production of selected secondary metabolites in transformed ...

in last years some ways for overproduction of carotenoids including modern methods of
molecular cloning and genetic engineering are studied.
Erwinia carotovora is nonphotosynthetic bacterium pathogenic for higher plants. Beside
its agricultural significance, Erwinia strains are of increasing interest as industrial microbes
producing pectinolytic, cellulolytic and proteolytic enzymes as well as antileukaemic
asparaginase. Erwinia strains form carotenoids which cause yellow-orange coloured
phenotype.
METHODS
Bacterial strains. For production of oxytetracyclines bacterium Streptomyces rimosus 4018
was used. For production of carotenoids bacterium Erwinia carotovora CCM 1008 was used.
For transformation DH5α, DH10β and ET 12567 Escherichia coli competent cells were
prepared.
Cultivation. Escherichia coli was cultivated in 2TY medium at 37ºC. 2TY medium
contained, per litre: tryptone, 16 g; yeast extract, 10 g; NaCl, 5 g .
Plasmids. pHSG298, pGEM-T (for PCR product), pSGset2 (containing Erm, OriC, Apr,
OriT, attP, phi-C31 int), pTS55 (containing Amp, tsr, att, int, repSA) and pIJ 4026
(containing Erm, bla) were used as transformation vectors.
Isolation. Plasmid DNA isolation was performed using commercial kits (Gen Elute
Plasmid Miniprep Kit).
Transformation. Transformation of E. coli cells was done using electroporation by BioRad
GENEPULSER apparatus at following conditions: voltage 2500 V, resistance 200 Ω,
capacitance 25 μF.
Electrophoresis. DNA was analysed using agarose electrophoresis and pulsed field gel
electropohoresis (PFGE).
Analysis of carotenoids. Production of carotenoids by transformants was analysed
chromatographically. Carotenoids were extracted from E. coli transormant cells by ethanol.
Individua pigments were separated and quantified by RP-HPLC using a Nucleosil 100 C18
column and methanol (analysis of lycopene, lutein and carotenes) or mixture
acetonitril:methanol 95:5 (phytoene analysis) as eluent.
RESULTS
Presented work was focused on production of selected secondary metabolites in
transformed bacterial cells. First, regulation of polyketide antibiotik production in Escherichia
coli cells transformed by otc genes from Streptomyces rimosus was studied. Further, isolation
and cloning of crt gene cluster from bacteria Erwinia carotovora in E.coli DH5α cells was
tested. Most OF experiments were performed in co-operation with Biotechnical Faculty,
University of Ljubljana (Socrates/Erasmus exchange).
The DNA sequence of Streptomyces rimosus was analysed by FramePlot (FramePlot is a
web-based tool for predicting protein-coding regions in bacterial DNA with a high G+C
content, such as Streptomyces). Primer structure was derived from sequence analysis results.
The genes were amplified by PCR. Recommended sizes of PCR products were 714 bp and
470 bp. The sequence of 714 bp was named CEL and the sequence of 470 bp was named
MUT. After purification by Gen Elute PCR Clean-Up Kit the PCR products were ready to be
cloned. CEL and MUT PCR products were ligated into the pGEM-T and introduced into
E. coli competent cells. The cells with CEL or MUT were selected on LB agar plates
Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006
Sekce DSP 2006, strana 198