Halohydrin dehalogenases (HHDHs) are powerful enzymes for the asymmetric diversification of oxyfunctionalized synthons. Based on phylogenetic relationship, they share high sequence and structural homology to short-chain dehydrogenases and reductases. Thus, their unambiguous identification from public sequence databases so far relied on the presence of two HHDH-specific sequence motifs, covering residues which are important for catalytic activity as well as structural integrity. Here we show that an impressive number of HHDH-homologous proteins exist in nature that carry diverse motif variations while still catalyzing HHDH-typical reactions. Most compelling is the exchange of the catalytic tyrosine residue by alanine or proline, which still does not abolish HHDH activity. This highlights that the HHDH family is even more diverse than previously expected. Moreover, the herein determined crystal structures of selected enzymes carrying motif variations reveal unique insights into the conformational dynamics of this enzyme family.

Vanillin is one of the most important aroma compounds, naturally occurring in vanilla pods. Many routes to access natural vanillin from various renewables have been investigated, including a natural five-step microbial transformation of eugenol to vanillin. Readily available eugenol was also the starting material for a chemical two-step sequence to vanillin employed in the 19 th century. Here we show that a two-step sequence can also be realized using biocatalysts only and run it in one-pot simultaneously. This was achieved by isomerizing the C=C double bond of eugenol by oxidation to coniferyl alcohol followed by oxidative C=C cleavage catalyzed by newly identified enzymes. Thus, two oxidative steps catalyzed by two different biocatalysts - one containing flavin and the other a non-heme iron(II) cofactor - were successfully run simultaneously just requiring molecular oxygen as oxidant for each step. Using natural eugenol sources, e. g. clove oil, vanillin was obtained with 91 % product formation. This study shows that natural pathways like the microbial transformation of eugenol to vanillin involving five steps can be shortened, hereto just two simultaneous steps, by exploiting and combining the repertoire of promiscuous enzymatic activities present in different organisms leading to new-to-nature cascades.

5-Hydroxymethylfurfural (HMF) has emerged as a crucial bio-based chemical building block in the drive towards developing materials from renewable resources, due to its direct preparation from sugars and its readily diversifiable scaffold. A key obstacle in transitioning to bio-based plastic production lies in meeting the necessary industrial production efficiency, particularly in the cost-effective conversion of HMF to valuable intermediates. To address the challenge of developing scalable technology for oxidizing crude HMF to more valuable chemicals, we have integrated process and enzyme engineering to provide a galactose oxidase (GOase) variant with remarkably high activity toward HMF, improved O2 binding and excellent productivity (>1,000,000 TTN). The process concept presented here for GOase catalysed selective oxidation of HMF to 2,5-diformylfuran offers a productive and efficient platform for further development, thereby laying the groundwork for a biocatalytic route to scalable production of furan-based chemical building blocks from sustainable feedstocks.

Biocatalysis gains significant industrial interest due to its controlled stereoselectivity and use of mild process conditions. Here, a biosynthetic route is proposed for the synthesis of filbertone (5-methyl-2-hepten-4-one, 1), which is the principal flavor compound of hazelnut. The enantiomeric purity of the industrially synthesized compound is defined, contrary to the natural aroma, which varies according to the source of the nut, extraction conditions, and treatments. The novel synthetic pathway for a hazelnut aroma precursor proposed here consists of a multienzyme cascade, which starts from the two amino acids D-Ile and L-Thr that are individually converted by enzyme catalysts, i.e., D-amino acid oxidase and threonine deaminase, followed by CC ligation of the obtained products, allowing a potentially sustainable production of the natural aroma. The most critical step is CC ligation, which uses two carbonyl compounds as starting material. This step is catalyzed by a regioselective transketolase (TK) that originates from Geobacillus stearothermophilus. The approach holds promise for the industrial production of natural hazelnut aroma precursors, addressing the growing demand in the aroma industry for synthesis methods that sustain the regulatory claims for natural compounds.

