Posters

This is the poster session page. For the abstract, please tap on the title.

1. Lignin valorization by Thermus thermophilus laccase

Lignin valorization by Thermus thermophilus laccase
Shams T. A. Islam, Tijn Sewandono, Jie Zhang, Peter-Leon Hagedoorn

Lignin has great potential as a renewable (and abundant) feedstock for various fuels and chemicals but is currently being utilized commercially only in a limited manner (2%). The limited use is due mainly to its inconsistent composition/ structure and may be overcome by engineering enzymes which are naturally able to convert lignin in a specific manner. Hence, research is being conducted on Thermus thermophilus laccase (Tt laccase) whose central T1 copper atom can be manipulated by mutagenesis to alter its activity (catalytic scope and rate) via altering its redox potential. Thermus thermophilus laccase has been structurally characterized (PDB 2XU9) and is thermostable. Moreover, the enzyme can be functionally expressed in Escherichia coli.

A bottleneck in the engineering approach is screening for the best lignin conversion rate. Here we explore enthalpy measurements by enzyme calorimetry and thermographic imaging to solve the screening bottleneck. The variants will be placed in multi- microtiter plates to carry out reactions on model substrates (ABTS) to give a ∆H value which can be used to calculate the rate of enzyme reactions, ultimately leading to evaluation by Michaelis-Menten kinetics. The extremely thermostable ~53 kDa Tt laccase has a yet unreported redox potential. Since, the redox potential is related to oxidation efficiency (expressed as kcat/Km) and range of substrates, work emphasis will be placed on: 1. determination of the T1 redox potential of the laccase and 2. determine the type of lignin valorization achieved by size exclusion chromatography, light scattering, LC-MS and NMR. Emphasis will also be placed on discovery and structural and EPR spectroscopic characterization of the new biocatalysts developed in this study.

2. Artificial metalloenzyme catalyzed new-to-nature reaction in the cytoplasm of a bacterial cell

Artificial metalloenzyme catalyzed new-to-nature reaction in the cytoplasm of a bacterial cell
Shreyans Chordia, Aditya Iyer, Gerard Roelfes

Artificial metalloenzymes (ArM) can combine the cutting edge of homogeneous catalysis with the attractive facets of biological enzymes. The Roelfes group has focused on the Lactococcal multi-drug resistance regulator, a transcription factor with a large hydrophobic pocket, to create a novel class of Artificial enzymes by using diverse anchoring strategies. Integrating abiological transformations catalyzed by ArMs inside living cells remains a big challenge. This study describes the incorporation of an ArM catalyzed Enantioselective Friedel-Crafts Alkylation in the cytoplasm of E. coli. Despite the well documented Cu toxicity and weak binding (micromolar) of Cu(II)-phenanthroline complex to the hydrophobic pocket of LmrR protein, the straightforward supramolecular assembly of the artificial metalloenzyme proved compatible with the cytoplasm of E. coli. The in vivo assembled de novo enzyme increases the reaction rate and affords enantioselective product formation. This presented the opportunity to apply the biotechnological approach of directed evolution. With one round of directed evolution we were able to improve the reaction rate about 3 fold. The supramolecular anchoring strategy for Artificial metalloenzyme assembly seems to be the most favorable for in vivo incorporation. We envision that this approach can contribute to achieving the ultimate goal of integrating Artificial metalloenzyme chemistry with the metabolism of a cell.

3. Sequential aldol condensation catalyzed by 2-Deoxy-D-Ribose-5-Phosphate Aldolase (DERA)

Sequential aldol condensation catalyzed by 2-Deoxy-D-Ribose-5-Phosphate Aldolase (DERA)
Eman M. M. Abdelraheem, Fabio Tonin, Peter-Leon Hagedoorn, Ulf Hanefeld

2-deoxy-d-ribose-5-phosphate aldolase (DERA) is a class 1 aldolase that offers access to several building blocks for organic synthesis by catalyzing a stereoselective C-C bond formation between acetaldehyde and numerous other aldehydes [1]. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of acetaldehyde.
Here, we describe our studies with the acetaldehyde tolerant DERA lb [2]. The gene encoding DERA lb from Lactobacillus brevis ECU8302 was expressed in Escherichia coli and a point mutation E78K was introduced to improve both the acetaldehyde resistance of the enzyme and its thermostability. The structures and activities of the two enzymes were characterized and compared with those of E. coli DERA. The two DERA lb variants showed much greater activity in the sequential aldol condensation reaction (using acetaldehydes and 2-substituted acetaldehydes as substrates) than the E. coli ortholog. At 25 °C and 300 mM acetaldehyde, the chiral lactol intermediate was obtained in good yield. This intermediate is a synthon for the preparation of a large variety of optically pure super-statins, such as Rosuvastatin and Pitavastatin.

