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

[2] Mohebali, G. & A. S. Ball (2008): Biocatalytic Desulfurization (BDS) of petrodiesel fuels.Microbiol. 154, 2169-2183.[3] Oldfield, C. et al (1997): Elucidation of the metabolic pathway for dibenzothiophenedesulfarization by Rhodococcus sp. Strain IGTS8 (ATCC 53968). Microbiol. 143, 2961-2973.GWP024Identification and characterization of a 1,3-propanedioloxidoreductase from Pectobacterium atrosepticumS. Elleuche*, B. Klippel, G. AntranikianUniversity of Technology, Hamburg, GermanyThe compound 1,3-propanediole (1,3-PD) is a valuable chemical forpolyester production used in textile fiber, film and plastic industry. It isroutinely converted from acrolein or ethylene oxide via 3-hydroxypropionaldehyde (3-HPA) using chemical approaches. Since thechemical synthesis of 1,3-PD produces toxic intermediates and is highlyexpensive, much effort has been spent on its microbiological production. Innature, anaerobic microbial metabolism of glycerol involves a reductivepathway, enabling the NADH-dependent formation of 1,3-PD and a coupledoxidative pathway, which generates the reducing power for the reductivebranch. In a first reaction step, the conversion of glycerol to 3-HPA iscatalyzed by the enzyme glyceroldehydratase. Finally, 1,3-propanedioloxidoreductase (PDOR) reduces 3-HPA to 1,3-PD.To identify biocatalysts with novel properties for the production of 1,3-PD,we performed BLAST searches using the sequences of PDOR from bacteriaof the genera Citrobacter, Clostridium and Klebsiella, which are known toconvert glycerol to 1,3-PD. In addition, the sequence of the gene yqhD fromEscherichia coli was used as query. This open reading frame encodes aNADPH-dependent aldehyde reductase capable of catalyzing the formationof 1,3-PD from 3-HPA. Homologues of PDOR and YqhD were identified inthe genome of the facultative anaerobic bacterium Pectobacteriumatrosepticum. Both genes were cloned into pQE30 and pQE80 expressionvectors and were purified after heterologous production in E. coli. Resultson the characterization of the enzyme, including physicochemical andkinetic properties, will be presented.GWP025Expression of metagenomic membrane-bounddehydrogenases from acetic acid bacteria: The design ofnew oxidative catalysts.B. Peters*, D. Kostner, M. Mientus, A. Junker, W. Liebl, A. EhrenreichDepartment for Micriobiology, Technical University Munich, Freising,GermanyAcetic acid bacteria are used in biotechnology due to their ability toincompletely oxidize a great variety of carbohydrates, alcohols and relatedcompounds in a regio- and stereo-selective manner. Most of these reactionsare catalyzed by membrane-bound dehydrogenases with a broad substraterange.Acetic acid bacteria contain a multitude of such dehydrogenases and manyof them cannot be grown as pure cultures. Therefore we expect habitats richin acetic acid bacteria, such as a mother of vinegar to be good sources ofuncharacterized metagenomic dehydrogenases of potential value forbiotechnology. We investigated the diversity of several mothers of vinegarby 16S rDNA sequencing as a preparation to construct metagenomiclibraries.The metagenomic membrane bound dehydrogenases will be screened bysequence similarity and functionally expressed in tailor made Gluconobacteroxydans strains devoid of their own dehydrogenases to avoid overlappingspecificities. Expression in an acetic acid bacterium should facilitatefunctional integration in the membrane physiology of these organisms.To achieve this goal we developed a clean deletion system forGluconobacter strains based on 5' fluorouracil counter selection. Thissystem was used to delete various genes. Furthermore we developed theshuttle vector (E. coli-G. oxydans) system pKOS4 that is needed for theexpression of metagenomic dehydrogenases controlled by naturalconstitutive and inducible promoters of such enzymes.As currently very little is known about the promoters of membrane-bounddehydrogenases we investigated the regulation and transcription start pointof different dehydrogenases in G. oxydans 621H.GWP026Modification of the fatty acid composition of the bacterialmembrane of Rhodobacter capsulatusN. Katzke*, V. Svensson, K.-E. Jaeger, T. DrepperInstitute of Enzyme Biotechnology Heinrich-Heine-University, Jülich,GermanyUnsaturated or functionalized fatty acids are used for a multitude ofbiotechnological applications. As these compounds can only be found intrace amounts in their natural source and chemical synthesis is notefficiently feasible, they are usually produced via biocatalytic processes. Toproduce fatty acids with potentially high biological activity, fatty acidmodifyingenzymes are widely used for their functionalization. However,enzymatic functionalization of fatty acids is mostly limited by theavailability of substrates as many fatty acid modifying enzymes onlyspecifically convert defined acyl chains of membrane phospholipids.Recently we developed a novel expression system which is based on thephotosynthetic bacterium R. capsulatus [1-3]. In contrast to standardexpression hosts, R. capsulatus is particularly suited for the heterologousexpression of membrane proteins because it forms an extensive system ofintracytoplasmic membranes (ICM) during phototrophic growth. Since ICMformation basically allows accommodation of heterologous membraneproteins as well as efficient storage of phospholipids, we now tested if thefatty acid composition of the bacterial membrane can be modified in order tobiotechnologically produce functionalized fatty acids in high amounts. Herewe demonstrate that fatty acids of different chain length and degree ofunsaturation that have been supplemented to the growth medium wereefficiently integrated into the R. capsulatus membrane. Furthermore, theincorporation efficiency of foreign fatty acid could be significantly increasedby specific inhibition of the biosynthesis of endogenous unsaturated fattyacids.[1] Drepper, T. et al (2008): Verfahren und Vektor zur heterologen Genexpression, Patent applicationDE 10 2013 2304.2003, 2008.[2] Katzke N, Arvani S, Bergmann R, Circolone F, Markert A, Svensson V, Jaeger KE, Heck A,Drepper T: A novel T7 RNA polymerase dependent expression system for high-level proteinproduction in the phototrophic bacterium Rhodobacter capsulatus. Protein Expr Purif. 2010;69(2):137[3] Katzke, N.et al (2010): High-level gene expression in the photosynthetic bacterium Rhodobactercapsulatus. Methods Mol Biol.; in press.GWP027A pathway transfer system that facilitates theheterologous expression of large gene clusters in a broadrange of bacterial hostsA. Loeschcke* 1 , A. Markert 2 , K.-E. Jaeger 1 , T. Drepper 11 Institute for Molecular Enzyme Technology (IMET), Heinrich-Heine-University, Research Center Jülich, Jülich, Germany2 Radiology Department, University Hospital, Heidelberg, GermanyTo access valuable natural substances synthesized by microorganisms it isnecessary to establish complex biosynthetic pathways in heterologousbacterial hosts. However, several limitations associated with cloning,transfer, stable maintenance and functional expression of all pathway genesretain this process challenging. In order to overcome these limitations wedeveloped a novel biosynthetic pathway transfer and expression system,which facilitates the expression of unmodified large gene clusters indifferent heterologous hosts.The novel in vivo auto cloning and expression system (IVAC) consists oftwo different cassettes, named L- and R-IVAC. The two cassettes comprisestructural elements allowing (i) the conjugational transfer of large DNAfragments encompassing all genes of interest into the expression host, (ii)the integration of the IVAC-labelled gene cluster into the host chromosomevia transposition, and (iii) the expression of all target genes irrespective oftheir orientation and natural DNA elements that might affect theircoordinated expression in the respective host strain.Using a carotenoid biosynthetic gene cluster we could demonstrate that theIVAC-system is a powerful tool that allows the concerted functionalexpression of clustered genes in different bacterial hosts.spektrum | Tagungsband 2011