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Rapid Engineering of the Geldanamycin Biosynthesis Pathway by Red/ET Recombination and Gene Complementation

Rapid Engineering of the Geldanamycin Biosynthesis Pathway by Red/ET Recombination and Gene... Rapid Engineering of the Geldanamycin Biosynthesis Pathway by Red/ET Recombination and Gene Complementation Leandro Vetcher , Zong-Qiang Tian , Robert McDaniel , Andreas Rascher , W. Peter Revill , C. Richard Hutchinson and Zhihao Hu * Kosan Biosciences Inc., Hayward, California 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied and Environmental Microbiology American Society For Microbiology

Rapid Engineering of the Geldanamycin Biosynthesis Pathway by Red/ET Recombination and Gene Complementation

Rapid Engineering of the Geldanamycin Biosynthesis Pathway by Red/ET Recombination and Gene Complementation

Applied and Environmental Microbiology , Volume 71 (4): 1829 – Apr 1, 2005

Abstract

Rapid Engineering of the Geldanamycin Biosynthesis Pathway by Red/ET Recombination and Gene Complementation Leandro Vetcher , Zong-Qiang Tian , Robert McDaniel , Andreas Rascher , W. Peter Revill , C. Richard Hutchinson and Zhihao Hu * Kosan Biosciences Inc., Hayward, California 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.

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References (30)

Publisher
American Society For Microbiology
Copyright
Copyright © 2005 by the American society for Microbiology.
ISSN
0099-2240
eISSN
1098-5336
DOI
10.1128/AEM.71.4.1829-1835.2005
pmid
15812008
Publisher site
See Article on Publisher Site

Abstract

Rapid Engineering of the Geldanamycin Biosynthesis Pathway by Red/ET Recombination and Gene Complementation Leandro Vetcher , Zong-Qiang Tian , Robert McDaniel , Andreas Rascher , W. Peter Revill , C. Richard Hutchinson and Zhihao Hu * Kosan Biosciences Inc., Hayward, California 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.

Journal

Applied and Environmental MicrobiologyAmerican Society For Microbiology

Published: Apr 1, 2005

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