The actin-binding protein CAP1 represses MRTF-SRF-dependent gene expression in mouse cerebral cortex.

Serum response factor (SRF) is an essential transcription factor for brain development and function. Here, we explored how an SRF cofactor, the actin monomer-sensing myocardin-related transcription factor MRTF, is regulated in mouse cortical neurons. We found that MRTF-dependent SRF activity in vitro and in vivo was repressed by cyclase-associated protein CAP1. Inactivation of the actin-binding protein CAP1 reduced the amount of actin monomers in the cytoplasm, which promoted nuclear MRTF translocation and MRTF-SRF activation. This function was independent of cofilin1 and actin-depolymerizing factor, and CAP1 loss of function in cortical neurons was not compensated by endogenous CAP2. Transcriptomic and proteomic analyses of cerebral cortex lysates from wild-type and Cap1 knockout mice supported the role of CAP1 in repressing MRTF-SRF-dependent signaling in vivo. Bioinformatic analysis identified likely MRTF-SRF target genes, which aligned with the transcriptomic and proteomic results. Together with our previous studies that implicated CAP1 in axonal growth cone function as well as the morphology and plasticity of excitatory synapses, our findings establish CAP1 as a crucial actin regulator in the brain relevant for formation of neuronal networks.

Mechanism and structural dynamics of sulfur transfer during de novo [2Fe-2S] cluster assembly on ISCU2.

Maturation of iron-sulfur proteins in eukaryotes is initiated in mitochondria by the core iron-sulfur cluster assembly (ISC) complex, consisting of the cysteine desulfurase sub-complex NFS1-ISD11-ACP1, the scaffold protein ISCU2, the electron donor ferredoxin FDX2, and frataxin, a protein dysfunctional in Friedreich's ataxia. The core ISC complex synthesizes [2Fe-2S] clusters de novo from Fe and a persulfide (SSH) bound at conserved cluster assembly site residues. Here, we elucidate the poorly understood Fe-dependent mechanism of persulfide transfer from cysteine desulfurase NFS1 to ISCU2. High-resolution cryo-EM structures obtained from anaerobically prepared samples provide snapshots that both visualize different stages of persulfide transfer from Cys381NFS1 to Cys138ISCU2 and clarify the molecular role of frataxin in optimally positioning assembly site residues for fast sulfur transfer. Biochemical analyses assign ISCU2 residues essential for sulfur transfer, and reveal that Cys138ISCU2 rapidly receives the persulfide without a detectable intermediate. Mössbauer spectroscopy assessing the Fe coordination of various sulfur transfer intermediates shows a dynamic equilibrium between pre- and post-sulfur-transfer states shifted by frataxin. Collectively, our study defines crucial mechanistic stages of physiological [2Fe-2S] cluster assembly and clarifies frataxin's molecular role in this fundamental process.

Targeting the High-Density Lipoprotein Proteome for the Treatment of Post-Acute Sequelae of SARS-CoV-2.

Here, we target the high-density lipoprotein (HDL) proteome in a case series of 16 patients with post-COVID-19 symptoms treated with HMG-Co-A reductase inhibitors (statin) plus angiotensin II type 1 receptor blockers (ARBs) for 6 weeks. Patients suffering from persistent symptoms (post-acute sequelae) after serologically confirmed SARS-CoV-2 infection (post-COVID-19 syndrome, PCS, n = 8) or following SARS-CoV-2 vaccination (PVS, n = 8) were included. Asymptomatic subjects with corresponding serological findings served as healthy controls (n = 8/8). HDL was isolated using dextran sulfate precipitation and the HDL proteome of all study participants was analyzed quantitatively by mass spectrometry. Clinical symptoms were assessed using questionnaires before and after therapy. The inflammatory potential of the patients' HDL proteome was addressed in human endothelial cells. The HDL proteome of patients with PCS and PVS showed no significant differences; however, compared to controls, the HDL from PVS/PCS patients displayed significant alterations involving hemoglobin, cytoskeletal proteins (MYL6, TLN1, PARVB, TPM4, FLNA), and amyloid precursor protein. Gene Ontology Biological Process (GOBP) enrichment analysis identified hemostasis, peptidase, and lipoprotein regulation pathways to be involved. Treatment of PVS/PCS patients with statins plus ARBs improved the patients' clinical symptoms. After therapy, three proteins were significantly increased (FAM3C, AT6AP2, ADAM10; FDR < 0.05) in the HDL proteome from patients with PVS/PCS. Exposure of human endothelial cells with the HDL proteome from treated PVS/PCS patients revealed reduced inflammatory cytokine and adhesion molecule expression. Thus, HDL proteome analysis from PVS/PCS patients enables a deeper insight into the underlying disease mechanisms, pointing to significant involvement in metabolic and signaling disturbances. Treatment with statins plus ARBs improved clinical symptoms and reduced the inflammatory potential of the HDL proteome. These observations may guide future therapeutic strategies for PVS/PCS patients.

A Synthetic Pathway for the Production of Benzylsuccinate in Escherichia coli.

(R)-Benzylsuccinate is generated in anaerobic toluene degradation by the radical addition of toluene to fumarate and further degraded to benzoyl-CoA by a ß-oxidation pathway. Using metabolic modules for benzoate transport and activation to benzoyl-CoA and the enzymes of benzylsuccinate ß-oxidation, we established an artificial pathway for benzylsuccinate production in Escherichia coli, which is based on its degradation pathway running in reverse. Benzoate is supplied to the medium but needs to be converted to benzoyl-CoA by an uptake transporter and a benzoate-CoA ligase or CoA-transferase. In contrast, the second substrate succinate is endogenously produced from glucose under anaerobic conditions, and the constructed pathway includes a succinyl-CoA:benzylsuccinate CoA-transferase that activates it to the CoA-thioester. We present first evidence for the feasibility of this pathway and explore product yields under different growth conditions. Compared to aerobic cultures, the product yield increased more than 1000-fold in anaerobic glucose-fermenting cultures and showed further improvement under fumarate-respiring conditions. An important bottleneck to overcome appears to be product excretion, based on much higher recorded intracellular concentrations of benzylsuccinate, compared to those excreted. While no export system is known for benzylsuccinate, we observed an increased product yield after adding an unspecific mechanosensitive channel to the constructed pathway.

Conserved Characteristics of NMPylation Activities of Alpha- and Betacoronavirus NiRAN Domains.

Coronavirus genome replication and expression are mediated by the viral replication-transcription complex (RTC) which is assembled from multiple nonstructural proteins (nsp). Among these, nsp12 represents the central functional subunit. It harbors the RNA-directed RNA polymerase (RdRp) domain and contains, at its N terminus, an additional domain called NiRAN which is widely conserved in coronaviruses and other nidoviruses. In this study, we produced bacterially expressed coronavirus nsp12s to investigate and compare NiRAN-mediated NMPylation activities from representative alpha- and betacoronaviruses. We found that the four coronavirus NiRAN domains characterized to date have a number of conserved properties, including (i) robust nsp9-specific NMPylation activities that appear to operate largely independently of the C-terminal RdRp domain, (ii) nucleotide substrate preference for UTP followed by ATP and other nucleotides, (iii) dependence on divalent metal ions, with Mn2+ being preferred over Mg2+, and (iv) a key role of N-terminal residues (particularly Asn2) of nsp9 for efficient formation of a covalent phosphoramidate bond between NMP and the N-terminal amino group of nsp9. In this context, a mutational analysis confirmed the conservation and critical role of Asn2 across different subfamilies of the family Coronaviridae, as shown by studies using chimeric coronavirus nsp9 variants in which six N-terminal residues were replaced with those from other corona-, pito- and letovirus nsp9 homologs. The combined data of this and previous studies reveal a remarkable degree of conservation among coronavirus NiRAN-mediated NMPylation activities, supporting a key role of this enzymatic activity in viral RNA synthesis and processing. IMPORTANCE There is strong evidence that coronaviruses and other large nidoviruses evolved a number of unique enzymatic activities, including an additional RdRp-associated NiRAN domain, that are conserved in nidoviruses but not in most other RNA viruses. Previous studies of the NiRAN domain mainly focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggested different functions for this domain, such as NMPylation/RNAylation of nsp9, RNA guanylyltransferase activities involved in canonical and/or unconventional RNA capping pathways, and other functions. To help resolve partly conflicting information on substrate specificities and metal ion requirements reported previously for the SARS-CoV-2 NiRAN NMPylation activity, we extended these earlier studies by characterizing representative alpha- and betacoronavirus NiRAN domains. The study revealed that key features of NiRAN-mediated NMPylation activities, such as protein and nucleotide specificity and metal ion requirements, are very well conserved among genetically divergent coronaviruses, suggesting potential avenues for future antiviral drug development targeting this essential viral enzyme.

