Chair of Chemistry of Biogenic Raw Materials (Prof. Dr. Volker Sieber)
Today’s chemical production is based almost exclusively on crude oil and natural gas as raw materials. In the course of a more sustainable economy, it is essential to increasingly produce basic chemicals from renewable raw materials, i.e. from biomass, in order to create the basis for a new “green” chemistry. The aim of the work at the chair is to develop technical processes that allow established basic materials for chemical production and fine chemicals to be produced efficiently and thus economically from biomass and to establish new relevant substances that can be obtained from renewable raw materials.
Fields of research
Biomass digestion and fermentation
As a rule, biogenic raw materials contain a large number of different ingredients. In order to obtain raw materials from them for future industrial use, the biomasses must first be suitably prepared and, depending on the desired ingredients, fractionated. These can then be further converted using chemical and/or biotechnological methods in order to substitute petrochemical products or synthesise new functional substances. The research area “Biomass Digestion & Fermentation” deals both with the development of processes for the targeted processing of biogenic raw and residual materials and with the development of biobased processes for the conversion of ingredients into valuable raw materials for the chemical industry or energy sources. The R&D work focuses on the digestion and fractionation of lignocellulose-containing raw and residual materials with the aim of developing biorefinery systems for the most complete possible use of raw materials. The focus is also on microalgae. Mechanical, physical-chemical (thermal hydrolysis) and chemical digestion processes are used in combination with biotechnological (enzymatic) processes. Suitable analytical methods are developed or adapted for the identification of ingredients or digestion products. By means of a GC-MS-based analysis method developed further at the chair, lignin-derived phenolic inhibitors, for example, can be reliably identified and quantified. The development of bioconversion and fermentation processes focuses on the conversion of saccharides into lignocellulose hydrolysates. Activities in biogas technology concern methods to improve hydrolytic biomass digestion and to optimize the efficiency of process biology.
Transformations for renewable raw materials
Today’s chemical production is still largely based on crude oil and natural gas. Their limited availability requires the use of renewable raw materials and, at the same time, corresponding research to expand the current possibilities of use. Due to this fact, the replacement of the raw material oil in the production of basic and fine chemicals is one of the central challenges for the chemistry of the 21st century. Raw material change requires environmentally friendly, efficient and economical processes based on new synthesis methods and strategies. We are dedicated to this field of research, with a focus on chemical and enzymatic synthesis methods and the combination of both. Our goal is to create new methods and develop them into technical processes for the efficient and economical synthesis of basic and fine chemicals. To achieve this, we make use of the synthetic arsenal of organic and inorganic chemists and also in Mother Nature’s supermarket. Properties not found there are created by our own hands through the application of methods of protein evolution and enzyme engineering and tools for combining chemical and biochemical catalysts. Chemo-enzymatic synthesis Use of biocatalysts and organic/inorganic catalysts for the synthesis of basic and fine chemicals from renewable raw materials. There is an abundance of organic and inorganic chemical catalysts, which exhibit an extraordinary variety and complexity as well as selectivity in the reactions they catalyse. Nevertheless, they are generally incompatible with enzymes and biogenic substrates and the associated solvents. On this basis, we investigate chemo-enzymatic processes for the efficient and targeted conversion of complex biogenic raw materials. We are working to enable the combination of different catalysts in order to advance raw material change in chemical production. The growing demand for new catalysis strategies for the conversion of biogenic substrates is leading to increased research into the combined application of bio- and chemocatalysis. So far, we have focused on the combination of chemical oxidation by molecular oxygen with biocatalytic dehydration reactions. As an example, four different 2-keto-3-deoxy sugar acids could be obtained by combining a gold catalyst retained in a continuous reactor with an immobilized dehydratase. We are currently working on further chemo-enzymatic synthesis approaches that lead to other products and that include further reaction steps. We are also developing new strategies for the combination of catalysts in compartments based on continuous systems. Enzyme engineering Nature provides us with a variety of catalysts, all of which intrinsically have the ability to convert biogenic substrates – the enzymes. However, approaches for the conversion of biomass into basic and fine chemicals require biochemical parameters that differ significantly from those in the natural habitats of the enzymes. The enzymes have to be compatible with each other, but must also be able to cope with all intermediates that occur and also with high product concentrations – a difficult task! To prepare them for this task and to optimize the corresponding biotransformations, we have to tailor many enzyme properties such as (thermo)stability, specific activity, substrate specificity, cofactor specificity and inhibition (also by high product concentrations). Our focus is on the optimization of dehydratases, oxidoreductases, hydroxylases and aminotransferases. Enzyme engineering is an important part of our research, which is currently developing very rapidly. We research new tools for enzyme engineering and use them to obtain improved catalysts for the production of basic and fine chemicals from carbohydrates.
