Use of synthetic scaffolds for the production of biosynthetic pathway products
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 method of producing a product or a product precursor of a biosynthetic pathway in a genetically modified host cell, the method comprising culturing a genetically modified host cell under suitable conditions, wherein the genetically modified host cell is genetically modified with one or more nucleic acids 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 3 provides for at least 25% increased production of the product or product precursor compared to the level of the product or product precursor produced in the corresponding control cell not genetically modified with a nucleic acid comprising a nucleotide sequence encoding the scaffold polypeptide; 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, and wherein the first enzyme produces an intermediate that is a substrate for the second enzyme,
said culturing providing for synthesis of said scaffold polypeptide and said enzymes in the genetically modified host cell, resulting in production of said product or product precursor.
2. The method of claim 1 , wherein said biosynthetic pathway is a biosynthetic pathway that converts acetyl-CoA to isopentenyl diphosphate via a mevalonate intermediate, wherein said pathway comprise two or more enzymes that produce an isoprenoid product or an isoprenoid product precursor.
3. The method of claim 2 , wherein the isoprenoid product precursor is mevalonate.
4. The method of claim 1 , wherein the peptide binding element comprises a protein-protein interaction motif 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 Bc1-2 homology domain, a coiled-coil domain, and a bZIP domain.
5. The method of claim 1 , wherein the scaffold polypeptide comprises at least three different peptide binding elements, and wherein said at least three peptide binding elements comprise an SH3 domain, a PDZ domain, and a GTPase binding domain.
6. The method of claim 1 , wherein the host cell is a prokaryotic cell.
7. The method of claim 1 , wherein the host cell is a eukaryotic cell.