Characterization of the Hansenula polymorpha PUR7 gene and its use as selectable marker for targeted chromosomal...

The Hansenula polymorpha genes encoding the putative functional homologs of the enzymes involved in the seventh and eighth step in purine biosynthesis, HpPUR7 and HpPUR8, were cloned and sequenced. An overexpression vector designated pHIPA4 was constructed, which contains the HpPUR7 gene as selectable marker and allows expression of genes of interest via the strong, inducible alcohol oxidase promoter. An ade11 auxotrophic mutant that is affected in the activity of the HpPUR7 gene product was used to construct strain NCYC495 ade11.1 leu1.1 ura3. This strain grew on methanol at wild-type rates (doubling time of approximately 4 h) and is suitable for independent introduction of four expression cassettes, each using one of the markers for selection, in addition to the zeocin resistance marker. It was subsequently used as a host for overproduction of two endogenous peroxisomal matrix proteins, amine oxidase and catalase. Efficient site-specific integration of pHIPA4 and overproduction of amine oxidase and catalase is demonstrated. The expression cassette appeared to be pre-eminently suited to mediate moderate protein production levels. The advantages of pHIPA4 and the new triple auxotrophic strain in relation to the use of H. polymorpha as a versatile cell factory or as a model organism for fundamental studies on the principles of peroxisome homeostasis is discussed.