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Abstract Enzymes are natures’ catalysts and will have a lasting impact on (organic) synthesis as they possess unchallenged regio- and stereo selectivity. On the downside, this high selectivity limits enzymes’ substrate range and hampers their universal application. Therefore, substrate scope expansion of enzyme families by either modification of known biocatalysts or identification of new members is a key challenge in enzyme-driven catalysis. Here, we present a streamlined approach to rationally select enzymes with proposed functionalities from the ever-increasing amount of available sequence data. In a case study on 4-phenol oxidoreductases, eight enzymes of the oxidase branch were selected from 292 sequences on basis of the properties of first shell residues of the catalytic pocket, guided by the computational tool A2CA. Correlations between these residues and enzyme activity yielded robust sequence-function relations, which were exploited by site-saturation mutagenesis. Application of a peroxidase-independent oxidase screening resulted in 16 active enzyme variants which were up to 90-times more active than respective wildtype enzymes and up to 6-times more active than the best performing natural variants. The results were supported by kinetic experiments and structural models. The newly introduced amino acids confirmed the correlation studies which overall highlights the successful logic of the presented approach.

ABSTRACT The glutathione S-transferases carried on the plasmid for the styrene-specific degradation pathway in the Actinobacterium Gordonia rubripertincta CWB2 were heterologously expressed in Escherichia coli. Both enzymes were purified via affinity chromatography and subjected to activity investigations. StyI and StyJ displayed activity toward the commonly used glutathione S-transferase model substrate 1-chloro-2,4-dinitrobenzene (CDNB) with Km values of 0.0682 ± 0.0074 and 2.0281 ± 0.1301 mM and Vmax values of 0.0158 ± 0.0002 and 0.348 ± 0.008 U mg−1 for StyI and StyJ, respectively. The conversion of the natural substrate styrene oxide to the intermediate (1-phenyl-2-hydroxyethyl)glutathione was detected for StyI with 48.3 ± 2.9 U mg−1. This elucidates one more step in the not yet fully resolved styrene-specific degradation pathway of Gordonia rubripertincta CWB2. A characterization of both purified enzymes adds more insight into the scarce research field of actinobacterial glutathione S-transferases. Moreover, a sequence and phylogenetic analysis puts both enzymes into a physiological and evolutionary context. IMPORTANCE Styrene is a toxic compound that is used at a large scale by industry for plastic production. Bacterial degradation of styrene is a possibility for bioremediation and pollution prevention. Intermediates of styrene derivatives degraded in the styrene-specific pathways are precursors for valuable chemical compounds. The pathway in Gordonia rubripertincta CWB2 has proven to accept a broader substrate range than other bacterial styrene degraders. The enzymes characterized in this study, distinguish CWB2s pathway from other known styrene degradation routes and thus might be the main key for its ability to produce ibuprofen from the respective styrene derivative. A biotechnological utilization of this cascade could lead to efficient and sustainable production of drugs, flavors, and fragrances. Moreover, research on glutathione metabolism in Actinobacteria is rare. Here, a characterization of two glutathione S-transferases of actinobacterial origin is presented, and the utilization of glutathione in the metabolism of an Actinobacterium is proven.

Recent studies have shown that the metal adaptation of Actinobacteria offers a rich source of metal inducible environmentally relevant bio-compounds and molecules. These interact through biosorption towards the unique cell walls or via metal chelating activity of metallophors with trace elements, heavy metals and even with lanthanides to overcome limitations and toxic concentrations. Herein, the purpose is to investigate the adaptation potential of Gordonia rubripertincta CWB2 in dependence of the rare earths and to determine if we can utilize promising metallophore metal affinities for metal separation from aquatic solutions. For details on data interpretation and applicability of siderophores we refer to the related article entitled “Cultivation dependent formation of siderophores by Gordonia rubripertincta CWB2” [1].The respective workflow comprises a metal adaptation method to demonstrate effects on bacterial growth, pH, metallophore production, and metabolic change. All this was evaluated by LC-MS/MS and effects on biosorption of rare earths was verified by ICP-MS. Furthermore, we were able to carry out batch metal adsorption and desorption studies of metallophores entrapped in inorganic polymers of tetramethoxysilane (TMOS) to determine metal chelating capacities and selective enrichment effects from model solutions. The adaptation potential of strain CWB2 at increased erbium and manganese concentrations was verified by increased chelating activity on agar plates, in liquid assays and demonstrated by the successful enrichment of erbium by metallophore-functionalized TMOS-polymers from an aquatic model solution. Furthermore, the number of detected compounds in dependency of rare earths differ in spectral counts and diversity compared to the wild type. Finally, the biosorption of rare earths for the selected adaptation was increased significantly up to 2-fold compared to the wild-type. Overall a holistic approach to metal stress was utilised, integrating a bacterial erbium adaptation, metal chelating, biosorption of lanthanides and immobilization as well as enrichment of metals using metallophore functionalized inorganic TMOS polymers for separation of metals from aquatic model solutions.

