Transformation of methylotrophic yeast Hansenula polymorpha: Cloning and expression of genes

✍ Scribed by Dr. L. P. Tikhomirova; R. N. Ikonomova; E. N. Kuznetsova; I. I. Fodor; L. V. Bystrykh; L. R. Aminova; Yu. A. Trotsenko

• Publisher: John Wiley and Sons
• Year: 1988
• Tongue: English
• Weight: 671 KB
• Volume: 28
• Category: Article
• ISSN: 0233-111X
• DOI: 10.1002/jobm.3620280509

✦ Synopsis

We developed a host-vector system for transformation and gene cloning experiments using the methylotrophic yeast Hansenula p o l y w p h a . Regeneration of protoplasts in a medium containing polyethylene glycol before plating made transformation more efficient and reproducible (2 to 3 -lo4 pg DNA). The frequency of transformation was significantly lower when dominant resistance marker Cuplr was used for transformant selection. The transformation system developed was used t o clone the DNA fragment which complements functionally the defect in the dihydroxyacetone kinase (DHAK*) activity of a H. polymorpha mutant strain. The DNA insert isolated was shown to increase by up to ten times the activity of DHAK in transformants carrying recombinant plasmids. When recombinant plasmids were introduced into S . cerevkiae, the transformants obtained acquired the ability to grow on the medium with dihydroxyacetone as a sole carbon source and the activity of DHAK was observed.