SVIBOR - Project code: 1-08-045

MINISTRY OF SCIENCE AND TECHNOLOGY

Strossmayerov trg 4, HR - 10000 ZAGREB
tel.: +385 1 459 44 44, fax: +385 1 459 44 69
E-mail: ured@znanost.hr

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Project code: 1-08-045


STUDY, CONSTRUCTION AND CLONING IN ORGANISMS FOR CONVERSION OF GLUCOSE TO KETOACIDS


Main researcher: DELIĆ, VLADIMIR (9215)



Assistants
Type of research: basic
Duration from: 01/01/91. to 12/15/95.

Papers on project (total): 28
Papers on project quoted in Current Contents: 4
Institution name: Pliva - Istraživački institut, Zagreb (60)
Department/Institute: Biosynthesis and Biotechnology
Address: B.Filipovića 89
City: 10000 - Zagreb, Croatia
Communication
Phone: 385 (0)1 181-600
Fax: 385 (0)1 576-690
Fax: 385 (0)1 181-606

Summary: Bacteria from genus Erwinia and Gluconobacter converted D-glucose (up to 20 %) to different keto-acids which accumulated in the medium. Optimal conditions were found for determination by thin-layer chromatography, D-gluconic, 2-keto-D-gluconic, 5-keto-D-gluconic and 2,5 diketo-D-gluconic acid in the medium. To find and construct suitable vector for gene cloning in these genera, plasmid isolation revealed that E. citreus ATCC31623 had cryptic plasmid of 3.8 kb (pPZG500). Partial restriction map have shown position of HindIII, DraI, EcoRV, BglII, XbaI and EcoRI sites. Enzymes AcyI, Asp718, AvaI, BalI, BamHI, ClaI, HpaI, KpnI, MroI, EclXI, NsiI, PstI, PvuI, PvuII, SacI, SalI, ScaI and SmaI did not cut pPZG500. Bacterium E. herbicola ATCC21998 had two cryptic plasmids: pPZG600 (7.5 kb) and pPZG601 (8.6 kb) respectively, which were partially characterized by restriction enzymes. Plasmid pPZG500 was cut open by HindIII and ligated to pSELECT to bring tetracycline resistance gene suitable as a marker for genus Erwinia. After transformation recombinant plasmids showed two orientation of pPZG500 (pMB1A, pMB1B) in pSELECT. Plasmid pMB1AA inserted dimeric pPZG500 in A orientation. Recombinant plasmids replicated in E. coli and Erwinia citreus and were useful for cloning 2,5-DKG reductase gene. From plasmid pE194, SacI/NsiI fragment of 906 bp containing erythromycin resistance gene was ligated to plasmid pUC19 and new recombinant plasmid pPZG650 of 3.6 kb (ApR, EmR) was constructed and proved by hybridization. Transformants in E coli JM109 and Erwinia citreus were selected on azithromycin. Insertion of EmR fragment into pPZG500 and by deletion, new constructs pPZG502 (3.20 kb) and pPZG503 (2.50 kb) were obtained. Segregational stability study during 90 generations revealed that region for autonomous replication (ori) and partition (par) were on 2.1 kb EcoRI-EcoRV fragment. Plasmid pPZG503 disappeared from the population at three times higher rate than pPZG502.

Keywords: Glucose bioconversion, Erwinia and Gluconobacter plasmids, vector construction

Research goals: The aim of research is to study molecular biology of less known microorganisms from genus Erwinia and Gluconobacter. The goal was to examine the ability of these organisms in conversion of D-glucose to keto-acids and to find out analytical methods for determination of oxido-reduction intermediates. For gene cloning in these bacteria, plasmids will be isolated and characterized for useful vector construction and gene cloning and expression. The results will help in understanding at the molecular level,the biology of these phytopathogenic and industrially important group of microorganisms.

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Last update: 10/09/95
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