Twin-arginine translocation in
Described herein are methods to enhance protein secretion in a host cell. In preferred embodiment, the host cell is a gram-positive microorganism such as a Bacillus . In another preferred embodiment, the host cell is a gram-negative microorganism. Preferably the gram-negative microorganism is an Escherichia coli or a member of the genus Pantoea . Protein secretion may be enhanced by the overexpression of protein components of the Tat pathway. Alternatively, secretion of foreign proteins can be selectively enhanced by forming a chimeric polypeptide comprising a tat signal sequence and the protein of interest. In a preferred embodiment, the tat signal sequence is selected from phoD or LipA.
1. An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a Bacillus subtilis lipase LipA signal sequence operatively linked to a second nucleotide sequence encoding a heterologous polypeptide.
2. A recombinant expression vector comprising a first DNA sequence encoding a Bacillus subtilis lipase LipA signal sequence operatively linked to a second DNA sequence encoding a heterologous polypeptide.
3. A host cell containing a recombinant expression vector comprising a first DNA sequence encoding a Bacillus subtilis lipase LipA signal sequence operatively linked to a second DNA sequence encoding a heterologous polypeptide.
4. The host cell of claim 3 , wherein said polypeptide is not naturally associated with a secretion signal peptide.
5. A method for producing a polypeptide, comprising culturing a host cell containing a recombinant expression vector comprising a first DNA sequence encoding a Bacillus subtilis lipase LipA signal sequence operatively linked to a second DNA sequence encoding a heterologous polypeptide is produced by the host cell, wherein the host cell comprises a nucleic acid encoding TatAd protein and a nucleic acid encoding TatCd protein.
6. The method of claim 5 , wherein the polypeptide is secreted by the host cell into a culture medium.
7. The method of claim 6 , further comprising recovering the polypeptide from the culture medium.
8. A method for producing a heterologous polypeptide in bacteria comprising:
(a) culturing bacterial cells that (i) lack a functional TatCy gene and (ii) contain a recombinant expression vector comprising a first DNA sequence encoding a Bacillus subtilis lipase LipA signal sequence operatively linked to a second DNA sequence encoding a heterologous polypeptide such that the heterologous polypeptide is produced by the cells, wherein the cells comprise a nucleic acid encoding TatAd protein and a nucleic acid encoding TatCd protein; and
(b) recovering the heterologous polypeptide from the periplasm or the culture medium.
9. A process for producing a heterologous polypeptide in bacteria comprising:
(a) culturing bacterial cells that (i) overexpress one or more B. subtilis Tat system genes encoding membrane-bound components thereof and (ii) contain a recombinant expression vector comprising a first DNA sequence encoding a Bacillus subtilis lipase LipA signal sequence operatively linked to a second DNA sequence encoding a heterologous polypeptide such that the heterologous polypeptide is produced by the cells, wherein the cells comprise a nucleic acid encoding TatAd protein and a nucleic acid encoding TatCd protein; and
(b) recovering the heterologous polypeptide from the periplasm or the culture medium.
10. The nucleic acid molecule of claim 1 , wherein the heterologous polypeptide is an enzyme.
11. The nucleic acid molecule of claim 10 , wherein the enzyme is selected from the group consisting of a protease, cellulase, amylase, lipase, and hydrolase.
12. The nucleic acid molecule of claim 11 , wherein the enzyme is a protease.
13. The nucleic acid molecule of claim 11 , wherein the enzyme is a cellulase.
14. The nucleic acid molecule of claim 11 , wherein the enzyme is an amylase.
15. The nucleic acid molecule of claim 11 , wherein the enzyme is a lipase.
16. The nucleic acid molecule of claim 11 , wherein the enzyme is a hydrolase.
17. The recombinant expression vector of claim 2 , wherein the heterologous polypeptide is an enzyme.
18. The recombinant expression vector of claim 17 , wherein the enzyme is selected from the group consisting of a protease, cellulase, amylase, lipase, and hydrolase.
19. The recombinant expression vector of claim 18 , wherein the enzyme is a protease.
20. The recombinant expression vector of claim 18 , wherein the enzyme is an amylase.