Derivation of Aromatic Amino Acid Mutants from a Methanol-Utilizing Yeast, Hansenula polymorpha

Three classes of mutants, deregulated to enhance the flow of aromatic intermediates through the tryptophan biosynthetic branch, were obtained. 5-Fluorotryptophan, an antimetabolite of tryptophan, was employed to obtain one class of deregulated mutants. By sequential resistance development, three resistant mutants were isolated. Hansenula polymorpha strains showed greater sensitivity to 5-fluorotryptophan when growing on methanol than when growing on glucose. Yeast extract stimulated the production of total indole metabolites (indoles) by wild-type and mutant strains, with each 5-fluorotryptophan mutant producing higher amounts of these metabolites than its predecessor. Two other mutant classes were isolated: (i) a mutant resistant to anthranilate (an inhibitory intermediate in the tryptophan biosynthetic branch) and (ii) a phenylalanine-plus-tyrosine bradytroph. Each of these produced a higher extracellular titer of total indoles than its immediate parent. With respect to the overproduction of indoles, resistance to 5-fluorotryptophan was a more useful selection method than were anthranilate resistance and phenylalanine-plus-tyrosine bradytrophy.