Integration of genes into the chromosome of
View Patent ↗This invention includes a process for the identification and validation of a neutral polynucleotide integration site within the S. spinosa genome. In addition, the invention includes the use of the neutral site and methods for the integration of a polynucleotide containing a gene expression cassette, which is stably maintained and expressed over subsequent generations. The invention includes neutral integration sites that can be disrupted without negatively impacting spinosyn production, growth or other desired metabolic characteristics.
1. A method for producing spinosyn, the method comprising;
cloning two genomic DNA fragments from an obscurin polyketide synthase gene locus to generate a plasmid comprising a target gene, wherein one genomic fragment is cloned upstream of the target gene and the second genomic fragment is cloned downstream of the target gene;
integrating the plasmid comprising the target gene into a native obscurin polyketide synthase gene locus in a strain of S. spinosa to produce a strain of S. spinosa comprising the target gene;
culturing the strain of S. spinosa comprising the target gene;
allowing the strain of S. spinosa comprising the target gene to produce spinosyn; and
expressing the target gene,
wherein the target gene comprises a coding sequence functionally linked to a promoter heterologous to the strain of S. spinosa.
2. The method of claim 1 , wherein the integration of the plasmid comprising the target gene into the native obscurin polyketide synthase locus of the strain of S. spinosa does not negatively impact spinosyn production, growth, or other desired metabolic characteristics.
3. The method of claim 1 , wherein cloning two genomic DNA fragments from an obscurin polyketide synthase gene locus comprises cloning two genomic DNA fragments from an obsA obscurin polyketide synthase gene locus, and
wherein integrating the plasmid comprising the target gene into a native obscurin polyketide synthase gene locus in a strain of S. spinosa comprises integrating the plasmid comprising the target gene into an obsA gene of the native obscurin polyketide synthase locus of the strain of S. spinosa.
4. The method of claim 1 , wherein the target gene further comprises a selectable marker.
5. The method of claim 4 , wherein the selectable marker comprises an apramycin resistance gene, aac3(IV).
6. The method of claim 1 , wherein integration comprises a homologous recombination mediated integration.
7. A method for fermentation comprising:
cloning two genomic DNA fragments from an obscurin polyketide synthase gene locus to generate a plasmid comprising a target gene, wherein one genomic fragment is cloned upstream of the target gene and the second genomic fragment is cloned downstream of the target gene;
integrating the plasmid comprising the target gene into a native obscurin polyketide synthase gene locus in a strain of S. spinosa to produce a strain of S. spinosa comprising the target gene;
fermenting the strain of S. spinosa comprising the target gene;
allowing the strain of S. spinosa comprising the target gene to produce spinosyn; and
expressing the target gene,
wherein the target gene comprises coding sequence functionally linked to a promoter heterologous to the strain of S. spinosa.
8. The method of claim 7 , wherein the integration of the plasmid comprising the target gene into the native obscurin polyketide synthase locus of the strain of S. spinosa does not negatively impact spinosyn production, growth, or other desired metabolic characteristics.
9. The method of claim 7 , wherein cloning two genomic DNA fragments from an obscurin polyketide synthase gene locus comprises cloning two genomic DNA fragments from an obsA obscurin polyketide synthase gene locus, and
wherein integrating the plasmid comprising the target gene into a native obscurin polyketide synthase gene locus in a strain of S. spinosa comprises integrating the plasmid comprising the target gene into an obsA gene of the native obscurin polyketide synthase locus of the strain of S. spinosa.
10. The method of claim 7 , wherein the target gene further comprises a selectable marker.