acib – Innovations from Nature ............................................................................ 03 About acib – Fields of Expertise ........................................................................... 06 Homage to Coco Chanel ..................................................................................... 09 Liver in a Test Tube as a New Pharma Key Technology .......................................... 10 „Pine Aroma“ Against Beetle Invasion ................................................................. 13 Greenhouse Gas CO2 as a Raw Material: Aspirin From Carbon Dioxide.................. 15 Cell Aging Delayed .............................................................................................. 17 Millions of Liters of Juice from One Grapefruit ..................................................... 19 Advanced Biofuels Without Food Use .................................................................. 20 Enzymes from Austria Revolutionize the World of Paints ...................................... 22 New Value for Old Plastics ................................................................................... 25 “Enzyme-Google” Reveals Hidden Possibilities of Nature ..................................... 27 The Sugar Side of Biotechnology.......................................................................... 28 Plant Defense as a Biotech Tool ............................................................................ 31 High-Temperature Reductase for Organic Synthesis Available ............................... 32 Styrian Horse Radish Visualizes Active Enzymes .................................................... 35 Bodyguards for Precious Seeds ............................................................................. 36 Enzymes Against Fungal Toxins in Animal Feed .................................................... 38 Virus in the Service of Health ............................................................................... 40 Transparent Bioprocesses by Analyzing the Respiratory Air of Microorganisms ..... 42 Newly Discovered Metabolism Certifies Evolutionary Advantage for Yeast ........... 44 Microparticles for Superefficient Protein Purification ............................................ 47 World‘s First Method for Continuous Purification of Valuable Antibodies ............. 49 Genome of the Chinese Hamster Deciphered ...................................................... 50 Green Chemistry for the Pharmaceutical Industry ................................................. 53 facts & figures ..................................................................................................... 57 acib areas ............................................................................................................ 58
acib partners ........................................................................................................ 60 acib owners ......................................................................................................... 61 project design ...................................................................................................... 63
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acib â&#x20AC;&#x201C; Innovations from Nature There is and will always be much to learn from nature. The Austrian Centre of Industrial Biotechnology (acib) is a biotech research institution with the vision to replace traditional industrial technologies by new, more ecological and economic methods and to find new beneficial products that are based on natural processes. Therefor we connect the experience of 200+ highly qualified employees at acib with the additional knowledge of 50+ key researchers and their research groups at our international scientific partners to fulfill the needs of international biotech, pharma and chemical companies. Altogether, with 25+ years of experience in biotechnology acib is a perfect research partner for change in industrial production â&#x20AC;&#x201C; focused on biocatalysis, industrially used cells, synthetic biology, enzymes and (pharmaceutical) protein production and purification. acib translates academic knowledge into new industrial applications and products with higher values, lower costs and an optimized environmental balance. Our biotech stories show a part of our repertoire in the world of biotechnology that we jointly developed with 150+ international companies and scientific institutions within the acib network.
We are highly grateful to all partners who allowed us to create this brochure.
About acib – Fields of Expertise
HOST CELL & VECTOR ENGINEERING
Our expertise on bacteria, filamentous fungi, yeast, animal (CHO) and human cells in combination with the know-how in vector engineering allows us to address a huge diversity of scientific and industrial needs and to provide customized solutions for production or biological plant protection.
The combination of expertise on bioseparation processes with know-how on mathematical modelling and analysis of biophysical properties of macromolecules is the basis for the design of alternative approaches such as continuous bioseparation as well as their prediction and better understanding.
PROTEIN CHARACTERISATION & OPTIMISATION
BIOINFORMATICS AND MODELING
Our awarded „Catalophor System“ allows us to identify unknown enzyme functions. The further characterisation of proteins and their functionality is the basis for targeted engineering to provide tailor-made sulitions meeting the needs of modern biotechnology.
Experts at acib provide solutions that use rational design to reduce the resources that need to be invested into empirical approaches. acib methods facilitate the development of optimised processes or the search for new enzyme functionalities dramatically.
NOVEL COMPOUNDS & REACTION CONDITIONS
Novel routes providing innovative alternatives for reactions that are currently economically and/or ecologically problematic – this key expertise of acib provides solutions for critical steps in biocatalysis that can lead to substances that could not be synthesized previously.
The integrated approach of monitoring, modelling and fine tuning of bioprocesses – acibs expertise therein helps to improve efficiency and reduce costs of biotechnological production processes and to design new production strategies.
Our know-how in structural biology in combination with innovations in bioinformatics to model the active sites of enzymes as well as the expertise in synthetic biology using non-canonical amino acids to modify enzyme functionality offer innovative routes to novel enzymes. New synthetic pathways with 10+ steps allow us to produce highly valuable compounds biotechnologically.
Besides a broad panel of standard methods and all ”omics-technologies” acib provides access to highend analytical tools established within the centre and sophisticated instrumentation for chemical- and bioanalysis which are operated by highly-trained employees.
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Homage to Coco Chanel
Special aldehyde-based compounds are available with an ecofriendly note since researchers at acib have mastered the asymmetric C = C reduction selectively and in high yield thanks to a selected ene-reductase. As a result, important odorants are accessible in highest purity. According to the manufacturer Chanel, it’s most famous perfume “No. 5” sums up for “secret opulence and imperishable femininity”. What does that have to do with biotechnology? A lot, considering the Austrian Centre of Industrial Biotechnology (acib). Many perfumes smell particularly well because of an odorant from lily of the valley. Unfortunately, this component is available in the plants only in spring and in minute amounts. Using an enzymatic approach, a team headed by Prof. Kurt Faber (University of Graz and acib) was able to produce valuable fragrances in highest purity for the first time. The scientific challenge was to carry out the asymmetric reduction of C = C bonds selectively and with high purity while gaining only one of two possible chiral products; in this case lysmeral/Lilial™. That is impossible using classical chemical synthesis. Up to now, the odor had to be obtained in poor yield directly from the plants. acib researcher Clemens Stückler used biocatalysis, which is based on environmentally friendly, enzymatic natural processes that are adapted for industrial application.
This way, nonracemic aryl substituated α-methyldihydrocinnamaldehyde derivatives employed as olfactory principles in perfumes (Lilial™, Helional™) were obtained via enzymatic reduction of the corresponding cinnamaldehyde precursor using cloned and overexpressed ene-reductases. The product’s yield and quality are more than satisfactory: The odorant used in many perfumes is available with a purity of 99%. Ultimately, the research project won an Excellence Award from the Austrian Federal Ministry for Science and Research, which was awarded to Stückler for his PhD thesis “Asymmetric Reduction of C = C bonds”. All results were possible because of the cooperation of acib, the University of Graz and BASF as an industry partner. “acib provided the enzymes which originate from tomatoes or brewer’s yeast”, says the chemist Stückler, while University of Graz assisted with infrastructure and additional knowledge. BASF contributed financially to the research project.
During the past years, the asymmetric bioreduction of alkenes (bearing an electron-withdrawing, activating group) catalysed by enzymes/flavoproteins has emerged as a highly valuable tool for the biocatalytic synthesis of nonracemic – or – substituted aldehydes, ketones, carboxylic acids/esters, nitriles and nitroalkanes. Especially the aldehydes are interesting compounds not only for fragrance industry. Thanks to the acib-method optimized by Stückler it is now possible to produce the appropriately selected chiral molecules in pure form. 9
Liver in a Test Tube as a New Pharma Key Technology Together with F. Hoffmann LaRoche and Novartis acib-researchers developed a new key technology for the pharmaceutical industry: The first test system in a preparative scale simulating in vitro the metabolization of drugs in the human body. “For effects and unwanted side effects read the patient information leaflet ...” - every one of us is knowing a sentence like this by heart from the days of our early childhood. A key technology for the investigation of effects and unwanted side effects is subject of a research project carried out by the Austrian Centre of Industrial Biotechnology (acib). Together with our industrial partners Novartis and F. Hoffmann LaRoche the acib scientists have developed a technology, which enables to simulate in vitro the metabolization of drugs in the human body - a “liver in a test tube”. The liver is the primary organ for metabolizing pharmaceuticals; the test uses non-Cytochrome-P450 enzymes for the simulation of the metabolic processes in the lab. Pharmaceutical testing is required to ensure that the drug is not only effective but ideally doesn’t produce unwanted side effects, either. To date, pharmaceutical industry needs to predict all possible breakdown products in the body resulting from the use of the drug, produce them chemically and test all of them for their effects. “At the worst all predictions prove wrong because the metabolic processes in the body don’t go off as expected”, explains Margit Winkler, scientist at acib. The new acib method uses endogenous enzymes and in vitro simulates the metabolizing of the drugs in the body. The pharmaceutical industries keep developing new drug candidates to enhance the treatment possibilities of doctors and thus improve our health. The question is: What happens to pharmaceutical agents in the body? “On the one hand the compounds bring about the desired effects, on the other hand they are degraded so that they can be released more quickly”, says project manager Winkler. In addition to reductases/dehydrogenases and hydrolases, to date five other oxidative enzyme classes have been known as possibly responsible for the first metabolic step: Cytochrome P450 enzymes (CYPs), flavin monooxygenases (FMOs), monoamine oxidases (MAOs), aldehyde oxidase (AO) and xanthin oxidases (XO). Which of those enzymes in the end degrades a special agent depends on its chemical structure. 10
During the past years research has mainly concentrated on the CYP family and comparably little attention has been paid to the other four enzyme classes, the so-called nonCYP metabolizing enzymes (NCMEs). In cooperation with F. Hoffmann LaRoche and Novartis acib has now dedicated itself to these enzymes. “We intend to have available each single enzyme from the human degradation metabolism in order to simulate special functions of the human liver”, explains the acib scientist. With this “artificial liver” enzyme set the pharmaceutical industries are able to test new agents and know, at a very early stage, which breakdown products are formed.
