Sains Malaysiana 40(4)(2011): 323–329
Characterising Yeast Isolates from Malaysia towards the
Development of Alternative Heterologous Protein Expression Systems
(Pencirian Pencilan Yis dari Malaysia untuk Pembangunan
Sistem Pengekspresan Protein Heterolog Alternatif)
Douglas Law Sie Nguong1, Lim Yao Jun1, Nor Idaya Yatim1, Sheila Nathan2, Abdul Munir Abdul Murad1, Nor Muhammad Mahadi2 & Farah Diba Abu Bakar1*
1School of Biosciences and
Biotechnology, Faculty of Science and Technology
Universiti
Kebangsaan Malaysia, 43600 Bangi, Selangor D.E., Malaysia
2Malaysia Genome Institute,
Heliks Emas Block, UKM-MTDC Technology Centre
Universiti
Kebangsaan Malaysia, 43600 Bangi, Selangor D.E., Malaysia
Diserahkan:
20 Julai 2010 / Diterima: 18 Ogos 2010
ABSTRACT
Yeasts with GRAS (Generally Regarded as Safe)
status are commonly used as hosts for heterologous protein production. Yeasts
are suitable expression hosts as they have been extensively characterised
genetically. The objective of this project was to isolate yeasts from Malaysian
food sources and subsequently to develop these as alternative hosts for
heterologous protein production. Yeasts were isolated from Malaysian
traditional fermented food namely ‘tapai’, ‘tuak’ and ‘ragi’. A total of 23
isolates were obtained and subjected to molecular identification by
amplification and sequencing of the universally conserved ribosomal internal
transcribed spacer (ITS), 26S rDNA and 18S rDNA sequences.
We identified three species of yeasts, Saccharomyces cerevisiae, Hanseniaspora guilliermondii and Pichia
anomala, which have a long tradition of usage in food production and have no
adverse effects on humans. To test the feasibility of the yeasts as
heterologous expression hosts, we have constructed an integrative vector,
p1926Zeo containing the yeast 26S rDNA and Zeocin® resistance
cassette. The p1926Zeo vector was linearised and transformed into both P.
anomala and H. guilliermondii isolates via electroporation. Both
hosts were successfully transformed at a relatively high efficiency. The
transformants obtained had a growth profile similar to the respective wild
type, indicating that integration of the plasmids into the host chromosome did
not affect growth. These transformants were stable as they exhibited resistance
to Zeocin even after 20 generations. Thus, both P. anomala and H.
guilliermondii isolates exhibited the potential to be further developed as
alternative heterologous protein expression hosts.
Keywords: Hanseniaspora
guilliermondii; heterologous protein; Pichia anomala; yeast
expression systems
ABSTRAK
Yis berstatus GRAS (Generally Regarded as Safe) merupakan hos
yang kerap digunakan dalam penghasilan protein heterolog. Yis merupakan hos pengekspresan
yang sesuai kerana maklumat genetiknya telah difahami secara mendalam. Objektif
projek ini adalah untuk memencilkan yis daripada sumber makanan di Malaysia dan
seterusnya digunakan dalam pembangunan hos alternatif bagi penghasilan protein
heterolog. Yis dipencilkan daripada makanan terfermentasi seperti tapai, ragi
dan tuak. Sebanyak 23 pencilan yis telah dikenal pasti berdasarkan pada
pengecaman molekul yang melibatkan amplifikasi dan penjujukan kawasan yang
terpulihara iaitu penjarak jujukan dalaman (ITS),
jujukan 26S rDNA serta jujukan 18S rDNA. Tiga pencilan yis telah dikenalpasti
mempunyai potensi untuk dijadikan hos alternatif bagi penghasilan protein
heterolog iaitu Saccharomyces
cerevisiae, Hanseniaspora guiiliermondii dan Pichia anomala yang telah lama digunakan dalam proses
penghasilan makanan dan tidak mendatangkan kemudaratan kepada manusia. Untuk
menguji kesesuaian yis tersebut sebagai hos pengekspresan, kami telah membina
vektor terintergrasi p1926Zeo. Vektor p1926Zeo mengandungi jujukan 26SrDNA yis
dan kaset keringtangan terhadap antibiotik Zeocin®. Vektor p1926Zeo telah
dilinearkan dan di transformasikan ke dalam kedua-dua pencilan P. anomala dan H. guilliermondii melalui kaedah elektroporasi. Kedua-dua hos tersebut
berjaya ditransformasi-kan pada kecekapan yang agak tinggi. Profil pertumbuhan
transforman mempunyai kesamaan dengan profil pertumbuhan yis asal tersebut. Ini
menunjukkan intergrasi plasmid ke dalam kromosom hos tidak menjejaskan
pertumbuhan yis tersebut. Transforman ini juga menunjukkan kerintangan terhadap
Zeocin walaupun selepas 20 generasi. Oleh itu, kedua-dua pencilan P. anomala dan H. guilliermondii mempunyai potensi untuk dibangunkan sebagai hos pengekspresan protein heterlog
alternatif.
