Wageningen Universiteit en Researchcentrum. Food & Biobased Research (voorheen A&F) Business Unit: Biobased Products, Dept

Wageningen Universiteit en Researchcentrum Food & Biobased Research (voorheen A&F) Business Unit: Biobased Products, Dept. Bioconversion http://www.biobasedproducts.wur.nl/NL/

Stage- en afstudeerplaatsen 2010/2011

Wageningen Universiteit en Researchcentrum Food & Biobased Research (voorheen A&F) Business Unit: Biobased Products, Dept. Bioconversion http://www.biobasedproducts.wur.nl/NL/ Aard van de opdrachten: Research en ontwikkeling

Contact:

H. Mooibroek tel. 0317 480214 J. Springer tel. 0317 480215

[email protected] [email protected]

Stage- en afstudeerplaatsen 2010/2011

Stage- en afstudeeronderwerpen, met een focus op moleculaire biologie, fermentatietechnologie of beide, enzymologie en immunologie kunnen worden geformuleerd in de volgende onderzoekslijnen:

1. Productie van waterstof door anaerobe thermofiele bacteriën 2. Productie van farmaceutica via gemodificeerde enzymen 3. Tweede generatie Biobrandstoffen: Optimalisatie van de productie van biobutanol uit afval reststromen 4. Rapid immunochemical, molecular-biological and enzymatic one-step- and micro-array-test methods 5. Informer Genes technology for detection of contaminants, toxins and nanoparticles 6. Internships EU-PEARLS: EU-based Production and application of Alternative Rubber and Latex Sources (end 2010) 7. Productie van waterstof m.b.v. geïmmobiliseerde enzymen 8. Algae identification and culture dynamics (AlgiCoat; Internship mid 2010) 9. Methods to measure ‘functional’ lectins in animal feed 10. Pichia pastoris as a cell factory for designed proteins

1.

Productie van waterstof door anaerobe thermofiele bacteriën

Achtergrond: Waterstof wordt beschouwd als de brandstof van de toekomst omdat het kan worden gebruikt voor de productie van elektriciteit met behulp van een brandstofcel. Dit proces heeft een veel hogere efficiëntie dan conventionele processen en is daarnaast ook erg schoon met alleen water en warmte als bijproducten. Brandstofcellen zouden onder andere toegepast kunnen worden in de transportsector. Prototypes van dergelijke vervoersmiddelen rijden al rond. Momenteel wordt het merendeel van de waterstof geproduceerd uit aardgas. Dit is geen duurzaam proces doordat er een netto uitstoot van CO2 plaatsvindt (bijdrage aan het broeikaseffect) en doordat aardgas geen hernieuwbare grondstof is. Wanneer waterstof op grote schaal gebruikt zal gaan worden als energiebron is het van groot belang dat waterstof in de toekomst geproduceerd wordt uit hernieuwbare bronnen. Eén van de duurzame mogelijkheden hiervoor is de productie van waterstof uit biomassa met behulp van anaerobe bacteriën. Onderzoeksproject: Binnen de bioconversiegroep van Food & Biobased research wordt al geruime tijd onderzoek gedaan naar de productie van waterstof uit biomassa met behulp van anaerobe, thermofiele bacteriën. In samenwerking met een andere groep binnen Food & Biobased Research wordt de biomassa eerst geschikt gemaakt voor fermentaties, bijvoorbeeld door de biomassa om te zetten in fermenteerbare suikers met behulp van enzymen. Daarna vinden de microbiële fermentaties plaats. Studenten zijn van harte welkom om te participeren in dit onderzoek, waarin de microbiële fermentaties worden bestudeerd en geoptimaliseerd. Hierbij wordt gebruik wordt gemaakt van verschillende soorten bacteriën, al dan niet in mengpopulaties. Gekeken wordt onder andere naar de waterstofopbrengst, de omzettingsnelheid, het substraatgebruik, de nutriëntenbehoefte, en de waterstoftolerantie van de verschillende bacteriën onder verschillende kweekomstandigheden. De genoomsequentie van één van de meest effectieve waterstofproducenten is bekend, en deze informatie is zeer bruikbare bij ons onderzoek. Studenten zullen tijdens hun afstudeeronderzoek of stage onder andere anaerobe, thermofiele fermentaties uitvoeren, zowel in bioreactoren als in kweekflesjes. Ook zal gebruik worden gemaakt van analytische technieken zoals HPLC en GC.

