Biopolym. Cell. 2005; 21(6):525-530.
Molecular and Cell Biotechnologies
Construction of methylotrophic yeast Hansenula polymorpha strains over-producing formaldehyde dehydrogenase
- Institute of Cell Biology, NAS of Ukraine
14/16, Drahomanov Str., Lviv, Ukraine, 79005
Abstract
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).
Keywords: glutathione-dependent formaldehyde dehydrogenase, methylotrophic yeast Hansenula polymorpha, genetic construction
Full text: (PDF, in Ukrainian)
References
[1]
Hansen H, Hollenberg CP. Hansenula polymorpha (Pichia angusta). In: Nonconventional Yeasts in Biotechnology . Springer 1996;293–311.
[2]
Aggelis G, Margariti N, Kralli C, Flouri F. Growth of Candida boidinii on methanol and the activity of methanol-degrading enzymes as affected from formaldehyde and methylformate. J Biotechnol. 2000;80(2):119-25.
[3]
Formaldehyde. In: Environmental Health Criteria 89. Geneva: World Health Organization, 1989. (Polish Transl: Formaldehyd. Kryteria zdrowne srodowiska. T. 89. Jydu: Instytut Medycyny Pracy, 1993 1-191.
[4]
Szende B, Tyih?k E, Sz?k?n G, K?tay G. Possible Role of Formaldehyde in the Apoptotic and Mitotic Effect of 1-Methyl-Ascorbigen. Pathol Oncol Res. 1995;1(1):38-42.
[5]
Tyihak E, Trezl L, Szende B. Formaldehyde Cycle and the Phases of Stress Syndrome. Ann NY Acad Sci.; 1998;851:259-70.
[6]
Gonchar MV. [Alternative mechanisms of detoxication of formaldehyde, formate, and hydrogen peroxide in methylotrophic yeasts]. Mikrobiol Z. 2000;62(1):30-9.
[7]
Maidan NN, Gonchar MV, Sibirny AA. Oxidation of exogenous formaldehyde in methylotrophic and nonmethylotrophic yeast cells. Biochemistry (Mosc). 1997;62(6):636-40.
[8]
Feron VJ, Til HP, de Vrijer F, Woutersen RA, Cassee FR, van Bladeren PJ. Aldehydes: occurrence, carcinogenic potential, mechanism of action and risk assessment. Mutat Res. 1991;259(3-4):363-85.
[9]
Yu PH. Deamination of methylamine and angiopathy; toxicity of formaldehyde, oxidative stress and relevance to protein glycoxidation in diabetes. J Neural Transm Suppl. 1998;52:201-16.
[10]
Stasyk OV, Ksheminskaya GP, Kulachkovskii AR, Sibirnyi AA. Mutants of the methylotrophic yeast Hansenula polymorpha with impaired catabolite repression. Mikrobiologiia. 1997; 66(6):755-60.
[11]
Shen S, Sulter G, Jeffries TW, Cregg JM. A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. Gene. 1998;216(1):93-102.
[12]
Baerends RJ, Sulter GJ, Jeffries TW, Cregg JM, Veenhuis M. Molecular characterization of the Hansenula polymorpha FLD1 gene encoding formaldehyde dehydrogenase. Yeast. 2002;19(1):37-42.
[13]
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Lab. press, 1989. 452 p.
[14]
Faber KN, Haima P, Harder W, Veenhuis M, AB G. Highly-efficient electrotransformation of the yeast Hansenula polymorpha. Curr Genet. 1994;25(4):305-10.