4. Rapid enzyme stabilization by computationally designed libraries of HMF oxidase

Rapid enzyme stabilization by computationally designed libraries of HMF oxidase
Caterina Martin, Hein Weijma, Marco W. Fraaije

HMF oxidase (HMFO) from Methylovorus sp. is a recently characterized flavoprotein oxidase. HMFO is able to oxidize 5-(hydroxymethyl)furfural (HMF) into 2,5-furandicarboxylic acid (FDCA). Because HMF can be formed from fructose or other sugars and FDCA is a polymer building block, the oxidase has attracted attention as industrially relevant biocatalyst. The dicarboxylic acid FDCA can be polymerized with ethylene glycol to produce polyethylene furanoate (PEF). This renewable and bio-based polyester can be a valid alternative to the petroleum-based polyethylene terephthalate (PET) thanks to its similar characteristics. 
The first step to the development of an HMFO with improved catalytic properties is the engineering of the enzyme to enhance its thermostability using the recently developed FRESCO method.
FRESCO (Framework for Rapid Enzyme Stabilization by Computational libraries) is a computational approach to determine thermostabilizing point mutations in a protein structure. FRESCO has the potential to become a more valid alternative to random approaches like direct evolution when the protein structure is known. The first steps consist of in silico screening of single variants. After this screening the selected mutants are subject to experimental verification for improved TM and preserved catalytic activity. Finally, the combination of stabilizing mutations are combined with a novel Golden Gate based technique to lead to highly stabilized variants.
I will present the results obtained by using the FRESCO method combined with the new gene shuffling technique developed: the stability and activity profiles of the generated HMFO mutants.

5. Detoxifying chlorite contaminated wastewater: Kinetic investigations of a Chlorite Dismutase

Detoxifying chlorite contaminated wastewater: Kinetic investigations of a Chlorite Dismutase
Julia Püschmann, Duga Mahor, Marc J.F. Stampraad, Peter-Leon Hagedoorn

Anthropogenic activities like chemical industries are releasing chlorinated species, like perchlorate (ClO4-) or chlorite (ClO2-), into our environment. These substances are toxic and difficult to treat in the wastewater. However, nature offers a way to solve this problem. Perchlorate-respiring bacteria possess an enzyme, namely called Chlorite dismutase (Cld). It can detoxify chlorite (ClO2-), which is a by-product of the perchlorate respiration, to chloride (Cl-) and molecular oxygen (O2). The heme b-dependent Cld disproportionates the chlorite by a unique O=O bond formation. Only photosystem II was known so far to catalyse these rare O=O bond formations. Therefore, it is interesting to characterize Clds in more detail to resolve its reaction mechanism and to investigate its potential for wastewater treatment.
Cld from Azospira oryzae (AoCld) has been structurally and functionally characterized in some detail. The crystal structure of AoCld has revealed that the heme is solvent accessible from two sides which makes the cofactor susceptible for binding a wide range of ligands. Imidazole, a contaminant after purification of the recombinant enzyme, was found to be removed only after extensive dialysis. Furthermore, the kinetic properties of AoCld were found to be strongly pH and buffer dependent. An optimal pH of AoCld of 5.8 was found, which was characterized by a smaller Km-value compared to other pH values. Further structural, spectroscopic and (pre) steady state kinetic results are presented.

6. Redesign of flavin-monooxygenase for bio-indigo production

Redesign of flavin-monooxygenase for bio-indigo production
Hugo L. van Beek, Nikola Lončar, Marco W. Fraaije

Current industrial indigo dyeing processes involve the chemical synthesis of insoluble indigoid dyes that are reduced to become water soluble (leuco-indigo) after which they are applied onto textile. It would be highly attractive to develop a biotechnological process to produce these popular dyes. Recently, a microbial flavin-containing monooxygenase (MeFMO) was discovered that is able to oxidize indole, resulting in the formation of indigo blue.
MeFMO is able to convert indole into indigo blue, but the turnover rate and substrate recognition is rather poor. This work aims to improve the catalytic performance of the monooxygenase by rational enzyme engineering. To achieve a better performing monooxygenase, the Multichange Isothermal mutagenesis (MISO) approach was used to generate a focused library targeting three residues close to the active site. Mutants have been produced, purified and characterized in detail. Mutants were compared according to their catalytic efficiency towards indole, TMA and 6 bromoindole. This resulted in an improved biocatalyst, but also provides an insight into the structural elements that tune the activity and specificity of MeFMO and provides hints for further improvement of the monooxygenase as a biocatalyst.

7. Going from natural to the unnatural: unique anomeric selectivity of trehalose transferases

Going from natural to the unnatural: unique anomeric selectivity of trehalose transferases
Hessel van der Eijk, Luuk Mestrom, Stefan R. Marsden, Ulf Hanefeld, Peter-Leon Hagedoorn

Trehalose (α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside) is a non-reducing disaccharide and is utilised in the food –, pharma- and cosmetic industry. Despite receiving the GRAS (Generally Recognizes As Safe) status by the FDA a recent study showed the correlation between the increase of dietary usage of trehalose and an increase in nosocomial infections with Clostridium difficile. Therefore, the development of structural derivatives of trehalose is highly desired. Here we describe the substrate scope and enantioselectivity of a thermostable and soluble fusion protein of trehalose transferase from Thermoproteus uzoniensis and mCherry (mCherry TuTreT).