Aspartate ammonia lyases catalyze the reversible amination of fumarate to l-aspartate. Recent studies demonstrate that the thermostable enzyme from Bacillus sp. YM55-1 (AspB) can be engineered for the enantioselective production of substituted beta-amino acids. This reaction would be attractive for the conversion of acrylic acid to beta-alanine, which is an important building block for the preparation of bioactive compounds. Here we describe a bioinformatics and computational approach aimed at introducing the beta-alanine synthesis activity. Three strategies were used: First, we redesigned the alpha-carboxylate binding pocket of AspB to introduce activity with the acrylic acid. Next, different template enzymes were identified by genome mining, equipped with a redesigned alpha-carboxylate pocket, and investigated for beta-alanine synthesis, which yielded variants with better activity. Third, interactions of the SS-loop that covers the active site and harbors a catalytic serine were computationally redesigned using energy calculations to stabilize reactive conformations and thereby further increase the desired beta-alanine synthesis activity. Different improved enzymes were obtained and the best variants showed k cat values with acrylic acid of at least 0.6-1.5 s-1 with K M values in the high mM range. Since the beta-alanine production of wild-type enzyme was below the detection limit, this suggests that the k cat/K m was improved by at least 1000-fold. Crystal structures of the 6-fold mutant of redesigned AspB and the similarly engineered aspartase from Caenibacillus caldisaponilyticus revealed that their ligand-free structures have the SS-loop in a closed (reactive) conformation, which for wild-type AspB is only observed in the substrate-bound enzyme. AlphaFold-generated models suggest that other aspartase variants redesigned for acrylic acid hydroamination also prefer a 3D structure with the loop in a closed conformation. The combination of binding pocket redesign, genome mining, and enhanced active-site loop closure thus created effective beta-alanine synthesizing variants of aspartase.

Many relevant metabolites, as well as chemical commodities, contain at least one sulfate ester group. Consequently, biocatalytic strategies to attach sulfate to a molecule under mild conditions are of high interest. In order to expand the enzymatic toolbox available, five new arylsulfate sulfotransferases (ASSTs) were identified in this study. Overexpression in Escherichia coli and enzyme purification resulted in soluble proteins which catalyzed the sulfate transfer to an acceptor substrate using p-nitrophenyl sulfate (pNPS) as sulfate donor. Optimal reaction conditions were established with respect to temperature and pH, as well as their tolerance to organic co-solvents and melting temperature. Additionally, the kinetic parameters (Vmax, KM, and kcat) were determined. The substrate scope for the acceptor showed that a structurally diverse spectrum of alcohols is accepted. The substrates included phenolic alcohols with one, two, and three hydroxy groups, linear and cyclic aliphatic alcohols, and amines. The phenolic substrates were accepted reaching activities of up to 154 U/mg purified enzyme. Additionally, also the aliphatic alcohols (both linear and cyclic) were accepted at reduced activity, showing that these enzymes are not limited to phenolic alcohols. Moreover, catalytic activity was detected when using aniline as an acceptor substrate implying their ability to sulfate also amino groups. Finally, the consecutive sulfation of di- and trihydroxy compounds was observed, resulting in the detection of the corresponding disulfated molecules. • Five novel arylsulfate sulfotransferases were identified and characterized. • Accepted substrates included aromatic and aliphatic alcohols, as well as aniline. • Disulfation of di- and trihydroxy aromatic compounds was studied and confirmed.

On the occasion of Professor Bernhard Hauer & PRIME;s(partial)retirement, we reflect on and highlight his distinguished career inbiocatalysis. Bernhard, a biologist by training, has greatly influencedbiocatalysis with his vision and ideas throughout his four-decadecareer. The development of his career went hand in hand with the evolutionof biocatalysis and the application and development of enzymes forchemical processes. In this Account, we present selected examplesof his early work on the development of enzymes and their applicationin an industrial setting, with a focus on his specific contributionsto harnessing the catalytic power of enzymes for novel reactions andthe understanding and engineering of flexible loops and channels oncatalysis.

