Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of (E)-Citral and (Z)-Citral

Wang, Tairan; Wei, Ran; Feng, Yingting; Jin, Lijun; Jia, Yunpeng; Yang, Duxia; Liang, Zuonan; Han, Minghui; Li, Xia; Lu, Chenze; Ying, Xiangxian

doi:10.3390/molecules26165040

Cited by 6 publications

(7 citation statements)

References 45 publications

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“…Interestingly, although the use of ERED for the stereoselective reduction of double bonds is well documented, [8b,15,24] to the best of our knowledge this is the first example of employing ene‐reductases to discriminate between stereoisomers of the non‐reduced conjugated double bond. Only another very recent work described the possibility to apply a previously engineered ERED, OYE3 W116A/S296F, for the production of ( R )‐citronellal, discriminating ( E )‐citral and ( Z )‐citral isomers [25] . However in this case the selectivity of the variant depends on the steric configuration of the double bond to be reduced instead of the non‐reduced conjugated double bond.…”

Section: Resultsmentioning

confidence: 99%

“…Only another very recent work described the possibility to apply a previously engineered ERED, OYE3 W116A/S296F, for the production of (R)-citronellal, discriminating (E)-citral and (Z)citral isomers. [25] However in this case the selectivity of the variant depends on the steric configuration of the double bond to be reduced instead of the non-reduced conjugated double bond. This feature could be applied for the kinetic resolution of conjugated dienes based on the (E/Z) isomer configuration of the γ,δ-double bond.…”

Section: Evaluation Of Ncr For Comparison Due To the Observed High St...mentioning

confidence: 99%

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Ene‐Reductase Catalyzed Regio‐ and Stereoselective 1,4‐Mono‐Reduction of Pseudoionone to Geranylacetone

et al. 2021

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The regio-and stereoselective mono-reduction of a particular C=C bond of conjugated C=C double bonds is a very challenging task. Here the regio-and stereoselective 1,4reduction of pseudoionone, an α,β,γ,δ-bisunsaturated ketone, was demonstrated to give geranylacetone, an industrially relevant molecule. OYE1 from Saccharomyces pastorianus was identified as the most suitable biocatalyst for this reaction. Elevated substrate concentrations of up to 200 mM were tolerated allowing still to reach excellent conversions (> 99 % and 80 % for 100 or 200 mM pseudoionone concentration, respectively). Interestingly, the organic cosolvent often required for substrate solubilization in aqueous buffer can be avoided for pseudoionone when using permeabilized E. coli cells containing the overexpressed enzyme instead of purified enzyme, reaching still > 99 % conversion at 100 mM (19.2 g/L) substrate concentration. Performing this reaction at a 0.5 g scale allowed to run the reaction to completion (> 99 %) and pure product was isolated with 80 % yield. Additionally, the bis-unsaturated ketone 6-methyl-3,5-heptadien-2-one was transformed under similar conditions giving the floral compound sulcatone with excellent conversion (97 %) and 77 % isolated yield. Finally, the stereoselective reduction of the (E,E)-over the (E,Z)-pseudoionone isomer was enabled by the ene-reductase from Zymomonas mobilis (NCR). Thus, both (E)-geranylacetone and (E,Z)pseudoionone were obtained with isomeric excess above 60 %.

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Section: Resultsmentioning

confidence: 99%

Section: Evaluation Of Ncr For Comparison Due To the Observed High St...mentioning

confidence: 99%

Ene‐Reductase Catalyzed Regio‐ and Stereoselective 1,4‐Mono‐Reduction of Pseudoionone to Geranylacetone

et al. 2021

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“…It was found that mutation of the residue W116 to larger amino acids decreased the ( R )-enantioselectivity while replacing W116 with smaller residues enlarged substrate binding space and favored a flipped binding mode for ( Z )-citral, thereby promoting the ( R )-enantioselectivity to >99%. A final double point mutant S296F/W116G was obtained, exhibiting strict ( R )-enantioselectivity towards ( E )-citral and ( E/Z )-citral without any ( Z )-citral catalytic activity ( Wang et al, 2021 ). The rational design strategy based on steric hindrance can also be used to modify enzyme enantioselectivity in biocatalytic desymmetrization of meso substrates.…”

Section: Rational Design Strategy Based On Steric Hindrancementioning

confidence: 99%

Rational design of enzyme activity and enantioselectivity

Song

Zhang

Wu³

et al. 2023

Front. Bioeng. Biotechnol.

