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Ziel dieser Doktorarbeit war es, die Bedeutung der Kristallstrukturbestimmung aus Pulverdaten (SDPD) herauszuarbeiten und etwaige Grenzen durch neue Methodenentwicklungen zu erweitern, insbesondere bei Analyse der Paarverteilungsfunktion (PDF).
Die Effizienz der SDPD konnte anhand der erfolgreich gelösten Kristallstruktur von Carmustin (1,3 Bis-2-chlorethyl-1-nitrosoharnstoff, C5H9Cl2N3O2) aufgezeigt werden. [CS01]
Die Grenzen der SDPD wurden ausgelotet und erfolgreich erweitert. Nach weit verbreiteter kristallographischer Meinung ist die Strukturlösung mittels des simulierten Temperns (simulated annealing, SA) bei mehr als 25 zu bestimmenden Parametern problematisch oder unmöglich. Die pharmazeutischen Salze Lamivudin-Camphersulfonat (LC) und Aminogluthethimid-Camphersulfonat (AC) konnten, trotz ihrer hohen Anzahl an Freiheitsgraden von 31 für LC bzw. 37 für AC erfolgreich bestimmt werden. Die Strukturlösung von AC war herausfordernd und nicht direkt bei Anwendung der SA-Methode möglich. Nach einer intensiven Fehleranalyse stellte sich heraus, dass nicht die Grenzen der SA-Methode ausschlaggebend für das anfängliche Scheitern der Strukturlösung waren, sondern falsch extrahierte Intensitäten des vorangegangenen Pawley-Fits. Nach Behebung dieser Fehlerquelle war die Strukturlösung von AC problemlos. [CS02]
Mittels SDPD kann die absolute Konfiguration chiraler Verbindungen nicht direkt bestimmt werden. Durch Kristallisation der zu bestimmenden chiralen Verbindung mit einem chiralen Gegenion bekannter Konformation in einer simplen Säure-Base-Reaktion zu einem diastereomeren Salz und nachfolgender SDPD konnte eine neue Methode entwickelt werden, um die Konfigurationsbestimmung aus Pulverdaten zu ermöglichen. Diese Methode wurde anhand der drei pharmazeutischen Salze (R)-Flurbiprofen-(R)-Chinin (FQ), (2R5S)-Lamivudin-(R)-Camphersulfonat (LC) und (R)-Aminogluthethimid-(R)-Camphersulfonat (AC) aufgezeigt: In allen drei Fällen konnte die korrekte Konfiguration des pharmazeutischen Wirkstoffes mit den hierfür entwickelten Kriterien erfolgreich bestimmt werden. [CS03, CS04]
Durch Kombination der klassischen SDPD mit neuen methodischen Ansätzen konnten die Kristallstrukturen der schlecht kristallinen organischen Pigmente 2-Monomethylchinacridon (MMC, C21H14N2O2) und 4,11-Difluorchinacridon (DFC, C20H10N2O2F2) bestimmt werden, obwohl aufgrund ihrer geringen Kristallqualität keine sinnvolle Indizierung möglich war.
Für die Kristallstrukturbestimmung von DFC lieferte der neu entwickelte Global-Fit des Programms FIDEL mögliche Strukturmodelle mit ähnlich guter Übereinstimmung an das experimentelle Pulverdiagramm. Die Rietveld-Verfeinerung der Strukturmodelle in Kombination mit der Anpassung der Kristallstruktur an die PDF-Daten und kraftfeldbasierter Gitterenergieminimierung konnte einen geeigneten Strukturrepräsentanten von DFC liefern. [CS05, CS06]
Im Fall von MMC war eine Kombination der Methoden von Rietveld-Verfeinerung, Verfeinerung an die PDF-Daten und Gitterenergieminimierung zielführend zur Bestimmung der Orientierungs-Fehlordnung von MMC im Kristall. MMC ist hierbei die erste organische Verbindung, deren Fehlordnung durch Anpassung an die PDF bestimmt werden konnte. [CS07]
Große Erfolge konnten bei der Methodenentwicklung der PDF-Analyse erzielt werden. Die Bestimmung von Kristallstruktur organischer Verbindungen durch Anpassung an die PDF ohne vorherige Kenntnis der Gitterparameter oder Raumgruppe wurde durch die Entwicklung des PDF-Global-Fits erreicht. Lediglich die PDF-Kurve und eine Molekülstruktur werden als Input benötigt. Die Strukturlösung beruht auf einem globalen Optimierungs-Ansatz, bei welchem in ausgewählten Raumgruppen Zufallsstrukturen erzeugt werden. Die Zufallsstrukturen werden mit den experi¬mentellen Daten verglichen und entsprechend ihres Ähnlichkeitsindexes, basierend auf der Kreuz-Korrelation, sortiert. [CS08, CS09] Die vielversprechendsten Kandidaten werden in einem einge¬schränkten simulierten annealing-Ansatz an die experimentelle PDF angepasst. Eine nachfolgende Strukturverfeinerung der besten Strukturmodelle liefert die korrekte Kristallstruktur. Der Erfolg des PDF-Global-Fits wurde am Beispiel der Barbitursäure aufgezeigt: Ausgehend von 300 000 Zufallsstrukturen konnte die korrekte Kristallstruktur von Barbitursäure bestimmt werden. Barbitursäure ist hierdurch die erste organische Verbindung, deren Lokalstruktur durch Anpassung an die PDF bestimmt wurde, ohne Input oder Vorgabe von Gitterparametern oder Raumgruppe.[CS10]
