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Megasynthases are large multienzyme proteins that produce a plethora of important natural compounds by catalyzing the successive condensation and modification of precursor units. Within the class of megasynthases, polyketide synthases (PKS) are responsible for the production of a large spectrum of bioactive polyketides (PK), which have frequently found their way into therapeutic applications. Rational engineering approaches have been performed during the last 25 years that seek to employ the "assembly-line synthetic concept" of megasynthases in order to deliver new bioactive compounds. Here, we highlight PKS engineering strategies in the light of the newly emerging structural information on megasynthases, and argue that fatty acid synthases (FAS) are and will be valuable objects for further developing this field.
Fatty acids (FAs) are considered strategically important platform compounds that can be accessed by sustainable microbial approaches. Here we report the reprogramming of chain-length control of Saccharomyces cerevisiae fatty acid synthase (FAS). Aiming for short-chain FAs (SCFAs) producing baker’s yeast, we perform a highly rational and minimally invasive protein engineering approach that leaves the molecular mechanisms of FASs unchanged. Finally, we identify five mutations that can turn baker’s yeast into a SCFA producing system. Without any further pathway engineering, we achieve yields in extracellular concentrations of SCFAs, mainly hexanoic acid (C6-FA) and octanoic acid (C8-FA), of 464 mg l−1 in total. Furthermore, we succeed in the specific production of C6- or C8-FA in extracellular concentrations of 72 and 245 mg l−1, respectively. The presented technology is applicable far beyond baker’s yeast, and can be plugged into essentially all currently available FA overproducing microorganisms.
Background: The ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase-mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway.
Results: The previously engineered short-chain acyl-CoA producing yeast Fas1R1834K/Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involvement of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized octanoyl-CoA was converted to 1-octanol up to a titer of 26.0 mg L−1 in a 72-h fermentation. The additional accumulation of 90 mg L−1 octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was supplied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escherichia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L−1. However, in growth tests concentrations even lower than 50.0 mg L−1 turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake.
Conclusions: By providing chain length control via an engineered octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae. Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells.
The yeast fatty acid synthase (FAS) is a barrel-shaped 2.6 MDa complex. Upon barrel-formation, two multidomain subunits, each more than 200 kDa large, intertwine to form a heterododecameric complex that buries 170,000 Å2 of protein surface. In spite of the rich knowledge about yeast FAS in structure and function, its assembly remained elusive until recently, when co-translational interaction of the β-subunit with the nascent α-subunit was found to initiate assembly. Here, we characterize the co-translational assembly of yeast FAS at a molecular level. We show that the co-translationally formed interface is sensitive to subtle perturbations, so that the exchange of two amino acids located in the emerging interface can prevent assembly. On the other hand, assembly can also be initiated via the co-translational interaction of the subunits at other sites, which implies that this process is not strictly site or sequence specific. We further highlight additional steps in the biogenesis of yeast FAS, as the formation of a dimeric subunit that orchestrates complex formation and acts as platform for post-translational phosphopantetheinylation. The presented data supports the understanding of the recently discovered prevalence of eukaryotic complexes for co-translational assembly, and is valuable for further harnessing FAS in the biotechnological production of aliphatic compounds.
Global distributions of profiles of sulphur hexafluoride (SF6) have been retrieved from limb emission spectra recorded by the Michelson Interferometer for Passive Atmospheric Sounding (MIPAS) on Envisat covering the period September 2002 to March 2004. Individual SF6 profiles have a precision of 0.5 pptv below 25 km altitude and a vertical resolution of 4–6 km up to 35 km altitude. These data have been validated versus in situ observations obtained during balloon flights of a cryogenic whole-air sampler. For the tropical troposphere a trend of 0.230±0.008 pptv/yr has been derived from the MIPAS data, which is in excellent agreement with the trend from ground-based flask and in situ measurements from the National Oceanic and Atmospheric Administration Earth System Research Laboratory, Global Monitoring Division. For the data set currently available, based on at least three days of data per month, monthly 5° latitude mean values have a 1 o standard error of 1%. From the global SF6 distributions, global daily and monthly distributions of the apparent mean age of air are inferred by application of the tropical tropospheric trend derived from MIPAS data. The inferred mean ages are provided for the full globe up to 90° N/S, and have a 1 o standard error of 0.25 yr. They range between 0 (near the tropical tropopause) and 7 years (except for situations of mesospheric intrusions) and agree well with earlier observations. The seasonal variation of the mean age of stratospheric air indicates episodes of severe intrusion of mesospheric air during each Northern and Southern polar winter observed, long-lasting remnants of old, subsided polar winter air over the spring and summer poles, and a rather short period of mixing with midlatitude air and/or upward transport during fall in October/November (NH) and April/May (SH), respectively, with small latitudinal gradients, immediately before the new polar vortex starts to form. The mean age distributions further confirm that SF6 is destroyed in the mesosphere to a considerable degree. Model calculations with the Karlsruhe simulation model of the middle atmosphere (KASIMA) chemical transport model agree well with observed global distributions of the mean age only if the SF6 sink reactions in the mesosphere are included in the model.
