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Institute
The composition of cellular membranes is extremely complex and the mechanisms underlying their homeostasis are poorly understood. Organelles within a eukaryotic cell require a non-random distribution of membrane lipids and a tight regulation of the membrane lipid composition is a prerequisite for the maintenance of specific organellar functions. Physical membrane properties such as bilayer thickness, lipid packing density and surface charge are governed by the lipid composition and change gradually from the early to the late secretory pathway. As the endoplasmic reticulum (ER) is situated at the beginning of the cells secretory pathway, it has to accept and accommodate a great variety and quantity of secretory and transmembrane proteins, which enter the ER on their way to their final cellular destination. Secretory proteins can be translocated into the lumen of the ER co- or posttanslationally and membrane proteins are being inserted and released into the ER membrane. In the oxidative milieu of the ER-lumen, supported by a variety of chaperones, proteins can fold into their native form.
If the folding capacity of the ER-lumen is exceeded, an accumulation of mis- or unfolded proteins in the lumen of the ER occurs, consequently triggering the unfolded protein response (UPR). This highly conserved program activates a wide-spread transcriptional response to restore protein folding homeostasis. In fact, 7 – 8% of all genes in the yeast Saccharomyces cerevisiae (S. cerevisiae) are regulated by the UPR. The mechanism underlying the activation of the UPR by protein folding stress has been investigated thoroughly in the last decades and many of its mechanistic details have been elucidated. Recently, it became evident that aberrant lipid compositions of the ER membrane, collectively referred to as lipid bilayer stress, are equally potent in activating the UPR. The underlying molecular mechanism of this membrane-activated UPR, however, remained unclear.
This study focuses on the UPR in S. cerevisiae and characterizes the inositol requiring enzyme 1 (Ire1) as the sole UPR sensor in S. cerevisiae. Active Ire1 forms oligomers and, collaboratively with the tRNA ligase Rlg1, splices immature mRNA of the transcription factor HAC1, which results in the synthesis of mature HAC1 mRNA and the production of the active Hac1 protein, which binds to UPR-elements in the nucleus and activates the expression of UPR target genes. Here, the combination of in vivo and in vitro experiments is being used, which is supplemented by molecular dynamics (MD) simulations performed by Roberto Covino and Gerhard Hummer (MPI for Biophysics, Frankfurt), aiming to identify the molecular mechanism of Ire1 activation by lipid bilayer stress. This study focuses on the analysis of the juxta- and transmembrane region of Ire1. Bioinformatic analyses revealed a putative ER-lumenal amphipathic helix (AH) N-terminally of and partially overlapping with the transmembrane helix (TMH). This predicted AH contains a large hydrophobic face, which inserts into the ER membrane, forcing the TMH into a tilted orientation within the membrane. The resulting unusual architecture of Ire1’s AH and TMH constitutes a unique structural element required for the activation of Ire1 by lipid bilayer stress.
To investigate the function of the AH in the physiological context, different variants of Ire1 were produced under the control of their endogenous promoter and from their endogenous locus. The functional role of the AH was tested, by disrupting its amphipathic character by the introduction of charged residues into the hydrophobic face of the AH. The role of a conserved negative residue between the TMH and the AH (E540 in S. cerevisiae) was tested by substituting it by a unipolar, polar, or positively charged residue. These variants were intensively characterized using a series of assays:
This thesis provides evidence that the AH is crucial for the function of Ire1: Mutant variants with a disrupted (F531R, V535R) or otherwise modified AH (E540A) exhibited a lower degree of oligomerization and failed to catalyze the splicing of the HAC1 mRNA as the Wildtype control. Likewise, the induction of PDI1, a target gene of the UPR, was greatly reduced in mutants with a disrupted or defective AH. These data revealed an important functional role of the AH for normal Ire1 function.
