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 MURAD1*
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
Diserahkan: 15 September 2017/Diterima:
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
RUJUKAN
Andréasson, C., Neve,
E.P.A. & Ljungdah, P.O. 2004. Four permeases import proline and the toxic
proline analogue azetidine-2-carboxylate into yeast. Yeast 21: 193-199.
Bharudin, I., Zaki,
N.Z., Bakar, F.D.A., Mahadi, N.M., Najimudin, N., Illias, R.M. & Murad,
A.M.A. 2014. Comparison of RNA extraction methods for transcript analysis from
the psychrophilic yeast, Glaciozyma antarctica. Malaysian Applied
Biology 43: 71-79.
Boer, V.M., Amini, S. &
Botstein, D. 2008. Influence of genotype and nutrition on survival
and metabolism of starving yeast. Proceedings of the National
Academy of Sciences of the United States of America 105:
6930-6935.
Conesa, A., Götz, S.,
García-Gómez, J.M., Terol, J., Talón, M. & Robles, M. 2005. Blast2GO: A
universal tool for annotation, visualization and analysis in functional
genomics research. Bioinformatics 21: 3674-3676.
Coutts, G., Thomas, G.,
Blakey, D. & Merrick, M. 2002. Membrane sequestration of the signal
transduction protein GlnK by the ammonium transporter AmtB. The EMBO Journal 21: 536-545.
D'Amico, S., Collins, T.,
Marx, J.C., Feller, G., Gerday, C. & Gerday, C. 2006. Psychrophilic
microorganisms: Challenges for life. EMBO Reports 7: 385-389.
Ewing, B. & Green,
P. 1998. Base-calling of automated sequencer traces using phred. II. Error
probabilities. Genome Research 8(3): 186-194.
Finley, D., Ulrich,
H.D., Sommer, T. & Kaiser, P. 2012. The ubiquitin-proteasome system of Saccharomyces
cerevisiae. Genetics 192: 319-360.
Firdaus-Raih, M.,
Hashim, N.H.F., Bharudin, I., Abu Bakar, M.F., Huang, K.K., Alias, H., Lee,
B.K.B., Mat Isa, M.N., Mat- Sharani, S., Sulaiman, S., Tay, L.J., Zolkefli, R.,
Muhammad Noor, Y., Law, D.S.N., Abdul Rahman, S.H., Md-Illias, R., Abu Bakar,
F.D., Najimudin, N., Abdul Murad, A.M. & Mahadi, N.M. 2018. The Glaciozyma
antarctica genome reveals an array of systems that provide sustained
responses towards temperature variations in a persistently cold habitat. PLoS
One 13: e0189947.
George, R.A. 2001.
StackPACK clustering system. Briefings in Bioinformatics 2: 394-397.
Good, M.C., Zalatan,
J.G. & Lim, W.A. 2011. Scaffold proteins: Hubs for controlling the flow of
cellular information. Science 332: 680-686.
Gretzmeier, C.,
Eiselein, S., Johnson, G.R., Engelke, R., Nowag, H., Zarei, M., Küttner, V.,
Becker, A.C., Rigbolt, K.T.G., Høyer-Hansen, M., Andersen, J.S., Münz, C.,
Murphy, R.F. & Dengjel, J. 2017. Degradation of protein translation
machinery by amino acid starvation-induced macroautophagy. Autophagy 13:
1064-1075.
Hashim, N.H.F.,
Bharudin, I., Nguong, D.L.S., Higa, S., Bakar, F.D.A., Nathan, S., Rabu, A.,
Kawahara, H., Illias, R.M., Najimudin, N., Mahadi, N.M. & Murad, A.M.A.
2013. Characterization of Afp1, an antifreeze protein from the psychrophilic
yeast Glaciozyma antarctica PI12. Extremophiles 17: 63-73.
Iraqui, I., Vissers, S.,
Bernard, F., de Craene, J.O., Boles, E., Urrestarazu, A. & André, B. 1999.
Amino acid signaling in Saccharomyces cerevisiae: A permease-like sensor
of external amino acids and F-box protein Grr1p are required for
transcriptional induction of the AGP1 gene, which encodes a
broad-specificity amino acid permease. Molecular and Cellular Biology 19:
989-1001.
Jørgensen, M.U., Bruun,
M.B., Didion, T. & Kielland-Brandt, M.C. 1998. Mutations in five loci
affecting GAP1- independent uptake of neutral amino acids in yeast. Yeast 14:
103-114.
Kitzing, K., Auweter,
S., Amrhein, N. & Macheroux, P. 2004. Mechanism of chorismate synthase.
Role of the two invariant histidine residues in the active site. Journal of
Biological Chemistry 279(10): 9451-9461.
Klasson, H., Fink, G.R.
& Ljungdahl, P.O. 1999. Ssy1p and Ptr3p are plasma membrane components of a
yeast system that senses extracellular amino acids. Molecular and Cellular
Biology 19: 5405-5416.
Kohlhaw, G.B. 2003.
Leucine biosynthesis in fungi: Entering metabolism through the back door. Microbiology
and Molecular Biology Reviews 67: 1-15.