Biocatalysis that produces economically interesting compounds can be carried out by using free enzymes or microbial cells. However, often the cell metabolism does not allow the overproduction or secretion of activated sugars and thus downstream processing of these sugars is complicated. Here enzyme immobilization comes into focus in order to stabilize the enzyme as well as to make the overall process economically feasible. Besides a robust immobilization method, a highly active and stable enzyme is needed to efficiently produce the product of choice. Herein, we report on the identification, gene expression, biochemical characterization as well as immobilization of the uridine-5′-diphosphate-glucose (UDP-glucose) pyrophosphorylase originating from the thermostable soil actinobacterium Thermocrispum agreste DSM 44070 (TaGalU). The enzyme immobilization was performed on organically modified mesostructured cellular foams (MCF) via epoxy and amino group to provide a stable and active biocatalyst. The soluble and highly active TaGalU revealed a Vmax of 1698 U mg–1 (uridine-5′-triphosphate, UTP) and a Km of 0.15 mM (UTP). The optimum reaction temperature was determined to be 50°C. TaGalU was stable at this temperature for up to 30 min with a maximum loss of activity of 65%. Interestingly, immobilized TaGalU was stable at 50°C for at least 120 min without a significant loss of activity, which makes this enzyme an interesting biocatalyst for the production of UDP-glucose.

The fate of plastic waste and a sustainable use of synthetic polymers is one of the major challenges of the twenty first century. Waste valorization strategies can contribute to the solution of this problem. Besides chemical recycling, biological degradation could be a promising tool. Among the high diversity of synthetic polymers, polyurethanes are widely used as foams and insulation materials. In order to examine bacterial biodegradability of polyurethanes, a soil bacterium was isolated from a site rich in brittle plastic waste. The strain, identified as Pseudomonas sp. by 16S rRNA gene sequencing and membrane fatty acid profile, was able to grow on a PU-diol solution, a polyurethane oligomer, as the sole source of carbon and energy. In addition, the strain was able to use 2,4-diaminotoluene, a common precursor and putative degradation intermediate of polyurethanes, respectively, as sole source of energy, carbon, and nitrogen. Whole genome sequencing of the strain revealed the presence of numerus catabolic genes for aromatic compounds. Growth on potential intermediates of 2,4-diaminotoluene degradation, other aromatic growth substrates and a comparison with a protein data base of oxygenases present in the genome, led to the proposal of a degradation pathway.

Siderophores are of high interest for biotechnological, pharmaceutical, agricultural and industrial applications. Although they are synthesized by various organisms, the yield is usually low which hindrances their suitability for broad range uses. Thus, it is necessary to identify novel producers and to increase the understanding of the biosynthesis pathways. Herein we report the isolation of two novel Pseudomonas strains and the identification of the gene clusters for the biosynthesis of pseudomonine as well as pyochelin and pyoverdine. Keywords: Pseudomonas, Siderophore production, Beech wood hydrolysate, Pyochelin, Pyoverdine, Pseudomonine

The dye-decolorizing peroxidases (DyP) are a family of heme-dependent enzymes present on a broad spectrum of microorganisms. While the natural function of these enzymes is not fully understood, their capacity to degrade highly contaminant pigments such as azo dyes or anthraquinones make them excellent candidates for applications in bioremediation and organic synthesis. In this work, two novel DyP peroxidases from the organism Rhodococcus opacus 1CP (DypA and DypB) were cloned and expressed in Escherichia coli. The enzymes were purified and biochemically characterized. The activities of the two DyPs via 2,2′-azino-bis [3-ethylbenzthiazoline-6-sulphonic acid] (ABTS) assay and against Reactive Blue 5 were assessed and optimized. Results showed varying trends for DypA and DypB. Remarkably, these enzymes presented a particularly high tolerance towards H2O2, retaining its activities at about 10 mM H2O2 for DypA and about 4.9 mM H2O2 for DypB.

