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Construction of flavocytochrome b 2-overproducing strains of the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta)

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Abstract

L-Lactate cytochrome c oxidoreductase (flavocytochrome b 2, FC b 2) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b 2 producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b 2. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b 2 activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b 2 producer characterized by a sixfold increased (to 3 μmol min−1 mg−1 protein in cell-free extract) activity of the enzyme.

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Authors and Affiliations

  1. Institute of Cell Biology, National Academy of Sciences of Ukraine, ul. Drahomanova 14/16, Lviv, 79005, Ukraine

    K. V. Dmitruk, O. V. Smutok, M. V. Gonchar & A. A. Sibirnyi

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  1. K. V. Dmitruk
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  2. O. V. Smutok
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  3. M. V. Gonchar
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  4. A. A. Sibirnyi
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Correspondence to A. A. Sibirnyi.

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Original Russian Text © K.V. Dmitruk, O.V. Smutok, M.V. Gonchar, A.A. Sibirnyi, 2008, published in Mikrobiologiya, 2008, Vol. 77, No. 2, pp. 213–218.

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Dmitruk, K.V., Smutok, O.V., Gonchar, M.V. et al. Construction of flavocytochrome b 2-overproducing strains of the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta). Microbiology 77, 181–185 (2008). https://doi.org/10.1134/S0026261708020100

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