Selection and characterization of mutants of Phanerochaete chrysosporium Exhibiting Ligninolytic activity under nutrient-rich conditions

Synthesis of the ligninolytic system of the wood-degrading fungus Phanerochaete chrysosporium is induced during secondary metabolism, brought about by nitrogen, carbon, or sulfur starvation. We describe here a strategy for selection of mutants which are ligninolytic (lignin → CO₂) and overproduce lignin-degrading enzymes (ligninases) under nitrient-rich conditions (during primary metabolism). The strategy is based on using an aduct of lysine and a lignin model compound. Ligninase-dependent oxidation of this adduct releases free lysine, which complements the lysine requirements of a lysine auxotroph. Accordingly, a lysine auxotroph was mutagenized by UV irradiation and survivors were plated onto medium containing the adduct and high ammonia nitrogen. Four mutants which overproduce the ligninase isozymes were isolated by this procedure. Further characterization of one of the mutants, PSBL-1, indicated that the predominant isozymes produced are H1 (pI = 4.7) and H2 (pI = 4.4). The ligninase activity of PSBL-1, measured by veratryl alcohol oxidation, peaks on day 5 at over 1,000 U · liter^-1. The mutant PSBL-1 was also able to degrade [¹⁴C]lignin to ¹⁴CO₂, indicating that the complete ligninolytic system is deregulated.