Rapid Assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae

Microbial production of natural products using metabolic engineering and synthetic biology approaches often involves the assembly of multiple gene fragments including regulatory elements, especially when using eukaryotes as hosts. Traditional cloning strategy using restriction enzyme digestion and l...

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Main Authors:	Ahmad Bazli Ramzi, Ku Nurul Aqmar Ku Bahaudin, Syarul Nataqain Baharum, Muhammad Lutfi Che Me, Goh, Hoe-Han, Maizom Hassan, Normah Mohd Noor
Format:	Article
Language:	English
Published:	Penerbit Universiti Kebangsaan Malaysia 2018
Online Access:	http://journalarticle.ukm.my/12917/ http://journalarticle.ukm.my/12917/ http://journalarticle.ukm.my/12917/1/05%20Ahmad%20Bazli%20Ramzi.pdf

id	ukm-12917
recordtype	eprints
spelling	ukm-129172019-05-15T10:58:54Z http://journalarticle.ukm.my/12917/ Rapid Assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae Ahmad Bazli Ramzi, Ku Nurul Aqmar Ku Bahaudin, Syarul Nataqain Baharum, Muhammad Lutfi Che Me, Goh, Hoe-Han Maizom Hassan, Normah Mohd Noor, Microbial production of natural products using metabolic engineering and synthetic biology approaches often involves the assembly of multiple gene fragments including regulatory elements, especially when using eukaryotes as hosts. Traditional cloning strategy using restriction enzyme digestion and ligation are laborious and inflexible owing to the high number of sequential cloning steps, limited cutting sites and generation of undesired ‘scar’ sequences. In this study, a homology-based isothermal DNA assembly method was carried out for one-step simultaneous assembly of multiple DNA fragments to engineer plant phenylpropanoid biosynthesis in Saccharomyces cerevisiae. Rapid construction of yeast plasmid harboring dual gene expression cassettes was achieved via isothermal assembly of four DNA fragments designed with 20 bp overlapping sequences. The rate-limiting enzyme of phenylpropanoid pathway, cinnamate 4-hydroxylase encoded by C4H gene from Polygonum minus was cloned in tandem with yeast promoter and terminator elements of S. cerevisiae for efficient construction of phenylpropanoid biosynthetic pathway in recombinant yeast. The assembled pAG-CAT (C4H-ADH1t-TEF1p) shuttle plasmid and transformation of S. cerevisiae with the plant C4H gene were confirmed via PCR analysis. Based on these findings, the yeast shuttle plasmid harboring P. minus phenylpropanoid biosynthesis gene was efficiently constructed to be the starting platform for the production of plant natural products in genetically-engineered S. cerevisiae. Penerbit Universiti Kebangsaan Malaysia 2018-12 Article PeerReviewed application/pdf en http://journalarticle.ukm.my/12917/1/05%20Ahmad%20Bazli%20Ramzi.pdf Ahmad Bazli Ramzi, and Ku Nurul Aqmar Ku Bahaudin, and Syarul Nataqain Baharum, and Muhammad Lutfi Che Me, and Goh, Hoe-Han and Maizom Hassan, and Normah Mohd Noor, (2018) Rapid Assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae. Sains Malaysiana, 47 (12). pp. 2969-2974. ISSN 0126-6039 http://www.ukm.my/jsm/malay_journals/jilid47bil12_2018/KandunganJilid47Bil12_2018.html
repository_type	Digital Repository
institution_category	Local University
institution	Universiti Kebangasaan Malaysia
building	UKM Institutional Repository
collection	Online Access
language	English
description	Microbial production of natural products using metabolic engineering and synthetic biology approaches often involves the assembly of multiple gene fragments including regulatory elements, especially when using eukaryotes as hosts. Traditional cloning strategy using restriction enzyme digestion and ligation are laborious and inflexible owing to the high number of sequential cloning steps, limited cutting sites and generation of undesired ‘scar’ sequences. In this study, a homology-based isothermal DNA assembly method was carried out for one-step simultaneous assembly of multiple DNA fragments to engineer plant phenylpropanoid biosynthesis in Saccharomyces cerevisiae. Rapid construction of yeast plasmid harboring dual gene expression cassettes was achieved via isothermal assembly of four DNA fragments designed with 20 bp overlapping sequences. The rate-limiting enzyme of phenylpropanoid pathway, cinnamate 4-hydroxylase encoded by C4H gene from Polygonum minus was cloned in tandem with yeast promoter and terminator elements of S. cerevisiae for efficient construction of phenylpropanoid biosynthetic pathway in recombinant yeast. The assembled pAG-CAT (C4H-ADH1t-TEF1p) shuttle plasmid and transformation of S. cerevisiae with the plant C4H gene were confirmed via PCR analysis. Based on these findings, the yeast shuttle plasmid harboring P. minus phenylpropanoid biosynthesis gene was efficiently constructed to be the starting platform for the production of plant natural products in genetically-engineered S. cerevisiae.
format	Article
author	Ahmad Bazli Ramzi, Ku Nurul Aqmar Ku Bahaudin, Syarul Nataqain Baharum, Muhammad Lutfi Che Me, Goh, Hoe-Han Maizom Hassan, Normah Mohd Noor,
spellingShingle	Ahmad Bazli Ramzi, Ku Nurul Aqmar Ku Bahaudin, Syarul Nataqain Baharum, Muhammad Lutfi Che Me, Goh, Hoe-Han Maizom Hassan, Normah Mohd Noor, Rapid Assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae
author_facet	Ahmad Bazli Ramzi, Ku Nurul Aqmar Ku Bahaudin, Syarul Nataqain Baharum, Muhammad Lutfi Che Me, Goh, Hoe-Han Maizom Hassan, Normah Mohd Noor,
author_sort	Ahmad Bazli Ramzi,
title	Rapid Assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae
title_short	Rapid Assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae
title_full	Rapid Assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae
title_fullStr	Rapid Assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae
title_full_unstemmed	Rapid Assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae
title_sort	rapid assembly of yeast expression cassettes for phenylpropanoid biosynthesis in saccharomyces cerevisiae
publisher	Penerbit Universiti Kebangsaan Malaysia
publishDate	2018
url	http://journalarticle.ukm.my/12917/ http://journalarticle.ukm.my/12917/ http://journalarticle.ukm.my/12917/1/05%20Ahmad%20Bazli%20Ramzi.pdf
first_indexed	2023-09-18T20:03:41Z
last_indexed	2023-09-18T20:03:41Z
_version_	1777407016273182720

Rapid Assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae

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