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The marine bacterium Alcanivorax borkumensis produces a surface-active glycine-glucolipid during growth with long-chain alkanes. A high-performance liquid chromatography (HPLC) method was developed for absolute quantification. This method is based on the conversion of the glycine-glucolipid to phenacyl esters with subsequent measurement by HPLC with diode array detection (HPLC-DAD). Different molecular species were separated by HPLC and identified as glucosyl-tetra(3-hydroxy-acyl)-glycine with varying numbers of 3-hydroxy-decanoic acid or 3-hydroxy-octanoic acid groups via mass spectrometry. The growth rate of A. borkumensis cells with pyruvate as the sole carbon source was elevated compared to hexadecane as recorded by the increase in cell density as well as oxygen/carbon dioxide transfer rates. The amount of the glycine-glucolipid produced per cell during growth on hexadecane was higher compared with growth on pyruvate. The glycine-glucolipid from pyruvate-grown cells contained considerable amounts of 3-hydroxy-octanoic acid, in contrast to hexadecane-grown cells, which almost exclusively incorporated 3-hydroxy-decanoic acid into the glycine-glucolipid. The predominant proportion of the glycine-glucolipid was found in the cell pellet, while only minute amounts were present in the cell-free supernatant. The glycine-glucolipid isolated from the bacterial cell broth, cell pellet, or cell-free supernatant showed the same structure containing a glycine residue, in contrast to previous reports, which suggested that a glycine-free form of the glucolipid exists which is secreted into the supernatant. In conclusion, the glycine-glucolipid of A. borkumensis is resident to the cell wall and enables the bacterium to bind and solubilize alkanes at the lipid-water interface. IMPORTANCE Alcanivorax borkumensis is one of the most abundant marine bacteria found in areas of oil spills, where it degrades alkanes. The production of a glycine-glucolipid is considered an essential element for alkane degradation. We developed a quantitative method and determined the structure of the A. borkumensis glycine-glucolipid in different fractions of the cultures after growth in various media. Our results show that the amount of the glycine-glucolipid in the cells by far exceeds the amount measured in the supernatant, confirming the proposed cell wall localization. These results support the scenario that the surface hydrophobicity of A. borkumensis cells increases by producing the glycine-glucolipid, allowing the cells to attach to the alkane-water interface and form a biofilm. We found no evidence for a glycine-free form of the glucolipid.

Organisms from all kingdoms of life represent a rich source for valuable natural products with various applications. Microbial systems are useful to implement the expression of respective biosynthetic genes and establish the production and effective access to natural products, or to elucidate the biochemistry of the underlying pathways. Pseudomonas putida, a Gram-negative soil bacterium, appears to be particularly suitable for natural product biosynthesis because of an advantageous intrinsic metabolism and a remarkable tolerance toward various xenobiotics. This article presents the current state of engineering P. putida for the biosynthesis of rhamnolipids, terpenoids, polyketides, non-ribosomal peptides, and products from amino acid metabolism. Technological advances facilitating chromosomal integration and expression of biosynthetic genes, and smart metabolic engineering are key to success.

The isolation and screening of bacteria and fungi for the production of surface-active compounds has been the basis for the majority of the biosurfactants discovered to date. Hence, a wide variety of well-established and relatively simple methods are available for screening, mostly focused on the detection of surface or interfacial activity of the culture supernatant. However, the success of any biodiscovery effort, specifically aiming to access novelty, relies directly on the characteristics being screened for and the uniqueness of the microorganisms being screened. Therefore, given that rather few novel biosurfactant structures have been discovered during the last decade, advanced strategies are now needed to widen access to novel chemistries and properties. In addition, more modern Omics technologies should be considered to the traditional culture-based approaches for biosurfactant discovery. This chapter summarizes the screening methods and strategies typically used for the discovery of biosurfactants and highlights some of the Omics-based approaches that have resulted in the discovery of unique biosurfactants. These studies illustrate the potentially enormous diversity that has yet to be unlocked and how we can begin to tap into these biological resources.

Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts. Species of the Pseudomonas clade thus represent especially valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chemicals. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compounds. Finally, toxic chemicals such as reactive aldehydes can be eliminated by oxidation and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective production of natural compounds and new chemicals. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnological context. We further highlight strategies for the identification of yet unknown tolerance-associated genes and their utilisation for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective production of natural compounds and novel biochemicals.

The FEBS journal 288(11), 3570 - 3584 (2020). doi:10.1111/febs.15680, Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone-sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio-, regio-, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme–substrate or enzyme–inhibitor complexes are less frequent although important to understand the molecular basis of enzyme–substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen, which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81 Å resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an α-helical cap domain. The substrate analogue methyl 4-methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4-methylumbelliferone was found remaining in the active site cavity, and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme–substrate interaction of esterases belonging to family IV., Published by Wiley-Blackwell, Oxford [u.a.]

The isolation and screening of bacteria and fungi for the production of surface-active compounds has been the basis for the majority of the biosurfactants discovered to date. Hence, a wide variety of well-established and relatively simple methods are available for screening, mostly focused on the detection of surface or interfacial activity of the culture supernatant. However, the success of any biodiscovery effort, specifically aiming to access novelty, relies directly on the characteristics being screened for and the uniqueness of the microorganisms being screened. Therefore, given that rather few novel biosurfactant structures have been discovered during the last decade, advanced strategies are now needed to widen access to novel chemistries and properties. In addition, more modern Omics technologies should be considered to the traditional culture-based approaches for biosurfactant discovery. This chapter summarizes the screening methods and strategies typically used for the discovery of biosurfactants and highlights some of the Omics-based approaches that have resulted in the discovery of unique biosurfactants. These studies illustrate the potentially enormous diversity that has yet to be unlocked and how we can begin to tap into these biological resources.

Transportation networks underpin socioeconomic development by enabling the movement of goods and people. However, little is known about how flooding disrupts transportation systems in urban areas in developing country cities, despite these natural disasters occurring frequently. This study documents the channels through which regular flooding in Kinshasa, the Democratic Republic of Congo, impacts transport services, commuters' ability to reach their jobs, and the associated economic opportunity costs from travel delays. This assessment is based on transit feed specification data sets collected specifically for this analysis under normal and flooded conditions. These data sets were combined with travel survey data containing travelers' socioeconomic attributes and trip parameters, as well as a high-resolution flood maps. The results show that (1) flood disruptions cause increases in public transit headways and transit re-routing, decreases in travel speeds, and thus travel time delays, which translate into substantial economic costs to local commuters; (2) accessibility to jobs decreases under flooded conditions, hindering the establishment of an integrated citywide labor market; (3) there are spatial clusters where some of the poorest commuters experience among the highest travel delays, highlighting socio-spatial equity aspects of floods; (4) certain road segments are critical for the transport network and should be prioritized for resilience measures; and (5) the estimated daily cost of flood disruption to commuters’ trips in Kinshasa is $1,166,000. The findings of this assessment provide disaster mitigation guidance to the Office des Voiries et Drainage under the Ministry of Infrastructure, as well as strategic investment recommendations to the Ministry of Housing and Planning.

Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs., Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by carboxylic ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of carboxylic ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant esterase from Pseudomonas aestusnigri. In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard ester hydrolase CalB from Candida antarctica. IMPORTANCE Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.