Kata kunci: Hanseniaspora
guiiliermondii; Pichia anomala; protein heterolog; sistem
pengekspresan yis
RUJUKAN
Agaphonov, M.O.,
Beburov, M.Y., Ter-Avanesyan, M.D. & Smirnov, V.N. 1995. A
distruption-displacement approach for the targeted integration of foreign genes
in Hansenula polymorpha. Yeast 10: 509-513.
Beburov, M.Y., Ter-Avanesyan, M.D. &
Smirnov, V.N. 1995. A distruption-displacement approach for the targeted
integration of foreign genes in Hansenula polymorpha. Yeast 10: 509-513.
Bergkamp, R.J.M., Kool, I.M., Geerse,
R.H. & Planta, R.J. 1992. Multi-copy integration of the
α-galactosidase gene from Cyamopsis tetragonoloba into the
ribosomal DNA of Kluyveromyces lactis. Current Genetics 21: 365-370.
Brito, N., Perez, M.D., Perdomo, G.,
Gonzalez, C., Garcia-Lugo, P. & Sivero, J.M. 1999. A set of Hansenla
polymorpha integrative vectors to construct lacZ fusions. Journal
of Applied Microbiology and Biotechnology 53: 23-29.
Calmels, T., Parriche, M., Burand, H.
& Tiraby, G. 1991. High efficiency transformation of Tolypocladium
geodes conidiospores top resistance. Current Genetics 2: 309-314.
Carlson, M., Taussig, R., Kustu, S.
& Olsen, M.V. 1983. The secreted form of invertase in Saccharomyces
cerevisiae is synthesized from an mRNA encoding a signal sequence. Molecular
and Cellular Biology 3: 439-447.
Cereghino, L. & Cregg, J.M. 2000.
Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews 24: 45-66.
Cox, H., Mead, D., Sudbery, P., Elands,
M. & Evans, L. 2000. Constitutive expression of recombinant proteins in the
methylotrophic yeast Hansenula polymorpha using the PMA 1 promoter. Yeast 16: 1191-1203.
Cregg, J.M. 1999. Expression in
methylotrophic yeast Pichia pastoris. In Gene expression systems:
using nature for the art of expression, edited by Fernandez, J.M., Hoefftler,
J.P. San Diego, CA: Academic press.
Dietrich, F.S., Voegeli, S., Brachat,
S., Lerch, A., Gates, K., Steiner, S., Mohr, C., Pohlmann, R., Luedi, P., Choi,
S., Wing, R.A., Flavier, A., Gaffney, T.D. & Philippsen, P. 2004. The Ashbya
gossypii genome as a tool for mapping the ancient Saccharomyces
cerevisiae genome. Science 304: 304-307.
Gellisen, G. 2005. Production
of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems. Germany:
Wiley-VCH Verlag GmbH & Co.
Gellissen, G. 2000.
Heterologous protein production in methylotrophic yeasts. Applied
Microbiology and Biotechnology 54: 741-750.