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The strategy of rational design to engineer enzymes is to predict the potential mutants based on the understanding of the relationships between protein structure and function, and subsequently introduce the mutations using the site-directed mutagenesis. Rational design methods are universal, relatively fast and have the potential to be developed into algorithms that can quantitatively predict the performance of the designed sequences. Compared to the protein stability, it was more challenging to design an enzyme with improved activity or selectivity, due to the complexity of enzyme molecular structure and inadequate understanding of the relationships between enzyme structures and functions. However, with the development of computational force, advanced algorithm and a deeper understanding of enzyme catalytic mechanisms, rational design could significantly simplify the process of engineering enzyme functions and the number of studies applying rational design strategy has been increasing. Here, we reviewed the recent advances of applying the rational design strategy to engineer enzyme functions including activity and enantioselectivity. Five strategies including multiple sequence alignment, strategy based on steric hindrance, strategy based on remodeling interaction network, strategy based on dynamics modification and computational protein design are discussed and the successful cases using these strategies are introduced.

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“…In contrast, the NADH-dependent cyclohexenone ene-reductase (NCR) from Zymomonas mobilis 23 and the N-ethylmaleimide reductase from Providencia stuartii (NemR-PS) 24,25 reduced (E/Z)-citral exclusively to (S)-citronellal, indicating that the substrate bound with a flipped orientation in the active site. 11,18 F I G U R E 4 The optimal reaction pH and temperature for the reduction of (R)-carvone (A and C) and (S)-carvone (B and D) catalyzed by old yellow enzyme 3 (OYE3) and its variants. The standard reaction system (1 mL) consisting of 20 mM substrate, 2 mg/mL enzyme, 0.6 mM NAD + , 50 mM glucose, 1 U/mL glucose dehydrogenase, and 50 mM buffer (phosphate buffer pH 5.5-6.…”

Section: Site-directed Mutagenesis Of W116 F250 P295 and Y375mentioning

confidence: 99%

Modulation of the catalytic performance of OYE3 by engineering key residues at the entrance of the catalytic pocket

Wang

et al. 2023

Biotech and App Biochem

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The amino acid residues at the entrance of the catalytic pocket may impose steric hindrance on the substrate to enter the active center of the enzyme. Based on the analysis of the three‐dimensional structure of the Saccharomyces cerevisiae old yellow enzyme 3 (OYE3), four bulky residues were chosen and mutated to small amino acids. The results showed that mutation of the W116 residue had interesting impacts on the catalytic performance. All four variants became inactive for the reduction of (R)‐carvone and (S)‐carvone, but inverted the stereoselectivity for the reduction of (E/Z)‐citral. The mutation of the F250 residue had a more positive effect on the activity and stereoselectivity. Two variants, F250A and F250S, showed excellent diastereoselectivity and activity for the reduction of (R)‐carvone (de > 99%, c > 99%) and increased diastereoselectivity and activity for the reduction of (S)‐carvone (de > 96%, c > 80%). One variant of the P295 residue, P295G, displayed excellent diastereoselectivity and activity only for the reduction of (R)‐carvone (de > 99%, c > 99%). Mutation of the Y375 residue had a negative impact on the activity of the enzyme. These findings provide some solutions for rational enzyme engineering of OYE3.

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Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of (E)-Citral and (Z)-Citral

Cited by 6 publications

References 45 publications

Ene‐Reductase Catalyzed Regio‐ and Stereoselective 1,4‐Mono‐Reduction of Pseudoionone to Geranylacetone

Ene‐Reductase Catalyzed Regio‐ and Stereoselective 1,4‐Mono‐Reduction of Pseudoionone to Geranylacetone

Rational design of enzyme activity and enantioselectivity

Modulation of the catalytic performance of OYE3 by engineering key residues at the entrance of the catalytic pocket

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