Biodegradation of synthetic polymers, in particular polyethylene terephthalate (PET), is of great importance, since environmental pollution with PET and other plastics has become a severe global problem. Here, we report on the polyester degrading ability of a novel carboxylic ester hydrolase identified in the genome of the marine hydrocarbonoclastic bacterium Pseudomonas aestusnigri VGXO14T. The enzyme, designated PE-H, belongs to the type IIa family of PET hydrolytic enzymes as indicated by amino acid sequence homology. It was produced in Escherichia coli, purified and its crystal structure was solved at 1.09 Å resolution representing the first structure of a type IIa PET hydrolytic enzyme. The structure shows a typical α/β-hydrolase fold and high structural homology to known polyester hydrolases. PET hydrolysis was detected at 30°C with amorphous PET film (PETa), but not with PET film from a commercial PET bottle (PETb). A rational mutagenesis study to improve the PET degrading potential of PE-H yielded variant PE-H (Y250S) which showed improved activity, ultimately also allowing the hydrolysis of PETb. The crystal structure of this variant solved at 1.35 Å resolution allowed to rationalize the improvement of enzymatic activity. A PET oligomer binding model was proposed by molecular docking computations. Our results indicate a significant potential of the marine bacterium P. aestusnigri for PET degradation.

Cyclic triterpenes are a large group of secondary metabolites produced by plants, fungi and bacteria. They have diverse biological functions, and offer potential health benefits for humans. Although various terpenes from the mono-, sesqui- and diterpene classes are easy to produce in engineered bacteria, heterologous synthesis of cyclic triterpenes is more challenging. We have recently shown that the triterpene cycloartenol can be produced in Rhodobacter capsulatus SB1003 but initial titers were low with 0.34 mg L−1. To assess, if this phototrophic α-proteobacterium can be engineered for enhanced triterpene production, we followed two alternative strategies by comparing the performance of the R. capsulatus SB1003 wildtype strain with two recombinant strains carrying either a mevalonate pathway implemented from Paracoccus zeaxanthinifaciens or a deletion in the intrinsic carotenoid biosynthesis gene crtE. These strains are thus engineered for an enhanced isoprenoid biosynthesis or a suppressed precursor conversion by the competing carotenoid pathway. Moreover, three different cycloartenol synthase (CAS) genes from Arabidopsis thaliana or the myxobacterial strains Stigmatella aurantiaca Sg a15 and DW4/3-1 were tested for heterologous cycloartenol synthesis. We found that the heterologous expression of mevalonate pathway enzymes had little impact on cycloartenol levels irrespective of the chosen CAS. In contrast, the use of the newly constructed carotenoid-deficient crtE deletion strain showed threefold increased cycloartenol product titers. We conclude that R. capsulatus is a promising alternative host for the functional expression of triterpene biosynthetic enzymes from plants and microbes. Apparently, product titers can also be improved by suppression of competing precursor consumption.

Ribosomal RNA (rRNA) operons have recently been identified as promising sites for chromosomal integration of genetic elements in

Ribosomal RNA (rRNA) operons have recently been identified as promising sites for chromosomal integration of genetic elements in Pseudomonas putida, a bacterium that has gained considerable popularity as a microbial cell factory. We have developed a tool for targeted integration of recombinant genes into the rRNA operons of various Pseudomonas strains, where the native context of the rRNA clusters enables effective transcription of heterologous genes. However, a sufficient translation of foreign mRNA transcriptionally fused to rRNA required optimization of RNA secondary structures, which was achieved utilizing synthetic ribozymes and a bicistronic design. The generated tool further enabled the characterization of the six rRNA promoter units of P. putida S12 under different growth conditions. The presence of multiple, almost identical rRNA operons in Pseudomonas also allowed the integration of multiple copies of heterologous genetic elements. The integration of two expression cassettes and the resulting disruption of rRNA units only moderately affects growth rates, and the constructs were highly stable over more than 160 generations.