8. Laccase catalyzed oxidation of lignin: Calorimetric determination of kinetic parameters

Laccase catalyzed oxidation of lignin: Calorimetric determination of kinetic parameters
Jie Zhang, Peter-Leon Hagedoorn

Laccase (EC 1.10.3.2) is a multi-copper-containing enzyme, that catalyzes one-electron oxidation of a range of inorganic and aromatic substances, like ortho-and para-diphenols, aminophenols and aryl diamines, with the concomitant four-electron reduction of molecular oxygen to water. Isothermal titration calorimetry (ITC) was used to study the oxidation of lignin by Trametes versicolorlaccase. The initial ratemethod of ITC IrCal that was recently by us indicated that the reaction of lignin (alkali, low sulfonate, ~10 kDa Mw) with laccase can be measured. The total molar heat of reaction (ΔHITC) of the reaction of laccase with lignin was -462±12 kcal/mol which represents the oxidation of circa 50 phenolic groups per lignin molecule.The IrCaldata were fitted to the Michaelis-Menten equation. The assay resulted in a Km of 6.6±1.3 μM and kcat of 0.0078±0.0007 s-1 at 25°C, pH 6.5.

9. A traffic light enzyme: Acetate binding switches the colour of heme enzyme Chlorite dismutase from red to green

A traffic light enzyme: Acetate binding switches the colour of heme enzyme Chlorite dismutase from red to green
Durga Mahor, Julia Püschmann, Marc J.F. Strampraad, Peter-Leon Hagedoorn*

Chlorite dismutase (Cld) is a unique heme b dependent enzyme catalyzing the disproportionation of chlorite into chloride and molecular oxygen in an O=O bond forming reaction. Enzymes that catalyze O=O bond formation are rare and therefore there is considerable interest in understanding the mechanism. Since the kinetic and spectroscopic properties of chlorite dismutase are strongly pH dependent, the structural basis for this pH dependence was investigated. We observed that chlorite dismutase from Azospira oryzae (AoCld) had a green colour in acetate buffer pH 5.0, while it remained red in other buffers at the same pH and at higher pH values. The colour change was found to be reversible after buffer exchange to KPi pH 7.0. This excludes an irreversible covalent modification of the heme cofactor, which has been observed for other green heme proteins such as myeloperoxidase.
UV-visible spectroscopy of AoCld showed a broad Soret peak at 400 nm and Q bands at 519 and 647 nm for the resting state ferric enzyme. This spectrum is characteristic of a five-coordinate high spin ferric heme. In acetate buffer at pH 5, this spectrum changed dramatically with a much sharper Soret band and higher absorptivity at 405 nm and Q bands at 502 and 626 nm. We believe that the more intense and red-shifted charge transfer band CT1 at 626 nm is the main contributor to the colour change. EPR spectroscopy showed that the enzyme is in a high spin ferric state, which is consistent with acetate being a weak field ligand (i.e. not changing the spin state). The activity of the enzyme was also strongly pH dependent, with optimal activity at pH 5.8.

10. The Use of a Micro-bioreactor as a High-Throughput Screening Tool for Immunotherapy Applications

The Use of a Micro-bioreactor as a High-Throughput Screening Tool for Immunotherapy Applications
Cristina Bernal Martinez, Applikon Biotechnology

Adoptive T-cell Therapy is a promising area of medicine where the lymphocytes are used to induce antitumor effects in patients with haematological malignancies. Despite the recent commercial approval of chimeric antigen receptor (CAR) T-cells to treat B-cell acute lymphoblastic leukaemia and non-Hodgkin lymphoma, the design space for the manufacturing of T-cell therapies is yet to be fully characterised and optimised. High throughput systems such as micro-Matrix (Applikon Biotechnology) in combination with Design of Experiment approach is a powerful tool for screening culture conditions for manufacture of T-cells at the micro-scale.

11. Optimization of Mushroom Substrate

Optimization of Mushroom Substrate
Gijs Kleine, Jimmy Burggraaf, Erik Postma, Jaap Rijfers, Jeno Bongers, Minke Noordermeer

CNC Holding BV has an annual production of 600,000 tons substrate (compost) for mushroom cultivation. This substrate is sold to mushroom producers and is ready for use. To be able to continuously improve the mushroom substrate, CNC Holding BV would like to get a better understanding of the chemical processes taking place during production.