This information at hand - together with an abundance of the right enzymes - makes it a child’s play to produce sufficient quantities of the degradation products for further investigations - no more guessing! The acib method was successfully used for the degradation of moclobemide - a monoamine oxidase A (MAO-A) inhibitor known under the brand names Aurorix or Manerix prescribed to patients with depressions and is finally being used in drug development. Together with a processes based on enzymes from almond trees and pig liver and others acib can look back at a series of new processes for the pharmaceutical industries.
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„Pine Aroma“ Against Beetle Invasion Researchers from the Austrian Centre of Industrial Biotechnology shorten the production of biological agents from 14 to 3 steps. Eco-friendly products not only to cope with chewing pests, bacteria or fungi can be produced easier and environmentally friendly. Pine trees and red ants have something in common: Both use alkaloids to banish enemies. These organic ingredients are more and more in demand because of their environmental friendliness and safety. The problem is that they are only present in minimal amounts in natural form. Chemical synthesis in turn is complicated and expensive. Researchers at the Austrian Centre of Industrial Biotechnology (acib) and at the University of Graz led by Prof. Wolfgang Kroutil have now developed a new key technology to produce a promising alkaloid variety much easier than ever using biocatalysis. So remarkable the about half an inch wide pine weevil is, so harmful it can be. The insects, which play dead at the slightest vibration, live on young spruce or pine trees, where they place their eggs into the stocks. Occurring in large numbers, the critters can cause great harm, because the infected trees are consistently condemned to death. “In the northern hemisphere, this could be a real problem”, says Prof. Kroutil. Natural alkaloids are perfect remedies, which expel the beetles in a biological manner. The function of the alkaloid is similar to territorial marking of predators: If a newbie finds a marking scent, he knows that there is already someone else in place and he stays clear of the area. One of these alkaloids is “Dihydropinidine” which belongs to the class of 2,6-dialkylpiperidines. The substance has a major drawback: In its natural form it is present only in minute quantities in some pine varieties. Until now the production of this substance in larger quantities has nearly been impossible, because up to 14 very sophisticated chemical synthetic steps were necessary. An acib-research group led by Prof. Kroutil found a new approach to this class of substances. “The problem with many syntheses is that you have to protect certain parts of the parent molecule to get the desired reaction only at a certain position”, explains the researcher.
Without protection, the result is an useless substance. For chemists, this means many reaction steps until the desired substance is eventually synthesized in minimal quantities. The acib-researchers have found an enzyme, which reduces the number of steps from 14 to only 3. The first and last steps of the synthesis are “chemical”, the central one will be accomplished by a highly specific “omega transaminase”. This enzyme yields the product avoiding the undesired byproducts occurring in the chemical approach. This saves time and energy and reduces the use of environmentally harmful organic solvents. Thus the new method is not only a step forward in the battle against the beetle but opens up new opportunities in the production of biologically highly active alkaloids. Using the new synthesis technique, the chemical industry can synthesize environmentally friendly products against pests based on dialkylpiperidin including antifeedants as that against the pine weevil but also agents against bacteria (bactericids) or fungi (fungicides) that can be produced on a commercial scale now. Just recently, the method has been adapted for producing “Isosolenopsin”. The substance is an alkaloid oozed by red ants for defense. It is interesting for industrial application because it has strong antibacterial properties. In addition, it acts anti-hemolytic (prevents the destruction of red blood cells) or anti-necrotic (helps against the death of tissue). The importance of the new concept developed within the internal acib-partnership is proven by its publication in “Angewandte Chemie”, goo.gl/4qD4dF. The method was engineered in a research project with Sandoz.
BIOTECHNOLOGY, BIOREFINERY AND SUSTAINABLE PROCESS DEVELOPMENT Contact and Address: TU Wien Institute of Chemical Engineering Director: Univ. Prof. Dipl.-Ing. Dr. Anton Friedl Tel. +43-1-58801-166200 email@example.com www.vt.tuwien.ac.at Getreidemarkt 9/166 1060 Vienna Austria
Technology no longer merely targets machines and instruments, but extends to life itself! TU WIEN is intensely engaged in research on biotechnology and in underlying fundamental disciplines such as microbiology and genetics. We have expertise in gene technology, systems biology, synthetic biology and microbial biodiversity as well as reactor engineering and downstream technologies. BIOREFINERY AT TU WIEN Institute of Chemical Engineering leads an interdisciplinary competence platform on BIOREFINERY to produce highquality pharmaceutical and food products from agricultural waste. Our aim is to expand chemical technology to the use of raw materials in the development of innovative products: BIOREFINERY process development and the implementation of a BIOREFINERY pilot plant to use agricultural waste such as raw cellulose substrates, C5 and C6 sugars and lignin fractions. BIOREFINERY product development through engineered microorganisms producing high-quality, fine chemicals for food, energy and pharmaceutical industries. Examples the production of the sugar replacer erythritol directly from wheat straw, screening for novel enzymes and genetic improvement of enzyme producing fungal cell factories. BIOREFINERY analytics as a structural and quantitative monitoring of raw materials for optimum control of the chemical and/or enzymatic degradation of raw materials. FUNGI AS A BIOTECHNOLOGY TOOL TU WIEN is developing genetically engineered microbes for industrial needs. We do rational design of proteins and invent molecular tools for their recombinant production in filamentous fungi, yeasts and bacteria. We study regulation of gene expression, design producing strains and construct of whole-cell biocatalysts. For example, our mutant strains can produce valuable medical compounds from abundant biopolymer chitin. Genomes of other fungi are modified for production of enzymes for biofuels and other industries.
TU WIEN FOR SUSTAINABLE SYSTEMS AND HIGH WATER QUALITY Modern technology of crop production requires reduced use of chemical pesticides and fertilizers as well as strict toxicological regulations. TU WIEN is conducting research on bioeffectors â&#x20AC;&#x201C; viable microorganisms that directly or indirectly influence plant health and thus may be used for the development of biofungicides and biofertilizers. We study the ecology, physiology and genomics of natural microbial antagonists across various ecosystems and transfer the mechanisms to biocontrol applications. To control undesirable microbes and their toxic metabolites in water and food products TU WIEN is developing bioanalytical methods for environmental monitoring based on contemporary DNA technologies and flash test systems.
Greenhouse Gas CO2 as a Resource
An enzymatic approach allows the use of carbon dioxide CO2 as a raw material for chemical synthesis. With the help of decarboxylases, industrially most interesting molecules can be produced “climate friendly” and highly specific. Carbon dioxide is the best known greenhouse gas. Since the introduction of CO2 pollution allowances, carbon dioxide is a dreaded thing for the industry. For scientists at the Austrian Centre of Industrial Biotechnology (acib) und the University of Graz the exhaust has a new meaning: they have developed a way to use CO2 as a raw material for chemical synthesis – an important step forward in the face of global climate change and dwindling resources. Researchers around Silvia Glück and Prof. Kurt Faber use the carbon in CO2 for the synthesis of valuable chemicals. The reaction is carried out using modified decarboxylases as biocatalysts. The acib-researchers used phenolic compounds as output material, but pyroles and indoles also can be used as a substrate. The resulting aromatic carboxylic acids have a huge importance for industrial application: Salicylic acid, a precursor of aspirin, was synthesized in this way as well as p-aminosalicylic acid, an active ingredient for the treatment of tuberculosis. Depending on the kind of enzyme, highly specific substances are accessible – even those who were not synthesizable with classical chemical methods – like compounds gained via asymmetric hydration.
Currently the acib-researchers optimize the enzymatic process to improve the specifity and selectivity of the reaction and to broaden the spectrum of substrates and reaction products. Another advantage of the enzymatic approach: The classical method (Kolbe-Schmitt reaction) is not only non-specific – it causes unwanted side products – it also requires a high energy input (high temperatures and pressures). The highly specific enzymatic variant operates at room conditions and meets environmental and economic requirements of a modern synthesis technology. The environmentally friendly process eventually opened up new possibilities in building innovative compounds for the pharmaceutical, cosmetics and chemical industries. The project was honored at the 2013 Houska-Prize, Austria’s most prestigious award for scientific progres.