Fettsäuresynthasen vom Typ I (FAS I), hier bezeichnet als Fettsäuremegasynthasen,sind Multienzymkomplexe, in denen sämtliche funktionellen Domänen für die de-novo-Synthese von Fettsäuren einen strukturellen Verbund eingehen. Auch das für den Transport von Edukten und Intermediaten nötige Acyl Carrier Protein (ACP) ist kovalent gebundener Teil dieses Komplexes, der so zu einer hocheffizienten molekularen Maschine zur Massenproduktion dieser grundlegend essentiellen Zellbausteine wird. Die FAS I aus Pilzen (fFAS), als Gegenstand dieser Arbeit, mit einer Masse von bis zu 2,7 MDa ist heute in ihrer Struktur durch Röntgenkristallographische sowie elektronenmikroskopische Methoden gut charakterisiert. 48 funktionelle Domänen sind zu einem geschlossenen Reaktionskörper angeordnet, indem sie in einer strukturgebenden Matrix aus Expansionen und Insertionen bzgl. der enzymatischen Kerndomänen eingebettet sind, die 50% des gesamten Proteins ausmacht. Neben den zahlreichen strukturellen Informationen über fFAS ist jedoch noch wenig über ihre Assemblierung verstanden. Dabei ist sie nicht nur als ein Beispiel für das generelle Verständnis von Assemblierungsmechanismen von Multienzymkomplexen interessant, sondern wird hier auch als Ziel eines inhibitorischen Eingriffs betrachtet, um eine neue antimykotische Wirkstrategie abseits des Ausschaltens aktiver Zentren zu evaluieren. Nur wenn die Mechanismen und Wechselwirkungen im Assemblierungsprozess offen gelegt sind, lassen sie sich später gezielt attackieren. Essentielle Sekundärstrukturmotive müssen identifiziert und bewertet werden, um sie einer weiteren Evaluation als Drug-Target-Kandidaten zugänglich zu machen. In dieser Arbeit werden Resultate aus in-vivo-Experimenten an rational mutierten fFAS-Konstrukten unter Zuhilfenahme einer evolutionären Betrachtung der fFAS gemeinsam mit Erkenntnissen aus andernorts geleisteten in-vitro-Experimenten an fFAS-Fragmenten zu einem geordneten Assemblierungsweg der fFAS zusammengeführt. Dabei werden Evidenzen aus den Kausaltäten zentraler Anforderungen an einen Assemblierungsmechanismus der fFAS zu drei konsequenten Schlüsselschritten verdichtet, die (i) eine frühe Interaktion zweier komplementärer Polypeptidketten zu einer Pseudo-Einzelkette, (ii) eine posttranslationale Modifikation von ACP und (iii) die geordnete Reifung zum fertigen Komplex durch Selbstassemblierung der beteiligten Domänen umfassen. Durch rationale Mutationen an den Schnittstellenmotiven für die Pseudo-Einzelkettenbildung, werden diese als Schwachstelle der Assemblierung unterschiedlicher fFAS-Typen charakterisiert, wobei für S. cerevisiae nicht weniger als zwei gezielte Punktmutationen ausreichen, um die Assemblierung des gesamten Komplexes zu verhindern. Darüber hinaus zeigen Experimente mit fFAS-Konstrukten, deren Schnittstellenmotive einer intramolekular kompetitiven Wechselwirkung ausgesetzt sind, prinzipiell die Möglichkeit zur Inhibierung der fFAS-Assemblierung durch Störung der Pseudo-Einzelkettenbildung.
