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Nearly 170 million people are chronically infected with HCV and thus at risk of developing liver cirrhosis and hepatocellular carcinoma. Although new and effective oral antiviral drugs are available, there is still the need for a preventive vaccine. In addition, in light of the high number of patients who are chronically infected with HCV the development of a therapeutic vaccine will present a support or even an alternative to the expensive medications.
To induce HCV-specific immune responses in a vaccine model, the HBV capsid is used as a carrier to deliver HCV antigens. Due to its icosahedral structure, the HBV capsid is highly immunogenic and helps to elicit a strong B cell response against the delivered antigens. In addition, the translocation motif (TLM) from the HBV surface protein is fused to the core protein. The TLM conveys membrane-permeability to the carrier capsid, enabling antigen transfer into the cytoplasm, and thus allows immunoproteasomal processing and MHC class I-mediated presentation of the antigen. To load the capsid with foreign antigens, a strep-Tag/streptavidin system is utilized. Recombinant capsids and antigens were purified from the E. coli production system. Detailed characterization of the carrier capsid demonstrated the proper assembly, adequate thermal stability and the successful loading of the foreign antigens onto the capsid surface.
As a further step, seven different HCV-derived proteins were produced and purified for the coupling on the surface of TLM-core particles. The characterization of their immunogenicity using this system is being performed.
Using ovalbumin as a model antigen, which is coupled to the carrier capsids via strep-Tag/streptavidin binding, shows that this system is suitable to efficiently deliver antigens into the cytoplasm of antigen-presenting cells (APCs), leading to the activation of APCs. This activation was assessed by measuring the secretion of IL-6 and TNF-α, in addition to the upregulation of activation markers (CD40, CD80, CD69, and MHC class I). Upon activation, the APCs were able to activate ova-specific CD8+ T cells measured by secreted IFN-γ, which was up to 20-folds more than IFN-γ secreted upon incubation with free ovalbumin. These data indicate that the TLM-capsid is suitable to serve as a carrier to deliver foreign antigens into the cytoplasm of APCs leading to MHC class I-mediated presentation and induction of an antigen-specific CTLs response.
This thesis describes the adaptation of Acinetobacter species to dry environments with the soil bacterium A. baylyi and the opportunistic hospital pathogen A. baumanii in its focus. The adaptation of A. baylyi and A. baumannii to osmotic stress was investigated. Compatible solutes that were uptaken from the environment or synthesized de novo to cope with the loss of water at high salinity were identified. The corresponding transporters and enzymes involved were characzerized. In addition, the desiccation resistance of A. baumannii was analyzed to elucidate its survival in hospital environments. The usage of compatible solutes during desiccation stress was analyzed and proteins that were produced were identified.
The availability of water is essential for bacterial life and if environmental conditions are awkward, bacteria have to cope with high salinitiy to prevent loss of water. In this thesis it was shown that A. baylyi synthesizes glutamate and mannitol de novo as compatible solutes in response to osmotic stress to balance the osmotic potential. The pathway for mannitol biosynthesis from Fructose-6-Phosphate (F-6-P) via Mannitol-1-Phosphate (Mtl-1-P) was elucidated and the isolation and characterization of a novel type of biofunctional enzyme was described. Interestingly, the unique bifunctional enzyme MtlD, acting as dehydrogenase and phosphatase, mediates both steps of the mannitol biosynthesis pathway. This enzyme catalyzes the reduction of F-6-P to Mtl-1-P with NADPH as reducing equivalent. The dehydrogenase activity of MtlD was salt dependent and the phosphatase activity was dependent on Mg2+ as cofactor. Phylogenetic analyses revealed that MtlD is broadly distributed among other Acinetobacter strains but not in other phylogenetic tribes.
In this thesis it is also described that, besides de novo synthesis of compatible solutes, A. baylyi takes up glycine betaine (GB) or its precursor choline by different transport systems and uses this solutes as osmoprotectants. The uptake of GB occurs via a secondary transporter (ACIAD3460) of the BCCT family. Choline is taken up as precursor and oxidized to GB by two dehydrogenases. The uptake and use of choline as GB precursor involves two transporters, whose genes are encoded in the bet cluster (BetT1, BetT2), two dehydrogenases (BetA, BetB) and a regulatory protein (BetI). Both transporters differ from each other in structure and function: BetT1 is osmo-independent and active independently of osmotic stress. BetT2 contains - in contrast to BetT1 - a long C-terminal domain for osmo-sensing and its activity highly increases in the presence of high osmolarity. The oxidation of choline occurs independently of the osmolarity of the medium but in the absence of salt stress, GB is exported. In contrast, in the presence of high salinity, GB is accumulated in the cytoplasm to balance the osmotic potential in order to prevent loss of water. The regulation of both transporters, the uptake of choline independently of the osmolarity and the export of GB under isoosmotic conditions are regulated by the transcriptional regulator BetI.
