of our Research Group
Research Group Yeast Genetics
Head: Prof. Dr. Gotthard Kunze | Dr. Martin Giersberg (temp.)
The main field of research in the yeast genetics unit is the non-conventional, non-pathogen yeast Arxula adeninivorans and its use for the production of recombinant proteins, as a gene-donor and biocatalyst for new biotechnological products. It is also utilised as the microbial component in biosensors for the analysis of waste water, urine and blood, for the monitoring of feed and food and for medical investigations.
The yeast A. adeninivorans was used already in the 1980's for the production of single cell protein in Malchin (Mecklenburg-Western Pomerania) because of its rapid growth, extraordinarily high cell titers and its braod substrate spectrum. Parallel to the biotechnological utilisation investigations took place regarding its taxonomical classification and its physiology. In cooperation with the department yeast genetics of the University of Greifswald molecular tools were developed for the production of mutants by chemical mutagenesis, protoplast fusion and mitotic segregation as the basis for further improvement of A. adeninivorans as host for the production of new biotechnological products and the construction, establishment and validation of microbial biosensors. Such biosensors for the monitoring of food and environmental conditions as well as medical and pharmaceutical purposes were developed starting from the 1990's in the working group yeast genetics under the direction of Professor Dr. habil. Gotthard Kunze.
The genome of A. adeninivorans
Beginning in the early 1990's the genome of A. adeninivorans was analysed in detail by DNA reassociation studies. At the same time physiological studies were accomplished concerning usable substrates as carbon and nitrogen sources and the corresponding metabolic pathways were examined. In this context the first transgenic A. adeninivorans strains were constructed. In the following years several A. adeninivorans genes and its 25S rDNA were isolated and characterised leading to establishment of a transformation/expression system for the production of recombinant proteins in transgenic yeast strains. This system called Xplor®2 was further developed to a modular platform (expression modules, selection marker modules, ARS modules, Chaperon modules) for fast and effective construction of desired plasmids. In order to make this platform usable for the construction of custom-made transgenic production strains with high acceptance, it was established in close cooperation with small and medium-sized enterprises (KMUs) like the ARTES Biotechnology GmbH in Langenfeld. The main advantage of this patent-registered platform is that only essential DNA sequences are transformed avoiding undesired E. coli sequences and resistance genes. Since the expression modules stably integrate in the A. adeninivorans genome the transformed yeast strains show a high mitotic stability, essential for biotechnological application.
In 2010 the complete genome of A. adeninivorans was sequenced and annotated at the IPK making the Xplor®2 system even more attractive.
Development of sensors for environmental survey
Due to the ease of genetic manipulation of A. adeninivorans several collaborations have been established especially with KMUs. Among other things A. adeninivorans cells were tested for the first time as microbial components of biosensors for the measurement of the biochemical oxygen demand (BOD). With this system the BOD in waste water, tap water, brackish water and sea water was determined within 70 seconds demonstrating the usability of the yeast for pollution control of aqueous samples.
During the last years we have focused on Arxula based biosensors for application to environmental survey, in the agriculture and for medical purposes. In several collaborations with industrial partners, research facilities and federal offices numerous biosensors were established for the determination of hormones, dioxin and pharmaceuticals in tap water, soda water, waste water, urine and blood serum. The used transgenic A. adeninivorans cells contain the genetic information for the constitutive expression of the particular human receptor which interacts with the appropriate ligand (estrogen, androgen, progesteron, glucocorticoids, dioxin, pharmaceuticals) resulting in expression of a reporter gene which encodes a recombinant enzyme detectable by simple biochemical or amperometrical measurements. Since the activity of the enzyme correlates directly with the concentration of the hormone or pharmaceutical, all substances interacting with the appropriate receptor can be measured as so-called sum parameters. First assays for the detection of estrogenic and androgenic activities in tap water, soda water and waste water were already commercialised by the industrial partner. Assays for the determination of estrogens, androgens, progesterone, glucocorticoids, dioxins and pharmaceuticals in waste water, urine and blood serum are in the phase of validation and expected to enter the market in 2012/13.
Development of sensors for medical purpose
Apart from the application in biosensors A. adeninivorans is an excellent host for the production of recombinant proteins like above mentioned human receptors. However, since some recombinant proteins require a different host to be synthesised in a functional manner, the Xplor®2 transformation/expression platform was further developed. Now additional yeasts like Hansenula polymorpha can be transformed and exploited as producers of recombinant proteins. The first functional recombinant receptors (e.g. human estrogen receptor α and human progesterone receptor) are synthesised at present for application in environmental analytics and, furthermore, in medical examinations. In the near future a surface plasmon resonance (SPR) platform for cancer analysis (e.g. breast cancer-interaction of Herceptin with the receptor HER2 [new]) and testing of novel drug-receptor-interactions will be developed in collaboration with the Fraunhofer Institute and industrial partners.
Additional fields of application for transgenic yeasts
In parallel to the above described applications transgenic yeasts, preferably A. adeninivorans, have been and will be designed for the following biotechnological applications:
- Recombinant tannase enzyme as admixture of animal feeds and/or as additive for the increase of the yield in biogas plants.
- Production of food with decreased purine content using an enzymatic procedure.
- Use of A. adeninivorans as biocatalyst for the production of n-butanol.
- Processing and recuperation of metals from tailings and waste dump material as well as for the processing of poor ores.
- Use of A. adeninivorans as microbial component of the fuel cell.
Nachweis von Weinkontaminanten Vor-Ort per Multiplex-Wein-Stick bzw. im Labor per Multiplex-Makrodot-Assay.
Ziel eines von der Investitionsbank Sachsen-Anhalt geförderten Vorhabens ist die Entwicklung neuer innovativer biologischer Testsysteme zum schnellen und sensitiven Nachweis von Weinkontaminanten. Hierzu werden durch Übertragung des biologischen Nachweissystems (DNA-RNA-Hybridisierung) auf einen Multiplex-Wein-Stick bzw. Multiplex-Makrodot-Assay im Mikrotiterplattenformat Systeme zum schnellen, sensitiven und eindeutigen Nachweis von Weinkontaminanten für die Weinindustrie etabliert und validiert. Der direkt für Vor-Ort Analysen ausgelegte Multiplex-Wein-Stick soll zukünktig in einer Ja/Nein-Aussage und semi-quantitativen Detektion dem Winzer einen ersten Überblick über die Qualität seines Produktes hinsichtlich Kontamination geben. Bei einer ja-Aussage kan anschließend der für das Labor ausgelegter Multiplex-Makrodot-Assay dieser Plattform als Bestätigungsverfahren zusätzlich.