Transformation of methylotrophic yeast Hansenula polymorpha: cloning and expression of genes.
We developed a host-vector system for transformation and gene cloning experiments using the methylotrophic yeast Hansenula polymorpha. Regeneration of protoplasts in a medium containing polyethylene glycol before plating made transformation more efficient and reproducible (2 to 3 x 10(4) micrograms DNA). The frequency of transformation was significantly lower when dominant resistance marker Cup1r was used for transformant selection. The transformation system developed was used to 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. cerevisiae, 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.
Tikhomirova
L P
LP
Institute of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, Pushchino, Moscow Region.
Ikonomova
R N
RN
Kuznetsova
E N
EN
Fodor
I I
II
Bystrykh
L V
LV
Aminova
L R
LR
Trotsenko YuA
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D005819:Genetic Markers; D005822:Genetic Vectors; D000432:Methanol; D010770:Phosphotransferases / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor); D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids; D004718:Saccharomycetales / Q000235:genetics*; D014170:Transformation, Genetic
D005819:Genetic Markers; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C023132:glycerone kinase; D000432:Methanol