Meer informatie kan worden verkregen bij Truus de Vrije ([email protected], tel. 0317-480213) of Astrid Mars ([email protected], tel. 0317-480226).

Biomassa

Hydrolyse van biomassa

Fermentatie

2.

Productie van farmaceutica via gemodificeerde enzymen.

Complexe koolhydraten zijn belangrijke componenten van farmaceutische- en voedingsmiddelen. De meeste daarvan zijn natuurlijke producten zoals heparine, hyaluronan, pectines, alginaten en verschillende gommen. Een specifieke groep van bio-actieve koolhydraat co-polymeren wordt gevormd door de glycosaminoglycans (GAG’s) die bestaan uit alternerende glucuron-zuur en (N-acetyl)-glucosamine disaccharide-eenheden die op specifieke plaatsen van sulfaatgroepen voorzien kunnen zijn. De bekendste stoffen uit deze groep zijn heparine (anti-stollingsmiddel) en hyaluronan (vooral gebruikt bij de behandeling van reumatische artritis). Verder worden chondroitin-sulfaat en dermatan-sulfaat gebruikt voor medische toepassingen en in cosmetica. De huidige productie van GAG’s is vooral uit natuurlijke bronnen waarvan slachthuisafval de belangrijkste is. Dit heeft grote nadelen zoals lage opbrengsten (gepaard gaande met grote afvalstromen) en risico van verontreiniging. Verder is inactivatie van het eindproduct als gevolg van het rigide opwerkingsproces een groot probleem. Productie van deze GAG’s met behulp van recombinante enzymen in bio-reactoren maakt niet alleen een schoner proces en schoner product mogelijk maar levert ook de technologie om veel preciezer te produceren. Er zijn aanwijzingen dat het werkingsspectrum van deze polysacchariden varieert met het molecuulgewicht. Specifieke molecuul-gewichtsklasses kunnen voor specifieke doelen worden gebruikt. Productie hiervan met de huidige technieken is moeilijk en duur. Bij AFSG zullen recombinante enzymen worden geproduceerd met behulp van microorganismen. Hiertoe worden genen uit verschillende organismen gekloneerd, waar nodig gemodificeerd en tot expressie gebracht in bacteriën en gisten. De enzymen waarvoor de genen coderen worden geïsoleerd en getest op functionaliteit. Andere partners (Wageningen Universiteit en Akzo Nobel) binnen het project zullen de enzymen gebruiken om bioreactoren te ontwerpen voor de productie van farmaceutische polysacchariden op maat. Aanpak en Technieken Isolatie en karakterisering van genen coderend voor de GAG synthetiserende enzymen uit geschikte donor organismen. Verschillende kloneringstechnieken, transformatie van E. coli en gisten, PCR in allerlei vormen, constructie van expressievectoren. Eiwit expressie in E. coli en in gisten op kleine schaal. SDS-PAGE, eiwitzuiveringstechnieken en eiwit analyses. Directed evolution van de bestaande enzymen en ontwikkeling van screeningstechnieken daarvoor. Informatie: Ing. Jan Springer, tel: 0317-480215 Email: [email protected] UDP-sugars

designer enzymes

UDP

Heparine

defined GAGs

3.

Tweede generatie Biobrandstoffen: Optimalisatie van de productie van biobutanol uit afval reststromen

