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

From these results, we conclude that the squalene-hopene cyclase ZmSHC1from Z. mobilis has a high bio-catalytic potential for a large variety ofindustrial applications.[1] Reipen, I. et al (1995): Microbiology, 141: 155-161.[2] Abe, I. et al (1993): Chem. Rev., 93: 2189-2206.[3] Wendt, K. (2000): Angew. Chem., 112: 2930-2952.[4] Neumann, S. and H. Simon (1986): Biol. Chem. Hoppe-Seyler, 367: 723-729.GWV016Rhamnolipids- Green Surfactants Based on RenewablesM.M. Müller*, B. Hörmann, C. Syldatk, R. HausmannProcess Engineering in Life Sciences, Karlsruhe Institute of Technology(KIT), Karlsruhe, GermanyBiosurfactants are green alternatives to conventional chemically-synthesizedsurfactants. Rhamnolipids are glycolipids with good physico-chemicalproperties. However, the main obstacles towards an economic productionare low productivity, high raw-material costs, relatively expensivedownstream processing, excessive foaming problems and a lack ofunderstanding the rhamnolipid regulation in bioreactor systems.A recent study shows that the sequenced Pseudomonas aeruginosa strainPAO1 is able to produce high quantities of up to 40 g/L of rhamnolipidduring 30 L batch bioreactor cultivations with sunflower oil as sole carbonsource [1]. The rhamnolipid production capacity of PAO1 was evaluated incomparison with the over-producer strains DSM7108 and DSM2874 [2].PAO1 not only seems to be an appropriate model, but surprisingly has thepotential as a strain of choice for actual biotechnological rhamnolipidproduction.The induction of rhamnolipid biosynthesis is known to be quorum sensingregulated. Nevertheless little is understood about the up- and downregulationof rhamnolipid production. In 30L bioreactor cultivations ofP.aeruginosa PAO1 the specific rhamnolipid productivity shows a distinctmaximum correlated with the transition of the logistic growth to a stationaryphase. In contrast to well-established production strains, PAO1 allowsknowledge-based „systems biotechnological” process developmentcombined with the frequently used heuristic bioengineering approach. ThusPAO1 could be an appropriate model for rhamnolipid production in pilotplant bioreactor systems. In a current project different fed-batch cultivationstrategies and an optimized down-stream procedure are evaluated. The aimis to develop an economic cultivation process where the cells are kept athigh levels of specific rhamnolipid productivity.[1] Müller, M.M. et al (2010): Pseudomonas aeruginosa PAO1 as a model for rhamnolipid productionin bioreactor cultivations. Appl Microbiol Biotechnol 87: 167 – 174.[2] Müller, M.M. et al (2010): Evaluation of rhamnolipid production capacity of Pseudomonasaeruginosa PAO1 in comparison to the rhamnolipid pver-prducer strains DSM7108 and DSM2874.Appl Microbiol Biotechnol Published Online. DOI: 10.1007/s00253-010-2901-z.GWV017Regioselective hydroxylation of medium-chain n-alkanesand primary alcohols by CYP153 enzymesD. Scheps*, S. Honda Malca, B. Nestl, B. HauerInstitute of Technical Biochemistry, University of Stuttgart, Stuttgart,GermanyCytochrome P450 monooxygenases are a very large and diverse superfamilyof heme-containing proteins found in all domains of life. The enzymescatalyze a variety of reactions including hydroxylation of C-H bonds,heteroatom oxygenation, heteroatom release (dealkylation), oxidativedeaminations, dehalogenations, desaturations and epoxide formation [1].They have long been the focus of biochemists because of their interestingability to introduce a single oxygen atom from O 2 into an organic substrate,that results in an one-step synthesis of complex molecules. Most of theseenzymes only work as part of a multiprotein complex with redox partnersproviding electrons from NADH cofactors to the heme domain.Selectively hydroxylated hydrocarbons are of great interest in the chemicalindustry, due to their role as intermediates for the synthesis of bulk and finechemicals. The selective terminal hydroxylation of alkanes is stillproblematic and there is to date no efficient chemical strategy to direct theintroduction of hydroxyl groups on primary non-activated C-H bonds.CYP153 enzymes are such enzymes catalyzing the terminal hydroxylationof aliphatic, alicyclic and alkyl-substituted compounds with high regio- andstereoselectivity under mild reaction conditions [2]. Two CYP153Aenzymes were cloned and expressed in Escherichia coli. The activity of eachP450 was reconstituted with artificial electron transfer partners [3]. TheCYP153A enzymes were assayed in vitro with purified proteins using C 5-C 12n-alkanes and C 6-C 12 primary alcohols as substrates.This work was performed within the "Systembiologie in Pseudomonas fürdie industrielle Biokatalyse" and financial support by the BMBF isgratefully acknowledged.[1] Isin, E.M. and F.P. Guengerich (2007): Biochim Biophys Acta. 1770, 314-329.[2] van Beilen, J.B. et al (2006): Appl Environ Microbiol.72, 59-65.[3] Koga, H. et al (1989): J Biochem. 106, 831-836.GWV018Whole-cell biotransformation for the stereospecifichydroxylation of the incompatible solute guanidinoectoineA. Meffert*, E.A. GalinskiInstitute for Microbiology and Biotechnology, Friedrich-Wilhelms-University, Bonn, GermanyThe Fe(II)/α-ketoglutarate-dependent ectoine hydroxylase (EctD) from thehalotolerant Halomonas elongata uses, besides ectoine, a broad spectrum ofunusual cyclic substrates, including synthetic ectoine derivatives [1]. In thisstudy, we investigated whether the enzyme is able to hydroxylate the socalled incompatible solute guanidino-ectoine (2-amino-3,4,5,6-tetrahydro-4-pyrimidine-carboxylic acid). During salt stress E. coli accumulates thischemically synthesized substrate via the osmotically induced proP and/orproU transport systems. But in contrast to the compatible version ectoine,guanidino-ectoine displayed a negative effect on the growth rate of E. coli[2]. In order to supply and investigate the hydroxylated form of guanidinoectoine,we employed a whole-cell biotransformation system in E. coliBL21, which is based on heterologous expression of the ectD-gene from H.elongata [3]. This biotransformation system enabled a 100% conversion ofguanidino-ectoine into its 5-hydroxy derivative (2-amino-5-hydroxy-3,4,5,6-tetrahydro-4-pyrimidine-carboxylic acid). The hydroxylated version itselfproved inhibitory to cellular metabolism, and therefore represents a newaddition to the group of cyclic incompatible solutes.[1] Stein, M. (2008): Whole-cell biocatalysis for the stereospecific hydroxylation of cyclic compatiblesolutes. Poster VAAM.[2] Sell, K. et al (2011): Guanidino-ectoine: a new member of the incompatible solute family. PosterVAAM.[3] Galinski, E.A. et al (2007): Stereospezifische Hydroxylierung von kompatiblen Soluten. DE 102007 052 900 A1, 07.11.2007; PCT/EP 2008/009414.GWV019Catalytic biofilms: Real time solvent tolerance analysis ofPseudomonas sp. strain VLB120ΔC and profiling of EPSmatrixB. Halan*, A. Schmid, K. BuehlerLaboratory of Chemical Biotechnology, Department of Biochemical andChemical Engineering, University of Technology, Dortmund, GermanyBiofilms are ubiquitous surface associated microbial communities, whichare embedded in an extra cellular polymeric matrix (EPS) responsible forbiofilm structural integrity and strength. Biofilm grown cells exhibitenhanced tolerance towards adverse environmental stress conditions (e.g.antimicrobial substances, toxic chemicals and heavy metals) and thus therehas been a growing interest in the recent years to use biofilms forbiotechnological applications [1]. We present a time and loci resolved, noninvasive,quantitative approach to study biofilm development and itsresponse to the toxic solvent styrene. Pseudomonas sp. strain VLB120ΔC-BT-gfp1, was grown in modified flow cell reactors and exposed to thesolvent styrene. Biofilm grown cells displayed stable catalytic activityproducing (S)-styrene oxide continuously during the experimental period.The pillar like structure and growth velocity of the biofilm was notinfluenced by the presence of the solvent. However, the cells experiencesevere membrane damage during the styrene treatment, although they areobviously able to adapt to the solvent as the amount of permeabilized cellsdecreased from 80% to 40% in 48 hours. Concomitantly the fraction ofConA stainable polysaccharides increased, substantiating the assumptionthat those polysaccharides play a major role in structural integrity andenhanced biofilm tolerance towards toxic environments. Compared tocontrol experiments with planktonic grown cells, the Pseudomonas biofilmadapted much better to toxic concentrations of styrene, as nearly 65% ofbiofilm cells were not permeabilized (viable) as compared to only 7% inanalogous planktonic cultures [2]. Preliminary biochemical profiling of thespektrum | Tagungsband 2011