Zbornik - Društvo genetičara Srbije

II-Uvo-2 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE 45
Subotica, 30. novembar - 4. decembar 2004.
ANALIZA GENA ZA BIOSINTEZU SEKUNDARNIH METABOLITA
KOD PRIPADNIKA RODA Streptomyces
M. Saviæ, Ivana Bratiæ i Branka Vasiljeviæ
Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd
Razlièiti mikroorganizmi proizvode sekundarne metabolite od kojih veæina pokazuje
toksièno delovanje na razlièite æelije i organizme. Ovi metaboliti, ili prirodni proizvodi,
predstavljaju izuzetno znaèajan resurs za otkrivanje novih jedinjenja sa specifiènim
fiziološkim delovanjem. Poliketidi su velika i strukturno veoma razlièita grupa bioaktivnih
jedinjenja za èiju biosintezu su odgovorne poliketid sintaze (PKS). Molekularna
karakterizacija gena ukljuèenih u biosintezu poliketidnih metabolita je ukazala na postojanje
kompleksnih enzimskih sistema èiji lokusi obièno zauzimaju desetine hiljada baznih parova
na hromozomu. Kod tipa I PKS enzimatski domeni su organizovani u module i genetièki
redosled modula je kolinearan sa redosledom biohemijskih reakcija. Svaki modul je
odgovoran za prepoznavanje i ugraðivanje specifiènog malog konstituenta u rastuæi
poliketidni lanac, kao i za prateæe reakcije redukcije. Ovakav mehanizam je u suprotnosti sa
tipom II PKS gde se istovetni set proteina, svaki zasebno katalizujuæi razlièitu enzimatsku
reakciju, koristi tokom nekoliko ciklusa. Analizirajuæi laboratorijsku kolekciju streptomiceta
izabran je soj Streptomyces sp. MS405. Odabrani soj proizvodi bioaktivno jedinjenje
mehanizma delovanja nalik na imunosupresor FK506. Poreðenjem sa poznatim poliketidnim
jedinjenjima, metodom tankoslojne hromatografije, kao što su FK506, FK520, rapamicin,
eritromicin i rifampicin, utvrðeno je da se analizirano jedinjenje razlikuje od referentnih.
Kloniranjem kratkog DNK fragmenta utvrðeno je da biosinteza ovog jedinjenja zavisi od
PKS tipa I. Filogenetske analize ukazuju na visok stepen homologije izmeðu kloniranog
fragmenta i fkbA PKS S. hygroscopicus, prozvoðaèa imunosupresora rapamicina.
ANALYSIS OF THE GENE CLUSTERS INVOLVED IN BIOSYNTHESIS
OF THE SECONDARY METABOLITES AMONG MEMBERS OF THE GENUS
Streptomyces
A variety of microorganisms produce secondary metabolites, many of which exhibit
toxic activity against different cells and organisms. These metabolites have proved to be
a valuable resource to identify compounds with specific biological effects. Polyketides
are a large and structurally diverse family of bioactive compounds whose biosynthesis is
catalysed by polyketide synthases (PKS). Molecular characterization of the genes involved
in formation of polyketide metabolites has demonstrated the involvement of complex
enzyme systems whose genetic loci usually span tens of thousands of base pairs on a
chromosome. In type I PKS enzymatic domains are organized into modules and genetic
order of the modules is collinear with the order of biochemical reactions. Each module is
responsible for recognition and incorporation of a specific small constituent into growing
polyketide chain as well as the subsequent reductive reactions. This is in contrast to type
II PKS where a set of discrete proteins, each catalyzing different enzymatic reaction, is
repeatedly employed. Analysing laboratory streptomycetes collection strain Streptomyces
sp. MS405 was selected. This strain produces bioactive compound with the same
mechanism of action as immunosuppressant FK506. Thin layer chromatography (TLC)
studies showed that the compound is chemically different compared to referent
polyketides: FK506, FK520, rapamycin, erythromycin and rifampycin. Cloned short
fragment of the biosynthetical gene cluster revealed that biosynthesis of this compound is
governed by type I PKS. Phylogenetical studies imply high sequence homology between
cloned sequence and fkbA PKS of S. hygroscopicus, producer of polyketide immunosuppressant
rapamycin.