Cell Aging Delayed Protection against cellular aging in a natural way: Austrian researchers biotechnologically produce a valuable antioxidant from olives that is more effective than Vitamin C. It can prevent cancer and has many positive effects on human health. Whether in salads, for frying fish or meat or just as a snack on white bread - for millennia olive oil has been one of the tastiest natural products. And it is one of the healthiest because of the high content of unsaturated fatty acids and additional substances, which are present in minute amounts in olives (and in virgin olive oil “extra virgine”). A particularly valuable one is 3-hydroxytyrosol. “The substance is said to protect the cells and thus delays aging and prevents various diseases”, says Margit Winkler, researcher at the Austrian Centre of Industrial Biotechnology (acib). This is due to the antioxidant effect, which is even stronger than that of ascorbic acid, the well-known antioxidant Vitamin C from citrus fruits. No wonder that the substance is always in demand as a natural food supplement or as a component for cosmetics. The crux of the matter is the availability of 3-hydroxytyrosol: olive trees grow in limited geographic areas where olives can be harvested once a year and should remain a valuable and tasty food and not become the raw material for a substance that is present only in minimal amounts. As to Winkler, the separation is difficult and expensive. A research group of acib and the Swiss industrial partner Lonza has found a way to produce the valuable substance biotechnologically. A patent has been filed and the project outcomes were scientifically published. The biotechnological route uses bacteria (Escherichia coli) as a cell factory. The researchers have integrated a new enzyme from another microorganism (called Nocardia), which is
able to convert a cheap, low-carbon acid (3,4-Dihydroxyphenylacetic acid, DOPAC) into the valuable 3-hydroxytyrosol. The entire reaction was improved so that the cofactors otherwise necessary for this complicated conversion are no longer needed - the high art of biotechnology. “Fed with DOPAC at laboratory scale there is a turnover of 100 percent using our modified Escherichia coli cell factories”, explains acib-scientist Winkler. What works in the lab could be upscaled into industrial mesures. ANTIOXIDANTS AND CANCER Oxidative stress damages living cells. They form very active “radicals”, which react with everything that comes their way. This causes cancer or an even faster cell death. For example, skin cells exposed to UV light show such oxidative stress. The skin ages faster, dries out, becomes wrinkled. In the worst case, skin cancer is the long-term consequence. Antioxidants neutralize the free radicals and decrease the risk of damages dramatically. 3-hydroxytyrosol has already been tested in many studies and showed “cytoprotective effect”: Protection of cells was shown with intestinal and brain cells, cells of the cardiovascular system, liver and various other cell types. For the authors of the studies, 3-hydroxytyrosol can prevent cancer, is anti-inflammatory and has a positive effect on the cardiovascular system. The results were finally published in “ChemCatChem”: goo.gl/vogdYl
Millions of Liters of Juice from One Grapefruit New method allows production of expensive grapefruit aroma Nootkatone biotechnologically from cheap sugar using a “turbo-yeast”. The versatile, healthy and tasty substance is used in drinks, pharmaceutical products or even as an insect repellent. The Austrian Centre of Industrial Biotechnology (acib) uses the positive aspects of synthetic biology for the ecofriendly production of a natural compound. The challenge of the biotechnologists Tamara Wriessnegger and Harald Pichler in Graz was to produce Nootkatone in large quantities. The substance is expensive (more than 4000 USD per kilogram) and can be found only in minute quantities in grapefruits. At the same time the need is great, because Nootkatone is used as a high quality, natural flavoring substance in millions of liters of soft and lifestyle drinks, as a biopharmaceutical component or as a natural insect repellent. FOUR NEW GENES “We have installed new genetic information in the yeast Pichia pastoris, so that our cells are able to produce Nootkatone from sugar”, says acib researcher Tamara Wriessnegger. The genome of the yeast cells has been extended with four foreign genes derived from the cress Arabidopsis thaliana, the Egyptian henbane Hyoscyamus muticus, the Nootka cypress Xanthocyparis nootkatensis and from baker’s yeast Saccharomyces cerevisiae. Ultimately, the genetic information of the aroma found in one grapefruit leads to millions of liters of tasty juice. With the help of the new genes the yeast is capable to synthesize the high-prized, natural flavor (more than 4000 euros per kilogram) in a cheap way and in useful quantities from sugar (one euro per kilogram).
As an insecticide it is effective against ticks, mosquitoes or bedbugs. In the medical field, the substance has shown activity against cancer cell lines. In cosmetics, people appreciate the good smell, in soft drinks a fine, subtle taste. Because the natural sources by far cannot meet the demands, the acib method replaces chemical synthesis an energy-consuming and anything but environmentally friendly process. The common biotech variant via Valencene and a chemical synthesis step is less ecofriendly, more difficult and expensive. Pichler: “With our method, the important and expensive terpenoid Nootkatone can be produced industrially in an environmentally friendly, economical and resource-saving way in useful quantities.” SYNTHETIC BIOLOGY Synthetic biology could be of vital importance to humanity, as Artemisinin shows. Thanks to this substance malaria is curable. Unfortunately, it could be found only in tiny quantities in the sweet wormwood – until the US researcher Jay Keasling was able to transfer the appropriate production route from the plant in bacteria. With these “synthetic” organisms the active ingredient is produced at lower costs. The acib research results were published in the journal “Metabolic Engineering”: goo.gl/xu2s0h
Nootkatone is an important substance for the food, pharmaceutical and chemical industries, says Harald Pichler.
Advanced Biofuels Without Food Use Today wheat or corn is mostly used to produce biofuels. But this misuse of food is not necessary at all. acib and an industrial partner developed methods, which use agricultural waste and straw as basis for new biofuel materials. “Fuel instead of food”, is currently a common question although this need not be. The first generation of biofuels is mostly made of corn, wheat or sugarcane. But that’s not exactly the ideal solution. Biofuels of the 2nd generation are made of agricultural waste – from wood chips, straw or specially cultivated “energy crops”. The Austrian competence centre acib (Austrian Centre of Industrial Biotechnology) has found ways to make these renewable sugar resources available for industry and for the production of biofuels. The enzymes that are used are called cellulases. They can cleave cellulose and hemicellulose - both components of wood (besides lignin) – into small sugar molecules, explains Professor Christian Kubicek (Technical University of Vienna). In the framework of the center of excellence acib, he worked together with researchers in Graz and at an industrial partner’s site on the access of new industrial sugars from renewable resources. “The enzymes work like choppers”, says Professor Anton Glieder, key researcher at acib, “the long cellulose chains are transported through the enzymes. Thereby, the enzyme cuts small sugar molecules off the comparatively huge cellulose chain, until the whole cellulose is digested into smaller sugars.” The best enzymes for the process are produced using the fungus Trichoderma reesei, which normally grows on decaying wood residues. In the Styrian project “MacroFun” at Graz University of Technology the fungal enzymes were improved by using the yeast Pichia pastoris to make the “molecular shredder” more robust, declares Glieder.
Admittedly, the general procedure is still extensive, says Kubicek. The plant remains or “energy crops” such as flower stalk grass (Miscanthus) or switchgrass (Panicum virgatum) must first be “unlocked” to separate the lignin and make the cellulose accessible. Then specially designed cellulases come into play and cleave the long cellulose chains into small sugars. These are finally converted – similar to the alcoholic fermentation in wine – from yeast to bioethanol, which can then be used for biofuels. The great advantage of this method: food remains completely unaffected and the carbon footprint looks a lot better. How promising this type of sugar and subsequently biofuel production is, shows the fact that alone in Europe 400 million tons of wheat straw per year accumulate. To ensure a sustainable use, 30% of them remain on the field to regenerate the soil, but the enormous residue can be processed further. Actually, about 1 liter of bioethanol could be made from 5 kg of straw. The goal for acib is to raise the yield by optimization of the degrading enzymes and to make more yet unused sugar sources available for biofuel production. 2nd generation biofuels are more or less ready to use.
Enzymes from Austria Revolutionize the World of Paints
Austrian scientists banish toxic heavy-metal catalysts from paints. For the first time pollutants are replaced by natural enzymes. The project has been knighted scientifically: on the front page of the journal “Green Chemistry”. Wood floors, garden furniture, paintings in various indoor and outdoor use – hardly anyone uses paints in the course of her or his life. Most commonly in use are so-called “ alkyd resins”; in Europe alone there are produced 700,000 tons per year. But hardly anyone was aware until now that the paint will dry, because the heavy metal cobalt accelerates the drying process. Recently it was discovered that cobalt is potentially carcinogenic which makes the paint industry think on alternatives. Cytec Austria and the Austrian Centre of Industrial Biotechnology (acib) found a solution to replace Cobalt by enzymes which make paints ecologically friendly. “We had the idea to use enzymes instead of metals in 2009”, says Professor George Gubitz, enzyme specialist at acib and professor at the Institute for Environmental Biotechnology at the University of Natural Resources and Life Sciences Vienna. The enzyme was derived from the tree sponge Trametes hirsute or “Hairy Bracket”, isolated from Gubitz’s garden in Graz. “We knew that the fungus contains the enzyme type we were looking for”, explains Gubitz. So a piece of mushroom was harvested, the active enzyme isolated and proliferated. The enzyme called “Laccase” is able to combine fatty acid molecules in the alkyd resin with the aid of oxygen from the air so that the paint dries and solidifies. Previously this was only possible with the help of heavy metals; particularly cobalt was used. Lately a new EU-regulation demands that only 10 ppm of cobalt are allowed in new in coatings as of 2013. Currently, a useful paint works only with a significantly higher amount of the potentially harmful substance!