The substrate is a mixture of horse manure, chicken slurry and gypsum and the production process consists of 3 phases:
- Pretreatment phase, 80 °C, 3-5 days
- Addition and growth of thermophile fungi, 48 °C, 5 days
- Addition and growth of mushroom mycelium, 25 °C, 16 days

It is known that the saccharides from the straw in the substrate are not completely used by the mushroom mycelium during mushroom production. This indicates that the pretreatment in phase 1 is currently not optimal and part of the (hemi)cellulose in the straw remains inaccessible for the fungi and mushroom mycelium. Third year students from the HU minor Food & Pharma developed a model system and studied the pretreatment efficiency under different conditions. By analyzing process samples from CNC it was found that pretreatment during phase 1 increases the amount of (hemi)cellulose available for next phases, 5 days being optimal. At the end of phase 1 part of the (hemi)cellulose is readily accessible for cellulase enzymes and in phase 2 most of these easily accessible saccharides are quickly used by the thermophilic fungi. At the end of phase 3 some (hemi)cellulose becomes accessible again, probably due to the action of lignases from the mushroom mycelium which further open up the lignin. Different ratios of chicken slurry and horse manure and different percentages of gypsum during phase 1 appeared to have little influence on pretreatment efficiency during phase 1.

12. Biobased production of valuable ester compounds by a novel type alcohol acetyl transferase; Construction of a complete CRISPR/CPF1 tool for Clostridium beijerinckii

Biobased production of valuable ester compounds by a novel type alcohol acetyl transferase; Construction of a complete CRISPR/CPF1 tool for Clostridium beijerinckii
Constantinos Patinios, John van der Oost, Serve Kengen, Ruud Weusthuis

Esters (e.g. ethyl acetate, butyl acetate, butyl butyrate) are very important molecules for the industry as they are used for many applications such as flavourings and fragrances and in myriads of products such as solvents, waxes, paints and coatings. Currently, production of ester compounds is heavily relied on petroleum-based feedstocks whereas only a small fraction is extracted from natural sources. The petroleum-based synthesis methods have many drawbacks, including harmful waste, by-product formation and high costs. Ester-producing industries are therefore looking for alternatives to switch from petrochemical to renewable feedstocks. Results and conclusions: We have recently identified a novel enzyme family called ethanol acetyltransferase 1 (Eat1) which is able to catalyse the condensation of an alcohol and an acyl-CoA for the formation of esters. An ideal microbe for the production of esters is Clostridium beijerinckii as it is a natural producer of alcohols (ethanol, butanol and isopropanol) and acyl-CoAs (acetyl-CoA and butyryl-CoA). Currently, we have successfully expressed Eat1 homologs in C. beijerinckii which showed the production of butyl acetate and butyl butyrate. However, the production of these esters is limited due to endogenous esterases which break down the produced esters. To further increase the production of esters in C. beijerinckii we have developed a complete CRISPR/Cpf1 tool which includes multiplex editing (knock-out or knock-in), multiplex silencing and multiplex base editing techniques, which will be used for identifying and eliminating the esterase(s) responsible for the degradation of esters in C. beijerinckii. Such a complete CRISPR/Cpf1 tool can be applied to other Clostridia species as well.

13. Unravelling genetic accessibility and physiological characterization of Clostridrium thermosuccinogenes using flow cytometry

Unravelling genetic accessibility and physiological characterization of Clostridrium thermosuccinogenes using flow cytometry
Joyshree Ganguly, Richard van Kranenburg

The transition towards bio-based economy demands the development of microbial fermentation processes as an alternative to unsustainable production processes. Economic production of green chemicals as building blocks for bioplastics has become a major target for sustainability. The use of thermophilic clostridia, that are able to consume and ferment biomass derived sugars, is an emerging niche in this field. However, the limited  availability of genetic tools for the manipulation of clostridial species is a major barrier for exploiting them. Hence, we intent to establish high-throughput genome engineering tools to develop thermophilic clostridia, in particular, Clostridium thermosuccinogenes,  into an improved microbial cell factory for production of green chemicals.

The project is classified into two major parts: (1) genetic accessibility and developing genetic toolbox for C. therrmosuccinogenes and, (2) study the sporulation mechanisms in the same organism and link it to the metabolism and product formation. In this work, the genetic accessibility of C. thermosuccinogenes was studied and transformants were obtained reproducibly by electroporation using shuttle vector pNW33n. Based on the identified restriction-modification systems of C. thermosuccinogenesE. coli derivatives carrying the host’s methylation genes are being created, the results of which will be discussed. The second part is on physiological characterization of C. thermosuccinogenes strains including the effect of temperature and pH on sporulation. Flow cytometry and Fluorescent-assisted cell sorting (FACS) demonstrated distinct populations of mature spores, endospores and vegetative cells.

14. Investigating the central metabolism of Clostridium thermosuccinogenes

Investigating the central metabolism of Clostridium thermosuccinogenes
Jeroen Girwar Koendjbiharie, Kilian Wiersma, Richard van Kranenburg

Clostridium thermosuccinogenes is a thermophilic anaerobic bacterium able to convert various carbohydrates to succinate and acetate as main fermentation products. Genomes of the four publicly available strains have been sequenced and the genome of the type-strain has been closed. The annotated genomes were used to reconstruct the central metabolism, and enzyme assays were used to validate annotations and to determine co-factor specificity. Genes for the pathways to all fermentation products were identified, as well as for the Embden-Meyerhof-Parnas pathway, and the pentose phosphate pathway. Notably, a candidate transaldolase was lacking and also transcriptomics during growth on glucose versus xylose did not provide any leads to potential transaldolase genes or alternative pathways connecting the C5 with the C3/C6 metabolism. Enzyme assays showed xylulokinase to prefer GTP over ATP, which could be of importance for engineering xylose utilization in related, thermophilic species of industrial relevance. Furthermore, the gene responsible for malate dehydrogenase was identified via heterologous expression in E. coli and subsequent assays with the cell-free extract, which has proven to be a simple and powerful method for basal characterization of thermophilic enzymes.

