title: Application of In Vitro Transposon Mutagenesis to Erythromycin Strain Improvement in Saccharopolyspora erythraea.
abstract: Transposon mutagenesis is an invaluable technique in molecular biology for the creation of random mutations that can be easily identified and mapped. However, in the field of microbial strain improvement, transposon mutagenesis has scarcely been used; instead, chemical and physical mutagenic methods have been traditionally favored. Transposons have the advantage of creating single mutations in the genome, making phenotype to genotype assignments less challenging than with traditional mutagens which commonly create multiple mutations in the genome. The site of a transposon mutation can also be readily mapped using DNA sequencing primer sites engineered into the transposon termini. In this chapter an in vitro method for transposon mutagenesis of Saccharopolyspora erythraea is presented. Since in vivo transposon tools are not available for most actinomycetes including S. erythraea, an in vitro method was developed. The in vitro method involves a significant investment in time and effort to create the mutants, but once the mutants are made and screened, a large number of highly relevant mutations of direct interest to erythromycin production can be found.
authors.affiliation: Fermalogic, Inc., 4222 N. Ravenswood Ave., Suite 208, Chicago, IL, 60613, USA. mark@fermalogic.com.
authors.forename: J Mark
authors.initials: JM
authors.lastname: Weber
authors.affiliation: Coskata, Inc., 4575 Weaver Parkway, Suite 100, Warrenville, IL, 60555, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Reeves
authors.affiliation: Fermalogic, Inc., 4222 N. Ravenswood Ave., Suite 208, Chicago, IL, 60613, USA.
authors.forename: William H
authors.initials: WH
authors.lastname: Cernota
authors.affiliation: Fermalogic, Inc., 4222 N. Ravenswood Ave., Suite 208, Chicago, IL, 60613, USA.
authors.forename: Roy K
authors.initials: RK
authors.lastname: Wesley
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D004917:Erythromycin / Q000096:biosynthesis*; D005798:Genes, Bacterial / Q000235:genetics; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D016930:Saccharopolyspora / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004917:Erythromycin
keywords: Actinomycetes; Erythromycin; Gene replacement; In vitro transposon mutagenesis; S. erythraea; Strain improvement