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

[1] Stegmann et al (2010): Glycopeptide biosynthesis in the context of basic cellular functions, Curr.Opin. Microbiol., 13: 595-602.[2] Wohlleben et al (2009): Chapter 18: Molecular genetic approaches to analyze glycopeptidebiosynthesis, Methods Enzymol, 458: 459-489.[3] Gallo et al (2010): Differentiell proteomic analysis reveals novel links between primarymetabolism and antibiotic production in Amycolatopsis balhimycina, Proteomics , 10:1336-1358.[4] Shawky, R. et al (2007): The border sequence of the balhimycin biosynthesis gene cluster fromAmycolatopsis balhimycina contains bbr, encoding a StrR-like pathway-specific regulator. J. Mol.Microbiol. Biotechnol., 13:76-88.GWP051Glycerol 3-phosphatase from Corynebacteriumglutamicum: evidence for the first bacterial glycerol 3-phosphataseS.N. Lindner, M. Panhorst, T.M. Meiswinkel*, J.-W. Youn, V.F. WendischGenetic of Prokaryotes (Bio VI), University of Bielefeld, Bielefeld, GermanyCorynebacterium glutamicum is a well known amino acid producer which isused e.g. to produce over 1.3 million tons of L-lysine per year. Duringgrowth and amino acid production glycerol may be formed as a byproduct.The C. glutamicum genome encodes a putative glycerol 3-phosphatase(GPP). Here, we characterize this enzyme as the first of its kind described inprokaryotes. Among the substrates tested, GPP was solely active withglycerol 3-phosphate. The enzyme prefers conditions of neutral pH andrequires Mg 2+ or Mn 2+ for its activity. The maximal activity of GPP wasestimated to be 0.67 U/mg and GPP possesses a K M of 2.9 mM for glycerol3-phosphate. Phenotypically, slow growth of the recombinant C. glutamicum(pVWEx1-glpFK) in the presence of glycerol, which is due to intracellularglycerol 3-phosphate accumulation, could be alleviated by gppoverexpression with concomitant reduction of the intracellular glycerol 3-phosphate concentration. GPP was shown to be involved in utilization ofglycerol 3-phosphate as sole source of phosphorus, since growth in glucoseminimal medium with glycerol 3-phosphate as sole phosphorus source wasreduced in the gpp deletion strain, but accelerated when gpp wasoverexpressed. Since glycerol formation during growth in fructose minimalmedium could be abolished by deletion of gpp, but was increased asconsequence of gpp overexpression, GPP plays a major role in formation ofglycerol as by-product in C. glutamicum.GWP052Metabolic engineering of Corynebacterium glutamicumfor the production of L-aspartateC. Ziert*, V.F. WendischGenetics of Prokaryotes, University of Bielefeld, Bielefeld, GermanyCorynebacterium glutamicum is used biotechnologically for the productionof L-glutamate and L-lysine, which are used as food and feed additives, butstrains for other products such as 1,4-diaminobutane and 1,5-diaminopentane have also been developed (1). The amino acid L-aspartate isused for the sweetener aspartame, and its derivatives are promisingprecursors for polymer synthesis. Therefore, we are aiming on engineeringC. glutamicum for producing L-aspartate.In bacteria, L-aspartate is the precursor for the amino acids threonine,isoleucine, methionine and lysine and for the cell wall precursordiaminopimelate. The first step of conversion of aspartate is catalyzed byaspartokinase (encoded by lysC) (2). Deletion of lysC is not possible asdiaminopimelate is essential for growth. A downregulation of the gene viapromoter exchange appears to be more promising and is thought to lead toan accumulation of L-aspartate.Synthesis of L-aspartate from oxaloacetate depends on transamination e.g.by glutamate-aminotransferase (aspB). Oxaloacetate supply can be improvedby enhancing the expression of the genes coding for the anapleroticphosphoenolpyruvate carboxylase (ppc) and pyruvate carboxylase (pyc).Deletion of the aspartate-α-decarboxylase (panD) avoids conversion of L-aspartate, but yields a pantothenate auxotrophy. Limiting pantothenatesupplementation indirectly improves oxaloacetate supply as flux via thepyruvate dehydrogenase complex is reduced and carboxylation of pyruvateor PEP may be enhanced.L-aspartate may also be synthesized by reductive amination of fumarate.Heterologous expression of aspartate ammonia lyase genes (aspA) may yieldL-aspartate if the level of fumarate is increased due to deletion of the genecoding for the fumarate hydratase (fumC) (3). Recombinant aspartase genesfrom Escherichia coli and Bacillus subtilis were expressed in C. glutamicumand high specific activities were obtained. Expression of the aspartase genesin C. glutamicum wild type did not lead to an accumulation of aspartate insupernatants of batch cultures unless the cells were supplied with sufficientamounts of fumarate and nitrogen.[1] Schneider J. and VF. Wendisch (2010): Putrescine production by engineered Corynebacteriumglutamicum. Appl Microbiol Biotechnol. 88:859-68.[2] Cremer, J. et al (1991): Control of the lysine biosynthesis sequence in Corynebacteriumglutamicum as analyzed by overexpression of the individual corresponding genes. Appl. Microbiol.Biotechnol. 43: 482-488.[3] Menkel, E. et al (1989): Influence of Increased Aspartate Availability on Lysine Formation by aRecombinant Strain of Corynebacterium glutamicum and Utilization of Fumarate. Appl EnvironMicrobiol. 55: 684-688.GWP053Production of rhamnolipids in Pseudomonas putida byheterologous expression of rhl-genes from Pseudomonasaeruginosa PA01A. Wittgens* 1 , L.M. Blank 2 , B. Santiago-Schuebel 3 , S. Wilhelm 1 ,F. Rosenau 41 Institute for Molecular Enzyme Technology, Research Center Jülich,Jülich, Germany2 Laboratory of Chemical Biotechnology, University of Technology,Dortmund, Germany3 Central Division of Analytical Chemistry / BioSpec, Research CenterJülich, Jülich, Germany4 Institute of Pharmaceutical Biotechnology, University of Ulm, Ulm,GermanyRhamnolipids are biosurfactants with great potential for applications inindustry. They can be used as detergents in laundry products, dish washingliquids or as emulsifiers in cosmetics and foods. Furthermore they exhibitantimicrobial properties. The production of rhamnolipids is wellcharacterized for the opportunistic human pathogen P. aeruginosa. They arecomposed of one or two rhamnose-molecules linked to hydroxyfatty acidswith different chain length between C 8 and C 12. The biosynthesis occurs inthree steps: RhlA is responsible for the production of 3-(3-hydroxyalkanoyloxy)alkanoates (HAAs) by linking two hydroxyfatty acidstogether. RhlB bonds a rhamnose-molecule to HAA and creates monorhamnolipid.The genes rhlAB are arranged as an operon. RhlC isresponsible for the production of di-rhamnolipid and bonds a secondrhamnose to the mono-rhamnolipid. The rhlC gene forms an operon togetherwith the gene PA1131, which function is still unknown.However, especially for the uses of rhamnolipids in the cosmetic and foodindustry it is problematical when they are produced in a human pathogen.We here present the production of both mono- and di-rhamnolipids byheterologous expression using the non-pathogenic strain P. putida KT2440as a host. Moreover, P. putida is able to grow with high concentrations ofrhamnolipid (>100 g/L) in the culture media. The production in P. putida ispossible, because precursors for rhamnolipid production are available in thisbacterium. For the heterologous expression we cloned the rhlAB operon aswell as the rhlC gene as single or in combination in the pVLT33 expressionvector. Expression of rhlAB in P. putida resulted in the production of monorhamnolipidand if all three genes were expressed, P. putida is also able toproduce di-rhamnolipids. Characterization of rhamnolipids produced by P.putida via HPLC-ESI-MS showed that it produced the same species like theP. aeruginosa wild type.GWP054Biological Hydrogen Production with MicroalgaeM. MorweiserTechnical Biology, Karlsruhe Institute of Technology (KIT), Karlsruhe,GermanyMicroalgae are capable of producing a variety of products, includingcompounds of high energy content such as sugars, lipids and hydrogen. Thelatter is becoming a product of interest in the context of renewable energyproduction. Hydrogen may be used as energy carrier, ready for powergeneration in fuel cells for mobile and stationary applications.Certain strains of microalgae are long known to produce hydrogen underanaerobic conditions [2]. The oxygen inhibited hydrogenase recombineselectrons from the chloroplast electron transport chain with protons to formhydrogen directly inside the chloroplast. Melis and coworkers obtained asustained hydrogen production under low sulfur conditions inChlamydomonas reinhardtii [4], reducing the net oxygen evolution andthereby overcoming the inhibition of the hydrogenase. The development ofspecially adapted hydrogen production strains led to higher yields andoptimized biological process preconditions [1, 3].spektrum | Tagungsband 2011