The level and distribution of methyl-esters influence the impact of pectin on intestinal T cells, microbiota, and Ahr activation.

Pectins are dietary fibres that modulate T cell immunity, microbiota composition, and fermentation profiles, but how this is influenced by the degree of methyl-esterification (DM) and degree-of-blockiness (DB) of pectin is unknown. Here, we demonstrate that supplementation of DM19(high-DB), DM49(low-DB) and DM43(high-DB) pectins at a low dose increased the frequencies of intestinal T-helper (Th)1 and Th2 cells after 1 week of pectin supplementation in mice, whereas DM18(low-DB) did not. After 4 weeks of supplementation with those pectins, Th1 and Th2 frequencies returned to control levels, whereas Rorγt+ regulatory T-cell frequencies increased. These structure-dependent effects could derive from induced shifts in microbiota composition that differed between DM18(low-DB) pectin and the other pectins. T-cell-modulating effects were not short-chain-fatty acid-dependent, but rather through an increase in Aryl-hydrocarbon-receptor-activating components. Thus, pectins with a specific combination of DM and DB have an impact on intestinal T cell-immunity in mice, when supplemented at a low dose.

The impact of the level and distribution of methyl-esters of pectins on TLR2-1 dependent anti-inflammatory responses.

Pectins have anti-inflammatory effects via Toll-like receptor (TLR) inhibition in a degree of methyl-esterification-(DM)-dependent manner. However, pectins also vary in distribution of methyl-esters over the galacturonic-acid (GalA) backbone (Degree of Blockiness - DB) and impact of this on anti-inflammatory capacity is unknown. Pectins mainly inhibit TLR2-1 but magnitude depends on both DM and DB. Low DM pectins (DM18/19) with both low (DB86) and high DB (DB94) strongly inhibit TLR2-1. However, pectins with intermediate DM (DM43/DM49) and high DB (DB60), but not with low DB (DB33), inhibit TLR2-1 as strongly as low DM. High DM pectins (DM84/88) with DB71 and DB91 do not inhibit TLR2-1 strongly. Pectin-binding to TLR2 was confirmed by capture-ELISA. In human macrophages, low DM and intermediate DM pectins with high DB inhibited TLR2-1 induced IL-6 secretion. Both high number and blockwise distribution of non-esterified GalA in pectins are responsible for the anti-inflammatory effects via inhibition of TLR2-1.

Characterization of an AA9 LPMO from Thielavia australiensis, TausLPMO9B, under industrially relevant lignocellulose saccharification conditions.

BACKGROUND: The discovery of lytic polysaccharide monooxygenases (LPMO) has changed our perspective on enzymatic degradation of plant biomass. Through an oxidative mechanism, these enzymes are able to cleave and depolymerize various polysaccharides, acting not only on crystalline substrates such as chitin and cellulose, but also on other polysaccharides, such as xyloglucan, glucomannan and starch. Despite their widespread use, uncertainties related to substrate specificity and stereospecificity, the nature of the co-substrate, in-process stability, and the nature of the optimal reductant challenge their exploitation in biomass processing applications. RESULTS: In this work, we studied the properties of a novel fungal LPMO from the thermophilic fungus Thielavia australiensis, TausLPMO9B. Heterologous expression of TausLPMO9B in Aspergillus niger yielded a glycosylated protein with a methylated N-terminal histidine showing LPMO activity. High sequence identity of the AA9 domain to that of MtLPMO9B (MYCTH_80312) from Myceliophthora thermophila (84%) indicated strictly C1-oxidizing activity on cellulose, which was confirmed experimentally by the analysis of products released from cellulose using HPAEC. The enzyme was stable and active at a pH ranging from 4 to 6, thus matching the conditions commonly used in industrial biomass processing, where a low pH (between 4 and 5) is used due to the pH-optima of commercial cellulases and a desire to limit microbial contamination. CONCLUSION: While the oxidative cleavage of phosphoric acid swollen cellulose (PASC) by TausLPMO9B was boosted by the addition of H2O2 as a co-substrate, this effect was not observed during the saccharification of acid pretreated corn stover. This illustrates key differences between the lab-scale tests with artificial, lignin-free substrates and industrial settings with lignocellulosic biomass as substrate.

