The Development and Opportunities of Predictive Biotechnology.

Recent advances in bioeconomy allow a holistic view of existing and new process chains and enable novel production routines continuously advanced by academia and industry. All this progress benefits from a growing number of prediction tools that have found their way into the field. For example, automated genome annotations, tools for building model structures of proteins, and structural protein prediction methods such as AlphaFold2TM or RoseTTAFold have gained popularity in recent years. Recently, it has become apparent that more and more AI-based tools are being developed and used for biocatalysis and biotechnology. This is an excellent opportunity for academia and industry to accelerate advancements in the field further. Biotechnology, as a rapidly growing interdisciplinary field, stands to benefit greatly from these developments.

Preface to Special Issue on Biocatalysis as Key to Sustainable Industrial Chemistry.

In their Editorial for the Special Issue on Biocatalysis as Key to Sustainable Industrial Chemistry, Guest Editors Andrés Alcántara, Pablo Domínguez de María, Jennifer Littlechild, and Roland Wohlgemuth and their co-workers on the European Society of Applied Biocatalysis' (ESAB) Working Group on Sustainable Chemistry Martin Schürmann and Roger Sheldon discuss the Special Issue and the importance of biocatalysis in carrying out cutting-edge industrial chemistry in a sustainable way, as well as the future prospects for the field.

Biocatalysis as Key to Sustainable Industrial Chemistry.

Invited for this month's cover is the Working Group Sustainable Chemistry of the European Society of Applied Biocatalysis (ESAB). The image shows the significant contributions of Biocatalysis to science, industry, society, and environment as a technology of first choice for Sustainable Chemistry in the 21st century. The Perspective itself is available at 10.1002/cssc.202102709.

Biocatalysis as Key to Sustainable Industrial Chemistry.

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.

Expanding the Toolbox of R-Selective Amine Transaminases by Identification and Characterization of New Members.

Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. R-selective ATAs belong to the fold type IV PLP-dependent enzymes, and different sequence-, structure- and substrate scope-based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R)-ATAs, with additional bias towards fungal origin. We aimed to expand the toolbox of (R)-ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R)-ATAs. The ATA from Exophiala sideris contains a motif characteristic for d-ATAs, which was previously believed to be a disqualifying factor for (R)-ATA activity. The crystal structure of the ATA from Shinella is the first from a Gram-negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R)-ATAs with a shortened/missing N-terminal helix. The active-site charges vary significantly between the new and known ATAs, correlating with their diverging substrate scope.

Cofactor Generation Cascade for α-Ketoglutarate and Fe(II)-Dependent Dioxygenases.

Fe(II)- and α-ketoglutarate dependent dioxygenases have emerged as important catalysts for the preparation of non-natural amino acids. The stoichiometric supply of the cosubstrate α-ketoglutarate (αKG) is an important cost factor. A combination of the N-succinyl amino acid hydroxylase SadA with an l-glutamate oxidase (LGOX) allowed for coupling in situ production of αKG to stereoselective αKG-dependent dioxygenases in a one-pot/two-step cascade reaction. Both enzymes were used as immobilized enzymes and tested in a preparative scale setup under process-near conditions. Oxygen supply, enzyme, and substrate loading of the oxidation of glutamate were investigated under controlled reaction conditions on a small scale before upscaling to a 1 L stirred tank reactor. LGOX was applied with a substrate concentration of 73.6 g/L (339 mM) and reached a space-time yield of 14.2 g/L/h. Additionally, the enzyme was recycled up to 3 times. The hydroxylase SadA reached a space-time yield of 1.2 g/L/h at a product concentration of 9.3 g/L (40 mM). For both cascade reactions, the supply with oxygen was identified as a critical parameter. The results underline the robustness and suitability of α-ketoglutarate dependent dioxygenases for application outside of living cells.

Optimization of Alcohol Dehydrogenase for Industrial Scale Oxidation of Lactols.