De productie van aceton, butanol en ethanol door middel van fermentatie, de ABE fermentatie, is een bekend en vroeger veel toegepast proces. Ondanks dat het proces in het verleden is afgestoten, is er sinds kort hernieuwde interesse in het produceren van aceton en butanol op basis van agrarisch en GFT-afval. Butanol is een alcohol die zeer geschikt is voor het mengen met diesel, en het maken van biodiesel. Daarom is er nu een veel interesse voor dit proces, en lopen er nu in Nederland een aantal projecten in dit gebied. Het doel van de projecten waar wij nu aan werken is de ontwikkeling van een continu fermentatieproces met een hoge productiviteit en waar de bijproducten van de fermentatie worden gerecycled in het proces. Er wordt op labschaal (1-20 L fermentoren) gewerkt met geselecteerde ABE producerende bacteriestammen. Deze stammen zijn anaëroob, er wordt dan ook onder zuurstofvrije omstandigheden gewerkt. Verschillende procestechnologische aspecten van de fermentatie worden bestudeerd, onder andere continue productwinning (gasstrippen en pervaporatie). Daarnaast zal ook de productie stabiliteit van de bacteriestam op lange termijn worden bestudeerd. Stammen met een verhoogde butanol productiviteit of met een verhoogde stabiliteit zullen worden verkregen via screening van verschillende bronnen of via het toepassen van genetische technieken (e.g. mutagenesis) op bestaande stammen. Technieken Moleculaire biologische (o.a. DNA isolatie, kloneren in E.coli) en biochemische (o.a. eiwit zuivering, enzymatische activiteit, SDS-PAGE). Microbiologische en Fermentatie technologieën. Werken met anaërobe micro-organismen. Analytische technieken zoals High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC) Informatie: Dr. Ana López Contreras, tel. 0317-481314 Email: [email protected]

4.

Rapid immunochemical, molecular-biological and enzymatic one-step and micro-array test methods

At AFSG rapid one-step Lateral flow immunoassays (LFIAs; compare with the pregnancy hormone one-step assay) have been developed for over 15 years. The LFIAs are applied to detect ligands such as antibiotics, toxins, allergens and micro-organisms in raw materials and in food and feed products. Also human diagnostic applications are being developed (Schistosomiasis, TB). The assays are simple and straightforward and the results are easily interpretable and visible in a few minutes. No expensive equipment nor special skills are necessary and, therefore, the assays are especially suited for low-facility laboratory and field settings. Colloidal carbon particles, the immuno-labels applied, show an excellent signal-tonoise ratio (black on a white membrane), making it for example an ideal label for the blood matrix. However, development of such assays is often a real challenge, since in these homogeneous, one-step assays all reagents and the sample are combined simultaneously in the assay device, thereby easily giving rise to background and other non-specific signals. A new development is the combination of mRNA/DNA amplification and LFIA technology. In this Nucleic Acid LFIA (NALFIA) amplified genetic material (PCR, NASBA) is directly transferred to a one-step LFIA device and the result, i.e., the presence of one or more amplicon(s), is indicated by black lines at particular locations in the reading window in 15 minutes. Recently, a (semi-)quantitative colloidal particles micro-array assay has been developed. In this method specifically bound target molecules (DNA and/or proteins) on a nitrocellulose surface are detected by ligand labeled colloidal carbon particles. The signals in the (NA)LFIA and micro-array assays can be analysed, and if required quantified, using a flatbed scanner and image analysis software. As compared to conventional quantification of DNA micro-array results this approach is rapid and inexpensive. Examples of target organisms/species are Salmonella, E .coli and B. cereus and genetically modified soy. The focus of the programme is shifting to the field of bionanotechnology, which is exemplified by development of very small diagnostic devices and silica based sol-gel matrices that contain entrapped enzymes for biosensor applications. Depending on specific projects running students will be responsible for a sub-project. The following tasks may be involved: Characterisation of antigens Various immunoassay formats such as ELISA, (NA)LFIA, micro-arrays. Development / Application of immuno-labels; colloidale nanoparticles and conjugates with specific proteins such as antibodies Development / Assemblage of (NA)FIAs and micro-arrays; optimalising composition / performance of various filters and membranes, buffers and buffer components, etc. Amplification of genetic material of target organisms Silica based sol-gel matrices to entrap enzymes with respect to biosensor interface development Techniques/Expertises (a.o.): FPLC, PAGE, affinity chromatography, dia-filtration, ELISA, (NA)LFIA, micro-array, amplification, hybridisation, sol-gel preparation.

Information: Dr. Aart van Amerongen, tel: 0317-480164 Email: [email protected]

5.