Sensing and transduction of nutritional and chemical signals in filamentous fungi: Impact on cell development and secondary metabolites biosynthesis.

Filamentous fungi respond to hundreds of nutritional, chemical and environmental signals that affect expression of primary metabolism and biosynthesis of secondary metabolites. These signals are sensed at the membrane level by G protein coupled receptors (GPCRs). GPCRs contain usually seven transmembrane domains, an external amino terminal fragment that interacts with the ligand, and an internal carboxy terminal end interacting with the intracellular G protein. There is a great variety of GPCRs in filamentous fungi involved in sensing of sugars, amino acids, cellulose, cell-wall components, sex pheromones, oxylipins, calcium ions and other ligands. Mechanisms of signal transduction at the membrane level by GPCRs are discussed, including the internalization and compartmentalisation of these sensor proteins. We have identified and analysed the GPCRs in the genome of Penicillium chrysogenum and compared them with GPCRs of several other filamentous fungi. We have found 66 GPCRs classified into 14 classes, depending on the ligand recognized by these proteins, including most previously proposed classes of GPCRs. We have found 66 putative GPCRs, representatives of twelve of the fourteen previously proposed classes of GPCRs, depending on the ligand recognized by these proteins. A staggering fortytwo putative members of the new GPCR class XIV, the so-called Pth11 sensors of cellulosic material as reported for Neurospora crassa and some other fungi, were identified. Several GPCRs sensing sex pheromones, known in yeast and in several fungi, were also identified in P. chrysogenum, confirming the recent unravelling of the hidden sexual capacity of this species. Other sensing mechanisms do not involve GPCRs, including the two-component systems (HKRR), the HOG signalling system and the PalH mediated pH transduction sensor. GPCR sensor proteins transmit their signals by interacting with intracellular heterotrimeric G proteins, that are well known in several fungi, including P. chrysogenum. These G proteins are inactive in the GDP containing heterotrimeric state, and become active by nucleotide exchange, allowing the separation of the heterotrimeric protein in active Gα and Gßγ dimer subunits. The conversion of GTP in GDP is mediated by the endogenous GTPase activity of the G proteins. Downstream of the ligand interaction, the activated Gα protein and also the Gß/Gγ dimer, transduce the signals through at least three different cascades: adenylate cyclase/cAMP, MAPK kinase, and phospholipase C mediated pathways.

Self-Regulated Plasma Heat Flux Mitigation Due to Liquid Sn Vapor Shielding.

A steady-state high-flux H or He plasma beam was balanced against the pressure of a Sn vapor cloud for the first time, resulting in a self-regulated heat flux intensity near the liquid surface. A temperature response of the liquid surface characterized by a decoupling from the received heating power and significant cooling of the plasma in the neutral Sn cloud were observed. The plasma heat flux impinging on the target was found to be mitigated, as heat was partially dissipated by volumetric processes in the vapor cloud rather than wholly by surface effects. These results motivate further exploration of liquid metal solutions to the critical challenge of heat and particle flux handling in fusion power plants.

Advanced Thomson scattering system for high-flux linear plasma generator.

An advanced Thomson scattering system has been built for a linear plasma generator for plasma surface interaction studies. The Thomson scattering system is based on a Nd:YAG laser operating at the second harmonic and a detection branch featuring a high etendue (f/3) transmission grating spectrometer equipped with an intensified charged coupled device camera. The system is able to measure electron density (n(e)) and temperature (T(e)) profiles close to the output of the plasma source and, at a distance of 1.25 m, just in front of a target. The detection system enables to measure 50 spatial channels of about 2 mm each, along a laser chord of 95 mm. By summing a total of 30 laser pulses (0.6 J, 10 Hz), an observational error of 3% in n(e) and 6% in T(e) (at n(e) = 9.4 × 10(18) m(-3)) can be obtained. Single pulse Thomson scattering measurements can be performed with the same accuracy for n(e) > 2.8 × 10(20) m(-3). The minimum measurable density and temperature are n(e) < 1 × 10(17) m(-3) and T(e) < 0.07 eV, respectively. In addition, using the Rayleigh peak, superimposed on the Thomson scattered spectrum, the neutral density (n(0)) of the plasma can be measured with an accuracy of 25% (at n(0) = 1 × 10(20) m(-3)). In this report, the performance of the Thomson scattering system will be shown along with unprecedented accurate Thomson-Rayleigh scattering measurements on a low-temperature argon plasma expansion into a low-pressure background.

