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

Received: 20 July 2010 / Accepted: 18 August 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

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*Corresponding author; e-mail: fabyff@ukm.my