Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Asgard archaea capable of anaerobic hydrocarbon cycling
Seitz, Kiley W. ; Dombrowski, Nina ; Eme, Laura ; Spang, Anja ; Lombard, Jonathan ; Sieber, Jessica R. ; Teske, Andreas P. ; Ettema, Thijs J.G. ; Baker, Brett J. - \ 2019
Nature Communications 10 (2019). - ISSN 2041-1723

Large reservoirs of natural gas in the oceanic subsurface sustain complex communities of anaerobic microbes, including archaeal lineages with potential to mediate oxidation of hydrocarbons such as methane and butane. Here we describe a previously unknown archaeal phylum, Helarchaeota, belonging to the Asgard superphylum and with the potential for hydrocarbon oxidation. We reconstruct Helarchaeota genomes from metagenomic data derived from hydrothermal deep-sea sediments in the hydrocarbon-rich Guaymas Basin. The genomes encode methyl-CoM reductase-like enzymes that are similar to those found in butane-oxidizing archaea, as well as several enzymes potentially involved in alkyl-CoA oxidation and the Wood-Ljungdahl pathway. We suggest that members of the Helarchaeota have the potential to activate and subsequently anaerobically oxidize hydrothermally generated short-chain hydrocarbons.

Proposal of the reverse flow model for the origin of the eukaryotic cell based on comparative analyses of Asgard archaeal metabolism
Spang, Anja ; Stairs, Courtney W. ; Dombrowski, Nina ; Eme, Laura ; Lombard, Jonathan ; Caceres, Eva F. ; Greening, Chris ; Baker, Brett J. ; Ettema, Thijs J.G. - \ 2019
Nature Microbiology 4 (2019). - ISSN 2058-5276 - p. 1138 - 1148.

The origin of eukaryotes represents an unresolved puzzle in evolutionary biology. Current research suggests that eukaryotes evolved from a merger between a host of archaeal descent and an alphaproteobacterial endosymbiont. The discovery of the Asgard archaea, a proposed archaeal superphylum that includes Lokiarchaeota, Thorarchaeota, Odinarchaeota and Heimdallarchaeota suggested to comprise the closest archaeal relatives of eukaryotes, has helped to elucidate the identity of the putative archaeal host. Whereas Lokiarchaeota are assumed to employ a hydrogen-dependent metabolism, little is known about the metabolic potential of other members of the Asgard superphylum. We infer the central metabolic pathways of Asgard archaea using comparative genomics and phylogenetics to be able to refine current models for the origin of eukaryotes. Our analyses indicate that Thorarchaeota and Lokiarchaeota encode proteins necessary for carbon fixation via the Wood–Ljungdahl pathway and for obtaining reducing equivalents from organic substrates. By contrast, Heimdallarchaeum LC2 and LC3 genomes encode enzymes potentially enabling the oxidation of organic substrates using nitrate or oxygen as electron acceptors. The gene repertoire of Heimdallarchaeum AB125 and Odinarchaeum indicates that these organisms can ferment organic substrates and conserve energy by coupling ferredoxin reoxidation to respiratory proton reduction. Altogether, our genome analyses suggest that Asgard representatives are primarily organoheterotrophs with variable capacity for hydrogen consumption and production. On this basis, we propose the ‘reverse flow model’, an updated symbiogenetic model for the origin of eukaryotes that involves electron or hydrogen flow from an organoheterotrophic archaeal host to a bacterial symbiont.

DNA polymerases at work: single-molecule observations of DNA synthesis in real time
Fijen, Carel - \ 2018
Wageningen University. Promotor(en): H. van Amerongen, co-promotor(en): J.C. Hohlbein. - Wageningen : Wageningen University - ISBN 9789463437431 - 174

This thesis focuses on the characterization of DNA polymerases with single-molecule techniques. More specifically, I aimed to study polymerase processivity and fidelity-related conformational changes using assays based on Förster Resonance Energy Transfer (FRET) on a total internal reflection fluorescence (TIRF) microscope.

Chapter 2 reviews some of the recent applications of single-molecule FRET (smFRET) to study DNA and DNA binding proteins, in particular DNA polymerases. The chapter begins with an introduction of FRET, employed to measure distance changes in the 1-10 nm region, and introduces the two most common fluorescence-based implementations of single-molecule techniques: confocal microscopy and TIRF microscopy. The chapter concludes with a short discussion on FRET-based structural modelling, parts of which are applied in practice later in this thesis.

In chapter 3, I report the development of a short, fluorescently labelled DNA sensor to probe DNA polymerization at the single-molecule level. The sensor is a simple primer-template combination labelled with donor and acceptor fluorophores suitable for FRET. The advantage of this assay is that polymerases do not need to be labelled with any fluorophore. I show that the FRET efficiency of the sensors changes significantly upon polymerization of the 25 nucleotide template, and I present time traces showing polymerization of single sensors by three different polymerases (E. coli DNA Polymerase I (KF), human Polymerase Beta (POLB) and the α subunit of bacterial Polymerase III (POLIIIα)). Based on these traces, I can measure polymerase speed and pausing: KF and POLIIIα extended the primer in ~1.0-1.5 s, but POLB was far slower (tens of seconds). I foresee applications for these sensors in the single-molecule field, where they can be used to characterize the processivity of other polymerases, but also for ensemble experiments in which native polymerases need to be tested for activity.

I take a closer look at POLB in chapter 4. This polymerase is involved in DNA repair, and I address the question whether resolving the conformational dynamics of the enzyme can shed new light on fidelity-related mechanisms. Previous work on both KF and POLB showed that the polymerase “fingers” domain binds a nucleotide and subsequently transfers it to the active site (a conformational change known as “fingers closing”). For KF, it was shown that the fingers domain does not entirely close when non-complementary nucleotides are present, suggesting that nucleotides are screened for complementarity with the templating base during fingers closing. To see whether POLB employs a similar mechanism, I designed an smFRET assay with an immobile donor fluorophore on the DNA primer and an acceptor fluorophore on the fingers domain. Using this approach, I can visualize fingers closing in the presence of the correct nucleotide in single POLB-DNA complexes. Incorrect nucleotides (non-complementary dGTPs and complementary rUTPs) did not induce fingers closing. Instead, we observed a slight shift in the mean FRET efficiency of the open conformation (from E* ≈ 0.55 to E* ≈ 0.62), while a fully closed conformation corresponds to E* ≈ 0.75. I find evidence for a partially closed, fidelity-related conformation of the fingers subdomain. Simultaneously, I find that high concentrations of incorrect nucleotides (1 mM and 3 mM) stabilize the POLB-DNA complex by lowering the POLB dissociation rate. In contrast, for KF, a destabilizing effect was shown previously. The mechanism behind this stabilization remains unknown, but I hypothesize that with the abundance of incorrect nucleotides in the cell, DNA repair is much faster if high levels of these nucleotides do not promote dissociation.

In chapter 5, I introduce novel nanofluidic devices for high-throughput single-molecule imaging. These devices are completely made of glass. I present two designs: one design with a parallel array of nanochannels for equilibrium studies, and another with a single, T-shaped nanochannel for mixing studies allowing access to non-equilibrium conditions. A channel height of 200 nm confines movement of the molecules such that they do not move out of focus. With the implementation of parallel flow control, the devices can be driven with conventional syringe pumps. I achieve a high temporal resolution on our emCCD camera due to stroboscopic excitation (1.5 ms excitation in 10 ms frame time). I show that we can track single molecules at low concentrations for extended periods of time. The track length depends on the flow speed, but ranges from several frames to tens of frames. Moreover, at higher concentrations, I achieve hundreds of thousands of localizations within 10 minutes. These localizations allowed me to construct flow profiles, which confirms that the flow in the nanochannels is laminar. I also calculate that, at low flow rates and with the small DNA molecules I used, motion due to flow is of the same order of magnitude as motion due to diffusion. I illustrate this concept by mixing DNA hairpins in a primarily open configuration with a high-salt solution in the mixing channel: the FRET signature of the hairpins changes abruptly towards an equilibrium of primarily closed DNA hairpins. After fine-tuning the conditions, this so-called “diffusive” mixing is employed to trigger single-molecule reactions: I successfully polymerize my previously described DNA sensor inside the channel. I believe that these nanofluidic devices are a promising platform for studying non-immobilized single molecules at high throughput and high temporal resolution.

Moderate and high intensity pulsed electric fields : Effect on microbial inactivation, shelf life and quality of fruit juices
Timmermans, Rian Adriana Hendrika - \ 2018
Wageningen University. Promotor(en): M.A.J.S. van Boekel, co-promotor(en): H.C. Mastwijk; M.N. Nierop Groot. - Wageningen : Wageningen University - ISBN 9789463438155 - 241

Pulsed Electric Field (PEF) processing has gained a lot of interest the last decades as mild processing technology as alternative to thermal pasteurisation, and is suitable for preservation of liquid food products such as fruit juices. PEF conditions typically applied at industrial scale for pasteurisation are high intensity pulsed electric fields aiming for minimal heat load, with an electric field strength (E) in the range of 15 − 20 kV/cm and pulse width (τ) between 2 − 20 μs. Alternatively, moderate intensity pulsed electric fields with an electric field strength in the order of E = 0.5 − 5.0 kV/cm are used as a pre-step to disintegrate plant cells or electroporate micro-organisms for mass transfer in food and biotechnological, but not as an alternative preservation technology. The work described in this thesis investigated how moderate and high intensity PEF processing conditions, product matrix and characteristics of target species affect microbial inactivation, shelf life, enzyme activity and quality of fruit juices. This was performed by a systematic evaluation of the impact of most important process parameters in PEF processing, including electric field strength, pulse width and temperature, in a continuous-flow configuration. Microbial inactivation after PEF treatment was compared to that of an equivalent thermal process to distinguish the electroporation and thermal effects responsible for inactivation.

In Chapter 1, an overview of the current status in the scientific field on fruit juice processing, pulsed electric field processing and kinetic modelling is provided, knowledge gaps were identified and the aim and research questions following from that are described.

In Chapter 2, the impact of a high intensity PEF process (E = 20 kV/cm and τ = 2 μs) was studied on the inactivation of Escherichia coli, Salmonella Panama, Listeria monocytogenes and Saccharomyces cerevisiae in apple, orange and watermelon juice. Kinetic data showed that for identical process and matrix conditions, the yeast S. cerevisiae was the most sensitive micro-organism, followed by S. Panama and E. coli, which displayed comparable inactivation kinetics. L. monocytogenes was most resistant towards the treatment conditions tested. A synergistic effect between temperature and electric pulses was observed at inlet temperatures above 35 °C, hence less electrical energy for inactivation was required at higher temperatures. The different juice matrices resulted in a different degree of inactivation, predominantly determined by pH, where more acidic conditions led to more inactivation.

In Chapter 3, the effects of high intensity PEF processing conditions (E = 13.5 − 24.0 kV/cm) and storage temperature on the outgrowth of surviving yeast and mould populations naturally present in a fresh fruit smoothie were assessed over time. Results showed that untreated smoothie was predominantly spoiled by the outgrowth of yeasts, typically after 8 days (stored at 4 or 7 °C), while initial number of moulds present in the smoothie declined during storage. PEF inactivated most yeasts present in the smoothie, thereby providing outgrowth opportunities for the moulds, which were visually observed after 14 days (stored at 7°C) or 18 days (stored at 4°C). The intensity of the electric field strength both affected the log10 reduction of yeasts and the lag-time, the period the cell required to grow out. A similar effect of electric field strength on the degree of inactivation has been observed for moulds, although electric field strength did not influence the period to visual mould growth.

Chapter 4 and 5 focussed on the development of a model to be used to fit and predict non-linear log-time inactivation of a thermal process at a holding time comparable to a PEF process. In Chapter 4, a rational thermodynamic model based on Gaussian distribution and Eyrings rate constant has been developed that can accurately model non-linear log-time inactivation kinetics of enzymes and micro-organisms exposed to a thermal and/or chemical (acid) treatment. This so-called Gauss-Eyring model is a bivariate log-normal distribution with temperature and time as independent variables. Model parameters standard activation enthalpy and entropy are directly related to reference temperature Tr and Z-value, commonly used in kinetic analysis in food microbiology. An essential feature of the kinetic model is that its parameters are treated as stochastic variables, owing to the underlying physics, based on the Lumry-Eyring model for unfolding of proteins using transition state theory. The performance of the model was evaluated using published data on enzyme inactivation and microbial inactivation including a wide range of temperatures and pH.

In Chapter 5, this Gauss-Eyring model was used to fit inactivation data of E. coli, L. monocytogenes, Lactobacillus plantarum, Salmonella Senftenberg and S. cerevisiae in orange juice. Thermal inactivation data was collected by exposing capillary tubes with target organisms to different temperature time combinations using a water bath to obtain inactivation kinetics, either via isothermal (a fixed temperature with varying holding time) or isotime (a fixed holding time with varying temperature) series. The model fitted well to the inactivation data of individual cultures. Variability between the different cultures of the five tested micro-organisms was observed. Therefore, an average value of the parameters of the individual cultures was used to predict inactivation as a function of temperature for a chosen (short) holding time.

In Chapter 6, a systematic evaluation of the individual effects of electric field strength and pulse width in combination with heat on the inactivation of E. coli, L. monocytogenes, S. Senftenberg, L. plantarum and S. cerevisiae in orange juice was carried out. A wide range of conditions has been tested, including both moderate intensity as well as high intensity PEF. Both electric field strength and pulse width were shown to be important for microbial inactivation. Inactivation kinetics of the tested conditions were compared to an equivalent thermal reference process, based on parameter estimates obtained in Chapter 5. A nonthermal pulse effect was observed for three specific sets of conditions in addition to the thermal effect responsible for inactivation. A non-thermal pulse effect was found for high intensity PEF treatment at E = 15 or 20 kV/cm and τ = 2 μs, but also at moderate intensity PEF condition of E = 2.7 kV/cm and τ = 1000 μs. The effectivity of this moderate intensity PEF condition was evaluated for E. coli and L. monocytogenes in watermelon juice and coconut water, varying in pH and conductivity. Interestingly, this moderate intensity PEF condition showed the same effectivity for all matrices in the pH range of 3.8 to 6.0, while high intensity PEF conditions at E = 20 kV/cm did show a strong dependence on product pH for microbial inactivation (Chapter 2). This suggests that a different mechanism is responsible for inactivation at moderate intensity conditions compared to high intensity conditions. Speculations on the mechanism responsible for inactivation are made in Chapter 8.

In Chapter 7, the impact of moderate intensity PEF (E = 0.9 − 2.7 kV/cm) and long pulse width (τ = 1000 μs) at variable maximum temperatures was evaluated on quality attributes of freshly squeezed orange juice, and compared to the impact of two thermal processes using either mild or severe pasteurisation conditions. No differences for pH and soluble solids were found after application of any treatment, and only small differences were observed for colour and vitamin C content after PEF and thermal treatment, mainly for the conditions applied at higher temperature. A large processing effect was measured in the enzyme activity of pectin methylesterase (PME), responsible for undesired cloud instability. Reduction of the remaining enzyme activity depended on the maximum applied temperature, and levels below the critical value to obtain shelf stable juices were found, showing that it is possible to select moderate intensity PEF conditions for adequate pasteurisation of fruit juices, both with respect to microorganisms and enzymes. The impact of processing on volatile flavour compounds was moderate when compared to untreated, although some deviations between moderate intensity PEF treated and thermally processed orange juice were found for individual compounds, with a better retention of the flavour compounds after application of moderate intensity PEF.

In Chapter 8, the main results of this thesis were discussed and concluding remarks and recommendations were presented. In conclusion, this thesis provided novel insight in the use of pulsed electric field processing as a mild pasteurisation process with improved quality as alternative to thermal pasteurisation of fruit juice. The work described in this thesis resulted in more insight in the individual effects of electric field strength and pulse width, and presented an effective combination of long pulse width and moderate intensity electric field strength as alternative PEF condition to the currently used high intensity electric field strength conditions for preservation. In addition, these moderate intensity/long pulse duration conditions are promising for industrial application, as they showed to be less sensitive for differences in the characteristics of the micro-organisms than high intensity PEF conditions and they are effective in both high-acid as well as low-acid products.

The Relationship between Hydro-Climatic Variables and E. coli Concentrations in Surface and Drinking Water of the Kabul River Basin in Pakistan
Shahid Iqbal, Muhammad ; Nauman Ahmad, Muhammad ; Hofstra, Nynke - \ 2017
AIMS Environmental Science 4 (2017)5. - ISSN 2372-0344 - p. 690 - 708.
Microbial water contamination is a risk for human health, as it causes waterborne diseases like diarrhea. E. coli is a faecal indicator microorganism. Climate variables, such as temperature and precipitation, influence E. coli concentrations in surface and drinking water resources. We measure and statistically analyse E. coli concentrations in drinking and surface water in the Kabul River Basin. E. coli concentrations are very high in the basin. Drinking and bathing water standards are violated. Water temperature, surface air temperature, discharge and precipitation were positively correlated with E. coli concentrations. Precipitation induced runoff transports of E. coli from agricultural lands to Kabul River and high temperature coincides with high precipitation and discharge. A linear regression model was developed to assess the net effect of the climate variables on E. coli concentrations. We found that climate variables accounted for more than half of the observed variation in E. coli concentrations in surface (R2 = 0.61) and drinking water (R2 = 0.55). This study indicates that increased precipitation together with higher surface air temperature, as expected in this region with climate change, were significantly correlated with increased E. coli concentrations in the future. Waterborne pathogens are expected to respond similarly to hydro-climatic changes, indicating that disease outbreaks could well become more frequent and severe.
Probing the bacterial cell wall with chemical biology tools
Sminia, Tjerk J. - \ 2017
Wageningen University. Promotor(en): H. Zuilhof; W.M. de Vos, co-promotor(en): T. Wennekes. - Wageningen : Wageningen University - ISBN 9789463437080 - 196
bioengineering - sugars - labelling - synthesis - biochemical techniques - akkermansia muciniphila - gastrointestinal microbiota - carbohydrates - bioengineering - suikers - etiketteren - synthese - biochemische technieken - akkermansia muciniphila - microbiota van het spijsverteringskanaal - koolhydraten

After DNA and proteins, carbohydrates are the third language of life. Chapter 1 introduces the reader to this class of biomolecules, also called sugars or glycans, that can be found on the outer surface of almost all cells and plays a critical role as the social messengers of a cell. Although our knowledge about the role of glycans in eukaryotic cells has increased considerably in recent decades, our understanding of the glycan layer on bacterial cells is still very limited. Besides the carbohydrates that are present in both eukaryotes and prokaryotes an additional wide range of unique (e.g. microbial sialic acid), often very complex (e.g. pseudaminic acid), carbohydrates is present in prokaryotes. This chapter briefly introduces two research fields, carbohydrate chemistry and chemical biology, that when combined provide a powerful way to investigate the biological role of these unique bacterial carbohydrates at the molecular level. This chemistry-based approach, termed chemical microbiology, often starts with the development of a chemical synthesis for a target bacterial carbohydrate. Subsequently, the synthetic route towards this target allows for the introduction of unnatural functional groups, like chemical reporters, that result in the molecular tools needed to study their biological function. The studies described in this thesis, focus on developing such molecular tools to study the role of glycans and glycoconjugates in human gut bacteria and human-associated bacteria.

Chapter 2 provides an overview of metabolic oligosaccharide engineering (MOE) a popular chemical biology technique to label glycans in living cells. In MOE, carbohydrates derivatives are synthesised with unnatural chemical reporters and used to study their incorporation in glycans of eukaryote to prokaryote species. The progress in this field over the last 6 years is reviewed in detail with a special emphasis on the synthesis of the unnatural carbohydrates from commercially available sources. The principle behind MOE is that these unnatural carbohydrates with e.g. azide, alkyne, cyclopropene, or isonitrile chemical reporter groups, are still recognised by the endogenous enzymes in the cell that salvage this new carbohydrate. In this way they can enter the associated biochemical pathways and end up in newly biosynthesised cellular glycans. Subsequent labelling techniques, such as strain promoted azide alkyne cycloaddition or tetrazine ligation, enable the visualisation of these incorporated unnatural carbohydrates with for instance fluorescence microscopy.

Metabolic labelling is further explored in chapter 3. Key cell envelope glycoconjugates in the mucin-degrading gut microbiota member, Akkermansia muciniphila, were subjected to chemistry-based functional analysis, with Escherichia coli being used as a control species. Two novel non-toxic peptidoglycan (PG) probes were designed and synthesised to investigate the presence of PG in this species. Their design was based on the natural d-alanine dipeptide motif found in PG. Inspired by the fact that d-alanine dipeptide-derivatives were previously reported to be incorporated in newly synthesised PG, we synthesised a cyclopropene and isonitrile d-alanine dipeptide. Our probes proved to be non-toxic, as shown by growth and viable count analysis, and were therefore superior over existing PG probes. Another beneficial property was that the probes also did not influence the specific growth rate of A. muciniphila or E. coli. The PG probes were successfully incorporated into the peptidoglycan layer of A. muciniphila and visualised using a tetrazine click-ligation with a fluorophore. Our analysis proved for the first time that A. muciniphila has a PG layer. Besides PG labelling, we also investigated metabolic labelling of other glycoconjugates on the outer surface of A. muciniphila. This part of the study showed that azido-monosaccharide derivatives of N-acetylglucosamine, N-acetylgalactosamine, and fucose are successfully processed by A. muciniphila salvage pathways and incorporated into its surface glycoconjugates. Especially 6-azido-fucose was readily processed by the recently discovered l-fucose salvage pathway of A. muciniphila. The two compatible labelling techniques were next combined in a dual labelling experiment. Our isonitrile dipeptide peptidoglycan probe and 6-azido-fucose were successfully incorporated into A. muciniphila. Subsequent fluorescent labelling with bio-orthogonal techniques resulted in dual labelling of peptidoglycan and fucose-containing glycans in live A. muciniphila cells.

With the positive results of MOE in A. muciniphila in hand, chapter 4 describes the further investigation of MOE. After successful validation of our Ac4FucAz probe for MOE in Bacteroides fragilis we continued their application in other human gut microbiota members, including the butyrate-producing Anaerostipes rhamnosivorans, Intestimonas butyriciproducens, and Eubacterium hallii. Labelling of these human gut microbes proved to be rather challenging with a-specific cellular labelling with the fluorophore being the major problem. Initial results, however, did show that a 6-azido-l-rhamnose probe resulted in fluorescent labelling of A. rhamnosivorans, which provides initial evidence for the existence of an as of yet undocumented salvage pathway. In this species the 6-azido-fucose probe was not salvaged. Via confocal microscopy and flow cytometry analysis we observed that the 6-azido-rhamnose probe was selective for A. rhamnosivorans in the presence of A. muciniphila. Such a co-culture experiment is a first step in mimicking the complex human gut microbiome. For E. hallii Ac4GalNAz gave clear metabolic labelling and the majority of the cell population could be labelled with the fluorescent dye after a strain-promoted azide alkyne cycloaddition. Other glycan probes (Ac4GlcNAz, Ac4FucAz, and Neu5Az) also resulted in labelling, but not as prominent as Ac4GalNAz. Surprisingly, MOE has never been reported for the common lab strain Escherichia coli MG1655. Curious to investigate this in more detail we started MOE in E. coli. However, no labelling was obtained when Ac4GlcNAz probe was added to E. coli, most likely due to the fast growth, metabolism and turnover. Only, when fresh Ac4GlcNAz probe was added every 30 minutes, metabolic labelling in E. coli was observed. To further investigate the influence of GlcNAc metabolism in E. coli on MOE, single-gene knock-outs of E. coli GlcNAc metabolism from the Keio collection were investigated. Labelling was observed for NagA (N-acetyl glucosamine 6 P deacetylase) and NagK (N-acetyl-d-glucosamine kinase) E. coli mutants. Both enzymes are involved in the last step of the biosynthesis towards UDP-N-acetylglucosamine. When the overall E. coli metabolism was inhibited, after addition of the respiration inhibitor sodium azide, no metabolic labelling was observed. These results indicate that MOE in E. coli is possible, but challenging and can only be performed under specific circumstances.

An investigation into the total synthesis of pseudaminic acid, a sialic acid produced by specific human-associated prokaryotes, is described in chapter 5. Sialic acids are typically found at the terminal positions of surface glycoconjugates in both eukaryotes and prokaryotes. Other related microbial sialic acids are legionaminic and acinetaminic acid. The total synthesis of these microbial sialic acids is notoriously difficult, as exemplified by the fact that only a few chemical synthesis routes towards them are currently known. Our total synthesis of pseudaminic acid started from the readily available amino acid l-threonine that was transformed into a key versatile Garner aldehyde derivative intermediate. With this aldehyde in hand, the Henry nitro-aldol condensation reaction was investigated. After studying numerous conditions, such as asymmetric catalysis or elongated reaction times, and extensive optimisation efforts we were never able to obtain the Henry reaction product to continue with this route. As an alternative, a tethered aminohydroxylation was investigated for its ability to introduce the key functional group and stereochemistry onto an intermediate obtained from the Garner aldehyde derivative. This reaction indeed gave the desired amino-alcohol motif in the correct stereochemistry, but another diastereomer proved very difficult to separate from the desired product. After some additional transformations and protection steps we obtained a derivative in which the primary alcohol could be oxidised to provide a hexose intermediate that resembles the hexose intermediate present in pseudaminic acid biosynthesis. This key hexose intermediate will likely enable a subsequent Barbier reaction, a chain elongation step, in future studies. With most of the key transformations accomplished, the completion of a pseudaminic total synthesis based on l-threonine should soon be possible. Besides finishing the total synthesis, future work should also focus on adapting this synthesis route to allow installation of chemical reporter groups on pseudaminic acid for its application in MOE.

Chapter 6 is the general discussion about all the work mentioned in the other chapters. It also contains additional information and suggestions for further research in the field of chemical microbiology.

Production of medium-chain, a, omega-bifunctional monomers from fatty acids and n-alkanes
Nuland, Youri M. - \ 2017
Wageningen University. Promotor(en): G. Eggink; J.P.M. Sanders, co-promotor(en): R.A. Weusthuis. - Wageningen : Wageningen University - ISBN 9789463436809 - 161

In chapter 1, we give an introduction to bifunctional monomers that play an important role in the chemical industry. Briefly, the conventional production processes of α,ω-dicarboxylic acids and α,ω-diols are discussed. Strategies for more sustainable alternatives for production of medium-chain bifunctional monomers are discussed. Monooxygenase-based processes seem promising, if the problem of poor diterminal oxidation capacities of monooxygenases is solved. Esterification could be a tool to solve this problem.

In chapter 2 we have investigated the ω-oxidation activities of E. coli expressing AlkBGT or AlkBGTL, with various esters having an alkyl chain >1. These strains were able to ω-oxidize ethyl, propyl and butyl esters of C6-C10 fatty acids. Using esters with a longer alkyl chain enhanced ω-oxidation activities for C6 and C7 fatty acids. The major products were ω-hydroxy fatty acid esters, but over oxidation to the aldehyde and carboxylic acid also occurred. AlkL improved whole-cell ω-oxidation activities for substrates with a logPo/w above 4.

Since the major products were ω-hydroxy fatty acid esters in chapter 2, we investigated further conversion of these compounds to mono-esterified dicarboxylic acids in chapter 3. Alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH were functionally expressed in E. coli. AlkJ is functional with 9-hydroxy ethyl nonanoate as substrate, AlkH is functional with 9-oxo methyl nonanoate. Expansion of the AlkBGTL system with AlkJ and AlkH yielded strain E. coli AlkBGTHJL. This strain accumulated mono-ethyl azelate exclusively from ethyl nonanoate. Adding the substrate dissolved in a carrier solvent increased final product titers.

Subsequently, we investigated if in vivo esterification could enhance the ω-oxidation of AlkB in chapter 4. E. coli expressing AlkBGTHJL can ω-oxidize octanoate and nonanoate, but not efficiently. When acyl-CoA ligase AlkK and acyltransferase AtfA or Eeb1 were also expressed, ω-oxidation was more efficient. Furthermore, complete oxidation to the carboxylic acid was much more efficient when also in vivo esterification was achieved. Also di-ethyl esters were produced, meaning that esterification occurred twice.

Since ω-oxidation of fatty acids was improved with in vivo esterification in chapter 4, we were interested to investigate whether this system could also work with n-alkanes in chapter 5. Mono-esters of dicarboxylic acids were produced from n-alkanes by E. coli expressing AlkBGTHJKL and either AtfA or Eeb1. Starting from n-alkanes would also allow production of alcohols if overoxidation could be prevented. Application of a different alcohol acyltransferase (Atf1), limited the overoxidation by AlkB. ω-Oxidation of the formed ester resulted in the production of ω-alcohols, which were again esterified by Atf1.

Chapter 6 is the general discussion of this thesis, which evaluates the combination of esterification and terminal oxidation. Suggestions for improvements of the biocatalytic pathway are provided and critical factors for experiments in bioreactors are identified.

Adapting to change : on the mechanism of type I-E CRISPR-Cas defence
Künne, Tim A. - \ 2017
Wageningen University. Promotor(en): J. van der Oost, co-promotor(en): S.J.J. Brouns. - Wageningen : Wageningen University - ISBN 9789463436649 - 239
immunity - defence mechanisms - rna - bacteria - escherichia coli - analytical methods - priming - immuniteit - verdedigingsmechanismen - rna - bacteriën - escherichia coli - analytische methoden - zaadbevochtiging

Host-pathogen interactions are among the most prevalent and evolutionary important interactions known today. The predation of prokaryotes by their viruses is happening on an especially large scale and had a major influence on the evolutionary history of prokaryotes. Since most viruses are lytic at some point in their life-cycle, there is a high selection pressure for prokaryotes to develop defense mechanisms. As described in Chapter 1, the CRISPR-Cas system is a relatively recently discovered defense system and is also the first adaptive defense system discovered in prokaryotes. CRISPR-Cas systems are widespread, occurring in the majority of archaea and also a considerable fraction of bacteria. This diversity is also reflected in the diversity of different types of CRISPR-Cas systems, currently being divided into 6 major types with a large number of subtypes. The type I-E system of Escherichia coli is a well-studied model system and of high relevance, since it is a major subtype of type I systems which make up around 50 % of all discovered CRISPR-Cas systems. CRISPR-Cas systems basically comprise the CRISPR array, made up of repeats and foreign derived spacers, and a set of cas genes. Immunity is commonly divided into three functional stages, adaptation, expression and interference. Adaptation is the acquisition of new spacers from the foreign nucleic acid and its incorporation into the CRISPR array. During expression, the CRISPR array is transcribed, processed and assembled with Cas proteins into CRISPR RNA (crRNA) guided ribonucleoprotein complexes (crRNP). Interference is the detection, binding and destruction of foreign nucleic acids by the crRNP and in type I systems the Cas3 nuclease. The type I-E system contains another function, called primed adaptation. Primed adaptation is a more rapid and efficient version of regular (naïve) adaptation. In addition to the adaptation machinery, primed adaptation also requires the interference machinery.

Chapter 2 describes and compares a fundamental feature of most, if not all, CRISPR-Cas systems and also many other small RNA based systems. The mode of action of small RNAs relies on protein-assisted base pairing of the guide RNA with target mRNA or DNA to interfere with their transcription, translation or replication. Several unrelated classes of small non-coding RNAs have been identified including eukaryotic RNA silencing associated small RNAs, prokaryotic small regulatory RNAs and prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) RNAs. All three groups identify their target sequence by base pairing after finding it in a pool of millions of other nucleotide sequences in the cell. In this complicated target search process, a region of 6 to 12 nucleotides of the small RNA termed the ‘seed’ plays a critical role. The seed is often a structurally pre-ordered region that increases accessibility and lowers the energy barrier of RNA-DNA duplex formation. Furthermore, the length of the seed is optimally chosen to allow rapid probing and also rejection of potential target sites. The seed is a perfect example of parallel evolution, showing that nature comes up with the same strategy independently multiple times.

Chapter 3 provides a description and protocol of the Electrophoretic Mobility Shift Assay (EMSA) and its use for studying crRNPs. EMSA is a straightforward and inexpensive method for the determination and quantification of protein–nucleic acid interactions. It relies on the different mobility of free and protein-bound nucleic acid in a gel matrix during electrophoresis. Nucleic acid affinities of crRNPs can be quantified by calculating the dissociation constant (Kd ). Protocols for two types of EMSA assays are described using the Cascade ribonucleoprotein complex from Escherichia coli as an example. One protocol uses plasmid DNA as substrate, while the other uses short linear oligonucleotides. Plasmids can be easily visualized with traditional DNA staining, while oligos have to be radioactively labelled using the 32Phosphate isotope. The EMSA method and these protocols are applied throughout the other chapters of this thesis.

Chapter 4 focusses on the processes of interference and primed adaptation, specifically on their tolerance of mutations. Invaders can escape Type I-E CRISPR-Cas immunity in E. coli by making point mutations in the protospacer (especially in the seed) or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive feedback process termed priming. Here, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6 nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of utilizing degenerate target regions with up to at least eleven mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position and is nucleotide dependent. Our findings imply that even out-dated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the co- evolutionary arms race with their invaders.

In Chapter 5 we elucidate the mechanism of priming. Specifically, we determine how new spacers are produced and selected for integration into the CRISPR array during priming. We show that priming is directly dependent on interference. Rapid priming occurs when the rate of interference is high, delayed priming occurs when the rate of interference is low. Using in vitro assays and next generation sequencing, we show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30–100 nt enriched for thymine-stretches in their 3’ ends. By reconstituting the spacer integration process in vitro, we show that the Cas1-2 complex further processes these fragments and integrates them sequence- specifically into CRISPR repeats by coupling of a 3’ cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers.

In Chapter 6 we look deeper into a nucleotide specific effect on priming that was discovered in Chapter 4. Immunity is based on the complementarity of host encoded spacer sequences with protospacers on the foreign genetic element. The efficiency of both direct interference and primed acquisition depends on the degree of complementarity between spacer and protospacer. Previous studies focused on the amount and positions of mutations, not the identity of the substituted nucleotide. In Chapter 4, we describe a nucleotide bias, showing a positive effect on priming of C substitutions and a negative effect on priming of G substitutions in the basepairing strand of the protospacer. Here we show that these substitutions rather directly influence the efficiency of interference and therefore indirectly influence the efficiency of interference dependent priming. We show that G substitutions have a profoundly negative effect on interference, while C substitutions are readily tolerated when in the same positions. Furthermore, we show that this effect is based on strongly decreased binding of the effector complex Cascade to G mutants, while C mutants only minimally affect binding. In Chapter 5 we showed a connection between the rate of interference and the time of occurrence of priming. Here, we also quantify the extent of priming and show that priming is very prevalent in a population that shows intermediate levels of interference, while high or low levels of interference lead to a lower prevalence of priming.

Chapter 7 describes an attempt to make use of our knowledge about the Cascade complex and develop it into a genome editing tool. The development of genome editing tools has made major leaps in the last decade. Recently, RNA guided endonucleases (RGENs) such as Cas9 or Cpf1 have revolutionized genome editing. These RGENs are the hallmark proteins of class II CRISPR-Cas systems. Here, we have explored the possibility to develop a new genome editing tool that makes use of the Cascade complex from E. coli. This RNA guided protein complex is fused to a FokI nuclease domain to sequence specifically cleave DNA. We validate the tool in vitro using purified protein and two sets of guide RNAs, showing specific cleavage activity. The tool requires two target sites of 32 nt each at a distance of 30-40 nt and inward facing three nucleotide flexible PAM sequences. Cleavage occurs in the middle between the two binding sites and primarily creates 4 nt overhangs. Furthermore, we show that an additional RFP can be fused to FokI-Cascade, allowing visualization of the complex in target cells. Unfortunately, we were not able to successfully apply the tool in vivo in eukaryotic cells.

Assessing the impact of socio-economic development and climate change on faecal indicator bacteria in the Betna River, Bangladesh
Islam, Majedul - \ 2017
Wageningen University. Promotor(en): R. Leemans, co-promotor(en): N. Hofstra. - Wageningen : Wageningen University - ISBN 9789463436304 - 137
climatic change - environmental impact - water quality - rivers - contamination - bacteria - coliform bacteria - faecal coliforms - bangladesh - south asia - klimaatverandering - milieueffect - waterkwaliteit - rivieren - besmetting - bacteriën - coliformbacteriën - fecale coliformen - bangladesh - zuid-azië

Consumption of water that is contaminated with pathogens still causes high numbers of death and disease. Understanding the factors that influence the dynamic distribution of waterborne pathogens is important, as this will help understanding improvements and possible solutions. Such understanding is particularly important in a developing country like Bangladesh, where large proportions of the population often have little or no access to clean water. Despite the high relevance for public health, few studies currently exists on the fate and transport of pathogens and the so-called Faecal Indicator Bacteria (FIB, e.g. E. coli, enterococci) in (sub)tropical systems. FIB are susceptible to shifts in water flow and quality. The predicted increases in rainfall and floods due to climate change will exacerbate the faecal contamination scenarios. This could be further compounded by the rapid change in socio-economic conditions (population growth, urbanization, sanitation and agricultural management) in the developing countries. Therefore, to reduce future health risks, understanding the influence of changes in socio-economic conditions and climate on microbial dynamics is important.

Very few studies have quantified the relationship between the waterborne pathogens/FIB concentrations and climate and socio-economic changes. In this study a process-based model was developed and a scenario analysis was performed based on the new combined climate and socio-economic changes scenarios, to assess the present and future river hydrodynamics, FIB sources, die-off processes and concentrations. We used FIB, because measuring FIB are cheaper than pathogens. FIB are usually not pathogenic but their presence indicates the likely presence of waterborne pathogens. These pathogens are expected to respond to climate change in a comparable way to FIB. The present study is based on the Betna River basin in southwestern Bangladesh, where faecal contamination is not monitored and very little knowledge exists on the distribution of contaminants.

First of all, FIB concentrations of the river water were measured to identify the river’s faecal contamination levels that can be used to validate the water-quality model. In the study area, wastewater is not treated and this untreated wastewater is discharged directly into the river. This is evident from the measured FIB data. In 88% of the E. coli and all enterococci samples, the USEPA bathing water quality standards were violated (Chapter 2). Such violation indicates potential health risks associated with the use of the river water for domestic, bathing and irrigation purposes. The correlation between environmental variables (water temperature, precipitation and salinity) and FIB concentrations was also determined. A positive correlation was found with water temperature and precipitation, and a negative correlation with salinity. The positive correlation with temperature is due to the co-occurrence of high summer temperature with abundant monsoon rainfall. The positive correlation with precipitation can be explained by the increased runoff from agricultural lands and urban areas. This runoff contains many bacteria. In the study area, during the rainy season (July to September) precipitation increases and as a result water salinity decreases. The observed negative correlation with salinity is more likely due to the typical weather patterns during the rainy season when low salinity coincides with increased precipitation and high temperature, than to salinity dependent die-off of bacteria. A regression model was applied that explained almost half of E. coli and enterococci variability in river water. This, however, only considers water temperature and precipitation (Chapter 2).

Then, the present and future hydrodynamics of the river were simulated using a two dimensional hydrodynamic model (MIKE 21 FM). Although the main goal of this thesis is to assess the river’s present and future FIB concentrations, the reasons for this hydrodynamic modelling are twofold. Firstly, outputs of the hydrodynamic model are used as input into the water-quality model (Chapter 4). Secondly, hydrodynamics (i.e. water level and discharge) are simulated because increased water level and discharge together with sea level rise stimulate floods in the river basin. These floods are related to outbreaks of waterborne diseases. The modelled results corresponded very well with the measured water levels and discharges. The model was applied to simulate baseline and future water levels and discharge for Representative Concentration Pathway RCP4.5 and RCP8.5 scenarios using bias-corrected downscaled data from two climate models (IPSL-CM5A and MPI-ESM). The model results showed an expected increase in water level up to 16% by the 2040s and 23% by the 2090s (Chapter 3). The monsoon daily maximum discharge was expected to increase up to 13% by the 2040s and 21% by the 2090s. These model results also showed that the duration of the water level above the danger level and extreme discharge periods can increase by half a month by the 2040s and over a month by the 2090s. The coincidence of the water danger level with extreme discharge may cause disastrous floods in the study area.

Next, the hydrodynamic model was coupled with a water-quality module (ECOLab). The fate and transport of FIB was simulated, the influence of different processes tested and the contribution from different sources to the total contamination quantified (Chapter 4). The model outputs corresponded very well with the measured FIB data. The present river microbial water quality based on measured and simulated results indicated, once again, noncompliance with bathing water standards. Primary and secondary levels of wastewater treatment were not sufficient to reach the standards most of the time, and discharges from sewer drains and incoming concentrations from the upstream boundary were found to be a major cause of water contamination. Tide, wind and diffuse sources (urban and agricultural runoff) contributed little. The high FIB inputs from the upstream open boundary come from untreated point source discharges from upstream urban areas and accumulation of diffuse contaminants from the large upstream areas. Therefore, this study underlines the need for establishment of wastewater treatment plants both in the studied basin and upstream urban areas. This study provides insight into bacterial fate and transport mechanisms, contribution of different sources to the faecal contamination and applicability of wastewater treatment in a river of a subtropical developing country where this type of study is lacking. Uncertainties are related to the lack of high temporal resolution measured FIB data and the lack of available data for contaminant loads from septic tank leakages, open defecation and sediment resuspension. However, the model well captured the measured FIB variability, suggesting that it can be applied for microbial water quality assessments in other watersheds of the world with similar characteristics.

The developed model could be an ideal tool to forecast future impacts of climate and socioeconomic changes on FIB fate, transport and dynamics. Finally, future FIB concentrations were simulated using the coupled hydrodynamic and microbial model (MIKE 21 FM-ECOLab) and scenario analysis (Chapter 5). Scenarios have been developed building on the most recent Shared Socio-economic Pathways (SSPs) and Representative Concentration Pathways (RCPs) scenarios from the Intergovernmental Panel on Climate Change (IPCC). We developed a baseline scenario (October 2014–September 2015) reflecting the current conditions and two future scenarios, S1 (sustainability scenario) and S2 (uncontrolled scenario) mimicking different future developments of socio-economic (population, urbanization, sanitation, wastewater treatment development, land use) and climate-change factors (temperature, precipitation and sea-level rise). In S1 RCP4.5 was combined with socio-economic scenarios SSP1, and for S2 RCP8.5 was combined with SSP3 (S2). Assumptions on sanitation, waste water treatment and agricultural management in line with the storylines were made to quantify future changes in FIB concentrations and consequent health risk. Different future scenarios were found to have substantial impact on FIB concentrations in the river. By the 2090s, FIB concentrations are expected to decrease by 98% or increase by 75% for the sustainability scenario and uncontrolled scenario respectively. An uncontrolled future resulted in a deterioration of microbial water quality due to socio-economic developments, such as higher population growth, land-use change and increased sewage discharges and changes in rainfall patterns. Microbial water quality strongly improved under a sustainable climate and improved sewage treatment. FIB concentrations were much more sensitive to changes in socio-economic factors than to changes in climatic factors. This underlines the importance of socio-economic factors in assessing and improving microbial water quality.

The results show the importance of improvements in sanitation and wastewater treatment in the Bangladeshi Betna River basin to ensure that future FIB concentrations in the river comply with the US-EPA bathing water quality standards. Major investments to construct wastewater treatment plants are necessary to compensate for the population growth and increased the volume of wastewater treatment. Although the current level of contamination is already too high, without wastewater treatment the water quality will further deteriorate.

The thesis assesses the present and future FIB dynamics in the Betna River through sampling, statistical and process-based modelling, and scenario analysis. The results contribute to increase the knowledge base on the dynamic distributions of the FIB in surface water in a developing country and in a subtropical system, where this type of study is lacking. It also reduces the knowledge gaps regarding future flooding scenarios at the local scale. While some earlier studies focused on only assessing climate-change impacts on microbial water quality, this study for the first time assessed the influence of combined climate and socio-economic scenarios (using scenarios based on the new SSP-RCP scenario matrix) on river FIB concentrations. This combined modelling and scenario approach enables the assessment of faecal contamination sources and dynamics at present and in the future. The developed model and scenario analysis approach provides a basis for the water managers to reduce the widespread faecal contamination and the risks of waterborne disease outbreaks, which are still a leading cause of deaths in developing countries.

Tuning for light and more : engineering phototrophy and membrane proteins in Escherichia coli
Claassens, Nicolaas J.H.P. - \ 2017
Wageningen University. Promotor(en): John van der Oost; Willem de Vos, co-promotor(en): Vitor Martins dos Santos. - Wageningen : Wageningen University - ISBN 9789463430920 - 328
escherichia coli - phototropism - membranes - proteins - light - photosystem i - gene expression - escherichia coli - fototropie - membranen - eiwitten - licht - fotosysteem i - genexpressie

The application of microbial and plant photosynthesis for biobased production on the one hand has a huge potential but on the other hand photosynthesis has serious limitations regarding its efficiency. Hence, studying both fundamental features of photosynthetic processes and engineering of photosystems is of paramount interest, exploring the engineering of photosystems is the overarching aim of this thesis. As described in Chapter 1, natural photosystems may be modified or transplanted to allow for more efficient conversion of solar light energy into biochemical energy. Hereto ambitious proposals to engineer photosystems have been made, and to realize those endeavors the disciplines of synthetic and systems biology are required. To explore how to apply and improve those disciplines hereto, the work described in this thesis has focused on the transplantation of simple photosystems (proton-pumping rhodopsins; PPRs) into the cell membrane of the heterotrophic model bacterium Escherichia coli. Both in silico analyses, including metabolic and thermodynamic modeling (Chapter 3) and a series of experimental studies on transplanting PPR photosystems (Chapters 4,6 and 7) were performed, which identified several challenges, limitations and most importantly opportunities. This thesis also describes the application of novel tools to substantially improve the functional production of PPRs and a variety of other membrane proteins in E. coli.

Chapter 2 provides more details on previously reported examples of heterologous expression of PPRs in several hosts, and on the physiological impact of these transplanted photosystems. Based on this evaluation, some suggestions are made to improve and further exploit the transplantation of these photosystems.

In Chapter 3 a systematic, integrated in silico analysis is made of anaerobic, photo-electro-autotrophic synthetic metabolism in E. coli, consisting of (i) a PPR photosystem for ATP regeneration, (ii) an electron uptake pathway, and (iii) a natural or synthetic carbon fixation pathway. Constraint-based metabolic modelling of E. coli central metabolism is used, in combination with kinetic and thermodynamic pathway analyses. The photo-electro-autotrophic designs are predicted to have a limited potential for anaerobic, autotrophic growth of E. coli, given the relatively low ATP regenerating capacity of the PPR photosystems, and the relatively high ATP consumption due to maintenance. In general these analyses illustrate the potential of in silico analyses to identify potential bottlenecks and solutions in complex designs for autotrophic and photosynthetic metabolism, as a basis for subsequent experimental implementation.

To tackle a main bottleneck of PPR systems: their functional membrane-embedded production level, the heterologous production in E. coli of the proton-pumping rhodopsins from Gloeobacter violaceus (GR) and from Thermus thermophilus JL18 (TR) is quantified and experimentally optimized in Chapter 4. High constitutive production of both rhodopsin proteins is achieved by fine-tuning transcription and translation. Besides the canonical retinal pigment, the GR system has the ability to bind a light-harvesting antennae pigment, echinenone. After optimization of the heterologous pigment biosynthesis pathways for either retinal or echinenone production, appropriate quantities of retinal or echinenone for PPR reconstitution were detected in E. coli. Association of echinenone with GR broadens its absorption spectrum in E. coli, broadening the potential for light-harvesting also to blue light. Optimization of the branched pathway for simultaneous biosynthesis of both retinal and echinenone has been attempted by using a smart library of variable Ribosome Binding Sites (RBSs) with varying strengths (RedLibs). In general, the here described approaches towards improved functional production of rhodopsin photosystems in E. coli and their pigments may prove more widely applicable for heterologous production of more complex photosystems and other systems.

In Chapter 5 an up-to-date overview is provided on how codon usage can influence functional protein production. The fact that all known organisms have an incomplete set of tRNAs, indicates that biased codon usage could act as a general mechanism that allows for fine-tuning the translation speed. Although translation initiation is the key control step in protein production, it is broadly accepted that codon bias, especially in regions further downstream of the start codon, can contribute to the translation efficiency by tuning the translation elongation rate. Modulation of the translation speed depends on a combination of factors, including the secondary structure of the transcript (more or less RNA hairpins), the codon usage landscape (frequent and more rare codons) and for bacteria also RBS-like sequences at which ribosomes can pause. The complex combination of interdependent factors related to codon usage that can influence translation initiation and elongation. This complexity makes that the design of synthetic genes for heterologous expression is still in its infancy, and despite the availability of some codon usage algorithms, it is often as well a matter of trial and error.

In Chapter 6 the effect of different codon usage algorithms (optimization and harmonization) has been experimentally tested for heterologous production of membrane proteins. Apart from the codon usage algorithms also the combined effect of transcriptional fine-tuning in E. coli LEMO21(DE3) was assessed. The overproduction of 6 different membrane-embedded proteins, including 4 PPR variants (from bacteria, archaea and eukaryotes), was tested. For production of tested PPR variants, the different codon usage algorithms hardly influenced production, while transcriptional tuning had a large impact on production levels. Interestingly, for the other two tested non-PPR membrane proteins, some codon usage variants significantly improved production on top of transcriptional tuning. For both these proteins the codon-optimization algorithm reduced functional production below that of the wild-type codon variant, while the harmonization algorithm gave significantly higher production, equal or even higher than for the wild-type variant.

In Chapter 7 it is demonstrated that a translational-tuning system can be used to successfully optimize the expression of several membrane proteins, based on initial findings presented in Chapter 4. The employed, recently developed Bicistronic Design (BCD) system is based on translational coupling of a gene encoding a short leader peptide and the gene of interest that is under control of a variable ribosome binding site. A standardized library of 22 RBSs allows for precise, gene context-independent, fine-tuning of expression of this second gene, here encoding a membrane protein. For all four membrane proteins tested in this study the BCD approach resulted in 3 to 7-fold higher protein levels than those obtained by two other recently developed methods for optimizing membrane protein production. The presented approach allows for inducer-free, constitutive, high-level production of membrane proteins in E. coli, which can be widely applicable for both membrane protein purification studies as well as for synthetic biology projects involving membrane proteins.

In Chapter 8 a broad review and perspectives are provided on the potential of microbial autotrophs for the production of value-added compounds from CO2. Both photoautotrophic and chemolithoautotrophic production platforms are discussed, and recent progress in improving their efficiency and production potential is highlighted. Transplantation efforts for photosystems, but also for CO2 fixation pathways and electron uptake systems are discussed. An overview is provided on novel in silico and experimental approaches to engineer components related to autotrophy in heterotrophic and autotrophic model hosts, including approaches applied in this thesis. Future avenues are discussed for realizing more efficient autotrophic production platforms.

Finally, in Chapter 9 and 10 the work in this thesis is summarized and a general discussion is provided on future avenues for engineering of PPR photosystems, photosystems in general and on the optimization of membrane protein production.

Trimming proline dehydrogenase : protein and cofactor minimization
Huijbers, Mieke M.E. - \ 2017
Wageningen University. Promotor(en): Willem van Berkel. - Wageningen : Wageningen University - ISBN 9789463430517 - 181
proline - thermus thermophilus - enzymes - amino acids - binding proteins - catalysts - proline - thermus thermophilus - enzymen - aminozuren - bindende eiwitten - katalysatoren

Proline is one of the proteinogenic amino acids and one of the most abundant amino acids in the cell. Next to serving as one of the non-essential amino acids, proline also has a central role in metabolism. In Chapter 1, the different functions of this imino acid are described, as well as the proline metabolic enzymes. The focus is on the enzyme proline dehydrogenase (ProDH), which catalyzes the flavin-dependent conversion of L-proline to Δ1-pyrroline-5-carboxylate (P5C). Malfunctioning of this enzyme has severe implications for human health and has been associated with tumorigenesis and schizophrenia.

This thesis deals with the engineering and biochemical characterization of Thermus thermophilus ProDH (TtProDH) in order to gain more insight into the structure-function relationship of this thermo-resistant flavoenzyme. TtProDH is a membrane-associated protein and recombinant soluble forms of the enzyme have only been obtained in limited amounts. Chapter 2 describes the heterologous production of TtProDH in Escherichia coli. Using maltose-binding protein (MBP) as solubility tag, high yields of active holoenzyme are obtained. The MBP-tag can be efficiently removed from the fusion protein with trypsin, yielding native TtProDH. This enzyme is thermotolerant as well as solvent tolerant; however, both fused and clipped TtProDH are prone to aggregation. In Chapter 3, we show that the hydrophobic N-terminal helix of TtProDH is responsible for this non-native self-association. Phe10 and Leu12, located at the protein surface, were replaced by glutamates, generating the F10E/L12E (EE) variant of MBP-TtProDH. This more polar variant exclusively forms tetramers and exhibits excellent catalytic features. Specific removal of the MBP-tag of the EE variant is less easy than for WT, as trypsinolysis of the fusion enzyme leads to degradation of TtProDH. Since the MBP tag does not influence the spectral and catalytic properties of the enzyme, further experiments were performed with MBP-tagged variants of TtProDH.

ProDH has a distorted (βα)8 TIM-barrel fold which is conserved throughout the PutA/ProDH family. In contrast, the N-terminal sequence of ProDH is poorly conserved. TtProDH contains, next to the distorted TIM-barrel, three N-terminal helices, αA, αB and αC, of which the function is not well understood. In Chapter 4, we describe the characterization of helical arm-truncated variants, lacking respectively one (ΔA), two (ΔAB), or three (ΔABC) N-terminal helices. All three variants show flavin properties that are highly similar to EE, indicating no changes in the microenvironment of the flavin isoalloxazine ring. ΔA and ΔAB are highly active tetramers, whereas removal of the complete N-terminal arm (ΔABC) results in poorly active dimers. Furthermore, EE, ΔA and ΔAB rapidly react with the suicide inhibitor N-propargylglycine, while ΔABC is not capable of forming a flavin adduct with N-propargylglycine. This indicates that helix αC has a crucial role in both the oligomerization and activity of TtProDH. Closer examination revealed an ionic interaction as well as a hydrophobic patch between helices αC and α8, the latter helix being crucial for substrate recognition. To investigate the functional role of helix αC in further detail, additional enzyme variants were created that disrupt the interactions between both helices. While disrupting the ionic interaction had minor effects, disrupting the hydrophobic patch leads to dimer formation, loss of activity and decreased reactivity with N-propargylglycine. This supports that helix αC is crucial for TtProDH catalysis and tetramerization through positioning of helix α8.

The quaternary structure of TtProDH was investigated in more detail in Chapter 5. Two ionic interactions at the dimeric interface were selectively disrupted by changing Asp205 and Glu207 of TtProDH variants EE, ΔA, ΔAB and ΔABC into lysines. These KK-variants form monomers (except for EE KK, which forms dimers) and have improved catalytic properties at moderate temperatures compared to their non-KK counterparts. However, their melting temperatures are decreased by more than 20 °C. This indicates that a trade-off is made between thermostability and catalytic activity.

In Chapter 6, we studied the cofactor binding of TtProDH. Flavoenzymes contain either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. To study the cofactor binding of TtProDH, we produced MBP-TtProDH EE in its apoform using a riboflavin auxotrophic E. coli strain. Reconstitution of the enzyme with either FAD or FMN revealed that MBP-TtProDH has no preference for FAD as cofactor. Kinetic parameters of both holo-FAD and holo-FMN are similar, as are the dissociation constants for FAD and FMN release. We show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In addition, we obtained the crystal structure TtProDH ΔABC, which shows no electron density for an AMP moiety of the cofactor. This indicates the presence of mainly FMN in the enzyme. The capability of TtProDH to display equal properties with both cofactors is unique for flavoenzymes, and classification of TtProDH as an FAD-containing enzyme should be reconsidered.

In Chapter 7, we discuss the novel findings described in this thesis and put them in a broader perspective. We have created a minimalist ProDH that is an excellent catalyst, but is deprived of all structural features that are unnecessary for in vitro functioning. Our results expand the knowledge on the structure-function relationship of ProDHs, and give insight into enzyme functionality from an industrial perspective. We also discuss how this knowledge might be used in future studies for a better understanding of the properties of eukaryotic ProDHs, with a special interest in the human enzyme.

Antibiotic resistance reservoirs : the cases of sponge and human gut microbiota
Versluis, Dennis - \ 2016
Wageningen University. Promotor(en): Hauke Smidt, co-promotor(en): Mark van Passel; Detmer Sipkema. - Wageningen : Wageningen University - ISBN 9789462579057 - 197
antibiotic resistance - reservoirs - intestinal microorganisms - luffa - forest soils - sediment - escherichia coli - penicillium - faecal examination - antibioticaresistentie - reservoirs - darmmicro-organismen - luffa - bosgronden - sediment - escherichia coli - penicillium - fecesonderzoek

One of the major threats to human health in the 21st century is the emergence of pathogenic bacteria that are resistant to multiple antibiotics, thereby limiting treatment options. An important route through which pathogens become resistant is via acquisition of resistance genes from environmental and human-associated bacteria. Yet, it is poorly understood to what extent and by what mechanisms these so-called reservoirs contribute to emerging resistance. Therefore, the work described in this thesis focussed on generating novel insights into different niches as sources of resistance, with a particular focus on the human gut microbiota as well as on microbial communities associated with marine sponges, especially because the latter have been described as one of the richest sources of bioactive secondary metabolites, including a broad range of antimicrobials. Cultivation-based methods were complemented with culture-independent approaches in order to study bacterial taxa that are not readily cultivated.

Using metatranscriptomics it was found that clinically relevant antibiotic resistance genes are expressed in a broad range of environmental niches including human, mouse and pig gut microbiota, sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment. The diversity of resistance gene transcripts differed greatly per niche indicating that the environment contains a rich reservoir of functional resistance that could be accessible by pathogens. Even though resistance gene expression might be linked to the presence of natural antibiotics, we did not detect expression of the corresponding secondary metabolite biosynthesis clusters.

Thirty-one antibiotic-resistant bacteria, amongst which three belonging to potentially novel Flavobacteriaceae spp., were isolated from the Mediterranean sponges Aplysina aerophoba, Corticium candelabrum and Petrosia ficiformis. Isolates were identified in a high throughput manner by double-barcoded 16S rRNA gene amplicon sequencing. Furthermore, analysis of sponge tissue-derived bacterial biomass growing on agar media showed that many novel bacterial taxa can still be isolated by conventional cultivation methods. Genomic DNA from the 31 antibiotic resistant bacteria was interrogated with respect to the presence of active resistance genes by functional metagenomics. In addition, we also screened metagenomic libraries prepared from DNA directly isolated from sponge tissue in order to circumvent the need for cultivation. In total, 37 unique resistance genes were identified, and the predicted gene products of 15 of these shared <90% amino acid identity with known gene products. One resistance gene (blaPSV-1), which was classified into a new β-lactamase family, was found to be exclusive to the marine specific genus Pseudovibrio. These findings raised questions as to the functional roles of these genes in sponges, but more importantly, the functionality of these genes in E. coli shows that they can potentially be harnessed by phylogenetically distinct bacteria in other environments, including human pathogens. As such, it is a wake-up call as to the significance of marine resistance reservoirs.

Pseudovibrio, a genus of α-Proteobacteria, was studied in more detail by comparative genomics as it comprises bacteria that potentially play a role as sponge symbionts and marine hubs of antibiotics resistance. Based on gene content, members of the genus Pseudovibrio were found to cluster by sponge sampling location indicating geographic speciation. Furthermore, Pseudovibrio spp. isolated from sponges near the Spanish coast clustered by sponge, suggesting host-specific colonization or adaptation. Strong support for Pseudovibrio spp. forming symbiotic relations with sponges came from the presence of a plethora of (predicted) conserved symbiosis-related functions in their genomes.

A final study aimed to isolate novel antibiotic resistant reservoir species from the human gut microbiota using a targeted approach. Faecal samples from hospitalized patients that received Selective Digestive Decontamination (SDD), a prophylactic treatment with a cocktail of different antibiotics (tobramycin, polymyxin E, amphotericin B and cefotaxime), were inoculated anaerobically on agar media, after which bacterial biomass was analysed by 16S rRNA gene amplicon sequencing. Six novel taxa were identified that, based on their growth on media supplemented with the SDD antibiotics, could serve as clinically relevant reservoirs of antibiotic resistance. For one of these six taxa a member was obtained in pure culture by targeted isolation. The abundance of antibiotic resistant uncultivated taxa in the human gut microbiota warrants further research as to their potential roles in resistance dissemination.

In conclusion, this thesis provides deeper insights into different environmental niches as reservoirs of antibiotic resistance. The results can serve to prime and inspire future research.

Improvement of methods for the detection of Gram-negative foodborne pathogens
Margot, H.F.T. - \ 2016
Wageningen University. Promotor(en): Marcel Zwietering; Han Joosten, co-promotor(en): R. Stephan. - Wageningen : Wageningen University - ISBN 9789462578708 - 158
gram negative bacteria - pathogens - foodborne pathogens - detection - real time pcr - salmonella - escherichia coli - mung beans - gramnegatieve bacteriën - pathogenen - voedselpathogenen - detectie - real time pcr - salmonella - escherichia coli - mungbonen

Foodborne diseases are a major source of morbidity and mortality worldwide. In most cases, these diseases are caused by contaminated food products, but transmission can also subsequently occur via person to person contact. The ability to detect the pathogens is an important aspect in the verification of food safety. A major proportion of foodborne disease is caused by Gram-negative bacteria. In this thesis, the detection of Gram-negative foodborne pathogens is addressed by looking at the successive steps from enrichment to detection with Salmonella, Shiga toxin-producing E. coli and Cronobacter spp. as example pathogens. The detection of foodborne pathogens using microbiological culture media aiming at the resuscitation and growth of bacteria is still regarded as the gold standard and included in many reference methods. However, cultural methods are time and labour-intensive. Since an immediate response is required in case of contamination and during outbreaks there is a strong interest in methods that deliver information on the microbiological status of the product as quickly and reliable as possible. Rapid cultural methods and commercially available real-time PCR systems for the detection of Salmonella and STEC were compared with regards to their sensitivity and specificity. It was shown that most of the marketed systems are as reliable as the standard methods. However, false-positive results were obtained with real-time PCR systems for the detection of Salmonella. Rapid cultural methods that were based on procedures without the pre-enrichment step, reduced the time to detection but did show some ambiguous results with difficult matrices such as tea. Of the seven rapid tests for the detection of STEC, one did not detect relevant Stx subtypes.

In order to be detected, pathogens need to multiply to reach a minimum threshold level. However, because they are often sublethally injured due to hostile processing and storage conditions, they first need to be resuscitated. For most pathogens, (Salmonella, STEC and Cronobacter spp.) the first step in the detection is an enrichment including resuscitation in a non-selective medium such as BPW. Modifications to BPW were compared with respect to their ability to promote growth of unstressed and stressed Gram-negative pathogens. The aim was to develop a medium that could be used for the enrichment of pathogens in horizontal methods using only one enrichment step. The resuscitation of stressed Cronobacter cells was improved in BPW supplemented with an additional iron source and sodium pyruvate along with low levels of compounds for the inhibition of Gram-positive bacteria. However, it was observed that BPW containing these supplements allowed for less resuscitation of STEC when compared to regular BPW. Based on these results it was concluded that the application of a one-broth enrichment in food products with a high number of competing bacteria is not recommended due to the overgrowth of the target bacteria. Limitations of the current method for the detection of STEC from sprouted seeds were noticed. Therefore, the growth of stressed STEC cells from different serotypes was assessed in media used for the enrichment of Enterobacteriaceae. In addition, the growth of STEC was examined in the enrichment of sprouts using different media and incubation temperatures. It was shown that the high level of competitors was inhibiting the detection of the target pathogen and that the similarity of target and competing bacteria prevents the design of a selective enrichment procedure. In order to get a better insight in the enrichment ecology, the microbiome of mungo bean sprouts was analysed using Illumina HiSeq sequencing prior to and during the enrichment in BPW and EE-broth at different temperatures. The majority of the sprout flora was composed of bacteria belonging to the phylum Proteobacteria. Enrichment in BPW increased the proportion of Firmicutes whereas the incubation in EE-broth enriched Proteobacteria. The results point out that with the application of a selective medium like EE-broth, growth of the competitive microflora that complicates the detection of STEC is promoted. It was shown that EE-broth also resulted in good growth of STEC however, the problematic situation of low maximum population densities of the target strain in the matrix is still present. The probability of detection is not only influenced by the natural flora of a food product, but also by the physiological state of the pathogen. The influence of stress on the lag time of single cells and the resulting probability of detection were determined for Cronobacter spp. in powdered infant formula. Lag time was calculated from optical density measurement data and different scenarios were modelled. Lag time was longest after acid stress and lag time increase coincided with increased lag time variability. The probability of detection, however, depended both on the sampling plan and on the duration of the lag phase.

This thesis provides a critical evaluation of rapid methods and valuable new insights on enrichment procedures, the role of competitors in bacterial enrichment procedures and the limitations of selective agents. This information will be of great help to further improve microbiological methods and thereby contribute to more effective management of food safety.

Biomarkers and mechanisms of natural disease resistance in dairy cows
Altena, S.E.C. van - \ 2016
Wageningen University. Promotor(en): Huub Savelkoul, co-promotor(en): Edwin Tijhaar. - Wageningen : Wageningen University - ISBN 9789462578005 - 158
dairy cows - biomarkers - disease resistance - immunity - antibodies - proteomics - immune response - dendritic cells - immunology - melkkoeien - biomarkers - ziekteresistentie - immuniteit - antilichamen - eiwitexpressieanalyse - immuniteitsreactie - dendritische cellen - immunologie

The aim of this thesis was to define and test biomarkers for disease resistance in dairy cows and to determine the underlying mechanism in natural disease resistance. The health status of the cows is an important issue in dairy farming. Due to the mandatory reduction in the use of antibiotics, alternatives are required to prevent the development and expression of illness in dairy cows. The identification of biomarkers associated with such disease offers the opportunity to adapt the management of cows at risk, and in this way, prevent them from developing overt disease. Previously, natural antibodies (NAbs) in serum and milk were used as candidate biomarkers for natural disease resistance in cows. In this thesis, we continue on the occurrence and mode of action of NAbs and also focus on their source: the B-1 cells. We performed a literature study on the identification and function of B-1 cells in different species and defined the limitations in the current identification of these cells in pigs, sheep and cows (Chapter 2). B-1 cells were described in cows by using widely accepted cell surface markers CD5 and CD11b. However, in literature several findings suggest that these cell surface markers are not unique markers for B-1 identification. The similarities between mice and veterinary animals in foetal B-cell development and antibody production, implies that B-1 cells are present in cows. In chapter 3, we carefully studied new markers to selectively identify B-1 cells in cows. The combination of B-1 cell markers IgM++ and pSYK++ (indicator constitutive intracellular signalling) identifies a distinct cell population with essential B-1 characteristics such as high CD80 expression. In addition, the development of these B-1 cells in calves before colostrum intake and 3 weeks afterwards shows the same kinetics as the development of NAbs represented by IgM antibodies binding to the well-accepted NAb-antigen phosphatidylcholine (PtC). In calves up to half a year of age, it is shown that the production of such NAbs increases from birth and stabilises from 6 weeks onwards. This implies an endogenous NAb production, which follows the same age-related kinetics as can be expected from B-1 cell development. In contrast, the development of total IgM antibody levels in calves shows a bimodal distribution, which is caused by the uptake and breakdown of maternally-derived IgM and simultaneous endogenous production of specific and natural IgM. Chapter 4 describes the role of such NAbs in bovine immunity. NAbs were represented by the binding of IgM to the naïve antigen keyhole limpet hemocyanin (KLH). Cows with high serum NAb levels were shown to have more IgM and IgG antibodies binding to common microbial structures LPS, LTA and PGN, than cows with low serum NAb levels. In addition, they also have more IgM antibodies binding to intact, fixed E. coli and S. Typhimurium bacteria. However, the killing of live E. coli and S. Typhimurium bacteria via antibody-mediated complement killing does not differ between cows with high and low NAb levels. The antibody-mediated complement killing was determined in a newly developed serum bactericidal test. Cows that performed less in the bactericidal test were more likely to develop mastitis in the future. This association was observed for the killing of E. coli and S. Typhimurium and the development of mastitis within the next one year. For S. Typhimurium the association was still present for the cases of mastitis occurring within four years after testing. Alternative biomarkers for disease resistance in cows were defined in chapter 5 by using a contemporary proteomics approach. Milk samples from high and low disease resistant cows were selected from the “Resilient Cattle” (Weerbaar Vee) biobank. Comparing the spectrum of milk proteins of high and low disease-resistant cows showed potential candidate biomarkers that were elevated in the milk of low-resistant cows. Two candidate marker proteins were validated with ELISA in a new and larger group of high- and low-resistant cows. Lactoferrin (LF) levels were significantly increased in milk of low-resistant cows. In addition, LF levels in milk were associated with clinical manifestations of lameness and had a predictive value for subsequent culling.

In conclusion, we found that also in cows NAbs are produced by B-1 cells that can be identified based on the combined expression of cell surface IgM and internal pSYK. In addition, the frequency of these B-1 cells after birth follows a similar kinetics as described before in mice. These NAbs can be more precisely identified based on their PtC binding ability and their functional activity in a bactericidal test. However, the true predictive value of B-1 cells and NAbs for the health status and immunocompetence of dairy cattle remains to be established. Proteomics turned out to be a useful approach for identifying potential new biomarkers for health and disease in milk of cows. Application and further development of their predictive capacity is dependent on the availability of robust, sensitive and quantitative assays. This project was part of the “Resilient Cattle” project providing biological samples and essential data on the health status during respective lactation periods of individual dairy cows. The impact of this research now requires translation into management tools and principles for the individual farmer impacting on the overall health status and economic performance of his herd of dairy cattle.

Tospovirus : induction and suppression of RNA silencing
Hedil, Marcio - \ 2016
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Richard Kormelink. - Wageningen : Wageningen University - ISBN 9789462577848 - 137
tospovirus - rna - plants - immunity - gene silencing - biochemical pathways - rna interference - viral proteins - plant viruses - tospovirus - rna - planten - immuniteit - uitschakelen van genexpressie - biochemische omzettingen - rna-interferentie - viruseiwitten - plantenvirussen

While infecting their hosts, viruses must deal with host immunity. In plants the antiviral RNA silencing pathway is an important part of plant innate immunity. Tospoviruses are segmented negative-stranded RNA viruses of plants. To counteract the antiviral RNA silencing response in plants, tospoviruses have evolved a silencing suppressor function via its NSs protein. This viral protein has previously been shown to bind dsRNA that likely arises from secondary RNA folding structures in viral RNAs. The aim of the present research was to further investigate the interaction between tospoviruses and the plant antiviral RNA silencing response, including the target sequences in the viral RNA and the further role of the NSs protein as part of the tospovirus counterdefence strategy.

In order to identify the target and inducer for RNA silencing against tospoviruses, small RNAs purified from plants infected with three tospoviral species, tomato spotted wilt virus (TSWV), groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), were probed against the viral RNA segments of these three different tospoviruses (Chapter 3). Virus-derived siRNAs (vsiRNAs) were found to be derived from all three genomic RNA segments but predominantly the ambisense M and S RNAs. Further profiling on the S RNA sequence revealed that vsiRNAs were found from almost the entire S RNA sequence, except the predicted AU-rich hairpin (HP) structure encoded by the intergenic region (IGR) from where hardly any vsiRNAs were found. Similar profiles were observed with the closely related GRSV as well as the distantly related TYRV. Dicer cleavage assays using Drosophila melanogaster embryo extracts showed that synthetic transcripts of the IGR-HP region were recognized as substrate for Dicer. Transient agroinfiltration assays of a GFP-sensor construct containing the IGR-HP sequence at its 3′-UTR did not show more rapid/strong silencing, and profiling of the corresponding siRNAs generated outside the context of a viral infection still revealed relatively low levels of IGR-HP-derived siRNAs. These data support the idea that the IGR-HP region/structure is a weak inducer of RNA silencing and plays a minor role in the amplification of a strong antiviral RNA silencing response.

Next, a biochemical analysis was performed using E. coli-expressed and purified NSs from GRSV and TYRV. The binding of both purified NSs proteins to small and long dsRNA indicated that this is likely a generic feature of all tospoviral NSs proteins (Chapter 4). Binding of siRNAs to NSs furthermore revealed two shifts on polyacrylamide gels i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When the NSs protein of TSWV resistant breaker (RB) isolates (of Tsw-gene based resistance), which lack RSS activity when transiently expressed, were analyzed using extracts from infected plants still a major (second) shift of siRNAs was observed, similar to the case with extracts containing TSWV resistant inducer (RI) isolates. In contrast, plant extracts containing transiently expressed NSs proteins alone (no infection) showed only the smaller, first shift for NSsRI but no shift for NSsRB.

The ability of NSs to suppress systemic silencing is demonstrated for the NSs proteins of TSWV, GRSV and TYRV, and their relative strengths to suppress local and systemic silencing were compared (Chapter 5). A system was developed to quantify suppression via GFP silencing constructs, allowing comparison of relative RNA silencing suppressor strength. In this case NSs proteins of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Moreover, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream of the biogenesis of siRNAs and their sequestration.

In summary, evidence is presented showing that sequences from all three genomic segments from tospovirus are targeted by the plant RNA silencing machinery. The predicted hairpin sequence in the IGR is poorly targeted. Biochemical experiments with purified NSs proteins further support the view that binding to small and long dsRNA is a characteristic common to all tospovirus NSs proteins. Furthermore, tospovirus NSs proteins suppress systemic silencing and there are indications that local and systemic silencing suppression can be uncoupled in NSs. Collectively, these results add to our current understanding of the tospovirus-plant interaction involving antiviral RNA silencing and the viral counter-defence (NSs protein). Lastly, the results of the research presented in this thesis are discussed in light of the current knowledge on RNA silencing and to present some future perspectives and questions that remain open and/or resulted from this thesis (Chapter 6).

Beehold : the colony of the honeybee (Apis mellifera L) as a bio-sampler for pollutants and plant pathogens
Steen, J.J.M. van der - \ 2016
Wageningen University. Promotor(en): Huub Rijnaarts, co-promotor(en): Tim Grotenhuis; Willem Jan de Kogel. - Wageningen : Wageningen University - ISBN 9789462577510 - 206
apis mellifera - honey bees - honey bee colonies - biological indicators - sampling - instruments - pollution - pollutants - heavy metals - plant pathogenic bacteria - erwinia amylovora - erwinia pyrifoliae - analytical methods - apis mellifera - honingbijen - honingbijkolonies - biologische indicatoren - bemonsteren - instrumenten (meters) - verontreiniging - verontreinigende stoffen - zware metalen - plantenziekteverwekkende bacteriën - erwinia amylovora - erwinia pyrifoliae - analytische methoden

Bio-sampling is a function of bio-indication. Bio-indication with honeybee colonies (Apis mellifera L) is where the research fields of environmental technology and apiculture overlap. The honeybees are samplers of the environment by collecting unintentionally and simultaneously, along with nectar, pollen, water and honeydew from the flowers or on the leaves, other matter (in bio-indication terms: target matter) and accumulating this in the colony. Collected target matter, in this thesis heavy metals, the plant pathogens Erwinia pyrifoliae and Erwinia amylovora and the soil pollutant γ-HCH, is collected from the colony by subsampling. Subsampling the honeybee colony is done by taking and killing bees from the hive (sacrificial) or by collecting target matter from the bee’s exterior without killing the bee (non-sacrificial). In environmental technology terms the application of the honeybee colony is a Passive Sampling Method (PSM). In this thesis the possibilities and restrictions of the PSM honeybee colony are explored.

Bio-indication is a broad research field with one common factor: a living organism (bio) is applied to record an alteration of the environment (indication). The environment may be small such as a laboratory or big such as an ecosystem. Alterations in the organism may vary from detecting substances foreign to the body to mortality of the organism. In environmental technology the concept Source-Path-Receptor (SPR) is applied to map the route of a pollutant. It describes where in the environment the pollution is, how it moves through the environment and where it ends. This environment is the same environment of all living organisms, ergo also honeybees. Honeybees depend on flowers for their food. In the SPR concept, a flower can be a source, path or receptor. Along with collecting pollen, nectar, water and honeydew, target matter is collected by honeybees. Each honeybee functions as a micro-sampler of target matter in the environment, in this case the flower. Each honeybee is part of a honeybee colony and in fact the honeybee colony is the bio-sampler. The honeybee colony is a superorganism. The well-being of the colony prevails over the individual honeybee. Food collection is directed by the colony’s need. Foragers are directed to the most profitable food sources by the bee dance and food exchange (trophallaxis). The result of this feature is that mainly profitable sources are exploited and poor food sources less or not at all. During the active foraging period hundreds to thousands of flowers are visited daily. The nectar, pollen, water and honeydew plus the unintentionally collected target matter is accumulated in the honeybee colony. In order to obtain target matter the colony must be subsampled. This is done by picking bees from the hive-entrance (hive-entering bees) or inside the hive (in-hive bees) and processing them for analysis (sacrificial). This is the most commonly applied method. However, it is possible to subsample the colony without picking and processing the bees by collecting target matter from the hive-entering bee’s exterior (non-sacrificial). For non-sacrificial subsampling of the honeybee colony the Beehold device with the sampling part Beehold tube has been developed. The results of bio-indication with honeybee colonies are qualitative and indicative for follow up study (Chapter 1).

Six bio-indication studies with honeybee colonies for bio-indication of heavy metals, the plant pathogens Erwinia pyrifoliae and Erwinia amylovora and the soil pollutant γ-HCH are presented. Chapter 2 describes how the concentration of eighteen heavy metals in honeybees fluctuate throughout the period of July, August and September (temporal) at the study sites: the city of Maastricht, the urban location with an electricity power plant in Buggenum and along the Nieuwe Waterweg at Hoek van Holland (spatial). A number of the metals have not been previously analysed in honeybees. To study whether honeybees can be used for bio-indication of air pollution, the concentrations of cadmium, vanadium and lead were compared to concentrations found in honeybees. The honeybee colonies were placed next to the air samplers. Only significant differences of metal concentrations in the ambient air also show in honeybees. This was the case with vanadium in ambient air and honeybees. The spatial and temporal differences of cadmium and lead were too futile to demonstrate a correspondence (Chapter 3). In a national surveillance study in 2008 the concentration of eighteen metals in honeybees has been analysed. The results showed a distinct regional pattern. Honeybees in the East of the Netherlands have higher concentrations of heavy metals compared to the bees in the West. Besides regional differences local differences were also recorded. An approximate description of the land use around 148 apiaries (> 50% agriculture, > 50% wooded area, > 50% urban area and mixed use) indicated the impact of land use on metal concentrations in honeybees. In areas with > 50% wood significantly higher concentrations of heavy metals were detected (Chapter 4). Subsampling of the honeybee colonies in Chapter 2, 3 and 4 was done sacrificially. In the studies presented in Chapter 5, 6, and 7 the honeybee colonies were subsampled non-sacrificially or simultaneously non-sacrificially and sacrificially. The plant pathogen E. pyrifoliae causes a flower infection in the strawberry cultivation in greenhouses. In greenhouse strawberry cultivation honeybees are applied for pollination. In Chapter 5 the combination pollination / bio-indication by honeybee colonies is studied. This proved to be a match. E. pyrifoliae could be detected on in-hive bees prior to any symptom of the infection in the flowers. In the Beehold tube, the bacterium was detected at the same time as the first tiny symptoms of the infection. In Chapter 5 the principles on which the Beehold tube is based are presented and discussed. The plant pathogen E. amylovora causes fireblight in orchards. The combination pollination / bio-indication has also been applied in this study performed in Austria in 2013. It is known that E. amylovora can be detected on honeybees prior to any symptom in the flower or on the fruit tree. A fireblight outbreak depends on flowering period, humidity and temperature. In 2013 no fireblight infection emerged in the orchards where the study was performed. Therefore, the bacterium could not be detected on the honeybees. γ-HCH (Lindane) is one of the soil pollutants in the Bitterfeld region in Saxony-Anhalt in Germany. It is the result of dumping industrial waste around the production locations. Although γ-HCH is bound to soil particles there is a flux to groundwater and surface water. Consequently, the pollution may end up in the sediments of the streambed and flood plains. The study objective was to investigate the hypothetic route of γ-HCH from polluted soil (source), via soil erosion and atmospheric deposition (route) to the receptor (flowering flowers) by detecting γ-HCH in the Beehold tube. Although on average over 17000 honeybees passed through the Beehold tube daily for a maximal period of 28 days, no γ-HCH has been detected. The pollen pattern in the Beehold tube revealed where the bees collected the food (Chapter 7).

The application of the honeybee colony has pros and cons. Distinctive pros are many micro samplers, the extensive collection of matter (both food and target matter) and the accumulation in the colony. For successful bio-indication with honeybee colonies, determining factors are: the target matter, location of the target matter, distance between target matter and the honeybee colony, individual or pooled subsampling, the minimal sampling frequency and sample size, and sacrificial or non-sacrificial subsampling applied solely or in combination. Taking bees from a colony impacts upon the colony’s performance and consequently the passive sampling method. Based on a long-years’ experience and inter-collegial discussion it is stated that 3% of the forager bees (hive-entering) and 1.5% of the in-hive bees can be sampled safely without impacting upon the colony. This restriction does not apply when carrying out non-sacrificial subsampling of the honeybee colony (Chapter 8).

Performing bio-indication with honeybee colonies has more applications than have been exploited so far. Further research can make a change. In particular I mention here the combination of pollination and bio-indication and the application of non-sacrificial subsampling solely or in combination with sacrificial subsampling.

Everywhere Apiculture is practiced (all over the world except the polar areas) bio-indication with honeybee colonies can be applied in a simple, practical and low cost way.

Transmission of antibiotic resistance from animals to humans : Broilers as a reservoir of ESBL-producing bacteria
Huijbers, P.M.C. - \ 2016
Wageningen University. Promotor(en): Mart de Jong; Lisette Graat; E. van Duijkeren. - Wageningen : Wageningen University - ISBN 9789462576216 - 156
broilers - man - disease transmission - antibiotic resistance - bacteria - enterobacteriaceae - poultry farming - epidemiology - vleeskuikens - mens - ziekteoverdracht - antibioticaresistentie - bacteriën - enterobacteriaceae - pluimveehouderij - epidemiologie

Huijbers, P.M.C. (2016). Transmission of antibiotic resistance from animals to humans: Broilers as a reservoir of ESBL-producing bacteria. PhD thesis, Wageningen University, Wageningen, the Netherlands.

Antibiotic resistance in animals becomes a public health issue when there is transmission of antibiotic resistant bacteria, or their resistance genes, from animals to humans. β-lactam antibiotics are critically important for the treatment of human bacterial infections. Resistance to this class of antibiotics, mediated by extended-spectrum β-lactamases (ESBL) has emerged. Broilers might contribute to transmission to humans due to the high prevalence of ESBL-producing Enterobacteriaceae among their intestinal biome, compared to other livestock species, companion animals, and wildlife. Transmission to humans might occur via the food chain, by direct contact or via the environment. The aim was to investigate transmission of antibiotic resistant bacteria between animals and humans, and more specifically transmission of ESBL-producing E. coli between broilers, and between broilers and humans in varying degrees of contact with these animals. Systematically collected and categorised evidence from literature showed that clinically relevant antibiotic resistant bacteria were present in the natural environment, that is in soil, water, air and wildlife. It was therefore hypothesised that humans in areas with high broiler densities might have an increased risk for carriage of ESBL-producing Enterobacteriaceae. This hypothesis was rejected, as the observed risk was lower for these individuals. The situation might be different for individuals living on broiler farms as ESBL-producing E. coli were detected on all investigated farms. Among broilers, the within farm prevalence approached 100%, and there was no difference between conventional and organic farms at five weeks, i.e. just before slaughter on conventional farms. On organic farms, the prevalence decreased to 80.0% at 70 days, i.e. slaughter age. Not only transmission to humans via the farm environment, but close physical contact with broilers might, therefore, lead to increased risk for carriage. Prevalence among farmers, their family members and employees on both conventional (19.1%) and organic (18.5%) broiler farms was higher compared to humans in the general population (5.1%). Moreover, people in close contact with live broilers showed the highest risk (27.1 vs. 14.3%). Evidence for clonal transmission of ESBL-producing E. coli between humans and broilers was found on conventional farms, and horizontal gene transfer was suspected on both conventional and organic farms. Even without selection pressure from antibiotics ESBL-producing E. coli were able to transmit and persist in an organic broiler flock, which shows that broilers form a reservoir of antibiotic resistance genes. This leads to an increased risk of carriage of humans on farms through direct contact with broilers and possibly via the direct farm environment. As only a very small percentage of the general population is exposed to live broilers, direct contact with broilers does not appear to be important for carriage in the general human population.

Biofunctionalized nanoporous aluminum oxide culture chips : for capture and growth of bacteria
Debrassi, A. - \ 2016
Wageningen University. Promotor(en): Han Zuilhof; Willem de Vos; Tom Wennekes. - Wageningen : Wageningen University - ISBN 9789462576179 - 218
aluminium oxide - porous media - unimolecular films - immobilization - bacteria - binding - antibodies - aluminiumoxide - poreus medium - unimoleculaire films - immobilisatie - bacteriën - binden - antilichamen

Porous aluminum oxide (PAO) is a nanostructured material used for various biotechnological applications, including the culturing microorganisms and other types of cells. The ability to chemically modify the PAO surface and tailor its surface properties is a promising way to expand and refine its applications. The immobilization of biomolecules on PAO that specifically interact with and bind to target bacteria would enable the capture and subsequent growth of bacteria on the same surface, and this was the ultimate goal of the research presented in this thesis.

After a general introduction to the overall subject of this thesis, presented in Chapter 1, the most commonly used and recent methods to prepare glycosurfaces are reviewed and compared on their merits and drawbacks in Chapter 2. Although there are a great number of techniques, the main challenge that still remains is to develop an accessible, reproducible and inexpensive approach that produces well-defined and stable glycosurfaces using as few steps as possible. The most used analytical techniques for the characterization of glycosurfaces and several applications of these surfaces in the binding, capture, and sensing of bacteria and bacterial toxins were also discussed in Chapter 2.

Biofunctionalization of surfaces in general requires a stepwise approach, in which it is very important to have a stable monolayer as the first step. At the beginning of this research it was known that various functional groups were able to react with (porous) aluminum oxide, but there was no comprehensive study comparing the stability of these modified surfaces under the conditions that are important for microbiological applications. In Chapter 3, the PAO surface was modified with various functional groups known to react with PAO (carboxylic acid, α-hydroxycarboxylic acid, alkyne, alkene, phosphonic acid, and silane), and the stability of these modified surfaces was assessed over a range of pH and temperatures that are relevant for microbial growth. Silane and phosphonate-modified PAO surfaces with a hydrophobic monolayer proved to be the most stable ones, but the phosphonate modification was both more easily applied and reproducible. This modification was stable for at least two weeks in buffer solutions with pH values between 4 and 8, and at temperatures up to 40 °C. Only at elevated temperatures of 60 °C and 80 °C under hydrolytic conditions it was observed that the stability of the same monolayer on PAO decreased gradually. As a proof-of-principle for the biofunctionalization and bacterial capture on this PAO phosphonate monolayer, an alkyne-terminated monolayer was biofunctionalized via a CuAAC click reaction with an azido-mannoside and the binding and growth of Lactobacillus plantarum was successfully demonstrated.

In Chapter 4 various approaches to install reactive groups onto the phosphonate-modified PAO surface were developed, creating a (bio)functionalization “tool-box”. PAO surfaces presenting different terminal reactive groups were prepared, such as azide, alkyne, alkene, thiol, isothiocyanate, and N-hydroxysuccinimide (NHS), starting from a single, straightforward and stable initial modification with a bromo-terminated phosphonic acid. These reactive surfaces were then used to immobilize (bio)molecules, including carbohydrates and proteins. Fluorescently labeled bovine serum albumin (BSA) was covalently immobilized on the PAO surface as a proof-of-principle, and it was shown that a range of bacteria could still grow on the BSA-functionalized PAO surface.

With a PAO (bio)functionalization tool-box in hand, the successful proof-of-principle mannoside-dependent binding and growth of L. plantarum on PAO (Chapter 3) was further investigated and expanded upon (Chapter 5). The parameters involved in the preparation of these surfaces and in the binding with L. plantarum were investigated in more detail in Chapter 5, such as the nature of the spacer connected to the mannoside derivative and the presence of soluble carbohydrates and bovine serum albumin (BSA) in the medium. The surfaces with the azido-mannoside with the long hydrophobic spacer showed the best binding of L. plantarum when compared to a long PEG-based hydrophilic spacer and a short hydrophobic one. The presence of a soluble a-glucoside did not prevent the binding of the bacteria to the mannose-presenting PAO, and similar results were obtained when BSA was present. Additionally, a mutant strain of L. plantarum that does not have the mannose-specific adhesion was not able to bind to the mannose-presenting PAO. When taken together, this proves that the mannoside–adhesin interaction is the main mechanism of binding the bacteria to the mannose-biofunctionalized PAO in this system.

In Chapter 6, the NHS-terminated PAO developed in Chapter 4 was used for the immobilization of antibodies against Escherichia coli. After an extensive optimization of the modification chemistry of the surfaces and the incubation conditions, commercially available anti-E. coli antibodies were immobilized on the PAO surface. Binding and washing experiments indeed demonstrated increased binding of E. coli on the antibody-presenting PAO surfaces, providing avenues for testing other bacteria such as Lactobacillus rhamnosus GG widely used in probiotic formulations worldwide.

In Chapter 7, the most important achievements of this project are discussed, together with additional ideas and recommendations for further research. Most notably some preliminary results are presented on the immobilization of two antibodies against L. rhamnosus GG: anti-L. rhamnosus GG, against the whole bacterial cell, and anti-SpaC, against only the SpaC part of the pili present on the cell surface of L. rhamnosus GG. Anti-L. rhamnosus GG antibody showed promising but not yet optimal increased binding of L. rhamnosus GG. Finally, some reflections on PAO and its (bio)functionalization are provided in the context of a risk analysis and technology assessment.

Bio-filtration of helminth eggs and coliforms from municipal sewage for agricultural reuse in Peru
Yaya Beas, R.E. - \ 2016
Wageningen University. Promotor(en): Grietje Zeeman; Jules van Lier; Katarzyna Kujawa. - Wageningen : Wageningen University - ISBN 9789461734945 - 187
waste water treatment - waste water treatment plants - anaerobic treatment - helminth ova - anaerobic conditions - filtration - public health - afvalwaterbehandeling - afvalwaterbehandelingsinstallaties - anaërobe behandeling - wormeneitjes - anaërobe omstandigheden - filtratie - volksgezondheid

Where fresh water resources are scarce, treated wastewater becomes an attractive alternative for agricultural irrigation. However, the presence of large amounts of pathogens, even in treated wastewater, constraints its productive use, which is aggravated when sanitation and public health are poor. Among pathogenic indicators, helminth eggs are one of the most persistent microorganisms in treated effluents that may survive for several months in the irrigated fields. Application of upflow anaerobic sludge blanket (UASB) reactors could contribute to decrease the pathogenic content in wastewater due to physical and biological interactions with the anaerobic sludge bed, such as filtration and entrapment. In this thesis, the potential of the anaerobic sludge bed to particularly remove helminth eggs, was investigated in four phases. In the first phase, a temperature of 4° C was fixed in the UASB reactors in order to reduce the biological activity of the sludge. Hence, the anaerobic sludge filtration capacity at different upflow velocities was studied. This phase of the research was performed in two experiments. The first one using latex beads, simulating helminth eggs, and the second one using real helminth eggs, predominating in Peruvian wastewater. First experimental results show that increasing the upflow velocity led to a decrease in the removal efficiency of latex beads. At the lowest upflow velocity of 0.3 m·h−1, 100% removal of latex beads was reached. At an upflow velocity higher than 1 m·h−1, the removal efficiency dropped under 90 %. The degree of stabilisation of the sludge nor the sludge bed volume did not have a significant effect. Second experiment's results show that with upflow velocities below 1.5 m·h−1 real helminth eggs removal is greater than 70 %. Simultaneously tested, total and faecal coliforms removal was less than 83 %. The most common helminth eggs species found in the studied wastewater were Ascaris lumbricoides, Trichuris spp. and Strongyloides spp. The second phase was performed using two lab-scale UASB reactors at average ambient temperatures between 16.7 °C and 28.5 °C in the city of Lima (Peru). Ascaris suum eggs originating from infected pigs were selected as model organisms, considering their similarity, in terms of size and morphology, with Ascaris lumbricoides, a human pathogen. The sludge filtration capacity was determined, applying upflow velocities between 0.09 and 0.68 m·h−1. Average helminth eggs removals varied between 26 and 93 %, depending on upflow velocity and sludge bed height. 93 % removal was achieved when applying an upflow velocity of 0.09 m·h−1 and a sludge bed height reaching 19-25 % of the total reactor height. The third phase was conducted to test the effect of lower operational temperatures in the UASB reactor on the pathogen removal from domestic wastewater. Thus, a lab scale UASB reactor in the city of Puno (Peru), treating wastewater with temperatures varying between 11.3 and 14.3 °C for a period of 22 weeks after the start-up of the reactor, was used. Upflow velocities varied between 0.12 and 0.41 m·h−1. Results confirmed outcomes of the first phase of this research concerning helminth eggs removal, and consequently show that the sludge bed filtration capacity varied between 89 and 95 %. Faecal coliform removal varied between 0.9 and 2.1 log10 and E. coli removal between 0.8 and 1.6 log10. In general, removal efficiencies regarding helminth eggs and faecal coliforms, are not sufficient to comply with reuse standards. Finally, the capacity of Down Flow Hanging Sponge (DHS) reactors for removing faecal coliforms from domestic UASB reactor effluent for agricultural reuse in developing countries was investigated. Applied reactors were the cube type DHS (G1) without recirculation, the cube type DHS (G1) with recirculation and the curtain type DHS (G2). Results reveal an average faecal coliform removal of 4.74, 3.42 and 1.25 log10 respectively. These results comply with categories A, B and C of WHO (1989) standards, correspondingly. Therefore, treatment trains consisting of UASB-DHS reactors can possibly be applied when agricultural reuse is contemplated.

Metabolic engineering of Escherichia coli for itaconate production
Vuoristo, K.S. - \ 2016
Wageningen University. Promotor(en): Gerrit Eggink; Johan Sanders, co-promotor(en): Ruud Weusthuis. - Wageningen : Wageningen University - ISBN 9789462576001 - 162
fermentation - escherichia coli - aspergillus niger - biobased chemistry - bioengineering - acids - organic acids - glutamates - tca - production - chemicals - fermentatie - escherichia coli - aspergillus niger - chemie op basis van biologische grondstoffen - bioengineering - zuren - organische zuren - glutamaten - tca - productie - chemicaliën

Interest in sustainable development together with limited amounts of fossil resources have increased the demand for production of chemicals and fuels from renewable resources. The market potential for bio-based products is growing and a transition from petrochemicals to biomass-based chemicals is ongoing. Itaconic acid is a C5-dicarboxylic acid which can be produced by microbial conversion processes. It can be easily polymerized and is an appealing building block for the chemical industry with many potential applications. However, biobased chemicals have to compete with their petrochemical counterparts, and yield and productivity of the microbial processes are therefore of the utmost importance. Traditionally itaconic acid is produced using the ascomycete Aspergillus terreus. This process is not competitive with petrochemical processes due to high production costs caused by low yields, and difficult and expensive product recovery. Maximizing product yield is important to lower production costs. This thesis looked at ways to reach theoretical maximum yield in a recombinant production host, Escherichia coli.

Chapter 2 describes the construction of an itaconate biosynthesis pathway in E. coli. The key enzyme of microbial itaconate production is cis-Aconitate decarboxylase (CadA) that converts the citric acid cycle intermediate cis-aconitate into itaconate. We focused on optimizing heterologous expression of cadA from Aspergillus terreus in E. coli. Initially this resulted in low CadA activities and production of trace amounts of itaconate. CadA was primarily present as inclusion bodies, explaining the low activity. The activity was significantly improved by using lower cultivation temperatures and mineral medium and this resulted in enhanced itaconate titres. The itaconate titre was further increased in aerobic bioreactor cultures by introducing citrate synthase and aconitase from Corynebacterium glutamicum and by deleting genes encoding phosphate acetyltransferase and lactate dehydrogenase. The maximum itaconate yield from glucose obtained in this study was only 0.09 mol/mol, due to high flux of carbon to by-products such as acetate and pyruvate. Pyruvate is a precursor molecule for itaconate biosynthesis and its accumulation suggested that the activity of CadA might be one of the rate limiting steps. It was concluded that further optimization of cadA expression, and reduction of acetate formation should be achieved to obtain higher itaconate yield.

As sufficient cis-aconitate decarboxylase activity is crucial for itaconate production, in chapter 3 ways to increase the activity of CadA were investigated. A recently characterized cis-aconitate decarboxylase of mammalian origin was therefore expressed in E.coli. The novel cis-aconitate decarboxylase from Mus musculus encoded by immunoresponsive gene 1 (irg1) produced comparable amounts of itaconate as CadA from A. terreus. In addition, the effects of codon optimization and harmonization on enzymatic activities of heterologously expressed cadA and irg1 were studied. Codon harmonization increased the activity of CadA in cell free extracts, but this did not result in higher itaconate production in bioreactor cultures. This suggests that other factors such as itaconate transport may limit the production.

In chapter 4, proof of principle for an anaerobic fermentation process for the production of itaconic acid was obtained by using the mixed acid fermentation pathway of E. coli. Itaconic acid production was redox balanced by co-producing succinate or ethanol with H2 and CO2. Expression of cadA together with citrate synthase (gltA) and aconitase (acnA) from Corynebacterium glutamicum resulted in 0.66 mM (1.2 % Cmol) itaconate under anaerobic conditions. Unexpectedly, strains started to produce significant amounts of glutamate when the itaconate pathway was introduced. As glutamate production depends on the availability of nitrogen in the medium, a nitrogen-limited medium was tested to diminish glutamate production. This enhanced the production of itaconate to up to 2.9 mM (5.4 % C mol %). Here, anaerobic production of itaconate from glucose was reported for the first time. The observed itaconate yields and productivities were still modest. Eliminating the pathways to major by-products like glutamate, succinate, and acetate, and enhancing the pathway between pyruvate and itaconate is crucial to obtain a cost-competitive anaerobic itaconic acid process production.

To investigate how itaconate production can be improved, the insights from the previous chapters together with existing scientific literature were combined with our pathway design proposals in chapter 5. The tricarboxylic acid (TCA) cycle is an important source of precursors for biobased chemicals. The opinion article takes a closer look at the metabolic engineering of TCA cycle for the production of chemicals high yield. For most TCA cycle products the maximum pathway yield is much lower than the theoretical maximum yield. For succinate, this was solved by creating two pathways to the product, using both branches of the TCA cycle, connected by the glyoxylate shunt. A similar solution cannot be applied directly for production of compounds from the oxidative branch of the TCA cycle because irreversible reactions are involved: the conversion of acetyl-CoA and glyoxylate to malate in the glyoxylate shunt and the conversion of 2-oxoglutarate into succinyl-CoA in the TCA cycle. This way, the pathway yield for products originating from the oxidative TCA cycle branch such as citrate, itaconate and L-glutamate becomes identical to the theoretical maximum. Future research should focus on implementing these solutions in suitable production hosts, and increasing the ATP yield of the production pathways. This will minimize the oxygen requirement of the process, or even allow for anaerobic operation, and should lead to reduced operational costs and maximal product yields.

In chapter 6 the implications of the overall results of this thesis for the current research status of itaconate production are presented. Solutions to optimize itaconate production strains and production process were proposed.

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