The paint manufacturer Cytec Austria with laboratories in Graz and the production unit in Werndorf is a pioneer in environmentally friendly technologies. The development of water-soluble resins and varnishes in the end of the 20th century was a major contribution to the banning of toxic and hazardous solvents from these products. Now Cytec goes a step further and eliminates the next health hazard by replacing cobalt by biocatalysts. “The attributes “free of heavy metals” and “biocatalyzed” are perfect marketing arguments for the sale of new products”, states project manager Roland Feola from Cytec Austria. In the project partnership Cytec Austria provides the alkyd resin while acib handles the enzymes and provides the scientific basis specifically in the area of biotechnology. Besides Cytec handles the application-oriented issues of the paint manufacturing process and brings in it’s knowhow about product- and market requirements. The partnership finally led not only to a more environmentally friendly paint, but also to a new measurement method for monitoring the hardening of the resin. “We have developed an optical measurement system to monitor the decrease of the oxygen content in the coating film”, says acib-scientist Katrin Greimel. Oxygen decreases during the hardening process. This is the first high-precision observation method of paint hardening in a supersmall scale which facilitates research a lot. The project has been filed for a patent. Because of a shared knowledge business model, acib is open for further cooperations In specific painting applications.
ABOUT ALKYD RESINS Alkyd paints consist of fatty acid molecules, color pigments and “siccatives” (the dryer). When a handyman spreads the paint on the furniture, the siccatives accelerate the connection of the fatty acid molecules by the incorporation of atmospheric oxygen and thus the drying of the paint. Previously, this task was taken over by metal compounds based on cobalt which turned out to be potentially harmful. Now it is possible to replace cobalt by enzymes – by natural and environmentally friendly biocatalysts.
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New Value for Old Plastics
Every year huge amounts of plastic are globally converted into minor valued materials, ending up in the waste incineration or pollute the environment; for example in the form of floating garbage islands. Researchers at acib have found ways to disassemble old plastic into valuable starting molecules and develop new bioplastics, which are fully degradable in nature. Industry produces 250 million tons of plastics per year; for packaging, clothing, decoration and much more. A good part of it is not utilized in a savvy way. In Europe, only a third of 14 million tons of textile waste per year are used for recycling. The rest is burned, processed into low-grade plastics or ends up in the environment, where industrially produced plastic hardly rots. Huge plastic islands diving in the oceans for decades testify about that. This problem could be solved. Biotechnology, with the help of microorganisms or enzymes, it is possible to degrade polymers into their building blocks - under environmentally friendly conditions, without the addition of chemicals or energy. acib scientists led by Prof. Georg Gübitz (University of Natural Resources and Life Sciences and acib) do research on biological recycling for many years. Meanwhile four patents are pending. The acib technology is based on the adaption of natural enzymes with the ability of cleaving polymers similar to their natural substrates. “Naturally occurring cutins are akin to synthetic polyesters according to their bonds and water repellent properties. That is why cutinases also can dismantle plastics into their original molecules”, explains Gübitz. Using targeted mutagenesis the biotechnologists have optimized cutinases so that they can better degrade synthetic polyesters. The enzymatic degradation works with PE (polyethylene terephthalate) as well es PU (polyurethane). More plastics are under investigation. Because nowadays many plastics are composite products, this way it is possible to selectively dissolve the desired molecules from plastic waste. Gübitz: “With the help of the enzymes we are obtaining the starting molecules from various plastics in high purity.”
For waste treatment, no longer used plastics have to be cleaned, crushed and finally broken down using specifically enzymes. “Our process has the advantage that plastic waste no longer needs to be further sorted”, says acib-researcher Enrique Herrero-Acreo. The acib research not only deals with the degradation of polymers, but also with their modification and the creation of bio-plastics that are degradable in nature. “We use enzymes to alter the surfaces of plastics specifically and gently. We think of manufacturing durable membranes, which are both breathable and anti-static”, explains biotechnologist Gübitz. When talking about decomposition of synthetic material, Gübitz draws a comparison with wood: “This biopolymer is very durable with proper care, but in nature it rots in a few years.” As micro plastic and other hardly degradable polymers are known to pollute the terrestrial aquatic systems massively, acib researchers Doris Ribitsch and Karolina Härnvall are investigating whether and how plastics are being degraded in aqueous systems such as rivers or lakes. Conclusions about the microbial and enzymatic degradation allow the design of new plastics that are broken down in an aquatic environment into harmless substances and not longer burden the environment as those hardly degradable plastic islands that have formed in the oceans. The project won the Austrian Neptun Award 2015, which is donated by the Austrian Federal Ministry of Agriculture, Forestry, Environment and Water Management once in two years.
“Enzyme-Google” Reveals Hidden Possibilities of Nature
A new search engine, including a database with more than 100.000 proteins, opens up new possibilities in the search for industrial usable enzymes or for alternatives to patented biocatalysts. The method was published in Nature Communications. On the basis of this script the “Catalophor System” browses 110.000+ database entries for similarities. The result is a weighted list of possible candidates. In the next step the most promising candidates are manufactured and tested in the lab. The preliminary work on the computer saves countless experiments and screenings for new enzyme functionality. The database itself is constantly being expanded. “Every week about 150 new structures are added,” says Georg Steinkellner from acib. “We also refine the entire system to answer more complex search queries.” As high-precision miniature tools from nature, enzymes can solve certain tasks perfectly. The search for new industrial usable enzyme functions is very complex. A project of the Austrian Centre of Industrial Biotechnology (acib) and the University of Graz opens up a new approach: The “Catalophor System” – a combination of database and search engine – filters desired enzyme functions out of tens of thousands of protein structure data and can even track functionalities that have not been discovered yet. The procedure is similar to a typical search engine type search, although the input of data is a bit more complicated. All starts with the question for the required enzyme function. “We focus on the active site of the enzyme sought-after and write a program which specifies the positions and distances of the most important amino acids as well as important structural features in the vicinity of the active site”, explains acib researcher Christian Gruber.
The “Catalophor System” has a high practical benefit for science and industry. “Based on the protein structures we can discover new possible reaction pathways of enzymes that have not been described yet. For the chemical industry our approach opens up new reaction pathways that were not possible until now”, says Prof. K. Gruber. The opportunity to replace conventional industrial processes with environmentally friendly and more economic enzymatic methods increases. And it can offer alternatives to patented industrial enzymes. The method was applied for a patent and published in “Nature Communications” (goo.gl/eK3HAJ) and finally won an Innovation Award for process development at the CPhI 2014 – the world’s largest fair for the pharmaceutical and chemical industries.
The Sugar Side of Biotechnology Sweetening power , UV protection, sugars against spoilage of food or even for cancer protection – EU research partnership about the power of sugar relies on knowledge of the Austrian Centre of Industrial Biotechnology (acib) in glycoside chemistry. Sugar is not the same as sugar. Sucrose, the white substance which we use to sweeten or for cooking, is just one example of a variety of sugar compounds that have a special meaning in nature; for example in the form of cellulose as a stabilizing structure for plants. Or as a prebiotic oligosaccharide in mother’s milk, which helps that a baby’s intestinal flora can develop perfectly. The EU research project SUSY deals with producing particularly valuable sugars more easily. The starting material is our retail sugar – sucrose. The tools used for modification of glycosides are three enzymes – highly specific instruments in micro format: Sucrose synthase turns sucrose into an activated sugar. A glycosyltranferase transmits the activated sugar onto an industrially desired molecule. The overall objective is to produce a new, effective sugar compound. “Finally fructan sucrase makes the process more diverse”, explains acib researcher Christiane Luley, “because other activated sugars are accessible that can be transferred to specific target molecules”. For industry, the resulting bioactive molecules are very interesting, the scientist explains: “We are able to synthesize compounds that occur in nature only in minute amounts. They could be used as improved UV protection in cosmetics. There is just as much potential at classic sugar substitutes with a high sweetening intensity – but without calories or tooth-damaging properties – as with new sugar substances that are even effective against cancer.” The antioxidant potential of the new sugar molecules makes them ideal preservatives, which increase the storability of food – all based on the concepts of nature. The EU-funded project SUSY deals with improving these three enzymes, manufacturing them in large quantities
and finally optimizing the biochemical conversions. The EU-subsidized total budget until the project ending in August 2017 is five million euro. 800,000 euro will be turned over at acib in Graz, the rest from seven other project partners in Austria (TU Graz), Germany, Spain, Belgium and the Netherlands. The acib is responsible for the process development for industrial enzyme production of large quantities. HEALTHY OLIGOSACCHARIDES Sugar chemistry is a speciality of acib and Graz University of Technology (TU Graz). Since more and more studies show that breast milk is supporting babys health more than milk substitutes, research attempts to investigate the benefits of breast milk ingredients. More than 100 oligosaccharides are described to have highest effectiveness, eg in brain development or in binding pathogens in the intestinal tract. The complex production of these molecules is best achieved enzymatically. For example with a sialyltransferase that was modified by researchers led by Prof. Bernd Nidetzky (TU Graz and CSO acib) in order to be able to synthesize two important oligosaccharides from human milk in useful amounts. Normally, sialyltransferase transfers a sialylic acid group from CPM-N-acetylneuraminic acid to various saccharides with a terminal galactosyl group (like lactose). “In the original reaction, a 2,3-link is formed whereas in our modified reaction a 2,6link comes about”, explains Nidetzky. Both products are found in human milk, but have different characteristics. Actually researchers from acib and TU Graz are developing and modifying more enzymes for glycosylation and the production of valuable oligosaccharide molecules occurring in human milk.