In this thesis, we characterized megasynthases such as fatty acid synthases (FASs) and polyketide synthases. The obtained insights into structure and function were used to engineer such systems to produce new-to-nature compounds.
The in vitro characterization of megasynthases requires reproducible access to these enzymes in high quality. Therefore, we established purification strategies for the yeast FAS and the methylsalicylic acid synthase (MSAS) from Saccharopolyspora erythraea (SerMSAS) and applied the latter one on MSAS from Penicillium patulum (PenPaMSAS) and on 6-deoxyerythronolide B synthase (DEBS) module 6. With the purified samples, we were able to obtain initial structural data for SerMSAS and solve the complete structure of the yeast FAS (PDB: 6TA1). On the example of the yeast FAS, we could show that the sample can suffer from adsorption to the water-air interface during the grid preparation for electron microscopy and presented how the use of graphene-based grids can overcome this problem. The combined structural and functional analysis of the yeast FAS showed that the structural domains trimerization module and dimerization module 2 are not essential for the assembly of the whole system. Therefore, they can potentially be used for domain exchange approaches. The in-depth functional analysis of SerMSAS revealed that not SerMSAS itself releases the product, but a 3-oxoacyl-(acyl-carrier protein) synthase like enzyme within the gene cluster transfers 6-methyl salicylic acid from SerMSAS to another carrier protein for subsequent modifications. In contrast, we showed that PenPaMSAS can release its product by hydrolysis and that non-native substrates can be incorporated although at significantly slower turnover rates compared to the native starter substrate. Our further investigation demonstrated that the substrate specificity of the acyltransferase (AT) is a critical factor for the incorporation of non-native substrates.
With the insight from the functional and structural characterization, we engineered megasynthases for the biosynthesis of natural product derivatives. We targeted the AT of PenPaMSAS for active site mutagenesis and discovered a mutant which can transfer non-native substrates significantly faster (~200-300%). Additionally, the malonyl/acetyl transferase (MAT) of the mammalian FAS was used as a promising target for protein engineering because of its previously reported properties including polyspecificity, fast transfer kinetics, robustness, and plasticity. We showed that the MAT can transfer fluorinated substrates and accept the acyl carrier protein of DEBS module 6. By exchanging the substrate specific AT of DEBS with the polyspecific MAT of the mammalian FAS, we demonstrated an efficient DEBS/FAS hybrid and an optimal truncation site for the applied ATs. In contrast to the wild type system, the DEBS/FAS enzyme was able to synthesize demethylated and fluorinated derivatives. The production and purification of a fluoro-methyl-disubstituted polyketide was of particular interest, as it has a high potential for the generation of new drugs and shows the potential of protein engineering. Furthermore, the incorporation of the disubstituted substrate had important implication in the mechanistic details of the ketosynthase-mediated C-C bond formation.
Non-ribosomal peptide synthetase docking domains : structure, function and engineering strategies
(2021)
Non-ribosomal peptide synthetases (NRPSs) are known for their capability to produce a wide range of natural compounds and some of them possess interesting bioactivities relevant for clinical application like antibiotics, anticancer, and immunosuppressive drugs. The diverse bioactivity of non-ribosomal peptides (NRPs) originates from their structural diversity, which results not only from the incorporation of non-proteinogenic amino acids into the growing peptide chain, but also the formation of heterocycles or further peptide modifications like methylation, hydroxylation and acetylation.
