Sains Malaysiana 47(8)(2018): 1675–1684

http://dx.doi.org/10.17576/jsm-2018-4708-06

Pengenalpastian dan Profil Pengekspresan Gen Biosintesis Asid Amino Yis Psikrofil, Glaciozyma antarctica

(Identification and Expression Profiles of Amino Acid Biosynthesis Genes from Psychrophilic Yeast, Glaciozyma antarctica)

IZWAN BHARUDIN¹, RADZIAH ZOLKEFLI¹, MOHD FAIZAL ABU BAKAR², SHAZILAH KAMARUDDIN¹, ROSLI MD. ILLIAS³, NAZALAN NAJIMUDIN⁴, NOR MUHAMMAD MAHADI², FARAH DIBA ABU BAKAR¹ & ABDUL MUNIR ABDUL MURAD^1*

¹Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

²Malaysia Genome Institute, Jalan Bangi Lama, 43000 Kajang, Selangor Darul Ehsan, Malaysia

³Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor Darul Takzim, Malaysia

⁴School of Biological Sciences, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia

Received: 15 September 2017/Accepted: 12 April 2018

ABSTRAK

Mekanisme pengambilan dan penghasilan asid amino bagi mikroorganisma psikrofil yang bermandiri dan berpoliferasi pada persekitaran sejuk melampau masih belum difahami sepenuhnya. Objektif kajian ini ialah untuk mengenal pasti gen yang terlibat dalam penjanaan asid amino bagi yis psikrofil, Glaciozyma antarctica serta menentukan pengekspresan gen tersebut semasa kehadiran dan kekurangan asid amino dalam medium pertumbuhan. Pengenalpastian gen telah dilakukan melalui penjanaan penanda jujukan terekspres (ESTs) daripada dua perpustakaan cDNA yang dibina daripada sel yang dikultur dalam medium pertumbuhan kompleks dan medium pertumbuhan minimum tanpa asid amino. Sebanyak 3552 klon cDNA daripada setiap perpustakaan dipilih secara rawak untuk dijujuk menghasilkan 1492 transkrip unik (medium kompleks) dan 1928 transkrip unik (medium minimum). Analisis pemadanan telah mengenl pasti gen mengekod protein yang terlibat di dalam pengambilan asid amino bebas, biosintesis asid amino serta gen yang terlibat dengan kitar semula asid amino berdasarkan tapak jalan yang digunakan oleh yis model, Saccharomyces cerevisiae. Analisis pengekspresan gen menggunakan kaedah RT-qPCR menunjukkan pengekspresan gen mengekod protein yang terlibat di dalam pengambilan asid amino bebas iaitu permease adalah tinggi pada medium kompleks manakala pengekspresan kebanyakan gen mengekod protein yang terlibat dalam kitar semula dan biosintesis asid amino adalah tinggi di dalam medium minimum. Kesimpulannya, gen yang terlibat dalam penjanaan dan pengambilan asid amino bagi mikroorganisma psikrofil adalah terpulihara seperti mikroorganisma mesofil dan pengekspresan gen-gen ini adalah diaruh oleh kehadiran atau ketiadaan asid amino bebas pada persekitaran.

Kata kunci: Biosintesis asid amino; Glaciozyma antarctica; penanda jujukan terungkap; psikrofil

ABSTRACT

The mechanism of amino acid uptake and synthesis in the psychrophilic microorganism lives and proliferate in the extreme low-temperature environment is still not well understood. The aim of this study was to identify genes involved in amino acid generation for psychrophilic yeast, Glaciozyma antarctica and to determine their expression profiles when cells grow in media rich in amino acids or with limited amount of amino acids. The identification of genes was carried out by generating expressed sequence tags (EST) from two cDNA libraries generated from cells grown in complex growth medium and minimal growth medium without amino acids. A total of 3552 cDNA clones from each library was randomly picked and sequenced, generating 1492 unique transcripts (complex medium) and 1928 unique transcripts (minimal medium). Homology analyses have identified genes encoding proteins required for free amino acid uptake, biosynthesis of amino acids and recycling of amino acids based on the pathway used in the model yeast, Saccharomyces cerevisiae. Gene expression analysis by RT-qPCR showed that genes required for free amino acid uptake showed a higher expression profile in the complex medium, whereas the expression of most genes encode for proteins essential for biosynthesis and recycling of amino acids are higher in the minimal medium. In summary, genes that are involved in the generation and the uptake of amino acids for psychrophilic microorganism are conserved as in their mesophilic counterparts and the expression of these genes are regulated in the presence or absent of free amino acids in the surrounding.

Keywords: Amino acid biosynthesis; expressed sequence tag; Glaciozyma antarctica; psychrophiles

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*Corresponding author; email: munir@ukm.edu.my