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Plant Defense as a Biotech Tool
Against voracious beetles or caterpillars plants protect themselves with cyanide. Certain enzymes release the toxic substance when the plant is chewed. These HNL-called enzymes are also important for industry. acib found a new biocatalyst in a fern which outshines all other HNL-type enzymes on the market. Defense strategies are not only important in chess or military tactics but also in nature. Especially plants are masters in this discipline. Some stone fruit, almond trees or even ferns defend their young buds against feeding pests with cyanide. The poison expels the greatest enemy. This is due to an enzyme called hydroxynitrile lyase (HNL), which can release molecularly stored hydrogen cyanide. What is useful for the plants, is also in demand in industry where the reverse reaction of the HNL-enzymes allows to bind cyanide to different molecules. This creates a double benefit. On the one hand, it is possible to recycle unwanted cyanide wastes, which for example are generated during the production of acrylonitrile. Acrylonitrile is not only used in adhesives, it is also the raw material for polyacrylonitrile or “acrylic”, an important fiber for textiles. On the other hand, industry gains valuable building blocks for pharmaceutical agents or the vitamin synthesis. The extremely high specificity of the HNL-enzymes makes them so useful for industrial application. Ideally, through biocatalysis valuable products are derived from inexpensive precursors. HNL-enzymes have a fine tradition in industry. The first HNL enzymes have been successfully developed in the mid-1990s at Graz University of Technology (TU Graz) and were used industrially for the production of insect repellents. Important improvements in the synthesis of high-value products aroused more industrial interest. The early enzymes certainly can’t meet all of today’s requirements, so researchers are searching for new HNL biocatalysts. Within the framework of the EU project KYROBIO, which deals with production technologies for new molecules, acib-researchers have successfully looked for those bio-tools.
SMELL ENZYME ACTIVITY The acib-researchers Margit Winkler, Elisa Lanfranchi and Anton Glieder finally made a find in the white rabbit’s foot fern, where the scientists sniffed enzyme activity. “When you rub a young fern croizer between the fingers, it smells of hydrocyanic acid and benzaldehyde (similar to marzipan), indicating that there is enzyme activity”, explains Margit Winkler, “knowing that ferns show the desired activity, we have been searching in the woods and in commercially available plants”. In three and a half years the acib-researchers in Graz have kept a close eye on eligible enzymes, examined their structure, produced the biocatalysts biotechnologically and tested their activity. Finally, the enzymes of a commercial rabbit’s-foot fern from the hardware store were the most promising. The Styrian bracken fern also showed activity and is currently being studied in more detail. The new enzyme has an extremely high activity, although it is not even optimized. “Our new HNL is more efficient and simpler to handle than those previously used, because it is a small, uncomplicated enzyme”, says Anton Glieder (TU Graz, acib). These results are a perfect basis for the industrial utilization. The range of applications is huge: It includes everything from crop protection to the production of repellents against mosquitoes and Co. The acib-method used for bioprospecting of enzyme activities was published in the “Current Biotechnology”
High-Temperature Reductase for Organic Synthesis Available acib researchers created a temperature resistant nitrile reductase for organic synthesis. The enzymes converts pyrrolopyrimidines at temperatures up to 65 °C and can be adapted to specific substrates to meet industrial requirements. “We have used an enzyme from Geobacillus kaustophilus”, explains the acib researcher. G. kaustophilus is a thermophilic microorganism, which is found in hot environments. Its enzymes are therefore particularly resistant to heat. Knowing the coding sequence of the nitrile reductase, the genetic information was transferred to Escherichia coli – the standard production bacteria in biotechnology. Because of its resistance to high temperatures up to 65 °C, the purification is highly facilitated: Using heat and ultrasound allows the separation in high purity; without complicated cleaning processes. Active site mutagenesis enhanced the enzyme potential. While more and more antibiotics are less effective and bacteria develop unimagined resisting forces, the pharmaceutical industry is in demand for alternative substances. Substances based on pyrrolopyrimidine seem to be promising, because these chemicals can suppress the genome synthesis in microorganisms by inhibiting certain enzymes. This could be an interesting opportunity to fight against resistant microorganisms. A new, high temperature resistant nitrile reductase discovered and modified at acib is able to generate new pyrrolopyrimidine based molecules to meet these requirements. The research project about new nitrile reductases is a big step for acib and a big step for organic synthesis, since acib researcher Birgit Wilding has managed to reduce a nitrile to an amine for the first time using a high-temperature enzyme. The reaction step is frequently used in organic synthesis and is in a classical manner only feasible with considerable effort compared to the smooth enzymatic approach. But that’s not all – acib’s enzyme has a number of advantages.
Another advantage is the wide range of applications for this nitrile reductase. It converts those nitriles particularly well, which are in turn starting materials for compounds with activity against bacteria and even tumors cells. The enzyme may therefore be a key to new drugs or substitutes for no longer effective antibiotics. Research expects a lot especially from pyrrolopyrimidines - with this class of substances, acib’s enzyme is good deal. Finally, the enzyme from G. kaustophilus functions with a broad substrate range and a higher transformation rate than known from nitrile reductases. “We have already tested 22 major substrates, but not all can be converted satisfactorily. If there is interest, we could easily improve the enzyme so that it meets the requirements of the pharmaceutical and chemical industry”, explains Birgit Wilding. The project was published in the scientific journal “Advanced Synthesis and Catalysis”: goo.gl/SRtThT
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Styrian Horse Radish Visualizes Active Enzymes Biocatalysts are being increasingly used in manufacturing of pharmaceuticals. While supporting the upscaling of the production of transaminases, acib researchers developed a method to quantify enzyme activity. The test is based on a color reaction that is catalyzed by an enzyme from horseradish. Transaminases are important enzymes in every organism; they are used in the metabolism of proteins. Since the pharmaceutical industry searches for more environmentally friendly and efficient production methods for active ingredients, biocatalysts are highly asked industrial tools. This also applies to transaminases - in this case branched chain aminotransferases - which transfer the amino groups from one molecule to another; more specifically, efficiently and ecologically compared to chemical synthesis.
Transaminases are not only indispensable for prokaryote and eukaryote metabolism; they can also be used for synthesizing precursors of active pharmaceutical ingredients more environmentally friendly and more specifically compared to the classical chemistry. The mechanism provided by transaminases is important for example in the large scale synthesis of amino acids or chiral amines that finally simplify the production of drugs for AIDS therapy, knows Katrin Weinhandl, researcher at the Austrian Centre of Biotechnology (acib). A project of acib and a company partner deals with the production of transaminases in a large scale. acib has facilitated the manufacturing process of the enzymes essentially by developing a new test. It allows determining whether the biotechnological production has led to useful transaminases of a high quality or not.