A. baumannii ATCC 19606 was also shown to cope with high salinity. Analogously to A. baylyi, A. baumannii ATCC19606 synthesizes glutamate and mannitol de novo in response to osmotic stress. The genes for the synthesis of these compatible solutes are identical to those found in A. baylyi. This suggests that the solute biosynthesis pathways of A. baumannii and A. baylyi are identical. A. baumannii was also able to take up GB and choline in response to osmotic stress and growth at high salinity was restored upon addition of GB and its precursor choline. The bet cluster was also present in the genome A. baumannii and also contains the two different choline transporters BetT1 and BetT2.
Our suggestion that choline or GB or the utilization of phosphatidylcholine as carbon source led to an increase in the survival under desiccation stress was not confirmed. However, 2D analysis of proteins produced during desiccation stress in A. baumannii led to elevated amounts of proteins implicated in biofilm formation, regulation, cell morphology and general stress response, such as Hsp60 or superoxide dismutase, both might play a role in general stress protection.
In den vergangenen Jahren haben ökologische Fragen in der Naturstoffforschung mehr und mehr an Bedeutung gewonnen. Naturstoffe bilden dabei einen wichtigen Aspekt in der Aufrechterhaltung symbiotischer Systeme.
Symbiosen stellen eine der treibenden Kräfte der Evolution dar. Diese artenübergreifende Interaktion zweier Organismen ermöglicht die Evolution in wechselseitiger Anpassung, wobei per Definition in die Kategorien Mutualismus, Kommensalismus und Parasitismus unterschieden wird. Teilweise führt die obligatorische Abhängigkeit eines Organismus zum partiellen Merkmals- und Stoffwechselwegverlust, der durch seinen Symbiose-Partner kompensiert wird. In den meisten Fällen stellt Symbiose ein komplexes Netzwerk aus mehr als zwei Lebewesen dar.
Diese Arbeit beschreibt die Anwendung der Klonierungsmethode ExRec ("overlap extension PCR-yeast homologous recombination") für die vereinfachte Bereitstellung von Naturstoffen. Es konnte ein 45 kb großes Gencluster erfolgreich kloniert und zwei neue Peptide Ambactin und Xenolindicin aus Xenorhabdus charakterisieren werden, wobei letztgenanntes von einem stillen Gencluster stammt. ExRec stellt eine sehr effiziente und wichtige Methode für die Klonierung großer Gencluster als auch für die Klonierung aus Metagenombibliotheken und RNA Pools dar...
As fossil resources are diminishing, environmental concerns arise and chemical synthesis often involves expensive catalysts or extensive extraction procedures, the demand for production of industrially relevant compounds from renewable resources increases. In this context, engineering microorganisms for production of specialty chemicals, such as 3-alkylphenols, presents an attractive, environmental-friendly approach. 3-alkylphenols have various applications: due to their antiseptic and stabilizing properties many 3-alkylphenols, including 3-methylphenol (3-MP), are utilized as additives in disinfectant reagents and biological products, while they can be also implemented as platform chemicals for production of lubricating oil additives or flavors. Some 3-akylphenols have potential for transmission control of the disease sleeping sickness that is transmitted by tsetse flies in sub-saharan Africa, since 3-ethylphenol (3-EP) and 3-propylphenol (3-PP) and to a lesser degree 3-MP were found to attract tsetse flies and improved catch rates in impregnated tsetse fly traps. Microbial fermentation of 3-alkylphenols would provide a simple and inexpensive way for local communities in Africa to produce these compounds and prepare their own tsetse fly traps.
Some molds synthesize 3-MP as an intermediate during biosynthesis of the mycotoxin patulin. However, the heterologous host Saccharomyces cerevisiae has advantageous traits for industrial application, since it is well characterized, robust, simple to handle and easily genetically accessible. In this thesis, genetical engineering approaches were utilized to establish the yeast S. cerevisiae for biotechnological production of 3-alkylphenols. As a proof of concept, the iterative polyketide synthase from Penicillium patulum, 6-methylsalicylic acid synthase (MSAS), and 6-methylsalicylic acid (6-MSA) decarboxylase PatG from Aspergillus clavatus were heterologously expressed in S. cerevisiae resulting in the first reported de novo biosynthesis of 3-MP via 6-MSA in yeast from sugars (Hitschler & Boles, 2019). It was shown that codon-optimization and genomic integration of heterologous genes, high initial cell densities and a balanced expression of PatG were beneficial for heterologous production of up to 589 mg/L 3-MP in S. cerevisiae. However, toxicity of 3-MP limited higher product accumulation.
