Objective Selection of B. subtilis strains producing new types of lipopeptidesOptimization of the fermentation parametersBiotechnological and pharmaceutical activites of the lipopeptide productsCloning and sequencing of lipopeptide biosynthetic genesRegulation of lipopeptide biosynthesis in Bacillus subtilisAnalysis of the competence development in Bacillus subtilisRearrangement of amino acid activating modules and protein engineering of surfactin synthetaseLipopeptide production of B. subtilis was optimized. In particular, the influence of oxygen transfer in fermentation of this organism was improved for high lipopeptide production. The lipopeptide spectrum of B. subtilis was studied in detail. Fengycin was characterized as a potent antifungal agent showing much lower hemolytic activities than iturin. The antiviral potential of surfactin has been evaluated. This lipopeptide specifically inactivates viruses that are equipped with a lipid envelope, as herpes- and retro- viruses, for example. Its antiviral activity is mainly due to its ability to disrupt the viral lipid envelope. Surfactin seems to be qualified for applications in virus safety of cell cultures and biotechnological products. As a membrane active agent it exhibits only a low risk for resistence development due to its physico-chemical mode of action in contrast to other drugs which are involved in intracellular effects of viruses.Important progress has been obtained in cloning and sequencing of genes coding for the biosynthesis of lipopeptides in B. subtilis. The pps operon which obviously codifies the production of fengycin was completely sequenced and genetically characterized. Parts of the putative operons for the formation of iturin and mycosubtilin were cloned and sequenced. In this way the complete elucidation of these gene structures was initiated. The regulation of the genetic competence of B. subtilis is mediated through a complex signal transduction network sensing information on cell density, medium composition and growth stage. Two loci which are indispensible for the proper functioning of the pathway leading to competence were investigated in detail: srfA and comK. The srfA operon specifies the production of the biosurfactant surfactin, while comK codes for the competence transcription factor involved in the expression of the late competence genes. By deletion analysis a small ORF, called comS, which is essential for competence development has been detected in the gene segment coding for the fourth, valine activating module of srfA. ComS represents the gene of a small protein of 46 amino acids. Preliminary evidence was obtained that ComS is capable of counteracting the combined effect of MecA and MecB in inactivating the affinity of ComK to the promoters of the late competence genes. Definite proof has been obtained that comK encodes the competence transcription factor. From gel retardation experiments it was established that four molecules of comK are attached to the upstream region of its target gene. MecA and MecB which are also components of the competence signal transduction network inactivate ComK on the protein level presumably through sequestration of the MecA/MecB complex. Two types of DNases produced by B. subtilis have been characterized. One is membrane bound, the other is secreted. Although neither of these genes were important for competence development, expression of the membrane-bound nuclease seemed to be dependent on the previous expression of ComK and, therefore, also on the expression of the srfA operon.Investigating peptide synthetase domains in B. subtilis associated with activities necessary for peptide synthesis and secretion recombinant peptide forming multienzymes were constructed by domain swapping of amino acid activating modules of the srfA operon enabling the synthesis of new peptide products by recombinant technology. This achievement represents an important milestone for the design of novel biotechnologically and pharmaceutically important peptide products. In addition, by protein engineering structural elements determining the specificity for amino acid selection and binding as well as the role of the thioester domain located at the C-terminal end of peptide synthetases has been elucidated.MAJOR SCIENTIFIC BREAKTHROUGHS:Comprehensive survey on the lipopeptides produced by Bacillus subtilis/ Characterization of some of their biological activities/ Complete sequencing and characterization of the pss operon putatively encoding fengycin synthesis/ Characterization of comS as an essential component for competence development/ Detailed knowledge on the loci important for the regulation of competence development of B. subtilis/ Construction of recombinant peptide synthetases by rearrangements of modules of the srfA operon. Fields of science natural sciencesbiological sciencesmicrobiologyvirologynatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsnatural sciencesbiological sciencesbiochemistrybiomoleculeslipidsnatural scienceschemical sciencesorganic chemistryaminesengineering and technologyindustrial biotechnologybioprocessing technologiesfermentation Programme(s) FP3-BIOTECH 1 - Specific research and technological development programme (EEC) in the field of biotechnology, 1990-1994 Topic(s) 2.2 - Metabolism of animals, plants and microbes: essential physiological traits Call for proposal Data not available Funding Scheme Data not available Coordinator Technische Universität Berlin EU contribution No data Address 10587 Berlin Germany See on map Total cost No data Participants (3) Sort alphabetically Sort by EU Contribution Expand all Collapse all EniTecnologie S.p.A. Italy EU contribution No data Address Via Felice Maritano 20097 Milano - San Donato Milanese See on map Total cost No data University of Groningen Netherlands EU contribution No data Address 9751 NN Haren See on map Total cost No data Université de Liège Belgium EU contribution No data Address 4000 Liège See on map Total cost No data
