Application of robust ketoreductase from Hansenula polymorpha for the reduction of carbonyl compounds

Petrovičová, Tatiana; Gyuranová, Dominika; Plž, Michal; Myrtollari, Kamela; Smonou, Ioulia; Rebroš, Martin

doi:10.1016/j.mcat.2020.111364

Cited by 6 publications

(7 citation statements)

References 82 publications

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“…A sudden spike in dissolved oxygen in the 22nd hour of cultivation indicated the depletion of glycerol and was therefore used as a signal for feeding. The final concentration of biomass was 35.6 g•dm −3 , which is comparable to the previously reported protocol for ketoreductase production (38.7 g•dm −3 for induction at 30 °C), even though the applied temperature was lower (18 °C) [28].…”

Section: High Cell Density Cultivation Of E Coli Expressing Loxsupporting

confidence: 82%

“…While LOX is a quite ordinary enzyme, for this purpose, it is usually applied as a soyabean powder with LOX content or as a commercial soyabean native enzyme [10,14,18,19]. The reliable recombinant production with high cell density cultivation for this process was therefore adapted from Petrovičová et al [28]. A semi-defined glycerolbased medium and a fed-batch strategy with temperature reduction (18 °C) before the induction (0.5 mM IPTG) were applied (Figure 2).…”

Section: High Cell Density Cultivation Of E Coli Expressing Loxmentioning

confidence: 99%

“…Semi-defined medium for high cell density biomass preparation (expression of LOX): Medium (and feeding medium) was that used by Petrovičová [28] Autoinduction media (expression of HPL): glycerol 5 g•dm −3 ; tryptone 8.4 g•dm −3 ; yeast extract 4.65 g•dm −3 ; (NH4)2SO4 5.5 g•dm −3 ; glucose 0.25 g•dm −3 ; lactose 1 g•dm −3 ; KH2PO4 0.136 g•dm −3 ; K2HPO4 0.174 g•dm −3 ; MgSO4•7H2O 0.246 g•dm −3 (sterilized separately); ampicillin 50 µg•cm -3 , sterilized by filtration.…”

Section: Materials and Mediamentioning

confidence: 99%

“…The procedure described by Petrovičová [28] was used with minor modifications. Biomass was prepared in DASbox bioreactors (350 cm 3 , mini bioreaktor DASbox ® system, Eppendorf, Germany) starting with 150 cm 3 of SD media.…”

Section: High Cell Density Cultivation Of E Coli Expressing Loxmentioning

confidence: 99%

“…Negative control for LOX and HPL activity was performed with E.coli BL21(DE3) strains [28]. No LOX and HPL activity was determined (data not shown).…”

Section: Determination Of Hpl Activitymentioning

confidence: 99%

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Optimization of Hydroperoxide Lyase Production for Recombinant Lipoxygenase Pathway Cascade Application

et al. 2021

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Cis-3-hexenal and its more stable isomer, trans-2-hexenal, are highly valued chemicals used in the food and perfume industries. They are produced by the plant lipoxygenase pathway, where two enzymes, lipoxygenase (LOX) and hydroperoxide lyase (HPL), are involved. However, the application of this pathway is limited, especially due to the instability of HPL. This enzyme belongs to the cytochrome P450 enzyme family and needs heme as a prosthetic group. Its synthesis must be effectively performed by a host organism in order to produce an active protein. In this work, Pseudomonas aeruginosa LOX was expressed in Escherichia coli BL21(DE3), and whole cells were used for the synthesis of 13(S)-hydroperoxy-(Z,E,Z)-9,11,15-octadecatrienoic acid (13-HPOT) as a substrate for HPL. Expression of Psidium guajava HPL was carried out by recombinant E. coli JM109(DE3) in autoinduction media, and the influence of the addition of heme precursors δ-ALA and FeII+ was studied. Specific activity of whole cells expressing HPL was measured by the direct use of a synthesized 13-HPOT solution (2.94 mM of total hydroperoxides, 75.35% of 13-HPOT (2.22 mM)) and increased 2.6-fold (from 61.78 U·mg−1 to 159.95 U·mg−1) with the addition of 1 mM FeII+ to the autoinduction media. Productivity and activity were further enhanced by an increase in the expression temperature, and a total of 3.30·105 U·dm−3 of culture media was produced in the optimized process.

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Section: High Cell Density Cultivation Of E Coli Expressing Loxsupporting

confidence: 82%

Section: High Cell Density Cultivation Of E Coli Expressing Loxmentioning

confidence: 99%

Section: Materials and Mediamentioning

confidence: 99%

Section: High Cell Density Cultivation Of E Coli Expressing Loxmentioning

confidence: 99%

“…Negative control for LOX and HPL activity was performed with E.coli BL21(DE3) strains [28]. No LOX and HPL activity was determined (data not shown).…”

Section: Determination Of Hpl Activitymentioning

confidence: 99%

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Optimization of Hydroperoxide Lyase Production for Recombinant Lipoxygenase Pathway Cascade Application

et al. 2021

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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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Biocatalytic characterization of an alcohol dehydrogenase variant deduced from Lactobacillus kefir in asymmetric hydrogen transfer

Rudzka,

Zdun,

Antos

et al. 2023

Commun Chem

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Hydrogen transfer biocatalysts to prepare optically pure alcohols are in need, especially when it comes to sterically demanding ketones, whereof the bioreduced products are either essential precursors of pharmaceutically relevant compounds or constitute APIs themselves. In this study, we report on the biocatalytic potential of an anti-Prelog (R)-specific Lactobacillus kefir ADH variant (Lk-ADH-E145F-F147L-Y190C, named Lk-ADH Prince) employed as E. coli/ADH whole-cell biocatalyst and its characterization for stereoselective reduction of prochiral carbonyl substrates. Key enzymatic reaction parameters, including the reaction medium, evaluation of cofactor-dependency, organic co-solvent tolerance, and substrate loading, were determined employing the drug pentoxifylline as a model prochiral ketone. Furthermore, to tap the substrate scope of Lk-ADH Prince in hydrogen transfer reactions, a broad range of 34 carbonylic derivatives was screened. Our data demonstrate that E. coli/Lk-ADH Prince exhibits activity toward a variety of structurally different ketones, furnishing optically active alcohol products at the high conversion of 65–99.9% and in moderate-to-high isolated yields (38–91%) with excellent anti-Prelog (R)-stereoselectivity (up to >99% ee) at substrate concentrations up to 100 mM.

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Application of robust ketoreductase from Hansenula polymorpha for the reduction of carbonyl compounds

Cited by 6 publications

References 82 publications

Optimization of Hydroperoxide Lyase Production for Recombinant Lipoxygenase Pathway Cascade Application

Optimization of Hydroperoxide Lyase Production for Recombinant Lipoxygenase Pathway Cascade Application

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

Biocatalytic characterization of an alcohol dehydrogenase variant deduced from Lactobacillus kefir in asymmetric hydrogen transfer

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