Land plants are engaged in intricate communities with soil bacteria and fungi indispensable for plant survival and growth. The plant-microbial interactions are largely governed by specific metabolites. We employed a combination of lipid-fingerprinting, enzyme activity assays, high-throughput DNA sequencing and isolation of cultivable microorganisms to uncover the dynamics of the bacterial and fungal community structures in the soil after exposure to isothiocyanates (ITC) obtained from rapeseed glucosinolates. Rapeseed-derived ITCs, including the cyclic, stable goitrin, are secondary metabolites with strong allelopathic affects against other plants, fungi and nematodes, and in addition can represent a health risk for human and animals. However, the effects of ITC application on the different bacterial and fungal organisms in soil are not known in detail. ITCs diminished the diversity of bacteria and fungi. After exposure, only few bacterial taxa of the Gammaproteobacteria, Bacteriodetes and Acidobacteria proliferated while Trichosporon (Zygomycota) dominated the fungal soil community. Many surviving microorganisms in ITC-treated soil where previously shown to harbor plant growth promoting properties. Cultivable fungi and bacteria were isolated from treated soils. A large number of cultivable microbial strains was capable of mobilizing soluble phosphate from insoluble calcium phosphate, and their application to Arabidopsis plants resulted in increased biomass production, thus revealing growth promoting activities. Therefore, inclusion of rapeseed-derived glucosinolates during biofumigation causes losses of microbiota, but also results in enrichment with ITC-tolerant plant microorganisms, a number of which show growth promoting activities, suggesting that Brassicaceae plants can shape soil microbiota community structure favoring bacteria and fungi beneficial for Brassica plants.

Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases’ substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein–ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets., C.C. thanks the Spanish Ministry of Economy, Industry and Competitiveness for a Ph.D. fellowship (Grant BES-2015-073829). This project received funding from the European Union’s Horizon 2020 research and innovation program [Blue Growth: Unlocking the potential of Seas and Oceans] under grant agreement no. 634486 (project acronym INMARE). This research was also supported by the European Community Projects MAGICPAH (FP7-KBBE-2009-245226), ULIXES (FP7-KBBE-2010-266473), and KILLSPILL (FP7-KBBE2012-312139) and grants BIO2011-25012, PCIN-2014-107, BIO2014-54494-R, and CTQ2016-79138-R from the Spanish Ministry of Economy, Industry and Competitiveness. The present investigation was also funded by the Spanish Ministry of Economy, Industry and Competitiveness within the ERA NET IB2, grant no. ERA-IB-14-030 (MetaCat), the UK Biotechnology and Biological Sciences Research Council (BBSRC), grant no. BB/M029085/1, and the German Research Foundation (FOR1296). R.B. and P.N.G. acknowledge the support of the Supercomputing Wales project, which is part-funded by the European Regional Development Fund (ERDF) via the Welsh Government. O.V.G. and P.N.G. acknowledge the support of the Centre of Environmental Biotechnology Project funded by the European Regional Development Fund (ERDF) through the Welsh Government. A.Y. and A.S. gratefully acknowledge funding from Genome Canada (2009-OGI-ABC-1405) and the NSERC Strategic Network grant IBN. A.I.P. was supported by the Counseling of Economy and Employment of the Principality of Asturias, Spain (Grant FC-15-GRUPIN14-107). V.G. acknowledges the joint BSC-CRG-IRB Research Program in Computational Biology. The authors gratefully acknowledge financial support provided by the European Regional Development Fund (ERDF)., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

The biosynthesis of natural products by heterologous expression of biosynthetic pathways in amenable production strains enables biotechnological access to a variety of valuable compounds by conversion of renewable resources. Pseudomonas putida has emerged as a microbial laboratory work horse, with elaborated techniques for cultivation and genetic manipulation available. Beyond that, this bacterium offers several particular advantages with regard to natural product biosynthesis, notably a versatile intrinsic metabolism with diverse enzymatic capacities as well as an outstanding tolerance to xenobiotics. Therefore, it has been applied for recombinant biosynthesis of several valuable natural products. This review provides an overview of applications of P. putida as a host organism for the recombinant biosynthesis of such natural products, including rhamnolipids, terpenoids, polyketides and non-ribosomal peptides, and other amino acid-derived compounds. The focus is on de novo natural product synthesis from intrinsic building blocks by means of heterologous gene expression and strain engineering. Finally, the future potential of the bacterium as a chassis organism for synthetic microbiology is pointed out.

The Gram-negative proteobacterium Pseudomonas oleovorans DSM 1045 is considered a promising source for enzymes of biotechnological interest, e.g., hydrolases and transaminases. Here, we present a draft sequence of its 4.86-Mb genome, enabling the identification of novel biocatalysts.