15. Production of ethanol from carbon monoxide by Clostridium autoethanogenum is enhanced in co-culture with Clostridium kluyveri

Production of ethanol from carbon monoxide by Clostridium autoethanogenum is enhanced in co-culture with Clostridium kluyveri
Martijn DienderIvette Parera Olm, Jasper J. Koehorst, Marten Gelderloos, Peter J. Schaap, Alfons J.M. Stams, Diana Z. Sousa

In a previous study, we established a synthetic co-culture of Clostridium autoethanogenum and Clostridium kluyveri with the ability to convert carbon monoxide (CO) to butyrate, caproate and their respective alcohols. In this co-culture, C. kluyveri converts ethanol and acetate, produced by C. autoethanogenum from syngas, to the medium-chain fatty acids butyrate and caproate, which can be further reduced by C. autoethanogenum to the respective alcohols. The co-culture was stable over several months of reactor operation, but how the two species interact and maintain an efficient flux of electrons from CO to the final products was unclear. CO conversion efficiency was higher in co-cultures than in mono-cultures of C. autoethanogenum. In addition, ethanol production by C. autoethanogenum was stimulated when grown in co-culture with C. kluyveri. In this work, we further studied the interactions between the two microorganisms to unravel the mechanism behind this behaviour. Results indicate that the transcriptome of the central carbon and energy metabolism of C. autoethanogenum remains unaltered in co-culture and that, instead, the metabolic boost observed is caused by thermodynamics of the system.

16. The impact of mixtures of xylose and glucose on the microbial diversity and fermentative metabolism of sequencing-batch or continuous enrichment cultures

The impact of mixtures of xylose and glucose on the microbial diversity and fermentative metabolism of sequencing-batch or continuous enrichment cultures
Julius L. Rombouts, Galvin Mos, David G. Weissbrodt, Robbert Kleerebezem, Mark C.M. van Loosdrecht

Glucose and xylose are the two most abundant monomers found in lignocellulosic waste streams Microbial diversity plays an important role in the functioning of mixed-culture biotechnologies. Complexity in the carbohydrate source could be one of the governing factors. The effect of feeding equivalent substrates to a microbial community, such a xylose and glucose is not well understood in terms of number of dominant species and how these species compete for substrate. We compared the metabolism and microbial community structure in a continuous-flow stirred tank reactor (CSTR) and a sequencing batch reactor (SBR) fed with a mixture of xylose and glucose. We hypothesise that a CSTR will select for generalist species, taking up both substrates. We used 16S rRNA gene sequencing and fluorescent in situ hybridisation to accurately determine the microbial community structures. Both enrichments were stoichiometrically and kinetically characterised. The CSTR enrichment culture was dominated by Clostridium intestinale (91%±2%), which confirms the generalist hypothesis. The closest related strain (99% identity of the 16 rRNA gene) showed presence of both a high affinity xylose and glucose transporter. The SBR showed a large fraction of Enterobacteriaceae (75%±8%), which was dominated by Citrobacter freundii and a minor fraction of Raoultella ornithinolytica. Citrobacter freundii ferments xylose and glucose in a non-diauxic fashion. Clearly, a non-diauxic generalist outcompetes specialists and diauxic generalists in a SBR environment. A non-diauxic generalist fermenting xylose and glucose simultaneously is thus a good candidate organism when designing an industrial fermentation process using a lignocellulosic feed.

17. EV Biotech: Evolution by Revolution

EV Biotech: Evolution by Revolution
Sonal Sengupta, Dmitry Bachin, Paula van Mourik, Sergey Lunev, Agnieszka Wegrzyn, Linda Dijkshoorn

Over the past decade, scientists have considerably expanded the understanding of microorganisms using a vast array of omics data. However, the functioning of a single cell is more than the sum of its genes, proteins, and metabolites. It is the synergistic effect that comes from the activity of all those components and their relationships to one another that add up to a living organism. From this, a new field of systems biology was born, which attempts to use the power of mathematics, engineering, and computer science to analyse and integrate data from all the omics to create models of entire biological systems.
EV Biotech introduces a specific industrial segment that leverages systems biology to design microbial strains to transform modern industries. At the very core of our efforts is a data-centric, predictive modelling based approach to designing our microcellular factories. We implement state-of-the-art computational tools for each step, from genetic design, protein engineering, to optimisation of metabolic pathways. Our in silico research is validated by the in vivo experimental data generated in our laboratory. Additionally, generated data are integrated and utilised in a feedback loop to develop our methods further, resulting in better predictions for future designs. Our multidisciplinary approach enables us to work on a wide variety of products, ranging from flavours to fibres. Empowered by our methods, we at EV Biotech, envision a world where sustainability will not come at the expense of, but rather as an increase in profitability.