Different in vivo detoxification strategies were implemented to face this bottleneck. Growth tests revealed that 3-methylanisole (3-MA) is less toxic to the yeast cells than 3-MP. Expression of an orcinol-O-methyltransferase from chinese rose hybrids (OOMT2) was combined with in situ extraction converting the toxic 3-MP product into the volatile 3-MA and accumulating up to 211 mg/L 3-MA in the dodecane phase. Alternatively, up to 533 mg/L 3-MP glucoside were synthesized by expression of a UDP-glycosyltransferase (UGT72B27) from Vitis vinifera in the 3-MP producing strain, revealing saccharose as beneficial carbon source and ethanol growth phase as essential for high 3-MP production, although 3-MP conversions were not yet complete. Both detoxification strategies allowed circumvention of the toxicity imposed limited product accumulation. This was demonstrated when both detoxification strategies were combined with redirection of the carbon flux through deletion of phosphoglucose isomerase gene PGI1 and feeding a mixture of fructose and glucose leading to majorly improved product formation, with up to 899 mg/L 3-MA/3-MP and 873 mg/L 3-MP/3-MP glucoside, compared to less than 313 mg/L product titers in the wild type controls (Hitschler & Boles, 2020).
For provision of the tsetse fly attractants 3-EP from propionyl-CoA and 3-PP from butyryl-CoA, the substrate promiscuities of MSAS and PatG were exploited. However, slower formation rates with the alternative substrates propionyl-CoA and butyryl-CoA suggested that competing formation of 6-MSA from the preferred priming unit acetyl-CoA was dominating in vivo. Indeed, 3-EP or 3-PP formation was not observed in 3-MP producing yeast strains. Assuming that intracellular levels of propionyl-CoA and butyryl-CoA were limiting 3-EP and 3-PP formation, different strategies were implemented to raise the supply of these alternative priming units and successfully compete with acetyl-CoA for MSAS priming.
Supplementation of propionate increased propionyl-CoA levels by endogenous pathways sufficiently to enable 3-EP formation in yeast mediated by MSAS and PatG. Deletion of the 2-methylcitrate synthases CIT2 and CIT3 revealed that degradation of propionyl-CoA was not limiting 3-EP formation at this stage. In order to raise propionyl-CoA levels further, a heterologous propionyl-CoA synthase (PrpE) was expressed in the 3-MP producing yeast strain leading to up to 12.5 mg/L 3-EP with propionate feeding and blockage of degradation. Moreover, PrpE enabled also 3-EP formation without propionate supplementation suggesting that an endogenous supply of propionate existed that was reactivated by PrpE. As threonine or 2-ketobutyrate feeding increased 3-EP titers in combination with PrpE, this indicated that threonine degradation via 2-ketobutyrate was responsible for the endogenous propionate supply. Moreover, expression of branched-chain ketoacid dehydrogenase complex from Pseudomonas putida combined with PrpE provided propionyl-CoA from endogenous 2-ketobutyrate and raised 3-EP titers up to 5.9 mg/L compared to 2.8 mg/L with only PrpE indicating a potential route for optimization of 3-EP titers independent of propionate or threonine feeding.
For 3-PP production from butyryl-CoA, a heterologous ‘reverse ß-oxidation’ pathway was introduced in the 3-MP producing yeast strain providing sufficient butyryl-CoA for biosynthesis of up to 2 mg/L 3-PP. Degradation of the precursor via ß-oxidation was slightly limiting, since deletion of fatty acyl-CoA oxidase POX1 increased 3-PP titers slightly to 2.6 mg/L.
As the concentrations of 3-alkylphenols are close to the concentrations implemented in tsetse fly traps, the engineered yeast strains have the potential for simple and inexpensive on-site production of 3-alkylphenols as tsetse fly attractants by local rural communities in Africa. In spite of this success, 3-MP remained the main product in the developed yeast strains. Since 3-EP and 3-PP are more efficient tsetse fly attractants, a shift in substrate specificities of MSAS and PatG is desirable for a more favorable 3-EP/3-MP and 3-PP/3-MP product ratio regarding tsetse fly attraction. During rational engineering of MSAS, the MSASQ625A/I752V mutant showed a beneficial shift of product ratios with up to 11 mg/L 3-EP/63 mg/L 3-MP and 4.5 mg/L 3-PP/116 mg/L 3-MP, compared to a higher proportion of 3-MP with up to 343 mg/L, 11 mg/L 3-EP and 1.5 mg/L 3-PP in the wild type controls. Further engineering of MSAS and PatG might majorly improve production of 3-EP and 3-PP.
In summary, this thesis successfully established the yeast S. cerevisiae as cell factory for production of different 3-alkylphenols optimizing expression of the heterologous production pathway, elucidating means to detoxify products and establishing different approaches to increase intracellular levels of acyl-CoA precursors. The engineered yeast strains can be potentially implemented for simple and inexpensive fermentation of tsetse fly attractants in Africa.
