Figure 2.

Breaking down the barriers to genetic manipulation and engineering of actinomycetes. Numerous methods and tools were developed to facilitate the genetic manipulation of actinomycetes. Suitable vectors and other vehicles (e.g., integrative and replicative systems) as well as fast and easy cloning strategies were developed to assemble genetic constructs, which are introduced into the host by using different transfer methods (e.g., conjugation and protoplast transformation). Protocols for avoiding DNA-degradation (e.g., treatment of the DNA with NaOH) and solutions for ensuring correct transcription/translation (e.g., codon usage optimization) were established. These innovations led to the generation of many gene deletions and (over)expression mutants (e.g., deletion of competitive biosynthetic pathways, overexpression of activators for activation of silent biosynthetic gene cluster (BGC)). This enormous progress facilitated the metabolic engineering of actinomycetes chassis for manufacturing bioactive natural products, including antibiotics. (Single elements of the figure (e.g., schematic representation of plasmid backbone) were re-used from a previous publication [74] with permission from the Royal Society of Chemistry.).