Nucleic acids encoding synthetic scaffolds and host cells genetically modified with the nucleic acids
The present invention provides methods of producing a product or product precursor of a biosynthetic pathway in a genetically modified host cell. The present invention also provides genetically modified host cells comprising nucleic acids encoding a scaffold polypeptide and nucleic acids comprising nucleotide sequences encoding two or more enzymes in a biosynthetic pathway. The present invention further provides nucleic acids comprising nucleotide sequences encoding scaffold polypeptides, for use in a subject method.
1. A nucleic acid comprising:
a) a nucleotide sequence encoding a scaffold polypeptide of the formula (X 1 ) n1 (Y 1 )(X 2 ) n2 (Y 2 )(X 3 ) n3 , wherein X 1 , X 2 , and X 3 are different peptide binding elements, wherein each of n 1 , n 2 , and n 3 is independently an integer from one to about 10, and wherein Y 1 and Y 2 , if present, are linker peptides, wherein the ratio of n 2 to n provides for at least 25% increased production of a product or product precursor of a biosynthetic pathway compared to the level of the product or product precursor produced in a cell not genetically modified with said nucleic acid; and
b) nucleotide sequences encoding at least a first and a second enzyme in a biosynthetic pathway, wherein the first enzyme comprises a first heterologous peptide that binds a first peptide binding element in the scaffold polypeptide, wherein the second enzyme comprises a second heterologous peptide that binds a second peptide binding element in the scaffold polypeptide, and wherein the first enzyme produces an intermediate that is a substrate for the second enzyme.
2. The nucleic acid of claim 1 , wherein each X is a peptide binding element selected from an SH3 domain, a PDZ domain, a GTPase binding domain, a leucine zipper domain, an SH2 domain, a PTB domain, an FHA domain, a WW domain, a 14-3-3 domain, a death domain, a caspase recruitment domain, a bromodomain, a chromatin organization modifier, a shadow chromo domain, an F-box domain, a HECT domain, a RING finger domain, a sterile alpha motif domain, a glycine-tyrosine-phenylalanine domain, a SNAP domain, a VHS domain, an ANK repeat, an armadillo repeat, a WD40 repeat, an MH2 domain, a calponin homology domain, a Dbl homology domain, a gelsolin homology domain, a PB1 domain, a SOCS box, an RGS domain, a Toll/IL-1 receptor domain, a tetratricopeptide repeat, a TRAF domain, a Bcl-2 homology domain, a coiled-coil domain, and a bZIP domain.
3. The nucleic acid of claim 1 , wherein X 1 is a GTPase-binding domain, wherein X 2 is an SH3 domain, wherein X 3 is a PDZ domain, wherein n1 is one, wherein n2 is 1 or 2, and wherein n3 is an integer from 2 to 6.
4. The nucleic acid of claim 1 , wherein the first and second enzymes are mevalonate pathway enzymes.
5. An expression construct comprising the nucleic acid of claim 1 .
6. A genetically modified host cell comprising the nucleic acid of claim 1 .
7. The genetically modified host cell of claim 6 , wherein said host cell is a prokaryotic cell.
8. The genetically modified host cell of claim 6 , wherein said host cell is a eukaryotic cell.
9. A genetically modified host cell comprising the expression construct of claim 5 .
10. The genetically modified host cell of claim 9 , wherein said host cell is a prokaryotic cell.
11. The genetically modified host cell of claim 9 , wherein said host cell is a eukaryotic cell.