Isolation and characterization of temperature-sensitive mutants of Hansenula

For investigation of the feasibility of obtaining yeast strains with high protein and low nucleic acid content or high nucleic acid and low protein content for industrial production of single cell protein or nucleotide, the methanol-utilizing Hansenula polymorpha NTU-AM-P5 was chosen for isolation of temperature-sensitive mutants. Approximately 70 temperature-sensitive (ts) mutants were obtained. All of the ts mutants increased in cell size and aggregated when they were cultivated at permissive temperature and shifted to restrictive temperature in the late log phase or in the stationary phase. 86.1% of ts mutants had a lower growth rate than that of their wild-type. Only ts mutants, NTU-AM-L2, NTU-AM-E10, NTU-AM-E19, NTU-AM-E25 and NTU-AM-E30 had same or slightly higher than that of their wild-type strain. Cycloheximide at 1 mg/ml inhibited the growth of ts mutant NTU-AM-E19, but inhibited one-third of the other ts mutants, and their wild-type strain. Rifampicin at 0.1 mg/ml had an inhibitory activity on wild-type strain but not on ts mutant NTU-AM-E1. ts mutants NTU-AM-L2 and NTU-AM-E19 converted methanol to cell mass more efficiently than others. The ts mutants divided into two groups. One included absolute ts mutants, such as NTU-AM-E15 and NTU-AM-E20, which did not grow even in enriched media at restrictive temperature; the other group, such as NTU-AM-L2, NTU-AM-L3, NTU-AM-E10, NTU-AM-E19, NTU-AM-N37 and NTU-AM-m5, was auxotrophic ts mutants, which grew slightly in enriched media at restrictive temperature.