Octanoic acid (C8 FA) is a medium-chain fatty acid which, in nature, mainly occurs in palm kernel oil and coconuts. It is used in various products including cleaning agents, cosmetics, pesticides and herbicides as well as in foods for preservation or flavoring. Furthermore, it is investigated for medical treatments, for instance, of high cholesterol levels. The cultivation of palm oil plants has surged in the last years to satisfy an increasing market demand. However, concerns about extensive monocultures, which often come along with deforestation of rainforest, have driven the search for more environmentally friendly production methods. A biotechnological production with microbial organisms presents an attractive, more sustainable alternative.
Traditionally, the yeast Saccharomyces cerevisiae has been utilized by mankind in bread, wine, and beer making. Based on comprehensive knowledge about its metabolism and genetics, it can nowadays be metabolically engineered to produce a plethora of compounds of industrial interest. To produce octanoic acid, the cytosolic fatty acid synthase (FAS) of S. cerevisiae was utilized and engineered. Naturally, the yeast produces mostly long-chain fatty acids with chain lengths of C16 and C18, and only trace amounts of medium-chain fatty acids, i.e. C8-C14 fatty acids. To generate an S. cerevisiae strain that produces primarily octanoic acid, a mutated version of the FAS was generated (Gajewski et al., 2017) and the resulting S. cerevisiae FASR1834K strain was utilized in this work as a starting strain.
The goal of this thesis was to develop and implement strategies to improve the production level of this strain. The current mode of quantification of octanoic acid includes labor-intensive, low-throughput sample preparation and measurement – a main obstacle in generating and screening for improved strain variants. To this end, a main objective of this thesis was the development of a biosensor. The biosensor was based on the pPDR12 promotor, which is regulated by the transcription factor War1. Coupling pPDR12 to GFP as the reporter gene on a multicopy plasmid allowed in vivo detection via fluorescence intensity. The developed biosensor enabled rapid and facile quantification of the short- and medium-chain fatty acids C6, C7 and C8 fatty acids (Baumann et al., 2018). This is the first biosensor that can quantify externally supplied octanoic acid as well as octanoic acid present in the culture supernatant of producer strains with a high linear and dynamic range. Its reliability was validated by correlation of the biosensor signal to the octanoic acid concentrations extracted from culture supernatants as determined by gas chromatography. The biosensor’s ability to detect octanoic acid in a linear range of 0.01-0.75 mM (≈1-110 mg/L), which is within the production range of the starting strain, and a response of up to 10-fold increase in fluorescence after activation was demonstrated.
A high-throughput FACS (fluorescence-activated cell sorting) screening of an octanoic acid producer strain library was performed with the biosensor to detect improved strain variants (Baumann et al., 2020a). For this purpose, the biosensor was genomically integrated into an octanoic acid producer strain, resulting in drastically reduced single cell noise. The additional knockout of FAA2 successfully prevented medium-chain fatty acid degradation. A high-throughput screening protocol was designed to include iterative enrichment rounds which decreased false positives. The functionality of the biosensor on single cell level was validated by adding octanoic acid in the range of 0-80 mg/L and subsequent flow cytometric analysis. The biosensor-assisted FACS screening of a plasmid overexpression library of the yeast genome led to the detection of two genetic targets, FSH2 and KCS1, that in combined overexpression enhanced octanoic acid titers by 55 % compared to the parental strain. This was the first report of an effect of FSH2 and KCS1 on fatty acid titers. The presented method can also be utilized to screen other genetic libraries and is a means to facilitate future engineering efforts.
In growth tests, the previously reported toxicity of octanoic acid on S. cerevisiae was confirmed. Different strategies were harnessed to create more robust strains. An adaptive laboratory evolution (ALE) experiment was conducted and several rational targets including transporter- (PDR12, TPO1) and transcription factor-encoding genes (PDR1, PDR3, WAR1) as well as the mutated acetyl-CoA carboxylase encoding gene ACC1S1157A were overexpressed or knocked out in producer or non-producer strains, respectively. Despite contrary previous reports for other strain backgrounds, an enhanced robustness was not observable. Suspecting that the utilized laboratory strains have a natively low tolerance level, four industrial S. cerevisiae strains were evaluated in growth assays with octanoic acid and inherently more robust strains were detected, which are suitable future production hosts.
...