Metabolic labeling and LC-MS/MS-based identification of interleukin-1α-induced secreted proteomes from epithelial cells in the presence or absence of serum.

The unbiased identification of cytokine-induced, secreted proteins from cells cultured in serum-containing medium is challenging. Here, we describe an experimental and bioinformatics workflow to label interleukin-1α-regulated proteins in living cells with the methionine analogue L-homopropargylglycine. We detail their purification and identification by means of CLICK-chemistry-based biotinylation followed by nanoHPLC-MS/MS. A side-by-side comparison of enriched proteins and their ontologies to serum-free conditions demonstrates the sensitivity and specificity of this approach to study the inducible secreted proteomes of epithelial cells.

Phosphorylation of Influenza A Virus Matrix Protein 1 at Threonine 108 Controls Its Multimerization State and Functional Association with the STRIPAK Complex.

The influenza A virus (IAV)-encoded matrix protein 1 (M1) acts as a master regulator of virus replication and fulfills multiple structural and regulatory functions in different cell compartments. Therefore, the spatiotemporal regulation of M1 is achieved by different mechanisms, including its structural and pH-dependent flexibility, differential association with cellular factors, and posttranslational modifications. Here, we investigated the function of M1 phosphorylation at the evolutionarily conserved threonine 108 (T108) and found that its mutation to a nonphosphorylatable alanine prohibited virus replication. Absent T108, phosphorylation led to strongly increased self-association of M1 at the cell membrane and consequently prohibited its ability to enter the nucleus and to contribute to viral ribonucleoprotein nuclear export. M1 T108 phosphorylation also controls the binding affinity to the cellular STRIPAK (striatin-interacting phosphatases and kinases) complex, which contains different kinases and the phosphatase PP2A to shape phosphorylation-dependent signaling networks. IAV infection led to the redistribution of the STRIPAK scaffolding subunits STRN and STRN3 from the cell membrane to cytosolic and perinuclear clusters, where it colocalized with M1. Inactivation of the STRIPAK complex resulted in compromised M1 polymerization and IAV replication. IMPORTANCE Influenza viruses pose a major threat to human health and cause annual epidemics and occasional pandemics. Many virus-encoded proteins exert various functions in different subcellular compartments, as exemplified by the M1 protein, but the molecular mechanisms endowing the multiplicity of functions remain incompletely understood. Here, we report that phosphorylation of M1 at T108 is essential for virus replication and controls its propensity for self-association and nuclear localization. This phosphorylation also controls binding affinity of the M1 protein to the STRIPAK complex, which contributes to M1 polymerization and virus replication.

Import and Export of Mannosylerythritol Lipids by Ustilago maydis.