Together with a company partner acib investigated a way to produce transaminases in large quantities, so that this type of enzyme can be used efficiently in the industrial production. “We use a transaminase originating from E. coli bacteria, which is finally produced harnessing the yeast Pichia pastoris. Our experiments showed that only Pichia can handle the required enzyme quantities”, explains Katrin Weinhandl. To determine whether enough active transaminases are present after biotechnological production - or to identify optimal production clones -, acib researchers developed a colorimetric test that visualizes the activity of transaminases and makes it measurable. The new assay couples the transaminase reaction with two other enzyme reactions. An enzyme from the Styrian horseradish catalyzes the last step: A peroxidase that finally leads to a green color, if the transaminase activity is sufficient. Green light for the new assay was given in the form of a publication in the “Tetrahedron” journal: goo.gl/NCVJjg
Bodyguards for Precious Seeds
Naturally occurring bacteria as a crop protection agent are now available for use in crop protection to alleviate the contamination of soil with pesticides - arguably the most environmentally friendly way of plant protection developed to date. The fungi (Rhizoctonia solani) is stealthy blight, infesting beets or corn at their roots and becoming visible only shortly before the harvest. The fungal rot begins early in the season, working its way from the inside out, and only becoming visible in the fall, destroying the possibility of a harvest. Year after year crop failures due to attacks by pests and pathogens are reported in the media despite their being treated with pesticides. Crop failure is further exacerbated by pesticide treatments which cause the death of insects such as bees through neonicotinoids. “There are also more extreme environmental conditions such as periods of hot weather, drought or flood disasters, to be taken into account when considering the extent of damage caused by pesticide use alone”, states acib researcher Christin Zachow. The Austrian Centre of Industrial Biotechnology (acib) is perfecting protective methods which will make the use of “chemical mace” measures obsolete. One such research project currently underway involves biological plant protection, in which microorganisms (bacteria) work as bodyguards for the safeguarding of seeds like corn, canola, tomato, sorghum, or sugar beet. The underlying principle 36
is that special bacteria are planted in combination with the plant seeds in fields. Together with Graz University of Technology and five international industry partners, acib engages the growth promoting properties of microorganisms: While the seed germinates, the microorganisms are simultaneously developing and supplying the plant with nutrients, promoting growth, warding off pests, reducing the stress on the crop and increasing their resilience. STRESSED CROPS “Crops are challenged by climate change, drought, and high salted floors. These nutrient deficiencies occur as a result of monoculture practices”, states Christin Zachow. Since 2011, she has been managing project research and delivery in collaboration with Prof. Gabriele Berg - a pioneer in this research field - from the Institute for Environmental Biotechnology at Graz University of Technology. Their primary task has been to find bacteria that are adapted to extreme environmental conditions. Every plant needs specific bacteria, and every soil type harbors specific bacteria. For example, researchers who work in the areas of moss and lichen research have found that the
former tolerate acidic pH values and nutrient deficiencies, while the latter accept UV-light and drought. Bacteria that promote growth are identified, characterized and tested for their resistance to stress. Zachow: “We want to know which genes are activated by which particular environmental conditions in order to ensure that the bacteria will be ideally matched with crops under the local conditions.” When a promising population of bacteria is found, it is deliberately improved by researchers so that the “microbiome” will have an optimal outcome under their given environmental conditions. Success has been recorded with the bacterial species Pseudomonas poae and Stenotrophomonas rhizophila. While Stenotrophomonas caused an enormous burst of growth of crops in the salty steppe of Uzbekistan (300 % more than without microbiome treatment), Pseudomonas made a similarly positive impact in the sugar beet test field from acib’s industry partner in Germany. Five international, industrial enterprises are currently involved in the research project. HEALTHY DIET After finalizing the preliminary investigations, target bacteria have come into interaction with their prospective host plants. Zachow: “Plants are searching for exactly those types of bacteria that they need for perfect growth.” The bacteria-host-interaction is similar to that which occurs in the human intestine, in which a specific
microflora is beneficial to human health. The culmination of the research and development of a commercial product has led to a bacterial-seed-combination. In moist soil types, the bacteria grow along with the germinating seed and at the same time protect it. Our goal: “We strive to develop plants with optimal health to provide consumers with an optimal basis for a healthy diet”, says the scientist, “a functioning biological plant protection system offers a viable alternative to pesticides.” Biological plant protection is undeniably a significant step towards ensuring a biologically sound agricultural industry producing healthier food. Ultimately, the acib method is in competition with conventional systems of pesticide driven “plant protection”. The chemical industry turns over about 40 billion euros a year in chemical plant protection products worldwide, and a third of these chemicals ends up on the fields in the EU alone. There is a lot of money to be made using biological plant protection when more and more customers call for healthier food.
Enzymes Against Fungal Toxins in Animal Feed Fungal toxins in animal feed are regularly causing troubles in the food industry. Nevertheless extremely poisonous mycotoxins in feed grain must not be a problem. Within the acib-consortium, a method for the industrial production of enzymes has been developed, which can degrade fungal toxins. Thus, the feed is safe - and our food as well. The natural, frequently occurring mycotoxins in grains such as corn, rye, wheat or barley are not only a natural danger for chickens, cattle and pigs which eat contaminated feed grains. Certain types of these poisons - around 300 are currently known - can even reach the consumers of milk, meat or eggs. Just think of ergot, which repeatedly lead to deaths until the 20th century. It is no wonder then that the Food & Agriculture Organization FAO classifies the contamination with mycotoxins as a main threat to humans and animals. FAO estimates that a total of around a quarter of the world’s food production contains mycotoxins. This fact, however, wouldn’t have to be a threat. The precautionary treating of animal feed with enzymes that can break down this natural fungal toxins completely and without residues, plays an important role in the quest for a healthy animal feed - and thus also for food security for consumers. The Lower Austrian company BIOMIN can fall back on many years of experience in the use of enzymes against various fungal toxins. Dieter Moll, research group leader at the Biomin Research Center in Tulln: “Although we use the enzymes in a very gentle way to produce valuable feed, they are highly effective and efficient in fighting against pollutants. Since the toxins are completely eliminated by these enzymes, they can no longer exert their toxic effect.” The removal of toxins happens in the digestive tract of animals, where the feed admixed enzymes have full effect against fumonisin, deoxynivalenol or zearalenone. On the other hand, afaltoxins are being removed by binding to clay minerals in the feed mixture.
For the production of enzymes Biomin utilizes the yeast Pichia pastoris, which is routinely used in biotechnology. “Yeast can not only ferment wine or soften dough, special varieties can also produce enzymes that are of a high industrial interest. Like the biocatalysts that are used to inactivate fungal toxins”, explains project leader Prof. Diethard Mattanovich (University of Natural Resources and Life Sciences Vienna (BOKU) and head of “Systems Biology & Microbial Cell Factories” at acib). The scientists at acib, Biomin and BOKU finally developed a yeast strain, which allows producing these enzymes quickly and in large quantities. In addition, the production organism has been adapted to the technical requirements and the process paths were optimized to produce the enzymes as cost-effective as possible. The project finally was awarded with an Austrian Science2Business Award in 2013. Mycotoxins in animal feed should be no longer a problem - however, the technology already exists. And it is improved in Austria. “Our cooperation continues. We are optimizing the production system in order to expand it to other enzymes”, says Mattanovich. The goal is to be able to degrade enzymatically as many mycotoxins as possible.
Virus in the Service of Health The per se harmless enzyme “Npro” plays a major role during the attack of the swine fever virus. Nevertheless, the enzyme can be used perfectly for manufacturing medical drugs. The acib research network revealed its secrets and thus not only opened up new possibilities for the production of protein drugs, but also for controlling the virus. Swine fever is one of the world’s most dangerous animal diseases and was previously difficult to control. The international research network of the Austrian Centre of Industrial Biotechnology (acib) demonstrates how (harmless) parts of a dangerous virus can be transformed into something very useful. Scientists at the Universities of Innsbruck, Salzburg and the University of Natural Resources and Life Sciences Vienna and the company partners Sandoz and Boehringer Ingelheim revealed the mystery of the enzyme “Npro” after eight years of research. The autoprotease plays a major role in infection by the swine fever virus. The research team led by Prof. Bernhard Auer (University of Innsbruck) successfully deciphered the structure of the enzyme. Knowing the 3-dimensional shape of the enzyme, it was possible to understand the method of the
virus for attacking its targets. This is the basis for new methods to control the infection and to combat the virus. Biotechnologically more important are the enzyme’s special properties. The extraordinary abilities of Npro - to from inclusion bodies and to split itself off from the viral protein – makes it an ideal vehicle for the biotechnological production of therapeutic proteins or other valuable protein based substances. Using Npro for the formation of fusion proteins cumulated in inclusion bodies simplifies the bacterial protein production and purification extremely. “The method can be improved and adapted to new pharmaceutical needs”, explains Auer, “resulting in higher quality medicines, which can be produced cheaper”. This project is yet another example of how joint research at acib leads to new technologies and competitive advantages for the company partners in the acib partnership.
ABOUT NPRO The comparable small enzyme Npro has the extraordinary ability to cleave itself of from an attached protein. In the biotechnological production using Escherichia coli as a cell factory, this is quite convenient, because the bacteria not only produce the desired macromolecule, but also hundreds of other proteins from which the desired product must be separated without any residue. Causing E. coli to synthesize the desired protein coupled to Npro as a fusion protein, all becomes easier. Npro then forms inclusion bodies, small definite structures in the microorganism in which the product is concentrated. The inclusion bodies are easier to separate from the rest. Because of Npro’s ability to split itself off subsequently, biotech industry saves a lot of effort during the purification process. acib’s industrial partners have access to this smart process already.
Fachbereich Bioengineering Bachelor- und Masterstudieng채nge www.fh-campuswien.ac.at/bioe_b
Transparent Bioprocesses by Analyzing the Respiratory Air of Microorganisms A new method allows analyzing the exhaust of those microorganisms in real time, which produce active agents for the pharmaceutical industry. This facilitates process control. The air we breathe contains much more information than how many drinks you had at the last party. You can even draw conclusions about a person’s state of health. In humans, thanks to the most sensitive analytical instruments, even cancer signals can be found in the breath. A cigarette leaves signs in the exhaled air a week after smoking. Not only people, but also microorganisms “breathe”. After several years of joint research of acib and the Tyrolean business partner Ionicon on the “breath” of microorganisms, the sensitive analysis of the components of respiratory air has become possible. For the first time the health status of bacteria or yeasts, which are grown in a fermentation vessel to produce active agents for the pharmaceutical industry can be observed in real-time. “Our analysis method detects individual substances which are directly related to the metabolism of the cell,” explains Gerald Striedner, acib-project leader at the University of Life Sciences Vienna (BOKU). With this information, process engineers can quickly intervene in the production process if something does not go according to plan. Preventing an overuse of the cells, it is at the same time possible to avoid too few or inferior products. “The challenge was to develop a technology that meets the strict requirements of the pharmaceutical industry and at the same time leads the “breath” to the analyser”
as unchanged as possible”, says Rene Gutmann from acib-partner Ionicon. The analyzing unit – a highly sensitive proton transfer mass spectrometer – must be connected to the sterile production vessel without any chance for infections. In addition, the information conveyed in the exhaust must reach the analyzer unchanged to enable reliable conclusions. To this end, the researchers in the acib-network developed a suitable interface between fermenter and analyzing unit. For the first time the analysis of industrial fermentation processes in a production scale of several 1000 liters is possible without interfering with the sterile areas. The safety in manufacturing active pharmaceutical ingredients rises while costs decrease because production losses can be prevented and the processes can be improved immediately – based on the statements of the on-line analysis of the cell metabolism. Previously, for process control technicians had to draw samples, work them up und finally analyze them – in comparison a slow and costly method. “Our new technology once again highlights the innovation performance of Austrian biotechnology”, says Mathias Drexler, director of acib. The innovation has been awarded the Vienna INiTS Award 2012 in the category “Life Science”. The award was handed over to acib-researcher Markus Luchner for enabling the “trial and error” method in fermentation processes to be replaced by a substantial on-line analysis.