An in vitro system was established to analyze the membrane-mediated oligomerization of Ire1. This system enabled the isolated functional analysis of the AH and TMH during Ire1 activation by lipid bilayer stress. A fusion construct, coding for the maltose binding protein (MBP) from Escherichia coli (E. coli), N-terminally to the AH and TMH of Ire1 was produced. The heterologous production in E. coli, the purification and reconstitution of this minimal sensor of Ire1 in liposomes was established as part of this study. To analyze the oligomeric status of the minimal sensor in different lipid environments, continuous wave electron paramagnetic resonance (cwEPR) spectroscopic experiments were performed. These experiments revealed that the molecular packing density of the lipids had a significant influence of the oligomerization of the spin-labeled membrane sensor: increasing packing densities resulted in sensor oligomerization. The AH-disruptive F531R mutant, in which the amphipathic character of the AH was destroyed, showed no membrane-sensitive changes in its oligomerization status.
Thus, the activation of Ire1 by lipid bilayer stress is achieved by a membrane-based mechanism. According to the current model, the AH induces a local membrane compression by inserting its large hydrophobic face into the membrane. As membrane thickness and acyl chain order are interconnected, this compression simultaneously results in an increased local disordering of lipid acyl chains. Supporting MD simulations performed by Roberto Covino and Gerhard Hummer revealed that the bilayer compression is significantly more pronounced in a densely packed lipid environment, than in a lipid environment of lower lipid packing density. Hence, the energetic cost of the local compression increases with the packing density of the membrane, but is compensated for by the oligomerization of Ire1. This minimization of energetic cost induced by the membrane deformation of Ire1 forms the basis for the activation of Ire1 by lipid bilayer stress.
RNA research is very important since RNA molecules are involved in various gene regulatory mechanisms as well as pathways of cell physiology and disease development.1 RNAs have evolved from being considered as carriers of genetic information from DNA to proteins, with the three major types of RNA involved in protein synthesis, including messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).2 In addition to the RNAs involved in protein synthesis numerous regulatory non-coding RNAs (ncRNAs) have been discovered in the transcriptome. The regulatory ncRNAs are classified into small ncRNAs (sncRNAs) with transcripts less than 200 nucleotides (nt) and long non-coding RNAs (lncRNAs) with more than 200 nt.3
LncRNAs represent the most diverse and versatile class of ncRNAs that can regulate cellular functions of chromatin modification, transcription, and post-transcription through multiple mechanisms.4 They are involved in the formation of RNA:protein, RNA:RNA and RNA:DNA complexes as part of their gene regulatory mechanism.4,5 The RNA:DNA interactions can be divided into RNA:DNA heteroduplex formation, also called R-loops, and RNA:DNA:DNA triplex formation. In triplex formation, RNA binds to the major groove of double-stranded DNA through Hoogsteen or reverse Hoogsteen hydrogen bonding, resulting in parallel or anti-parallel triplexes, respectively. In vitro studies have confirmed the formation of RNA:DNA:DNA triplexes.6 However, the extent to which these interactions occur in cells and their effects on cellular function are still not understood, which is why these structures are so exciting to study (Chapter I RNA:DNA:DNA Triplexes).
This cumulative thesis investigates several functional and regulatory important RNAs. The first project involves the improved biochemical and biophysical characterization of RNA:DNA:DNA triplex formation between lncRNAs of interest and their target genes. Triplex formation was confirmed by a series of experiments including electromobility shift assays (EMSA), thermal melting assays, circular dichroism (CD), and liquid state nuclear magnetic resonance (NMR) spectroscopy. The following is a summary of the main findings of these publications.
In research article 5.1, the oxygen-sensitive HIF1α-AS1 was identified as a functionally important triplex-forming lncRNA in human endothelial cells using a combination of bioinformatics techniques, RNA/DNA pulldown, and biophysical experiments. Through RNA:DNA:DNA triplex formation, endogenous HIF1α-AS1 decreases the expression of several genes, including EPH receptor A2 (EPHA2) and adrenomedullin (ADM), by acting as an adaptor for the repressive human silencing hub (HUSH) complex, which has been studied by our collaborators in the groups of Leisegang and Brandes.