The regio- and stereoselective mono-reduction of a particular C=C bond of conjugated C=C double bonds is a very challenging task. Here the regio- and stereoselective 1,4-reduction of pseudoionone, an alpha,beta,gamma,delta-bisunsaturated ketone, was demonstrated to give geranylacetone, an industrially relevant molecule. OYE1 from Saccharornyces postorianus was identified as the most suitable biocatalyst for this reaction. Elevated substrate concentrations of up to 200 mM were tolerated allowing still to reach excellent conversions (>99% and 80% for 100 or 200 mM pseudoionone concentration, respectively). Interestingly, the organic cosolvent often required for substrate solubilization in aqueous buffer can be avoided for pseudoionone when using permeabilized E. coli cells containing the overexpressed enzyme instead of purified enzyme, reaching still >99% conversion at 100 mM (19.2 g/L) substrate concentration. Performing this reaction at a 0.5 g scale allowed to run the reaction to completion (> 99%) and pure product was isolated with 80% yield. Additionally, the bis-unsaturated ketone 6-methyl-3,5-heptadien-2-one was transformed under similar conditions giving the floral compound sulcatone with excellent conversion (97%) and 77% isolated yield. Finally, the stereoselective reduction of the (E,E)- over the (E,Z)-pseudoion-one isomer was enabled by the ene-reductase from Zyrnornonas mobilis (NCR). Thus, both (E)-geranylacetone and (E,Z)-pseudoionone were obtained with isomeric excess above 60%.

5-Hydroxymethylfurfural (HMF) has emerged as a crucial bio-based chemical building block in the drive towards developing materials from renewable resources, due to its direct preparation from sugars and its readily diversifiable scaffold. A key obstacle in transitioning to bio-based plastic production lies in meeting the necessary industrial production efficiency, particularly in the cost-effective conversion of HMF to valuable intermediates. To address the challenge of developing scalable technology for oxidizing crude HMF to more valuable chemicals, we have integrated process and enzyme engineering to provide a galactose oxidase (GOase) variant with remarkably high activity toward HMF, improved O2 binding and excellent productivity (>1,000,000 TTN). The process concept presented here for GOase catalysed selective oxidation of HMF to 2,5-diformylfuran offers a productive and efficient platform for further development, thereby laying the groundwork for a biocatalytic route to scalable production of furan-based chemical building blocks from sustainable feedstocks.

ZusammenfassungMuttermilch ist eine beeindruckende „biologische Flüssigkeit“, die sich über Millionen von Jahren zu einer entscheidenden Ernährungs‐ und Immunschutzquelle für Säuglinge entwickelt hat. Die funktionalen humanen Milcholigosaccharide (HMO) der menschlichen Muttermilch sind in Struktur und Zusammensetzung weitaus komplexer als die der Milch vieler anderer Säugetierspezies. Die Forschung nach den Ursachen, warum der Mensch unter Energieeinsatz solch eine Vielfalt an HMO produziert, ist hochaktuell und hat gerade in den letzten zehn Jahren an Bedeutung gewonnen. Moderne Synthesemethoden ermöglichen den Zugang zu einer Vielzahl an HMO – auch, um die Struktur‐Aktivitäts‐Wechselwirkungen aufzuklären. Die industrielle Produktion einzelner HMO wurde bereits entwickelt, Zulassungen für die Zugabe erster HMO in Säuglingsmilchnahrungen wurden erteilt und die ersten Produkte sind auf dem Markt erhältlich. Damit wurde in den letzten zehn Jahren ein wichtiger Schritt gemacht, die Zusammensetzung moderner Säuglingsmilchnahrungen weiter an das natürliche Original anzunähern.Stillen ist dennoch nach wie vor der Goldstandard für die Babyernährung, denn es gibt mehr als 200 gute (HMO)‐Gründe für das Stillen.