ABOUT PTR-MS The PTR-MS method can detect dozens of volatile products that are “exhaled” by microorganisms during fermentation, including acetone, acetaldehyde, indole, isoprene, ethanol or methanol. If Escherichia coli (in the biotech industry most widely used species of bacteria) “exhales”, for example, tiny traces of acetaldehyde in a fermentation that is an indication that the desired sugar breakdown no longer takes place (along with the production of the target product), but the microorganisms have changed to an unwanted metabolic pathway (a kind of acid fermentation). On the basis of the results of the air analysis, the process can be redirected.
Pichia pastoris Protein Expression Excellence Manifold expression and production strategies Record-breaking productivities MeOH-induced and MeOH-free processes Tailor-made glycoproteins â&#x20AC;&#x201C; VTU Pichia boosting Pichia GlycoSwitchÂŽ
Newly Discovered Metabolism Certifies Evolutionary Advantage for Yeast Duplicate copies of genes safeguard survival of the biotech yeast Pichia when only methanol is present as feed. A recently elucidated metabolism is similar to that used by plants for the utilization of CO2. Metabolic models of last decades are wrong. Yeast is being used by mankind for longer time than any other microorganism. Bread, beer, wine - all of these could not be produced without Saccharomyces cerevisiae (baker’s yeast) and other yeast species. Over the last decades yeast has become indispensable for industrial biotechnology as a reliable cell factory. Valuable products ranging from enzymes to active pharmaceutical ingredients are industrially produced using yeast, mostly by a species called Pichia pastoris that is particularly productive. Because of its long and varied utilization, yeast is one of the best studied organisms. Besides its industrial application Pichia pastoris is also used by scientists as a model organism for studying cell structures. Everything seemed familiar – until this year. Researchers of the Austrian Centre of Industrial Biotechnology (acib) and the University of Natural Resources and Life Sciences Vienna (BOKU) have elucidated a new pathway that makes the yeast Pichia pastoris unique. “We were able to show that the assumptions and models that have been used in the last 30 years are not right”, explains Prof. Diethard Mattanovich (BOKU and head of the research area “Systems Biology & Microbial Cell Factories” at acib). The new pathway explains the utilization of methanol as “feed”. Yeasts such as Pichia pastoris belong to the rare kind of microorganisms that are able to utilize this simple alcohol as nutrient. Mattanovich: “The cells use that
option, for example, when they grow naturally in the sap of trees, where methanol is present.” The researchers around project leader Dr. Brigitte Gasser discovered amazing similarities with plants. These use carbon dioxide (CO2) as a nutrient and recycle the greenhouse gas in cell organelles called chloroplasts. Eventually CO2 is converted to biomass. Pichia works similarly: It converts methanol, which consists of one carbon atom like CO2, in a cell organelle called peroxisome. The decisive role in both processes is the formation of chemical bonds between carbon atoms and the rearrangement into sugar molecules and other substances, which are necessary for the synthesis of biomass. “So far we did not know where these rearrangements are performed in the cells, and which genes control them”, says Brigitte Gasser. Just as little was known about the genetic encoding of this metabolism. Most cells have one gene available per protein and metabolic step. Pichia is evolutionarily on the safe side. All genes for methanol manipulation are duplicated, as Mattanovich and Gasser have discovered together with 13 scientists who were involved in this research project. The genes do not only have an additional genetic information so that the appropriate reactions are located to the peroxisome. They are active only when methanol is present as a nutrient source.
For these findings, the researchers have re-evaluated the entire data, which have emerged in the recent years while improving Pichia pastoris biotechnologically at acib and BOKU. “The interpretation of our systems biology data revolutionized the understanding of cell biology,” says Brigitte Gasser, delighted about the new knowledge of life processes on earth. The work was recently published in the prestigious journal BMC Biology. The results demonstrate the leading role of Vienna researchers when it comes to the biotech yeast Pichia pastoris.
The work was recently published in the journal “BMC Biology”: goo.gl/BYZZeR The results demonstrate the leading role of Vienna researchers when it comes to the biotech yeast Pichia pastoris.
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Microparticles for Superefficient Protein Purification Today most medicines are produced biotechnologically. A new cleaning method developed at acib combines five purification steps and leads to an extremely facilitated, more economical and ecological manufacturing process. Seven of the ten best-selling drugs in 2012 were biopharmaceuticals; biotechnology in pharmaceutical production is still growing. A severe problem is the cleaning of products; up to 80% of the total production costs are spent for protein purification. The Austrian Centre of Industrial Biotechnology (ACIB) has developed a method based on microparticles, which reduces five purification steps to only one; a revolution in the cleaning process of biopharmaceuticals. “Our micro-particle system comprises a new cleaning method especially for proteins for medical application, which are produced by microorganisms. Using our microparticles, the manufacturing process of biopharmaceuticals becomes qualitatively better and faster”, explains Prof. Bernd Nidetzky, CSO of acib. The new process was developed within a cooperation of acib and Boehringer Ingelheim RCV. “FIVE IN ONE” “The economic relevance of this new purification method lies in its simplicity and speed. We aggregated five process steps into a single one”, says Georg Klima, head of Process Development Biopharma at Boehringer Ingelheim in Vienna, “in addition, we expect an even better quality because the microparticles directly absorb the product out of the cells with high specifity.” A key issue was the transfer of technology from a research level into corporate laboratories, which was completed within a few weeks.
ABOUT THE MICROPARTICLE TECHNOLOGY Currently, chromatography is used for standardized protein purification. Industry fights with slow binding rates, clogged or damaged material, product losses and other obstacles.The new acib system works differently: Scientists prepared charged microparticles based on the ion exchange principle with a size of one to two microns. The entire binding operation is completed within 30 seconds. Additionally, a continuous process based on the micro particle technology has been established to further accelerate the biopharmaceutical purification.
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World‘s First Method for Continuous Purification of Valuable Antibodies Scientists at acib develop world’s first continuous purification method for valuable drugs. This will lead to significantly reduced production costs and to cheaper pharmaceuticals that are affordable for non-privileged health care systems. Imagine a loved relative suffering from cancer – and you could not afford a treatment because the drugs are too expensive. The Austrian Centre of Industrial Biotechnology (acib) developed a method with the power to reduce production costs of highly valued drugs significantly. Without antibodies we would be at the mercy of pathogens or cancer cells. Therapeutic antibodies are used as passive vaccines, for cancer treatment or for controlling autoimmune diseases such as multiple sclerosis. According to “bccresearch.com”, the global market for antibody drugs was worth 70 billion USD in 2014 and should rise to 122 billion USD until 2019.
according to the speed of operation. A further advantage is the transferability of the operation parameters from the actually used batch to the continuous approach. “Our method shows great potential as a new platform technology for the pharmaceutical industry”, says Prof. Alois Jungbauer, who is in negotiations with several international companies about building pilot plants based on acib’s technology. The purification method was published in the “Biotechnology Journal”: goo.gl/KYvWLD An external scientific comment emphasizes the relevance of the project: goo.gl/q89aAt
Two thirds of those molecules are produced biotechnologically using Chinese hamster ovary cells (CHO cells). Actually the major cost factor for industry is purification using “protein A” affinity chromatography where tens of thousands of liters of culture volume have to be processed annually. About 80 % of the production costs fall upon purification. Here the new purification method comes into play. Researchers from the acib and the University of Life Sciences Vienna developed the first downstream processing method for recombinant antibodies from clarified CHO cultures. A feasibility study exemplified by the purification of immune globulin G (IgG) shows that the method can compete with “protein A” affinity chromatography in terms of yield and outperforms chromatography
Genome of the Chinese Hamster Deciphered Because ovarian cells of the Chinese hamster are the most popular vehicles for making valuable therapeutics, the genome decrypted within the Austrian Centre of Industrial Biotechnology (acib) enables developing of cheaper, more effective therapies.
not only of new biopharmaceuticals and treatments but also of cheaper medicines that are affordable ideally even in underprivileged countries. Because the hamster genome is comparable in its size to the human one, it was necessary to cope with huge amounts of data. “We produced 1.4 billion short segments of DNA”, says Karina Brinkrolf. She was responsible for sequencing at CeBiTec. The challenge was to assemble these parts like a puzzle to get the entire genome, which is spread over 11 pairs of chromosomes.