Speciation despite globally overlapping distributions in Penicillium chrysogenum: the population genetics of Alexander Fleming's lucky fungus.

Eighty years ago, Alexander Fleming described the antibiotic effects of a fungus that had contaminated his bacterial culture, kick starting the antimicrobial revolution. The fungus was later ascribed to a putatively globally distributed asexual species, Penicillium chrysogenum. Recently, the species has been shown to be genetically diverse, and possess mating-type genes. Here, phylogenetic and population genetic analyses show that this apparently ubiquitous fungus is actually composed of at least two genetically distinct species with only slight differences detected in physiology. We found each species in air and dust samples collected in and around St Mary's Hospital where Fleming worked. Genotyping of 30 markers across the genome showed that preserved fungal material from Fleming's laboratory was nearly identical to derived strains currently in culture collections and in the same distinct species as a wild progenitor strain of current penicillin producing industrial strains rather than the type species P. chrysogenum. Global samples of the two most common species were found to possess mating-type genes in a near 1:1 ratio, and show evidence of recombination with little geographic population subdivision evident. However, no hybridization was detected between the species despite an estimated time of divergence of less than 1MYA. Growth studies showed significant interspecific inhibition by P. chrysogenum of the other common species, suggesting that competition may facilitate species maintenance despite globally overlapping distributions. Results highlight under-recognized diversity even among the best-known fungal groups and the potential for speciation despite overlapping distribution.

Intermediate frequency band digitized high dynamic range radiometer system for plasma diagnostics and real-time Tokamak control.

An intermediate frequency (IF) band digitizing radiometer system in the 100-200 GHz frequency range has been developed for Tokamak diagnostics and control, and other fields of research which require a high flexibility in frequency resolution combined with a large bandwidth and the retrieval of the full wave information of the mm-wave signals under investigation. The system is based on directly digitizing the IF band after down conversion. The enabling technology consists of a fast multi-giga sample analog to digital converter that has recently become available. Field programmable gate arrays (FPGA) are implemented to accomplish versatile real-time data analysis. A prototype system has been developed and tested and its performance has been compared with conventional electron cyclotron emission (ECE) spectrometer systems. On the TEXTOR Tokamak a proof of principle shows that ECE, together with high power injected and scattered radiation, becomes amenable to measurement by this device. In particular, its capability to measure the phase of coherent signals in the spectrum offers important advantages in diagnostics and control. One case developed in detail employs the FPGA in real-time fast Fourier transform (FFT) and additional signal processing. The major benefit of such a FFT-based system is the real-time trade-off that can be made between frequency and time resolution. For ECE diagnostics this corresponds to a flexible spatial resolution in the plasma, with potential application in smart sensing of plasma instabilities such as the neoclassical tearing mode (NTM) and sawtooth instabilities. The flexible resolution would allow for the measurement of the full mode content of plasma instabilities contained within the system bandwidth.

Development and testing of a fast Fourier transform high dynamic-range spectral diagnostics for millimeter wave characterization.

A fast Fourier transform (FFT) based wide range millimeter wave diagnostics for spectral characterization of scattered millimeter waves in plasmas has been successfully brought into operation. The scattered millimeter waves are heterodyne downconverted and directly digitized using a fast analog-digital converter and a compact peripheral component interconnect computer. Frequency spectra are obtained by FFT in the time domain of the intermediate frequency signal. The scattered millimeter waves are generated during high power electron cyclotron resonance heating experiments on the TEXTOR tokamak and demonstrate the performance of the diagnostics and, in particular, the usability of direct digitizing and Fourier transformation of millimeter wave signals. The diagnostics is able to acquire 4 GHz wide spectra of signals in the range of 136-140 GHz. The rate of spectra is tunable and has been tested between 200,000 spectra/s with a frequency resolution of 100 MHz and 120 spectra/s with a frequency resolution of 25 kHz. The respective dynamic ranges are 52 and 88 dB. Major benefits of the new diagnostics are a tunable time and frequency resolution due to postdetection, near-real time processing of the acquired data. This diagnostics has a wider application in astrophysics, earth observation, plasma physics, and molecular spectroscopy for the detection and analysis of millimeter wave radiation, providing high-resolution spectra at high temporal resolution and large dynamic range.

