Isolation of auxotrophic mutants of Aspergillus niger by filtration enrichment and lytic enzymes

Summary More than 100 auxotrophic mutants of Aspergillus niger were isolated using filtration enrichment. The mutants obtained in this way were predominantly aminoacid requiring. Hardly any vitamin-deficient mutants were found. The limitations of the method turned out to be due to cross feeding in liquid medium and non-specific loss of ungerminated conidiospores. To circumvent these problems an enrichment method has been used in which conidiospores that germinated on solid medium were selectively killed by the lytic enzyme preparation Novozym 234. We found auxotrophic mutants at high frequencies and several new types of mutants. Optimal conditions for the enrichment procedures have been determined. Essential factors appeared to be segregation of the conidia after mutagenic treatment, in order to obtain synchronization of germination, and the incubation time of the conidia on minimal medium prior to enzymic treatment. Under appropriate conditions Novozym enrichment proved to be very efficient. Depending on the type of mutants desired, one or both procedures can provide an effective method for the enrichment of mutants with a metabolic defect. The Novozym method can be adapted to other fungi and some of the observations that are described may also be of importance to improve other enrichment methods that are based on the selective killing of germinating conidiospores.