Title

Mining for specialized metabolism genes in Red Sea brine pool prokaryotic metagenomes for antibacterial and anticancer activities

Author

Laila Ziko

Abstract

Antibiotic and anticancer drug resistance are current global health threats, thus new antibiotics and anticancer agents are required to treat the strains and cancers that are currently untreatable with the available drug spectrum. One way to search for new chemotherapeutics is to search nature, particularly metagenomes of un-cultured microbial communities. A subset of specialized metabolites (SMs) produced by microbes confer activities of pharmaceutical importance, such as antibacterial and anticancer effects, and are coded by specialized metabolism gene clusters (SMGCs) in the organisms’ genomes. We aimed to search for SMGCs in the metagenomes of Red Sea brine microbial communities by employing computational methods and functional screening methods. Metagenome mining was performed for shotgun metagenomic sequences from Atlantis II (ATII), Discovery Deep (DD), Kebrit Deep (KD) brine water and ATII, DD and Non-brine (NB) sediment samples of assembled metagenomic prokaryotic environmental DNA. SMGCs were detected by using the antibiotics and secondary metabolite analysis shell (antiSMASH) tool. A total of 2,751 SMGCs were detected, which belonged to 28 different SM classes. The SMGCs were thoroughly analysed for promising potential antibacterial and anticancer activity, taxonomic evaluation for all the sites was performed, and chemical structure prediction was also performed. The potential biotechnological applications of the detected Red Sea brine SMGCs was studied, as well as the potential role of the detected SMGCs in microbe-environment interactions, extremophile survival and microbial diversity in extreme environments. As a preliminary proof of concept, a detected polyketide synthase type III (PKSIII) enzyme was expressed. Functional screening was employed to detect antibacterial and anticancer activities from the Atlantis II Lower Convective Layer (ATII LCL) metagenomic fosmid library comprising 10,656 clones. A phenotypic assay was employed to detect clones of antibacterial effect against a marine Bacillus strain (Bacillus Cc6), yielding 11 positive clones. The top six clones were selected for sequencing and screening for anticancer activity. Whole extracts were prepared from the fosmid clones and added to MCF-7 (breast cancer cell line), U2OS (osteosarcoma cell line) and 1BR hTERT (non-cancerous fibroblasts) in concentrations (1, 5, 10, 15, 20 and 50%) for 48 hrs, after which cell viability was determined by 3-(4, 5-dimethylthiazolyl-2)- 2, 5-diphenyltetrazolium bromide (MTT). The whole cell extracts resulted into MCF-7 cell viability (38%  7 - 46.2%  9.9 at 50% v/v), (28.3%  1.7 - 79.9%  5.9 at 50% v/v) for U2OS cells and (48.1%  3.4 – 76.4%  4.8 at 50% v/v) for 1BR hTERT cells. Two putative orphan SMGCs were annotated for the clones 10-2G and 14-7E, with the latter being of archaeal origin. Additionally, putative proteases were annotated on 102-5A clone, while 88-1G clone harboured putative biosynthetic genes. The current project highlights the huge potential of Red Sea brine microbiome to harbour SMGCs that synthesize specialized metabolites of biotechnological applications, function in the microbial evolution of extremophiles, and also possess antibacterial and anticancer effects. Although it is a step towards understanding Red Sea brine SMs, further computational and experimental studies are recommended to understand and utilize the Red Sea brine SM dark matter.

Department

Biotechnology Program

Graduation Date

2-1-2019

Submission Date

January 2019

First Advisor

Siam, Rania

Committee Member 1

Gad, Mohamed

Committee Member 2

Breitinger, Hans-Georg

Extent

325 p.

Document Type

Doctoral Dissertation

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.

Institutional Review Board (IRB) Approval

Not necessary for this item

Comments

I would like to acknowledge the Youssef Jameel foundation for providing me the PhD fellowship. The project was funded by AUC student research grant. Also, I would like to thank EMBO short-term research fellowship for the three-month research visit to Germany.

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