Novel shuttle vectors named pEBP were constructed to allow the gene expression in different bacterial hosts including Escherichia coli, Bacillus subtilis and Pseudomonas putida. These vectors share the inducible promoters P(T7) and P(Xyl) and a cos site to enable packaging of plasmid DNA into phage, and carry different multiple cloning sites and antibiotic resistance genes. Vector pEBP41 generally replicates episomally while pEBP18 replicates episomally in Gram-negative bacteria only, but integrates into the chromosome of B. subtilis. Plasmid copy numbers determined for E. coli and P. putida were in the range of 5-50 per cell. The functionality of pEBP18 and pEBP41 was confirmed by expression of two lipolytic enzymes, namely lipase A from B. subtilis and cutinase from the eukaryotic fungus Fusarium solani pisi in three different host strains. Additionally, we report here the construction of a T7 RNA polymerase-based expression strain of P. putida.

The crystal structures of the ternary phases (RE) 2 (Ag, Al) 17 (RELa, Ce, Pr, Yb) contain puckered Kagome nets of silver and aluminium which are stacked identically along the [001] direction. La 2 Ag 17- x Al x ( x =7.3), Ce 2 Ag 17- x Al x ( x =8.2), Pr 2 Ag 17- x Al x ( x =7) and Yb 2 Ag 17- x Al x ( x =8) crystallize as a ternary variant of the Th 2 Zn 17 structure type (space group R3m ; lattice constants in the range a =940.0–945.1 pm; c =1366.1–1372.7 pm; Z =3) which contains channels resulting from the sharing of common basal faces by hexagonal prisms of silver and aluminium atoms. Two-thirds of these prisms are occupied by RE atoms and one-third by Ag 2 pairs. La 1.8 Ag 17- x Al x ( x =10.5), Ce 1.8 Ag 17- x Al x ( x =10.5), Pr 1.8 Ag 17- x Al x ( x =10.5) and Yb 1.8 Ag 17- x Al x ( x =10) form ternary variants of the Th 2 Ni 17 structure type (space group P 6 3 / mmc ; a =933.2–940.4 pm; c =911.0–915.3 pm; Z =3) with one-third of the channels being occupied by RE atoms and the remaining two-thirds alternately by Ag 2 pairs and RE atoms. A correlation between electron concentration and the various elements which occupy the hexagonal prismatic channels will be discussed.

We report the first measurements of the electrical resistivity, the magnetic susceptibility and the specific heat of the new hexagonal compound CePd 2 Al 3 . The results indicate that CePd 2 Al 3 h is a new heavy fermion system with an electronic specific heat coefficient (γ) of 380 mJ/mol·K 2 and antiferromagnetic ordering below T N =2.8 K.

Zusammenfassung Die Kristallstrukturen der intermediaren Phasen der ternaren Verbindungen Ca(Yb)-Ag-Al enthalten Kagome-Netze in unterschiedlicher Stapelfolge. YbAg0.3Al1.71 kristallisiert als ternare Variante des MgCu2-Strukturtyps mit Aluminium-Kagome-Netzen in der Abfolge ABC; in der Kristallstruktur von CaAg0.8Al1.22 (ternare Variante des MgZn2Strukturtyps) werden Aluminium-Kagome-Netze in der Abfolge AB gestapelt. Beide Verbindungen stellen damit direkte Abkommlinge der Lavesphasen dar. Die Kristallstrukturen von CaAg1.3A11.73, YbAg1.3A11.74, CaAg0.7Al2.35, YbAg0.7A12.36, CeAg0.9Al2.17, und PrAgAl28 zeigen deutliche Bezuge zu Friauf-Laves-Phasen; die Aluminium Kagome-Netze treten in 3 und 4 allerdings mit der Abfolge AABB, in 5, 6, 7 und 8 mit der Abfolge AABBCC auf (ternare Varianten des CeNi3-, bzw. des PuNi3-Typs). Korrelationen zwischen Elektronenkonzentration und der Netzabfolge werden diskutiert. Verbindung Gitterkonstanten (pm) Raumgruppe Z Strukturtyp Stapelfolge der Kagome-Netze a c YbAg0.3Al1.71 792,5(5) Fd3m 8 MgCu2 ABC CaAg0.8Al1.22 571,6(4) 913,3(6) P63/mmc 4 MgZn2 AB CaAg1.3Al1.73 559,7(4) 1800,2(7) P63/mmc 6 CeNi3 AABB YbAg1.3Al1.74 559,0(4) 1790,8(7) P63/mmc 6 CeNi3 AABB CaAg0.7Al2.35 561,4(1) 2678,8(5) R3m 9 PuNi3 AABBCC YbAg0.7Al2.36 555,5(4) 2638,4(7) R3m 9 PuNi3 AABBCC CeAg0.9Al2.17 563,7(1) 2691,3(8) R3m 9 PuNi3 AABBCC PrAgAl28 563,2(2) 2704,5(6) R3m 9 PuNi3 AABBCC