Gellissen, G., Hollenberg,
C.P. & Janowicz, Z.A. 1995. Gene expression in methylotrophic yeast. Bioprocessing
Technology 22: 195-239.
Gellissen, G. & Melber,
K. 1996. Methylotrophic yeast Hansenula polvmorpha as production
organism for recombinant pharmaceuticals. Drug Research 46: 943-948.
Guerra, G.O., Rubio, I.G.S.,
da Silva-Filho, C.G., Bertoni, R.A., dos Santos-Govea, R.C. & Vicente, E.J.
2006. A novel system of genetic transformation allows multiple integrations of
desired gene in Saccharomyces cerevisiae chromosomes. Journal of
Microbiological Methods 67: 437-445.
Hellwig, S., Stockmann, C.,
Gellissen, G. & Buchs, J. 2005. Comparative fermentation. In Production
of recombinant proteins: novel microbial and eukaryotic expression systems, edited
by Gellisen, G. Germany: Wiley-VCH Verlag GmbH & Co.
Ilgen, C., Lin-Cereghino, J.
& Cregg, J.M. 2005. Pichia pastoris. In Production of Recombinant
Proteins: Novel Microbial and Eukaryotic Expression Systems, edited by
Gellisen, G. Germany: Wiley-VCH Verlag GmbH & Co.
Jeyaram, K., Mohendro Singh,
W., Capece, A. & Romano, P. 2008. Molecular identification of yeast species
associated with ‘Hamei’- A traditional starter used for rice wine production in
Manipur, India. International Journal of Food Microbiology 124: 115-125.
Madzak, C., Gaillardin, C.
& Beckerich, J-M. 2004. Recombinant proteins and host cell physiology. Journal
of Biotechnology 109(1-2): 63-81.
Miller, T.K., Patel, C. &
Selitrennikoff, C.P. 2007. Construction of a Saccharomyces cerevisiae strain
expressing the Leishmania major nucleotide hydrolyse gene. International
Journal of Antimicrobial Agents 29: 103-107.
Murray, J. 1971. Paulesco and
the isolation of insulin. Journal of the History of Medicine and Allied
Sciences 26: 150-157.
Medlin, L., Elwood, H.J.,
Stickel, S. & Sogin, M.L. 1988. The characterization of enzymatically
amplified eukaryotic 16S-like rRNA-coding regions. Gene 71: 491-499.
Sohn, J.H., Choi, E.S., Kim,
C.H., Agaphonov, M.O., Ter-Avabesyan, M.D., Rhee, J.S. & Rhee, J.K. 1996. A
novel autonomously replicating sequences (ARS) for multiple integration in the
yeast Hansenula polymorpha DL-1. Journal of Bacteriology 178:
4420-4428.
Tamas, L. & Shewry, P.R.
2006. Heterlogous expression and protein engineering of wheat gluten proteins. Journal
of Cereal Science 43: 259-274.
Teixeira, R.M., Cavalheiro,
D., Ninow, J.L. & Furigo, A. Jr. 2002. Optimization of Acetion production
by Hanseniaspora guilliermondii using experimental design. Brazilian
Journal of Chemical Engineering 19(2): 181-186.
Watson, J.D. & Crick,
F.H.C. 1953. Molecular structure of nucleic acids. A structure for
deoxyribosenucleic acid. Nature 171: 737-738.
White, T., Burns, T., Lee, S.
& Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal
RNA genes for phylogenetics. In PCR Protocols Innis, M.A., Gelfand,
D.H., Sninsky, J.J., White, T.K. (eds), London: Academic Press, pp. 315-322.
Wu, S. & Letchworth, G.J.
2004. High efficiency transformation by electroporation of Pichia pastoris pretreated
with lithum acetate and dithiothreitol. BioTechniques 36: 152-154.
Yoshizawa, K. 1979. Treament
of waste-water discharge from a sake brewery using yeast. Fermentation
Technology 56: 389-395.
*Pengarang untuk surat-menyurat;
e-mail: fabyff@ukm.my
|