2) Triplex formation between HIF1α-AS1 and the target genes EPHA2 and ADM was investigated in biochemical and biophysical studies. The EMSA results indicated that HIF1α-AS1 forms a low mobility RNA:DNA:DNA triplex complex with the EPHA2 DNA target sequence. The CD spectrum of the triplex showed distinct features compared to the EPHA2 DNA duplex and the RNA:DNA heteroduplex. Melting curve analysis revealed a biphasic melting transition for triplexes, with a first melting point corresponding to the dissociation of the RNA strand with melting of the Hoogsteen hydrogen bonds. The second, higher melting temperature corresponds to the melting of stronger Watson-Crick base pairing. Stabilized triplexes were formed using an intramolecular EPHA2 DNA duplex hairpin construct in which both DNA strands were attached to a 5 nucleotide (nt) thymidine linker. This approach allowed improved triplex formation with lower RNA equivalents and higher melting temperatures. By NMR spectroscopy, the triplex characteristic signals were observed in the 1H NMR spectrum, the imino signals in a spectral region between 9 and 12 ppm resulting from the Hoogsteen base pairing. To elucidate the structural and sequence specific Hoogsteen base pairs 2D 1H,1H-NOESY measurements of the EPHA2 DNA duplex and the HIF1α-AS1:EPHA2 triplex were performed. The 1H,1H-NOESY spectrum of the HIF1α-AS1:EPHA2 triplex with a 10-fold excess of RNA was semi-quantitatively analyzed for changes in the DNA duplex spectrum. We discovered, strong and moderate attenuation of cross peak intensities in the imino region of the NOESY spectrum. This attenuation was proposed to result from weakening of Watson-Crick base pairing by Hoogsteen hydrogen bonding induced by RNA binding. The Hoogsteen interactions can be mapped based on the analysis of the cross peak attenuation in the NOESY spectra, which we used to generate a structural model of the RNA:DNA:DNA triplex. These biophysical results support the physiological function of HIF1α as a triplex-forming lncRNA that recruits the HUSH-epigenetic silencing complex to specific target genes such as EPHA2 and ADM, thereby silencing their gene expression through RNA:DNA:DNA triplex formation.
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.
This work characterizes the post-PKS modifications of AQ-256. Additionally, the second part describes the establishment of an AQ production platform for electrolyte generation that can be utilized in redox-flow-batteries. Lastly, a silent BGC that encodes the genes for terpenoid biosynthesis was described and characterized with regards to product formation and putative ecological function.
Die hier vorliegende Dissertation befasst sich mit der Synthese von Naturstoffen aus Xenorhabdus und Photorhabdus spp. Da 6,0 - 7,5% ihres Genoms Sekundärmetabolit Clustern zuzuordnen sind, gelten diese entomopathogenen Bakterien als vielversprechende Naturstoffproduzenten. Die Palette der von ihnen produzierten Naturstoffe reicht von Antibiotika über Insektizide bis hin zu potentiellen Zytostatika. Die im Rahmen dieser Arbeit synthetisierten und charakterisierten Substanzen lassen sich in vier Kategorien einteilen: kleine Sekundärmetabolite (Phurealipide), zyklische Makrolaktame (Xenotetrapeptide, GameXPeptide und Ambactin), zyklische Makrolaktone (Szentiamide, Xentrivalpeptide und Xenephematide) und methylierte lineare Peptide (Rhabdopeptide und Rhabdopeptid-ähnliche Moleküle).
This work deals with the characterization of three different type II polyketide synthase systems (PKS II) from the Gram-negative bacteria Xenorhabdus and Photorhabdus.
Particular attention was paid to a biochemically underexplored class of aryl polyene (APE) pigments. Bioinformatic analysis of enzymes involved in the biosynthesis and the in vitro reconstruction proved that the synthesis of APEs involves an unusual fatty acid-like elongation mechanism. Furthermore, the discovery of unexpected protein-protein interactions provided new insights into the multienzyme complex formation of this unusual PKS II system. Through collaboration with the groups from Prof. Michael Groll and junior Prof. Nina Morgner, two protein complexes were structurally solved and several native protein multimerization events were identified and allowed us to suggest a possible protein-interaction network. The results are summarized in publication ‘An Uncommon Type II PKS Catalyzes Biosynthesis of Aryl Polyene Pigments’ (first author; J. Am. Chem. Soc.).
