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GENETIC MANIPULATION OF THE ANAEROBE ZYMONONAS MOBILES 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.

THE EXPLORE THE APPLICABILITY OF ZYMOMONAS MOBILIS FOR INDUSTRIAL FERMENTATIONS PRODUCING HIGH RATES AND YIELD 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.
TRANSFER IN ZYMOMONAS MOBILIS OF GENES CODING FOR ENZYMES WHICH REDUCE SORBITOL OR RAFFINOSE OR MALTOSE OR ALTER SUGARS TO GLUCOSE.

IN PARTICULAR :
1. THE TRANSFORMATION SYSTEM OF ZYMOMONAS MOBILIS WILL BE OPTIMIZED.
2. TRANSFER BY CONJUGATION OF GENES CODING FOR TRANSFORMATION OF SORBITOL, RAFFINOSE OR MALTOSE INTO GLUCOSE.
3. CONSTRUCTION BY PROTOPLAST FUSION OF HYBRIDS BETWEEN STRAINS THAT FERMENT SORBITOL, RAFFINOSE OR MALTOSE.
4. NEW STRAINS WILL BE TESTED FOR ETHANOL PRODUCTION.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

UNIVERSITY OF IOANNINA
Address
Department Of Chemistry Division Of Organic Chemistry-biochemistry
45332 Ioannina
Greece

Participants (2)

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

Athens