Research

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

Biohybrid Enzyme Catalysis

The emerging field of biohybrid enzyme catalysis aims at combining catalytic methods from different fields with enzyme catalysis. Our main focus is on chemo-enzymatic synthesis and biohybrid electrocatalysis for the generation of basic and fine chemicals from renewable raw materials.

There are already a number of examples where such biohybrid approaches are being explored. A plethora of organic and inorganic chemical catalysts and electrochemically used electrode materials exist. Nevertheless, they generally lack compatibility with enzymes and biogenic substrates and their associated solvents. On this basis, we are exploring chemo-enzymatic and electro-enzymatic processes for the efficient and targeted conversion of complex biogenic feedstocks. We have had initial success in combining chemical oxidation by molecular oxygen with biocatalytic dehydration reactions. As an example, four different 2-keto-3-deoxy sugar acids could be obtained by the combinations of a gold catalyst retained in a continuous reactor with an immobilized dehydratase. Furthermore, the continuous combination of electrochemical reactions with bioenzymatic product generation is investigated in depth at CBR by process analyses and electrode development.

 

Figure 1: One-pot (A) and compartment setup for the chemoenzymatic synthesis of KDS. (B) Direct transfer of sugar acid solution to immobilized SsDHAD. (C) Adjustment of pH and removal of H2O2 by catalase treatment prior to loading of immobilized SsDHAD with sugar acid solution and subsequent purification of KDS via anion and cation exchange chromatography. Symbols and drawings are used in accordance with the literature. (DOI 10.1021/acscatal.6b01276)

Bioprocess engineering

Figure 1: 2L fermenter

In addition to molecular optimization of biotechnological processes at the enzyme or cell level, scaling and process optimization are equally important. Bioprocess engineering helps to replace conventional chemical processes, in particular in the utilization of

biogenic raw materials by producing a wide range of value-added substances. In addition to purely enzyme-based processes, fermentative processes that enable material production with the aid of microorganisms are still essential today.

The research area “Fermentative processes” is concerned with the development of these processes for the targeted production and refinement of valuable products.

Especially the integration of residual material flows is of central importance and an important aspect in terms of circular economic cycles.

In order to ensure economical production or to compete with conventional production procedures, processes must be continuously optimized and adapted to the latest biological findings.

Figure 2: 30 L fermenter

Coating

Microorganisms can be part of biohybrid materials. The properties of these materials can be specifically influenced by selecting and modifying the apllied microorganisms. At the Chair of Chemistry of Biogenic Resources, corresponding biohybrid materials are investigated and their properties optimized. By using microbial biomass, the materials are sustainable. For example, cells from industrial fermentation processes can be used to create a higher-value utilization option in addition to thermal/energy recovery. The cells can be considered as a platform for functionalization of the material. The function of natural or recombinant proteins is conserved in the material and becomes macroscopically usable.

Development of Biocatalysts - Enzyme Engineering

Figure 1: Crystal structure of an oxidoreductase. A substrate was docked into the structure (molecule in cyan) to identify hotspots for semi-rational engineering (turquoise area).

Nature provides a variety of biocatalysts (enzymes), all of which have the intrinsic ability to convert biogenic substrates. However, approaches for the conversion of biomass into basic and fine chemicals require biochemical parameters that differ significantly from those found in the enzymes’ natural habitats. The enzymes must be compatible with each other, but also be able to cope with any intermediates that occur and also with high product concentrations. To optimize them for this and for the corresponding biotransformations, we need to tailor many enzyme properties, such as (thermo)stability, specific activity, substrate specificity, cofactor specificity. While our focus is always on an enzyme’s use in a process, we also seek to understand the molecular mechanisms of catalytic processes through enzyme engineering. Successful examples from our work include stabilizing enzymes for function at high temperatures, optimizing general activity, changing cofactor specificity, and using non-natural cofactors.

Critical to the success of directed evolution are the quality of enzyme libraries and the efficiency and accuracy of screening methods. We develop new mutagenesis and recombination strategies as well as chemical, enzymatic or physical high-throughput assays and selection methods. The assays are established both in the classical 96-well format on our robotic platform and for ultrahigh throughput applications on our microfluidic systems.

Biomass Pretreatment and Fermentation

Biogenic raw materials contain a large number of different constituents. In order to obtain basic materials from them for future industrial use, the biomass must be suitably processed, which includes pretreatment, refining, and separation of the desired constituents. These can then be further converted via chemical or biotechnological routes to substitute petrochemical products or synthesize new functional chemicals or materials.