18. A superior method for membrane protein production in Escherichia coli

A superior method for membrane protein production in Escherichia coli
Max Finger-Bou, Nico J. Claassens, Bart Scholten, Frederieke Muis, Jonas J. de Groot, Jan-Willem de Gier, Willem M. de Vos and John van der Oost

Escherichia coli has been widely used as a platform microorganism for both membrane protein production and cell factory engineering. The current methods to produce membrane proteins the model organism require the induction of target gene expression and often result in unstable, low yields. We present a method combining a constitutive promoter with a library of BiCistronic Design (BCD) elements, which enables inducer-free, tuned translation initiation for optimal protein production. Our system mediates stable, constitutive production of bacterial membrane proteins at yields that outperform these obtained with E. coli Lemo21 (DE3), the current standard for bacterial membrane protein production. We envisage that the continuous, fine-tuneable and high-level production of membrane proteins by our method will greatly facilitate their study and their utilization in engineering cell factories.

19. Production of medium-chain α,ω-diols from carbohydrates by Pseudomonas putida KT2440

Production of medium-chain α,ω-diols from carbohydrates by Pseudomonas putida KT2440
Chunzhe Lu, Rene Wijffels, Vitor Martins dos Santos, Ruud A. Weusthuis

Diols are used in the synthesis of polyesters and polyurethanes. Mid-chain-length α,ω-alkanediols -as 1,4-butanediol (1.8 million tons/year) and 1,6-hexanediol (0.1 million tons/year)- are important bulk building blocks for the production of plastics. Since the onset of petrochemistry it has been attempted to develop methods for the chemical conversion of mid-chain-length alkanes into α,ω-alkanediols. Until recently these efforts were unsuccessful because aspecific subterminal oxidation and overoxidation occurred. Instead, industry uses other resources, e.g. benzene for the production of 1,6-hexanediol which result in highly energy intensive processes with high CO2 emission. Biomolecular chemistry and bioprocess technology can be applied to realize this conversion. Alkane monooxygenases are able to specifically oxidize the terminal methyl group of mid-chain-length alkanes. However, they also overoxidize and are not able to make diterminally functionalised alkanes. Recently monooxygenase activity was combined with esterification in E. coli cells. The esterification of the hydroxyl group by alcohol acetyltransferase Atf1 prevented overoxidation and also made diterminal oxidation possible. However, alkanes were used as substrates, which are mainly derived from two resources: petroleum and natural gas. In order to make this process more sustainable, we try to synthesise diols starting from carbohydrates in P. putida KT2440. This microorganism shows a very high robustness against extreme environment and is successfully used for the production of bio-based polymers and a broad range of chemicals.

20. Customized DNA screening devices: a business model

Customized DNA screening devices: a business model
Marisca Nieuwenweg, Carina Nieuwenweg, Vittorio Saggiomo

The laboratory of BioNanoTechnology has developed a DNA test which can detect this genetic trait for a few euros by using a modular device which we have built with cheap microcontrollers (costing less than 100 euros). The device, similar to PCR for the amplification and detection of DNA, is based on Loop Mediated Isotermal Amplification (LAMP) which is faster and sturdier compared to standard PCR. Now, we want to use this device to showcase the potential of our approach as a business model. With a real-world case scenario, we want to show that we can create a customized DNA screening device capable of fast, cheap and easy detection of any sequence. The test works on the amplification of a specific part of DNA, the target. If the target is found, then the DNA is amplified by enzyme and the outcome can be detected with the naked eye. Our device is built on an Arduino system and has temperature-controlled cells which allow the amplification of the target. It is cheap, sturdy, modular and portable, as it can be battery operated.

21. Improved protein production and codon optimization analyses in Escherichia coli by bicistronic design

Improved protein production and codon optimization analyses in Escherichia coli by bicistronic design
Thijs Nieuwkoop, Nico J. Claassens, John van der Oost

Different codon optimization algorithms are available that aim at improving protein production by optimizing translation elongation. In these algorithms, it is generally not considered how the altered protein coding sequence will affect the secondary structure of the corresponding RNA transcript, particularly not the effect on the 5′‐UTR structure and related ribosome binding site availability. This is a serious drawback, because the influence of codon usage on mRNA secondary structures, especially near the start of a gene, may strongly influence translation initiation. In this study, we aim to reduce the effect of codon usage on translation initiation by applying a bicistronic design (BCD) element. Protein production of several codon‐optimized gene variants is tested in parallel for a BCD and a standard monocistronic design (MCD). We demonstrate that these distinct architectures can drastically change the relative performance of different codon optimization algorithms. We conclude that a BCD is indispensable in future studies that aim to reveal the impact of codon optimization and codon usage correlations. Furthermore, irrespective of the algorithm used, using a BCD does improve protein production compared with an MCD. The overall highest expression from BCDs for both GFP and RFP is at least twofold higher than the highest levels found for the MCDs, while for codon variants having very low expression from the MCD, even 10‐fold to 100‐fold increases in expression were achieved by the BCD. This shows the great potential of the BCD element for recombinant protein production.

