STUDY, CONSTRUCTION AND CLONING IN ORGANISMS FOR CONVERSION OF GLUCOSE TO KETOACIDS
Main researcher
: DELIĆ, VLADIMIR (9215) Assistants
SOJAK, VLASTA (43924)
NOŽINIĆ, RANKA (33780)
GAMULIN, STJEPAN (13074)
PAVLOV, LJUBOMIR (35712)
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. Other information about the project.