Photorhabdus and Xenorhabdus bacteria live in a highly specific symbiosis with nematodes that belong to the genus of Heterorhabditis and Steinernema, respectively. These cruiser type nematodes actively search for soil-dwelling insects and infect them via natural openings. Inside of the insect, the bacteria are released into the hemocoel where they start producing an array of secondary metabolites to bypass the insect immune system and kill the prey within 48 hours. Many of those natural products possess bioactivities against other bacteria, fungi, protozoa or insects, which makes them interesting candidates for pharmaceutical applications. Even though advanced molecular biological methods in combination with bioinformatics tools can now be used to predict biosynthetic gene clusters (BGCs) and their products, there are still many BGCs with unknown products. Even for the plethora of natural products that were successfully identified in the last couple of years, the exact ecological function often remains elusive, as laboratory conditions can vary considerably from the natural environment of the bacteria. Knowledge about the natural conditions that stimulate, or repress production of certain natural products and their underlying regulatory mechanisms yield new approaches for natural product research and enables possibilities for selective manipulations of the regulatory cascades.
The overarching goal of this work was to examine the regulatory networks in Photorhabdus and Xenorhabdus strains. The first part of this work focused on the Hfq-dependent regulation of specialized metabolite production. In those genera, the RNA chaperone, Hfq, represses expression of hexA, which encodes for a global transcriptional regulator that acts as the master repressor for SM production. Multiple global approaches were used to identify the sRNA ArcZ, which targets a specific region in the 5’-untranslated region of the hexA mRNA and ultimately guides Hfq in order to repress its expression. It was shown that a deletion of arcZ led to a drastic reduction of SM production in Photorhabdus and Xenorhabdus, consistent with the phenotype of their respective hfq deletion mutants. Transcriptomic profiling revealed far-reaching effects on the transcriptome, with up to 735 coding sequences significantly affected in the arcZ deletion strain. Finally, it was shown that the resulting chemical background, devoid of SMs, in combination with targeted promotor exchange can be used to exclusively overproduce a desired natural product, representing an alternative route of genetic manipulation.
The second part of this work focused on the influence and identification of insect related compounds that affect SM production in P. laumondii, X. szentirmaii and X. nematophila. Insect homogenate was generated from G. mellonella larvae, a model host for these bacteria. Supplementation of the cultivation medium with homogenate induced considerable shifts in the SM profiles of those bacteria. A global effect on the transcriptional output was determined by transcriptomic profiling. The core response to the simulation of an insect environment consisted of ten CDS, eight of which are involved in the degradation of fatty acids or the import of maltose and maltodextrin into the cells. Two abundant components in the insect homogenate, trehalose and putrescin, were added to the cultivation medium of those strains and subsequent HPLC-MS analysis revealed a direct correlation of their concentration in the medium and the production titres of certain SMs. These results indicated that the bacteria sense the insect environment via different insect specific components in order to initiate a metabolic adjustment, which is probably required for adaptation to the insect host.
The last part of this work examined the influence of other, so far not directly related genes on SM production, based on the isolation of P. laumondii transposon-insertion mutants with clear phenotypic alterations. Re-sequencing and SM profiling of the mutant strains revealed that a transposon-insertion in the gene encoding for a putative DNA-adenine methyltransferase affected SM production. The phenotype was confirmed by deleting this gene. Based on Single-Molecule Real-Time sequencing, the complete methylome of the WT, deletion- and complementation mutant were analysed (experimental work performed by Sacha J. Pidot, Melbourne, Australia). No obvious alterations were detected in the methylation patterns of the strains, indicating that the dam gene product does not methylate the adenine in GATC-motifs, as it was described in literature for E. coli. This data raises the question what the function of the putative DNA-adenine methyltransferase is in P. laumondii and how it can influence the secondary metabolism. Even though there is currently no clear evidence, the potential role of epigenetic gene regulation mechanisms should be considered in further work.
Die Substitution von klassischen, mit der Nahrungsmittelproduktion in Konkurrenz stehenden, Substraten wie Glukose durch alternative Kohlenstoffquellen in der Biotechnologie ist sowohl aus ethischer, als auch aus ökonomischer Sicht erstrebenswert. Diese Arbeit beschreibt die Synthese von Bulkchemikalien in Form zweier Dicarboxylsäuren und einer Feinchemikalie in Form eines Sesquiterpens aus dem alternativen Substrat Methanol mit Hilfe genetisch veränderter Stämme des methylotrophen α-Proteobakteriums Methylobacterium extorquens.