We report measurements on polycrystalline and, for the first time, single-crystalline samples of UPd2Al3, a new heavy-fermion superconductor (HFS) with a record-Tc of 2K and an antiferromagnetic transition at TN = 14K.

Measurements of the resistivity, susceptibility and specific heat are reported which clearly show that the hexagonal compound UNi2Al3 is a heavy-fermion magnet belowT N=4.6 K and a heavy-fermion superconductor belowT c=1 K.

UPd2Al3 is a new heavy-fermion superconductor with a recordTc of 2 K. In addition, it shows a transition to long-range antiferromagnetic order atTN=14 K. Its Sommerfeld coefficient is reduced from γp=210mJ/K2 mole in the paramagnetic to γ0=150mJ/K2 mole in the antiferromagnetic phase.

[EN] Engineering dual-function single polypeptide catalysts with two abiotic or biotic catalytic entities (or combinations of both) supporting cascade reactions is becoming an important area of enzyme engineering and catalysis. Herein we present the development of a PluriZyme, TRE, with efficient native transaminase (k: 69.49±1.77 min) and artificial esterase (k: 3908–0.41 min) activities integrated into a single scaffold, and evaluate its utility in a cascade reaction. TRE (pH: 8.0–9.5; T: 60–65 °C) efficiently converts methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate into 3-(R)-amino-4-(2,4,5-trifluorophenyl)butanoic acid, a crucial intermediate for the synthesis of antidiabetic drugs. The reaction proceeds through the conversion of the β-keto ester into the β-keto acid at the hydrolytic site and subsequently into the β-amino acid (e.e. >99 %) at the transaminase site. The catalytic power of the TRE PluriZyme was proven with a set of β-keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions., FuturEnzyme Project funded by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 101000327. We also acknowledge financial support under Grants PCIN-2017-078 (within the Marine Biotechnology ERA-NET, GA No. 604814), BIO2017-85522-R (M.F.), PID2020 112758RB-I00 (M.F.), PID2019-106370RB-I00 (V.G.) and PDC2021-121534-I00 (M.F.), and PID2019-105838RB-C31 (F.J.P.) from the Ministerio de Economía, Industria y Competitividad, Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI) (Digital Object Identifier 10.13039/501100011033), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (“NextGenerationEU/PRTR”), and Grant 2020AEP061 (M.F.) from the Agencia Estatal CSIC. C. Coscolín thanks the Ministerio de Economía y Competitividad and FEDER for a PhD fellowship (Grant BES-2015-073829). J.L.G.-A. acknowledges support from the Spanish Ministry of Education, Culture and Sport through the National Program FPU (FPU17/00044). The Center for Structural Studies is funded by the Deutsche Forschungsgemeinschaft (DFG Grant number 417919780; INST 208/740-1 FUGG; INST 208/761-1 FUGG). S. R. thanks the Spanish Ministry of Science and Innovation for a PhD fellowship (FPU19/00608).