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Scheme 1. Geraniol Oxidation by a CRO-AlcOx (Here CgrAlcOx) and Subsequent Geranial Reduction to (R)-or (S)-Citronellal by an OYE a
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Biocatalytic pathways for the synthesis of (−)-menthol, the most sold flavor worldwide, are highly sought-after. To access the key intermediate (R)-citronellal used in current major industrial production routes, we established a one-pot bienzymatic cascade from inexpensive geraniol, overcoming the problematic biocatalytic reduction of the mixture o...
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... therefore evaluated the ability of CgrAlcOx to convert geraniol (10 mM), starting with previously established conditions on octan-1-ol, 25 which include catalase (CAT) for in situ H 2 O 2 dismutation, and horseradish peroxidase (HRP) for CgrAlcOx activation. 49 We observed the facile conversion of geraniol (>99%, turnover number TON 10,000) in only 15 min (turnover frequency TOF 11.1 s −1 ), at mild temperature (23 °C), and the formation of one isomer of citral ( Figure 1, Figures S12 and S13). This citral isomer was further identified as geranial by 1 H NMR analysis ( Figure S17) based on the study of Zeng et al. 55 The concentrations of accessory enzymes CAT and HRP were then further investigated. ...Context 2
... We observed the facile conversion of geraniol (>99%, turnover number TON 10,000) in only 15 min (turnover frequency TOF 11.1 s −1 ), at mild temperature (23 °C), and the formation of one isomer of citral ( Figure 1, Figures S12 and S13). This citral isomer was further identified as geranial by 1 H NMR analysis ( Figure S17) based on the study of Zeng et al. 55 The concentrations of accessory enzymes CAT and HRP were then further investigated. As expected, both accessory enzymes are required to sustain the CgrAlcOx activity. ...Context 3
... expected, both accessory enzymes are required to sustain the CgrAlcOx activity. A minimum of 0.5 μM HRP ( Figure 1A) and 0.5 μM CAT ( Figure 1B) were required to reach the maximum conversion efficiency. At least 1 μM CgrAlcOx was required for total conversion of geraniol in 15 min ( Figure 1C). ...Context 4
... expected, both accessory enzymes are required to sustain the CgrAlcOx activity. A minimum of 0.5 μM HRP ( Figure 1A) and 0.5 μM CAT ( Figure 1B) were required to reach the maximum conversion efficiency. At least 1 μM CgrAlcOx was required for total conversion of geraniol in 15 min ( Figure 1C). ...Context 5
... minimum of 0.5 μM HRP ( Figure 1A) and 0.5 μM CAT ( Figure 1B) were required to reach the maximum conversion efficiency. At least 1 μM CgrAlcOx was required for total conversion of geraniol in 15 min ( Figure 1C). Interestingly, the HRP requirement was much lower here compared with that for the conversion of octan-1-ol in our previous study, 25 which could be due to the activated nature of the substrate in this study, rendering its oxidation easier. ...Context 6
... We then investigated the second part of the cascade (Scheme 1) to establish suitable conditions for the OYEcatalyzed reduction step, preferably resulting in enantiopure (R)-citronellal. Given the exceptionally fast formation of geranial by CgrAlcOx (TOF 11.1 s −1 ; Figure 1), it was desirable to identify conditions for a fast reduction by an OYE. The reduction step was investigated using citral (commercial mixture of neral and geranial). ...Context 7
... Accordingly, we observed the formation of (R)-citronellal with an ee ≥ 95% in 2.5 h ( Figures 3A and S8). Parallel cascade experiments coupling CgrAlcOx with OYEs from Thermus scotoductus (TsOYE) 57 or Gluconobacter oxydans (GluER) 58 yielded the alternative (S)-citronellal product, with ≥99% ee ( Figure S16) and respective conversion yields of 37% and 95.3%. Extending the reaction time from 16 to 24 h for TsOYE did not allow further improvement of the conversion yield ( Figure S9), probably due to poor substrate affinity of TsOYE toward this β-substituted substrate. ...Context 8
... performing the full cascade in a concurrent one-pot system, we observed a proportion of geraniol that was not oxidized (Figures 3A and S14A). We conjectured that in the conditions we applied, CgrAlcOx could be partly inhibited by the final citronellal product. ...Context 9
