Characterisation of local yeast isolates towards developing alternative heterologous protein expression systems

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 is to isolate local 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 'tapa;', 'tuak' and 'ragi '. A total of 23 isolates were obtained and subjected to molecular identification, involving amplification and sequencing of the universally conserved ribosomal DNAs (rDNA), ribosomal internal transcribed spacer (ITS), 26S rDNA and 18S rDNA sequences. We identified three species of yeasts, namely, Saccharomyces cerevisiae, Hanseniaspora guiiliermondii and Pichia anomala which have a long tradition of usage in food production and have no adverse effects or harm 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 the Zeocin?? resistance cassette. The p1926Zeo vector was linearized and transformed into both P. anomala and H. guiiliermondii 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 are stable as they exhibit resistance to Zeocin even after 20 generations. Thus, the P. anomala and H. guiiliermondii isolates exhibit the potential to be further developed as alternative heterologous protein expression hosts.