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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
Received: 30 May 2018/Accepted:
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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*Corresponding
author; email: bazliramzi@ukm.edu.my
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