STUDIES OF THE ENDOMYCES FIBULIGER GLUCOAMYLASE GENE BY TRANSFORMATION IN YARROWIA LIPOLYTICA

Abstract

In early studies, Endomyces fibuliger and Yarrowia lipolytica were classified under the same genus, and in recent studies, stable intergeneric hybrids have been produced between the two yeasts. These intergeneric hybrids have the ability to express both the amylolytic activity of E. fibuliger as well as the lipolytic activity of Y. lipolytica. Hence, we can postulate that some of the genes of E. fibuliger might be able to be expressed and secreted in Y. lipolytica . This project is aimed at producing a strain of Y. lipolytica that is not only able to hydrolyse lipids, but also able to breakdown starch and other polysaccharides. To achieve this, the glucoamylase gene (GLUI) of E. fibuliger was transformed into Y. lipolytica using a constructed plasmid which carried both the GLUI gene of E. fibuliger as well as the LEU2 gene of Y. lipolytica. This plasmid, called pNLGluES1, was transformed into two strains of Y. lipolytica, namely, El22 and 21501-4 by targeting the integration of the plasmid pNLGluESl into the LEU2 gene locus of the Y. lipolytica chromosome. These transform ants contained the E. fibuliger glucoamylase gene in its native form, under its own promoter and regulatory DNA sequences. Integration of the plasmids at the LEU2 gene locus was confirmed by Southern blot analysis. However, no production of functional glucoamylase was detected from these transformants, either extracellularly or intracellularly. Furthermore, Northern blot analysis and reverse transcriptase PCR experiments demonstrated that no glucoamylase mRNA was detectable in these Y. lipolytica transformants, indicating that the GLUI gene was probably not transcribed. In a further study, plasmids were constructed to introduce the glucoamylase gene of E. fibuliger under the control of the Y. lipolytica XPR2 (alkaline extracellular protease) gene sequences. In the plasmid construct pNL354Giul, the DNA sequences of the GLUI gene coding for the structural protein were introduced downstream of the XPR2 promoter region. Here, the glucoamylasc protein still retains its own secretion signal peptide region, but the transcription of the GLUI gene, is under the control of the XPR2 promoter. In another constructed plasmid, pNL476Giul, only the GLUI DNA sequences coding for the mature part of the structural glucoamylase were cloned downstream of the XPR2 promoter-prepro- expression cassette. In other words, not only is the transcription of the GLUI gene under the control of the XP R2 promoter, but the secretion signals of any 'hybrid' glucoamylase protein expected to be produced, are those of the Y. lipolytic a alkaline extracellular protease protein. The plasmids pNL354Giul and pNL476Giul were transformed into Y. lipolytica El22 and 21501-4 by targeted integration into the XPR2 locus of the Y. lipolytica chromosomal DNA. Integration was again confirmed by Southern blot analysis of the chromosomal DNAs of the parent strains and the transformants. Glucoamylase assays carried out on all the transform ants, harbouring either plasmid, showed that no glucoamylase activity was detectable both extracellularly or intracellularly. Since no functional glucoamylase was able to be produced from any of the previous Y. lipolytica transformants, the GLUI gene was transformed into strain S5, a weakly amylolytic E. fibuliger -Y. lipolytica intergeneric hybrid. The plasmid pNLI826Giul, which carried both GLUI gene and the ribosomal DNA (rONA) sequences of E. fibuliger, was transformed by targeting integration into the rDNA locus of S5. By selecting the transformants on media containing starch, a number of transform ants were found to exhibit an increased ability to hydrolyse starch. Southern blot analysis was used to confirm the presence of the plasmid in the chromosomal DNAs of the transformants. The results of this study seem to indicate that at the molecular level, the transcription signals and post-translational pathways for E. fibuliger and Y. lipolytica arc probably distinctly dissimilar, and that the phenomenon of co-expression observed in the intergeneric hybrids probably requires the existence of components of both E. fibuliger and Y. lipolytica cellular systems in the hybrids.