Informer Genes technology for detection of contaminants, toxins and possibly nanoparticles

For the detection of small chemical contaminants and toxins a large number of methods have been developed that each should be applied in particular cases for particular (groups of) contaminants. Furthermore, with the increased use of nanoparticles huge efforts are necessary to develop methods for the detection of these particles and also to assess their toxicity to human beings and to the eco-system. Moreover, as far as toxicity testing is concerned official legislation / regulation often requires the use of experimental animals with death as end-point of the test. AFSG has developed a generally applicable informer-genes technology that can be applied as a generic methodology for the detection of, e.g., chemical contaminants, toxins and possibly nanoparticles. In addition, the technology shows great potential to replace animal experimentation in toxicity testing. The new methodology is based on cell biomonitoring and a transcriptomics approach ('diagnomics'). Steps in development and application are: 1. Selection of bacterial or mammalian cells / cell lines responsive to the target compound. 2. Assessment of responsiveness by molecular biological subtraction techniques by scoring specifically induced genes. 3. Analysis of the induced genes with respect to their predictability for the presence of the target compound. 4. Design of suitable primers for a set of correlative genes that will be used in a number of detection methods such as (real-time) PCR, simple and rapid assays like the nucleic acid lateral flow immunoassay (NALFIA), mini-/microarrays, lab-on-a-chip devices, etc.. Depending on specific projects running students will be responsible for a (sub-)project. Tasks involved will be related to (part of the) the steps mentioned above, from cell culturing and molecular-biological subtraction techniques to the development of molecular diagnostic methods.

Informatie: Dr. Aart van Amerongen, tel: 0317-480164 Email: [email protected]

6.

Internships EU-PEARLS: EU-based Production and application of Alternative Rubber and Latex Sources (end 2010)

Introduction Natural rubber is an elastic polymer which is processed from the latex of laticifers like Para Rubber Tree (Hevea brasiliensis) For economical reasons and the high quality standard rubber has to meet, alternative sources for natural rubber are needed. Cultivation of rubber producing crops other than the rubber tree fit to grow in parts of the world other than Southeast Asia, is seen as a potential alternative. Russian dandelion (Taraxacum koksaghyz) and guayule (Parenthenum argentatum) grow in moderate and dry climates, respectively, and produce high quality rubber. One part of this research program aims at creating more fundamental knowledge concerning the physiology and regulation of (poly) isoprene-biosynthesis. For this purpose, a yeast model-organism, such as Saccharomyces cerevisiae will be used to study poly-isoprene biosynthesis. Natural rubber, cis-1,4-polyisoprene, is formed by the condensation of isoprenyl diphosphate molecules (IPP-molecules) to an allylic initiator (farnesyl diphosphate) by action of a cisprenyltransferase (CPT). The rubber is formed in rubber particles surrounded by a monolayer membrane, on to which the CPT and other, SRPPs, REF and yet undefined, proteins are attached. Recent studies demonstrated that besides CPT, other undefined proteins and IPP/allylic initiator ratios play an important role in synthesis of high grade rubber. When expression of a CPT gene is established successfully, more complex interactions with other proteins and physical factors can be studied in more detail. In order to optimize the yeast model to investigate rubber biosynthesis, genetic engineering of metabolic pathways involved in poly-isoprene formation, enabling control of substrate levels for optimal biosynthetic rate, will be addressed. As such methods to monitor substrate and product levels have to be developed. Internship 1: Yeast isoprenoid metabolic pathway engineering. The yeast IPP metabolic pathway and further downstream synthesis are target in engineering. To influence the stoichiometry and metabolite levels of IPP/FPP, and reduce leakage into FPP downstream products, the promoter of the fpps gene in S. cerevisiae BY4742 has been replaced by the S. cerevisiae met1 promoter (pMet3). This rF strain (regulated Farnesyl) shows a reduced growth rate with methionine supplemented to the medium.The S. cerevisiae BY4742 has strain has been transformed with a ura3-tHMGR::Ty integration construct expressing a truncated HMGR upon galactose induction. The tHMGR is supposed to be not susceptible to feedback inhibition (degradation) by downstream metabolites (FPP) and herewith should increase the flux through the MVA pathway even when high levels of downstream metabolites are present. Work: -Strain characterization: -DNA level -Southern blot – positive and negative label hybridization -PCR out total integration product and sequence -RNA level -Q-PCR on levels of expression of FPP upon methionine repression, key enzymes in IPP synthesis -growth and induction of CPT of strains during methionine repression -transform a BY4742/ Ty::tHMGR-Uri3 strain with pMet3-His3 and subsequent characterization -Strain characterization of the tHMGR strain analogous with the rF strain. -construction of a uri3 negative mutant through 5FOA selection -curing of Epy208

