Mìkrobìologìâ ì Bìotehnologìâ (Sep 2013)
CONSTRUCTION OF METHYLOTROPHIC YEAST HANSENULA POLYMORPHA STRAINS OVERPRODUCING FORMALDEHYDE REDUCTASE
Abstract
The aim of the presented work was to obtain recombinant strains of methylotrophic yeast Hansenula polymorpha overproducing formaldehyde reductase (FR) which could be used bioanalyticaly. Methods. The yeast cells were grown in a synthetic Burkholder medium containing the following (g/l): КН2РО4 – 1; (NH4)2SO4 – 3.5; MgSO4½7H2O – 0.5; CaCl2 – 0.1; with the standard content of trace elements and 0.05% (wt/vol) yeast extract. 1% (wt/vol) glucose or 1% methanol (vol/vol) served as the sources of carbon. Enzyme activities in the cell-free extracts were determined by spectrophotometry (Shimadzu-UV-1650) at 340 nm by the rates of NADH formation for alcohol dehydrogenase (ADH), or NADH uptake for formaldehyde reductase (FR) at room temperature (20–25 °C). The activity (A) for each enzyme (in μmol∙min–1∙mg–1 protein) was calculated by the formula: A = A+substrate – A–substrate, taking into account the nonspecific background reactions. Results. We constructed the expression plasmid р21ADH1Sc contained ADH1 Saccharomyces cerevisiae gene under the strong constitutive promotеr of the H. polymorpha glyceraldehyde 3-phosphate dehydrogenase gene (GAP1). H. polymorpha NCYC 495 (leu1-1) yeast cells were transformed by electroporation. Selection of the integrants was carried out in accordance with their resistance to zeocine. Presence of the recombinant plasmid, containing the ADH1 S. cerevisiae gene, in genome of transformants was checked using polymerase chain reaction (PCR). Conclusions. Recombinant strains were characterized by 4–6 times higher activity of alcohol dehydrogenase (ADH) and 2–3 times higher activity of FR in cellfree extracts, compared to the parental strain. Stable transformants have elevated resistance to formaldehyde (up to 10 mM) in methanol containing medium.
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