Phosphopantetheinyl transferases and uses thereof
View Patent ↗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.
1. A method for phosphopantetheinylating a substrate in a cell, comprising transforming the cell with a first nucleic acid molecule comprising an expressible form of a nucleotide sequence encoding a heterologous Bacillus phosphopantetheinyl transferase, and a second nucleic acid molecule comprising an expressible form of a nucleotide sequence encoding a heterologous protein which is a substrate of the phosphopantetheinyl transferase, such that phosphopantetheinylation of the substrate in the cell occurs, wherein the heterologous Bacillus phosphopantetheinyl transferase comprises:
(a) an amino acid sequence of an enzyme selected from the group consisting of Sfi. Psf-1, Gsp and Lpa-14; or
(b) an amino acid sequence at least 90% identical to the amino acid sequence of an enzyme selected from the group consisting of Sfp, Psf-1, Gsp and Lpa-14 and comprising the amino acid sequences set forth in SEQ ID NO: 57 and SEQ ID NO:59,
and wherein the substrate comprises the amino acid sequence shown in SEQ ID NOs: 70 or 71,
and is capable of being phosphopantetheinylated by an enzyme selected from (a) or (b).
2. A method for phosphopantetheinylating a substrate in a cell, comprising transforming the cell with a first nucleic acid molecule comprising an expressible form of a nucleotide sequence encoding a heterologous Bacillus phosphopantetheinyl transferase comprising an amino acid sequence selected from the group consisting of:
(a) an amino acid sequence of Sfp;
(b) an amino acid sequence set forth in SEQ ID NO:8, and
(c) an amino acid sequence at least 90% identical to the amino acid sequence of Sfp and comprising amino acid sequences set forth in SEQ ID NO: 57 and SEQ ID NO:59,
and a second nucleic acid molecule comprising an expressible form of a nucleotide sequence encoding a heterologous protein which is a substrate of the Bacillus phosphopantetheinyl transferase, such that phosphopantetheinylation of the substrate in the cell occurs, wherein the substrate comprises the amino acid sequence shown in SEQ ID NOs:70 or 71, and is capable of being phosphopantetheinylated by the heterologous Bacillus phosphopantetheinyl transferase of (a), (b) or (c).
3. The method of claim 1 , wherein the substrate is a type I acyl carrier protein.
4. The method of claim 1 , wherein the substrate is a type II acyl carrier protein.
5. The method of claim 1 , wherein the cell is selected from the group consisting of a bacterial cell, a yeast cell, a mammalian cell, a plant cell, and an insect cell.
6. The method of claim 1 , wherein the cell is an E. coli cell.
7. The method of claim 1 , wherein the Bacillus phosphopantetheinyl transferase is Sfp.
8. The method of claim 1 , wherein the cell is a yeast cell.
9. The method of claim 1 , wherein the cell is a plant cell.
10. The method of claim 1 , wherein the Bacillus phosphopantetheinyl transferase and the substrate are not naturally expressed in the same cell.
11. The method of claim 1 , wherein the substrate is a protein that is activated by phosphopantetheinylation.
12. The method of claim 1 , or 2 , wherein the heterologous Bacillus phosphopantetheinyl transferase further comprises the amino acid sequence motif 2a as shown in SEQ ID NO:58.
13. The method of claim 1 , or 2 , wherein the heterologous Bacillus phosphopantetheinyl transferase further comprises the amino acid sequence G-X-E (motif 1b), wherein X represents any amino acid residue.
14. The method of claims 1 , or 2 , wherein the amino acid sequences set forth in SEQ ID NOs:57 and 59 are separated by about 45 amino acids.
15. The method of any one of claims 1 , or 2 , wherein the amino acid sequence identity is at least 95%.