VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

GWP047Production of microbial biosurfactants with nonpathogenicstrainsB. Hörmann*, M.M. Müller, C. Syldatk, R. HausmannTechnical Biology, Karlruhe Institute of Technology (KIT), Karlsruhe,GermanyBiosurfactants are biodegradable, have low toxicity and can be producedwith biological waste materials or renewable resources. They are used asdetergents or surfactants in the pharmaceutical, cosmetic and food industry.Rhamnolipids produced by Pseudomonas aeruginosa belong to the verylimited group of commercially available biosurfactants. However, theopportunistic human pathogen Pseudomonas aeruginosa is up to now theconventional organism used for the production of rhamnolipids.The aim of this work is to increase the productivity of a non-pathogenicrhamnolipid production strain by optimizing culture conditions. This strainproduces a rhamnolipid which is composed of two rhamnose molecules andtwo hydroxy fatty acids with a chain length of 14 carbon atoms.Fermentations of the production strain are carried out with a sixfold parallelbioreactor system. By now, an average volumetric productivity of about 1.83mg/hL in rich medium was found.The rhamnolipid was purified with a gradient flash system using achloroform/methanol and a chloroform/methanol/acetic acid solventmixture. Successful purification was proven by nuclear magnetic resonancespectroscopy. The purified rhamnolipid had a more than 95% purity.A minimal medium on the basis of an elemental composition analysis of thenon-pathogenic production strain was designed.The next step will be further development of the minimal medium tooptimize the rhamnolipid yield.GWP048Establishment of an alternative carbohydrate metabolismpathway in R. eutropha.C. Fleige*, J. Kroll, A. SteinbüchelInstitute for Molecular Microbiology and Biotechnology, WestphalianWilhelms-University, Münster, GermanyThe β-proteobacterium Ralstonia eutropha H16 utilizes fructose andgluconate as carbon sources for heterotrophic growth exclusively via theEntner-Doudoroff-pathway with its key enzyme 2-keto-3-desoxy-6-phosphogluconate (KDPG) aldolase. By deletion of the eda gene, whichencodes this enzyme, we constructed a KDPG aldolase-negative strain,which is disabled to supply pyruvate for energy metabolism originated fromfructose or gluconate as sole carbon source. To restore the fructosecatabolism, an alternative pathway, similar to the fructose-6-phosphate shuntof heterofermentative bifidobacteria, was established. For this, the gene xfpfrom Bifidobacterium animalis, coding for a bifunctional xylulose-5-phosphate/fructose-6-phosphate-phosphoketolase (Xfp; [2]) was expressedin R. eutropha H16 PHB - 4 Δeda. Xfp catalyzes the phosphorolytic cleavageof fructose-6-phosphate to erythrose-4-phosphate and acetylphosphate aswell as of xylulose-5-phosphate to glyceralaldehyde-3-phosphate andacetylphosphate. The recombinant strain exhibited phosphoketolase (PKT)activity on both substrates and was able to use fructose as sole carbon sourcefor growth, due to the fact, that the PKT is the only enzyme that is missingin R. eutropha H16 to establish the artificial fructose-6-phosphate shunt. R.eutropha H16 PHB - 4 Δeda pBBR1MCS-3::xfp should be applicable for anovel variant of a plasmid addiction system to maintain episomal encodedgenetic information during fermentative production processes. Plasmidaddiction systems are often used to ensure plasmid stability inbiotechnological relevant microorganisms and processes without the need toapply external selection pressure like antibiotics [1]. By episomal expressionof Xfp in a R. eutropha H16 mutant lacking KDPG-aldolase-activity andcultivation in minimal media with fructose as sole carbon source, the growthof the cells will be addicted to the use of the xfp-containing plasmid. Thisnovel selection principle extends the range of further biotechnologicalprocesses using R. eutropha H16 as production platform.[1] Kroll, J. et al (2010): Plasmid addiction systems: Perspectives and applications in biotechnology.Microbial Biotechnology Vol. 3, Issue 6, 634-657.[2] Meile, L. et al (2001): Characterization of the D-xylulose 5-phosphate/D-fructose 6-phosphatephosphoketolase gene (xfp) from Bifidobacterium lactis. J Bacteriol 183: 2929-2936.GWP049Novel Lipases: Tools for biochemical synthesis and fat/oilprocessingJ. Modregger 1 , N. Wehofsky 2 , K. Türk* 1 , A. Monte 1 , D. Pérez-López 1 ,S. Sroka 1 , M. Cirefice 1 , R. Pandjaitan 3 , H. Kalisz 11 Eucodis Bioscience GmbH, Vienna, Austria2 Eucodis Bioscience GmbH, Halle, Germany3 Eviagenics SARL, Paris, FranceLipases are versatile tools in biotechnology, catalyzing a broad range ofhydrolytic and/or (trans-)esterification reactions. Due to thesecharacteristics, their industrial use is steadily expanding. For opening novelapplication solutions, developing lipases with new properties becomes morecrucial than ever. EUCODIS Bioscience therefore attaches great importanceto diversity and application-related characterization of their lipase portfolio,serving customers in the chemical, pharmaceutical, food, feed and otherindustries.Here, we investigated the characteristics of a number of novel lipases withregard to potential use in the synthesis of (bio-)chemical or pharmaceuticalproducts, and in the oil and fat processing industry. Candidate genes wereselected, expressed, the enzymes processed and supplied to interested partiesfor application testing. The enzymes were characterised with respect tosubstrate specificity (chain length, preference for saturated or(poly-)unsaturated fatty acids), regio- and enantioselectivity and optimalconditions for hydrolysis or (trans-)esterification reactions. Lipases withlong chain fatty acid acceptance as well as enzymes useful for the removalof short chain fatty acids were identified. Together with their preferencestowards saturated or unsaturated fatty acids, these lipases are valuable toolsfor the development of novel processes in the oil and fat industry. Toexplore the suitability of the Eucodis lipases in the synthesis of (bio-)chemical and pharmaceutical products, lipases were in particular tested fortheir capability to catalyze (trans-)esterification reactions.Here, we will introduce the Eucodis library of lipid modifying enzymes,their catalytic properties and reaction characteristics, and discuss theirpotential in industrial biocatalysis.GWP050Glycopeptide resistance in the producer strainAmycolatopsis balhimycinaH.-J. Frasch* 1 , G. Gallo 2 , T. Schäberle 1 , P. Steimle 1 , L. Kalan 3 , A.-M. Puglia 2 , G. Wright 3 , W. Wohlleben 1 , E. Stegmann 11 Department of Microbiology/Biotechnology, Eberhard-Karls-University,Tübingen, Germany2 Department of Cell and Developmental Biology, University of Pavia,Pavia, Italy3 Health Science Receiving, MacMAster University, Hamilton, CanadaGlycopeptides are the drugs of last resort for treatment of severe infectionscaused by gram positive pathogens. They impair bacterial growth by bindingto the terminal d-Ala-d-Ala residues of cell wall precursors and thus blockcell wall biosynthesis. However, the number of glycopeptide resistantbacteria rose steadily over the last two decades.The most common resistance mechanism of bacteria against glycopeptides isto reprogram the murein synthetic machinery resulting in resistant cell wallprecursors ending on d-Ala-d-Lac. This modification is catalyzed byenzymes encoded by the vanHAX operon. Their transcription is activated inthe presence of glycopeptides by the two component system VanRS.The genome of Amycolatopsis balhimycina, the producer of thevancomycin-like glycopeptide balhimycin, contains genes with highhomology to the enterococci vanRS and vanHAX genes.The constructed vanHAX b-deletion mutant in A. balhimycina, shows aglycopeptide sensitive phenotype. Surprisingly, the mutant strain stillproduces balhimycin after 42h of growth in balhimycin production medium.Therefore, A. balhimycina needs an additional set of genes which enable themutant to synthesize a resistant cell wall. The synthesis of cell wallprecursors ending on d-Ala-d-Lac was further confirmed by LC-MSanalysis. In non-production medium the mutant strain exclusively producescell wall precursors for a sensitive cell wall.The occurance of resistance in A. balhimycina is independent of the VanRStwo component system. The glycopeptide production is also not directlycontrolled by VanRS. Since we could recently show that the vanRS-systemcan activate the vanHAX-genes after their heterologous expression in S.coelicolor DvanRS, we intend to analyze which cellular functions areregulated by this two component system.spektrum | Tagungsband 2011