We conducts research on the targeted and customized processing of biogenic raw materials and residues, as well as on the development of biobased processes for the further conversion of contents into valuable basic materials for the chemical industry or into energy sources.

Research activities:

  • Biorefinery systems for the most complete and sustainable use of raw materials.
  • Pretreatment and processing of lignocellulosic biomass, such as straw, grasses, wood
  • Pretreatment and processing of microalgae
  • Mechanical, physico-chemical (thermal hydrolysis), and chemical pretreatment in combination with enzymatic digestion
  • Bioconversion of saccharides in lignocellulosic hydrolysates
  • Biogas technology: microbiological and enzymatic hydrolysis, optimization of process biology
  • Development of fermentation processes for the biosynthesis of chemicals and biobased polymers (alcohols, diols, carboxylic acids, esters, PHA, …)
Laboratory automation

Figure 1: Robot platform with 96 pipetting head, as well as integrated incubator, centrifuge and photometer.

With the know-how and equipment available at the chair, a wide variety of laboratory work is automated and realized in high throughput.

Established procedures are for example

  • Screening of strain collections for polysaccharide producers
  • Screening of enzyme libraries for activity, stability or substrate specificity
  • extraction and purification of proteins, RNA and DNA
  • absorptive and chromatographic separation methods in 96-well format (e.g. SEC or IMAC)
  • enzymatic and chemical quantification methods, e.g. for glucose, pyruvate, proteins

    Figure 2: Colony picker and rearray system.

  • enzyme activity assays
  • enzyme kinetics
  • optimization experiments, e.g. medium optimization
Microfluidics

Figure 1: Droplet generation.

Our focus lies on developing biochemical assays suited for ultrahigh throughput droplet microfluidics (Lab on a Chip). We apply droplet microfluidics when screening very large libraries in directed evolution experiments as well as in synthetic biology approaches for metagenomic mining of novel enzymes.

Droplet microfluidics allows the downscaling of reactions to picoliter scale by generating monodisperse droplets using microfabricated chips. Every droplet acts as a confined reaction chamber – similar to wells in microtiterplates – and millions of droplets can be generated on a single chip within minutes. These droplets can undergo various unit operations, like droplet fusion or adding fluids into existing droplets (picoinjection). In specialized sorting chips it is possible to screen and sort droplets based on absorbance or fluorescence signals in up to kHz frequencies. In biochemistry, sustainability and throughput can be increased dramatically by downscaling reaction volumes from 300 µL in a conventional 96-well-plate-format to 4 pL droplets and sorting 1000 droplets per second on a cm² sized microfluidic chip instead of screening several microplates per day with liquid handling robots.

Figure 2: Sifting and sorting of Droplets.

Multi-enzyme catalysis

Multi-enzyme catalysis means using several enzymes at the same time without the need to isolate intermediates at great expense before using them for the next reaction. This is particularly possible with enzymes, in contrast to chemical catalysts, because they can work effectively under similar conditions or can be modified to be compatible with each other. Our goal is to use multi-enzyme catalysis to produce platform and fine chemicals from sugars or other molecules that can be obtained from renewable resources.

Figure 1: Utilization of renewable raw materials through the use of enzyme cascades.

For the development and optimization of multi-enzyme catalysis we are investigating:

  • The intrinsic properties of the enzymes involved (activity, specificity, stability) and optimize them, if necessary.
  • Analytical methods such as HPLC, GC, MS, NMR or enzymatic assays, for the detection and quantification of intermediates, products and, if necessary, by-products, to understand the interplay of the enzymes in multi-enzyme catalysis and to identify their weaknesses and strengths.
  • Mathematical models (in cooperation) to make predictions about bottlenecks and optimal conditions for multi-enzyme catalysis according to the research question.

 

Several multi-enzyme cascades have already been established at the chair and are under continuous development:

Figure 2: Enzyme cascades from glucose via pyruvate to isobutanol or ethanol.

In a newly developed artificial cascade pyruvate is produced from glucose using only four of enzymes instead of the ten of the classical glycolysis. Pyruvate, in turn, is converted into various chemicals with additional enzymes in different cascades. Other starting molecules for our multi-enzyme catalyses are xylose, which can be obtained from hemicellulose, or glycerol.

In the context of the multi-enzyme catalysis, we also deal with the coupling of enzymatic with chemical or electrochemical reactions, the design of artificial cofactors and for the cascade construction also with enzyme engineering.