We constructed an enzymatic network composed of three different enzymes for the synthesis of valuable ether amines. The enzymatic reactions are interconnected to catalyze the oxidation and subsequent transamination of the substrate and to provide cofactor recycling. This allows production of the desired ether amines from the corresponding ether alcohols with inorganic ammonium as the only additional substrate. To examine conversion, individual and overall reaction equilibria were established. Using these data, it was found that the experimentally observed conversions of up to 60% observed for reactions containing 10 mM alcohol and up to 280 mM ammonia corresponded well to predicted conversions. The results indicate that efficient amination can be driven by high concentrations of ammonia and may require improving enzyme robustness for scale‐up. Biotechnol. Bioeng. 2016;113: 1853–1861. © 2016 Wiley Periodicals, Inc.

Burkholderia glumae is a Gram-negative phytopathogenic bacterium known as the causative agent of rice panicle blight. Strain B. glumae PG1 is used for the production of a biotechnologically relevant lipase, which is secreted into the culture supernatant via a type II secretion pathway. We have comparatively analyzed the genome sequences of B. glumae PG1 wild type and a lipase overproducing strain obtained by classical strain mutagenesis. Among a total number of 72 single nucleotide polymorphisms (SNPs) identified in the genome of the production strain, two were localized in front of the lipAB operon and were analyzed in detail. Both mutations contribute to a 100-fold overproduction of extracellular lipase in B. glumae PG1 by affecting transcription of the lipAB operon and efficiency of lipase secretion. We analyzed each of the two SNPs separately and observed a stronger influence of the promoter mutation than of the signal peptide modification but also a cumulative effect of both mutations. Furthermore, fusion of the mutated LipA signal peptide resulted in a 2-fold increase in secretion of the heterologous reporter alkaline phosphatase from Escherichia coli.

α-Chiral amines are key intermediates for the synthesis of a plethora of chemical compounds at industrial scale. We present a biocatalytic hydrogen-borrowing amination of primary and secondary alcohols that allows for the efficient and environmentally benign production of enantiopure amines. The method relies on a combination of two enzymes: an alcohol dehydrogenase (from Aromatoleum sp., Lactobacillus sp., or Bacillus sp.) operating in tandem with an amine dehydrogenase (engineered from Bacillus sp.) to aminate a structurally diverse range of aromatic and aliphatic alcohols, yielding up to 96% conversion and 99% enantiomeric excess. Primary alcohols were aminated with high conversion (up to 99%). This redox self-sufficient cascade possesses high atom efficiency, sourcing nitrogen from ammonium and generating water as the sole by-product.

To smoothen the process of n‐butanol formation in Pseudomonas putida KT2440, detailed knowledge of the impact of this organic solvent on cell physiology and regulation is of outmost importance. Here, we conducted a detailed systems biology study to elucidate cellular responses at the metabolic, proteomic, and transcriptional level. Pseudomonas putida KT2440 was cultivated in multiple chemostat fermentations using n‐butanol either as sole carbon source or together with glucose. Pseudomonas putida KT2440 revealed maximum growth rates (μ) of 0.3 h−1 with n‐butanol as sole carbon source and of 0.4 h−1 using equal C‐molar amounts of glucose and n‐butanol. While C‐mole specific substrate consumption and biomass/substrate yields appeared equal at these growth conditions, the cellular physiology was found to be substantially different: adenylate energy charge levels of 0.85 were found when n‐butanol served as sole carbon source (similar to glucose as sole carbon source), but were reduced to 0.4 when n‐butanol was coconsumed at stable growth conditions. Furthermore, characteristic maintenance parameters changed with increasing n‐butanol consumption. 13C flux analysis revealed that central metabolism was split into a glucose‐fueled Entner–Doudoroff/pentose‐phosphate pathway and an n‐butanol‐fueled tricarboxylic acid cycle when both substrates were coconsumed. With the help of transcriptome and proteome analysis, the degradation pathway of n‐butanol could be unraveled, thus representing an important basis for rendering P. putida KT2440 from an n‐butanol consumer to a producer in future metabolic engineering studies.

