Biopolymers and cell. 2005; 21 (6): 525 - 530

Construction of methylotrophic yeast Hansenula polymorpha strains over-producing formaldehyde dehydrogenase

O. M. Demkiv, S. Ya. Paryzhak, E. S. Krasovs'ka, O.V. Stasyk, G. Z. Gayda, A. A. Sibirny, M.V. Gonchar

The thermotolerant methylotrophic yeast H. polymorpha NCYC 495 (leu1-1) was chosen for genetic construction of the strain, over-producing glutathione-dependent formaldehyde dehydrogenase (FdDH). FLD1 gene with its own promoter was inserted into an integrative plasmid pYT1 containing LEU2 gene of Saccharomyces cerevisiae (as a selective marker) and the constructed vector was used for multi-copy integration of the target gene into genome   of leu1-1 recipient cells. Selection of the strains was carried out by leucine prototrophy, as well as by resistance to elevated concentrations of formaldehyde in the medium (up to 15 mM). The optimal cultivation conditions for the selected strains were established which resulted in maximal synthesis of the target enzyme. The best recombinant strain was chosen as a perspective over-producer of FdDH (with the protein activity of 4 mmoles × min^-1 ×mg^-1 in cell-free extract).

Key words: glutathione-dependent formaldehyde dehydrogenase, methylotrophic yeast Hansenula polymorpha, genetic construction.