Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Abstract

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.

Fig. 1

Riboflavin productivity of Pichia guilliermondii wild-type strain and mutants Δfes1–77, Δvma1–17 and Δfra1–45. Cultures of P. guilliermondii wild-type strain R-66 (WT) and mutants were grown for 5 days (stationary phase) aerobically at 30 °C in a synthetic Burkholder medium supplemented with 3.6 μM of iron added as ammonium ferrous sulphate. Values are means ± SE of three independent experiments.

Fig. 2

Iron content in the cells of the Pichia guilliermondii wild-type strain and mutants Δfes1–77, Δvma1–17 and Δfra1–45. Cultures of P. guilliermondii wild-type strain R-66 (WT) and mutants were grown aerobically at 30 °C in a synthetic Burkholder medium supplemented with 3.6 μM of iron added as ammonium ferrous sulphate. Cells from the middle exponential growth phase were used to measure the iron content. Values are means ± SE of three independent experiments.

Fig. 3

Sensitivity of Pichia guilliermondii wild-type strain and mutants Δfes1–77, Δvma1–17 and Δfra1–45 to H₂O₂. Cultures of P. guilliermondii wild-type strain R-66 (WT) and mutants were grown aerobically in YPD medium for 16 h, diluted to an OD_{600 nm} of 0.2 and allowed to grow for 3.5 h. Aliquots (2 mL) were treated with 1 mM H₂O₂ for 1.5 h at 30 °C. Cells were pelleted at 3000 g for 10 min and resuspended in a fresh YPD medium. Suspensions were diluted 100–1000-fold in a complete medium and plated on YPD agar plates. Colonies were counted after 3 days of incubation at 30 °C. The quantity of colonies obtained with untreated cultures was assumed to be 100%. Values are means ± SE of three independent experiments.

Fig. 4

Growth of Pichia guilliermondii wild-type strain, and mutants Δfes1–77, Δvma1–17 and Δfra1–45 under different temperature conditions. Cultures of P. guilliermondii wild-type strain R-66 (WT) and mutants were grown aerobically in YPD medium for 48 h. Cells were harvested, washed with water and resuspended in water to an OD_{600 nm} = 0.2. Serial fivefold dilutions were made. Five microlitre aliquots of each dilution were plated onto YPS medium. Plates were incubated at 30 and 37 °C for 4 days. The results of a typical representative experiment are shown.