SIDEROPHORES FOR SELECTIVE SOLID PHASE EXTRACTION OF STRATEGIC ELEMENTS

All over the world, industrial mining is leaving contaminated areas and dumps that, although being full of valuable metals, have high concentrations of toxic heavy metals that pollute the environment. The development of sustainable alternative biomining and bioremediation processes offers the potential to fully exploit these unexploited mining sites

pShuffle: A Plasmid for in vitro Evolution

Multi-gene shuffling is a powerful method used to combine and optimize attributes of various proteins. Here we report on the design and construction of the plasmid “pShuffle” which is suited for a variety of in vitro DNA-recombination techniques. The multiple cloning site (MCS) of pShuffle was designed to allow for the cloning of genes as well as their expression under control of either a lac- or a T7-promoter. As a specific feature, this MCS allows for the fusion of special linker sequences to both ends of cloned genes. After subsequent DNA-recombination steps, these linkers facilitate reamplification of generated gene variants, and thus may be used to construct clone libraries for activity screenings. The suitability of pShuffle for multi-gene shuffling applications was further shown with a set of styrene monooxygenase genes originating from proteo- and actinobacteria

Cell‐free protein synthesis for the screening of novel azoreductases and their preferred electron donor

Azoreductases are potent biocatalysts for the cleavage of azo bonds. Various gene sequences coding for potential azoreductases are available in databases, but many of their gene products are still uncharacterized. To avoid the laborious heterologous expression in a host organism, we developed a screening approach involving cell-free protein synthesis (CFPS) combined with a colorimetric activity assay, which allows the parallel screening of putative azoreductases in a short time. First, we evaluated different CFPS systems and optimized the synthesis conditions of a model azoreductase. With the findings obtained, 10 azoreductases, half of them undescribed so far, were screened for their ability to degrade the azo dye methyl red. All novel enzymes catalyzed the degradation of methyl red and can therefore be referred to as azoreductases. In addition, all enzymes degraded the more complex and bulkier azo dye Brilliant Black and four of them also showed the ability to reduce p-benzoquinone. NADH was the preferred electron donor for the most enzymes, although the synthetic nicotinamide co-substrate analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) was also accepted by all active azoreductases. This screening approach allows accelerated identification of potential biocatalysts for various applications

Vanillyl alcohol oxidase from Diplodia corticola:Residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives

Vanillyl alcohol oxidases belong to the 4-phenol oxidases family and are found predominantly in lignin-degrading ascomycetes. Systematical investigation of the enzyme family at the sequence level resulted in discovery and characterization of the second recombinantly produced VAO member, DcVAO, from Diplodia corticola. Remarkably high activities for 2,6-substituted substrates like 4-allyl-2,6-dimethoxy-phenol (3.5 ± 0.02 U mg -1) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3 ± 0.5 U mg -1) were observed which could be attributed to a Phe to Ala exchange in the catalytic center. In order to rationalize this rare substrate preference among VAOs, we resurrected and characterized three ancestral enzymes and performed mutagenesis analyses. The results indicate that a Cys/Glu exchange was required to retain activity for ɣ-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0 ± 0.1 U mg -1). Our findings contribute to the understanding of the functionality of VAO enzyme group, and with DcVAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts. </p

Neue Wege in der Weißen Biotechnologie

Mikroorganismen sind in der Lage, zahlreiche Xenobiotika abzubauen. Dazu nutzen sie unter aeroben Bedingungen oft einleitend Oxygenasen. Durch diese kann molekularer Luftsauerstoff aktiviert und auf organische Moleküle übertragen werden. Danach können die Verbindungen in den Metabolismus der Mikroorganismen eingeschleust und teils oder vollständig abgebaut werden. Am Beispiel des Styrols zeigen wir hier eine solche Abbauroute und wie wir diese biotechnologisch nutzen können, um interessante Verbindungen zu synthetisieren. Zielmoleküle der gesamten Enzymkaskade sind dabei diverse Phenylessigsäurederivate

Asymmetric azidohydroxylation of styrene derivatives mediated by a biomimetic styrene monooxygenase enzymatic cascade

Enantioenriched azido alcohols are precursors for valuable chiral aziridines and 1,2-amino alcohols, however their chiral substituted analogues are difficult to access. We established a cascade for the asymmetric azidohydroxylation of styrene derivatives leading to chiral substituted 1,2-azido alcohols via enzymatic asymmetric epoxidation, followed by regioselective azidolysis, affording the azido alcohols with up to two contiguous stereogenic centers. A newly isolated two-component flavoprotein styrene monooxygenase StyA proved to be highly selective for epoxidation with a nicotinamide coenzyme biomimetic as a practical reductant. Coupled with azide as a nucleophile for regioselective ring opening, this chemo-enzymatic cascade produced highly enantioenriched aromatic α-azido alcohols with up to >99% conversion. A bi-enzymatic counterpart with halohydrin dehalogenase-catalyzed azidolysis afforded the alternative β-azido alcohol isomers with up to 94% diastereomeric excess. We anticipate our biocatalytic cascade to be a starting point for more practical production of these chiral compounds with two-component flavoprotein monooxygenases. A one-pot enzymatic cascade for the asymmetric azidohydroxylation of styrenes leads to chiral 1,2-azido alcohols with up to two stereocenters

Editorial: Actinobacteria, a Source of Biocatalytic Tools

Actinobacteria (Actinomycetes) represent one of the largest and most diverse phyla among the Bacteria. The characteristics and phylogeny of actinobacteria have been well-described throughout the years (Anteneh and Franco; Embley et al., 1994; Stackebrandt et al., 1997a,b; Stach and Bull, 2005; Stackebrandt and Schumann, 2006; Ventura et al., 2007; Gao and Gupta, 2012; Goodfellow, 2012a,b; Schrempf, 2013; Lawson, 2018; Lewin et al., 2016). Still actinobacteria are hotspots for discovery of new biomolecules and enzyme activities, fueling an active field of research. The remarkable diversity is displayed by various lifestyles, distinct morphologies, a wide spectrum of physiological and metabolic activities, as well as genetics

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin