Title Promoter Affiliations Abstract "Accelerating evolution: engineering invertase enzymes to enhance fructan metabolism in Musa spp. (banana)" "Wim Van den Ende" "Molecular Biotechnology of Plants and Micro-organisms" "The equatorial countries of sub-saharan Africa have the highest worldwide mortality rates, primarily caused by infectious disease. Those who are immunosuppressed, including children, the elderly and the sick, are particularly at risk. Improving sanitation, and boosting the immune system with vaccination or improved diet, have been key for continuing to combat this issue.Short-chain low-digestible carbohydrates (LDCs), act as human prebiotics, selectively stimulating the growth and activity of selected intestinal bacteria, creating a symbiotic relationship between the gut and its microflora. In this way, LDCs act as immunomodulators, and significantly ameliorate the onset of type II diabetes, coronary heart disease, GI diseases, cancers, dental cavities and obesity.Of particular interest is fructan as a LDC. Fructans have roles in the plant stress response, as both ROS scavengers and cellular stabilizers. Abiotic stress is estimated to be the leading cause of crop loss, exceeding 50% worldwide. Genetic engineering has been suggested as a means of generating hardier plants - for example - heterologous over-expression of a fructosyltransferase in tobacco greatly improved the survival rate of plants submitted to abiotic stresses.Musa spp. (bananas) are the staple food crop in Uganda, Tanzania, Burundi, Rwanda and other surrounding countries. Banana organs, especially the fruit, contain small inulin-type fructans (kestoses and small inulo-n-oses) as reserve polysaccharides. Preliminary findings suggest that banana lacks genuine fructosyltransferases for the production of longer fructans, but instead produces small fructans via promiscuous vacuolar invertase (VI) enzymes. This suggests that the “fructan syndrome” is already naturally evolving in banana, but residing at a premature stage. It is believed that all fructosyltransferases evolved from VIs within the family 32 of glycoside hydrolases.The project aim is to identify and characterize the different banana vacuolar invertase enzymes, first by bioinformatic search, and then by heterologous expression in Pichia pastoris. The best fructan producing enzyme, as determined by in vitro assays, will be chosen to further enhance the fructosyltransferase properties of the enzyme through site directed mutagenesis, based on our extended previous expertise on structure-function relationships within family GH32. Mutations will then be introduced into this sequence to enhance the fructosyltransferase properties of the enzyme, for eventual re-introduction into banana, thereby creating a fruit with increased fructan content and hence a better prebiotic and immunomodulatory potential. Such fruits will have the greatest impact in the countries most susceptible to infectious disease where bananas are eaten as a staple crop. This project also provides the groundwork for engineering plants more resistant to abiotic stresses, which could be applied to other crops, as environmental extremes threaten increasing amounts of arable land.The methods of this project encompass classic molecular biology and biochemistry, with future aims to employ newer techniques, such as CRISPR/Cas9. The research will be carried out in the Plant Molecular Biology lab at the University KU Leuven." "Accelerating evolution: engineering invertase enzymes to enhance fructan metabolism in Musa spp. (banana)" "Wim Van den Ende" "Molecular Biotechnology of Plants and Micro-organisms" "Bananas are produced in more than 120 countries and around 85% of the crop is retained as a vital food source for 400 million people. With a worldwide production of 145 million tons, banana represents one of the most important food crops. The major threat to banana cultivation is drought, which causes yield losses up to 65%. Climate change is a leading human and environmental crisis of the 21st century. Banana fruits can accumulate smaller amounts of inulin-type fructans, fructose containing oligoand polysaccharides that are well-known because of their prebiotic, immunomodulatory and antioxidant properties contributing to human health. Additionally, inulin-type fructans are emerging as important signals during plant stress responses. In banana, fructans are produced by one or more vacuolar invertase enzymes (VIs) that are “on their way” to becoming “genuine” fructan biosynthetic enzymes or fructosyl transferases (FTs), as occurring in plants that accumulate fructans more abundantly. It is widely accepted that FTs evolved from VIs in plants. In this project we will use the CRISPR/Cas 9 technology to edit the banana genome in such a way that evolution is accelerated by transforming two banana VIs into FTs, with the aim of: (1) increasing fructan levels in banana fruit, as a strategy to launch these health-improving compounds into human diets, and (2) to increasing fructan levels in other parts of the banana plants to counteract stresses (specially drought stress)." "Enzyme functionality during the baking phase of bread making" "Christophe Courtin" "Food and Microbial Technology (CLMT)" "Enzymes are used in a wide array of applications, including cereal-based food systems. It is therefore not surprising that the global demand for enzymes is growing rapidly. In addition, there is a constant hunt for new and/or better performing enzymes in terms of substrate selectivity, inhibition sensitivity, chiral selectivity, and tolerance against extreme pH and temperature conditions. In order to obtain sufficient bread quality, yeast has to consume sugars to produce CO2 and flavour compounds. The majority of the sugars which are consumed by yeast during fermentation, are generated by enzymatic hydrolysis of damaged starch and fructan. The enzymes responsible for this hydrolysis can be of cereal or yeast origin, or be supplemented. Major enzymes that are of interest for the baking industry are amylases, invertases and inulinases. Depending on the thermal stability of the enzymes, sugar production can continue to some extend during the baking phase. Detailed information about the balance between the activity, possible inhibition and thermal stability of the enzymes during the baking phase is however lacking. This work will therefore unravel the stability, activity and inhibition of the endogenous wheat-flour enzymes, endogenous yeast enzymes and exogenous commercial enzymes in a bread model system during the baking phase. In addition, this project is part of the the intercluster project Enzymares, which is supported by Catalisti, Blauwe Cluster and Flanders’FOOD. The aim of the Enzymares project is to develop an enzyme prediction toolbox to speed up enzyme discovery and reduce the time-to-market for new enzymes. Three relevant enzymes with novel, commercially relevant properties will be selected and benchmarked against already commercially available enzymes." "Study of yeast invertases for changing fructan levels in bread" "Christophe Courtin" "Microbial and Plant Genetics (CMPG), Food and Microbial Technology (CLMT)" "Saccharomyces cerevisiae invertase (ScInv) is a β-fructofuranosidase encoded by different, closely related SUC genes and classified within the family 32 of glycoside hydrolases. ScInv prefers sucrose as a substrate but also hydrolyses raffinose, fructo-oligosaccharide (FOS) and short-chain fructans.  Invertase produced by baker’s yeast plays and important role during breadmaking. It is responsible for setting free those sugars that yield most of the CO2 produced during the first stages of fermentation. Despite its importance during breadmaking and several other industrial processes, little is known about the variability in ScInv activity and specificity and the factors determining this variability. Hence, the main goal of this research was to gain more insight into the activity and specificity of ScInv as it is present in yeast, and not just as an isolated enzyme. In the end, these insights can be used to develop yeast-based strategies to modulate fructan hydrolysis during breadmaking.A set of 28 S. cerevisiae strains was investigated for invertase activity and specificity. The strains displayed high variability in invertase activity towards sucrose and FOS, while the variability in FOS specificity was less pronounced. A comparison of 29 different SUC alleles showed a sequence similarity from 89 to 100%. The phylogenetic tree showed three distinct clades of SUC alleles, indicating a certain variability in SUC gene sequences. Further experiments showed that minor sequence differences impacted yeast invertase activity and specificity towards sucrose, FOS and MCI. More specifically, the F102Y substitution in Suc-enzymes lowers yeast invertase activity toward fructo-oligosaccharides by 36% and the specificity factor by 43%. The A409P substitution increased the capacity of yeast to hydrolyse FOS and mixed-chain inulin, likely because of a change in the loop conformation resulting in a wider active site.Other factors that can influence ScInv activity and specificity were also investigated. A clear correlation between invertase activity and ploidy, or invertase activity and the amount of SUC genes was not observed. The impact of the industrial provenance of the strains was more pronounced. Indeed, the mean invertase activity of bioethanol and bakery strains was higher than the other strains, in line with the ecological niches of the different yeasts. ScInv invertase activity increases with an increasing temperature, and an optimum was observed around 60°C, which was higher than for a purified enzyme. Furthermore, higher incubation temperatures resulted in a higher specificity towards FOS, probably due to higher molecular mobility in the enzyme. The overall impact of yeast background was investigated as well, and although this research clearly showed an overall effect of yeast background on invertase activity and specificity, it was hard to specify the underlying factors further.Using the variability in yeast invertase activity and specificity seemed to be a simple yet effective mean to modulate fructan hydrolysis during breadmaking. Strains with high invertase activities towards FOS hydrolysed wheat grain fructans faster and to a greater extent, resulting in a lower final fructan level and thus a lower FODMAP level in bread. Strains with a lower invertase activity could increase the fermentable dietary fibre levels in bread by retaining more fructan during fermentation. The use of non-bakery S. cerevisiae strains resulted in a lower CO2 production and different saccharide consumption and production dynamics compared to a reference bakery strain. However, the addition of glucose or amyloglucosidase could prevent these adverse effects. The selection of yeast strains for the modulation of fructan hydrolysis during fermentation can also be made based on SUC gene sequence. Indeed, the sequences of yeast SUC genes influence their ability to hydrolyse fructan and sucrose during dough fermentation. More specifically, yeast strains with Tyr-102 in the invertase hydrolysed less fructan during dough fermentation, especially during the first hour of fermentation. In contrast, Pro-409 in the invertase resulted in slightly higher fructan hydrolysis after 3 h of fermentation, but the sucrose hydrolysis was slower compared to invertases without Pro at this position. These differences in fructan and sucrose hydrolysis could be related to the differences in invertase activity and specificity and to differences in the capacity to hydrolyse FOS and MCI. Another approach is to use these insights to make small adaptations to the SUC gene sequence of a commonly used baker’s yeast to improve or reduce its fructan hydrolysing capacity. Using this approach, the same yeast strain can be used, and the impact on CO2 production, bread texture and flavour is limited.In a final part, the activity and specificity of Kluyveromyces marxianus inulinase (KmInu) was compared to those of ScInv by expressing INU1, encoding KmInu, and SUC alleles in an invertase negative S. cerevisiae strain. The fructosidase activity towards sucrose of the strains expressing INU1 was lower compared to strains expressing SUC alleles. In comparison both fructosidase activity and specificity on FOS and mixed- and long-chain inulin were higher for strains expressing INU1. The fructosidase activity and specificity of the dimeric enzyme secreted into the growth medium were determined as well. This revealed that the longer the substrate, the bigger the share of the free enzyme to the total fructosidase activity. Moreover, the free enzyme showed a higher specificity towards longer substrates, which can be explained by its higher accessibility for these substrates, as there is no cell wall barrier. Finally, wheat dough fermentation experiments showed that expressing an INU1 allele in S. cerevisiae might be a good alternative for the use of K. marxianus during breadmaking. Indeed, similar to K. marxianus, a S. cerevisiae strain expressing INU1 hydrolysed all wheat grain fructans after 3 h of fermentation.In summary, this PhD study provided more insight into the molecular basis for natural differences in invertase substrate specificity between S. cerevisiae strains and other factors influencing invertase activity and specificity. Hence, it opens the door for the selection or engineering of yeasts and Suc-enzymes with specific activities that allow controlling fructan hydrolysis. New yeast-based strategies to modulate fructan hydrolysis during dough fermentation were developed.." "Fructans as microbe- or damage-associated molecular patterns: Evolutionary perspectives and apoplastic dynamics in Cichorium intybus and Eruca sativa." "Wim Van den Ende" "Molecular Biotechnology of Plants and Micro-organisms" "In order to sustain the necessary crop production rates, which are necessary to meet the demands of our growing global population, agriculture still relies on the extensive use of pesticides. Nevertheless, the harmful effects pesticides on health and environment are well known. A lot of research effort goes into the discovery and development of more sustainable alternatives to reduce the negative effects of biotic and abiotic stresses. One of these alternatives, priming of the plant immune system using natural compounds, has received a lot of attention in the last two decades. A priming stimulus allows plants to respond better and faster against future stresses. In line with integrated pest management strategies, the use of priming agents in combination with other biocontrol methods and pesticides can help reduce the extensive use of the latter. To find potential natural priming compounds, it is important to look to the physiology of the plant and study compounds that have a natural function against stress exposure. Besides amino acids, organic acids and other compounds, several carbohydrates have been tested as priming agents, often with good results. A role of sugars in plant immune responses was more clearly defined in the sweet immunity concept. By extension, some oligo- and polysaccharides should also be considered, including fructans.Fructans are oligo-and polysaccharides built from Fru residues. They are found in ca. 15% of flowering plant species, where Fru moieties are added to Suc by different fructosyltransferases. On the other hand, fructan hydrolysis relies on plant fructan exohydrolases (FEHs). Fructans are synthesized and stored in the vacuole of the plant. Based on linkage type between the Fru residues different fructan types are found in plants. Inulin (β-2,1) is mostly found in dicots, while levan (β-2,6) and graminan (mixed linkage type) are more common in monocots. Several studies have indicated positive effects of fructans against abiotic stress, exemplified by many transgenic studies, as well as comparison studies between fructan and non-fructan accumulators. Recent studies also highlighted their potential use as priming agent, showing promising effects of plant inulin and levan in lettuce and apple, respectively. In Arabidopsis thaliana levan oligosaccharides (LOS) of microbial origin, low degree of polymerization (DP) levan produced by partial hydrolysis of higher DP levan, proved effective in reducing susceptibility of the plant to Botrytis cinerea infection. Since microbial fructan production, mainly of the levan-type, is common in bacteria and produced as an exopolysaccharide, this fructan may be sensed by the host plant as a microbe-associated molecular pattern (MAMP), triggering defence responses. Different fructans can activate toll-like receptors in animals to stimulate immune responses. Because of the high DP nature of microbial fructans, it is expected that low DP, more mobile LOS are produced as a primary signal. This production may rely on plant and microbial fructanase enzymes, playing a central role in apoplastic fructan dynamics.In a first part of the thesis we identified the most promising fructan priming agents and studied their influence on carbohydrate metabolism in the plant. Two plant species were chosen, rocket (Eruca sativa) and chicory (Cichorium intybus), both with agricultural value. Rocket belongs to the Brassicaceae, allowing us to compare our results with the previous results obtained in A. thaliana. Chicory was chosen as a fructan-accumulating plant, as it produces high amount of inulin in the taproot and has served as a model plant organism in past fructan research. This allowed us to study not only the effect of non-self fructan priming, but also the effect of exogenous priming with endogenous fructans in the case of chicory. In this species, inulin-type FOS (fructooligosaccharides) may be sensed as damage-associated molecular patterns (DAMPs). B. cinerea was chosen as a fungal pathogen to infect both host species. This necrotrophic pathogen has a very broad host range and has economically devastating effects.Since previous research on priming in these species is limited or absent, priming (leaf spraying) and disease assays were optimised using the necessary control treatments. Then, a broad array of different fructan types was used, some commercially available, while others were prepared and/or further purified in the lab. Besides testing higher DP fructans, enzymatic hydrolysis was used to produce FOS and LOS. A sulfated levan, as well as sulfated LOS produced from partial hydrolysis, were also considered due to the positive effects observed in animal studies. As hypothesized, mostly low DP fructans could induce an increased resistance to B. cinerea after leaf priming. Most importantly, microbial LOS priming greatly improved disease resistance in both rocket and chicory. FOS priming was only effective in chicory, which may indicate DAMP-specific type responses that are absent in rocket. Inulin and branched fructans had no clear positive effects, while microbial levan marginally improved resistance. Sulfated LOS also improved resistance in both species. Sulfated LOS induced a direct ROS burst in the plant, typical for elicitors, while non-sulfated LOS and FOS behaved as a genuine priming compound. In addition, sulfated LOS significantly inhibited growth of B. cinerea on plate, while no negative effects were observed for LOS. In rocket, total leaf sugars increased significantly after LOS priming as well as subsequent infection. In both species, LOS priming induced a temporal peak in apoplastic small soluble sugars. This indicates that during LOS priming, LOS and/or glucose dependent signalling may be activated for downstream immune responses. Although similar dynamics were observed for FOS, high levels of Glc and FOS in the apoplast may benefit B. cinerea, since this fungus can hydrolyse and utilise inulin-type fructan.In the second part of the thesis, we focussed on two fructanase enzymes. First, we characterized a novel chicory FEH. So far, no 6-FEH was identified from chicory, although such enzymes had been discovered in non-fructan plants such as A. thaliana and sugar beet. After a detailed study on protein sequence alignments, we identified a potential 6-FEH that was wrongly annotated as a cell wall invertase. The enzyme was expressed in Pichia pastoris and characterized on a functional level. The enzyme had high affinity towards levan-type fructan and a confirmed apoplastic localization. We showed that it can hydrolyse a typical fructan profile produced from microbial levan synthesis. Based on these data it is proposed that this enzyme has a key role in the apoplastic production of LOS from microbial levan, which may be central in interactions of the plant with beneficial microbes in the rhizosphere context. In depth modelling studies and in vitro assays provided important insights into the substrate specificity and catalytic behaviour of this enzyme. Second, we characterized a B. cinerea inulinase that we identified from the genome. This endo-inulinase is different from previously characterized fungal inulinases, since it harbours an additional N-terminal domain, resembling an extra β-sandwich. We expressed this form, as well as a shortened form missing the extra domain. The results indicated a strong difference in kinetic behaviour, with the long form following Hill kinetics, while Michaelis-Menten kinetics were observed for the short form. Using this B. cinerea inulinase, in depth modelling and docking studies were performed to gain important insights into structural differences between endo- and exo-type fructanase reactions. After a similar analysis of microbial levanases, an updated terminology is proposed for fructanase enzymes.In conclusion, the results of the thesis highlight low DP fructans, LOS and FOS, as promising priming agents to improve plant resistance to biotic stress. Probably all higher plants are able to sense LOS as a MAMP through yet unidentified receptors, while the inulin accumulator chicory likely evolved an extra receptor to detect endogenous inulin-type FOS as a DAMP, leaking out of damaged cells. The observed carbohydrate dynamics after LOS priming will allow future studies to go deeper into understanding the specific signalling pathways involved. Furthermore, detailed investigation of key fructan-degrading enzymes highlights the importance of apoplastic 6-FEHs in plant-microbe interactions, and new mechanistic insights into fructanase reaction mechanisms obtained from studying the B. cinerea inulinase provide a central starting point for further research going deeper into the unravelling of the interaction and signalling cascades involved in sweet priming of plants and beneficial plant bacteria (e.g. Bacilli; emerging levan detour concepts emerging from sucrose in root exudates). All these fundamental insights will boost the development of priming and/or biological control micro-organism formulations, reducing the widespread use of agrochemicals. " "Sugar binding in the active site of plant GH32 proteins." "Wim Van den Ende" "Molecular Biotechnology of Plants and Micro-organisms, Biochemistry, Molecular and Structural Biology" "Recently, many interesting discoveries were made on the structure-function relationships within the plant glycoside hydrolase family GH32, containing invertases, FEH's and FT's. Yet, a number of issues remained unsolved. In this project, we tackle these remaining issues. This project aims at 3 specific goals: 1. The discovery that some GH32 members can still bind sucrose without hydrolysing it, led to the hypothesis that some of these FEH enzymes in non-fructan plants might in fact represent catalytically defective invertases fulfilling important regulatory functions throughout plant growth and development. Perhaps some might even act as sucrose sensors involved in sucrose-specific signalling. In this project, we want to compare the sucrose (and fructan) binding and degradation capacity in defective and closely related fully active invertase enzymes. Moreover, we will compare the sucrose binding modi in the two systems and identify which amino-acid residues are responsible for the observed differences. 2. We want to better understand the difference between hydrolases (water as acceptor) and transferases (sucrose or fructans as acceptor) within family GH32, and use this information to develop FTs with superior kinetics for specific applications. 3. A last challenge is understanding the molecular differences in acceptor substrate specificity between FTs showing an extended difference in the fructan chain length produced ." "The role of sugar sensing and sugar metabolism during developmental and induced leaf senescence." "Wim Van den Ende" "Molecular Biotechnology of Plants and Micro-organisms" "Controlling leaf senescense is a hot research area in modern plant physiology research, in order to be able to extend the period of active photosynthesis and overall biomass production. Sugars play an important role during natural and induced senescence. However, the exact roles of the sugar signalling processes involved in plant senescence remain unclear. This project focuses on sugars and sugar metabolizing enzymes in Arabidopsis leaves. More in particular, special attention will be given go to the expression, localization and putative function of neutral invertases (9 genes in Arabidopsis) during senescence and to the regulatory role of defective invertases (3 genes in Arabidopsis)."