Mesacon- und (2S)-Methylsuccinsäure sind Dicarboxylsäurederivate der CoA-Ester Mesaconyl- und (2S)-Methylsuccinyl-CoA, die als Intermediate im Ethylmalonyl-CoA- Weg (EMCP) vorkommen. M. extorquens nutzt den EMCP für die Regeneration von Glyoxylat, das für das Wachstum auf C1-Substraten wie Methanol obligatorisch ist. In dieser Arbeit konnte erstmals Mesacon- und (2S)-Methylsuccinsäure de novo durch die Expression einer für die Vorstufen Mesaconyl- und (2S)-Methylsuccinyl-CoA aktiven Thioesterase produziert werden. Ein kobaltlimitiertes Wachstum von M. extorquens führte aufgrund mangelnder Cofaktorversorgung zweier Vitamin-B12-abhäniger Mutasen im EMCP zu einer Akkumulation der beiden CoA-Ester-Vorstufen, womit eine Produktion von 0.65 g/l Mesacon- und (2S)-Methylsuccinsäure erreicht wurde. Weitergehende Untersuchungen belegten außerdem einen positiven Effekt eines ausgeschalteten PHB-Zyklusses auf die Produktion der beiden EMCP- Dicarboxylsäurederivate.
Diese Arbeit beinhaltet zusätzlich grundlagenwissenschaftliche Untersuchungen zur Substitution der EMCP-katalysierten Glyoxylatregeneration durch einen heterologen Glyoxylatzyklus in EMCP-negativen M. extorquens-Stämmen. Dabei konnte erstmals ein methanolverwertendes, methylotrophes Bakterium identifiziert werden, das einen Serin-Zyklus in Kombination mit dem Glyoxylat-Zyklus zur Kohlenstoffassimilation verwendet, ohne dabei zusätzliche Stoffwechselwege zur CO2-Fixierung wie den EMCP, RuMP oder CBB-Zyklus zu verwenden.
Die Präsenz einer nativen C30-Carotinoidbiosynthese, ausgehend von der Vorstufe Farnesylpyrophosphat (FPP), empfiehlt M. extorquens als Produktionsorganismus für (Sesqui-)Terpene. In dieser Arbeit wurde mit Hilfe einer induzierbar gesteuerten Expression einer Terpensynthase in Form einer α-Humulen-Synthase, einer FPP-Synthase und eines prokaryontischen Mevalonatweges, erstmals die de novo Synthese eines Terpens aus Methanol am Beispiel des α-Humulens etabliert. Durch optimierte Expressionen der Terpensynthase, FPPS und einzelner MVA-Gene mit Hilfe angepasster Translationsinitiationsraten der jeweiligen ribosomalen Bindestellen und der Verwendung eines in der nativen Carotinoidbiosynthese inhibierten M. extorquens-Stammes wurden finale Produkttiter von bis zu 1.65 g/l α-Humulen in Fed-Batch-Fermentationen erreicht.
Diese kumulative Dissertation beinhaltet außerdem einen Reviewartikel, in dem der verwendete Mikroorganismus M. extorquens in mikrobiologischer, genetischer, biochemischer und auch biotechnologischer Hinsicht ausführlich beschrieben wird. Zudem gibt ein Buchkapitel eine Übersicht über die Verwendung von Methanol in der Biotechnologie.
Metabolic engineering can serve to convert microorganisms to
microbial cell factories with the goal of producing various chemicals. Commonly used strategies to modify metabolic pathways include deletions and overexpression of genes, as well as the introduction of heterologous genes or genes which have been optimized for the host organism or for a reaction of interest. Aside from these classic metabolic engineering strategies, researchers have also implemented pathway compartmentalization strategies, which mimic nature’s strategies of colocalizing enzymes for pathway optimization.
In this thesis, classic metabolic engineering strategies were combined with pathway compartmentalization strategies. For pathway compartmentalization, mitochondria and peroxisomes were harnessed, and additionally a new strategy to create artificial subcellular organelles was evaluated. In the latter approach, the so-called Zera peptide was fused to the enzymes of interest. Zera consists of the first 113 amino acids of the plant storage protein γ-Zein (Zea mays). Natively, plant storage proteins accumulate in endoplasmic reticulum (ER)-derived vesicles in plant seeds and serve as an amino acid source for the germinating plant. In this thesis, it was shown that Zera also induces the formation of artificial, ER-derived vesicles in Saccharomyces cerevisiae. Furthermore, it was shown that Zera fusion enzymes remained active, albeit with sometimes reduced activity.
In line with the goal of compartmentalizing pathways in these artificial, Zera-induced vesicles, a new tool was developed to determine the pH in the ER of S. cerevisiae and in the ER-derived vesicles. pHluorin, a pH-sensitive green fluorescent protein (GFP) variant, is commonly used to analyze the cytosolic pH or the pH of subcellular organelles. In this thesis, it was shown that pHluorin has very low fluorescence intensity and pH sensitivity in the ER and in Zera-induced ER-derived vesicles. Therefore, a superfolder variant of pHluorin was developed which allows reliable pH measurements in these compartments and can be used to analyze whether the organellar or vesicular pH suits a pathway of interest....
