A Structural View on the Stereospecificity of Plant Borneol‐Type Dehydrogenases

The development of sustainable processes for the valorization of byproducts and other waste streams remains an ongoing challenge in the field of catalysis. Racemic borneol, isoborneol and camphor are currently produced from alpha-pinene, a side product from the production of cellulose. The pure enantiomers of these monoterpenoids have numerous applications in cosmetics and act as reagents for asymmetric synthesis, making an enzymatic route for their separation into optically pure enantiomers a desirable goal. Known short-chain borneol-type dehydrogenases (BDHs) from plants and bacteria lack the required specificity, stability or activity for industrial utilization. Prompted by reports on the presence of pure (-)-borneol and (-)-camphor in essential oils from rosemary, we set out to investigate dehydrogenases from the genus Salvia and discovered a dehydrogenase with high specificity (E>120) and high specific activity (>0.02 U mg(-1)) for borneol and isoborneol. Compared to other specific dehydrogenases, the one reported here shows remarkably higher stability, which was exploited to obtain the first three-dimensional structure of an enantiospecific borneol-type short-chain dehydrogenase. This, together with docking studies, led to the identification of a hydrophobic pocket in the enzyme that plays a crucial role in the stereo discrimination of bornane-type monoterpenoids. The kinetic resolution of borneol and isoborneol can be easily integrated into the existing synthetic route from alpha-pinene to camphor thereby allowing the facile synthesis of optically pure monoterpenols from an abundant renewable source

CryoEM analysis of small plant biocatalysts at sub 2 resolution

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High resolution structural analysis of these enzymes is crucial in order to understand their mechanisms and modulate their properties by targeted engineering. Although cryo electron microscopy cryoEM has revolutionized structural biology, its applicability to high resolution structural analysis of comparatively small enzymes has so far been largely unexplored. Here, it is shown that cryoEM can reveal the structures of plant borneol dehydrogenases of amp; 8764;120 kDa at or below 2 resolution, paving the way for the rapid development of new biocatalysts that can provide access to bioactive terpenes and terpenoid

Engineering of NADPH Supply Boosts Photosynthesis-Driven Biotransformations

was reached, allowing the complete conversion of a 60 mM substrate solution within 4 h

Biochemical characterization of the carotenoid 1,2-hydratases (CrtC) from Rubrivivax gelatinosus and Thiocapsa roseopersicina

Two carotenoid 1,2-hydratase (CrtC) genes from the photosynthetic bacteria Rubrivivax gelatinosus and Thiocapsa roseopersicina were cloned and expressed in Escherichia coli in an active form and purified by affinity chromatography. The biochemical properties of the recombinant enzymes and their substrate specificities were studied. The purified CrtCs catalyze cofactor independently the conversion of lycopene to 1-HO- and 1,1′-(HO)2-lycopene. The optimal pH and temperature for hydratase activity was 8.0 and 30°C, respectively. The apparent Km and Vmax values obtained for the hydration of lycopene were 24 μM and 0.31 nmol h−1 mg−1 for RgCrtC and 9.5 μM and 0.15 nmol h−1 mg−1 for TrCrtC, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed two protein bands of 44 and 38 kDa for TrCrtC, which indicate protein processing. Both hydratases are also able to convert the unnatural substrate geranylgeraniol (C20 substrate), which functionally resembles the natural substrate lycopene

Structural insight into molecular mechanism of poly (ethylene terephthalate) degradation

Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis, was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase (IsPETase) at 1.5 angstrom resolution. IsPETase has a Ser-His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins

Production of Macrocyclic Sesqui- and Diterpenes in Heterologous Microbial Hosts: A Systems Approach to Harness Nature’s Molecular Diversity

Macrocyclic sesqui- (C15) and diterpenes (C20) are a functionally diverse group of natural products with versatile bioactivities encompassing anticancer, antimicrobial and insecticidal agents. Structural complexity prevents economically efficient total synthesis of these higher terpenoids. Heterologous production in recombinant whole-cell biocatalysts is an emerging alternative. Conventional cell systems (i.e., Escherichia coli and Saccharomyces cerevisiae) frequently suffer from low volumetric yields. However, recent combinations of metabolic, enzyme and process engineering in conjunction with systems biology allow significant improvements towards economically viable processes. This Review analyzes research trends in the dynamic fields of terpene-centered microbial cell systems and enzyme engineering. An outlook is given on emerging microbial hosts, which may simplify cellular engineering towards higher product titers

Molecular cloning and functional characterization of a two highly stereoselective borneol dehydrogenases from Salvia officinalis L

Enzymes for selective terpene functionalization are of particular importance for industrial applications. Pure enantiomers of borneol and isoborneol are fragrant constituents of several essential oils and find frequent application in cosmetics and therapy. Racemic borneol can be easily obtained from racemic camphor, which in turn is readily available from industrial side-streams. Enantioselective biocatalysts for the selective conversion of borneol and isoborneol stereoisomers would be therefore highly desirable for their catalytic separation under mild reaction conditions. Although several borneol dehydrogenases from plants and bacteria have been reported, none show sufficient stereoselectivity. Despite Croteau et al. describing sage leaves to specifically oxidize one borneol enantiomer in the late 70s, no specific enzymes have been characterized. We expected that one or several alcohol dehydrogenases encoded in the recently elucidated genome of Salvia officinalis L. would, therefore, be stereoselective. This study thus reports the recombinant expression in E. coli and characterization of two enantiospecific enzymes from the Salvia officinalis L. genome, SoBDH1 and SoBDH2, and their comparison to other known ADHs. Both enzymes produce preferentially (+)-camphor from racemic borneol, but (−)-camphor from racemic isoborneol