System and method for the heterologous expression of polyketide synthase gene clusters
A system and method for heterologous expression of polyketide biosynthetic pathways from streptomycetes hosts in Escherichia coli for the production and discovery of secondary metabolites. Genomic DNA from Streptomyces rimosus encoding the oxytetracycline biosynthetic pathway is inserted into the genome of the surrogate host Myxococcus xanthus . The M. xanthus transcriptional machinery recognizes and uses the streptomycetes promoter regions to express the biosynthetic enzymes. Co-expression in E. coli of S. rimosus oxytetracycline biosynthesis enzymes and M. xanthus σ 54 , a key piece of the M. xanthus transcriptional machinery, enables E. coli to recognize and use the promoters from the S. rimosus oxytetracycline biosynthetic pathway, facilitating production of oxytetracycline.
1. A genetically modified host organism for expressing a target polyketide, comprising:
a first modification comprising the addition of a non-native gene cluster that encodes for the target polyketide biosynthetic pathway;
a second modification comprising the addition of an rpoN gene from Myxococcus xanthus ; and
a third modification comprising the addition of an inducible promoter positioned upstream of and operatively associated with the rpoN gene added to said host organism.
2. The organism of claim 1 , further comprising a gene encoding an RNA polymerase.
3. The organism of claim 1 , wherein the organism is at least one organism selected from the group consisting of Escherichia coli, Streptomyces rimosus, Streptomyces lividans, Streptomyces coelicolor Myxococcus xanthus, Pseudomonas putida , and Bacillus subtilis.
4. The organism of claim 1 , wherein the rpoN gene comprises SEQ. ID. No. 1.
5. The organism of claim 1 , wherein the promoter comprises a T7 promoter.
6. The organism of claim 1 , wherein the target polyketide comprises oxytetracycline.