VAAM-Jahrestagung 2012 18.–21. März in Tübingen

95MEP011Development of Fed-Batch Strategies for AntibioticProduction of Actinoplanes friuliensisA. Steinkämper* 1 , A. Wolf 2 , R. Masuch 2 , J. Hofmann 1,2 , K. Mauch 3 ,J. Schmid 3 , D. Schwartz 1 , R. Biener 11 University of Applied Sciences Esslingen, Natural Sciences,Biotechnology, Esslingen, Germany2 micro-biolytics, Esslingen, Germany3 Insilico Biotechnology, Stuttgart, GermanyActinoplanes friuliensis, a rare actinomycete, is the producer strain offriulimicin, a lipopeptide antibiotic which is active against a broad range ofmultiresistant gram-positive bacteria such as methicillin-resistantEnterococcus spec. and Staphylococcus aureus (MRE, MRSA) strains(Aretz, 2000).In order to improve the understanding of the complex metabolic networkof the friulimicin biosynthesis in A. friuliensis, a genome-scale networkmodel will be developed and characterized (Insilico Biotechnology). Tovalidate the model and to perform metabolic flux analysis, data fromcultivations of A. friuliensis are collected and applied to this model. Thecultivations are carried out in a bioreactor under defined and controlledconditions. A chemically defined production medium, especiallydeveloped for A. friuliensis, is used. This defined medium is a prerequisitefor the quantitative analysis of cell metabolism during the cultivations andis also necessary to verify a new developed middle infrared spectroscopymethod (AquaSpec Technology, micro-biolytics GmbH). With thismethod, all known substrates and metabolites can be measured in onesample.By developing fed-batch cultivation strategies, the production of thefriulimicin inhibiting by-product ammonium could be prevented.The validated flux model, combined with data of cultivation andtranscription analysis, will subsequently give hints for directed geneticmodifications and optimization of process control strategies with theobjective to redirect metabolic fluxes towards friulimicin production.Aretz, W.; Meiwes, J.; Seibert, G.; Vobis, G.; Wink, J., J Antibiot (Tokyo), 2000, 53, 807-815.7MEP012The catalytic and regulatory role of aconitase AcnA inStreptomyces viridochromogenes Tü494.E. MichtaUniversity of Tübingen, MicrobiologyBiotechnology, Tübingen, GermanyIn many organisms, aconitases have dual functions: they serve as primarymetabolisms enzymes in the tricarboxylic acide cycle and as regulators ofiron metabolism and oxidative stress response. Inactivation of theaconitase AcnA in Streptomyces viridochromogenes Tü494, the producerof herbicide antibiotic phosphinothricyl-alanyl-alanin (phosphinothricintripeptide=PTT), leads to strong defects in physiological andmorphological differentiation. This mutant (MacnA) fails in sporulationand antibiotic production which are characteristic secondary metabolismspecific properties of sreptomyces. Furthemore, AcnA, in addition to itscatalytic function, is capable of binding to iron responsive elements (IREs)thus altering the m-RNA stability in a similar mechanism described for theiron regulatory proteins (IRPs). A mutation preventing the formation of the[4Fe-4S] cluster of the aconitase (HisacnA1(C538A)) abolishes itscatalytic activity, but does not inhibit its RNA-binding ability. In contrast,HisacnA2(125-129)in which 5 highly conserved aminoacids of AcnA aredeleted shows an higher affinity to IREs than HisacnA. Furthermore,expression of HisacnA2 (125-129) instead of native acnA gene results in astrain that sporulates earlier and has increaseg PTT production than wild type.This correlates with the improved RNA-binding ability of HisacnA2(125-129). In silico analysis of the S. viridochromogenes genome revealed severalIRE-like structures e.g. upstream of recA gene, involved in the bacterial SOSresponse, ftsZ gene, required for the onset of sporulation in streptomyces. Thebinding of AcnA to these IREs is confirmed in gel shift assays. In conclusion,the demonstrated regulatory function of AcnA on the posttranscriptional levelprovides a new, so far unknown and unexploited form of regulation ofsecondary metabolism in streptomyces which might serve as possibility tooptimize antibiotic production.MEP013Metabolic engineering of Corynebacterium glutamicum for theproduction of -alanineJ.P. Krause* 1 , D. Rittmann 2 , A. Hadiati 1 , C. Ziert 1 , V.F. Wendisch 11 Uni Bielefeld, Genetics of Prokaryotes, Bielefeld, Germany2 Forschungszentrum Jülich, Institut für Bio- und Geowissenschaften,Jülich, Germany-alanine is commercially available as a nutrition supplement for athletesand is a possible intermediate for the fermentative production of acrylicacid. Here, we report about the metabolic engineering of Corynebacteriumglutamicum for the production of -alanine. Biomass formation andgrowth rate of C. glutamicum cultivated in glucose minimal media werenot altered by supplementation with up to 200 mM -alanine. Productionof -alanine with C. glutamicum was achieved by overexpression of theaspartate 1-decarboxylase gene panD. During growth in glucose minimalmedia -alanine accumulated in the culture supernatant of cellsoverexpressing panD, but not of the empty vector control strains. Toenhance production of -alanine the panBC-operon coding for 3-methyl-2-oxobutanoate hydroxymethyltransferase and pantoate--alanine ligase wasdeleted in C. glutamicum to avoid the drain of -alanine into thepantothenate/Coenzyme A-pathway, thereby causing a pantothenateauxotrophy. Deletion of panBC in C. glutamicum R127 led to a 12 % increaseof -alanine production. However, supplementation of the auxotrophic strainwith less than 3 M pantothenate resulted in decreased biomass formation andfavored production of -alanine over -alanine. -alanine occurred as abyproduct in all production experiments. To lower the byproduct formation thegene alaT coding for the main -alanine-synthesizing transaminase in C.glutamicum was deleted in combination with panBC. The resulting so far mostpromising strain C. glutamicum ATCC13032panBCalaT(pVWEx1-panD)produced 20 mM -alanine and 2 mM -alanine as byproduct from CGXIImedia with 4 % glucose as carbon and energy source.MEP014Secondary metabolites of fungi from the German Wadden SeaJ. Silber*, B. Ohlendorf, A. Erhard, A. Labes, J.F. ImhoffKieler Wirkstoff-Zentrum am GEOMAR, Marine Microbiology, Kiel, GermanyThe Wadden Sea forms an interesting habitat since it underlies permanentchanges due to the tidal influence. Fungi living in such an environmentpresumably need a high metabolic versatility in order to survive. Becausemetabolic versatility also may relate to secondary metabolite biosynthesis,fungal strains isolated from the German Wadden Sea were investigatedwith regard to secondary metabolite production. The 109 strains isolatedfrom sediments were grown under varying culture conditions, in shaken orstatic cultures and in different media. Cultures were extracted applyingliquid-liquid extraction, and extracts were analysed by HPLC-DAD/MS.The results displayed a strong influence of the media composition onmetabolite production. One of the fungal strains showed exceptionallyattractive metabolite profiles and was selected for detailed investigations.The structures of several of the purified compounds of this strain wereidentified by NMR spectroscopy as the known substances tricinonoic acid(Bashyal and Gunatilaka, 2010), 6-hydroxymellein, 6-methoxymellein(Dunn et al. 1979), orbuticin, 32-hydroxyorbuticin, antibiotic 15G256-2,15G256-2, and 15G256 (Schlingmann et al. 2002). More importantly,six new compounds were elucidated in structure and bioactivity assays ofthese substances exhibited antibacterial and cytotoxic properties with thepotential of possible biotechnological application.Bashyal, B.P., Gunatilaka, A.A.L. (2010). Tricinonoic acid and tricindiol, two new irregularsesquiterpenes from an endophytic strain of Fusarium tricinctum. Nat. Prod. Res. 24: 349-356Dunn, A.W., Johnstone, R.A.W., King, T.J.,Lessinger, L., Sklarz, B. (1979). Fungal Metabolites.Part 7. Structures of C25 Compounds from Aspergillus variecolor. J.C.S. Perkin I: 2113-2117Schlingmann, G., Milne, L., Carter, G.T. (2002). Isolation and identification of antifungalpolyesters from the marine fungus Hypoxylon oceanicum LL-15G256. Tetrahedron 58: 6825-6835MEP015Terpenoids from Corynebacterium glutamicumS.A.E. Heider*, M. Metzler, V. Erdmann, P. Peters-Wendisch, V.F. WendischUniversität Bielefeld, Faculty of Biology, Bielefeld, GermanyTerpenoids are the most diverse class of natural products comprising morethan 40,000 of structurally different compounds. They naturally occur inmicrobes, animals and a wide range of plant species, where terpenes oftenare produced as secondary metabolites. Terpenoids exert a huge variety ofbiochemical properties and physiological functions. Therefore theircommercial applicability is not fully explored. At present terpenoidproducts are used in cancer therapy, treatment of infectious diseases, cropprotection, food additives, flavors and cosmetics, but the large-scalechemical synthesis is often difficult or costly due to their structuralcomplexity and the isolation from the natural sources usually does notyield the desired quantities. For that reason the microbial biosynthesis is apromising approach for the production. Moreover, all terpenoids derivefrom the same universal precursor molecule isopenthenyl pyrophosphate(IPP) or its isomer dimethylallyl pyrophosphate (DMPP).In this work, the bacterium Corynebacterium glutamicum is analysed withrespect to the production of carotenoids, terpene pigments of greatcommercial interest. The Gram positive C. glutamicum is used for theannual production of more than 3,000,000 tons of amino acids. Thepredominant carotenoids in C. glutamicum are C 50-terpenedecaprenoxanthin and its glucoside. The yellow pigmented C. glutamicumpossesses a carotenogenic gene cluster for the complete pathway ofdecaprenoxanthin synthesis starting from the precursors IPP and DMPP. Aseries of single gene deletions verified the proposed pathway leading todecaprenoxanthin as the respective precursor carotenoids accumulatedwhich sometimes resulted in a changed cell color. Overexpression of dxs,encoding 1-deoxy-D-xylulose-5-phosphat synthase, the first enzyme of theendogenous non-mevalonate pathway, slightly enhanced accumulation oflycopene in -crtXYXYX mutant. The possible influence of accumulatingBIOspektrum | Tagungsband 2012