Cloning and characterization of glucokinase from a methylotrophic yeast Hansenula polymorpha: different effects on glucose repression in H. polymorpha and Saccharomyces cerevisiae.

Abstract Glucokinase gene ( HPGLK1 ) was cloned from a methylotrophic yeast Hansenula polymorpha by complementation of glucose-phosphorylation deficiency in a H . polymorpha double kinase-negative mutant A31-10 by a genomic library. An open reading frame of 1416 nt encoding a 471-amino-acid protein with calculated molecular weight 51.6 kDa was characterized in the genomic insert of the plasmid pH3. The protein sequence deduced from HPGLK1 exhibited 55 and 46% identity with glucokinases from Saccharomyces cerevisiae and Aspergillus niger , respectively. The enzyme phosphorylated glucose, mannose and 2-deoxyglucose, but not fructose. Transformation of HPGLK1 into A31-10 restored glucose repression of alcohol oxidase and catalase in the mutant. Transformation of HPGLK1 into S . cerevisiae triple kinase-negative mutant DFY632 showed that H . polymorpha glucokinase cannot transmit the glucose repression signal in S . cerevsiae : synthesis of invertase and maltase in respective transformants was insensitive to glucose repression similarly to S . cerevisiae DFY568 possessing only glucokinase.