Upon nitrogen starvation, the basidiomycete Ustilago maydis, which causes smut disease on corn, secretes amphipathic glycolipids, including mannosylerythritol lipids (MELs). MELs consist of a carbohydrate core whose mannosyl moiety is both acylated with fatty acids of different lengths and acetylated. Here, we report the transport of MELs into and out of the cell depending on the transport protein Mmf1, which belongs to the major facilitator superfamily. Analysis of mmf1 mutants and mutants lacking the acetyltransferase Mat1 revealed that Mmf1 is necessary for the export of acetylated MELs, while MELs without an acetyl group are secreted independently of this transporter. Upon deletion of mmf1, we detected novel MEL species lacking the acyl side chain at C-3'. With the help of feeding experiments, we demonstrate that MELs are taken up by U. maydis in an mmf1-independent manner. This leads to catabolism or rearrangement of acetyl and acyl side groups and subsequent secretion. The catabolism of MELs involves the presence of Mac2, an enzyme required for MEL biosynthesis. In cocultivation experiments, mutual exchange of MELs between different mutants was observed. Thus, we propose a novel function for fungal glycolipids as an external carbon storage. IMPORTANCE Fungi produce and secrete various secondary metabolites that can act as weapons against competitors, help in accessing nutrients, or assist in development and communication. One group of secondary metabolites are surface-active glycolipids, which have significant biotechnological potential as biodegradable detergents. While the biosynthesis of several fungal biosurfactants is well characterized, their biological functions and transport routes are less understood. We developed a cocultivation assay to show that a class of glycolipids from Ustilago maydis called mannosylerythritol lipids (MELs) can be exchanged between cells and modified or even degraded by recipient cells. Feeding assays with purified MELs led to similar results. These data provide insight into the surprising biological role of MELs as putative external carbon sources. Applying feeding and cocultivation experiments on MEL biosynthesis mutants turned out to be a valuable strategy for systematically studying the import routes and degradation pathways of glycolipids. By using these assays, we demonstrate the function of the transport protein Mmf1 as a specific exporter of acetylated MELs. We propose that these assays may be applied more generally, thereby opening novel areas of research.

Photoelectron Circular Dichroism of Electrosprayed Gramicidin Anions.

Many sophisticated approaches for analyzing properties of chiral matter have been developed in recent years. But in general, the available chiroptical methods are limited to either solvated or small gaseous molecules. Studying the chirality of large biopolymers in the gas phase, including aspects of the secondary structure, becomes accessible by combining the electrospray ionization technique with chiroptical detection protocols. Here, laser-induced photodetachment from gramicidin anions, a peptide consisting of 15 amino acids has been investigated. The angular distribution of photoelectrons is demonstrated to be sensitive to the substitution of protons by cesium ions, which is accompanied by a conformational change. The photoelectron circular dichroism (PECD) is -0.5% for bare gramicidin, whereas gramicidin with several Cs+ ions attached exhibits a PECD of +0.5%. The results are complemented and supported by ion mobility studies. The presented approach offers the prospect of studying chirality and the secondary structure of various biopolymers.

Elucidation of the Translation Initiation Factor Interaction Network of Haloferax volcanii Reveals Coupling of Transcription and Translation in Haloarchaea.

Translation is an important step in gene expression. Initiation of translation is rate-limiting, and it is phylogenetically more diverse than elongation or termination. Bacteria contain only three initiation factors. In stark contrast, eukaryotes contain more than 10 (subunits of) initiation factors (eIFs). The genomes of archaea contain many genes that are annotated to encode archaeal homologs of eukaryotic initiation factors (aIFs). However, experimental characterization of aIFs is scarce and mostly restricted to very few species. To broaden the view, the protein-protein interaction network of aIFs in the halophilic archaeon Haloferax volcanii has been characterized. To this end, tagged versions of 14 aIFs were overproduced, affinity isolated, and the co-isolated binding partners were identified by peptide mass fingerprinting and MS/MS analyses. The aIF-aIF interaction network was resolved, and it was found to contain two interaction hubs, (1) the universally conserved factor aIF5B, and (2) a protein that has been annotated as the enzyme ribose-1,5-bisphosphate isomerase, which we propose to rename to aIF2Bα. Affinity isolation of aIFs also led to the co-isolation of many ribosomal proteins, but also transcription factors and subunits of the RNA polymerase (Rpo). To analyze a possible coupling of transcription and translation, seven tagged Rpo subunits were overproduced, affinity isolated, and co-isolated proteins were identified. The Rpo interaction network contained many transcription factors, but also many ribosomal proteins as well as the initiation factors aIF5B and aIF2Bα. These results showed that transcription and translation are coupled in haloarchaea, like in Escherichia coli. It seems that aIF5B and aIF2Bα are not only interaction hubs in the translation initiation network, but also key players in the transcription-translation coupling.