Pine Wilt Disease (PWD) is caused by Bursaphelenchus xylophilus, the pinewood nematode, and affects several species of pine trees worldwide. The ecosystem of the Pinus pinaster trees was investigated as a source of bacteria producing metabolites affecting this ecosystem: P. pinaster trees as target-plant, nematode as disease effector and its insect-vector as shuttle. For example, metals and metal-carrying compounds contribute to the complex tree-ecosystems. This work aimed to detect novel secondary metabolites like metallophores and related molecules produced under iron limitation by PWD-associated bacteria and to test their activity on nematodes. After screening 357 bacterial strains from Portugal and United States, two promising metallophore-producing strains Erwinia sp. A41C3 and Rouxiella sp. Arv20#4.1 were chosen and investigated in more detail. The genomes of these strains were sequenced, analyzed, and used to detect genetic potential for secondary metabolite production. A combinatorial approach of liquid chromatography-coupled tandem mass spectrometry (LC-MS) linked to molecular networking was used to describe these compounds. Two major metabolites were detected by HPLC analyses and described. One HPLC fraction of strain Arv20#4.1 showed to be a hydroxamate-type siderophore with higher affinity for chelation of Cu. The HPLC fraction of strain A41C3 with highest metal affinity showed to be a catecholate-type siderophore with higher affinity for chelation of Fe. LC-MS allowed the identification of several desferrioxamines from strain Arv20#4.1, in special desferrioxamine E, but no hit was obtained in case of strain A41C3 which might indicate that it is something new. Bacteria and their culture supernatants showed ability to attract C. elegans. HPLC fractions of those supernatant-extracts of Erwinia strain A41C3, enriched with secondary metabolites such as siderophores, were able to kill pinewood nematode. These results suggest that metabolites secreted under iron limitation have potential to biocontrol B. xylophilus and for management of Pine Wilt Disease.

p-Hydroxybenzoate hydroxylase (PHBH; EC 1.14.13.2) is a microbial group A flavoprotein monooxygenase that catalyzes the ortho-hydroxylation of 4-hydroxybenzoate to 3,4-dihydroxybenzoate with the stoichiometric consumption of NAD(P)H and oxygen. PHBH and related enzymes lack a canonical NAD(P)H-binding domain and the way they interact with the pyridine nucleotide coenzyme has remained a conundrum. Previously, we identified a surface exposed protein segment of PHBH from Pseudomonas fluorescens involved in NADPH binding. Here, we report the first amino acid sequences of NADH-preferring PHBHs and a phylogenetic analysis of putative PHBHs identified in currently available bacterial genomes. It was found that PHBHs group into three clades consisting of NADPH-specific, NAD(P)H-dependent and NADH-preferring enzymes. The latter proteins frequently occur in Actinobacteria. To validate the results, we produced several putative PHBHs in Escherichia coli and confirmed their predicted coenzyme preferences. Based on phylogeny, protein energy profiling and lifestyle of PHBH harboring bacteria we propose that the pyridine nucleotide coenzyme specificity of PHBH emerged through adaptive evolution and that the NADH-preferring enzymes are the older versions of PHBH. Structural comparison and distance tree analysis of group A flavoprotein monooxygenases indicated that a similar protein segment as being responsible for the pyridine nucleotide coenzyme specificity of PHBH is involved in determining the pyridine nucleotide coenzyme specificity of the other group A members.

To guarantee the supply of critical elements in the future, the development of new technologies is essential. Siderophores have high potential in the recovery and recycling of valuable metals due to their metal-chelating properties. Using the Chrome azurol S assay, 75 bacterial strains were screened to obtain a high-yield siderophore with the ability to complex valuable critical metal ions. The siderophore production of the four selected strains Nocardioides simplex 3E, Pseudomonas chlororaphis DSM 50083, Variovorax paradoxus EPS, and Rhodococcus erythropolis B7g was optimized, resulting in significantly increased siderophore production of N. simplex and R. erythropolis. Produced siderophore amounts and velocities were highly dependent on the carbon source. The genomes of N. simplex and P. chlororaphis were sequenced. Bioinformatical analyses revealed the occurrence of an achromobactin and a pyoverdine gene cluster in P. chlororaphis, a heterobactin and a requichelin gene cluster in R. erythropolis, and a desferrioxamine gene cluster in N. simplex. Finally, the results of the previous metal-binding screening were validated by a proof-of-concept development for the recovery of metal ions from aqueous solutions utilizing C18 columns functionalized with siderophores. We demonstrated the recovery of the critical metal ions V(III), Ga(III), and In(III) from mixed metal solutions with immobilized siderophores of N. simplex and R. erythropolis.

