Sains Malaysiana 47(12)(2018): 2969–2974

http://dx.doi.org/10.17576/jsm-2018-4712-05

 

Rapid Assembly of Yeast Expression Cassettes for Phenylpropanoid Biosynthesis in Saccharomyces cerevisiae

(Pemasangan Pantas Gen Kaset Yis untuk Penghasilan Fenilpropanoid dalam Saccharomyces cerevisiae)

 

AHMAD BAZLI RAMZI*, KU NURUL AQMAR KU BAHAUDIN, SYARUL NATAQAIN BAHARUM, MUHAMMAD LUTFI CHE ME, HOE-HAN GOH, MAIZOM HASSAN & NORMAH MOHD NOOR

 

Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

 

Diserahkan: 30 Mei 2018/Diterima: 18 September 2018

 

ABSTRACT

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.

 

Keywords: Phenylpropanoid biosynthesis; Polygonum minus; rapid DNA assembly; Saccharomyces cerevisiae; synthetic biology

 

ABSTRAK

Penghasilan produk semula jadi oleh mikrob melalui kaedah kejuruteraan metabolik dan biologi sintetik sering melibatkan pemasangan serpihan gen berganda termasuk elemen gen pengawalselia yang penting untuk sistem eukariot. Pengklonan gen secara tradisi menggunakan enzim pemotongan dan penyambungan DNA adalah sukar dan tidak fleksibel kerana bergantung kepada langkah pengklonan berjujukan yang sangat banyak, ketidaksesuaian tapak pemotongan dan penghasilan jujukan ‘parut’ yang tidak dikehendaki. Kajian ini melaporkan pemasangan berbilang fragmen DNA berganda secara serentak dan dalam satu langkah melalui kaedah pemasangan DNA isoterma untuk penghasilan fenilpropanoid dalam yis Saccharomyces cerevisiae. Pembinaan plasmid konstruk yis telah berjaya dilakukan dengan pantas melalui kaedah pemasangan isoterma empat fragmen DNA yang telah direka untuk mengandungi jujukan bertindih sebanyak 20 pasangan bes. Enzim sinamat 4-hidrolase (C4H) daripada Polygonum minus yang merupakan enzim pengehad kadar fenilpropanoid, telah dipasang bersama elemen penggalak dan penamat yis untuk pembinaan laluan fenilpropanoid dalam S. cerevisiae rekombinan secara cekap dan pantas. Hasil pemasangan plasmid lengkap pAG-CAT (C4H-ADH1t-TEF1p) dan transformasi gen C4H dalam S. cerevisiae telah disahkan melalui analisis tindak balas rantai polimerase (PCR). Berdasarkan hasil kajian ini, plasmid ulang-alik yis yang mengandungi gen biosintetik fenilpropanoid daripada P. minus telah berjaya dibina dengan cekap dan akan dijadikan sebagai landasan pemula untuk penghasilan produk semula jadi menggunakan S. cerevisiae yang terubah suai secara genetik.

 

Kata kunci: Biologi sintetik; biosintesis fenilpropanoid; pemasangan DNA pantas; Polygonum minus; Saccharomyces cerevisiae

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*Pengarang untuk surat-menyurat; email: bazliramzi@ukm.edu.my