Artificial fusion of dehydratase enzymes to improve production of fatty acids
AssigneeUniversity of Puerto Rico
InventorsCarlos Ralph Rullán Lind, Abel Juan Baerga Ortiz
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Abstract
The disclosure provides fused dimer constructs that enhance fatty acid production and methods for making the dimer constructs. The fused dimer comprises Fab A and/or FabZ enzymes.
Claims18
A heterodimer protein, comprising a FabA beta-hydroxyacyl-acyl carrier protein dehydratase subunit (FabA), a FabZ beta-hydroxyacyl-acyl carrier protein dehydratase subunit (FabZ) and a peptide linker, wherein the FabA and FabZ subunits are covalently or non-covalently linked, wherein the peptide linker comprises SEQ ID NO:
- 15, or a polypeptide with at least 75% identity to SEQ ID NO:
- 15.
A method for producing fatty acids comprising overexpressing a recombinant expression construct in a cell that encodes a FabA-peptide linker-FabZ or FabZ-peptide linker-FabA heterodimeric protein, wherein the peptide linker comprises SEQ ID NO:
- 15, or a polypeptide with at least 75% identity to SEQ ID NO:
- 15, wherein total fatty acid yield is increased compared with a cell expressing FabA or FabZ homodimeric proteins.
A method for making a fused heterodimer protein consisting of a FabA beta-hydroxyacyl-acyl carrier protein dehydratase subunit (FabA) a FabZ beta-hydroxyacyl-acyl carrier protein dehydratase subunit (FabZ) and a peptide linker, wherein the peptide linker comprises SEQ ID NO:
- 15, or a polypeptide with at least 75% identity to SEQ ID NO:
- 15, comprising the steps of a) amplifying the FabA gene, the FabZ gene, and a peptide linker gene;
- b) performing sequential PCR reactions to generate heterodimers;
- c) adding primers at the beginning of PCR reaction IV;
- d) cloning a construct into expression vectors;
- e) transforming the expression vectors into a cell;
- and f) verifying the sequence of the construct.
Dependencies are inferred from the claim text.