Construction of an hdfA Penicillium chrysogenum strain impaired in non-homologous end-joining and analysis of its potential for functional analysis studies.

The homologous recombination mechanism for DNA-repair is not predominant in most filamentous fungi, resulting in extremely low targeting efficiencies for molecular engineering. To increase the gene targeting efficiency, it is becoming common practice to inactivate the non-homologous end-joining (NHEJ) pathway that causes random integration, by deleting the fungal homologs of the human KU70 and KU80 genes that encode proteins functioning in the NHEJ pathway. This has been described for several filamentous fungi, but limited knowledge on the physiological consequences is available. In this study we characterized targeting efficiency and physiology of penicillinG producing Penicillium chrysogenum strains, in which the KU70 or KU80 homologues hdfA and hdfB had been deleted. Targeting efficiency was increased from ca. 1% in the reference strain to 47% and 56% in the hdfA and hdfB mutant strains, respectively, using an ends-out construct. Physiological and transcriptome analysis of glucose-limited chemostat cultures of the hdfA deletion strain and the reference strain showed minimal differences. Although, in a direct competition experiment to assess strain fitness, the reference strain had a clear advantage over the deletion strain, the results demonstrate the potential of DeltahdfAP. chrysogenum strains for the functional analysis of the recently completed P. chrysogenum genome sequence and in further metabolic engineering of antibiotics production.

A line-of-sight electron cyclotron emission receiver for electron cyclotron resonance heating feedback control of tearing modes.

An electron cyclotron emission (ECE) receiver inside the electron cyclotron resonance heating (ECRH) transmission line has been brought into operation. The ECE is extracted by placing a quartz plate acting as a Fabry-Perot interferometer under an angle inside the electron cyclotron wave (ECW) beam. ECE measurements are obtained during high power ECRH operation. This demonstrates the successful operation of the diagnostic and, in particular, a sufficient suppression of the gyrotron component preventing it from interfering with ECE measurements. When integrated into a feedback system for the control of plasma instabilities this line-of-sight ECE diagnostic removes the need to localize the instabilities in absolute coordinates.

Compartmentalization and transport in beta-lactam antibiotics biosynthesis.

Classical strain improvement of beta-lactam producing organisms by random mutagenesis has been a powerful tool during the last century. Current insights in the biochemistry and genetics of beta-lactam production, in particular in the filamentous fungus Penicillium chrysogenum, however, make a more directed and rational approach of metabolic pathway engineering possible. Besides the need for efficient genetic methods, a thorough understanding is needed of the metabolic fluxes in primary, intermediary and secondary metabolism. Controlling metabolic fluxes can be achieved by adjusting enzyme activities and metabolite levels in such a way that the main flow is directed towards the desired product. In addition, compartmentalization of specific parts of the beta-lactam biosynthesis pathways provides a way to control this pathway by clustering enzymes with their substrates inside specific membrane bound structures sequestered from the cytosol. This compartmentalization also requires specific membrane transport steps of which the details are currently uncovered.

Engineering aspects of beta-lactam biosynthesis.

The adaptation of an existing industrial production line to boost production capacity (a hardware analogue of strain improvement) or to enable the production of a derivative of the original product is used as an example to explore the similarities between process design and engineering and metabolic pathway engineering. In the two fields the same principles appear to apply: for most process engineering solutions metabolic pathway engineering analogues can be found. Analogues are illustrated by reference to literature (e.g. overproduction of amino acid precursors at the 'bottom' of the pathway, relocalisation of proteins and overexpression of genes from the secondary metabolism). Some possible future applications are defined (e.g. supervisory manipulations as regulation by gene transcription factors).

