A biotransformation to produce (R)-Undecavertol has been developed and demonstrated on the pilot scale. The process is a racemic resolution using a highly (S)-selective alcohol dehydrogenase in the ...oxidative direction and an NAD(P)H oxidase for cofactor regeneration. Oxygen limitation is prevented by supplying pure oxygen, achieving a >400 g L–1 substrate concentration and a space-time yield of 14 g L–1 h–1 on the pilot scale and even up to 680 g L–1 and a space-time yield of 21 g L–1 h–1 on the lab scale. In three pilot-plant batches, more than 85 kg of (R)-Undecavertol was produced biocatalytically.
The role and power of biocatalysis in sustainable chemistry has been continuously brought forward step by step to its present outstanding position. The problem‐solving capabilities of biocatalysis ...have been realized by numerous substantial achievements in biology, chemistry and engineering. Advances and breakthroughs in the life sciences and interdisciplinary cooperation with chemistry have clearly accelerated the implementation of biocatalytic synthesis in modern chemistry. Resource‐efficient biocatalytic manufacturing processes have already provided numerous benefits to sustainable chemistry as well as customer‐centric value creation in the pharmaceutical, food, flavor, fragrance, vitamin, agrochemical, polymer, specialty, and fine chemical industries. Biocatalysis can make significant contributions not only to manufacturing processes, but also to the design of completely new value‐creation chains. Biocatalysis can now be considered as a key enabling technology to implement sustainable chemistry.
The golden age of biocatalysis: Biocatalysis can shorten chemical process routes and improve sustainability, as well as using cheaper and bio‐based raw materials, thereby reducing greenhouse gas emissions. Biocatalysis is well positioned to tackle synthetic challenges throughout process design and improve safety, health, economic and environmental aspects of large‐scale industrial production (difficulty levels: L=light; M=medium; D=difficult).
The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant ...material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208mgL−1 cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources.
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•A self-sufficient, whole-cell system for (+)-nootkatone production in Pichia pastoris.•Metabolic engineering enhanced (+)-nootkatone yield to 208mgL−1 cell culture.•P. pastoris is proposed as promising, novel host for sesquiterpenoid production.
The development of P450 platform technology has enabled the sustainable production of an oxygenated intermediate, 4-hydroxy-α-isophorone, on kilogram scale. Application of a cytochrome P450 enzyme ...resulted in an unprecedented product concentration of 10 g/L and a space–time yield of 1.5 g/L/h. These findings are highly relevant for the economical evaluation of cytochrome P450 technology and, additionally, provide access to an alternative and cost-effective route toward 4-hydroxy-α-isophorone.
Optimization and scaling up of the Baeyer–Villiger oxidation of 3,3,5-trimethyl-cyclohexanone to trimethyl-ε-caprolactones (CHLs) were studied to demonstrate this technology on a 100 L pilot plant ...scale. The reaction was catalyzed by a cyclohexanone monooxygenase from Thermocrispum municipale that utilizes the costly redox cofactor nicotinamide adenine dinucleotide phosphate (reduced form), which was regenerated by a glucose dehydrogenase (GDH). As a first stage, different cyclohexanone monooxygenase formulations were tested: cell-free extract, whole cells, fermentation broth, and sonicated fermentation broth. Using broth resulted in the highest yield (63%) and required the least biocatalyst preparation effort. Two commercial glucose dehydrogenases (GDH-105 and GDH-01) were evaluated, resulting in similar performances. Substrate dosing rates and biocatalyst loadings were optimized. On a 30 mL scale, the best conditions were found when 30 mM h–1 dosing rate, 10% (v/v) cyclohexanone monooxygenase broth, and 0.05% (v/v) of glucose dehydrogenase (GDH-01) liquid enzyme formulation were applied. These same conditions (with oxygen instead of air) were applied on a 1 L scale with 92% conversion, achieving a specific activity of 13.3 U gcell wet weight (cww) –1, a space time yield of 3.4 gCHL L–1 h–1, and a biocatalyst yield of 0.83 gCHL gcww –1. A final 100 L demonstration was performed in a pilot plant facility. After 9 h, the reaction reached 85% conversion, 12.8 U gcww –1, a space time yield of 2.7 g L–1 h–1, and a biocatalyst yield of 0.60 gCHL gcww –1. The extraction of product resulted in 2.58 kg of isolated final product. The overall isolated CHL yield was 76% (distal lactone 47% and proximal lactone 53%).
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In R-parity-violating (RPV) supersymmetric models, light neutralinos with masses from the GeV-scale down to even zero are still allowed by all laboratory constraints. They are further ...consistent with dark matter observations, as they decay via RPV couplings. These RPV couplings are in general constrained to be small. Hence, such light neutralinos, if produced, e.g., at a beam-dump or collider experiment, appear as displaced vertices or missing energy at the detector level. The same signatures have been extensively searched for at various experiments in the theoretical context of sterile neutrinos which mix with active neutrinos. In this work, we recast the sensitivity of both past and present experiments to sterile neutrinos to obtain new bounds on RPV couplings associated with a light neutralino. We find experiments such as T2K, BEBC, FASER, DUNE, and MoEDAL-MAPP can improve the current bounds on RPV couplings by up to 3–4 orders of magnitude in several benchmark scenarios.
