Simulation based Development of Industrial PERC Cell Production beyond 20.5% Efficiency

AbstractIn this work we present our approach to realize an industrial process that allows cell efficiencies up to and above 21%. Based on a loss analysis we systematically investigate the feasible options to improve the efficiency with device simulations and production experiments. Subsequently we perform sensitivity analyses particularly for various silicon wafer materials to ensure stable process capability. Our best prototype process with optimized front and rear side passivation and enhanced laser contact patterning has demonstrated a maximum efficiency of 20.9% with a very high VOC of 670mV on high-lifetime mono material. We were able to assemble 60-cell based modules with more than 305Wp

Modulating synthetic pathways in megasynthases

Polyketides are highly valuable natural products, which are widely used as pharmaceuticals due to their beneficial characteristics, comprising antibacterial, antifungal, immunosuppressive, and antitumor properties, among others. Their biosynthesis is performed by large and complex multiproteins, the polyketide synthases (PKSs). This study solely focuses on the class of type I PKSs, which arrange all their enzymatic domains on one or more polypeptides. Despite their high medical value, little is known about mechanistic details in PKSs. One central domain is the acyl transferase (AT), which is present in all PKSs and channels small acyl substrates into the enzyme. More precisely, the AT loads the substrates onto the essential acyl carrier protein (ACP), which subsequently shuttles the substrates and all intermediates for condensation and modification to additional domains to build the final polyketide. Some PKSs use their domains several times during biosynthesis and work iteratively – these are called iterative PKSs. Others feature several sets of domains, each being used only once during biosynthesis – these PKSs are called modular PKSs. All PKSs or PKS modules consist of minimum three essential domains to connect the acyl substrates. Three modifying domains are optional and can enlarge the minimal set. According to the domain composition, the acyl substrate is fully reduced, partly reduced, or not reduced at all. This variation of modifying domains accounts for the huge structural and therefore functional variety of polyketides. Even though the structure of fatty acids is not exactly reminiscent of polyketides, their biosynthetic pathways are closely related. Fatty acid biosynthesis is carried out by fatty acid synthases (FASs), which share many similarities with PKSs. Both megasynthases feature the same domains, performing the same reactions to connect and modify small acyl substrates. In contrast to PKSs, FASs always contain one full set of modifying domains which is used iteratively, leading to fully reduced fatty acids. The present thesis extensively analyzes the AT of different PKSs in its substrate selectivity, AT-ACP domain-domain interaction, and enzymatic kinetic properties. The following key findings are revealed through comparison: 1.) ATs of PKSs appear slower than the ones of FASs, which may reflect the different scopes of biosynthetic pathways. Fatty acids as essential compounds in all organisms are needed in high amounts for physiological functions, whereas polyketides as secondary metabolites only require basal concentrations to take effect. 2.) The slower ATs from modular PKSs do not load non-native substrates even in absence of the native substrates. This is different to the faster ATs from iterative PKSs and FASs, which indicates high substrate specificity solely for the ATs from modular PKSs and emphasizes their role as gatekeepers in polyketide synthesis. 3.) The substrate selectivity can emerge in either the first or the second step of the AT-mediated ACP loading and is not assured by a hydrolytic proofreading function. Moreover, a mutational study on the AT-ACP interaction in the modular PKS 6-deoxyerythronolide B synthase (DEBS) shows that single surface point mutations can influence AT-mediated reactions in a complex manner. Data reveals high enzyme kinetic plasticity of the AT-ACP interaction, which was also recently demonstrated for the interaction in a type II FAS. Based on these findings, the mammalian FAS is engineered towards a modular PKS-like as- sembly line with the long-term goal to rationally synthesize new products. Basically, three important aspects need to be considered: 1.) AT’s loading needs to be splitted in specific loading of a priming substrate by a priming AT and in specific loading of an elongation substrate by an elongation AT. 2.) FAS-based elongation modules need to be designed with varying domain compositions for introducing functional groups in the product. 