In addition to in vitro-analysis, in vivo-studies were used to investigate the APE compound produced by X. doucetiae in more detail. The activation of the silent biosynthetic gene cluster (BGC) led to the detection of the APE compound in the homologous host. Further combination of homologous expression and targeted deletions of the APE BGC revealed an APE-lipid-like structure. MS-based analyses and purification of intermediates allowed us to deduce structural building blocks of the APE-lipid, which is composed of an APE structural core, a glucosamine residue and an unusual long-chain fatty acid with unusual conjugated double bonds and a phosphoethanolamine head group. In combination with the above stated in vitro-data, we assumed a plausible biosynthetic mechanism of the APE-lipid. The results are summarized in the section ‘Additional Results: Tracing the Full-length APE’.
The biosynthesis of isopropylstilbene (IPS) has already been well-studied by the Bode laboratory and the group of Prof. Ikuro Abe. Studies with Photorhabdus laumondii TT01 by the Bode group revealed the distributed locations and functions of the genes involved in biosynthesis, which originate from two pathways. Particularly, the Bode group first demonstrated that an unusual ketosynthase/cyclase (StlD) catalyzes the condensation of 5-phenyl-2,4-pentadienoyl-ACP and isovaleryl-beta-ketoacyl-ACP via a Michael addition. Such a pathway for stilbene formation is distinct from those widespread in plants. The Abe group solved the structure and biochemical mechanism of StlD and further investigated the aromatization reaction of the aromatase StlC. However, the generation of the required cinnamoyl-precursor 5-phenyl-2,4-pentadienoyl-ACP as a Michael acceptor for this cyclization reaction remained elusive. In this work, we were able to reconstitute the synthesis of the Michael acceptor in vitro, by the action of enzymes from the fatty acid biosynthesis. With the knowledge about the crucial cross-talk from primary and specialized metabolism, we further determined the minimal endowment for stilbene production in a heterologous host. Here, the discovered AasS enzyme StlB is responsible for the generation of cinnamoyl-ACP and among others, plFabH plays a key role as gatekeeper enzyme for further processing. With this information in hand, we were able to obtain IPS production in E. coli. These results are presented in the manuscript ‘Biosynthesis of the Multifunctional Isopropylstilbene in Photorhabdus laumondii Involves Cross-talk Between Specialized and Primary Metabolism’ (co-first author, manuscript).
The biosynthesis of the orange-to-red-pigmented anthraquinones (AQs) is the best-studied type II PKS system according to preliminary results. While several investigations by Brachmann et al. discovered the BGC and the overall product spectrum of the main AQ-256 and its methylated derivatives, data of Quiqin Zhou (Bode group) performed biochemical in vitro analysis paired with in vivo heterologous expression of the ant-genes antA-I. This led to the identification of shunt products that indicated an AQ-scaffold derived from an octaketide intermediate that gets shortened to a heptaketide by the hydrolase AntI, resulting in the main anthraquinone AQ-256. This PKS-shortening mechanism was further confirmed by the protein crystal structure of AntI by the Groll group (publication, minor contributions, co-author, Chem Sci. ‘Molecular Mechanism of Polyketide Shortening in Anthraquinone Biosynthesis of Photorhabdus luminescens’). Further substrate analysis of the P. luminescens AQ-producer and mutants revealed an inhibitory effect of cinnamic acid against the hydrolase AntI. Cinnamic acid might therefore be involved in regulation of AQ biosynthesis (‘Anthraquinone Production is Influenced by Cinnamic Acid’, first author, manuscript).
Biochemical analysis from Quiqin Zhou with the minimal PKS of the AQ-synthase further revealed the exclusive activation of the AQ-ACP by the PPTase AntB. The PPTase is insoluble alone but gets stabilized by the CoA-ligase, most likely inactive, working as a chaperone. Thus, the minimal PKS endowment to produce the octaketide scaffold compromises, besides the ACP, the KS:CLF heterodimer and the MCAT, the co-occurrence of the PPTase AntB and the CoA-ligase AntG. For the first time, X-ray crystallography depicted a minimal PKS in action, by obtaining the structural data of native complexes from an ACP:KS:CLF, the KS:CLF alone and an ACP:MCAT in their non-active and active forms. It was possible to confirm a KS-bound hexaketide, which was built upon heterologous expression of the KS:CLF. Mutagenesis with amino-acids proposed to be involved in protein-protein interactions in the ACP:KS:CLF complex revealed some interesting protein-interaction sites. Additionally, an induced-fit mechanism of the MCAT with the ACP during the malonylation reaction confirmed a monodirectional transfer reaction (‘Structural Snapshots of the Minimal PKS System Responsible for Octaketide Biosynthesis’ co-author, manuscript under review).
