H. polymorpha strains used are derivatives of NCYC495 ade11.1 leu1.1 [17 (link)] and were grown at 37°C, 30°C or 25°C in either (i) rich complex media (YPD) containing 1% yeast extract, 1% peptone and 1% glucose, (ii) selective media containing 0.67% yeast nitrogen base without amino acids (DIFCO) supplemented with 0.5% glucose (YND), or (iii) mineral medium (MM) as described by Van Dijken et al. [18 (link)], supplemented with 0.25% ammonium sulphate using 0.5% glucose or 0.5% methanol as carbon source. For growth on plates, 2% granulated agar was added to the media. Whenever necessary, media were supplemented with 30 μg/ml leucine and 20 μg/ml adenine. For biochemical analysis, selected strains were pre-cultured at least three times in MM containing glucose and subsequently shifted to MM containing methanol to induce expression of genes under the control of the PAOX.
A high penicillin producing P. chrysogenum strain (DSM anti-infectives, Delft, The Netherlands) was used as a control and was grown for 48 hours on a defined penicillin production medium supplemented with 0.05% phenylacetic acid [19 (link)].
For cloning purposes, Escherichia coli DH5α (Gibco-Brl, Gaithesburg, MD) was used and grown at 37°C in LB medium (1% bacto-tryptone, 0.5% yeast extract, 0.5% NaCl), supplemented with 50 μg/ml kanamycin when required.
A high penicillin producing P. chrysogenum strain (DSM anti-infectives, Delft, The Netherlands) was used as a control and was grown for 48 hours on a defined penicillin production medium supplemented with 0.05% phenylacetic acid [19 (link)].
For cloning purposes, Escherichia coli DH5α (Gibco-Brl, Gaithesburg, MD) was used and grown at 37°C in LB medium (1% bacto-tryptone, 0.5% yeast extract, 0.5% NaCl), supplemented with 50 μg/ml kanamycin when required.
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