The biosynthesis of NRPs is achieved via the orchestrated interplay of distinct catalytic domains, which are grouped to modules that are located on one or more polypeptide chains. Each cycle starts with the selection and activation of a specific amino acid by the adenylation (A) domain, which catalyzes the aminoacyl adenylate formation under ATP consumption. This activated amino acid is then bound via a thioester bond to the 4’-phosphopantetheine cofactor (PPant-arm) of the following thiolation (T) domain. Before substrate loading, the PPant-arm is post-translationally added to the T domain by a phosphopantetheinyl transferase (PPTase), which converts the inactive apo-T domain in its active holo-form. In the last step of the catalytic cycle, two T domain bound peptide building blocks are connected by the condensation (C) domain, resulting in peptide bond formation and transfer of the nascent peptide chain to the following module. Each catalytic cycle is performed by a C-A-T elongation module until the termination module with a C-terminal thioesterase (TE) domain is reached. Here, the peptide product is released by hydrolysis or intramolecular cyclisation.
In comparison to single-protein NRPSs, where all modules are encoded on a single polypeptide chain, multi-protein NRPS systems must also maintain a specific module order during the peptide biosynthesis. Therefore, small C-terminal and N-terminal communication-mediating (COM) domains/docking domains (DD) were identified in the C- and N-terminal regions of multi-protein NRPSs. It was shown that these domains mediate specific and selective non-covalent protein-protein interaction, even though DD interactions are generally characterized by low affinities.
The first publication of this work focuses on the Peptide-Antimicrobial-Xenorhabdus peptide-producing NRPS called PaxS, which consists of the three proteins PaxA, PaxB and PaxC. Here, in particular the trans DD interface between the C-terminal attached DD of PaxB and N-terminal attached DD of PaxC was structurally investigated and thermodynamically characterized by isothermal titration calorimetry (ITC), yielding a dissociation constant (KD) of ~25 µM, which is a DD typical affinity known from further characterized DD pairs. The artificial linking of the PaxB/C C/NDD pair via a glycine-serine (GS) linker facilitated the structure determination of the DD complex by solution nuclear magnetic resonance (NMR) spectroscopy. In comparison to known docking domain structures, this DD complex assembles in a completely new fold which is characterized by a central α-helix of PaxC NDD wrapped in two V-shaped α-helices of PaxB CDD.
The first manuscript of this work focuses on the application of synthetic zippers (SZ) to mimic natural docking domains, enabling the easy assembly of NRPS building blocks encoded on different plasmids in a functional way. Here, the high-affinity interaction of SZs unambiguously defines the order of the synthetases derived from single-protein NRPSs in the engineered NRPS system and allows the recombination in a plug-and-play manner. Notably, the SZ engineering strategy even facilitates the functional assembly of NRPSs derived from Gram-positive and Gram-negative bacteria. Furthermore, the functional incorporation of SZs into NRPS modules is not limited to a specific linker region, so we could introduce them within all native NRPS linker regions (A-T, T-C, C-A).
The second publication and the second manuscript of this thesis again focus on the multi-protein PaxS, in particular on the trans interface between the proteins PaxA and PaxB on a molecular level by solution NMR. Therefore, the PaxA CDD adjacent T domain was included into the structural investigation besides the native interaction partner PaxB NDD. Before a three-dimensional structure could be obtained from NMR data, the NH groups located in the peptide bonds had to be assigned to the respective amino acids of the proteins (backbone assignment). Based on these backbone assignments, the secondary structure of PaxA T1-CDD and PaxB NDD in the absence and presence of the respective interaction partner were predicted.
The structural and functional characterization of the PaxA T1-CDD:PaxB NDD complex is summarized in manuscript two. The thermodynamic analysis of this complex by ITC determined a KD value of ~250 nM, whereas the discrete DDs did not interact at all. The high-affinity interaction allowed to determine the solution NMR structure of the PaxA T1-CDD:PaxB NDD complex without the covalent linkage of the interaction partners and an extended docking domain interface could be determined. This interface comprises on the one hand α-helix 4 of the PaxA T1 domain together with the α-helical CDD, and on the other hand the PaxB NDD, which is composed of two α-helices separated by a sharp bend.