The addition of water to non‐activated carbon–carbon double bonds catalyzed by fatty acid hydratases (FAHYs) allows for highly regio‐ and stereoselective oxyfunctionalization of renewable oil ...feedstock. So far, the applicability of FAHYs has been limited to free fatty acids, mainly owing to the requirement of a carboxylate function for substrate recognition and binding. Herein, we describe for the first time the hydration of oleic acid (OA) derivatives lacking this free carboxylate by the oleate hydratase from Elizabethkingia meningoseptica (OhyA). Molecular docking of OA to the OhyA 3D‐structure and a sequence alignment uncovered conserved amino acid residues at the entrance of the substrate channel as target positions for enzyme engineering. Exchange of selected amino acids gave rise to OhyA variants which showed up to an 18‐fold improved conversion of OA derivatives, while retaining the excellent regio‐ and stereoselectivity in the olefin hydration reaction.
Redefining the substrate spectrum: The highly regio‐ and stereoselective hydration of oleic acid derivatives by an oleate hydratase is possible. The carboxylate of a free fatty acid—previously considered essential—is not mandatory for the conversion, which thus expands hydration biocatalysis beyond inferred restrictions.
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•Biosynthesis of vanillin using EUGO is optimised.•The best conditions allow an organic solvent free process of 9.9 g prod L−1 h−1.•Immobilised EUGO allowed the biocatalyst to be ...reused up to 18 reaction cycles.•Optimised conditions increased the safety and reduced the environmental impact.
Vanilla is the second most expensive spice in the world. This issue triggered the necessity to obtain its flavour compound, vanillin, by alternative methods, most of which have focused on biosynthesis since it can be labelled as “natural” and sold at higher price. In this work, a process for the enzymatic synthesis of vanillin has been optimised, not only from a process metrics point of view but also from an environmentally sustainable perspective. The maximum biocatalyst activity and stability was taken into account for the selection of the reaction conditions. Soluble and immobilised eugenol oxidase (EUGO) was tested under both reaction conditions. The optimum conditions obtained lead to an organic solvent free process reaching 9.9 g prod L−1 h−1. Moreover, the use of immobilised EUGO allowed the biocatalyst to be reused up to 18 reaction cycles, improving the biocatalyst yield more than 12-fold, thus also reducing the biocatalyst associated cost.
Amine transaminases are frequently used for the production of chiral amines starting from prochiral ketones. These amines can be applied as active pharmaceutical ingredients or drug precursors. ...However, there are still limitations to the use of amine transaminases when it comes to bulky ketone substrates, such as biaryl ketones. Using data mining, an (R)-selective amine transaminase from Exophiala xenobiotica was identified which naturally converts biaryl ketone substrates to the corresponding amines with up to 85% conversion and excellent enantioselectivity (>99% ee). Its protein crystal structure was obtained with a resolution of 1.52 Å, which enabled us to explain this interesting substrate acceptance. Structure-guided protein engineering resulted in a quintuple variant with increased stability. Moreover, the amino acid exchange T273S increased the activity and broadened the substrate scope, enabling conversions of various biaryl ketones with up to >99%. A preparative biotransformation of 1-(4-(pyridin-3-yl)phenyl)ethenone at 75 mM (15 g/L) resulted in 96% of isolated yield of the respective amine.
In the neglect syndrome, the perceptual deficit for contra-lesional hemi-space is increasingly viewed as a dysfunction of fronto-parietal cortical networks, the disruption of which has been described ...in neuroanatomical and hemodynamic studies. Here we exploit the superior temporal resolution of electroencephalography (EEG) to study dynamic transient connectivity of fronto-parietal circuits at early stages of visual perception in neglect. As reflected by inter-regional phase synchronization in a full-field attention task, two functionally distinct fronto-parietal networks, in beta (15–25Hz) and theta (4–8Hz) frequency bands, were related to stimulus discrimination within the first 200 ms of visual processing. Neglect pathology was specifically associated with significant suppressions of both beta and theta networks engaging right parietal regions. These connectivity abnormalities occurred in a pattern that was distinctly different from what was observed in right-hemisphere lesion patients without neglect. Also, both beta and theta abnormalities contributed additively to visual awareness decrease, quantified in the Behavioural Inattention Test. These results provide evidence for the impairment of fast dynamic fronto-parietal interactions during early stages of visual processing in neglect pathology. Also, they reveal that different modes of fronto-parietal dysfunction contribute independently to deficits in visual awareness at the behavioural level.
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