การศึกษาคุณลักษณะทางพันธุกรรม และชีวเคมีของเชื้อรา Xyalaria sp. BCC 1067 สายพันธุ์กลายพันธุ์ที่มีความบกพร่องในการสังเคราะห์สาร depudecin

Name: ณมล แซ่เบ้

Organization : มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

keyword: Xylaria sp.

; Polyketide

; Combinatorial Biosynthesis

; Depudecin

; 19,20-epoxycytochalasin Q

; Mutation Lesion

Abstract: Family of fungi is a representative that can produce large amount of metabolites, whichhave cytotoxic or antibiotics properties. Xylaria sp. BCC1067, one isolated specie offungi in Thailand, shows to produce five bioactive compounds. Two of them, depudecinand 19,20-epoxycytochalasin Q, have been classified as polyketides by their chemicalstructures. These compounds have the potential as anticancer and antimalarial agentsbut have a very high toxic to the mammalian cell. To improve their properties thecombinatorial biosynthesis approach is used to engineer genes involve in thebiosynthetic pathway of these compounds, which aim to reduce toxicity or/and increasetherapeutic efficiency. Until now, the biosynthesis pathways of cytochalasin anddepudecin have never been understood at the biochemical or molecular levels. In orderto understand biosynthetic pathway of these compounds the blocked mutants thataccumulate various intermediates in the biosynthetic pathways were generated bytagged DNA mediated mutagenesis. Then they result almost 300 mutants of which 16strains were putative depudecin non-productive mutants.In this study, a suitable blocked mutant was selected from the putative depudecin nonproducerstrain for molecular and biochemical characterizations. Xylaria sp. BCC1067mutant strain T113, the suitable blocked mutant, is a depudecin non-producer and19,20-epoxycytochalasin Q defective stains, and it has a difference integration patternwith the other mutants. In the biochemical characterization of T113, the profile of mainmetabolites production of this mutant was compared with that profile of BCC1067 wildtype strain by HPLC analysis according to the time course. The result showed, thismutant was completely lost the depudecin production and it was reduced and delayedthe 19,20-epoxycytochalasin production. At the same time, this mutant has theaccumulation of a few unknown metabolites, which they would be related metaboliteswith 19,20-epoxycytochalasin Q biosynthesis pathways. In the molecularcharacterization of T113, the flaking regions of 2 disrupted lesions nearby theintegrative plasmid were identified by the plasmid rescued, PCR walking and plaquescreening techniques. Then, a flaking fragment of a disrupted lesion was found byplaque screening technique, and its sequence was used for functional prediction bycomputation methods. From the prediction, this fragment has no similarity with allknown sequences of Xylaria sp. BCC1067 and it has no similarity with all genomesequences in databases, and it would be a new sequence from Xylaria sp. BCC1067.However, this fragment has on coding sequence but it has many transcription binding sites for gene regulation and gene promoter. Anyhow, this fragment would be used asprobe for identification of a gene either in the biosynthetic pathway or regulatorypathway, and the other disruption lesion can be identified too. One or both of 2disruption lesions would be affected on the biosynthetic pathway or the regulatorypathway of depudecin that cause the complete loss production. At the same time, theother lesion would be affected on 19,20-epoxycytochalasin Q production and/or theaccumulation of unknown metabolites. This study will provide the opportunity forunderstanding both of biosynthetic pathways and regulatory pathways of depudecin and 19,20-epoxycytochalasin Q in Xylaria sp. BCC1067.

มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Address: กรุงเทพมหานคร (Bangkok)

Email: info.lib@mail.kmutt.ac.th

Name: Asst. prof.Dr. supapon cheevadhanarak

Role: Supervisor

Created: 2546

Issued: 2005-11-08

Modified: 2006-05-09

วิทยานิพนธ์/Thesis

ISBN: 9741833504

CallNumber: BIT472

tha

DegreeName: วิทยาศาสตรมหาบัณฑิต

Level: ปริญญาโท

Descipline: เทคโนโลยีชีวภาพ

Grantor: มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

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