Internship 2: Expression of CPT genes in S. cerevisiae. Characterization of the in vivo synthesis of poly-isoprenoids. Three CPT genes have been cloned in a pYes2.1 vector and are expressed from the pGal1 promoter upon induction with galactose. The expressed protein was visualized on SDS-PAGE after Western blotting with a poly-clonal antibody to the CPT protein. Expression of CPT specific for rubber synthesis is described in literature to result in the synthesis of long chain isoprenoids C50-100 range. Thorough literature study on homologous and heterologous expression of CPTs can identify the physiology concerning expression of the CPT. Also other carotenoids, lycopene and other terpenoids synthesizing enzymes (polymerase) can be included in the study. Work: -transformation of engineered host strains with the CPT expression vector. -characterize CPT expression at protein and product level in time -processing and purification (partly) of products of synthesis -optimization of expression of a CPT in yeast (different induction regimes have to be tested) -product analysis by RP-TLC, MALDI-TOF-MS and or GPC -further cloning and construction of host strains

Internship 3: Saccharmyces cerevisiae poly-isoprenoid biosynthesis – Systems biology. Five SRPPs and the REF are to be expressed in S. cerevisiae under the pGal1 of the pYes2.1 vector. The host is the S. cerevisiae strain carrying the CPTs in the pRS413 plasmid or a Chromosomal integration will be targeted for co-expression. Therefore the CPTs will be expressed under the Cup1 promoter in the pRS413 yeast expression vector. The cloning of the pCup1 and CPT in pRS413 has to be verified by sequencing. Yeast strains expressing REF, SRPPs and CPT upon induction will be characterized at protein and product level. Work: -further construction of a CPT expression vector with the Cup I promoter and subsequent strain characterization -cloning of SRPPs and REF available in yeast expression vector (pYes2.1). cloning of the CPTs, SRPPs and REF in different hosts. -strain characterization, expression analysis, at mRNA and protein level -induction characterization, product analysis Contact information: Wageningen University and Research Centre; Food and Biobased Research; Business Unit: Biobased Products (BbP) Contacts: Dr. Hans Mooibroek Dr. ir. Matthé Wagemaker Website:

tel. 0317 480214 tel. 0317 480214

[email protected] [email protected]

www.EU-PEARLS.eu\UK

Russian dandelion

Tyres produced from Russian dandelion rubber

7.

Production of hydrogen by immobilised enzymes.

Aim of the project is the assessment of a „proof of principle“ for the conversion of pentoseand/or hexose polysaccharides into hydrogen by immobilised enzymes of the pentose phosphate cycle. Enzymatic conversion of all polysaccharides from biomass aims at an efficiency of 9-11 mole H2 per mole hexose (or 7.5-9 mole H2 per mole pentose), which implies an improvement of efficiency by 10-30% when compared to hydrogen production through fermentation. Immobilisation of enzymes In enzymatic production processes the advantages of immobilisation of enzymes are obvious. Recycling of the enzyme is enabled because the enzyme can easily be separated from the reaction mixture. In addition, continuous processes are made possible because the immobilised enzyme and its solid phase stay behind in the reaction vessel. Monoliths are well suited for both immobilisation and stabilisation of enzymes. A monolith consists of solid, porous material without empty cavities, enabling flow of the mobile phase through the stationary phase. Monoliths can be prepared by polymerization of organic monomers (e.g. acrylamide) or by polycondensation of alkoxy-silan monomers resulting in the formation in silica-based sol-gels. Project - Investigation of possibilities to immobilise and stabilise enzymes by entrapment in monolitic sol-gel material based on alkoxysilans. Entrapment must lead to: possibilities to regenerate the enzyme increase of its specific activity elongation of its “shelf-life” - Characterisation of sol-gels (assessment of influence of conditions of pH, temperature and presence of additives on pore-size distribution) - Optimisation of “loading” of sol-gel with enzyme - Assessment of enzymatic activity before and after entrapment - Assessment of enzymatic stability/durability before and after entrapment Models: Glucose-6-phosphate dehydrogenase 6-Fosfogluconaat dehydrogenase Hydrogenase Expertises Biochemistry, organic chemistry, colloid chemistry, enzymologie Information: Ir. Jan Wichers, tel: 0317-481297 Email: [email protected]

8.

