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

phenol content (23% and 55% respectively) in combination with acharacteristic shift of the fragmentation pattern of water-soluble aromatics.Our results show that the phenol-rich biopolymer stimulates the secretion ofextracellular fungal biocatalysts (e.g. MnP), which finally leads to adecomposition effect of agricultural waste material suitable for agriculturalpurposes.GWP036Heterologous production of the non-proteinogenic aminoacid L-pipecolic acid in Corynebacterium glutamicumN. Wagner, N. Fischer*, A. Steinkämper, R. Biener, D. SchwartzApplied Sciences/Biotechnology, University of Applied Sciences, Esslingen,GermanyThe non-proteinogenic amino acid L-pipecolic acid is found as a buildingblock in the structure of many microbial secondary metabolites such as theimmunosuppressants rapamycin and FK506 or the antibiotics pristinamycinand friulimicin [1]. Due to its ability to introduce reverse turns in peptides,pipecolic acid increases the stability and potency of such compounds. Thenon-racemic production of the amino acid is catalyzed by lysinecyclodeaminases such as the Pip protein in the friulimicin biosynthesis (2).The pip gene of the friulimicin biosynthetic gene cluster was heterologouslyexpressed both in Escherichia coli and different strains of the industriallysine producer Corynebacterium glutamicum (wildtype, DlysE mutant,DM1730). The functionality of corresponding His- and MalE-taggedrecombinant enzymes was shown by the detection and quantification of L-pipecolic acid production by thin layer chromatography. The best result wasfound in C. glutamicum DM1730 carrying the MalE-tagged Pip protein. Dueto its positive characteristics, L-pipecolic acid represents a useful buildingunit for production of bioactive natural or synthetic peptides. According toour results, C. glutamicum DM1730 seems to be a suitable heterologous hostfor a prospective biotechnological production of this unusual amino acid.[1] Vertesy et al. (2000), J. Antibiot (Tokyo). 53, 816-827.[2] Müller et al. (2007), Antimicrob Agents Chemother. 51, 1028-1037.GWP037Enzymatic and chemical modification of biosurfactantsM. Gerlitzki* 1 , V. Recke 2 , M.M. Müller 1 , R. Hausmann 1 , C. Syldatk 1 ,S. Lang 21 Technical Biology, Karlsruhe Institute of Technology (KIT), Karlsruhe,Germany2 Institute for Biochemistry and Biotechnology, Department ofBiotechnology, University of Technology, Braunschweig, GermanyThere has been an increasing interest in biologically produced surfactantssuch as Sophorolipids and Rhamnolipids. Rhamnolipids are produced byPseudomonas aeruginosa when grown on glycerol, triglycerides or n-alkanes. Sophorolipids are produced by Candida bombicola in high yields[1]. These substances are able, e.g., to enhance the biodegradation ofhydrocarbons in soil [2].In this study we are interested in modifying microbial glycolipids in order toget additional interesting properties such as improved surface/ interfacialactivity or bioactivity.Starting with these biosurfactants we try to achieve the sophorose, dirhamnoseand mainly the uncommen fatty acids by hydrolysis. Herefore, wewant to use chemical hydrolysis to get the β-hydroxydecanoic (RL) acid and17-hydroxyoctadecenoic (SL). Enzymatic hydrolysis will be used for the 3-(3-hydroxydecanoyloxy) decanoic acid. These first products shall be used asbuilding blocks for the syntheses of new glycolipids using variousglycosidases and/or lipases to show if the special surface/interface activityand bioactivity is founded in the fatty acids or in the unusual sugars.The new glycolipids will be purified and afterwards characterizedconcerning their molecular structures (NMR, mass spectrometry, elementalanalysis). Additionally, we plan to determine their antimicrobial and otherbioactive properties, e.g. anti-tumor promoting activity (in cooperation withH. Tokuda, Kanazawa University, Japan).[1] Daniel, H-J. et al (1998): Production of sophorolipids in high concentration from deproteinizedwhey and rapeseed oil in a two stage fed-batch process using Candida bombicola ATCC 22214 andCryptococcus curvatus ATCC 20509. Biotechnol. Lett. 20: 1153-1156.[2] Kang, S-W. et al (2009): Enhanced biodegradation of hydrocarbons in soil by microbialbiosurfactant, sophorolipid. Appl. Microbiol. Biotechnol. DOI10.1007/s12010-009-8580-5.GWP038Three Novel Thermostable Lipases from DifferentMetagenomes Ranging from Soil Enrichments toHydrothermal VentsJ. Chow* 1 , C. Vollstedt 1 ,M.Perner 1 , O. Thum 2 , W. Streit 11 Microbiology and Biotechnology, University of Hamburg, Hamburg,Germany2 Evonik Goldschmidt GmbH, Biotechnology Research, Essen, GermanyMetagenomics reveal culture-independent insights into microbes´ diversityand the enzymes they feature [1, 3]. Lipolytic enzymes, namelycarboxylesterases (EC 3.1.1.1) and triacylglycerol lipases (EC 3.1.1.3),catalyze both hydrolysis and synthesis reactions on a broad spectrum ofsubstrates at various conditions rendering them especially suitable forbiotechnological applications. Most lipases used today originate frommesophilic organisms and are susceptible to thermal denaturation (Levissonet al. 2009). Here we report on the identification of novel thermostablearchaeal and bacterial lipases from three different microbial communities.Our metagenomic libraries were constructed from an enrichment usingheating water as inoculum, a long term soil enrichment culture and a deepseahydrothermal vent-derived enrichment. Cultures were maintained at 65°to 70°C and microbial communities characterized on a phylogenetic levelbased on 16S rRNA genes. Mainly thermophilic Firmicutes were identifiedin the soil enrichment after several months of incubation, while the heatingwater culture contained mostly novel Thermales. The hydrothermal ventculture consisted predominantly of archaeal species that are closely relatedto Thermococcales. The metagenomic libraries constructed from thedesignated enrichments comprised 800 to 8,500 clones. Screening of thelibraries on pNP-substrates (C 4 and C 12) at temperatures between 50°C and70°C resulted in the identification of 15 lipolytically active clones. Untilnow, three enzymes, LipS, LipT and LipZ have been expressedrecombinantly in E. coli and in P. antarctica and have been characterizedbiochemically. Current studies show a half life time of up to 48 h at 70°C(LipS) and 50 min at 90°C (LipZ). The temperature optima ranged between70°C (LipS) and 100°C (LipZ). All three enzymes are able to catalyze thehydrolysis of long-chain fatty acid esters like pNP-palmitate (C 16), -stearate(C 18) and -oleate (C 18:1; LipT), indicating lipase activity. Current workfocuses on further biochemical characterization with unusual substrates andsynthesis reactions in organic solvents as well as crystallographic analyses.[1] Handelsman, J. et al (1998): Molecular biological access to the chemistry of unknown soilmicrobes: a new frontier for natural products. Chem Biol 5(10): R245-9.[2] Levisson, M. et al (2009): Carboxylic ester hydrolases from hyperthermophiles. Extremophiles13(4): 567-81.[3] Steele, H. L. et al (2009):. Advances in recovery of novel biocatalysts from metagenomes. J MolMicrobiol Biotechnol 16(1-2): 25-37.GWP039Obtaining and selection haploids of distillery yeastsP. Patelski*, M. Balcerek, K. Pielech-Przybylska, J. Szopa, P. DziuganInstitute of Technology Fermentation and Microbiology, Biotechnology andFood Sciences, Technical University of Lodz, Lodz, PolandHaploidization is a crucial step during obtaining yeast strains with improvedtechnological properties by means of yeast sexual hybridization. This naturalmethod improving of industrial strains of yeast is used for over 60 years.Aim: The aim of this study was to obtain and isolate haploid cultures ofdifferent Saccharomyces cerevisiae strains, also evaluate them as a possiblestrains for conjugation and hybrids selection to obtain a new distillery yeastsfor fermentation of concentrated broths prepared from sugar beet juices.Methods: 9 strains of S.cerevisiae from our Pure Cultures Collection wereused in experiments: PA1, PA2, PA3, PS2, PS3, M2, M3, OH2, and BC16.2 strains: S.cerevisiae Ma and S.cerevisiae Mα - stable haploid markers withknown mating type were also used for mating type assay. Presporulationmedium containing sodium pyruvate, glucose, yeast extract, bacto-peptonewas used. Modified McClary medium: potassium acetate 10g/L, yeastextract 2,5g/L, glucose 0,2g/L, agar 25g/l, was used for sporulation. Haploidclones were obtained according to the procedure Johnston and Mortimerusing enzymes from Helix pomatia to asci walls digestion. Single sporeswere isolated from tetrads by using micromanipulator with glass needle.Yeast colonies grown out of individual spores were transferred to YPGslants.The criteria in selecting parent strains and their haploid clonesobtained from the spores were: morphological features, ability to fermentand assimilate selected sugars, ability to assimilate glycerol as well asfermentation of 20 and 25°Blg broths prepared from concentrated sugar beetjuice.spektrum | Tagungsband 2011