Monitoring the Levels of Cellular NF-κB Activation States.

The NF-κB signaling system plays an important regulatory role in the control of many biological processes. The activities of NF-κB signaling networks and the expression of their target genes are frequently elevated in pathophysiological situations including inflammation, infection, and cancer. In these conditions, the outcome of NF-κB activity can vary according to (i) differential activation states, (ii) the pattern of genomic recruitment of the NF-κB subunits, and (iii) cellular heterogeneity. Additionally, the cytosolic NF-κB activation steps leading to the liberation of DNA-binding dimers need to be distinguished from the less understood nuclear pathways that are ultimately responsible for NF-κB target gene specificity. This raises the need to more precisely determine the NF-κB activation status not only for the purpose of basic research, but also in (future) clinical applications. Here we review a compendium of different methods that have been developed to assess the NF-κB activation status in vitro and in vivo. We also discuss recent advances that allow the assessment of several NF-κB features simultaneously at the single cell level.

Multi-level inhibition of coronavirus replication by chemical ER stress.

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.

TRAF6 Phosphorylation Prevents Its Autophagic Degradation and Re-Shapes LPS-Triggered Signaling Networks.

The ubiquitin E3 ligase TNF Receptor Associated Factor 6 (TRAF6) participates in a large number of different biological processes including innate immunity, differentiation and cell survival, raising the need to specify and shape the signaling output. Here, we identify a lipopolysaccharide (LPS)-dependent increase in TRAF6 association with the kinase IKKε (inhibitor of NF-κB kinase subunit ε) and IKKε-mediated TRAF6 phosphorylation at five residues. The reconstitution of TRAF6-deficient cells, with TRAF6 mutants representing phosphorylation-defective or phospho-mimetic TRAF6 variants, showed that the phospho-mimetic TRAF6 variant was largely protected from basal ubiquitin/proteasome-mediated degradation, and also from autophagy-mediated decay in autolysosomes induced by metabolic perturbation. In addition, phosphorylation of TRAF6 and its E3 ligase function differentially shape basal and LPS-triggered signaling networks, as revealed by phosphoproteome analysis. Changes in LPS-triggered phosphorylation networks of cells that had experienced autophagy are partially dependent on TRAF6 and its phosphorylation status, suggesting an involvement of this E3 ligase in the interplay between metabolic and inflammatory circuits.

Engineering Ustilago maydis for production of tailor-made mannosylerythritol lipids.

Mannosylerythritol lipids (MELs) are surface active glycolipids secreted by various fungi. MELs can be used as biosurfactants and are a biodegradable resource for the production of detergents or pharmaceuticals. Different fungal species synthesize a unique mixture of MELs differing in acetyl- and acyl-groups attached to the sugar moiety. Here, we report the construction of a toolbox for production of glycolipids with predictable fatty acid side chains in the basidiomycete Ustilago maydis. Genes coding for acyl-transferases involved in MEL production (Mac1 and Mac2) from different fungal species were combined to obtain altered MEL variants with distinct physical properties and altered antimicrobial activity. We also demonstrate that a U. maydis paralog of the acyltransferase Mac2 with a different substrate specificity can be employed for the biosynthesis of modified MEL variants. In summary, our data showcase how the fungal repertoire of Mac enzymes can be used to engineer tailor-made MELs according to specific biotechnological or pharmaceutical requirements.

Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit.

