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MOLECULAR GENETICS AND BIOTECHNOLOGY OF METHYLOTROPHIC YEASTS Cornelis P Hollenberg Institute for Microbiology, Heinrich-Heine-University Duesseldorf, Universitaetsstr. 1, D 40225 Duesseldorf, Germany.
Code Number:BA96081
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In recent years an number of yeast species other than
Saccharomyces cerevisiae have become accessible for
molecular genetics and thereby for potential application in
biotechnology. In this respect the methylotrophic yeasts,
Hansenula polymorpha and Pichia stipitis have
already been proven to offer significant advantages over S.
cerevisiae for the production of certain heterologous
proteins. The methylotrophic yeasts share general pathways to
assimilate and catabolize methanol. Growth on methanol is
accompanied by a strong induction of peroxisomes and enzymes
involved in methanol metabolism. The strong inducible
promoters of the corresponding genes are used for the
expression of heterologous genes.
To improve the use of these promoters we have analyzed in great detail the regulation of the MOX promoter of the gene encoding methanol oxidase. We have found methods to circumvent the tight glucose repression of this promoter. In S. cerevisiae the MOX promoter can mediate a glucose repressible expression of a fused lac Z gene. This repression was mediated by MOX-B, a 240 bp promoter region which is also involved in catabolite repression in H. polymorpha. The negative regulation mediated by MOX-B was counteracted by Adr 1p, a transcription factor which has been shown to be involved in the derepression of ADH2 and, most remarkably, of genes encoding peroxisomal proteins. Details of the binding of Adr 1p to the MOX promoter and its action will be discussed. During Mox derepression, two different transcripts have been detected starting in the MOX promoter at -25 and -425, from which the smaller transcript accounts for the translation of methanol oxidase. Several small ORFs in the leader sequence of the larger transcript prevent efficient translation. A model for the function of the strong Mox promoter, involving the Adr 1p homologue and a coordinated switch between the two transcription points will be presented. Finally the suitability of H. polymorpha for application in biotechnology will be demonstrated by the discussion of promising developments of pharmaceutical proteins such as the production of hirudin and hepatitis B L- and S-antigenes. Moreover the use of recombinant H. polymorpha for bioconversion will be presented. Copyright 1996 Elfos Scientiae |
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