Transient mRNA responses in chemostat cultures as a method of defining putative regulatory elements: application to genes involved in Saccharomyces cerevisiae acetyl-coenzyme A metabolism.

To identify common regulatory sequences in the promoters of genes, transcription of 31 genes of Saccharomyces cerevisiae was analysed during the transient response to a glucose pulse in a chemostat culture. mRNA levels were monitored during the subsequent excess glucose, ethanol and acetate phases, while other conditions were kept constant. This setup allowed a direct comparison between regulation by glucose, ethanol and acetate. Genes with identical regulation patterns were grouped to identify regulatory elements in the promoters. In respect to regulation on glucose four classes were identified: no transcription under any of the conditions tested, no difference in regulation on glucose, induced on glucose and repressed on glucose. In addition, genes were found that were repressed or induced on ethanol or acetate. Sequence alignment of genes with similar regulation patterns revealed five new, putative regulatory promoter elements. (i) The glucose-inducible fermentation genes PDC1 and ADH1 share the sequence ATACCTTCSTT. (ii) Acetate-repression might be mediated by the decamer CCCGAG RGGA, present in the promoters of ACS2 and ACR1. (iii) A specific element (CCWTTSRNCCG) for the glyoxylate cycle was present in seven genes studied: CIT2, ICL1, MLS1, MDH2, CAT2, ACR1 and ACH1. These genes were derepressed on ethanol or acetate. (iv) The sequence ACGTSCRGAATGA was found in the promoters of the partially ethanol-repressed genes ACS1 and YAT1. (v) Ethanol induction, as seen for ACS2, ADH3 and MDH1, might be mediated via the sequence CGGSGCCGRAG.

Overproduction of acetyl-coenzyme A synthetase isoenzymes in respiring Saccharomyces cerevisiae cells does not reduce acetate production after exposure to glucose excess.

To investigate whether the production of acetate which occurs after exposure of respiring Saccharomyces cerevisiae cells to excess glucose can be reduced by overproduction of acetyl-CoA synthetase (ACS, EC 6.2.1.1), the ACS1 and ACS2 genes were introduced on multi-copy plasmids. For each isoenzyme, the level in glucose-limited chemostat cultures was increased by 3-6-fold, relative to an isogenic reference strain. However, ACS overproduction did not result in a reduced production of acetate after a glucose pulse (100 mmol l-1) to these cultures. This indicates that a limited capacity of ACS is not the sole cause of acetate accumulation in S. cerevisiae.

The Saccharomyces cerevisiae acetyl-coenzyme A synthetase encoded by the ACS1 gene, but not the ACS2-encoded enzyme, is subject to glucose catabolite inactivation.

In Saccharomyces cerevisiae, the structural genes ACS1 and ACS2 each encode an isoenzyme of acetyl-CoA synthetase (ACS; EC 6.2.1.1). Involvement of glucose catabolite repression in regulation of the two isoenzymes was investigated by following ACS activity after glucose pulses (100 mM) to ethanol-limited chemostat cultures. In wild-type S. cerevisiae and in an isogenic strain in which ACS2 had been disrupted, ACS activity decreased after a glucose pulse. No such inactivation was observed in a strain in which ACS1 was disrupted. Western blots demonstrated that the ACS1 product, but not the ACS2 product, was degraded after a glucose pulse. Inactivation kinetics of the ACS1 product resembled those of isocitrate lyase.

Expression cassettes for formaldehyde and fluoroacetate resistance, two dominant markers in Saccharomyces cerevisiae.

We employed two genes in constructing yeast expression cassettes for dominant, selectable markers. The Saccharomyces cerevisiae gene SFA1, encoding formaldehyde dehydrogenase, was placed under the control of the GPD1 promoter and CYC1 terminator. The Moraxella sp. strain B gene dehH1, encoding fluoroacetate dehalogenase, was placed under the control of both the GPD1 and CYC1 promoters. With these constructs it was possible to select directly for yeast strains resistant to either formaldehyde or fluoroacetate. Both selective agents are completely metabolized and inexpensive, making them very useful in the pursuit of yeast gene functions and for industrial applications. An additional advantage of the formaldehyde dehydrogenase marker is that it is an S. cerevisiae gene, thus allowing 'all yeast' constructs.