Stability of biocatalysts
In the laboratories at the TUM Central Institute for Catalysis Research, which have been available to the chair since the beginning of 2016, we are working on enzyme catalytic issues. Today, biocatalysts are used in many industrial processes. In addition to catalyst production costs, catalytic activity and the catalytic stability of the catalysts under process conditions play the most important role in the cost-effectiveness of such processes. At the Central Institute for Catalysis Research, we are therefore conducting research into improving the stability of biocatalysts in order to increase the yields of industry-relevant processes. Today, various enzyme immobilization processes are used to increase enzyme stability. However, since these methods usually have to be redefined and analysed empirically for each biocatalyst, these methods are often time-consuming and not always promising. In order to simplify this process, we investigate these classical methods with regard to the immobilisation of enzymes of entire enzyme reaction cascades to produce immobilised heterogeneous multienzyme complexes in order to increase the substrate enzyme binding affinities of all biocatalysts involved. The use of rational protein design methods will be used to investigate when which stability criteria or structural elements are important for which environmental parameters, e.g. in organic solvents. For this purpose, questions such as “Which methods are suitable for improving the stability of biocatalysts” and “Can uniform stabilisation measures be derived” will be investigated.
Metabolic engineering and microbial polysaccharides
Based on the high genetic diversity of production organisms, microbial polysaccharides represent a multifunctional class of valuable biogenic polymers. Their often complex structure makes them interesting for use in a variety of (food) technical and pharmaceutical applications. The aim of the research focus is to identify new microbial exopolysaccharide producers (bacteria, filamentous fungi) based on a steadily growing strain collection. By means of a high-throughput screening developed at the department itself, the monomer composition of the polymers formed can be determined. Genome sequencing of the newly identified production strains and approaches of synthetic biotechnology are used to elucidate the biosynthesis of the individual polysaccharides and to identify starting points for enzymatic and in vivo modifications. For this purpose, specially developed chassis organisms are used for the expression of synthetic biosynthesis clusters. In addition to the molecular biological investigations, a physicochemical characterisation (rheology, structure, molecular weight) of the polymers is also carried out. Promising EPS producers are then subjected to process optimisation. Comparative parallel bioreactors (8*0.7L) and 2L as well as 15L and 30L bioreactors are available for scale-up. Application tests in various areas round off the investigations. Genetic and metabolic engineering are used to develop tailor-made polymers for specific applications.
Development of new high-throughput methods for the optimization of enzymes
Development of new high-throughput methods for the optimization of enzymes
Enzymes are increasingly used in the biotechnological and chemical industry as well as in medical diagnostics and therapy. They improve conventional processes or enable completely new applications. These possibilities create a great demand for enzymes with new or improved properties. The optimization of enzymes by rational or computer-aided methods is still very limited, despite great progress in the last decade. A rather successful method to optimize enzyme properties, however, is the directed evolution of proteins. First, by varying one or more DNA sequences of the corresponding enzyme, a gene library is created from which the gene variants coding for enzyme variants with the desired, optimized properties are isolated by selection or screening. The advantage of this approach is that no information about the structures of the enzymes, their dynamics or their interactions with different substrates is required.
The success of a directed evolution depends above all on the quality of the libraries and on the effectiveness of the selection and screening strategies. The quality of a library is determined by the available sequence variation and depends to a large extent on the method by which the sequence variation is formed. Today, two main methods of sequence variation are used: in vitro DNA recombination and random mutagenesis.