Marine habitats are promising sources for the identification of novel organisms as well as natural products. Still, we lack detailed knowledge on most of the marine biosphere. In the last decade, a number of reports described the potential of identifying novel bioactive compounds or secondary metabolites from marine environments. This is, and will be, a promising source for candidate compounds in pharma research and chemical biology. In recent years, a number of novel techniques were introduced into the field, and it has become easier to actually prospect for natural products, such as enzyme inhibitors. These novel compounds then need to be characterized and evaluated in comparison to well-known representatives. A number of current research projects target the exploitation of marine organisms and thus the corresponding diversity of metabolites. These are often encountered as potential drugs or biological active compounds. Among these, the class of enzyme inhibitors is an important group of compounds. There is room for new discoveries, and some more recent discoveries are highlighted herein.

Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from Rhodococcus opacus 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (kcat) while showing increased KM values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but kcat was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted N-phenyl-2-methylmaleimide equally well (81–84%) with an enantiomeric excess (ee) of more than 99% for the R-product. With cyclic ketones, the highest conversion (89%) and ee (>99%) was observed for the reaction of WT with R-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.

Styrene is one of the most produced and processed chemicals worldwide and is released into the environment during widespread processing. But, it is also produced from plants and microorganisms. The natural occurrence of styrene led to several microbiological strategies to form and also to degrade styrene. One pathway designated as side-chain oxygenation has been reported as a specific route for the styrene degradation among microorganisms. It comprises the following enzymes: styrene monooxygenase (SMO; NADH-consuming and FAD-dependent, two-component system), styrene oxide isomerase (SOI; cofactor independent, membrane-bound protein) and phenylacetaldehyde dehydrogenase (PAD; NAD+-consuming) and allows an intrinsic cofactor regeneration. This specific way harbors a high potential for biotechnological use. Based on the enzymatic steps involved in this degradation route, important reactions can be realized from a large number of substrates which gain access to different interesting precursors for further applications. Furthermore, stereochemical transformations are possible, offering chiral products at high enantiomeric excess. This review provides an actual view on the microbiological styrene degradation followed by a detailed discussion on the enzymes of the side-chain oxygenation. Furthermore, the potential of the single enzyme reactions as well as the respective multi-step syntheses using the complete enzyme cascade are discussed in order to gain styrene oxides, phenylacetaldehydes, or phenylacetic acids (e.g., ibuprofen). Altered routes combining these putative biocatalysts with other enzymes are additionally described. Thus, the substrates spectrum can be enhanced and additional products as phenylethanols or phenylethylamines are reachable. Finally, additional enzymes with similar activities toward styrene and its metabolic intermediates are shown in order to modify the cascade described above or to use these enzyme independently for biotechnological application.

The styrene oxide isomerase (SOI) represents a membrane-bound enzyme of the microbial styrene degradation pathway and has been discussed as promising biocatalyst. It catalyzes the isomerization of styrene oxide to phenylacetaldehyde. In this study a styC gene, which encodes the SOI of Rhodococcus opacus 1CP, was optimized for optimal expression in Escherichia coli BL21(DE3) pLysS. The expression of this synthetic styC was investigated and subsequently optimized. Highly active biomass was obtained yielding an SOI activity of 44.5 ± 8.7 U mg−1 after 10 h. This represents the highest SOI activity reported for crude cell extracts of SOI-containing bacterial strains. Remarkably, this biomass can be applied as whole cell biocatalyst for the production of phenylacetic acids from styrene oxides. In the case of non-substituted styrene oxide, nearly 730 mg l−1 phenylacetic acid (∼85% yield) was formed over a period of 20 days.

