production of selected secondary metabolites in transformed ...

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