Bipolar disorder (BD) is a highly heritable neuropsychiatric disease characterized by recurrent episodes of mania and depression. BD shows substantial clinical and genetic overlap with other psychiatric disorders, in particular schizophrenia (SCZ). The genes underlying this etiological overlap remain largely unknown. A recent SCZ genome wide association study (GWAS) by the Psychiatric Genomics Consortium identified 128 independent genome-wide significant single nucleotide polymorphisms (SNPs). The present study investigated whether these SCZ-associated SNPs also contribute to BD development through the performance of association testing in a large BD GWAS dataset (9747 patients, 14278 controls). After re-imputation and correction for sample overlap, 22 of 107 investigated SCZ SNPs showed nominal association with BD. The number of shared SCZ-BD SNPs was significantly higher than expected (p = 1.46x10-8). This provides further evidence that SCZ-associated loci contribute to the development of BD. Two SNPs remained significant after Bonferroni correction. The most strongly associated SNP was located near TRANK1, which is a reported genome-wide significant risk gene for BD. Pathway analyses for all shared SCZ-BD SNPs revealed 25 nominally enriched gene-sets, which showed partial overlap in terms of the underlying genes. The enriched gene-sets included calcium- and glutamate signaling, neuropathic pain signaling in dorsal horn neurons, and calmodulin binding. The present data provide further insights into shared risk loci and disease-associated pathways for BD and SCZ. This may suggest new research directions for the treatment and prevention of these two major psychiatric disorders.
Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.
In non-hadronic axion models, which have a tree-level axion-electron interaction, the Sun produces a strong axion flux by bremsstrahlung, Compton scattering, and axiorecombination, the "BCA processes." Based on a new calculation of this flux, including for the first time axio-recombination, we derive limits on the axion-electron Yukawa coupling gae and axion-photon interaction strength ga using the CAST phase-I data (vacuum phase). For ma <~ 10 meV/c2 we find ga gae < 8.1 × 10−23 GeV−1 at 95% CL. We stress that a next-generation axion helioscope such as the proposed IAXO could push this sensitivity into a range beyond stellar energy-loss limits and test the hypothesis that white-dwarf cooling is dominated by axion emission.
The Born cross sections of the e+e− → D*+D*− and e+e− → D*+D− processes are measured using e+e− collision data collected with the BESIII experiment at center-of-mass energies from 4.085 to 4.600 GeV, corresponding to an integrated luminosity of 15.7 fb−1. The results are consistent with and more precise than the previous measurements by the Belle, Babar and CLEO collaborations. The measurements are essential for understanding the nature of vector charmonium and charmonium-like states.
We search for the di-photon decay of a light pseudoscalar axion-like particle, a, in radiative J/ψ decays, using 10 billion J/ψ events collected with the BESIII detector. We find no evidence of a signal and set upper limits at the 95% confidence level on the product branching fraction B(J/ψ→γa)×B(a→γγ) and the axion-like particle photon coupling constant gaγγ in the ranges of (3.7−48.5)×10−8 and (2.2−101.8)×10−4 GeV−1, respectively, for 0.18≤ma≤2.85 GeV/c2. These are the most stringent limits to date in this mass region.
We report the first measurements of the absolute branching fractions of D0 → K0 Lϕ, D0 → K0Lη, D0 → K0Lω, and D0 → K0Lη0, by analyzing 2.93 fb−1 of eþe− collision data taken at a center-of-mass energy of 3.773 GeV with the BESIII detector. Taking the world averages of the branching fractions of D0 → K0Sϕ, D0 → K0Sη, D0 → K0Sω, and D0 → K0Sη0, the K0S − K0L asymmetries RðD0; XÞ in these decay modes are obtained. The CP asymmetries in these decays are also determined. No significant CP violation is observed
The Cabibbo-allowed weak radiative decay Λ+c→Σ+γ has been searched for in a sample of Λ+cΛ¯−c pairs produced in e+e− annihilations, corresponding to an integrated luminosity of 4.5fb−1 collected with the BESIII detector at center-of-mass energies between 4.60 and 4.70 GeV. No excess of signal above background is observed, and we set an upper limit on the branching fraction of this decay to be B(Λ+c→Σ+γ)<4.4×10−4 at a confidence level of 90\%, which is in agreement with Standard Model expectations.
