Heterologous expression of an engineered biosynthetic pathway: functional dissection of type II polyketide synthase components in Streptomyces species.
AUTOR(ES)
Kim, E S
RESUMO
Polyketides are an extensive class of secondary metabolites with diverse molecular structures and biological activities. A plasmid-based multicomponent polyketide synthase expression cassette was constructed using a subset of actinorhodin (act) biosynthetic genes (actI-orf1, actI-orf2, actI-orf3, actIII, actVII, and actIV) from Streptomyces coelicolor which specify the construction of the anthraquinone product aloesaponarin II, a molecule derived from acetyl coenzyme A and 7 malonyl coenzyme A extender units. This system was designed as an indicator pathway in Streptomyces parvulus to quantify polyketide product formation and to examine the functional significance of specific polyketide synthase components, including the act beta-ketoacyl synthase (beta-KS; encoded by actI-orf1 and actI-orf2) and the act cyclase/dehydrase (encoded by actVII-orf4). Site-directed mutagenesis of the putative active site Cys (to a Gln) in the actI-orf1 beta-KS product completely abrogated aloesaponarin II production. Changing the putative acyltransferase active-site Ser (to a Leu) located in the actI-orf1 beta-KS product led to significantly reduced but continued production of aloesaponarin II. Replacement of the expression cassette with one containing a mutant form of actI-orf2 gave no production of aloesaponarin II or any other detectable polyketide products. However, an expression cassette containing a mutant form of actVII-orf4 gave primarily mutactin with low-level production of aloesaponarin II.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=176724Documentos Relacionados
- Functional replacement of genes for individual polyketide synthase components in Streptomyces coelicolor A3(2) by heterologous genes from a different polyketide pathway.
- Heterologous activation of the actinorhodin biosynthetic pathway in Streptomyces lividans.
- Functional analysis of putative beta-ketoacyl:acyl carrier protein synthase and acyltransferase active site motifs in a type II polyketide synthase of Streptomyces glaucescens.
- Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3(2).
- Analysis of type II polyketide beta-ketoacyl synthase specificity in Streptomyces coelicolor A3(2) by trans complementation of actinorhodin synthase mutants.
