Tracking down new natural products
AnalytiCon Discovery is a global market leader when it comes to libraries of natural products with fully elucidated chemical structures. This BRAIN subsidiary has access to about 15% of all known natural products and therefore provides key resources for the Group’s BioArchive. BLICKWINKEL spoke with Dr Karsten Siems and Dr Lars Ole Haustedt about the special attraction of plant and microbial secondary metabolites for sustainable natural foods and cosmetics.
BRAIN: You have one of the world’s largest libraries of natural products. What exactly does that mean?
Dr. Lars Ole Haustedt: Natural products is the term we use for secondary substances that are produced by various organisms. They are called secondary because they steer functions in the organism that go beyond pure survival. A primary substance, for instance, is a vital protein, which makes an essential contribution to cell division or energy metabolism.
Dr. Karsten Siems: Such substances, which are also known as secondary metabolites, are often much more complex than primary substances and much less research has been done on them. Their function cannot be immediately recognised and may be very varied. They may act as signal substances, for instance. Their production at cellular level often proceeds in several stages, and involves a great deal of effort. These substances are known to interact with other proteins, and they can be presumed to be responsible for specific properties both in the living organism as well as in its past evolutionary forms, i.e. they are useful for the organism in some manner. In most cases we don’t know what this specific usefulness is, though.
BRAIN: Why are these secondary natural products so interesting?
Dr. Lars Ole Haustedt: Our customers come from the food, cosmetics and pharmaceutical industries. They are constantly on the lookout for new substances they can use to carry out systematic screening tests. That means they take secondary products we have isolated and test whether they show useful effects on cells or enzymes that can be translated into new products such as optimised foods or skin creams.
One example is the DOLCE progamme, which wass launched in 2016 and in which we are developing natural sugar substitutes or sweet taste enhancers together with BRAIN and Roquette. Our so-called “SweetBox” now contains some 60 such natural products that have been proved by means of screening tests to activate sweetness receptors in human taste cells. This is also the focus of research within the Natural Life Excellence Network 2020 innovation alliance initiated by the German Federal Ministry of Education and Research (BMBF) and coordinated by BRAIN. We at AnalytiCon Discovery are also on board. The partners involved are looking for bioactive food ingredients with the specific aim of improving the taste of bitter foods. For this purpose alone, we have isolated around 4,000 natural products, including 2,000 secondary products from edible plant material, and provided them for screening tests. These are intended to act as healthy replacements for salt and fat.
BRAIN: Which organisms do you work with?
Dr. Karsten Siems: Half of the natural products we have isolated come from plants, and the other half from microorganisms we have isolated and fermented. Substances derived from edible plant material are of special interest as food additives. Other resources we use are fungi and selected bacteria. We use all of these organisms in exactly the form they occur in nature, without altering them. In these so-called wild types, we take a look at the substances they produce on a very small scale in their metabolism. Our core expertise lies in isolating and characterising these substances.
TOOLBOX OF NATURE
AnalytiCon Discovery and BRAIN have comprehensive biological substance collections at their disposal. Together with the group's technology portfolio, the archive allows the company to translate biological diversity into sustainable processes and products. This BioArchive provides, inter alia, access to some 53,000 characterised microorganisms, 49,500 substances that are either natural or derived from nature, as well as 13,000 plant parts for isolating natural substances.
BRAIN: How do you discover and isolate these substances?
Dr. Lars Ole Haustedt: That is a new challenge each time. For one thing, it takes state-of-the-art analytical equipment to analyse the metabolic products and molecular structures. Just as important are scientific experience and the knowledge we at AnalytiCon Discovery have accumulated over a period of 30 years. AnalytiCon was established in the mid-1980s by doctoral students from Berlin's Technical University, including our current managing director Dr Lutz Müller-Kuhrt. We are therefore considered a leading authority in the field of natural product libraries.
Dr. Karsten Siems: The conditions for growth and fermentation that we optimise each time for the relevant organisms are of crucial importance for our work. We want the organisms to develop in the best possible way to provide high yields of the secondary products we are interested in. Factors such the ambient temperature or oxygen supply play a key role. The composition of the nutrient media in which we grow the organisms also has a major influence. These and other factors play a crucial role in determining the secondary products that are finally obtained, as well as their quality.
BRAIN: What systems do these organisms grow in?
Dr. Lars Ole Haustedt: There are liquid fermentation systems in which organisms are cultured and raised in glass laboratory flasks. That is standard practice when working with bacteria. Next to these there are also solid-state fermentation systems. These act as a kind of incubator and are especially suited for culturing fungi. In these, the organisms grow on dry nutrient media. Membrane systems are the third option. These are fine-pore ceramic membranes on which the organisms grow and via which they are fed with liquid nutrients.
BRAIN: What is the scale of these operations?
Dr. Karsten Siems: With plants, the first step is to use small extracts to examine whether any interesting secondary metabolites are produced. This usually requires only 1 to 1.5 g of dried sample material in powder form. Using HPLC mass spectrometry1 (HPLC-MS), we can recognise how many metabolites have been produced in which quantity and quality. By comparing the specific structural data determined by means of HPLC-MS and using data we have filed for the over 25,000 natural products we have already isolated, we can identify many compounds just in the extract. It’s always exciting when we discover hits we hadn't encountered before. When that happens, we scale up to one kilogram of starting materials and then isolate the products we are interested in from this sample. We are especially interested in new substances, but there may also be “regulars” that we use to fill up our stocks in the archive.