Algae identification and culture dynamics (AlgiCoat; Internship mid 2010)

Algae cultures, for economic reasons, are often cultured in open production systems resulting in a mixed culture environment. In this study a species specific identification system based on 18S and rITS gene sequences are developed to monitor the species representation in time in such a production system. Lab-scale and upscaled outdoor culturing techniques are instrument to study intervention measures such as change in pH, light intensity or temperature. keys: -genotypic and phenotypic identification -dynamics of genotypic and phenotypic stability -mixed cultures dynamics Measuring culture stability and dynamics through species identification DNA sequence analysis of 18S and rITS (ribosomal Internal Transcribed Spacers) of ribosomal genes followed by quantification techniques (QPCR). Specific and selective primers designed to discriminate the presence and abundance of the species in culturing systems are used to characterize the algae ecosystem present. Monitoring culture stability and dynamics through growth in batch culture Axenic algae cultures will be mixed and grown in batch culture under different culturing regimes. Under controlled conditions, changes in pH, light intensity or regime, temperature can influence species ratios present in culture and can identify measures to stimulate one species’ propagation above the other. Growth can be monitored in time by measuring the OD in a reader at intervals. When DNA is isolated from samples, these can be analyzed on species composition by qPCR, microscopy or protein profiling depending on species present. Techniques Several techniques will be employed such as: - Literature study, 18S and rITS database study, update and report - alignment and primer design - subcloning and sequencing - PCR, QPCR - Cultivation of phototrophic algae in micro-titre-plate and or shake flask - DNA extraction (RNA) - Microscopy - Protein profiling (1D, 2D, Proteomics), zymographs Contact information: Wageningen University and Research Centre, Food and Biobased Research, Business Unit: Biobased Products (BbP) Contacts: Dr. Hans Mooibroek, tel. 0317 480214, email: [email protected] Dr.ir. Matthé Wagemaker, tel. 0317 480214, email: [email protected]

Cultivation systems for algae

9.

Methods to measure ‘functional’ lectins in animal feed

Introduction Anti-nutritional components are present in raw beans, lentils, peas etc. One important antinutritional component is lectin. Lectins can cause intestinal problems in animals and humans. It is very interesting to be able to quantify “food-borne”lectins, which are able to bind to mannose receptors in the small intestine and cause intestinal problems. Aim of the project To set up a Functional Lectin Immuno Assay (FLIA) and/or Enzyme Linked Immuno Sorbent Assay (ELISA) in order to measure/quantify lectins which are responsible for intestinal health problems in animals. Expertises Plant biotechnology, biochemistry, immunology

Information: Ir. Jan Wichers, tel: 0317-481297 Email: [email protected]

10. Pichia pastoris as a cell factory for designed proteins

Introduction Amino acids are Nature’s Lego blocks. With only a few number of pieces (there is only 20 of them!) an infinite number of structures can be built. Organisms like silk worms or spiders are Lego builders specialists. By placing specific amino acids in a particular order they can produce strong yet elastic protein polymers like silk or spider web. In our group, we were inspired by these structures and decided to create new protein polymers with unique properties. To produce them we have chosen the “user friendly” methylotrophic yeast, Pichia pastoris. (Figure 1) This organism has been proven to be an excellent cell factory for the production of simple polymers. However, when the structure complexity is increased the production and consequent secretion of these proteins is impaired due to a blockage inside the cell.

Approach In order to know where the secretion blockage is we are using model proteins that can form triple helices (Figure 2). Since this structure cannot be properly secreted it will probably cause a traffic jam within the secretory pathway. Then, by using the new colourful and fluorescent techniques available we will be able to visualize where the protein is and follow, in real time, its path along the different cellular organelles.

Thesis subjects We are now in the stage of the project where we want to look inside the cells. Fluorescence microscopy techniques will be use to determine in which organelle the secretion is blocked. Once we have the secretion bottleneck identified we can figure out possible ways to overcome it. So, if you want to know what it takes to convert P. pastoris into a cell factory for custom-made proteins come and join us. You will have the opportunity to work or create designed proteins with special properties and become acquainted with fermentation and protein purification techniques.

Expertise: Genetic Engineering/Protein Engineering

Information: Catarina Silva (Building 118, KT-1-14, Tel: 0317 480216) E-mail: [email protected]