Abstract

Natural polyketide products are one of the major secondary metabolites produced among bacteria, fungi, and plants. They vary from flavonoids, pyrones, and stilbenes to phloroglucinols and resorcinols that are involved in important functions as antimicrobial activity, defense mechanisms and pigmentation. They are biosynthesized from acyl-CoA precursors by polyketides synthases (PKSs) that are categorized into 3 types: I, II and III. PKS type III is considered the simplest in its structure. It was believed that PKS type III was exclusively encoded by higher plants until the enduring efforts of bacterial genomes sequencing revealed the presence of more and more PKSs type III among them. There is an urge to investigate novel PKSs type III due to their promising polyketides of great biological and pharmaceutical advantages. This allowed metagenomic approaches to be a valuable tool to explore diverse environments for PKSs type III. Extreme environments as deep sea brine pools could probe unique natural polyketides capable of functioning in such conditions with valuable biotechnological and pharmaceutical applications. In this study, screening of the Lower Convective Layer (LCL) of Atlantis II (ATII) deep brine pool in the Red Sea was done. It identified sequences belonging to bacterial PKSs type III. A candidate encoding sequence was amplified from the environmental DNA. Functional annotations were assigned to the translated open reading frame including the conserved catalytic triad, domains, motifs and 3D modelling. Preliminary structural analysis showed well-fitted superimposition with the flowering plant Medicago sativa PKS type III crystal structure and predicted the interaction of the catalytic triad with the most common substrate malonyl-CoA. Further optimization of heterologous expression is required to investigate this isolated PKS type III functional activity. In an approach to gain better insights into the enzyme’s unresolved evolutionary origin, a comprehensive phylogenetic analysis was conducted. The analysis pinpoints the possible involvement of symbiotic bacterium Parachlamydia acanthamoebae in horizontal gene transfer events to eukaryotes. On the other hand, the sequence isolated from ATII brine pool was clustered in a clade with related PKSs type III sequences belonging to alpha-proteobacteria. Environmental assessment of PKSs type III abundance in ATII and nearby Discovery Deep (DD) brine pool revealed the presence of PKSs type III in ATII only, where most sequences were located in the LCL. This could be attributed to the high aromatic content within the brine as possible substrates for the enzyme. Based on these analyses, we could propose ATII microbial community as a unique source for natural polyketides.

Department

Biotechnology Program

Degree Name

MS in Biotechnology

Date of Award

6-1-2014

Online Submission Date

February 2014

First Advisor

Moustafa, Ahmed

Second Advisor

Ferreira, Ari

Committee Member 1

Moustafa, Ahmed

Committee Member 2

Bos, Arthur

Document Type

Thesis

Extent

83 p.

Library of Congress Subject Heading 1

Biotechnology -- Red Sea -- Egypt.

Library of Congress Subject Heading 2

Microbiology -- Red Sea -- Egypt.

Rights

The author retains all rights with regard to copyright. The author certifies that written permission from the owner(s) of third-party copyrighted matter included in the thesis, dissertation, paper, or record of study has been obtained. The author further certifies that IRB approval has been obtained for this thesis, or that IRB approval is not necessary for this thesis. Insofar as this thesis, dissertation, paper, or record of study is an educational record as defined in the Family Educational Rights and Privacy Act (FERPA) (20 USC 1232g), the author has granted consent to disclosure of it to anyone who requests a copy.

IRB

Not necessary for this item

Comments

King Abdullah University of Science and Technology (KAUST), the participants in the spring 2010 expedition and lab colleagues for both collecting the samples and establishing the database. American University in Cairo (AUC) for awarding me a Laboratory fellowship

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