Characterization of a Carbonyl Reductase from Rhodococcus erythropolis WZ010 and Its Variant Y54F for Asymmetric Synthesis of (S)-N-Boc-3-Hydroxypiperidine

Ying, Xiangxian; Zhang, Jie; Wang, Can; Huang, Meijuan; Ji, Yuting; Cheng, Feng; Yu, Meilan; Wang, Zhao; Mao, Ying

doi:10.3390/molecules23123117

Cited by 24 publications

(23 citation statements)

References 41 publications

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“…So far, the application of OYE-mediated citral reduction still suffers from insufficient enantioselectivity and activity. Protein engineering has emerged as an attractive and powerful strategy for improving enzyme activity and selectivity [16,17,18,19]. The circular permutation of OYE1 from S. pastorianus yielded the variants exhibiting over an order of magnitude improved catalytic activity [20].…”

Section: Introductionmentioning

confidence: 99%

Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal

Ying

Huang

et al. 2019

Molecules

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The members of the Old Yellow Enzyme (OYE) family are capable of catalyzing the asymmetric reduction of (E/Z)-citral to (R)-citronellal—a key intermediate in the synthesis of L-menthol. The applications of OYE-mediated biotransformation are usually hampered by its insufficient enantioselectivity and low activity. Here, the (R)-enantioselectivity of Old Yellow Enzyme from Saccharomyces cerevisiae CICC1060 (OYE2y) was enhanced through protein engineering. The single mutations of OYE2y revealed that the sites R330 and P76 could act as the enantioselectivity switch of OYE2y. Site-saturation mutagenesis was conducted to generate all possible replacements for the sites R330 and P76, yielding 17 and five variants with improved (R)-enantioselectivity in the (E/Z)-citral reduction, respectively. Among them, the variants R330H and P76C partly reversed the neral derived enantioselectivity from 32.66% e.e. (S) to 71.92% e.e. (R) and 37.50% e.e. (R), respectively. The docking analysis of OYE2y and its variants revealed that the substitutions R330H and P76C enabled neral to bind with a flipped orientation in the active site and thus reverse the enantioselectivity. Remarkably, the double substitutions of R330H/P76M, P76G/R330H, or P76S/R330H further improved (R)-enantioselectivity to >99% e.e. in the reduction of (E)-citral or (E/Z)-citral. The results demonstrated that it was feasible to alter the enantioselectivity of OYEs through engineering key residue distant from active sites, e.g., R330 in OYE2y.

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Section: Introductionmentioning

confidence: 99%

Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal

Ying

Huang

et al. 2019

Molecules

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“…Various acyclic α-alkyl-β-keto amides were reduced, yielding the ( S )-selective products’ high yields and excellent ee [37]. Enantiopure 3,4-dihydroisocoumarins were easily accessible via a one-pot dynamic reductive kinetic resolution process catalyzed by ADH-A [38] as was the asymmetric synthesis of ( S )- N -Boc-3-hydroxypipridines ( 13 ) using a variant (Y54F) of a KRED from R. erythropolis WZ010 [39]. N -Boc-3-piperidone ( 12 ) was selectively reduced, thereby forming an important intermediate of ibrutinib, an inhibitor of Bruton’s tyrosine kinase.…”

Section: Promiscuous Redox-reactions In Rhodococcusmentioning

confidence: 99%

Rhodococcus as a Versatile Biocatalyst in Organic Synthesis

Busch

Hagedoorn

Hanefeld

2019

IJMS

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The application of purified enzymes as well as whole-cell biocatalysts in synthetic organic chemistry is becoming more and more popular, and both academia and industry are keen on finding and developing novel enzymes capable of performing otherwise impossible or challenging reactions. The diverse genus Rhodococcus offers a multitude of promising enzymes, which therefore makes it one of the key bacterial hosts in many areas of research. This review focused on the broad utilization potential of the genus Rhodococcus in organic chemistry, thereby particularly highlighting the specific enzyme classes exploited and the reactions they catalyze. Additionally, close attention was paid to the substrate scope that each enzyme class covers. Overall, a comprehensive overview of the applicability of the genus Rhodococcus is provided, which puts this versatile microorganism in the spotlight of further research.

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“…Compared with isolated enzymes, the use of whole-cell catalysts simplifies the procedure, reduces costs and improves the enzyme stability. Despite great potential, the number of characterized alcohol oxidases is very limited, in contrast to the wide array of alcohol dehydrogenases with various substrate specificities [ 7 , 13 – 16 ]. Alcohol dehydrogenases catalyze reversible oxidation reactions and require NAD(P) + , suggesting that efficient NAD(P) + regeneration is needed to shift the reaction equilibrium toward product formation [ 11 , 17 ].…”

Section: Introductionmentioning

confidence: 99%

Efficient whole-cell oxidation of α,β-unsaturated alcohols to α,β-unsaturated aldehydes through the cascade biocatalysis of alcohol dehydrogenase, NADPH oxidase and hemoglobin

et al. 2021

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Background α,β-Unsaturated aldehydes are widely used in the organic synthesis of fine chemicals for application in products such as flavoring agents, fragrances and pharmaceuticals. In the selective oxidation of α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes, it remains challenging to overcome poor selectivity, overoxidation and a low atom efficiency in chemical routes. Results An E. coli strain coexpressing the NADP+-specific alcohol dehydrogenase YsADH and the oxygen-dependent NADPH oxidase TkNOX was constructed; these components enabled the NADP+ regeneration and catalyzed the oxidation of 100 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal with a yield of 21.3%. The oxygen supply was strengthened by introducing the hemoglobin protein VsHGB into recombinant E. coli cells and replacing the atmosphere of the reactor with pure oxygen, which increased the yield to 51.3%. To further improve catalytic performance, the E. coli cells expressing the multifunctional fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB were generated, which completely converted 250 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal after 8 h of whole-cell oxidation. The reaction conditions for the cascade biocatalysis were optimized, in which supplementation with 0.2 mM FAD and 0.4 mM 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 through the selective oxidation of various α,β-unsaturated alcohols. Conclusions The construction of a strain expressing the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB achieved efficient NADP+ regeneration and the selective oxidation of various α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes. Among the available redox enzymes, the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB has become the most recent successful example to improve catalytic performance in comparison with its separate components.

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Characterization of a Carbonyl Reductase from Rhodococcus erythropolis WZ010 and Its Variant Y54F for Asymmetric Synthesis of (S)-N-Boc-3-Hydroxypiperidine

Cited by 24 publications

References 41 publications

Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal

Engineering the Enantioselectivity of Yeast Old Yellow Enzyme OYE2y in Asymmetric Reduction of (E/Z)-Citral to (R)-Citronellal

Rhodococcus as a Versatile Biocatalyst in Organic Synthesis

Efficient whole-cell oxidation of α,β-unsaturated alcohols to α,β-unsaturated aldehydes through the cascade biocatalysis of alcohol dehydrogenase, NADPH oxidase and hemoglobin

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