Isolation of various hyperactive mutants of phenylalanine ammonia-lyase containing yeasts

Abstract

Various strains of phenylalanine ammonia-lyase (PAL) containing yeasts were mutagenised using ultraviolet (UV) irradiation. Two of the yeast, SPA 1 and SPA 17, produced UV kill curves with a distinct shoulder reflecting their diploid nature. Of the analogues used, p-fluoro-D,L-phenylalanine and β-2-thienyl-D,L-alanine selected the greatest frequency of mutants with the highest PAL activities: one such mutant, FP10M6, exhibited five times the PAL activity of the parent SPA 10. Mutants constitutive for PAL synthesis could not be isolated using any selection regime, including resistance to relatively high concentrations of 2-deoxyglucose. Three of the hyperactive PAL mutants examined were not impaired for growth and their PAL-induction profiles were not different from the respective parents. Inactivation of PAL shortly after peak synthesis was found with all mutants examined, although this was not extensively investigated. Of the 21 strains mutagenised, mutants from three of the wild-type yeasts were found to exhibit moderate growth at 37 °C, while growth of the parent strains was greatly impaired. The latter temperature-resistant mutants exhibited a twofold increase in PAL activity as compared with the parent strains at either 30 or 37 °C. Under biotransformation reaction conditions, several of the mutants were capable of producing more than 15 g L-phenylalanine∙L−1. Although the stability of the PAL catalyst varied markedly among different mutants, some of the most productive mutants did exhibit good reuseability under sequentially repeated batch reaction conditions.