Development of cultivation strategies for friulimicin production in Actinoplanes friuliensis.

Actinoplanes friuliensis is a rare actinomycete which produces the highly potent lipopeptide antibiotic friulimicin. This lipopeptide antibiotic is active against a broad range of multi-resistant gram-positive bacteria such as methicillin-resistant Enterococcus sp. and Staphylococcus aureus (MRE, MRSA) strains. Antibiotic biosynthesis and regulation in actinomycetes is very complex. In order to study the biosynthesis of these species and to develop efficient production processes, standardized cultivation conditions are a prerequisite. For this reason a chemically defined production medium for A. friuliensis was developed. With this chemically defined medium it was possible to analyze the influence of medium components on growth and antibiotic biosynthesis. These findings were used to develop process strategies for friulimicin production. The focus of the project presented here was to develop cultivation strategies which included fed-batch and continuous cultivation processes. In fed-batch processes, volumetric productivities for friulimicin of 1-2 mg/l h were achieved. In a perfusion process, a very simple cell retention system, which works via sedimentation of the mycelial cell pellets, was used. With this system, stable continuous cultivations with cell retention were dependent on the dilution rate. With a dilution rate of 0.05 h(-1), cell retention worked well and volumetric productivity of friulimicin was enhanced to 3-5 mg/l h. With a higher dilution rate of 0.1 h(-1), friulimicin production ceased because cell retention was not possible any longer with this simple cell retention system. In order to support process development, cultivation data were used to characterize metabolic fluxes in the developed friulimicin production processes.

Complete genome sequence of the actinobacterium Actinoplanes friuliensis HAG 010964, producer of the lipopeptide antibiotic friulimycin.

Actinoplanes friuliensis HAG 010964 (DSM 7358) was isolated from a soil sample from the Friuli region in Italy and characterized as a producer of the antibiotic friulimycin. The complete genome sequence includes genomic information of secondary metabolite biosynthesis and of its lifestyle. Genbank/EMBL/DDBJ Accession Nr: CP006272 (chromosome).

Calorimetric control of the specific growth rate during fed-batch cultures of Saccharomyces cerevisiae.

The specific growth rate of a Saccharomyces cerevisiae strain with glucose as limiting C-source was estimated from the measured heat flow produced by the cells. For the cultivation a standard 30 l laboratory bioreactor was used, which was extended in such a way that heat balancing is possible. The feed rate was adjusted by a feedforward/feedback controller such that the specific growth rate was kept on the desired set-point value. On the basis of experimental investigations it was demonstrated that the specific growth rate can be controlled at a given set point value below the critical value to prevent the production of growth-inhibitory ethanol due to the Crabtree effect. With this control strategy high biomass concentrations of more than 110 g l(-1) can be obtained.

Calorimetric control for high cell density cultivation of a recombinant Escherichia coli strain.

In order to achieve maximum productivity of recombinant proteins in Escherichia coli high cell density cultivation (HCDC) strategies have been the subject of many studies. The aim of this work was the application of calorimetric methods to HCDC. The specific growth rate of a recombinant E. coli strain producing green fluorescent protein (GFP) was controlled during fed-batch cultivations by estimating the specific growth rate from the measured heat flow produced by the cells. For the cultivation a standard 30 l laboratory bioreactor was used, which was extended in such a way that heat balancing is possible. The feed rate was adjusted by an adaptive controller such that the specific growth rate was kept on the desired set point value. On the basis of experimental investigations with a recombinant E. coli strain using glucose as limiting C-source it was demonstrated that the specific growth rate can be kept on a given set point value and biomass concentrations of up to 120 g l(-1) can be obtained, reproducibly.