Two stages are also involved when working with microorganisms, which are cultivated in liquid or solid systems. Here we scale up the volume one thousand fold from 10 millilitres to 10 litres. In both cases, we end up with pure substances in the milligram range with a purity of over 90%, which are stored in freezers.
Dr. Lars Ole Haustedt: Before they are stored, the natural products are carefully characterised and their molecular structures are elucidated. For this we use nuclear magnetic resonance spectroscopy2 (NMR) and mass spectrometry (MS). All of the information is transferred to a database and can be retrieved at any time. Besides the precise chemical structure and the analytical data, this information includes the taxonomy and the plant part from which the material was taken.
BRAIN: How high is the number of hits, and how long does isolation take?
Dr. Karsten Siems: About one in ten organisms we examine on a small scale provides a hit, i.e. new secondary products we find so interesting, of which the biomaterial contains a sufficient quantity for us to launch the time-consuming process of isolating the pure substances. Depending on the organism, on average it takes us six to eight weeks to obtain the pure substances.
BRAIN: And where do you collect this never-ending supply of new sample material?
Dr. Karsten Siems: We can rely on our established cooperation arrangements with reliable partners to procure new samples. We cooperate with botanical institutes that collect plant material for us, obviously in accordance with all of the relevant international conventions such as the Convention on Biological Diversity (CBD), also known as the Rio Convention, and the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Based on our wealth of experience, we can limit the search somewhat. For example, most grasses are of no interest to us because their metabolism offers virtually no exciting substances. When searching for microorganisms, we mainly use soil samples collected either by AnalytiCon staff or by our partners.
Dr. Lars Ole Haustedt: Sometimes we also launch a targeted search. The DOLCE programme, for instance, is exploring natural products that provide a sweet taste. In such cases, literature studies can be carried out up front. Here we concentrate on processing those plants that are known for their intensely sweet taste to find potential candidates faster.
BRAIN: How many natural products can you access?
Dr. Lars Ole Haustedt: Our natural product libraries are highly dynamic collections. On average, we isolate 1,000 new natural products each year, so we have accumulated several thousand natural compounds in our many years of work. We have access to all the data. AnalytiCon Discovery’s current catalogue showing our range of products always contains a selection of these secondary metabolites. Some products can only be stored for a limited period, whereas others are sold out at some point. We do not reproduce them automatically, because there is rarely a need to do so. Our work and our customers’ interest focuses on new natural products they would like to use for their screening tests.
Dr. Karsten Siems: Up until 2015, publicly accessible databases listed about 220,000 such natural products. Their number is constantly growing. We have access to about 15% of these natural sources. We have also synthesised about 40,000 compounds similar to natural products that are mainly of interest for the pharmaceutical industry. This collection is based on natural products that have been modified by chemical synthesis methods to make them more suitable for developing pharmaceutical active ingredients. Here, we aim to use nature’s ideas but to place them in a new context that makes it possible to more efficiently identify biologically interesting starting points.
BRAIN: How do you keep an overview of this wealth of resources?
Dr. Lars Ole Haustedt: We are backed by an extensive logistics system and infrastructure. The containers of all samples have bar codes that are defined in the database. They are stored in a cutting-edge compound storage system. This looks like a huge drinks fridge that automatically adjusts the temperature, atmospheric humidity, oxygen content and other parameters. An integrated robot arm can quickly and reliably remove any desired samples from the collection and place them in the required part of the system for further processing steps. But there is also a lot of manual work behind all of this robotics and data logistics, which has to be performed very conscientiously.
BRAIN: How does cooperation work within the BRAIN Group?
Dr. Karsten Siems: AnalytiCon and BRAIN are old acquaintances. AnalytiCon Discovery with its current focus on natural products emerged from the previous AnalytiCon AG with its broader thematic background in 2000. BRAIN and AnalytiCon were already in contact then. The two companies have been working together for various industrial partners since 2004. In 2007, BRAIN and AnalytiCon Discovery launched a large cooperation project with Symrise AG. In this context, we identified natural products for cosmetic applications, which are now being successfully marketed. One of the innovations was the cosmetic active ingredient SymSitive® 1609, which reduces skin irritation and is used in Beiersdorf’s Eucerin products.
Joining the BRAIN Group in 2013 has stepped up our cooperation even further. This can be seen from our successful joint research within the strategic NatLifE 2020 alliance or in the DOLCE programme. Today we have many points of contact, wherever the aim is to identify new natural products for various industrial applications as part of research partnerships with other industrial companies or BRAIN’s own development programmes.
Dr. Karsten Siems
Dr. Karsten Siems studied chemistry at Berlin's Technical University and received his PhD in 1992. He joined AnalytiCon in 1993 where he mainly focused on isolation and structure elucidation of natural compounds. Since 2006 he has been Vice President Natural Products at AnalytiCon Discovery GmbH in Potsdam, Germany, and responsible for R&D projects with natural products.
Dr. Lars Ole Haustedt
Dr. Lars Ole Haustedt studied Chemistry at the University of Hannover and received his PhD in 2002. Following a two year postdoctoral stay at Stanford University he joined AnalytiCon in 2005 starting in library design and syn- thesis. Since 2010, he has been a member of AnalytiCon Discovery's Business Development Team as Projects & Innovation Director.
Analyticon Discovery GmbH