3.) Covalent and non-covalent linkers need to be designed for connection of priming and elongation modules. This study focuses on the first aspect, splitting loading of priming and elongation substrates. An elongation substrate-specific AT is installed in the mammalian FAS via domain swapping. Since ATs from modular PKSs were proven to be substrate specific, these are used to exchange the mammalian FAS AT. This work demonstrates that it is extremely challenging to create stable and functional chimeras, but first essential steps are taken. Proper domain boundaries for AT swapping are established and a stable chimera with 70 % wild type AT activity is created. However, this chimera is only of limited value for application in an elongation module due to the intrinsic slow turnover rate of the wild type AT. Using another PKS AT, a stable elongation module is designed and analyzed in its activity in combination with a priming module. These experiments demonstrate that the loading of priming substrates are successfully suppressed in the elongation module, but nonetheless only minor turnover rates are detected in the assembly line. ...Polyketide sind wichtige Naturstoffe, die aufgrund ihrer Eigenschaften vielfältige medizinische Anwendungen finden. Ihre Biosynthese wird von großen Enzymkomplexen, den Polyketidsynthasen (PKSs), katalysiert. Die vorliegende Thesis beschäftigt sich mit Typ I-PKSs, die ihre enzymatischen Domänen auf einer oder mehreren Polypeptidketten tragen. Trotz ihrer großen medizinischen Bedeutung ist nur wenig über mechanistische Details von PKSs bekannt. Eine wichtige Domäne ist die Acyltransferase (AT), die kleine Acylsubstrate in das System zur Bildung des Polyketids schleust. Hierbei belädt die AT das Acyl-Carrier-Protein (ACP), das die Substrate und Intermediate zu den weiteren Domänen zu deren Kondensation und Modifikation transportiert. Manche PKSs – die iterativen PKSs – nutzen ihre Domänen mehrfach während der Biosynthese. Andere PKSs – die modularen PKSs – bestehen aus mehreren Domänensätzen, sogenannten Modulen, deren Domänen nur genau einmal während der Biosynthese genutzt werden. Alle PKSs oder PKS-Module bestehen aus drei essentiellen Domänen, die die Acylsubstrate zum Polyketid verbinden. Drei Substrat-modifizierende Domänen können diesen Minimalsatz erweitern. Die Domänenzusammensetzung bestimmt den Grad der Substrat-Reduktion und ist somit für die strukturelle sowie funktionelle Variabilität der Polyketide verantwortlich. Auch wenn sich die Strukturen von Polyketiden und Fettsäuren auf den ersten Blick nicht stark ähneln, sind ihre Biosynthesewege dennoch eng miteinander verwandt. Fettsäuren werden wie Polyketide von großen Multienzymkomplexen, den Fettsäuresynthasen (FASs), hergestellt, die viele Eigenschaften mit PKSs teilen. Beide Megasynthasen bestehen aus den gleichen Domänen, die die gleichen Reaktionen katalysieren, um kleine Acylsubstrate zu verbinden und zu modifizieren. Im Gegensatz zu PKSs bestehen FASs aber immer aus dem vollen Domänensatz, der iterativ genutzt wird und zur vollständig reduzierten Fettsäure führt. Die vorliegende Thesis analysiert die AT verschiedener PKSs in ihrer Substratselektivität, der AT-ACP-Interaktion und ihren enzymkinetischen Eigenschaften. Der Vergleich mit FAS-ATs liefert die folgenden Erkenntnisse: 1.) PKS-ATs erscheinen langsamer als FAS-ATs. Dies mag in den Unterschieden ihrer Biosynthesewege begründet sein. Die essentiellen Fettsäuren werden in hohen Mengen für die Erfüllung zahlreicher physiologischer Aufgaben benötigt, während Polyketide als Sekundärstoffe schon bei niedrigeren Konzentrationen wirken können. 2.) Die langsameren ATs der modularen PKSs laden auch in Abwesenheit ihrer nativen Substrate keine nicht-nativen Substrate. Dies unterscheidet sie von den schnelleren ATs der iterativen PKSs und FASs und verdeutlicht ihre Rolle als Selektivitätsfilter in der Polyketid-Biosynthese. 3.) Diese beobachtete Substratselektivität kann im ersten oder zweiten Schritt der AT-vermittelten ACP-Beladung erfolgen und ist nicht über eine hydrolytische Korrekturfunktion sichergestellt. Zusätzlich zeigt eine Mutationsstudie einer AT-ACP-Interaktion in der modularen PKS 6-Desoxyerythronolid B-Synthase (DEBS), dass einzelne Oberflächenpunktmutationen die AT-vermittelten Reaktionen in komplexer Weise beeinflussen können. Hierbei zeigt sich eine hohe enzymkinetische Plastizität der AT-ACP-Interaktion. Aufbauend auf diesen Ergebnissen soll die Säuger-FAS in eine PKS-ähnliche Fertigungsstraße umgewandelt werden, um modifizierte Produkte zu generieren. Hierbei müssen drei Aspekte beachtet werden: 1.) Die Beladung durch die AT muss in spezifische Beladung mit Starter- und spezifische Beladung mit Elongationssubstrat getrennt werden. 2.) Verschiedene FAS-Module mit unterschiedlichem Domänensatz müssen entworfen werden, um den Erhalt funktioneller Gruppen zu ermöglichen. 3.) Starter- und Elongationsmodule müssen mit Linkern verbunden werden. Die vorliegende Studie widmet sich vor allem dem ersten Aspekt, der Einführung einer spezifischen Elongationssubstrat-Ladefunktion in die Säuger-FAS mittels Domänenaustausch. Substratspezifische AT-Domänen modularer PKSs werden hierfür in einem Subkonstrukt der Säuger-FAS installiert. Viele der Chimären weisen eine drastisch verminderte Stabilität auf. Eine Mutationsstudie zeigt jedoch die ausgeprägte Eignung der Säuger-FAS für einen AT-Austausch, die sich durch eine erstaunlich hohe Robustheit gegenüber Mutationen in den Schnittstellen äußert. Schließlich werden geeignete Domänengrenzen in der Säuger-FAS etabliert und eine stabile Chimäre erstellt, die 70 % AT-Aktivität im Vergleich zur Wildtyp-PKS aufweist. Da ihre AT sich aber durch eine intrinsisch niedrige Umsatzrate auszeichnet, ist sie für das Forschungsvorhaben, das eine effiziente Synthese neuer Produkte zum Ziel hat, nur bedingt geeignet. Schließlich wird ein chimäres Minimal-Elongationsmodul mit einer anderen PKS AT erstellt. Erste Versuche zeigen, dass die Beladung mit Startersubstraten erfolgreich verhindert wird. Experimente mit einem Startermodul zeigen, dass das gewünschte Produkt in der Fertigungsstraße hergestellt wird. Allerdings sind die Umsatzraten sehr gering, was verschiedene Ursachen haben kann: intrinsische Eigenschaften der AT, intra- oder intermodulare Kommunikationsprobleme. ..