Alcohol dehydrogenases (ADH) are widely used to enantioselectively reduce ketones to chiral alcohols, but their application in industrial scale oxidations is rare. Reasons are the need for an NAD(P)+ cofactor regeneration system, often low performance in oxidative reactions and the limited substrate scope of ADHs. ADHA from Candida magnoliae DSMZ 70638 is identified to efficiently catalyze the regio-selective hydroxy-lactone oxidations to hydroxy-lactones. Hydroxy-lactones are common intermediates in industrial processes to cholesterol lowering (va)statin drugs. A biocatalytic aliphatic hydroxy-lactone oxidation process is developed using pure oxygen as oxidant reaching volumetric productivities of up to 12 g L-1 h-1 , product concentrations of almost 50 g L-1 and 95% reaction yield. For co-factor recycling a previously engineered, water-forming NAD(P)H-oxidase from Streptococcus mutans is used. The process is scaled up to industrial pilot plant scale and it could be demonstrated that ADH catalyzed oxidations can be developed to efficient and safe processes. However, the ADHA wild-type enzyme is not productive enough in chlorolactol oxidation. Therefore, enzyme engineering and multi-parameter screening is successfully applied to optimize the enzyme for the target reaction. The optimized ADHA variant shows a 17-fold higher oxidative activity, a 26°C increased stability and is applied to develop an efficient chlorolactol oxidation process.

Exploring Castellaniella defragrans Linalool (De)hydratase-Isomerase for Enzymatic Hydration of Alkenes.

Acyclic monoterpenes constitute a large and highly abundant class of secondary plant metabolites and are, therefore, attractive low-cost raw materials for the chemical industry. To date, numerous biocatalysts for their transformation are known, giving access to highly sought-after monoterpenoids. In view of the high selectivity associated with many of these reactions, the demand for enzymes generating commercially important target molecules is unabated. Here, linalool (de)hydratase-isomerase (Ldi, EC 4.2.1.127) from Castellaniella defragrans was examined for the regio- and stereoselective hydration of the acyclic monoterpene ß-myrcene to (S)-(+)-linalool. Expression of the native enzyme in Escherichia coli allowed for identification of bottlenecks limiting enzyme activity, which were investigated by mutating selected residues implied in enzyme assembly and function. Combining these analyses with the recently published 3D structures of Ldi highlighted the precisely coordinated reduction-oxidation state of two cysteine pairs in correct oligomeric assembly and the catalytic mechanism, respectively. Subcellular targeting studies upon fusion of Ldi to different signal sequences revealed the significance of periplasmic localization of the mature enzyme in the heterologous expression host. This study provides biochemical and mechanistic insight into the hydration of ß-myrcene, a nonfunctionalized terpene, and emphasizes its potential for access to scarcely available but commercially interesting tertiary alcohols.

Neural responses to criticism and praise vary with schizotypy and perceived emotional support.

Schizotypy is a latent organisation of a cluster of personality styles, such as magical thinking, disorganisation and anhedonia, which are in the normal range of the psychosis continuum. Schizotypy relates to an increased likelihood of perceiving expressed emotion (EE). EE is characterised by criticism, rejection, and emotional over-involvement and less warmth from a close relative. Neuroimaging studies have found normal frontal lobe activation to EE-criticism in people with high schizotypy. Alternatively, electroencephalography measures emotion processing, such as frontal theta power and occipital alpha power. Frontal theta power responds to cognitive and affective processes and occipital alpha power denotes less consciousness and emotional attention. This study aimed to determine the relation of these electroencephalography responses during criticism and praise to perceived emotional support. Participants (n = 32) representing the full (low-to-high) range of positive schizotypy listened to and rated the self-relevance of EE-like criticism and praise and affectively neutral comments while undergoing electroencephalography. Participants completed self-report measures of schizotypy, depression and anxiety. A subset of those with a high positive schizotypy score (n = 22) completed a measure of perceived EE - lack of emotional support. Higher perceived EE - lack of emotional support correlated with lower frontal theta power and lower occipital alpha power during criticism and praise in schizotypal participants. The findings suggest that these neural responses may relate to less perceived emotional support in people with high schizotypy, of which a reduction of frontal theta power denotes less emotional arousal and lower occipital alpha power denotes more alertness to emotional information may relate to less perceived emotional support in people with high schizotypy.

