ABSTRACT
This chapter reviews the molecular genetic tools that are now available for use with filamentous fungi in relation to the cloning and use of antibiotic biosynthetic genes. In addition, an assessment of the way forward is provided, with particular emphasis on furthering our understanding of gene expression. Bacteriophage lambda vectors, such as EMBL4 are generally used where heterologous probing is to be employed, and Agtll for cDNA cloning and expression. The transforming sequence, along with the bacterial vector sequences (usually antibiotic resistance and plasmid origin of replication), complementing the mutation of interest, integrate into the nuclear DNA, but can be rescued by partial digestion of the total transformant DNA, religation, and transformation of E coli. A vector to be used as an expression cassette should possess a strong constitutive or regulatable fungal promoter, an efficient translation start, a signal peptide-coding sequence when secretion is the goal, cloning sites for the heterologous gene, and sequences for transcriptional termination and polyadenylation.
