Mutant Hansenula polymorpha strain with constitutive alcohol oxidase and peroxisome biosynthesis.
A mutant of the methylotrophic yeast Hansenula polymorpha with constitutive alcohol oxidase (AOX) and peroxisome biosynthesis was obtained after UV treatment followed by cell plating on a medium containing methanol and 2-deoxy-D-glucose (DOG). DOG-resistant colonies of mutants were insensitive to catabolic repression by glucose and methanol. A selection procedure is described that allows the isolation of a mutant exhibiting a constitutive phenotype of AOX involved in methanol utilization. Furthermore, additional features of the constitutive presence of peroxisomes are demonstrated. 562 DOG-resistant colonies were tested, 24 of them demonstrating constitutive AOX formation. Based on quantitative analysis, one of the strains--DOG-13 was selected and its growth, biochemical and ultrastructural characteristics were examined. Its specific enzyme activity when cultivated on a yeast nitrogen base + 1% glucose (YNB + 1% Glucose) was found to reach 145 nmol x min(-1) x mg(-1) protein (compared to zero of the parent strain) after he 20th hour of cultivation. This was confirmed by fine-structure analysis, showing typical peroxisomes, which number and size increased with the enzyme activity. This study demonstrates a constitutive AOX and peroxisome biosynthesis by the mutant strain H. polymorpha DOG-13 obtained.
Hristozova
Tsonka
T
Institute of Microbiology, Bulgarian Academy of Sciences, Plovdiv. tshristozova@yahoo.com
Michailova
Lilia
L
Tuneva
Dimka
D
Gotcheva
Velitchka
V
Angelov
Angel
A
Roshkova
Zlatka
Z
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000528:radiation effects; D000431:Ethanol / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D007700:Kinetics; D008854:Microscopy, Electron; D016296:Mutagenesis; D020675:Peroxisomes / Q000235:genetics* / Q000528:radiation effects / Q000648:ultrastructure; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000528:radiation effects; D014466:Ultraviolet Rays
D000431:Ethanol; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D005947:Glucose