JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2024
·
IF 5.7
Gege Ma,Xiangyu Zhu,Dongxin Zhang,Haoran Li,Jinping Lin,Dongzhi Wei
(R)-Citronellal is a key chiral precursor of high-value chemicals, such as the best-selling flavor compound (−)-menthol; however, the conventional synthesis suffers from low yield and unsatisfactory enantioselectivity. In this study, we developed a highly atom-efficient hydrogen-borrowing cascade for the synthesis of (R)-citronellal from geraniol using alcohol dehydrogenase from Escherichia coli K12 (AdhP) and ene-reductase from Saccharomyces cerevisiae YJM1341 (OYE2p). The key rate-limiting enzyme, AdhP, was subjected to structure-guided semirational engineering, and the triple mutant AdhP260T/284A/268P (M3) was obtained that demonstrated a 1.28-fold improvement in catalytic efficiency (kcat/Km) toward geraniol. After optimization of the reaction conditions, the hydrogen-borrowing cascade system achieved the conversion of 23.14 g/L geraniol into (R)-citronellal at a conversion rate of 98.23% with 96.7% ee. This work represents an alternative approach for the biosynthesis of (R)-citronellal without sacrificing a cosubstrate or additional enzymes.
研究领域:
生物化学
·
合成生物学
·
酶学
✓ 已验证
DOI
PubMed
高影响因子
Microbial Biotechnology
·
2021
·Vol. 15
·期 5
·1486-1498
·
IF 5.813
Baoqi Zhang,Han Du,Yanqiu Zheng,Jiale Sun,Yu Shen,Jinping Lin,Dongzhi Wei
Summary Bioproduction of optical pure ( R )-citronellal from ( E /Z)-citral at high substrate loading remains challenging. Low catalytic efficiency of ( R )-stereoselective ene reductases towards crude citral mixture is one of the major bottlenecks. Herein, a structure-based engineering strategy was adopted to enhance the catalytic efficiency and stereoselectivity of an ene reductase (OYE2p) from Saccharomyces cerevisiae YJM1341 towards ( E / Z )-citral. On basis of homologous modelling, molecular docking analysis and alanine scanning at the binding pocket of OYE2p, a mutant Y84A was obtained with simultaneous increase in catalytic efficiency and stereoselectivity. Furthermore, site-saturation mutagenesis of Y84 yielded seven mutants with improved activity and stereoselectivity in the ( E / Z )-citral reduction. Among them, the variant Y84V exhibited an 18.3% and 71.3% rise in catalytic efficiency ( k cat / K m ) for ( Z )-citral and ( E )-citral respectively. Meanwhile, the stereoselectivity of Y84V was improved from 89.2% to 98.0% in the reduction in ( E / Z )-citral. The docking analysis and molecular dynamics simulation of OYE2p and its variants revealed that the substitution Y84V enabled ( E )-citral and ( Z )-citral to bind with a smaller distance to the key hydrogen donors at a modified ( R )-selective binding mode. The variant Y84V was then co-expressed with glucose dehydrogenase from Bacillus megaterium in E . coli D4, in which competing prim -alcohol dehydrogenase genes were deleted to prevent the undesired reduction in the aldehyde moiety of citral and citronellal. Employing this biocatalyst, 106 g l −1 ( E / Z )-citral was completely converted into ( R )-citronellal with 95.4% ee value and a high space-time yield of 121.6 g l −1 day −1 . The work highlights the synthetic potential of Y84V, which enabled the highest productivity of ( R )-citronellal from ( E / Z )-citral in high enantiopurity so far.
研究领域:
生物催化
·
酶工程
·
分子生物学
·
生物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
·
2025
·
IF 1.1
Wu S. J.,Chen F.,Ma X. J.,Sun S. Y.,Yan H. D.,Li T. T.,Qiu L. Q.
( R )-citronellal is a key intermediate in the synthesis of L-menthol, one of the world’s three most important spices. It can be obtained by asymmetric reduction of ( E / Z )-citral using old yellow enzymes (OYEs), but catalysis by the wild-type enzyme suffers from poor enantioselectivity as well as low efficient conversion. A protein-directed evolution technique was used to enhance the activity and enantioselectivity of Ct OYE from Chroococcidiopsis thermalis . Upon the sequence alignment and molecular docking results, two amino acid residues at positions 103 and 351 were selected as saturation mutation sites. Finally, three single substitution variants of Ct OYE-W103E, Ct OYE-W103M, Ct OYE-W103P with improved enantioselectivity ( ee > 99%) and moderate conversion (>71%), and one double substitution variant Ct OYE-Y351Q/W103E with high enantioselectivity ( ee > 99%) and high conversion (83.2%) for reduction of ( E / Z )-citral were obtained.
✓ 已验证
产品关联
DOI
高影响因子
Bioresources and Bioprocessing
·
2018
·Vol. 5
·期 1
·1-12
·
IF 0
Zheng Liandan,Lin Jinping,Zhang Baoqi,Kuang Yuyao,Wei Dongzhi
A NAD(P)H-dependent enoate reductase (OYE2p) from Saccharomyces cerevisiae YJM1341 was discovered by genome data mining for asymmetric reduction of (E/Z)-citral to (R)-citronellal with high enantioselectivity.This enzyme was heterologously expressed in E. coli and characterized for its biocatalytic properties. OYE2p was identified with reduction activities toward a diverse range of ɑ,β-unsaturated compounds bearing conjugated aldehyde, ketone, imide, carboxylic acid and ester.OYE2p showed the highest specific activity at 40 °C and a pH optimum at 7.0–8.0. The stability of OYE2p was rather pH-independent, and the half-life time values of the enzyme at pH 6.0–8.0 were more than 257 h. With regard to the reduction of (E)-citral and (Z)-citral, OYE2p exhibited different selectivity patterns. (E)-citral was exclusively reduced to (R)-citronellal by OYE2p in ≥ 99% ee, which was independent on pH. OYE2p produced both enantiomers of citronellal from (Z)-citral, but showed (R)-citronellal formation tendency, and the ee value of (R)-citronellal was affected by pH in the reaction system. Accordingly, the ee values for (R)-citronellal formation increased with the increasing levels of E-isomer in the (E/Z)-citral mixture as well as the increase of pH. Under the reaction conditions (30 °C and pH 8.6), using purified OYE2p as catalyst, 200 mM (E/Z)-citral (an approximately 10:9 mixture of geometric E-isomer and Z-isomer) was efficiently converted to (R)-citronellal with 88.8% ee and 87.2% yield.All these positive features demonstrate high potential of OYE2p for practical synthesis of (R)-citronellal and in asymmetric reduction of activated alkenes.
研究领域:
生物化学
·
酶工程
·
合成生物学
✓ 已验证
产品关联
DOI
危险品化学品经营许可证（带存储）