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The deubiquitinase USP32 regulates non-proteolytic ubiquitination in the endosomal-lysosomal system
(2021)
The regulation of essential cellular processes requires tightly controlled and directed transport of proteins and membranes. The highly dynamic endosomal and lysosomal system forms the key network for exchange and trafficking of molecules with its early endosomes, recycling endosomes, late endosomes, lysosomes, and additionally autophagosomes.
In this system, the small GTPase Rab7 has an essential role at the late endosomal stage regulating vesicle transport, tethering, and fusion, and retromer mediated receptor recycling back to the trans-Golgi network (TGN). Thus, Rab7 is also important for autophagosomes and lysosomes.
Lysosomes do not only represent the end point of the degradation pathway with several feeder pathways. But these organelles are also a dynamic signaling hub for a variety of metabolic processes. The ever-important regulator of cellular biosynthetic pathways mTORC1 dynamically associates with lysosomes where it is activated. mTORC1 activation is a complex multi-step process where a series of signaling events converge in dependence of amino acid levels thereby enabling interactions between the lysosomal v-ATPase, Ragulator complex (consisting of LAMTOR1-5), and Rag GTPases.
Ubiquitin signals are involved in almost all cellular processes. With this, their regulatory mechanism is also described for the endosomal-lysosomal system as well as mTORC1 signaling. Deubiquitinases (DUBs) release conjugated ubiquitin from proteins and thereby maintain the dynamic state of the cellular ubiquitinome.
The ubiquitin-specific protease 32 (USP32) is a poorly characterized DUB with only emerging cellular function. However, its predicted domain structure includes two unique domains within the entire DUB family. It has been linked to the development of breast cancer and small cell lung cancer. Furthermore, overexpressed GFP-USP32 was localized at the TGN, and a global mass spectrometry-based DUB interactome study suggested an interaction with the retromer complex. Based on these data, USP32 was a very interesting candidate to study its cellular function in this PhD project.
To investigate the function without disease background, a polyclonal USP32 knockout (USP32KO) RPE1 cell line was generated using the CRISPR/Cas9 technology. First experiments revealed different protein expression levels in various cell lines, and a subcellular localization of USP32 at membranes of the Golgi and lysosomal compartments. In a subsequent SILAC-based ubiquitinome analysis potential substrates of USP32 were identified. Interestingly, various proteins of the endosomal-lysosomal system were detected with enriched non-proteolytic ubiquitination upon USP32 depletion.
The further characterization of Rab7 as USP32 substrate confirmed the USP32-sensitive ubiquitination of Rab7 at lysine (K) residues 191 and 194. The ubiquitination in USP32KO cells did not change the subcellular localization of Rab7, but enhanced the interaction with the effector protein RILP. This implied that Rab7 was either more active or RILP had higher affinity to ubiquitinated Rab7. The subsequent results verified this theory. The retromer mediated recycling of CI-M6PR back to the TGN was faster or more efficient in USP32-depleted cells.
Accompanying this, levels of hydrolases were enriched in lysosomes isolated from USP32KO cells. Notably, USP32 had no direct effect on expression level or assembly of the retromer complex itself.
The observed lysosomal phenotypes connected another identified substrate to the function of USP32 in the endosomal-lysosomal system: LAMTOR1. LAMTOR1 is a component of the Ragulator complex and thus involved in the activation of mTORC1 at the lysosomal surface. Similar as for Rab7, the first experiments to characterize LAMTOR1 as USP32 substrate confirmed the USP32-sensitive ubiquitination at K20 independent of amino acid availability. However, ubiquitination of LAMTOR1 decreased its lysosomal localization in untreated and amino acid starved USP32KO cells. The following label-free interactome study detected a reduced interaction of LAMTOR1 and subunits of the lysosomal v-ATPase upon loss of USP32. This resulted in a shifted subcellular localization of mTOR (subunit of mTORC1) away from lysosomes. Furthermore, direct substrates of mTORC1 were less or slower re-phosphorylated after long amino acid starvation and re-activation of mTORC1 in USP32KO cells indicating a reduced mTORC1 activity.
Both USP32-dependent regulations of Rab7 and LAMTOR1/Ragulator converged in enhanced autophagic processes analyzed by increased LC3 levels upon amino acid starvation and USP32 depletion.
In summary, the presented thesis described the diverse role of USP32 in the endosomal and lysosomal system, and contributes to the understanding of novel ubiquitin signals in this context.