... conjectured that in the conditions we applied, CgrAlcOx could be partly inhibited by the final citronellal product. Indeed, conversions of geraniol by CgrAlcOx performed in the presence of exogenously added citronellal resulted in an incomplete reaction ( Figure S10). Such observation is consistent with a hypothesis formulated previously on the possible inhibition of CgrAlcOx by hydrated alkyl-aldehydes. ...Context 10
... avoid initial CgrAlcOx inhibition with citronellal, we performed a sequential one-pot conversion (with OYE2) by running first a 15 min reaction with all reagents except BsGDH and leaving an additional 2.5 h of reaction after addition of BsGDH. Under these conditions, >99% of geraniol was converted and 95.1% of the intermediate geranial was converted to (R)-citronellal with 95.9% ee (Figures 3B and S14B). ...Context 11
... prior to starting the reaction, the headspace and reaction media were saturated with pure oxygen to circumvent potential oxygen limitation in the first step. The resulting (R)-citronellal was simply extracted with ethyl acetate without further purification and characterized by chiral GC ( Figure S15) and NMR spectroscopy ( Figures S18 and S19). Conversion of the geraniol was 98% with a final isolated yield of 72% with 44.3 mg of (R)-citronellal with 95.1% ee. 1 H NMR showed a highly pure product after extraction with ethyl acetate ( Figure S18). ...Context 12
... prior to starting the reaction, the headspace and reaction media were saturated with pure oxygen to circumvent potential oxygen limitation in the first step. The resulting (R)-citronellal was simply extracted with ethyl acetate without further purification and characterized by chiral GC ( Figure S15) and NMR spectroscopy ( Figures S18 and S19). Conversion of the geraniol was 98% with a final isolated yield of 72% with 44.3 mg of (R)-citronellal with 95.1% ee. 1 H NMR showed a highly pure product after extraction with ethyl acetate ( Figure S18). ...Context 13
... resulting (R)-citronellal was simply extracted with ethyl acetate without further purification and characterized by chiral GC ( Figure S15) and NMR spectroscopy ( Figures S18 and S19). Conversion of the geraniol was 98% with a final isolated yield of 72% with 44.3 mg of (R)-citronellal with 95.1% ee. 1 H NMR showed a highly pure product after extraction with ethyl acetate ( Figure S18). Comparison of the catalytic efficiencies of the enzymes showed a TON of 17,458 (TOF 4.85 s −1 ) for CgrAlcOx and 1,636 (TOF 0.09 s −1 ) for OYE2. ...Citations
... ;https://doi.org/10.1101https://doi.org/10. /2023 shown to catalyze the reduction of geraniol or citral to citronellol in other microbes (Yuan et al. 2011;Ribeaucourt et al. 2022;Richter, Gröger, and Hummel 2011). In C. glutamicum JBEI 1.1.2, ...
Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at nearly 1500 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.
Highlights
Design of a mevalonate-based monoterpene production platform in C. glutamicum
Highest production titers of geranoids, eucalyptol, and linalool reported in C. glutamicum to date
Identification of citronellal as an intermediate in the reduction of geraniol to citronellol by C. glutamicum
... An alternative one-pot cascade for the synthesis of citronellal (a menthol precursor), starting from inexpensive geraniol was also reported recently. [60] The cascade utilises a copper radical oxidase (CRO) to convert geraniol to (E)-geranial and subsequently an old yellow enzyme (OYE) to produce key intermediate (R)-citronellal, with a 95.1 % conversion and 95.9 % ee. In parallel, an alternative OYE from Gluconobacter oxydans (GluER) was used to produce (S)citronellal from geraniol with 95.3 % conversion and 99.2 % ee. ...
Current environmental and safety considerations urge innovation to address the need for sustainable high‐value chemicals that are embraced by consumers. This review discusses the concept of sustainable fragrances, as high‐value, everyday and everywhere chemicals. Current and emerging technologies represent an opportunity to produce fragrances in an environmentally and socially responsible way. Biotechnology, including fermentation, biocatalysis, and genetic engineering, has the potential to reduce the environmental footprint of fragrance production while maintaining quality and consistency. Computational and in silico methods, including machine learning (ML), are also likely to augment the capabilities of sustainable fragrance production. Continued innovation and collaboration will be crucial to the future of sustainable fragrances, with a focus on developing novel sustainable ingredients, as well as ethical sourcing practices.
... Based on these advantages, ASR has been successfully applied to answer fundamental questions on topics such as the evolution of hormone receptors (44), apicomplexan lactate dehydrogenases (45), coral pigments (46), and thermophilicity in RNases (47). To date, a few examples of enzyme pairs have been shown to afford two complementary stereoisomers such as (R)-and (S)-citronellal from OYE pairs (48) and (1′S,2′S)-and (1′R,2′R)-3-(2-nitrocyclopropyl)alanine from nonheme iron enzyme pairs (49,50). However, it is not well understood how stereoselectivity evolves quickly to produce different stereoisomers. ...
Controlling the selectivity of a reaction is critical for target-oriented synthesis. Accessing complementary selectivity profiles enables divergent synthetic strategies, but is challenging to achieve in biocatalytic reactions given enzymes' innate preferences of a single selectivity. Thus, it is critical to understand the structural features that control selectivity in biocatalytic reactions to achieve tunable selectivity. Here, we investigate the structural features that control the stereoselectivity in an oxidative dearomatization reaction that is key to making azaphilone natural products. Crystal structures of enantiocomplementary biocatalysts guided the development of multiple hypotheses centered on the structural features that control the stereochemical outcome of the reaction; however, in many cases, direct substitutions of active site residues in natural proteins led to inactive enzymes. Ancestral sequence reconstruction (ASR) and resurrection were employed as an alternative strategy to probe the impact of each residue on the stereochemical outcome of the dearomatization reaction. These studies suggest that two mechanisms are active in controlling the stereochemical outcome of the oxidative dearomatization reaction: one involving multiple active site residues in AzaH and the other dominated by a single Phe to Tyr switch in TropB and AfoD. Moreover, this study suggests that the flavin-dependent monooxygenases (FDMOs) adopt simple and flexible strategies to control stereoselectivity, which has led to stereocomplementary azaphilone natural products produced by fungi. This paradigm of combining ASR and resurrection with mutational and computational studies showcases sets of tools for understanding enzyme mechanisms and provides a solid foundation for future protein engineering efforts.
... This compound is cheap, readily available, biodegradable and non-toxic (Api et al. 2020). It is a versatile green reagent, which can be used as a chiral synthon to introduce a new stereogenic center in more complex structures (Lenardao et al. 2007;Ribeaucourt et al. 2022). It represents the most frequently utilized chiral pool monoterpene in the synthesis of various molecules like octahydroacridines (Acelas et al. 2017;Santoro et al. 2018), dihydro-oxazines (Dömling and Ugi 1993;Kumarn et al. 2005), 3-thiazolines (Schlemminger et al. 2000), hexahydrocanabinols (Lu Zhan Guo et al. 1992;Wang et al. 2000), chiral imines Solladié 1977, 1981;Rehina and Parameswaran 1999;Nardini et al. 2019), thiosemicarbazones (Ferrari et al. 2002;Tarasconi et al. 2000), hydrazones (Dehmlow and Sauerbier 1989;Kobayashi et al. 2002), imidazoles (Curini et al. 2004;Radatz et al. 2011), benzodifuranes (Forier et al. 1999;Goossens et al. 2000), isoxazolidines (LeBel et al. 1964LeBel and Banucci 1971;Yadav et al. 2003), pheromones (Pempo et al. 1996(Pempo et al. , 2000Muto et al. 2004;Ishmuratov et al. 2005), hormones (Odinokov et al. 1993), vitamins (Coffen et al. 1994;Schmida et al. 2004), and several chiral synthons such as 1,7-dienes Beifuss 1986, 1988), bicyclo[5.1.0]octanone ...
An efficient in situ condensation of citronellal, the main constituent of Eucalyptus citriodora essential oil (51%), with different amine derivatives of 2,3-diaminomaleonitrile and 3-[(2-aminoaryl)amino]dimedone has led to novel chiral benzodiazepine structures. All reactions were precipitated in ethanol and pure products were obtained in good yields (58–75%) without any purification. The synthesized benezodiazepines were characterized by spectroscopic techniques, namely 1H-NMR, 13C-NMR, 2D NMR and FTIR. Differential Scanning Calorimetry (DSC) and HPLC were used to confirm the formation diastereomeric mixtures of benzodiazepine derivatives
... This compound is cheap, readily available, biodegradable and non-toxic (Api et al. 2020). It is a versatile green reagent, which can be used as a chiral synthon to introduce a new stereogenic center in more complex structures (Lenardao et al. 2007;Ribeaucourt et al. 2022). It represents the most frequently utilized chiral pool monoterpene in the synthesis of various molecules like octahydroacridines (Acelas et al. 2017;Santoro et al. 2018), dihydro-oxazines (Dömling and Ugi 1993;Kumarn et al. 2005), 3-thiazolines (Schlemminger et al. 2000), hexahydrocanabinols (Lu Zhan Guo et al. 1992;Wang et al. 2000), chiral imines Solladié 1977, 1981;Rehina and Parameswaran 1999;Nardini et al. 2019), thiosemicarbazones (Ferrari et al. 2002;Tarasconi et al. 2000), hydrazones (Dehmlow and Sauerbier 1989;Kobayashi et al. 2002), imidazoles (Curini et al. 2004;Radatz et al. 2011), benzodifuranes (Forier et al. 1999;Goossens et al. 2000), isoxazolidines (LeBel et al. 1964LeBel and Banucci 1971;Yadav et al. 2003), pheromones (Pempo et al. 1996(Pempo et al. , 2000Muto et al. 2004;Ishmuratov et al. 2005), hormones (Odinokov et al. 1993), vitamins (Coffen et al. 1994;Schmida et al. 2004), and several chiral synthons such as 1,7-dienes Beifuss 1986, 1988), bicyclo[5.1.0]octanone ...
An efficient in situ condensation of citronellal, the main constituent of Eucalyptus citriodora essential oil (51%), with different amine derivatives of 2,3-diaminomaleonitrile and 3-[(2-aminoaryl)amino]dimedone has led to novel chiral benzodiazepine structures. All reactions were precipitated in ethanol and pure products were obtained in good yields (58–75%) without any purification. The synthesized benezodiazepines were characterized by spectroscopic techniques, namely 1H-NMR, 13C-NMR, 2D NMR and FTIR. Differential Scanning Calorimetry (DSC) and HPLC were used to confirm the formation diastereomeric mixtures of benzodiazepine derivatives.
... Redox enzymes (oxidoreductases) (see Glossary) have become of major interest in the field of synthetic chemistry [1-4]. Biocatalysts enable a wide range of synthetically useful oxidation or reduction reactions with excellent stereo-, regio-, and enantioselectivities and efficiencies under mild conditions [5][6][7][8]. These properties make oxidoreductases attractive candidates for environmentally friendly synthesis in pharmaceutical and chemical industries [9][10][11][12]. ...
Biocatalytic photosynthesis combines the distinctive characteristics of redox biocatalysis and photocatalysis for solar-to-chemical conversion. Photocatalytic materials harvest renewable solar light to activate oxidoreductases that offer nature-inspired routes for sustainable chemical synthesis with unparalleled reaction selectivity. Here we begin with a conceptual discussion on solar-powered redox biocatalysis, describe the underlying molecular mechanisms and thermodynamics, and highlight recent advances in: (i) photocatalytic activation of cell-free or whole-cell biocatalytic machineries (e.g., redox enzymes, acetogens, cyanobacteria); (ii) repurposing cofactor-dependent enzymes for their new-to-nature syntheses; (iii) chemical transformations by photoenzymes; and (iv) photoelectrocatalytic biosynthesis.
... The TsOYE was produced and purified identically to methods previously described 69 . Briefly, E. coli BL21(DE3) strains harboring the gene for TsOYE (grown at 37°C, 180 rpm) were induced at OD 600 of 0.6 with IPTG (0.1 mM, 30 min, 4°C), resuspended in MOPS-NaOH (20 mM, pH 7.0), disrupted using a Multi Shot Cell Disruption System (two cycles) and clarified by centrifugation (17,500 × g, 30 min, 4°C). ...
Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P) ⁺ ) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN ⁺ ). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN ⁺ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN ⁺ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA ⁺ ), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P) ⁺ .