The oleochemical and petrochemical industries provide diverse chemicals used in personal care products, food and pharmaceutical industries or as fuels, oils, polymers and others. However, fossil resources are dwindling and concerns about these conventional production methods have risen due to their strong negative impact on the environment and contribution to climate change.
Therefore, alternative, sustainable and environmentally friendly production methods for oleochemical compounds such as fatty acids, fatty alcohols, hydroxy fatty acids and dicarboxylic acids are desired. The biotechnological production by engineered microorganism could fulfill these requirements. The concept of metabolic engineering, which is the modification of metabolic pathways of a host organism for increased production of a target compound, is a widely used strategy in biotechnology to generate cell factories or chassis strains for robust, efficient and high production. In this work, the versatile model and industrial yeast Saccharomyces cerevisiae was manipulated by metabolic engineering strategies for increased production of the medium-chain fatty acid octanoic acid and de novo production the derived 8-hydroxyoctanoic acid.
Octanoic acid production was enabled by the fatty acid biosynthesis pathway by use of a mutated fatty acid synthase (FASRK) in a wild type FAS deficient strain. The yeast fatty acid synthase (FAS) consists of two polypeptides, α and β, which assemble to a α6β6 complex in a co-translational manner by interaction of the subunits. Because this step might be subject to cellular regulation, the α- and β- subunits of fatty acid synthase were fused to form a single-chain construct (fusFASRK), which displayed superior octanoic acid production compared with split FASRK. Thus, FASRK expression was identified as a limiting step of octanoic acid production. But the strains that produce octanoic acid have a severe growth defect that is undesirable for biotechnological applications and could lead to lower production titers. One reason is the strong
inhibitory effect of octanoic acid. Another possibility is that the mutant FAS no longer produces enough essential long-chain fatty acids. To compensate for this, the mutated split and fused FAS variants were co-expressed individually in a strain harboring genomic wild type FAS alleles. In
addition, mutant and wild type variants of fused and split FAS were co-expressed together in a FAS deficient strain. However, both cases resulted in decreased octanoic acid titers potentially by physical and/or metabolic crosstalk of the FAS variants.
The fatty acid biosynthesis relies on cytosolic acetyl-CoA for initiation and derived malonyl-CoA for elongation and requires NADPH for reductive power. To increase production of octanoic acid, engineering strategies for increased acetyl-CoA and NADHP supply were investigated. First, the flux through the native cytosolic acetyl-CoA and NADPH providing pyruvate dehydrogenase bypass was enhanced by overexpression of the target genes ADH2, ALD6 and ACSL461P from Salmonella enterica in combination or individually. Next, the acety-CoA forming heterologous phosphoketolase/phosphotransacetylase pathway was expressed and NADPH formation was increased by redirecting the flux of glucose-6-phosphate into the NADPH producing oxidative branch of the pentose phosphate pathway. In particular, the flux through glycolysis and pyruvate dehydrogenase bypass was reduced by downregulating the expression of the phosphoglucose isomerase PGI1 and deleting the acetaldehyde dehydrogenase ALD6. Glucose-6-phosphate was guided into the pentose phosphate pathway by overexpressing the glucose-6-phosphate dehydrogenase ZWF1. The first approach did not influence octanoic acid production but the latter increased yields in the glucose consumption phase by 65 %. However,
combining the superior fusFASRK with acetyl-CoA and NADPH supply engineering strategies did not result in additive production effects, indicating that other limitations hinder high octanoic acid accumulation. Limitations could be caused in particular by the strong inhibitory effects of octanoic acid or by intrinsic limitations of the FASRK mutant. To enlarge the octanoic acid production platform towards other derived valuable oleochemical compounds the de novo production of 8-hydroxyoctanoic acid was targeted. Since short- and medium-chain fatty acids have a strong inhibitory effect on Saccharomyces cerevisiae, the inhibitory effect of hydroxy fatty acid and dicarboxylic with eight or ten carbon atoms were compared and revealed only little or no growth impairment. Subsequently, the formation of 8-hydroxyoctanoic acid was targeted by a terminal hydroxylation of externally supplied octanoic acid in a bioconversion. For that, three heterologous genes, encoding for cytochromes P450 enzymes and their cognate cytochrome P450 reductases were expressed and 8-hydroxyoctanoic acid production was compared. In addition, the use of different carbon sources was compared.
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Investigating the influence of truffle´s microbiome and genotype on the aroma of truffle fungi
(2019)
Truffles (Tuber spp.) are belowground forming fungi that develop in association with roots of various host trees and shrubs. Their fruiting bodies are renowned for their enticing aromas which vary considerably, even within truffles of the same species. This aroma variability might be attributed to factors such as geographical origin, degree of fruiting body maturation, truffle genotype and microbiome (microbial communities that colonise truffle fruiting bodies) which often co-vary. Although the influence of specific factors is highlighted by several studies, discerning the contribution of each factor remains a challenge since it requires an appropriate experimental design. The primary purpose of this thesis was to gain insight into the influence of truffle’s genotype and microbiome on truffle aroma.