Evolving the Promiscuity of Elizabethkingia meningoseptica Oleate Hydratase for the Regio- and Stereoselective Hydration of Oleic Acid Derivatives.

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.

Shade Stability of Polymer-Infiltrated and Resin Nano Ceramics.

BACKGROUND: The esthetics plays an increasingly significant role in today's dentistry. OBJECTIVE: The objective was to investigate the shade stability of a polymer-infiltrated and a resin nano ceramic in comparison to a conventional feldspar ceramic and an acrylate polymer. METHODS: 20 specimens of each of the materials, CAD-Temp (CT), Mark II (M), VITA Enamic (VE) and Lava Ultimate (LU), were prepared using the standard method. These were divided into groups (n=5) and placed in the following stain solutions: Distilled water, coffee, Coca-Cola and red wine for 14 days. The shade of each specimen was measured before and after using a spectrophotometer (n=5). The data were analyzed using ANOVA and Tukey's test (p ≤ 0.05). RESULTS: The largest ΔE mean values were observed in CT by Coca-Cola (4.38 ± 0.41), in M by coffee (5.95 ± 0.62), in VE (6.02 ± 0.78) and LU (8.61 ± 0.30) by red wine. LU had the largest and strongest overall shade differences (total score: SSC=16.95) and CT the least (SSC=8.74). Overall shade differences of VE (SSC=12.03) and M (SSC=12.48) were statistically indistinguishable. After 14 days, only Coca-Cola caused clinically relevant shade differences in CT (ΔE > 2.7); this was also caused by coffee, Coca-Cola and red wine in M and coffee and red wine in VE and LU. CONCLUSION: On average, VE showed fewer shade differences than LU. After 14 days of immersion, shade differences which exceeded the clinical acceptance threshold of ΔE=2.7 were shown by CT in Coca-Cola, by M in coffee, Coca-Cola and red wine, and by VE and LU in coffee and red wine.

TMS over the supramarginal gyrus delays selection of appropriate grasp orientation during reaching and grasping tools for use.

Tool use, a ubiquitous part of human behaviour, requires manipulation control and knowledge of tool purpose. Neuroimaging and neuropsychological research posit that these two processes are supported by separate brain regions, ventral premotor and inferior parietal for manipulation control, and posterior middle temporal cortex for tool knowledge, lateralised to the left hemisphere. Action plans for tool use need to integrate these two separate processes, which is likely supported by the left supramarginal gyrus (SMG). However, whether this integration occurs during action execution is not known. To clarify the role of the SMG we conducted two experiments in which healthy participants reached to grasp everyday tools with the explicit instruction to use them directly following their grasp. To study the integration of manipulation control and tool knowledge within a narrow time window we mechanically perturbed the orientation of the tool to force participants to correct grasp orientation 'on-line' during the reaching movement. In experiment 1, twenty healthy participants reached with their left hand to grasp a tool. Double-pulse transcranial magnetic stimulation (TMS) was applied, in different blocks over left or right SMG at the onset of perturbation. Kinematic data revealed delayed and erroneous online correction after TMS over left and right SMG. In Experiment 2 twelve participants reached, in different blocks, with their left or right hand and TMS was applied over SMG ipsilateral to the reaching hand. A similar effect on correction was observed for ipsilateral stimulation when reaching with the left and right hands, and no effect of or interaction with hemisphere was observed. Our findings implicate a bilateral role of the SMG in correcting movements and selection of appropriate grasp orientation during reaching to grasp tools for use.

Recombinant expression, purification and biochemical characterization of kievitone hydratase from Nectria haematococca.