RNA-dependent RNA polymerases (RdRps) of the Nidovirales (Coronaviridae, Arteriviridae, and 12 other families) are linked to an amino-terminal (N-terminal) domain, called NiRAN, in a nonstructural protein (nsp) that is released from polyprotein 1ab by the viral main protease (Mpro). Previously, self-GMPylation/UMPylation activities were reported for an arterivirus NiRAN-RdRp nsp and suggested to generate a transient state primed for transferring nucleoside monophosphate (NMP) to (currently unknown) viral and/or cellular biopolymers. Here, we show that the coronavirus (human coronavirus [HCoV]-229E and severe acute respiratory syndrome coronavirus 2) nsp12 (NiRAN-RdRp) has Mn2+-dependent NMPylation activity that catalyzes the transfer of a single NMP to the cognate nsp9 by forming a phosphoramidate bond with the primary amine at the nsp9 N terminus (N3825) following Mpro-mediated proteolytic release of nsp9 from N-terminally flanking nsps. Uridine triphosphate was the preferred nucleotide in this reaction, but also adenosine triphosphate, guanosine triphosphate, and cytidine triphosphate were suitable cosubstrates. Mutational studies using recombinant coronavirus nsp9 and nsp12 proteins and genetically engineered HCoV-229E mutants identified residues essential for NiRAN-mediated nsp9 NMPylation and virus replication in cell culture. The data corroborate predictions on NiRAN active-site residues and establish an essential role for the nsp9 N3826 residue in both nsp9 NMPylation in vitro and virus replication. This residue is part of a conserved N-terminal NNE tripeptide sequence and shown to be the only invariant residue in nsp9 and its homologs in viruses of the family Coronaviridae The study provides a solid basis for functional studies of other nidovirus NMPylation activities and suggests a possible target for antiviral drug development.

Dietary cellulose induces anti-inflammatory immunity and transcriptional programs via maturation of the intestinal microbiota.

Although it is generally accepted that dietary fiber is health promoting, the underlying immunological and molecular mechanisms are not well defined, especially with respect to cellulose, the most ubiquitous dietary fiber. Here, the impact of dietary cellulose on intestinal microbiota, immune responses and gene expression in health and disease was examined. Lack of dietary cellulose disrupted the age-related diversification of the intestinal microbiota, which subsequently remained in an immature state. Interestingly, one of the most affected microbial genera was Alistipes which is equipped with enzymes to degrade cellulose. Absence of cellulose changed the microbial metabolome, skewed intestinal immune responses toward inflammation, altered the gene expression of intestinal epithelial cells and mice showed increased sensitivity to colitis induction. In contrast, mice with a defined microbiota including A. finegoldii showed enhanced colonic expression of intestinal IL-22 and Reg3γ restoring intestinal barrier function. This study supports the epidemiological observations and adds a causal explanation for the health promoting effects of the most common biopolymer on earth.

Noncoding RNA MaIL1 is an integral component of the TLR4-TRIF pathway.

RNA has been proposed as an important scaffolding factor in the nucleus, aiding protein complex assembly in the dense intracellular milieu. Architectural contributions of RNA to cytosolic signaling pathways, however, remain largely unknown. Here, we devised a multidimensional gradient approach, which systematically locates RNA components within cellular protein networks. Among a subset of noncoding RNAs (ncRNAs) cosedimenting with the ubiquitin-proteasome system, our approach unveiled ncRNA MaIL1 as a critical structural component of the Toll-like receptor 4 (TLR4) immune signal transduction pathway. RNA affinity antisense purification-mass spectrometry (RAP-MS) revealed MaIL1 binding to optineurin (OPTN), a ubiquitin-adapter platforming TBK1 kinase. MaIL1 binding stabilized OPTN, and consequently, loss of MaIL1 blunted OPTN aggregation, TBK1-dependent IRF3 phosphorylation, and type I interferon (IFN) gene transcription downstream of TLR4. MaIL1 expression was elevated in patients with active pulmonary infection and was highly correlated with IFN levels in bronchoalveolar lavage fluid. Our study uncovers MaIL1 as an integral RNA component of the TLR4-TRIF pathway and predicts further RNAs to be required for assembly and progression of cytosolic signaling networks in mammalian cells.