This doctoral thesis is comprised of four chapters. Chapter1 (Vahdatzadeh et al., 2018) aimed to exclusively elucidate the influence of truffle genotype on truffle aroma by investigating the aroma of nine mycelial strains of the white truffle Tuber borchii. We also assessed whether strain selection could be employed to improve the human- perceived truffle aroma. Quantitative differences in aroma profiles among strains could be observed upon feeding of amino acids. Considerable aroma variabilities among strains were attributed to important truffle volatiles, many of which might be derived from amino acid catabolism through the Ehrlich pathway. 13 C-labelling experiments confirmed the existence of the Ehrlich pathway in truffles for leucine, isoleucine, methionine, and phenylalanine. Sensory analyses further demonstrated that the human nose can differentiate among strains. Our results illustrated the influence of truffle genotype on truffle aroma and showed how strain selection could be used to improve the human-perceived truffle aroma.
In chapter 2 the existing knowledge on the composition of bacterial community of four truffle species was compiled using meta-analysis approach (Vahdatzadeh et al., 2015). We highlighted the endemic microbiome of truffle as well as similarities and differences in the composition of microbial community within species at various phases of their life cycle. Furthermore, the potential contribution of truffle microbiome in the formation of truffle odorants was studied. Our findings showed that truffle fruiting bodies harbour complex microbial community composed of bacteria, yeasts, filamentous fungi, and viruses with bacteria being the dominant group. Regardless of truffle species, the composition of endemic microbiome of fruiting bodies appeared very similar and was dominated by α-Proteobacteria class. However, striking differences were observed in the bacterial community composition at various stages of the life cycle of truffle.Our analyses further suggested that odorants common to many truffle species might be produced by both truffle fungi and microbes, whereas specific truffle odorants might be derived from microbes only. Nevertheless, disentangling the origin of truffle odorants is very challenging, since acquiring microbe-free fruiting bodies are currently not possible.
Chapter 3 (Splivallo et al., 2019) further characterises truffle-associated bacterial communities of fruiting bodies of the black truffle T. aestivum from two different orchards. It aimed at defining the native microbiome in this truffle species, evaluating the variability of their microbiome across orchards, and assessing factors that shape assemblages of the bacterial communities. The dominant bacterial communities in T. aestivum revealed to be similar in both orchards: although a large portion of fruiting bodies were dominated by the α-Proteobacteria class (Bradyrhizobium genus) similar to other so far-assessed truffle species, in few cases β-Proteobacteria (Polaromonas genus), or Sphingobacteria (Pedobacter genus) were found to be predominant classes. Moreover, factors shaping bacterial communities influenced the two orchards differently, with spatial location within the orchard being the main driver in Swiss orchard and collection season in the French one. Surprisingly, in contrast to other fungi, truffle genotype and the degree of fruiting body maturity seemed not to contribute in shaping the assembly of truffle microbiome. Altogether, our data highlighted the existence of heterogeneous bacterial communities in T. aestivum fruiting bodies which are dominated by either of the three bacterial classes and mainly by the α-Proteobacteria class, irrespective of geographical origin. They further illustrated that determinants driving the assembly of various bacterial communities within truffle fruiting bodies are site-specific. Truffles are highly perishable delicacies with a short shelf life (1-2 weeks), and their aroma changes profoundly upon storage. Since truffle aroma might be at least partially produced by the truffle microbiome, chapter 4 (Vahdatzadeh et al., 2019) focuses on assessing the influence of the truffle microbiome on aroma deterioration of T.aestivum during post harvest storage. Specifically, volatile profile and bacterial communities of fruiting bodies collected from four different regions (three in France and one in Switzerland) were studied over nine days of storage. Our findings demonstrated the gradual replacement of dominant bacterial classes in fresh truffles (α-Proteobacteria, β-Proteobacteria, and Sphingobacteria) by food spoilage bacteria (members of γ- Proteobacteria and Bacilli classes), regardless of the initial diversity of the bacterial classes. This shift in the bacterial community also correlated with changes in volatile profiles, and markers for truffle freshness and spoilage could be identified. Ultimately, network analysis illustrated possible links among those volatile markers and specific bacterial classes. Our data showed that storage deeply influenced the composition of bacterial community as well as aroma of truffle fruiting bodies. They also illustrated the correlation between the shift in truffle microbiome, from commensal to detrimental, and the change of aroma profile, possibly leading to the loss of fresh truffle aroma. Overall, the work undertaken in this thesis demonstrated that truffle genotype and microbiome had a stronger influence on truffle aroma than previously believed.