This cumulative dissertation examines learning in chemistry laboratories, focusing on the challenges and benefits of problem-based learning (PBL) for novices in the lab. It addresses the lack of consistent understanding about what should be learned in labs and why it's important. The research aims to understand what students learn, how they learn, and how lab learning can be improved.
A central concept in PBL labs is Information Literacy, defined as a sociocultural practice enabling learners to identify and use information sources within a specific context as legitimized by the practice community.
The first publication, Wellhöfer and Lühken (2022a), investigates the relationship between PBL and learner motivation. It identifies factors that can foster students' intrinsic motivation in a PBL lab. Autonomy is found to be a key factor, increasing student motivation and presenting a model of the autonomous scientific process. This model involves four steps: information acquisition, designing and applying experimental procedures, experimental feedback, and autonomous process optimization. The results suggest that intrinsic motivation in PBL labs can be enhanced by enabling students to independently execute these steps.
The second publication, Wellhöfer and Lühken (2022b), examines the information process students undergo during their first PBL lab. Using a sociocultural framework, it explores Information Literacy to understand students' handling of information and their perceptions of the information process. The findings reveal that in PBL labs, developing a practical, applicable experimental procedure is crucial for problem-solving and significantly shapes the information-acquisition process. This process is iterative, influenced by new information, leading to more precise information needs. Students assess information quality based on its usefulness for their problem, implementability (considering cognitive understanding, available equipment, and psychomotor skills), and safety.
Furthermore, the role of privileged knowledge forms in evaluating the quality of text sources is explored. Students viewed non-scientific sources as "poor" and scientific sources as "good," yet used both for information gathering. There were discrepancies between their assessment of source quality and actual use, indicating that perception of source quality doesn't always affect their practical decisions.
The third publication, Wellhöfer, Machleid, and Lühken (2023), investigates students' information practices in the lab, focusing on discourse between novice learners and experienced assistants. It shows that theoretical knowledge isn't sufficient for independent practical action, and students need actionable social information from experienced community members. The results highlight that information literacy in the lab for newcomers to a community of practice has distinctive features, and physical experience and tacit knowledge are crucial for learning the methods and group-specific knowledge of the practice community. The article demonstrates how learning information literacy in a practice community requires a social and physical experience and provides insights on how educators can support this process.
Development and implementation of novel optogenetic tools in the nematode Caenorhabditis elegans
(2016)
Optogenetics, though still only a decade old field, has revolutionized research in neurobiology. It comprises of methods that allow control of neural activity by light in a minimally-invasive, spatio-temporally precise and genetically targeted manner. The optogenetic actuators or the genetically encoded light sensitive elements mediate light driven manipulation of membrane potential, intracellular signalling, neuronal network activity and behaviour (Fenno et al. 2011; Dugué et al. 2012). These techniques have been particularly useful for dissecting neural circuits and behaviour in the transparent and genetically amenable nematode model system Caenorhabditis elegans (Husson et al. 2013; Fang-yen et al. 2015).
In fact, C. elegans was the first living organism in which microbial rhodopsin based optogenetic tools (Channelrhodopsin-2 or ChR2, and Halorhodopsin or NpHR) were successfully implemented and bimodal 'remote' control of behaviour was achieved (Nagel et al. 2005; Zhang et al. 2007). Since then it has been a prominent model for the development and application of novel optogenetic tools and techniques, especially in the nervous system which comprises of 302 neurons and is organised in a hierarchical organization. The environmental stimuli are sensed by the sensory neurons, leading to the processing of information by the downstream interneurons, that relay to motor neurons which in-turn synapse onto muscles that drive the movement-based responses.
The microbial rhodopsins like ChR2 and NpHR mediate light driven depolarization and hyperpolarization, respectively and thereby activate or inhibit neural activity. However, they do not allow local control of membrane potential as they are expressed all over the plasma membrane of the cell rather than being restricted to specific domains, for example synaptic sites. Moreover, they completely over-ride the intrinsic activity of the cell, completely bypassing the signal transduction processes inside the cell. Thus, in order to study intracellular signalling and to answer questions pertaining to the endogenous role of receptors and channels in an in-vivo context, the optogenetic tool-kit needs to be expanded.
This thesis aimed at developing and implementing novel optogenetic tools in C. elegans that allow for sub-cellular signalling control as well as endogenous receptor control. These are: two light activated guanylyl cyclases (bPGC and BeCyclOp) to modify cyclic guanosine monophosphate (cGMP) mediated signalling in the sensory neurons, as well as attempts towards rendering endogenous C. elegans receptors - glutamate receptor (GLR-3/-6), acetylcholine receptor (ACR-16), glutamate gated chloride channel (GLC-1) light switchable and to understand their biological function in-vivo.
Organisms respond to sensory cues by activation of a primary receptor followed by relay of information downstream to effector targets by secondary signalling molecules. cGMP is a widely used 2nd messenger in cellular signaling, acting via protein kinase G or cyclic nucleotide gated (CNG) channels. In sensory neurons, cGMP allows for signal modulation and amplification, before depolarization. Chemo-, thermo-, and oxygen-sensation in C. elegans involve sensory neurons that use cGMP as the main 2nd messenger. For example, ASJ is the pheromone sensing neuron regulating larval development, AWC is the chemosensory neuron responding to volatile odours and BAG senses oxygen and carbon dioxide in the environment. In these neurons, cGMP acts downstream of the GPCRs and functions by activating cationic TAX-2/-4 CNG channels, thereby depolarising the sensory neuron. Manipulating cGMP levels is required to access signalling between sensation and sensory neuron depolarization, thereby provide insights into signal encoding. We achieve this by implementing two photo-activatable guanylyl cyclases - 1) a mutated version of Beggiatoa sp. bacterial light-activated adenylyl cyclase, with specificity for GTP (Ryu et al. 2010), termed BlgC or bPGC (Beggiatoa photoactivated guanylyl cyclase) and 2) guanylyl cyclase rhodopsin (Avelar et al. 2014) from Blastocladiella emersonii (BeCyclOp).
bPGC is a BLUF (blue light sensing using flavin) domain containing cyclase which uses FAD as the co-factor and catalyses the synthesis of cGMP from GTP upon activation by blue light. Prior to implementation in sensory neurons, a simpler heterologous system with co-expression of the TAX-2/-4 CNG channel in C. elegans body wall muscle (BWM) was used. The cGMP generated by the light activated cyclases activates the CNG channel leading to the muscle depolarization, thereby causing changes in body length which can be easily scored.
Natural products are valuable sources for biologically active compounds, which can be utilized as pharmaceuticals. Thereby, the synthesis is based purely on biosynthetic grounds often conducted by so-called megaenzymes. One major biosynthetic pathway is the acetate pathway including polyketide and fatty acid synthesis, which encompass one of the largest classes of chemically diverse natural products. These have medicinal relevance due to their antibacterial, antifungal, anthelmintic, immunosuppressive and antitumor properties.
Due to the high structural and functional similarity between polyketide synthases and type I animal fatty acid synthases (FASs), FAS can serve as a paradigm for the whole class of multifunctional enzymes. To fully exploit the biosynthetic potential of FASs, a good access to the enzyme is of essential importance. In this regard, Escherichia coli remains an unchallenged heterologous host due to low culturing costs, particularly fast mutagenesis cycles and relatively easy handling. Surprisingly, no sufficient expression strategy for an animal FAS in E. coli has yet been reported, as it turned out that the only approach was not reproducible.
We commenced our analysis with searching for an appropriate FAS homolog that fulfills our requirements of high protein quality, sufficient yield and ensured functionality. After extensive screening of different variants, culturing conditions and co-expression strategies, we identified the murine FAS (mFAS) as our protein of choice. The established purification strategy using tags at both termini led to a reproducible and sufficient access to the protein in excellent quality. The enzyme was further biochemically characterized including an enzyme kinetic investigation of fatty acid synthesis and an examination whether different acyl-CoA substrates can serve as priming units. This adds mFAS to our repertoire of manageable megaenzymes paving the way to exploit the catalytic efficiency in regards of microbial custom-compound synthesis.