This work comprises the investigation of four different biosynthesis gene clusters from Xenorhabdus. Xenorhabdus is an entomopathogenic bacterium that lives in mutualistic symbiosis with its Steinernema nematode host and together they infect and kill insect larvae. Xenorhabdus is well known for the production of so-called specialised metabolites and many of these compounds are synthesised by non-ribosomal peptide synthetases (NRPSs) or NRPS-polyketide synthase (PKS)-hybrids. These enzymes are organised in a modular manner and produce structurally very diverse molecules, often with the help of modifying domains and tailoring enzymes. In general, the genes involved in the biosynthesis are organised in so-called biosynthetic gene clusters (BGCs) in the genome of the producing strain. Exchanging the native promoter with an inducible promoter, e.g. PBAD, allows the targeted activation of the BGC and in turn the analysis of the biosynthesis product via LC-MS analysis.
The first BGC investigated in this work is responsible for the biosynthesis of xenofuranones. Based on gene deletions, this work shows that the NRPS-like enzyme XfsA produces a carboxylated furanone intermediate which is subsequently decarboxylated by XfsB to yield xenofuranone B. The next step in xenofuranone biosynthesis is the O-methylation of xenofuranone B to yield xenofuranone A. A comparative proteomics approach allowed the identification of four methyltransferase candidates and subsequent gene deletions confirmed one of the candidates to be responsible for methylation of xenofuranone B. The proteome analysis was based on the comparison of X. szentirmaii WT and X. szentirmaii Δhfq because distinct levels of the methylated xenofuranone A were observed when the xfs BGC was activated in either WT or Δhfq strain. Hfq is a global transcriptional regulator whose deletion is associated with the down regulation of natural product biosynthesis in Xenorhabdus. The strong PBAD activation of the xfs BGC also allowed the detection of two novel xenofuranone derivatives which arise from incorporation of one 4-hydroxyphenylpyruvic acid as first or second building block, respectively.
PBAD based activation of the second BGC addressed in this work lead to the detection of a novel metabolite and compound purification allowed NMR-based structure elucidation. The molecule exhibits two pyrrolizidine moieties and was named pyrrolizwilline (pyrrolizidine + twin (German: “Zwilling”)). The BGC comprises seven genes and single gene deletions as well as heterologous expression in E. coli and NRPS engineering were conducted to investigate the biosynthesis. The first two genes xhpA and xhpB encode a bimodular NRPS and a monooxygenase which synthesise a pyrrolizixenamide-like structure, similar to PxaA and PxaB in pyrrolizixenamide biosynthesis. It is suggested that the acyl side chain incorporated by XhpA is removed by the α,β-hydrolase XhpG. The keto function is then reduced by two subsequent two electron reductions catalysed by XhpC and XhpD. One of these two reduced pyrrolizidine units most likely is extended with glyoxalate prior to non-enzymatic dimerisation with the second pyrrolizidine moiety. To finally yield pyrrolizwilline, L-valine is incorporated, probably by the free-standing condensation domain XhpF.
The third BGC investigated is responsible for the production of a tripeptide composed of β-D-homoserine, α-hydroxyglycine and L-valine and is referred to as glyoxpeptide. This work demonstrates that the previously observed glyoxpeptide derivative is derived from glycerol present in the culture medium. Furthermore, this work shows that the monooxygenase domain, which is found in an unusual position between motifs A8 and A9 within the adenylation domain, is responsible for the α-hydroxylation of glycine. It is suggested that the α-hydroxylation of glycine renders the tripeptide prone to hydrolysis via hemiacetal formation. Hence, the XgsC_MonoOx domain might be an interesting candidate for further NRPS engineering.
The fourth BGC addressed is responsible for the production of xildivalines and this work describes two additional derivatives which are detected only when the promoter is exchanged and activated in the X. hominickii WT strain but not in X. hominickii Δhfq. Deletion of the methyltransferase encoding gene xisE results in the production of non-methylated xildivalines. It remains to be determined when the N-methylation of L-valine takes place. It is discussed that the methyltransferase could act on the NRPS released product but also during the assembly. The peptide deformylase is not involved in the proposed biosynthesis as xildivaline production is detected in a ΔxisD strain. The PKS XisB features two adjacent, so-called tandem T domains. The inactivation of the first or the second T domain by point mutation causes decreased production titres of detected xildivalines in the respective mutant strain when compared to the wild type.
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.
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