Improved measurement of the branching fractions of the inclusive decays D⁺ → Kₛ⁰X and D⁰ → Kₛ⁰X
(2023)
By analyzing 2.93 fb−1 of 𝑒+𝑒− collision data taken at the center-of-mass energy of 3.773 GeV with the BESIII detector, the branching fractions of the inclusive decays 𝐷+→𝐾0 𝑆𝑋 and 𝐷0→𝐾0 𝑆𝑋 are measured to be (33.11±0.13±0.36)% and (20.75±0.12±0.20)%, respectively, where the first uncertainties are statistical and the second are systematic. These results are consistent with the world averages of previous measurements, but with much improved precision.
Using e+e− collision data, corresponding to an integrated luminosity of 892pb−1 collected at center-of-mass energies from 4.84 to 4.95\,GeV with the BESIII detector, we search for the process e+e−→K+K−ψ(3770) by reconstructing two charged kaons and one D meson from ψ(3770). No significant signal of e+e−→K+K−ψ(3770) is found and the upper limits of the Born cross sections are reported at 90\% confidence level.
Using 7.33 fb−1 of e+e− collision data collected by the BESIII detector at center-of-mass energies between 4.128 and 4.226~GeV, we observe for the first time the decay D±s→ωπ±η with a statistical significance of 7.6σ. The measured branching fraction of this decay is (0.54±0.12±0.04)%, where the first uncertainty is statistical and the second is systematic.
Der Anspruch in der Implantatprothetik, ein hohes Maß an Qualität bei der Funktionalität und Ästhetik zu erreichen, hat zur Entwicklung verschiedener Implantatsysteme geführt. Deren Komponenten sollten nicht nur belastbar, sondern zugleich möglichst gewebeverträglich sein. Große Bedeutung kommt hier dem Bindeglied zwischen enossalem Implantat und der implantatgetragenen Krone, dem Abutment, zu. Dieses macht zumeist lediglich ein kleines Segment des transmukosalen Übergangs aus, zeigt sich jedoch für multiple Risikofaktoren, die den Langzeiterfolg der Implantatversorgung bedingen, verantwortlich.
Zirkonoxidabutments bestechen durch ihre Ästhetik und sehr gute Gewebeverträglichkeit. Titanabutments sind solchen aus Zirkonoxid mechanisch vor allem durch ihr charakteristisches Verhalten im Bereich der IAV überlegen. Um deren Vorteile zu vereinen, werden Standardabutments aus Titan mit einer individualisierten Zirkonoxidsuprakonstruktion adhäsiv zu einer funktionellen Einheit verbunden. Zur Verklebung dieser Hybridabutments eignen sich moderne Befestigungskomposite. Die Belastbarkeit dieser Verbindung kann durch verschiedene In-vitro-Untersuchungen bestätigt werden. Die Verbundfestigkeit kann durch Parameter wie den Zementspalt oder die Höhe des Titanabutments divergieren. Der Prozess der Verklebung muss einen hydrolysestabilen adhäsiven Verbund im feuchten Milieu der Mundhöhle gewährleisten, ohne die physikalischen Eigenschaften der eingesetzten Komponenten zu verändern.
Die Vermeidung bakterieller Kontamination der Hybridabutments erfolgt durch die Sterilisation im Autoklav. Da die Hybridabutments der semikritischen Risikogruppe der MP zugeordnet werden konnten, wurden diese häufig vor dem klinischen Einsatz jedoch nicht sterilisiert. Die Anwendung eines Sterilisationsverfahrens begünstigt sowohl den Zustand des periimplantären Weichgewebes, als auch die Stabilität der IAV bei Hybridabutments, geht allerdings mit einer thermischen Belastung einher. Verfahren zum Lösen von Klebeverbindungen nutzen die Eigenschaft von Klebstoffen, bei kritischem Wärmeeintrag mit einer Schädigung der Polymermatrix zu reagieren. Daher wurde die Hypothese formuliert, dass die Sterilisation im Autoklav den adhäsiven Verbund schwächen und zu niedrigeren Haftwerten führen könnte.
Zur Beurteilung eines möglichen Einflusses der Sterilisation auf die mechanische Widerstandsfähigkeit der verklebten Hybridabutments wurden mehrere Befestigungskomposite in die Untersuchung miteinbezogen. Es wurden 80 Titanbasen angefertigt und adhäsiv mit Zirkonoxidsuprakonstruktionen befestigt. Bei den fünf Befestigungskompositen handelte es sich um den DTK Kleber (Bredent), G-CEM LinkAce (GC Germany), RelyX Unicem2 (3M Espe AG), Multilink Hybridabutment (Ivoclar Vivadent GmbH) und Panavia F2.0 (Kuraray Medical). Von den fünf Gruppen mit je 16 Prüfkörpern durchlief jeweils die Hälfte eine Sterilisation im Autoklav während die Kontrollgruppe bei Raumtemperatur gelagert wurde. Abschließend wurden die Prüfkörper mit einer Universalprüfmaschine in uniaxialer Richtung bis zum Versagen der Verbindung belastet. Die dabei auftretende Abzugskraft wurde aufgezeichnet und die jeweiligen Werte miteinander verglichen.