With a strong focus on deepening our understanding of the working mode of such megaenzymes, rather than analyzing respective biosynthetic products, we have addressed the question whether mFAS itself can be engineered towards PKSs or whether properties of mFAS can be exploited to engineer PKSs. This approach was conducted on three levels of complexity from function of individual domains via organization of domains to form modules to the interplay of two modules in bimodular constructs.
Fatty acid synthesis begins with the loading of acyl moieties onto the FAS, which is conducted by a domain called malonyl-/acetyltransferase (MAT). This domain was in-depth characterized due to its important role of choosing the substrates that are built in the final compound. Our analysis comprised structural and functional aspects providing crystal structures of two different acyl-bound states and kinetic parameters for the hydrolysis and transacylation reaction using twelve exemplary CoA-esters. For this purpose, we have successfully established a continuous fluorometric assay using the α-ketoglutarate dehydrogenase as a coupled enzyme, which converts the liberated coenzyme A into Nicotinamide adenine dinucleotide. These data revealed an extensive substrate ambiguity of the MAT domain, which had not been reported to that extent before. Further, we could demonstrate that the fold fulfills both criteria for the evolvability of an enzyme by expressing MAT in different structural arrangements (robustness) and by altering the substrate ambiguity within a mutagenesis study (plasticity). Taken these aspects together, we are persuaded that the MAT domain can serve as a versatile tool for PKSs engineering in potential FAS/PKS hybrid systems.
On the higher level of complexity, we investigated the architectural variability of the mFAS fold, which constitutes a fundamental basis for a broader biosynthetic application. We could rebuild all four module types occurring in typical modular PKSs confirming a high degree of modularity within the fold. Not only structural, but also functional integrity of these modules was validated by using triacetic acid lactone formation and ketoreductase activity. Especially the latter analysis, made it possible to quantify effects of the engineering within the processing part by respective enzyme kinetic parameters. Expanding our focus beyond a singular module, we have utilized the mFAS fold for designing up to 380 kDa large bimodular constructs. In this approach, a loading didomain was attached N-terminally containing an additional MAT and acyl carrier protein (ACP) domain. Two constructs could be expressed and purified in excellent quality to investigate the influence of an altered overall architecture on fatty acid synthesis. By comparison with appropriate controls, a functional effect of the additional loading module could indeed be proven in the bimodular systems. Those constructs allow a comprehensive analysis of the underlying molecular mechanism in the future and serve as a potential model system to study the transition from iterative to vectorial polyketide synthesis in vitro.
Polyketide synthases (PKSs) are large megaenzymes that occur in bacteria, fungi, and plants and produce polyketides, a class of secondary metabolites. Many polyketide natural products exhibit high biological activities e.g. as antibiotics or anti-fungal compounds. The modular architecture of assembly line PKSs makes them exciting targets for engineering approaches via the exchange of whole modules or single domains. Although many engineering attempts have been pursued over the last three decades, the resulting chimeric PKSs often exhibit decreased turnover rates or diminished product yields.
In this thesis, new approaches to engineer chimeric PKSs were explored, each targeting a different aspect of the chimeric system: First the relative contribution of protein-protein and protein-substrate recognition on the turnover of chimeric PKS was assessed, revealing the importance of protein-protein interactions between the acyl carrier protein (ACP) and the ketosynthase (KS) domain in the chain translocation step. Directed evolution experiments followed to optimize the protein-protein interaction across a chimeric interface. Additionally, different junction sites for the generation of chimeric PKSs were compared, showing the ability for recombination without interfering with the chain translocation reaction, and highlighting the use of SYNZIP domains to bridge PKS modules. To optimize chimeric PKSs even further, multipoint mutagenesis of KS domains was established, with positive effects on the activity of chimeric systems.
To support engineering attempts, several structure elucidation techniques were combined with in silico modeling to characterize the architecture of a PKS module and the domain-domain interactions within it. Preliminary results show a strong conformational flexibility of the PKS module and the great potential of these techniques to define the multitude of transient interactions in PKS modules.