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

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


Official URL: http://www.ukm.my/jsm/malay_journals/jilid47bil12_...


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.

Item Type:Article
Keywords:Phenylpropanoid biosynthesis; Polygonum minus; Rapid DNA assembly; Saccharomyces cerevisiae; Synthetic biology
Journal:Sains Malaysiana
ID Code:12917
Deposited By: ms aida -
Deposited On:09 May 2019 02:07
Last Modified:15 May 2019 10:58

Repository Staff Only: item control page