Heterologous expression of cannabidiolic acid synthase in a microbial host for cannabinoid production

Renewed interest in the pharmacological applications of Phytocannabinoids from Cannabis sativa (C. sativa) has created a need for an efficient and scalable biomanufacturing platform to replace resource intensive and largely illegal agricultural approaches. Despite success in reproducing the cannabin...

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Main Author: Toh, Wing Loon
Other Authors: Liang Zhao-Xun
Format: Final Year Project
Language:English
Published: 2019
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Online Access:http://hdl.handle.net/10356/77106
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Institution: Nanyang Technological University
Language: English
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spelling sg-ntu-dr.10356-771062023-02-28T18:06:58Z Heterologous expression of cannabidiolic acid synthase in a microbial host for cannabinoid production Toh, Wing Loon Liang Zhao-Xun School of Biological Sciences DRNTU::Science::Chemistry::Analytical chemistry::Proteins Renewed interest in the pharmacological applications of Phytocannabinoids from Cannabis sativa (C. sativa) has created a need for an efficient and scalable biomanufacturing platform to replace resource intensive and largely illegal agricultural approaches. Despite success in reproducing the cannabinoid biosynthetic pathway in various Eukaryotic hosts, Prokaryotic hosts with higher space time yields have been ignored as a suitable alternative. In this study, we attempted to re-engineer the cannabinoid biosynthetic pathway of C. Sativa for optimized prokaryotic heterologous expression. Utilizing ROSETTA overexpression strain E. coli as a host, heterologous protein expression of terminal enzyme cannabidiol synthase (CBDAS) was achieved and optimized. Despite successful expression in harvested total protein fractions CBDAS was not soluble and active. Consequently, fusion with several solubility tags such as glycosylation protein ApNGT, expression reporter mCherry and periplasmic protein DsbA was attempted. The ApNGT CBDAS fusion managed to be expressed was not soluble. DsbA CBDAS appears to have yielded some success in obtaining a soluble protein but further verification is required. Fusion with expression reporter mCherry produced a soluble, and correctly folded mCherry protein but appeared to show degradation of CBDAS despite a correctly constructed plasmid. Lastly, we created a fusion protein NphB CBDAS with dual functions as a substitute for Cannabigerolic Acid Synthase (CBGAS) and a solubility enhancement tag. However, NphB CBDAS remained insoluble despite successful expression in ROSETTA E. coli. Bachelor of Science in Biological Sciences 2019-05-08T07:41:19Z 2019-05-08T07:41:19Z 2019 Final Year Project (FYP) http://hdl.handle.net/10356/77106 en Nanyang Technological University 26 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Science::Chemistry::Analytical chemistry::Proteins
spellingShingle DRNTU::Science::Chemistry::Analytical chemistry::Proteins
Toh, Wing Loon
Heterologous expression of cannabidiolic acid synthase in a microbial host for cannabinoid production
description Renewed interest in the pharmacological applications of Phytocannabinoids from Cannabis sativa (C. sativa) has created a need for an efficient and scalable biomanufacturing platform to replace resource intensive and largely illegal agricultural approaches. Despite success in reproducing the cannabinoid biosynthetic pathway in various Eukaryotic hosts, Prokaryotic hosts with higher space time yields have been ignored as a suitable alternative. In this study, we attempted to re-engineer the cannabinoid biosynthetic pathway of C. Sativa for optimized prokaryotic heterologous expression. Utilizing ROSETTA overexpression strain E. coli as a host, heterologous protein expression of terminal enzyme cannabidiol synthase (CBDAS) was achieved and optimized. Despite successful expression in harvested total protein fractions CBDAS was not soluble and active. Consequently, fusion with several solubility tags such as glycosylation protein ApNGT, expression reporter mCherry and periplasmic protein DsbA was attempted. The ApNGT CBDAS fusion managed to be expressed was not soluble. DsbA CBDAS appears to have yielded some success in obtaining a soluble protein but further verification is required. Fusion with expression reporter mCherry produced a soluble, and correctly folded mCherry protein but appeared to show degradation of CBDAS despite a correctly constructed plasmid. Lastly, we created a fusion protein NphB CBDAS with dual functions as a substitute for Cannabigerolic Acid Synthase (CBGAS) and a solubility enhancement tag. However, NphB CBDAS remained insoluble despite successful expression in ROSETTA E. coli.
author2 Liang Zhao-Xun
author_facet Liang Zhao-Xun
Toh, Wing Loon
format Final Year Project
author Toh, Wing Loon
author_sort Toh, Wing Loon
title Heterologous expression of cannabidiolic acid synthase in a microbial host for cannabinoid production
title_short Heterologous expression of cannabidiolic acid synthase in a microbial host for cannabinoid production
title_full Heterologous expression of cannabidiolic acid synthase in a microbial host for cannabinoid production
title_fullStr Heterologous expression of cannabidiolic acid synthase in a microbial host for cannabinoid production
title_full_unstemmed Heterologous expression of cannabidiolic acid synthase in a microbial host for cannabinoid production
title_sort heterologous expression of cannabidiolic acid synthase in a microbial host for cannabinoid production
publishDate 2019
url http://hdl.handle.net/10356/77106
_version_ 1759858327251058688