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GENETIC MANIPULATION OF THE ANAEROBE ZYMOMONAS MOBILIS FOR FERMENTATION OF FRUIT JUICES

Objective

EXPLORATION OF THE POSSIBILITY OF USING GENETIC MANIPULATION TO IMPROVE THE USE OF AGRICULTURAL WASTES FROM PEACHES, ORANGES, APPLES, SUGAR BEETS FOR LARGE SCALE PRODUCTION OF ETHANOL BY USING THE ANAEROBE BACTERIA ZYMOMONAS MOBILIS.

TO EXPLORE THE APPLICABILITY OF ZYMOMONAS MOBILIS FOR INDUSTRIAL FERMENTATIONS PRODUCING HIGH RATES AND YIELDS OF ETHANOL WITHOUT COSTLY SETUPS FOR AGITATION AND CONTROLLED OXYGEN.
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.
GENETIC IMPROVEMENT OF THE ANAEROBE BACTERIUM ZYMOMONAS MOBILIS FOR PRODUCTION OF ETHANOL FROM AGRICULTURAL BY PRODUCTS. SORBITOL-LEVAN NON-PRODUCERS WILL BE ISOLATED AND NEW STRAINS WILL BE CONSTRUCTED BY MEANS OF MUTAGENESIS, CONJUGATION, TRANSDUCTION AND PROTOPLAST FUSION,

IN PARTICULAR :

1. MUTANTS WILL BE SELECTED, AFTER CHEMICAL AND UV-IRRADIATION, FOR HIGHER TOLERANCE TO ETHANOL, FOR SORBITOL-LEVAN NON PRODUCERS AND FOR FRUCTOSE NON-UTILIZERS,

2. CONJUGATION BETWEEN ZYMOMONAS SPECIES WILL BE OPTIMIZED,

3. SPECIALIZED TRANSDUCING PHAGE LINES WILL BE CONSTRUCTED USING A NATURALLY-OCCURING ZYMOMONAS SPECIFIC PHAGE RECENTLY ISOLATED FROM GRAPES AS WELL AS OTHER ORGANISMS PHAGES,

4. MUTANTS ABLE TO FERMENT DIFFERENT CARBON SOURCES WILL BE FORCED TO PROTOPLAST FUSE,

5. STRAINS WHICH CAN UTILIZE RAFFINOSE, SORBITOL OR MALTOSE WILL BE TESTED IN BIOREACTORS FOR ENZYME PRODUCTION ON THE CHEMICALLY DEFINED MINIMAL MEDIUM COMPARED TO THE FRUIT JUICE.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

UNIVERSITY OF ATHENS
Address
Panepistemiopolis
Athinai 15701
Greece

Participants (2)

FORSCHUNGSZENTRUM JUELICH GMBH
Germany
Address
Wilhelm Johnen Strasse
52425 Juelich
UNIVERSITY OF IOANNINA
Greece
Address

Ioannina