Lecker, S.H., Goldberg,
A.L. & Mitch, W.E. 2006. Protein degradation by the
ubiquitin–proteasome pathway in normal and disease states. Journal of
the American Society of Nephrology 17: 1807-1819.
Maeda, H. &
Dudareva, N. 2012. The shikimate pathway and aromatic amino acid biosynthesis
in plants. Annual Review of Plant Biology 63: 73-105.
Moyer, C.L. &
Morita, R.Y. 2007. Psychrophiles and Psychrotrophs. Encyclopedia of Life
Science. New Jersey: John Wiley & Sons, Ltd. pp. 1-6.
Murad, A.M.A., Badrun,
R., Shahabudin, S., Kamaruddin, S., Zairun, M.A., Khairuddin, F., Mahadi, N.M.,
Illias, R.M., Zainal, Z. & Bakar, F.D.A. 2013. Pengenalpastian dan
pencirian gen Trichoderma virens UKM1 mengekod enzim terlibat dalam
pencuraian kitin krustasea. Sains Malaysiana 42(6): 715-724.
Okamura, E. & Hirai,
M.Y. 2017. Novel regulatory mechanism of serine biosynthesis associated with
3-phosphoglycerate dehydrogenase in Arabidopsis thaliana. Scientific
Reports 7: 3533.
Payne, S.H. &
Loomis, W.F. 2006. Retention and loss of amino acid biosynthetic pathways based
on analysis of whole-genome sequences. Eukaryotic Cell 5: 272-276.
Petti, A.A., Crutchfield,
C.A., Rabinowitz, J.D. & Botstein, D. 2011. Survival of starving
yeast is correlated with oxidative stress response and nonrespiratory
mitochondrial function. Proceedings of the National Academy of
Sciences of the United States of America 108: E1089-E1098.
Schmidt, M.C.,
McCartney, R.R., Zhang, X., Tillman, T.S., Solimeo, H., Wölfl, S., Almonte, C.
& Watkins, S.C. 1999. Std1 and Mth1 Proteins interact with the glucose
sensors to control glucose-regulated gene expression in Saccharomyces
cerevisiae. Molecular and Cellular Biology 19: 4561-4571.
Spellman, P.T.,
Sherlock, G., Zhang, M.Q., Iyer, V.R., Anders, K., Eisen, M.B., Brown, P.O., Botstein,
D. & Futcher, B. 1998. Comprehensive identification of cell
cycle–regulated genes of the yeast Saccharomyces cerevisiae by
microarray hybridization. Molecular Biology of the Cell 9: 3273-3297.
Springael, J.Y. & André, B. 1998.
Nitrogen-regulated ubiquitination of the Gap1 permease of Saccharomyces
cerevisiae. Molecular Biology of the Cell 9: 1253-1263.
Stekel, D.J., Git, Y. & Falciani, F.
2000. The comparison of gene expression from multiple cDNA libraries. Genome
Research 10: 2055-2061.
Sterky, F. & Lundeberg, J. 2000.
Sequence analysis of genes and genomes. Journal of Biotechnology 76:
1-31.
Susko, E. & Roger, A.J. 2004.
Estimating and comparing the rates of gene discovery and expressed sequence tag
(EST) frequencies in EST surveys. Bioinformatics 20: 2279-2287.
Thevelein, J.M., Geladé, R., Holsbeeks,
I., Lagatie, O., Popova, Y., Rolland, F., Stolz, F., Van de Velde, S., Van
Dijck, P., Vandormael, P., Van Nuland, A., Van Roey, K., Van Zeebroeck, G.
& Yan, B. 2005. Nutrient sensing systems for rapid activation of the
protein kinase A pathway in yeast. Biochemical Society Transactions 33(1):
253-256.
Tang, C., Gong, M., Li, S. & Zhu, C.
2012. Construction of cDNA library of Aspergillus niger H1 and screening
of phosphate-dissolving related gene. Wei Sheng Wu Xue Bao 52(3):
311-317.
Tu, Y., Chen, C., Pan, J., Xu, J., Zhou,
Z.G. & Wang, C.Y. 2012. The ubiquitin proteasome pathway (UPP) in the
regulation of cell cycle control and DNA damage repair and its implication in
tumorigenesis. International Journal of Clinical and Experimental Pathology 5:
726-738.
Wittenberg, C. & Reed, S.I. 2005.
Cell cycle-dependent transcription in yeast: Promoters, transcription factors,
and transcriptomes. Oncogene 24(17): 2746-2755.
Yin, H., Zhang, R., Xia, M., Bai, X.,
Mou, J., Zheng, Y. & Wang, M. 2017. Effect of aspartic acid and glutamate
on metabolism and acid stress resistance of Acetobacter pasteurianus.
Microbial Cell Factories 16(1): 109.
Zabriskie, T.M. & Jackson, M.D. 2000.
Lysine biosynthesis and metabolism in fungi. Natural Product Reports 17(1):
85-97.
Zhang, P., Du, G., Zou, H., Chen, J.,
Xie, G., Shi, Z. & Zhou, J. 2016. Effects of three permeases on arginine
utilization in Saccharomyces cerevisiae. Scientific Reports 6:
20910.
*Pengarang untuk
surat-menyurat; email: munir@ukm.edu.my
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