IBT

In charge: Prof. C. Wandrey, IBT, c.wandrey@fz-juelich.de

R&D work at the Institute of Biotechnology is focused on the development of biotechnological processes for the production of pharmaceutical products and fine chemicals. These activities comprise both theory-oriented and application-oriented topics. Particular attention is given to enzymes, microorganisms and higher cell cultures as biocatalysts. In order to selectively improve the synthesizing activity of these different biological systems (protein design, metabolic engineering), extensive studies are necessary on the structure and function of enzymes as well as on metabolism and its regulation using state-of-the-art technologies.

Furthermore, reaction and process engineering work is performed for optimum use of the enzymes, microorganisms and animal cell cultures. A relatively new and promising field is the proliferation of blood-forming stem cells from the cord blood of neonates.

Significant Results in 2003

IBT-1

• In genome-wide expression analyses with DNA chips, it was possible to characterize the regulation mechanisms of the branched-chain amino acids isoleucine and leucine in Corynebacterium glutamicum. It was furthermore shown that, apart from the provision of ATP and pyruvate in glycolysis, the pyruvate kinase in C. glutamicum is essential for growth on some gluconeogenic carbon sources.
• A suitable strain of C. glutamicum was developed for L-serine production which not only prevented overexpression of the serine biosynthesis genes but also inhibits the internal cell degradation of these amino acids. Selective deletion of the serine dehydratase and a reduced expression of the serine hydroxymethyl transferase enabled a C. glutamicum strain to be constructed that excreted 10 g/l L serine into the medium. The culture parameters for this strain are currently being optimized by our industrial cooperation partner.
• Continuing work on the development of a regeneration system for the NADH coenzyme in whole-cell biotransformations, a recombinant E. coli strain was constructed having a redox cycle from a formate dehydrogenase for NADH regeneration and an NADH-dependent mannitol dehydrogenase for D-fructose reduction. By additionally feeding it with both these substrates (D-fructose + formate), this biocatalyst with a glucose facilitator for the uptake of D-fructose formed up to 750 mM of D-mannitol. Within the framework of a BMBF project in cooperation with the working group of Prof. Görisch (Berlin), a Gluconobacter oxydans strain was developed with which it was possible for the first time to produce high yields of 5-ketogluconic acid from glucose. Starting from a pyruvate production strain from E. coli, by transformation with the gene for L-alanine dehydrogenase from Bacillus subtilis a strain was obtained that efficiently converted glucose and ammonium into L-alanine under anaerobic conditions.
• Studies on the production of two carbohydrate-converting enzymes from the extremophile bacterium Anaerobranca gottschalkii in the mesophile host bacterium Staphylococcus carnosus showed that the secretion of these enzymes in a folded form via the Tat pathway led to higher yields than export via the Sec pathway. On the basis of the gene for the essential component SecA of the Sec export apparatus, a new selection system was developed that prevents the loss of the subtilisin gene during the industrial production of this detergent enzyme.

• In producing the amino acid L-lysine with E. coli the overexpression of the phosphoenolpyrovate synthase was identified as limiting by 13-C intracellular flux analysis.
• In investigating the biosynthesis of aromatics with E. coli via a glucose step function (glucose limitation … glucose saturation) the decisive bottleneck was identified by cytosolic metabolite analysis and eliminated by overexpression of the corresponding gene.
• Transdiols and aminocyclitols, which branch off from the biosynthetic pathway of aromatics, were produced on a kg scale by corresponding metabolic engineering and fermentation developments. · NADPH was produced continuously for 120 h from NADP by means of a hydrogenase of Pyrococcus furiosus using hydrogen. A space-time yield of 53 (g/(L x d)) was achieved.
• Reduced nicotinamide cofactors can also be produced electroenzymatically. A space-time yield of more than 1 (kg/(L x d)) was achieved here.
• The cell-specific productivity for the production of certain potential tumour therapeuticals can be more than doubled in comparison to unregulated glucose supply by selective glucose limitation in the cell culture.
• Human granulocytes (for the treatment of sepsis) were expanded by a factor of 4400 within 18 days (sufficient for clinical use) starting out from stem and precursor cells from cord blood.
• T lymphocytes were produced from monocytes for loading tumour cell lysate for an immunotherapy. In using an influenza virus peptide, an expansion factor of 4300 was achieved for the corresponding T cells.
• Competing adsorption and desorption processes during chromatography can be observed in the interior of adsorbers by means of confocal laser scanning fluorescence spectroscopy.
• Biopolymers can be characterized and isolated with the aid of the distribution in aqueous two-phase systems.