Some soil bacteria are able to metabolize styrene via initial side-chain oxygenation. This catabolic route is of potential biotechnological relevance due to the occurrence of phenylacetic acid as a central metabolite. The styrene-degrading strains Rhodococcus opacus 1CP, Pseudomonas fluorescens ST, and the novel isolates Sphingopyxis sp. Kp5.2 and Gordonia sp. CWB2 were investigated with respect to their applicability to co-metabolically produce substituted phenylacetic acids. Isolates were found to differ significantly in substrate tolerance and biotransformation yields. Especially, P. fluorescens ST was identified as a promising candidate for the production of several phenylacetic acids. The biotransformation of 4-chlorostyrene with cells of strain ST was shown to be stable over a period of more than 200 days and yielded about 38 mmolproduct gcelldryweight−1 after nearly 350 days. Moreover, 4-chloro-α-methylstyrene was predominantly converted to the (S)-enantiomer of the acid with 40% enantiomeric excess.

At present, there is great demand for new resistant and metabolically active strains of biodegrading bacteria capable of degrading high concentrations of petroleum pollutants. In this study, we undertook a series of pot-based biodegradation experiments on soil from a petroleum refinery lagoon heavily polluted with aliphatic hydrocarbons (81.6 ± 2.5 g·kg−1 dry weight) and metals. Periodical bioaugmentation with either a mixture of isolated degraders identified as Bacillus sp. and Ochrobactrum sp. or biostimulation with nutrient medium, singly or in combination, did not produce any significant decrease in hydrocarbons, even after 455 days. Inoculation with Gordonia rubripertincta CWB2 and Rhodococcus erythropolis S43 in iron-limited media, however, resulted in a significant decrease in hydrocarbons 45 days after bioaugmentation. These actinobacterial strains, therefore, show significant potential for bioremediation of such highly polluted soils.

Flavoprotein monooxygenases create valuable compounds that are of high interest for the chemical, pharmaceutical, and agrochemical industries, among others. Monooxygenases that use flavin as cofactor are either single- or two-component systems. Here we summarize the current knowledge about two-component flavin adenine dinucleotide (FAD)-dependent monooxygenases and describe their biotechnological relevance. Two-component FAD-dependent monooxygenases catalyze hydroxylation, epoxidation, and halogenation reactions and are physiologically involved in amino acid metabolism, mineralization of aromatic compounds, and biosynthesis of secondary metabolites. The monooxygenase component of these enzymes is strictly dependent on reduced FAD, which is supplied by the reductase component. More and more representatives of two-component FAD-dependent monooxygenases have been discovered and characterized in recent years, which has resulted in the identification of novel physiological roles, functional properties, and a variety of biocatalytic opportunities.

Herein we describe the first representative of an E2-type two-component styrene monooxygenase of proteobacteria. It comprises a single epoxidase protein (VpStyA1) and a two domain protein (VpStyA2B) harboring an epoxidase (A2) and a FAD-reductase (B) domain. It was annotated as VpStyA1/VpStyA2B of Variovorax paradoxus EPS. VpStyA2B serves mainly as NADH:FAD-oxidoreductase. A Km of 33.6 ± 4.0 µM for FAD and a kcat of 22.3 ± 1.1 s−1 were determined and resulted in a catalytic efficiency (kcat Km−1) of 0.64 s−1 μM−1. To investigate its NADH:FAD-oxidoreductase function the linker between A2- and B-domain (AREAV) was mutated. One mutant (AAAAA) showed 18.7-fold higher affinity for FAD (kcat Km−1 of 5.21 s−1 μM−1) while keeping wildtype NADH-affinity and -oxidation activity. Both components, VpStyA2B and VpStyA1, showed monooxygenase activity on styrene of 0.14 U mg−1 and 0.46 U mg−1, as well as on benzyl methyl sulfide of 1.62 U mg−1 and 3.11 U mg−1, respectively. The high sulfoxidase activity was the reason to test several thioanisole-like substrates in biotransformations. VpStyA1 showed high substrate conversions (up to 95% in 2 h) and produced dominantly (S)-enantiomeric sulfoxides of all tested substrates. The AAAAA-mutant showed a 1.6-fold increased monooxygenase activity. In comparison, the GQWCSQY-mutant did neither show monooxygenase nor efficient FAD-reductase activity. Hence, the linker between the two domains of VpStyA2B has effects on the reductase as well as on the monooxygenase performance. Overall, this monooxygenase represents a promising candidate for biocatalyst development and studying natural fusion proteins.