Kievitone hydratase catalyzes the addition of water to the double bond of the prenyl moiety of plant isoflavonoid kievitone and, thereby, forms the tertiary alcohol hydroxy-kievitone. In nature, this conversion is associated with a defense mechanism of fungal pathogens against phytoalexins generated by host plants after infection. As of today, a gene sequence coding for kievitone hydratase activity has only been identified and characterized in Fusarium solani f. sp. phaseoli. Here, we report on the identification of a putative kievitone hydratase sequence in Nectria haematococca (NhKHS), the teleomorph state of F. solani, based on in silico sequence analyses. After heterologous expression of the enzyme in the methylotrophic yeast Pichia pastoris, we have confirmed its kievitone hydration activity and have assessed its biochemical properties and substrate specificity. Purified recombinant NhKHS is obviously a homodimeric glycoprotein. Due to its good activity for the readily available chalcone derivative xanthohumol (XN), this compound was selected as a model substrate for biochemical studies. The optimal pH and temperature for hydratase activity were 6.0 and 35°C, respectively, and apparent Vmax and Km values for hydration of XN were 7.16 µmol min-1 mg-1 and 0.98 ± 0.13 mM, respectively. Due to its catalytic properties and apparent substrate promiscuity, NhKHS is a promising enzyme for the biocatalytic production of tertiary alcohols.

An Enzymatic Route to α-Tocopherol Synthons: Aromatic Hydroxylation of Pseudocumene and Mesitylene with P450 BM3.

Aromatic hydroxylation of pseudocumene (1 a) and mesitylene (1 b) with P450 BM3 yields key phenolic building blocks for α-tocopherol synthesis. The P450 BM3 wild-type (WT) catalyzed selective aromatic hydroxylation of 1 b (94 %), whereas 1 a was hydroxylated to a large extent on benzylic positions (46-64 %). Site-saturation mutagenesis generated a new P450 BM3 mutant, herein named "variant M3" (R47S, Y51W, A330F, I401M), with significantly increased coupling efficiency (3- to 8-fold) and activity (75- to 230-fold) for the conversion of 1 a and 1 b. Additional π-π interactions introduced by mutation A330F improved not only productivity and coupling efficiency, but also selectivity toward aromatic hydroxylation of 1 a (61 to 75 %). Under continuous nicotinamide adenine dinucleotide phosphate recycling, the novel P450 BM3 variant M3 was able to produce the key tocopherol precursor trimethylhydroquinone (3 a; 35 % selectivity; 0.18 mg mL-1 ) directly from 1 a. In the case of 1 b, overoxidation leads to dearomatization and the formation of a valuable p-quinol synthon that can directly serve as an educt for the synthesis of 3 a. Detailed product pattern analysis, substrate docking, and mechanistic considerations support the hypothesis that 1 a binds in an inverted orientation in the active site of P450 BM3 WT, relative to P450 BM3 variant M3, to allow this change in chemoselectivity. This study provides an enzymatic route to key phenolic synthons for α-tocopherols and the first catalytic and mechanistic insights into direct aromatic hydroxylation and dearomatization of trimethylbenzenes with O2 .

Lateralized occipito-temporal N1 responses to images of salient distorted finger postures.

For humans as social beings, other people's hands are highly visually conspicuous. Exceptionally striking are hands in other than natural configuration which have been found to elicit distinct brain activation. Here we studied response strength and lateralization of this activation using event-related potentials (ERPs), in particular, occipito-temporal N1 responses as correlates of activation in extrastriate body area. Participants viewed computer-generated images of hands, half of them showing distorted fingers, the other half showing natural fingers. As control stimuli of similar geometric complexity, images of chairs were shown, half of them with distorted legs, half with standard legs. The contrast of interest was between distorted and natural/standard stimuli. For hands, stronger N1 responses were observed for distorted (vs natural) stimuli from 170 ms post stimulus. Such stronger N1 responses were found for distorted hands and absent for distorted chairs, therefore likely unrelated to visuospatial processing of the unusual distorted shapes. Rather, N1 modulation over both hemispheres - but robustly right-lateralized - could reflect distorted hands as emotionally laden stimuli. The results are in line with privileged visual processing of hands as highly salient body parts, with distortions engaging neural resources that are especially activated for biological stimuli in social perception.