The dehydrogenation of 1-(4-hydroxyphenyl)-ethanol to 4-hydroxyacetophenone represents the second reaction step during anaerobic degradation of p-ethylphenol in the denitrifying bacterium ‘Aromatoleum aromaticum' EbN1. Previous proteogenomic studies identified two different proteins (ChnA and EbA309) as possible candidates for catalyzing this reaction [Wöhlbrand et al: J Bacteriol 2008;190:5699-5709]. Physiological-molecular characterization of newly generated unmarked in-frame deletion and complementation mutants allowed defining ChnA (renamed here as Hped) as the enzyme responsible for 1-(4-hydroxyphenyl)-ethanol oxidation. Hped [1-(4-hydroxyphenyl)-ethanol dehydrogenase] belongs to the ‘classical' family within the short-chain alcohol dehydrogenase/reductase (SDR) superfamily. Hped was overproduced in Escherichia coli, purified and crystallized. The X-ray structures of the apo- and NAD+-soaked form were resolved at 1.5 and 1.1 Å, respectively, and revealed Hped as a typical homotetrameric SDR. Modeling of the substrate 4-hydroxyacetophenone (reductive direction of Hped) into the active site revealed the structural determinants of the strict (R)-specificity of Hped (Phe187), contrasting the (S)-specificity of previously reported 1-phenylethanol dehydrogenase (Ped; Tyr93) from strain EbN1 [Höffken et al: Biochemistry 2006;45:82-93].

The enzyme aryl/alkenyl malonate decarboxylase (AMDase) catalyses the enantioselective decarboxylative protonation (EDP) of a range of disubstituted malonic acids to give homochiral carboxylic acids that are valuable synthetic intermediates. AMDase exhibits a number of advantages over the non-enzymatic EDP methods developed to date including higher enantioselectivity and more environmentally benign reaction conditions. In this report, AMDase and engineered variants have been used to produce a range of enantioenriched heteroaromatic α-hydroxycarboxylic acids, including pharmaceutical precursors, from readily accessible α-hydroxymalonates. The enzymatic method described here represents an improvement upon existing synthetic chemistry methods that have been used to produce similar compounds. The relationship between the structural features of these new substrates and the kinetics associated with their enzymatic decarboxylation is explored, which offers further insight into the mechanism of AMDase.

In addition to the traditional 1-butanol production by hydroformylation of gaseous propene and by fermentation of biomass, the cytochrome P450-catalyzed direct terminal oxidation of n-butane into the primary alcohol 1-butanol constitutes an alternative route to provide the high demand of this basic chemical. Moreover the use of n-butane offers an unexploited ubiquitous feed stock available in large quantities. Based on protein engineering of CYP153A from Polaromonas sp. JS666 and the improvement of the native redox system, a highly ω-regioselective (>96%) fusion protein variant (CYP153AP.sp.(G254A)-CPRBM3) for the conversion of n-butane into 1-butanol was developed. Maximum yield of 3.12g/L butanol, of which 2.99g/L comprise for 1-butanol, has been obtained after 20h reaction time. Due to the poor solubility of n-butane in an aqueous system, a high pressure reaction assembly was applied to increase the conversion. After optimization a maximum product content of 4.35g/L 1-butanol from a total amount of 4.53g/L butanol catalyzed by the self-sufficient fusion monooxygenase has been obtained at 15bar pressure. In comparison to the CYP153A wild type the 1-butanol concentration was enhanced fivefold using the engineered monooxygenase whole cell system by using the high-pressure reaction assembly.