic properties caused by a single charge reversal at the protein surface

Enhanced diastereoselective synthesis of t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate by using aldo‐keto reductase and glucose dehydrogenase co‐producing engineered Escherichia coli

Efficient Whole-Cell Oxidation of α,β-Unsaturated Alcohols to α,β-Unsaturated Aldehydes through Cascade Biocatalysis of Alcohol Dehydrogenase, NADPH Oxidase and Hemoglobin

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