Correction to: Using a marine microalga as a chassis for polyethylene terephthalate (PET) degradation.

The author's middle name is missed out in the original publication of the article [1]. The correct coauthor's name is Tobias J. Erb.

Exploiting Substrate Promiscuity of Ectoine Hydroxylase for Regio- and Stereoselective Modification of Homoectoine.

Extant enzymes are not only highly efficient biocatalysts for a single, or a group of chemically closely related substrates but often have retained, as a mark of their evolutionary history, a certain degree of substrate ambiguity. We have exploited the substrate ambiguity of the ectoine hydroxylase (EctD), a member of the non-heme Fe(II)-containing and 2-oxoglutarate-dependent dioxygenase superfamily, for such a task. Naturally, the EctD enzyme performs a precise regio- and stereoselective hydroxylation of the ubiquitous stress protectant and chemical chaperone ectoine (possessing a six-membered pyrimidine ring structure) to yield trans-5-hydroxyectoine. Using a synthetic ectoine derivative, homoectoine, which possesses an expanded seven-membered diazepine ring structure, we were able to selectively generate, both in vitro and in vivo, trans-5-hydroxyhomoectoine. For this transformation, we specifically used the EctD enzyme from Pseudomonas stutzeri in a whole cell biocatalyst approach, as this enzyme exhibits high catalytic efficiency not only for its natural substrate ectoine but also for homoectoine. Molecular docking approaches with the crystal structure of the Sphingopyxis alaskensis EctD protein predicted the formation of trans-5-hydroxyhomoectoine, a stereochemical configuration that we experimentally verified by nuclear-magnetic resonance spectroscopy. An Escherichia coli cell factory expressing the P. stutzeri ectD gene from a synthetic promoter imported homoectoine via the ProU and ProP compatible solute transporters, hydroxylated it, and secreted the formed trans-5-hydroxyhomoectoine, independent from all currently known mechanosensitive channels, into the growth medium from which it could be purified by high-pressure liquid chromatography.

Using a marine microalga as a chassis for polyethylene terephthalate (PET) degradation.

BACKGROUND: The biological degradation of plastics is a promising method to counter the increasing pollution of our planet with artificial polymers and to develop eco-friendly recycling strategies. Polyethylene terephthalate (PET) is a thermoplast industrially produced from fossil feedstocks since the 1940s, nowadays prevalently used in bottle packaging and textiles. Although established industrial processes for PET recycling exist, large amounts of PET still end up in the environment-a significant portion thereof in the world's oceans. In 2016, Ideonella sakaiensis, a bacterium possessing the ability to degrade PET and use the degradation products as a sole carbon source for growth, was isolated. I. sakaiensis expresses a key enzyme responsible for the breakdown of PET into monomers: PETase. This hydrolase might possess huge potential for the development of biological PET degradation and recycling processes as well as bioremediation approaches of environmental plastic waste. RESULTS: Using the photosynthetic microalga Phaeodactylum tricornutum as a chassis we generated a microbial cell factory capable of producing and secreting an engineered version of PETase into the surrounding culture medium. Initial degradation experiments using culture supernatant at 30 °C showed that PETase possessed activity against PET and the copolymer polyethylene terephthalate glycol (PETG) with an approximately 80-fold higher turnover of low crystallinity PETG compared to bottle PET. Moreover, we show that diatom produced PETase was active against industrially shredded PET in a saltwater-based environment even at mesophilic temperatures (21 °C). The products resulting from the degradation of the PET substrate were mainly terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalic acid (MHET) estimated to be formed in the micromolar range under the selected reaction conditions. CONCLUSION: We provide a promising and eco-friendly solution for biological decomposition of PET waste in a saltwater-based environment by using a eukaryotic microalga instead of a bacterium as a model system. Our results show that via synthetic biology the diatom P. tricornutum indeed could be converted into a valuable chassis for biological PET degradation. Overall, this proof of principle study demonstrates the potential of the diatom system for future biotechnological applications in biological PET degradation especially for bioremediation approaches of PET polluted seawater.