Bei der Auswertung lagen statistisch signifikante Unterschiede der Haftwerte zwischen einer Test- und Kontrollgruppe, sowie unter den Befestigungskompositen selbst vor. Die Ergebnisse zeigen eine große Varianz, entkräften aber die formulierte Hypothese, dass eine abschließende Sterilisation im Autoklav zu einer Reduktion der Haftkraft innerhalb der Hybridabutments führen könnte. Die Ergebnisse können im Gegenteil dahingehend interpretiert werden, dass die Widerstandsfähigkeit durch die Sterilisation sogar verstärkt wird. Dies stellt vor dem Hintergrund, dass die Sterilisation neben der Beseitigung einer bakteriellen Kontamination auch Debris reduziert und somit die Passung der Komponenten erhöht, eindeutige Vorteile gegenüber weniger effektiven Aufbereitungsmethoden dar. Die positiven Effekte der Sterilisation auf den Knochenerhalt und das periimplantäre Gewebe überwiegen die potentiellen Nachteile nach derzeitigem Kenntnisstand bei weitem.
Da in der vorliegenden Versuchsreihe jedoch nur die Abzugskräfte in axialer Richtung verglichen wurden und keine künstliche Alterung simuliert wurde, bedarf es weiterer Untersuchungen um die mechanische Belastbarkeit in vivo zu verifizieren.
Observation of resonance structures in e⁺e⁻ → π⁺π⁻ψ₂(3823) and mass measurement of ψ₂(3823)
(2022)
Using a data sample corresponding to an integrated luminosity of 11.3 fb−1 collected at center-of-mass energies from 4.23 to 4.70 GeV with the BESIII detector, we measure the product of the 𝑒+𝑒−→𝜋+𝜋−𝜓2(3823) cross section and the branching fraction ℬ[𝜓2(3823)→𝛾𝜒𝑐1]. For the first time, resonance structure is observed in the cross section line shape of 𝑒+𝑒−→𝜋+𝜋−𝜓2(3823) with significances exceeding 5𝜎. A fit to data with two coherent Breit-Wigner resonances modeling the √𝑠-dependent cross section yields 𝑀(𝑅1)=4406.9±17.2±4.5 MeV/𝑐2, Γ(𝑅1)=128.1±37.2±2.3 MeV, and 𝑀(𝑅2)=4647.9±8.6±0.8 MeV/𝑐2, Γ(𝑅2)=33.1±18.6±4.1 MeV. Though weakly disfavored by the data, a single resonance with 𝑀(𝑅)=4417.5±26.2±3.5 MeV/𝑐2, Γ(𝑅)=245±48±13 MeV is also possible to interpret data. This observation deepens our understanding of the nature of the vector charmoniumlike states. The mass of the 𝜓2(3823) state is measured as (3823.12±0.43±0.13) MeV/𝑐2, which is the most precise measurement to date.
Using data samples collected with the BESIII detector operating at the BEPCII storage ring, the cross section of the inclusive process e+e−→η+X, normalized by the total cross section of e+e−→hadrons, is measured at eight center-of-mass energy points from 2.0000 GeV to 3.6710 GeV. These are the first measurements with momentum dependence in this energy region. Our measurement shows a significant discrepancy from calculations with the existing fragmentation functions. To address this discrepancy, a new QCD analysis is performed at the next-to-next-to-leading order with hadron mass corrections and higher twist effects, which can explain both the established high-energy data and our measurements reasonably well.
Based on electron positron collision data collected with the BESIII detector operating at the BEPCII storage rings, the differential cross sections of inclusive π0 and K0S production as a function of hadron momentum, normalized by the total cross section of the e+e−→ hadrons process, are measured at six center-of-mass energies from 2.2324 to 3.6710 GeV. Our results with a relative hadron energy coverage from 0.1 to 0.9 significantly deviate from several theoretical calculations based on existing fragmentation functions, especially at lower energies.