Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/145184
Title: Optimizing streptomyces sp. MD102 for heterologous expression of the cannabinoid biosynthetic pathway
Authors: Chea, Rachel Andrea Yuen Fong
Keywords: Science::Biological sciences
Issue Date: 2020
Publisher: Nanyang Technological University
Source: Chea, R. A. Y. F. (2020). Optimizing streptomyces sp. MD102 for heterologous expression of the cannabinoid biosynthetic pathway. Master's thesis, Nanyang Technological University, Singapore.
Abstract: Natural products synthesized from various plants or microorganisms have contributed significantly to drug development and discovery. In many cases, these secondary metabolites are generated at low levels in native hosts and the extraction of these compounds from some of these hosts may be time-consuming and costly. In recent years, there has been a growing interest in the development of suitable heterologous hosts to circumvent this problem. Amongst the various microorganisms studied, the Streptomyces genus has been shown to be ideal for heterologous expression. Our research team has recently isolated over 400 Actinobacteria strains around Singapore, one of which, MD102, is a fast-growing strain that shows potential of becoming a functional proprietary heterologous host. In this study, we endeavored to optimize MD102 and explore its capacity as a chassis for the production of cannabinoids, a group of secondary metabolites innately produced by Cannabis sativa. The pharmaceutical properties of cannabinoids have been widely accepted for many years. However, research efforts have been impeded by several constraints, such as legal restrictions and the extensive time needed for compound extraction. Following the optimization of MD102, we proposed the use of this strain for the production of cannabinoid compounds. To optimize MD102, we first utilized the CRISPR-Cas9 genome editing tool to delete competing endogenous biosynthetic pathways and to obtain a strain with a clean metabolic profile. Concurrently, we evaluated the effectiveness of the base editor system in the silencing of some of these gene clusters. Further optimization was performed by the incorporation of genes involved in the mevalonate and cannabinoid biosynthetic pathways. While our results suggest that the necessary components were successfully integrated in the chromosome of MD102, the final product was not detected in the high-performance liquid chromatography (HPLC) profile of the cell extract, indicating the need for further troubleshooting and optimization.
URI: https://hdl.handle.net/10356/145184
DOI: 10.32657/10356/145184
Rights: This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).
Fulltext Permission: embargo_20221214
Fulltext Availability: With Fulltext
Appears in Collections:SBS Theses

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