Transacylation kinetics in fatty acid and polyketide synthases and its sensitivity to point mutations

Fatty acid and polyketide synthases (FASs and PKSs) synthesize physiologically and pharmaceutically important products by condensation of acyl building blocks. The transacylation reaction catalyzed by acyl transferases (ATs) is responsible for the selection of acyl-CoA esters for further processing by FASs and PKSs. In this study, the AT domains of different multidomain (type I) PKS systems are kinetically described in their substrate selectivity, AT−Acyl carrier protein (ACP) domain-domain interaction and enzymatic kinetic properties. We observe that the ATs of modular PKSs, intricate protein complexes occurring in bacteria and responsible for the biosynthesis of bioactive polyketides, are significantly slower than ATs of mammalian FASs, reflecting the respective purpose of the biosynthetic pathways within the organism and their metabolic context. We further perform a mutational study on the kinetics of the AT−ACP interaction in the modular PKS 6-deoxyerythronolide B synthase (DEBS) and find a high plasticity in enzyme properties, which we explain by a high plasticity in AT−ACP recognition. Our study enlarges the understanding of ATs in its molecular properties and is similarly a call for thorough AT-centered PKS engineering strategies

Mapping Remunicipalisation: Emergent Trends in the Global Deprivatisation Process

No abstract available

Public Futures Database Report

Public ownership of services including water, energy and healthcare is returning to the forefront of policy at the local level. The current pandemic has also prompted further debate about the boundaries of public and private in providing basic services. As earlier experiences of privatisation have failed to deliver on promises of improved effectiveness, investment and modernisation, public services are being brought back in-house in an increasing number of towns, cities and regions around the world. This trend is commonly referred to as remunicipalisation. The Public Futures database collates (as of February 2021) 1451 verified cases of remunicipalisation from 2000 to the present day. These are located across 56 countries on every continent, in sectors including water, energy, telecommunications, local government and healthcare. The database is fully interactive, enabling users to both submit and analyse cases

A connection between the ribosome and two S. pombe tRNA modification mutants subject to rapid tRNA decay.

tRNA modifications are crucial in all organisms to ensure tRNA folding and stability, and accurate translation. In both the yeast Saccharomyces cerevisiae and the evolutionarily distant yeast Schizosaccharomyces pombe, mutants lacking certain tRNA body modifications (outside the anticodon loop) are temperature sensitive due to rapid tRNA decay (RTD) of a subset of hypomodified tRNAs. Here we show that for each of two S. pombe mutants subject to RTD, mutations in ribosomal protein genes suppress the temperature sensitivity without altering tRNA levels. Prior work showed that S. pombe trm8Δ mutants, lacking 7-methylguanosine, were temperature sensitive due to RTD, and that one class of suppressors had mutations in the general amino acid control (GAAC) pathway, which was activated concomitant with RTD, resulting in further tRNA loss. We now find that another class of S. pombe trm8Δ suppressors have mutations in rpl genes, encoding 60S subunit proteins, and that suppression occurs with minimal restoration of tRNA levels and reduced GAAC activation. Furthermore, trm8Δ suppression extends to other mutations in the large or small ribosomal subunit. We also find that S. pombe tan1Δ mutants, lacking 4-acetylcytidine, are temperature sensitive due to RTD, that one class of suppressors have rpl mutations, associated with minimal restoration of tRNA levels, and that suppression extends to other rpl and rps mutations. However, although S. pombe tan1Δ temperature sensitivity is associated with some GAAC activation, suppression by an rpl mutation only modestly inhibits GAAC activation. We propose a model in which ribosomal protein mutations result in reduced ribosome concentrations, leading to both reduced ribosome collisions and a reduced requirement for tRNA, with these effects having different relative importance in trm8Δ and tan1Δ mutants. This model is consistent with our results in S. cerevisiae trm8Δ trm4Δ mutants, known to undergo RTD, fueling speculation that this model applies across eukaryotes

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have 21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph