Das Projekt "Genmanipulation des anaeroben Zymomonas mobilis fuer die Fermentierung von Staerkeabfaellen" wird vom Umweltbundesamt gefördert und von Forschungszentrum Jülich GmbH durchgeführt. Objective: Zymomonas bacteria could offer considerable potential for improved alcohol fermentation in comparison with yeasts. An efficient expression vector is, however, an absolute requisite to open the possibility of genetically engineered zymomonas. The bacteria could then be made able to use for example renewable carbon substrates abundant in agriculture wastes. General Information: An efficient expression vector shall be constructed for zymomonas mobilis. For this, the promotor of the pyruvate decarboxylase gene will be isolated, sequenced and built into a broad host range plasmid. Genes for the degradation of amylose (from aspergillus awamorii) or xylose (from klebsiella) shall be placed under control of this strong promoter and expression will be tested in suitable escherichia coli strains. Afterwards, these recombinant plasmids shall be introduced into zymomonas mobilis cells and their expression and stability will be assayed. The long range aim is to study the recombinant strains for continuous ethanol production from agricultural waste substrates as starch and hemicellulose (xylose). Achievements: Zymomonas mobilis is a Gram negative anaerobe producing ethanol 5 to 6 times faster than yeast. It can use only glucose, fructose and sucrose as carbon sources. Therefore, applications of Z mobilis at the industrial level require extention of its spectrum of usable carbohydrates. Genetic improvement was approached through formal genetics and genetic engineering. Chemical mutagenesis, transposon mutagenesis, gene transfer by aided conjugation, construction of recombinant expression vectors and transfer into Z mobilis genes responsible for xylose metabolism were established and can now be applied to increase the substrate range of Z mobilis. Recombinant plasmids were constructed by subcloning fragments of native Z mobilis plasmids in known vectors. These were transferred into Z mobilis by aided conjugation and were stably inherited, especially after the loss of native plasmids. Subcloned Z mobilis plasmid sequences were used to construct new expression vectors, which could be transferred into Z mobilis and induce expression of the xylose catabolism genes xylA and xylB. However, the engineered strains could not ferment xylose. Transformation of Z mobilis by plasmid deoxyribonucleic acid (DNA) occurred at low frequencies only when sequences of incoming plasmid were integrated into the host's genome. Transposon mutagenesis was accomplished by conjugal transfer of transposon bearing plasmids from Pseudomonas, as well as Escherichia coli donors to Z mobilis. All Z mobilis strains tested were able to ferment extracts of various fruits, such as apples, oranges, peaches, watermelons, etc.
Das Projekt "Veraenderung des Biosynthesespektrums zellwandlytischer Enzyme bei Pilzen durch homologe und heterologe Transformation" wird vom Umweltbundesamt gefördert und von Technische Universität Berlin, Fachbereich 15 Lebensmittelwissenschaft und Biotechnologie, Wissenschaftsbereich Mikrobiologie durchgeführt. Veraenderte Enzymbildung zellwandlytischer Pilze kann durch Veraenderungen auf Transkriptions-, Translations- oder Sekretionsebene verursacht sein. Ziel des Projektes ist es, durch Amplifikation vorhandener Gene und Transformation mit heterologen Genen die Enzymbildung gezielt zu beeinflussen. Dabei sind die Auswirkungen zusaetzlicher Gene bzw die Ausschaltung eigener Gene durch Insektionsinaktivierung auf die Expressionskapazitaet des von Penicillium janthinellum gebildeten zellwandabbauenden Enzymkomplexes von besonderem Interesse. Dazu soll ein wirksamer Expressionsvektor auf der Basis der Nourseothricin-Resistenz als dominantem Selektionsmarker konstruiert und in Verbindung mit Expressionskassetten fuer einzelne Gene zellwandlytischer Enzyme eingesetzt werden. Transformationsereignisse und Stabilitaet von Transformanten sollen ua durch Genomanalyse mittels Puls-Feld-Elektrophorese untersucht werden.