Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains

Abstract

Development of natural products for therapeutic use is often hindered by limited availability of material from producing organisms. The speed at which current technologies enable the cloning, sequencing, and manipulation of secondary metabolite genes for production of novel compounds has made it impractical to optimize each new organism by conventional strain improvement procedures. We have exploited the overproduction properties of two industrial organisms- Saccharopolyspora erythraea and Streptomyces fradiae, previously improved for erythromycin and tylosin production, respectively-to enhance titers of polyketides produced by genetically modified polyketide synthases (PKSs). An efficient method for delivering large PKS expression vectors into S. erythraea was achieved by insertion of a chromosomal attachment site ( attB) for phiC31-based integrating vectors. For both strains, it was discovered that only the native PKS-associated promoter was capable of sustaining high polyketide titers in that strain. Expression of PKS genes cloned from wild-type organisms in the overproduction strains resulted in high polyketide titers whereas expression of the PKS gene from the S. erythraea overproducer in heterologous hosts resulted in only normal titers. This demonstrated that the overproduction characteristics are primarily due to mutations in non-PKS genes and should therefore operate on other PKSs. Expression of genetically engineered erythromycin PKS genes resulted in production of erythromycin analogs in greatly superior quantity than obtained from previously used hosts. Further development of these hosts could bypass tedious mutagenesis and screening approaches to strain improvement and expedite development of compounds from this valuable class of natural products.

Chalcomycin Biosynthesis Gene Cluster from Streptomyces bikiniensis : Novel Features of an Unusual Ketolide Produced through Expression of the chm Polyketide Synthase in Streptomyces fradiae

“…For isolations of total DNA, S. bikiniensis was grown in tryptone soya broth (19). S. fradiae K342-45 was made by conjugal transfer of plasmid pKOS342-45 from E. coli DH5␣/pUB307/pKOS342-45 to S. fradiae K159-1 as described previously (34). S. fradiae was maintained on AS-1 agar (8).…”

Transcriptome Analysis of an Antibiotic Downregulator Mutant and Synergistic Actinorhodin Stimulation via Disruption of a Precursor Flux Regulator in Streptomyces coelicolor

“…It has been proposed that the expression of antibiotic-biosynthetic genes is tightly controlled through multiple different regulatory networks in Streptomyces species (18,19). Traditionally, random mutation has been one of the most widely used strategies for Streptomyces strain improvement with regard to the generation of secondary-metabolite-overproducing industrial mutants, even though the molecular genetic basis underlying such enhanced production remains largely unknown (17,20,21,23). Recent "-omics"-guided reverse engineering approaches, including comparative transcriptomics and proteomics were successfully used to identify alterations in gene expression associated with the overproduction of secondary metabolites in industrial streptomycete strains (14,17).…”

Interspecies DNA Microarray Analysis Identifies WblA as a Pleiotropic Down-Regulator of Antibiotic Biosynthesis in Streptomyces

“…Like the biosynthesis of other secondary metabolites in Streptomyces species, the synthesis of doxorubicin is believed to be tightly regulated, thereby limiting doxorubicin production in wild-type-S. peucetius cultures (6,12,17). Traditional strain improvement via recursive random mutagenesis has been used to increase doxorubicin synthesis, although the molecular genetic basis underlying such enhanced production remains largely unknown (13,14,15,18).…”