Vectors for use in an inducible coexpression system
The present invention provides expression vectors for use in an inducible coexpression system, capable of controlled induction of expression of each gene product.
1. An expression construct comprising a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence comprising a propionate-inducible promoter and having at least 97% sequence identity to at least 225 contiguous bases of nucleotides 4937 through 5185 of SEQ ID NO:15;
(b) a nucleotide sequence comprising a propionate-inducible promoter and having at least 80% sequence identity to at least 300 contiguous bases of nucleotides 4937 through 5304 of SEQ ID NO:15;
(c) a nucleotide sequence comprising an arabinose-inducible promoter and having at least 87% sequence identity to at least 350 contiguous bases of nucleotides 2818 through 3259 of SEQ ID NO:15; and
(d) a nucleotide sequence comprising a propionate-inducible promoter and having at least 90% sequence identity to at least 450 contiguous bases of nucleotides 10 through 1822 of SEQ ID NO:2.
2. The expression construct of claim 1 comprising SEQ ID NO:15.
3. A host cell comprising the expression construct of claim 1 .
4. The host cell of claim 3 wherein the host cell is a prokaryotic cell.
5. The host cell of claim 4 wherein the host cell is E. coli.
6. The host cell of claim 5 wherein the host cell has a reduced level of gene function of at least one gene encoding a protein that metabolizes an inducer of at least one inducible promoter.
7. The host cell of claim 6 wherein the gene encoding a protein that metabolizes an inducer of at least one said inducible promoter is selected from the group consisting of araA, araB, prpB, prpD, rhaA, rhaB, rhaD, xylA, and xylB.
8. The host cell of claim 6 , wherein the host cell has a reduced level of function of at least one gene selected from the group consisting of araA and araB, and wherein the expression construct comprises at least one arabinose-inducible promoter and further comprises a polynucleotide sequence encoding AraC.
9. The host cell of claim 6 , wherein the host cell has a reduced level of function of at least one gene selected from the group consisting of prpB and prpD, and further comprises a polynucleotide sequence encoding PrpR.
10. The host cell of claim 5 wherein the host cell has an alteration of gene function of at least one gene encoding a transporter protein for an inducer of at least one said inducible promoter.
11. The host cell of claim 10 wherein the gene encoding a transporter protein is selected from the group consisting of araE, araF, araG, araH, rhaT, xylF, xylG, and xylH.
12. The host cell of claim 11 wherein the gene encoding a transporter protein is araE, and the alteration of gene function of said araE gene is changing the promoter controlling expression of the host cell's chromosomal araE gene from an arabinose-inducible promoter to a constitutive promoter.
13. The host cell of claim 5 wherein the host cell has a reduced level of gene function of a gene that encodes a reductase.
14. The host cell of claim 13 wherein the gene that encodes a reductase is trxB.
15. The host cell of claim 5 wherein the host cell has an altered gene function of a gene selected from the group consisting of gor and gshB.
16. The host cell of claim 5 wherein the host cell expresses a mutant form of AhpC.
17. The host cell of claim 5 wherein the host cell further comprises one or more of the following: (a) a deletion of the araBAD genes; (b) an altered gene function of the araE and araFGH genes; (c) a lacY(A177C) gene; (d) a reduced gene function of the prpB and prpD genes; (e) a reduced gene function of the sbm/scpA-ygfD/argK-ygfGH/scpBC genes; (f) a reduced gene function of the gor and trxB genes; (g) a reduced gene function of the AscG gene; (h) a polynucleotide encoding a form of DsbC lacking a signal peptide; (i) a polynucleotide encoding Erv1p; and (j) a polynucleotide encoding a chaperone.
18. A host cell comprising the expression construct of claim 1 , wherein the expression construct further comprises at least one polynucleotide sequence encoding at least one gene product to be transcribed from an inducible promoter.
19. A kit comprising the host cell of claim 1 .