In vitro DNA recombination starts with a small repertoire of DNA sequences that are very similar but not identical. The differences (mutations) between the sequences may originate from natural evolution (family shuflling) or may have been introduced by the combination of random mutagenesis and selection. They therefore represent a preselection and therefore improve enzyme properties to a large extent or are at least neutral. In vitro DNA recombination produces a repertoire of DNA sequences whose variants contain new combinations of mutations present in the original repertoire. The advantage of this method is that it can simultaneously vary a larger number of positions in a gene and usually combines favourable mutations. The disadvantage, on the other hand, is that a recombination method is always limited to positions that have already shown variations within the original repertoire and also only to the mutations present at these positions.
Random mutagenesis, on the other hand, involves completely new mutations. Their advantage is that all possible variations can be incorporated at all positions of a gene, i.e. they are not limited to the union of already existing mutations. Two techniques for introducing random mutations into a gene are now used almost exclusively: defective PCR and site-specific (cassette) mutagenesis using degenerated oligonucleotides.
The currently by far dominant method of site-independent random mutagenesis is defective PCR. The gene to be mutated is subjected to a PCR using a thermostable polymerase (usually Taq polymerase) which, depending on the conditions of the reaction, inserts individual bases incorrectly into the synthesized genes. A frequent variant of this method involves the use of manganese(II) ions or nucleotide analogues in the PCR approach. This method is very inexpensive and easy to use. Nevertheless, it has the following significant disadvantages:
Due to the nature of the genetic code – three nucleotides encode one amino acid, each amino acid is encoded by up to 6 different codons – an average of only 6 new amino acids can be obtained from individual nucleotide exchanges. Many amino acid exchanges are only possible by two nucleotide exchanges and some are only possible by three nucleotide exchanges. For example, a codon for isoleucine (ATT, ATA or ATC) can only be formed by two nucleotide exchanges from the codon CAA for glutamine and only by three exchanges from the codon CAG for glutamine. Examples from the application of mutagenesis to improve enzymes show that variants with the required properties can often only be obtained by triplet exchanges, i.e. exchanges of all three nucleotides of a codon. A repertoire of variants of a protein in which all possible amino acids are exchanged individually at all positions must be very large if it is obtained by individual nucleotide exchanges (e.g. defective PCR). An average protein of 300 amino acids in length has exactly 300 × 19 = 5700 different variants, which differ in one amino acid from it. A repertoire of variants of this protein, obtained by an average of three nucleotide exchanges, has (900 × 3)^3 = 2 × 1010 different variants. Such a number of variants can only be treated by very few selection methods. The method of defective PCR for mutagenesis cannot therefore be used to obtain complete or high-quality repertoires of a protein.
2. not all nucleotides are exchanged in the same way during faulty PCR. Transversions and transitions occur at different frequencies, some mutations occur much more frequently than others. This introduces a further reduction of the effective size of a repertoire obtained by defective PCR.
Due to the high redundancy of the codons – several codons code for the same amino acid – the exchange of individual nucleotides leads to an average of 23% synonymous mutations, in which the mutated codon codes for the same amino acid as the original one. Although these mutations can lead to favourable changes in the expression of protein, important intrinsic properties of a protein, such as its enzymatic activity or stability, remain unaffected and the effective size of a repertoire is reduced.
On average, stop codons are introduced by 4% of all nucleotide exchanges.
If the mutagenesis rate is high enough to obtain a sufficiently high number of amino acid exchanges, a large number of stop codons can be introduced, resulting in abbreviated gene products. At an average mutation rate of only 3 exchanges per gene, which is theoretically necessary to exchange all possible amino acids at at least one position, more than 10% of the repertoire is already shortened by premature stop codons [1-(1-0.04)^3] = 0.115.
The incorporation of mutations is a largely statistical process in which the number of mutations in the individual variants follows a Poisson distribution. Thus, depending on the average mutation rate, considerable parts of the repertoire can be without mutations, while others have many mutations. This results in a further reduction of the effective size of the repertoire.
While the site-specific random mutagenesis method allows many of the above problems to be avoided, it is always limited to specific gene segments and is therefore unsuitable for site-specific random mutagenesis.
The limitations of the currently used methods of random mutagenesis clearly show the need for 1.) a simple method that allows whole codons (regardless of the number of bases per codon) to be randomly exchanged instead of single nucleotides in DNA strands, and a defined number of mutations (codon exchanges) to be incorporated per DNA strand, and 2.) a universal method that can analyze millions of variants in a few hours.
The chair is working on both topics.