22. Fit for purpose evaluation of a reduced scale model for a commercial manufacturing process

Fit for purpose evaluation of a reduced scale model for a commercial manufacturing process
Akshita Chordia, Alexiane Goujon, Elisabeth Meulenbroek

Process characterization is a part of late stage development used to identify process parameters and material attributes that impact product quality to enable establishing ranges in which the process can run with guaranteed product quality at commercial scale. The number of experiments to be performed during process characterization is generally high. Due to limited capacity at full-scale, process characterization at full scale is practically not feasible. We therefore developed reduced scale models (RSM) that are representative for the commercial scale. We compared the performance of the RSM to the performance of anticipated commercial manufacturing of drug substance by performing multiple RSM runs, which showed the reduced scale models are fit-for-purpose. This poster shows the results of the comparison of the reduced scale models to the commercial scale.

23. A decade of HP-RFC developments flourishing into an array of surprising applications

A decade of HP-RFC developments flourishing into an array of surprising applications
Bas Stevens, Hans May, Marcel Raedts

The start of Proxcys b.v. was primarily to bring the radial flow chromatography to the level of performance that it potentially had but was failing to express. Now more than 10 years later we can say “mission accomplished”. Today, an array of applications, some more simple than expected while as robust as required and others highly effective against the odds. 
In this presentation we will demonstrate a few exiting examples of unexpected possibilities that should change everybody’s perception of a technology that was invented in the sixties.

  • Linear scaling from a 20ml lab column into a 600L industrial scale, 30.000-fold scaleup.
  • Crude feed processing in a packed bed cTRAC column up to 50 million CHO-cell per ml culture loaded and just as simple as “normal” adsorptive chromatography ?
  • Crude feed cTRAC application but than even in a continuous mode ?
  • Variable bed-height processing in radial columns feasible ?
    The aim of the presentation is to offer insight in unknown Downstream Processing possibilities that were not expected to be possible but will allow the user to do things with less effort, faster and more reliable.

24. Syngas Mass Transfer in a Membrane Bioreactor

Syngas Mass Transfer in a Membrane Bioreactor
Marina Elisiário, Heleen De Wever, Wouter van Hecke, Henk Noorman, Adrie Straathof

Syngas fermentation to commodity and fine chemicals is an emergent technology in biobased economy and it is foreseen to have an important contribution against climate change, by the reduction of greenhouse gas emissions and simultaneous valorization of waste streams. At the process side, the mass transfer limitation is the major resistance for gaseous substrate diffusion, due to the low aqueous solubilities of the gaseous substrates and reduced mass transfer coefficients. Membrane bioreactors are a promising configuration for overcoming gas to liquid mass transfer limitations in syngas fermentation, so that sufficient productivity and product titers can be achieved. High gas-liquid interfacial areas and higher driving force for mass transfer can be achieved in hollow fiber membrane modules. The syngas substrates are fed in hollow fiber lumens and permeate through the membrane wall toward the shell surface. There, the microorganisms can form biofilms and convert the gaseous substrates into the product, which then diffuses to the liquid phase. Syngas fermentation in suitable membrane bioreactors is mathematically modeled in Comsol Multiphysics, using chemostat data and literature data. The model incorporates computational fluid dynamics calculations, mass transfer equations and black box stoichiometric and kinetic data. Model-based optimization will investigate the optimal parameters for the operation of membrane bioreactor and it will be used for design of experiments at lab scale. At sufficiently high membrane area, mass transfer kinetics does not remain limiting. Therefore, other relevant phenomena need to be taken into account, such as fluid dynamics, fermentation stoichiometry and kinetics, and biofilm formation kinetics.

25. Crafting the master key to genetic accessibility

Crafting the master key to genetic accessibility
iGEM Delft 2019: Hafsa Akaouche 1, Joel Clotet i Peretó 1, Osman Esen 1, Sagarika Govindaraju 1, Esther Hoogerwerf 1, Weronika Hoska 1, Dennis Kenbeek 1, Mu-En Lu 1, Huyen My 1, Renée van der Winden 1

[1] Delft University of Technology

Differences in cellular transcription and replication machinery make every synthetic biology and metabolic engineering discovery an organism-specific milestone, which later on will have to be adapted to be functional in another cell platform. This limitation restricts us to a small population of genetically accessible organisms for biotechnological applications and does not allow us to work with naturally “gifted” bacterial species, such as fast-growing or thermophilic bacteria.

The 2019 iGEM team from TU Delft aspires to remove these barriers by designing a universal gene expression toolkit. We aim to achieve this by making use of orthogonal replication machinery found in the bacteriophage Φ29, and orthogonal transcription machinery found in the phage T7.