Protein expression with synthetic biology

Biological cell factories are capable of producing a wide range of products, covering fuels to pharmaceuticals without the need for harsh chemistry. In other words, they support more environmentally friendly industrial processes, which is essential for the change towards a bio-based economy. However, the development of these cell factories is currently time-consuming and labor-intensive, since, among other things, the integrity of the organism used must be ensured. By transferring the reactions from the inside of the cell to the free solution, these limitations can be overcome. Therefore, the sufficient supply of proteins for synthetic enzyme cascades (in vitro) is an important field of research.

The yeast Pichia pastoris is one of the most important heterologous expression systems in biotechnology. In addition to the facilitated secretion of proteins, posttranslational modifications such as glycosylation are also possible. Since it is cheaper to produce secreted proteins, they can then be applied more economically on an industrial scale. Although P. pastoris is widely used for industrial applications and intensely studied already, an efficient strategy for systematical identification of optimal expression and secretion conditions was still missing. Therefore, the Chair of Chemistry of Biogenic Resources has applied a Synthetic Biology approach and developed a modular toolbox of standardized genetic elements and different secretion signals. These elements can be easily combined randomly by using of a hierarchical assembly method to build large and diverse libraries. These libraries are then screened by robot assisted high-throughput screening (HTS). The developed approach found new combinations of genetic parts that lead to improved secretion of several enzymes. The flexible design of the toolkit enables efficient high-throughput optimization of future challenges in protein expression and secretion in yeast.

PHB & biobased monomers
PHB

Many microorganisms are capable of producing polyhydroxybutyrate as a storage material. This natural, biodegradable polyester can replace fossil based polymers in some applications, but is not equivalent to these with all its properties. Through strain engineering or the synthesis of the chiral building blocks for chemical polymerization, we are developing new PHB variants with tailor-made properties for broader applications. For cost-effective production of these polymers, we exploit the possibility of PHB accumulation in cells from highly diluted biogenic waste streams offering a way for their valorization.

Biobased monomers:

The production of materials or polymers today plays a major role in the utilization of petroleum. In the same way, the use of biomass for the production of polymers offers great potential. Beside biogenic polymers, which are manufactured directly by microorganisms or plants, the production of the monomers from biogenic raw materials with subsequent chemical polymerization is an efficient way to obtain new materials and in addition for providing a genuine CO2 sink. It is important that not only so-called drop-in monomers are produced, which directly replace the petroleum-based monomers. While, for example, it is possible to produce ethylene for polyethylene from glucose, this rarely makes sense with a mass yield of only 30% and such this it hardly economically viable nor would it actually be ecological. Oxygenated monomers such as butanediol, succinic acid or furan dicarboxylic acid have a better mass balance and can be produced more efficiently from biomass than from fossil raw materials. It becomes particularly interesting when the biogenic raw materials bring along properties that are not found in the molecules of fossil raw materials, i.e. with which completely new polymer or material properties can be obtained.

 

The following examples should give an impression on different activities in this field:

  • In our work on synthetic chemo-enzymatic reaction pathways, we have developed new reaction routes to 1,4-butanediol, 2,3-butanediol, isobutanol or acetoin from sugars.
  • Based on waste streams from the pulp industry (terpenes), we have developed a new class of chiral polyamides that are superior to fossil polymers in terms of hardness and transparency.
  • With the optimization of integrated whole cell biotransformation processes we have provided long-chain dicarboxylic acids for biobased polyesters and polyamides.
Metabolic Engineering

Metabolic engineering describes a variety of different methods to optimize the production of high value target molecules from cheap and simple substrates by living microorganisms. To do so, metabolic fluxes are optimized to overcome bottleneck enzymes or reduce the production of undesired side-products. Alternatively, completely new artificial pathways are introduced into a production organism to generate new compounds of interest.

Our research focuses on the optimization of pathways in commonly used lab organisms, but also promising alternative hosts such as Paenibacillus polymyxa. We use highly efficient tools, such as CRISPR/Cas9 to rationally modify the genome of our bacterial production platforms. Furthermore, we are also developing new tools to regulate complex metabolic pathways.

Contact

Chair of Chemistry of Biogenic Resources

Schulgasse 16
D-94315 Straubing

Head

Prof. Dr. Volker Sieber

Phone: +49 (0) 9421 187-300
Phone: +49 (0) 8161 71 35 91
Mail: sieber@tum.de

Team Assistant

Elisabeth Aichner

Phone: +49 (0) 9421 187-301
Mail: elisabeth.aichner@tum.de