The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation.

Saccharomyces cerevisiae contains two structural genes, ACS1 and ACS2, each encoding an active acetyl-coenzyme A synthetase. Characterization of enzyme activities in cell-free extracts from strains expressing either of the two genes revealed differences in the catalytic properties of the two enzymes. The Km for acetate of Acs1p was about 30-fold lower than that of Acs2p and Acs1p, but not Acs2p, could use propionate as a substrate. Enzyme activity measurements and mRNA analyses showed that ACS1 and ACS2 were both expressed during carbon-limited growth on glucose, ethanol, and acetate in aerobic chemostat cultures. In anaerobic glucose-limited cultures, only the ACS2 gene was expressed. Based on these facts, the products of the ACS1 and ACS2 genes were identified as the previously described "aerobic" and "non-aerobic" forms of acetyl-coenzyme A synthetase, respectively. Batch and glucose-pulse experiments revealed that transcription of ACS1 is subject to glucose repression. A mutant strain lacking Acs2p was unable to grow on glucose in batch cultures, but grew readily in aerobic glucose-limited chemostat cultures, in which the low residual glucose concentration alleviated glucose repression. Experiments in which ethanol was pulsed to aerobic ethanol-limited chemostat cultures indicated that, in addition to glucose, ethanol also repressed ACS1 transcription, although to a lesser extent. In contrast, transcription of ACS2 was slightly induced by ethanol and glucose. Absence of ACS2 prevented complete glucose repression of ACS1, indicating that ACS2 (in)directly is involved in the transcriptional regulation of ACS1.

ACS2, a Saccharomyces cerevisiae gene encoding acetyl-coenzyme A synthetase, essential for growth on glucose.

In Saccharomyces cerevisiae, the conversion of pyruvate to acetyl-coenzyme A may proceed directly via the pyruvate dehydrogenase complex (PDH) or indirectly via the so-called PDH bypass, which requires the sequential action of pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-coenzyme A synthetase. The relative contribution of both pathways to the rate of acetyl-coenzyme A synthesis varies in an unknown way with cultural conditions. To determine the possible role of acetyl-coenzyme A synthetase in this central part of metabolism, we have analyzed the genes encoding this enzyme. Disruption of the recently cloned ACS1 gene [De Virgilio, C., Burckert, N., Barth, G., Neuhaus, J., Boller, T. & Wiemken, A. (1992) Yeast 8, 1043-1051] did not cause an apparent phenotype, except for a prolonged lag-phase during growth on glucose or C2 compounds such as acetate and ethanol. In fact, a product from a different gene is responsible for acetyl-coenzyme A formation in the acs1 mutant. We cloned a second gene encoding acetyl-coenzyme A synthetase, which we called ACS2. Inactivation of this gene caused inability to grow on media containing glucose, but not on media with acetate or ethanol as the sole carbon source. This indicates that ACS2 is essential for growth on glucose in batch cultures. The acs1-acs2 double mutant was not viable. The role of both genes in glucose metabolism and acetate or ethanol metabolism is discussed.

Characterization of Saccharomyces cerevisiae mutants lacking the E1 alpha subunit of the pyruvate dehydrogenase complex.

Pyruvate dehydrogenase mutants of Saccharomyces cerevisiae were isolated by disruption of the PDA1 gene. To this end, the PDA1 gene encoding the E1 alpha subunit of the pyruvate dehydrogenase complex was replaced by the dominant Tn5ble marker. Disruption of the PDA1 gene abolished production of the E1 alpha subunit and pyruvate dehydrogenase activity. Two additional phenotypes were observed in the Pdh-mutants: (a) a reduced growth rate in glucose medium which was partially complemented by the amino acid leucine; (b) an increase in formation of petites which lack mitochondrial DNA [rho0], during growth on glucose. Both phenotypes were shown to be a result of inactivation of the PDA1 gene. Explanations for these phenotypes are discussed.