This will pave the way for scientists to exploit new bacterial strains with advantageous characteristics without the need for previous, deep characterization of their regulatory elements and enables the use of a single genetic construct for a wide range of bacteria.

26. Bacteriophage-based diagnostics. Fast detection of bacterial pathogens with the use of specific bacteriophages and dCas9-split-Nanoluc

Bacteriophage-based diagnostics: Fast detection of bacterial pathogens with the use of specific bacteriophages and dCas9-split-Nanoluc
iGEM Eindhoven 2019: Jeroen Deckers, Harm van der Veer

Antimicrobial resistance (AMR) is a growing threat to public health, expected to claim 10 million lives by 2050, far exceeding cancer fatality rate. One of the key accelerators of the antibiotic resistance problem is the misuse of antibiotics. Improving diagnostics for bacterial infections could be a major part of the solution. Based on the exceptional specificity of bacteriophages for their target hosts, a rapid and modular detection method with the potential of being used in a point-of-care setting is developed. After binding the target host, lytic phages inject their genome, which is replicated and shed into solution following cell lysis, triggered shortly after infection. A paired dCas9-split-NanoLuc fusion protein sensor specifically binds the amplified phage DNA, resulting in NanoLuc activation, generating a bright bioluminescent signal.

27. QRoningen

QRoningen
iGEM Groningen 2019: Lieke van Iersel, Sander de Weerd

Privacy and encryption in this era of connectivity are becoming increasingly important. The internet is not a secure platform to share delicate information. We aim to develop a protocol to safely exchange sensitive data using genetically engineered bacteria. Additionally, QRoningen will make synthetic biology easily understandable for the layman. By combining two widespread and emerging technologies, QR codes and 3D printing, we intend to familiarize the public with a future where genetic engineering is common practice. Currently, genetically engineered organisms, whether they be infertile mosquitoes to fight malaria or tomatoes with extended shelf life, are taboo. This needs to be eschewed before we can make full use of the advantages that genetic engineering offers. So, through a combination of engineering, genetic engineering and (en)coding we will send a message.

28. A squid's secret to success: producing suckerin as a versatile biomaterial for novel applications

A squid's secret to success: producing suckerin as a versatile biomaterial for novel applications
iGEM Leiden 2019: Jonah Anderson, Maarten Lubbers

The tentacles of the Humboldt squid have sucker ring teeth that are built up from the protein suckerin. This protein occurs in various configurations, and as a result, the sucker ring teeth come in different strengths and flexibilities.
Suckerin has great promise as a biomaterial. It is strong, flexible, moldable and soluble, and due to its simple amino acids repeats it is relatively easy to synthesize the protein in bacteria like Escherichia coli. The Leiden 2019 iGEM team will produce suckerin as a biomaterial for various medical applications.

29. The Universal Assay

The Universal Assay
iGEM Rotterdam 2019: Ruben Sturkenboom, Nathalie van Wingerden

We are constructing a universal assay, in which you can perform the diagnostic test with a single droplet. This will require a combination of two synthetic zinc finger proteins that each bind a strand of DNA that also contains an aptamer region (DNA aptamer). By binding both aptamers to the same target molecule results in a bridge between the two zinc finger proteins. The zinc finger proteins are also fused to two different halves of TEV protease (split enzyme). The regenerated TEV protease will cleave of an inhibitory domain from beta-lactamase, which will then act on nitrocefin which will result in a color change from yellow to red. We will be able to switch between detection targets, by choosing zinc finger binding DNA’s that are coupled to custom made aptamers

30. Biocontrol of Xylella fastidiosa in Grapevines – Hijacking its Insect Vector System to deliver Phage Therapy

Biocontrol of Xylella fastidiosa in Grapevines – Hijacking its Insect Vector System to deliver Phage Therapy
iGEM Wageningen 2019: Niels Appelman, Cleo Bagchus, Alba Balletbó Canals, Santiago Castanedo Fontanillas, Marijn Ceelen, Robert Hooftman, Hetty Huijs, Sebastiaan Kuiper, Ben Kuipers, Paul Alexander Niederau & Dennie te Molder

Annually, the infection and spread of Xylella fastidiosa result in millions of euros in damages for both farmers and governments and is therefore on the EU’s list of most dangerous plant pathogens. X. fastidiosa is a xylem-colonizing plant pathogenic bacterium that forms a large threat to the cultivation of grapes, olives, citrus and coffee. It is spread by insects from one plant to another, where the bacteria impede the transport of water and nutrients by the obstruction of plant sap vessels. Our project aims to develop a biological system for the control of grape vine infecting X. fastidiosa, utilizing modified bacteriophages. We will equip the bacteriophages with the same spreading potential as X. fastidiosa, utilizing the same insect vectors. On top of this we want to develop a detection device for the presence of X. fastidiosa on the insects, enabling real-time detection and consequently the ability to administrate the phage therapy highly specifically. The use of this biological cure will limit the use of insecticides, provide a non-labour-intensive solution for famers and could eventually be further developed to be applicable on other plant species infected by X. fastidiosa.