title: Rapid engineering of the geldanamycin biosynthesis pathway by Red/ET recombination and gene complementation.
abstract: Genetic manipulation of antibiotic producers, such as Streptomyces species, is a rational approach to improve the properties of biologically active molecules. However, this can be a slow and sometimes problematic process. Red/ET recombination in an Escherichia coli host has permitted rapid and more versatile engineering of geldanamycin biosynthetic genes in a complementation plasmid, which can then be readily transferred into the Streptomyces host from which the corresponding wild type gene(s) has been removed. With this rapid Red/ET recombination and gene complementation approach, efficient gene disruptions and gene replacements in the geldanamycin biosynthetic gene cluster have been successfully achieved. As an example, we describe here the creation of a ketoreductase 6 null mutation in an E. coli high-copy-number plasmid carrying gdmA2A3 from Streptomyces hygroscopicus NRRL3602 and the subsequent complementation of a gdmA2A3 deletion host with this plasmid to generate a novel geldanamycin analog.
authors.affiliation: Kosan Biosciences Inc., 3832 Bay Center Place, Hayward, CA 94545, USA.
authors.forename: Leandro
authors.initials: L
authors.lastname: Vetcher
authors.forename: Zong-Qiang
authors.initials: ZQ
authors.lastname: Tian
authors.forename: Robert
authors.initials: R
authors.lastname: McDaniel
authors.forename: Andreas
authors.initials: A
authors.lastname: Rascher
authors.forename: W Peter
authors.initials: WP
authors.lastname: Revill
authors.forename: C Richard
authors.initials: CR
authors.lastname: Hutchinson
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Hu
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics; D016227:Benzoquinones; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005816:Genetic Complementation Test; D005818:Genetic Engineering / Q000379:methods*; D047029:Lactams, Macrocyclic; D010957:Plasmids; D011809:Quinones / Q000737:chemistry / Q000378:metabolism*; D011995:Recombination, Genetic; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D016227:Benzoquinones; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D047029:Lactams, Macrocyclic; D011809:Quinones; C084497:RecT protein, E coli; D005090:Exodeoxyribonucleases; C038225:recE protein, E coli; C001277:geldanamycin