The Chinese hamster ovary cells (CHO cells) are an indispensable part of modern medicine, because they are the most sought after vehicles for production in pharmaceutical industry. A group of researchers led by Prof. Nicole Borth has decrypted the genome of the Chinese hamster – a result of the research partnership between the Austrian Centre of Industrial Biotechnology (acib), the University of Natural Resources and Life Sciences and the University of Bielefeld (CeBiTec). “We now understand better how the cells function and can adjust them to the desired requirements”, explains the scientist and thinks 50
Whether antibodies, blood clotting factors, rheumatism therapy or anti-cancer drugs – the pharmaceutical industry brings more and more therapeutic proteins on the market. Unfortunately, agents in human medicine are not knit in a simple pattern. There are chemically simple varieties, consisting of single molecules with a few atoms. Therapeutic proteins, however, are complex structures made up of hundreds of amino acids. In contrast to the simple products, these proteins must be perfectly adapted to the human organism, so that no secondary or defensive reactions can happen. “Since 1987 the most common production vehicle for these substances have been artificially cultured Chinese hamster ovary cells”, says Nicole Borth. The first active agent produced this way was a drug administered to heart attack patients to stimulate the dissolution of blood clots. Actually 70% of the active pharmaceutical ingredients are produced via CHO cells. Hamsters need not die for this anymore, because industry and researchers reproduce the cells that were once isolated in 1957.
The in vitro cultivation, however, leads to difficulties: â&#x20AC;&#x153;These cells are subjected to natural changes over timeâ&#x20AC;?, says researcher Borth, â&#x20AC;&#x153;the activity of genes is different in all laboratories that develop and grow hamster cells. The original genetic material is subject to constant modification.â&#x20AC;? This is an advantage - considering the adaptability of cells - and a disadvantage, because it may happen that, for the specific purpose, important elements of the genetic material have mutated. The sequenced genome of the â&#x20AC;&#x153;original hamsterâ&#x20AC;? is the perfect reference to examine and adapt the genetic material of the production cells.
To enable access to the data for as many researchers as possible the scientist has founded the online platform www.chogenome.orgÂ together with two colleagues where a lot of work material regarding the Chinese hamster ovary cells is available. Nicole Borths vision: â&#x20AC;&#x153;We can produce agents more efficiently and cheaper â&#x20AC;&#x201C; at prices that any average health system can afford; ideally even in Third World countries.â&#x20AC;? The research results were published in August 2013 in the journal â&#x20AC;&#x153;Nature Biotechnologyâ&#x20AC;?: goo.gl/yAI82I
Green Chemistry for the Pharmaceutical Industry
In the 26 million Euro IMI EU project CHEM21 23 partners including acib are developing more environmentally friendly methods for industrial production of active pharmaceutical ingredients. The goal: Modifying microorganisms such that they are using new metabolic pathways and thus can produce important substances reliably, environmentally friendly and in a high quality. Producing medical drugs is a resource-devouring matter. “For one kilogram of a active pharmaceutical ingredient (API) in a traditional production processes industry is consuming typically 100 or more kilos of raw materials”, explains Anton Glieder, professor for biotechnology at Graz University of Technology and key researcher at the Austrian Centre of Industrial Biotechnology (acib). Not to mention the use of energy. In addition, the processes consume a lot of time and cause harmful waste that has to be eliminated. In addition, the pharmaceutical industry has to cope with limited and expensive resources. Platinum for example, a commonly used high-value catalyst, is becoming increasingly rare and expensive. Overall, green, environmentally friendly alternatives are required.
At half-time of the project, promising achievements were visible. The acib-scientists have succeeded in producing simultaneously several interesting substances such as carotene with yeast cells. The carrots’ colour is a result of intracellular built carotene. Additionally, the substance is a precursor of vitamin A. For this purpose it was necessary to stably incorporate four bacterial genes in Pichia pastoris, the most important yeast strain for biotech industry. “It is our vision to incorporate new genes in a way that our cells master a new pathway and are able to produce quality products from simple sugars. Products that occur in nature only in tiniest quantities or have to be synthesized complicated and absolutely not environmentally friendly”, says Martina Geier, yeast-specialist the acib.
Pharmaceutical industry is looking after these alternatives within the EU project CHEM21 - Chemistry for the 21st Century. In cooperation with scientific partners in England, Germany, Belgium and the Netherlands, acib has prevailed in the competition for this project and is since 2012 working with the pharmaceutical companies GSK, Pfizer, Sanofi, Bayer, Orion, Johnson & Johnson and several SMEs on new, “green” production processes. The acib’s contribution is based on the experience in biocatalysis and especially in synthetic biology. “Our main focus is the development of tools for environmentally friendly multistep syntheses that enable microorganisms to produce APIs in a consistent high quality instead of dirty and unspecific chemical methods”, explains acib researcher Birgit Wiltschi.
The main task is to establish new pathways based on up to 20 foreign genes in bacteria or yeast, so that the microorganisms are capable of producing valuable products for human medicine in complex cascade reactions. The researchers have already installed nine foreign genes successfully and thus were able to synthesize carotene from sugar as well as the antibiotic violacein. The patented techniques, which are developed for installing the new genes, can be utilized by the pharmaceutical industry to manufacture important products - like new antibiotics against resistant bacteria or substances for cancer therapy. Active ingredients in drugs (APIs) are a billion dollar business. More than 120 billion has been implemented worldwide in 2014 - a growth rate of around 7 percept per year. 53
Changing tomorrow today Every day you push the boundaries of human understanding through research to improve the world we live in. Every day we commit to simplifying technology, without sacrificing the output. We use our experience to design intuitive, dependable products and support to help you get going. You get the data and flexibility you need with less hassle and frustration, allowing you to spend more time on what counts. And change tomorrow today. Amersham I Biacore I ÄKTA I Whatman I Cytell I Xuri GE works.
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25+ years of experience in applied industrial science network of 250+ international researchers 70+ industrial projects in 6 fields of expertise international partnership of 150+ universities and leading companies • headquartered in Austria with scientific and economic network linking 30+ countriesa
BiocatalySis & Enzyme technology acib extends the search for novel reactions to replace inefficient (chemical) methodology towards still unsolved â&#x20AC;&#x17E;dream-reactionsâ&#x20AC;&#x153;: (I) asymmetric hydration of C=C bonds, (II) enzymatic activation of hydroxy-compounds to replace ecologically problematic Mitsunobu- and Appel-protocols, (III) biocatalytic, metal-free replacement of the traditional Friedel-Crafts-Acylations, (IV) novel enzymatic C-C, C-S, C-O and C-N bond forming reactions.
Polymer- & Environmental Biotechnology Our focus lies on the adaptation and application of biocatalytic processes to functionalize, modify, recycle and degrade polymers as well as on environmental biotech applications employing enzymes, living cells and also complex cell populations in biofilms or (immobilized) on mineral materials in an industrial environment. This involves the identification of novel enzymes and detailed mechanistic studies to allow a knowledge-based adaptation of these enzymes to the polymeric substrates.
Systems Biology & Microbial Cell Factories Our overall goal is the knowledge based engineering of microbial production systems for metabolites and recombinant proteins. We convert information from systems biological analysis and models into successful engineering strategies to adapt the cellular metabolism and its regulation efficiently for most efficient industrial production of biomolecules. Our further vision is to rebuild nature on the computer and set up a complete mathematical model of living cells in silico. 58
Bioprospecting & Synthetic Biology Our vision is to combine bioprospecting and whole cell systems development with synthetic biology; including protein engineering strategies. Major long-term goals are designing innovative enzymes based on metal-catalysis and creating new and engineered whole cell biocatalysts for specific enzymatic reactions. Novel chassis strains and novel highly valuable substances produced via incorporation of new pathways (up to 20 genes) in microorganisms are a priority of our synthetic biology efforts.
Bioprocess Engineering To control and monitor bioprocesses, we address three goals: (I) acceleration of process development, (II) consistent quality and (III) real time release or parametric release. Suitable monitoring and control is a prerequisite of continuous manufacturing, which will be investigated by acib and implemented in the industry in the next decade. A further challenge is to combine material science with bioprocess engineering in order to develop materials with new functions for industrial bioprocesses.
Animal cell technology and engineering Systems biology for mammalian production cells (CHO and others) is a major research field at acib, aiming to achieve the paradigm shift from empiricism to controlling the molecular basis of productivity and product quality in mammalian cells. The establishment of bioinformatics tools, statistical analyses and mathematical models enable the identification of relevant parameters and the prediction of cell behavior during bioprocesses, which, amongst other benefits, will lead to reduced costs for R&D, monitoring and control. 59
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GRAZ UNIVERSITY OF TECHNOLOGY Graz University of Technology was founded 1811 and pursues teaching and research in the fields of science and engineering. With 12.000+ students and 2300+ employees she is a leading university regarding research agreements with business and industry from basic research to industrial implementation. www.tugraz.at
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A PROJECT WITH ACIB 1. scientific discussion of industrial interests with acib key researchers and CSO 2. project design between acib scientists and industry 3. representatives 4. entrance of company into acib consortium 5. signature of consortium agreement 6. cooperation agreement 7. IP agreement for a specific project Partners from all over the world are welcome. The participation in funded EU-programs is highly appreciated.
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