PEDS Advance Access published online on April 5, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh032
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond VA, 23219; Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond VA, 23219
* To whom correspondence should be addressed. E-mail: kareynol{at}hsc.vcu.edu.
Bacterial type I polyketide synthases (PKSs) generate a structurally-diverse group of natural products with a wide range of biological activities. Hybrid type I PKSs in which domains of one multifunctional polypeptide are replaced with components from heterologous systems have generated significant interest over the past decade. Almost invariably only one or several specific hybrids are made at a time and tested for functionality. This approach is slow, dependent upon a fortuitous choice of specific fusions points, and often leads to inactive or minimally active hybrid systems. We describe herein a method for generating and screening a library of hybrid pikAI complementation plasmids (encoding the loading domain and first two extension domains of pikromycin PKS) able to restore pikromycin in a BB138 S. venezuelae pikAI-deletion mutant. In the first step the plasmid sequence encoding the loading domain AT0-ACP0 was replaced by a counter selectable marker, sacB. DNA family shuffling was then used to generate a diverse library of chimeric AT0-ACP0 fragments, which were used to replace sacB by Keywords:
type I polyketide synthase/pikromycin/erythromycin/hybrid
Revised March 18, 2004
Accepted March 24, 2004
Oxford University Press 1741-0134
Article
An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases
2 Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065
Abstract 
-Red mediated in vivo recombination in an E. coli host. This method resulted in the rapid and efficient generation of a large number of hybrid pikAI complementation plasmids, which were used to transform S. venezuelae BB138. A bioassay of over 4000 of these transformants successfully revealed 3 different PikAI hybrids which were able to lead to pikromycin production. The study suggests that most of the hybrids are not detectably functional, and underscores the need to generate and screen large and diverse libraries in which different fusion points are tried. The methodologies applied in this study address this need and can be used for directed evolution of any component of the PikPKS, and potentially other type I PKS systems.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. Yuan, I. Kurek, J. English, and R. Keenan Laboratory-Directed Protein Evolution Microbiol. Mol. Biol. Rev., September 1, 2005; 69(3): 373 - 392. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Vetcher, Z.-Q. Tian, R. McDaniel, A. Rascher, W. P. Revill, C. R. Hutchinson, and Z. Hu Rapid Engineering of the Geldanamycin Biosynthesis Pathway by Red/ET Recombination and Gene Complementation Appl. Envir. Microbiol., April 1, 2005; 71(4): 1829 - 1835. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Umeno, A. V. Tobias, and F. H. Arnold Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution Microbiol. Mol. Biol. Rev., March 1, 2005; 69(1): 51 - 78. [Abstract] [Full Text] [PDF]
|
|