production of selected secondary metabolites in transformed ...
production of selected secondary metabolites in transformed ...
production of selected secondary metabolites in transformed ...
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conta<strong>in</strong><strong>in</strong>g ampicill<strong>in</strong>, IPTG and x-gal (blue-white test). Plasmids <strong>in</strong>corporated <strong>in</strong>to<br />
transformants were isolated by Gen Elute Plazmid M<strong>in</strong>iprep Kit. To confirm that genes CEL<br />
and MUT were successfully cloned <strong>in</strong>to pGEM, CEL+pGEM and MUT+pGEM were<br />
digested with EcoRI. Electrophoretic separation showed that <strong>in</strong>serts CEL and MUT were<br />
present <strong>in</strong> <strong>transformed</strong> cells. Verification <strong>of</strong> CEL and MUT sequences was done <strong>in</strong> cooperation<br />
with Macrogen Inc. Company (Soul, Korea).<br />
The CEL gene was extracted from pGEM-T us<strong>in</strong>g Nde I. After electrophoretic separation<br />
and extraction from the gel genes were ligated <strong>in</strong>to the pSGset2/Nde I-deP and <strong>in</strong>troduced<br />
<strong>in</strong>to Escherichia coli competent cells. The cells conta<strong>in</strong><strong>in</strong>g CEL+pSGset2/NdeI were <strong>selected</strong><br />
on LB agar plates with apramyc<strong>in</strong>.<br />
The MUT gene was extracted from pGEM-T us<strong>in</strong>g Eco RI and after electrophoretic<br />
separation and extraction from the gel. After ligation <strong>in</strong>to the pIJ 4026/Eco RI-deP<br />
transformation vestors were <strong>in</strong>troduced <strong>in</strong>to ET 12567 Escherichia coli competent cells. The<br />
cells conta<strong>in</strong><strong>in</strong>g MUT+pIJ 4026/Eco RI were <strong>selected</strong> on LB agar plates with<br />
ampicill<strong>in</strong>+chloramphenicol.<br />
We can summarize, that <strong>in</strong> this part <strong>of</strong> work PCR amplification <strong>of</strong> obta<strong>in</strong>able sequence and<br />
clon<strong>in</strong>g and <strong>in</strong>corporation to expression vector was performed. Further, target sequences were<br />
<strong>in</strong>corporated <strong>in</strong>to transformation vectors and recomb<strong>in</strong>ant plasmids were then <strong>in</strong>corporated<br />
<strong>in</strong>to Escherichia coli recipient cells. The presence <strong>of</strong> recomb<strong>in</strong>ant plasmids was verified at the<br />
level <strong>of</strong> genotype and phenotype. Transformation <strong>of</strong> Streptomyces rimosus recipient cells by<br />
recomb<strong>in</strong>ant plasmids will be conduct <strong>in</strong> the future.<br />
In second part <strong>of</strong> this work, regulation <strong>of</strong> carotenoid <strong>production</strong> us<strong>in</strong>g genetic<br />
eng<strong>in</strong>eer<strong>in</strong>g was tested. Several methods <strong>of</strong> isolation and transfer <strong>of</strong> crt genes from bacteria<br />
Erw<strong>in</strong>ia carotovora to recipient stra<strong>in</strong> Escherichia coli DH5α cells were tested and<br />
optimized. Identification <strong>of</strong> carotenoids produced by recomb<strong>in</strong>ant cells was verified by HPLC<br />
analysis.<br />
In <strong>in</strong>dividual Escherichia coli transformants <strong>production</strong> <strong>of</strong> lute<strong>in</strong>, lycopene and βcarotene<br />
was demonstrated. Production <strong>of</strong> carotenoids <strong>in</strong> Escherichia coli cells <strong>transformed</strong><br />
by several recomb<strong>in</strong>ant vectors pHSG298/crt was substantially higher then those found <strong>in</strong><br />
Erw<strong>in</strong>ia carotovora cells. Further, the posibility <strong>of</strong> regulated high-yield carotenoid <strong>production</strong><br />
<strong>in</strong> laboratory fermentor was tested. Production <strong>of</strong> lute<strong>in</strong> <strong>in</strong> Escherichia coli transformants was<br />
about 8x higher then amount <strong>of</strong> lute<strong>in</strong> found <strong>in</strong> Erw<strong>in</strong>ia carotovora cells, which were<br />
cultivated <strong>in</strong> the same conditions.<br />
ACKNOWLEDGEMENTS<br />
I would like to thank Dr. Hrvoje Petkovič and Ms. Urška Lešnik for help and technical<br />
assistance. Both from Biotechnical Faculty, University <strong>of</strong> Ljubljana, Slovenia.<br />
REFERENCES<br />
1. Petkovič H., Thamchaipenet A., Zhou L-H., Hranueli D., Raspor P., Waterman P.G.,<br />
and Hunter I. S.: Disruption <strong>of</strong> an Aromatase/Cyclase from the Oxytetracycl<strong>in</strong>e<br />
Gene Cluster <strong>of</strong> Streptomyces rimosus Results <strong>in</strong> Production <strong>of</strong> Novel Polyketides<br />
with Shorter Cha<strong>in</strong> Lengths. The Journal <strong>of</strong> Biological Chemistry 46, p.32829<br />
-32834, 1999.<br />
Sborník soutěže Studentské tvůrčí č<strong>in</strong>nosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006<br />
Sekce DSP 2006, strana 199