N1 responses to images of hands in occipito-temporal event-related potentials.

Hands, much like faces, convey social information, instructions and intentions to an observer. While the neural processes of face perception have been widely studied, it was only recently that fMRI identified occipito-temporal areas sensitive to static images of hands as body parts. To complement these studies with fine-grained timing information, we measured event-related EEG potentials (ERPs) from 33 subjects who were presented with static images of hands versus faces, whole bodies, and inanimate objects as controls. Already at N1 latency, ~ 170ms, hand-related ERP patterns were manifest in two results: (1) significant differences in amplitudes for images of hands versus bodies in occipito-temporal N1 responses; (2) left lateralization of responses to images of hands, and also of the difference waveforms (hands minus bodies), quantifying hand-related responses. In line with fMRI studies of hand-sensitive areas distinct from extrastriate body area (EBA), the current findings provide electrophysiological evidence for hand-sensitive brain activation, occurring at a similarly early latency as N1 responses to faces.

Synchronization of fronto-parietal beta and theta networks as a signature of visual awareness in neglect.

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.

Discovery and structural characterisation of new fold type IV-transaminases exemplify the diversity of this enzyme fold.

Transaminases are useful biocatalysts for the production of amino acids and chiral amines as intermediates for a broad range of drugs and fine chemicals. Here, we describe the discovery and characterisation of new transaminases from microorganisms which were enriched in selective media containing (R)-amines as sole nitrogen source. While most of the candidate proteins were clearly assigned to known subgroups of the fold IV family of PLP-dependent enzymes by sequence analysis and characterisation of their substrate specificity, some of them did not fit to any of these groups. The structure of one of these enzymes from Curtobacterium pusillum, which can convert d-amino acids and various (R)-amines with high enantioselectivity, was solved at a resolution of 2.4 Å. It shows significant differences especially in the active site compared to other transaminases of the fold IV family and thus indicates the existence of a new subgroup within this family. Although the discovered transaminases were not able to convert ketones in a reasonable time frame, overall, the enrichment-based approach was successful, as we identified two amine transaminases, which convert (R)-amines with high enantioselectivity, and can be used for a kinetic resolution of 1-phenylethylamine and analogues to obtain the (S)-amines with e.e.s >99%.

Enhancing cytochrome P450-mediated conversions in P. pastoris through RAD52 over-expression and optimizing the cultivation conditions.

Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts. Recently, our group has identified CYP-mediated hydroxylation of (+)-valencene as a major bottleneck in the biosynthesis of trans-nootkatol and (+)-nootkatone in Pichia pastoris. In the current study, we aimed at enhancing CYP-mediated (+)-valencene hydroxylation by over-expressing target genes identified through transcriptome analysis in P. pastoris. Strikingly, over-expression of the DNA repair and recombination gene RAD52 had a distinctly positive effect on trans-nootkatol formation. Combining RAD52 over-expression with optimization of whole-cell biotransformation conditions, i.e. optimized media composition and cultivation at higher pH value, enhanced trans-nootkatol production 5-fold compared to the initial strain and condition. These engineering approaches appear to be generally applicable for enhanced hydroxylation of hydrophobic compounds in P. pastoris as confirmed here for two additional membrane-attached CYPs, namely the limonene-3-hydroxylase from Mentha piperita and the human CYP2D6.

Metabolic Engineering toward Sustainable Production of Nylon-6.

Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid. Both pathways require yet unreported novel biocatalytic steps. We demonstrated proof of these bioconversions by in vitro enzyme assays with a set of selected candidate proteins expressed in Escherichia coli. One of the biosynthetic pathways starts with 2-oxoglutarate and contains bioconversions of the ketoacid elongation pathway known from methanogenic archaea. This pathway was selected for implementation in E. coli and yielded 6-aminocaproic acid at levels up to 160 mg/L in lab-scale batch fermentations. The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.