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AssigneesPresident and Fellows of Harvard College, The Regents of the University of California
InventorsRalph H. Lambalot, Amy M. Gehring, Ralph Reid, Christopher T. Walsh
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The invention pertains to isolated phosphopantetheinyl transferases, such as the E. coli acyl carrier protein synthase, which transfer a phosphopantetheinyl group onto a substrate. The enzyme can be purified from a natural source, produced recombinantly, or synthetically. Accordingly, the invention provides compositions and kits including phosphopantetheinyl transferases and host cells expressing phosphopantetheinyl transferases. The invention also provides nucleic acids encoding phosphopantetheinyl transferases and vectors comprising such nucleic acids. The invention further provides methods for phosphopantetheinylating a substrate in vitro or in vivo and methods for producing antibiotics in vitro or in vivo.
A method for phosphopantetheinylating a substrate in a cell or increasing phosphopantetheinylation of a substrate in a cell, wherein the cell is an E. coli cell or a yeast cell, comprising transforming the cell with a first nucleic acid molecule comprising an expressible form of a nucleotide sequence encoding a phosphopantetheinyl transferase, wherein the phosphopantetheinyl transferase is E. coli acyl carrier protein synthase that comprises the amino acid sequence set forth in SEQ ID NO:10 or a fragment thereof retaining phosphopantetheinylating activity, and a second nucleic acid molecule comprising an expressible form of a nucleotide sequence encoding a protein which is a substrate of the phosphopantetheinyl transferase, wherein the substrate is selected from the group consisting of Lactobacillus casei D-Alanyl carrier protein (Dcp) and E. coli acyl carrier protein (ACP), such that phosphopantetheinylation of the substrate in the cell occurs or is increased.
A method for phosphopantetheinylating a substrate in a cell or increasing phosphopantetheinylation of a substrate in a cell, wherein the cell is an E. coli cell or a yeast cell, comprising transforming the cell with a first nucleic acid molecule comprising an expressible form of a nucleotide sequence encoding a phosphopantetheinyl transferase, wherein the phosphopantetheinyl transferase is Bacillus subtilis Sfp that comprises the amino acid sequence set forth in SEQ ID NO:8 or a fragment thereof retaining phosphopantetheinylating activity, and a second nucleic acid molecule comprising an expressible form of a nucleotide sequence encoding a protein which is a substrate of the phosphopantetheinyl transferase, wherein the substrate is selected from the group consisting of Bacillus subtilis surfactin synthetase B1 domain (SrfB1), E. coli acyl carrier protein (ACP) and peptidyl carrier protein (PCP) domain of E. coli TycA, such that phosphopantetheinylation of the substrate in the cell occurs or is increased.
A method for phosphopantetheinylating a substrate in a cell or increasing phosphopantetheinylation of a substrate in a cell, wherein the cell is an E. coli cell or a yeast cell, comprising transforming the cell with a first nucleic acid molecule comprising an expressible form of a nucleotide sequence encoding a phosphopantetheinyl transferase, wherein the phosphopantetheinyl transferase is E. coli EntD that comprises the amino acid sequence set forth in SEQ ID NO:9 or a fragment thereof retaining phosphopantetheinylating activity, and a second nucleic acid molecule comprising an expressible form of a nucleotide sequence encoding a protein which is a substrate of the phosphopantetheinyl transferase, wherein the substrate is selected from the group consisting of E. coli EntF, E. coli acyl carrier protein (ACP) and peptidyl carrier protein (PCP) domain of E. coli TycA, such that phosphopantetheinylation of the substrate in the cell occurs or is increased.
Dependencies are inferred from the claim text.