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US 2020/0263156 A1Published application
Improving Expression Of A Protease By Co-Expression With Propeptide
AssigneeNovozymes A/S
InventorsMichael Thomas, Steen Joergensen
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Abstract
The present invention relates to prokaryotic host cells and methods for improving expression of a mature protease by co-expression with a propeptide of said protease as two separate polypeptides.
Claims15
1:
- A prokaryotic host cell comprising in its genome at least one polynucleotide encoding a protease proenzyme comprising a mature protease and at least one polynucleotide encoding a propeptide of said mature protease, wherein the at least one polynucleotide encoding the protease proenzyme and the at least one polynucleotide encoding the propeptide are not expressed as a translationally fused protein.
2:
- The prokaryotic host cell according to claim 1, which is a Gram-positive host cell;
- preferably the prokaryotic host cell is a Bacillus host cell;
- more preferably, the prokaryotic host cell is selected from the group consisting of Bacillus alkalophilus, Bacillus altitudinis, Bacillus amyloliquefaciens, B. amyloliquefaciens subsp. plantarum, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus safensis, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells;
- even more preferably, the prokaryotic host cell is a Bacillus licheniformis host cell.
1 more element
3:
- The prokaryotic host cell according to claim 1, wherein the mature protease is selected from the group consisting of serine protease, cysteine protease, threonine protease, aspartic protease, glutamic protease, metalloprotease, and asparagine peptide lyase;
- preferably the mature protease is a serine protease selected from the group consisting of trypsin-like protease, chymotrypsin-like protease, thrombin-like protease, elastase-like protease, and subtilisin-like protease;
- more preferably the mature protease is an S8 protease;
- most preferably the mature protease is an S8A protease.
1 more element
4:
- The prokaryotic host cell according to claim 1, wherein the mature protease is a native or an exogenous protease.
5:
- The prokaryotic host cell according to claim 1, wherein the mature protease comprises a C- or N-terminal propeptide and/or an N-terminal signal peptide.
6:
- The prokaryotic host cell according to claim 1, wherein the mature protease has an amino acid sequence that has at least 80% sequence identity to the mature polypeptide of SEQ ID NO:2;
- OR wherein the mature protease has an amino acid sequence that has at least 80% sequence identity to the mature polypeptide of SEQ ID NO:6.
7:
- The prokaryotic host cell according to claim 1, wherein the polynucleotide encoding the protease proenzyme comprises a polynucleotide encoding the mature protease that has at least 80% sequence identity to SEQ ID NO:1;
- OR wherein the polynucleotide encoding the protease proenzyme comprises a polynucleotide encoding the mature protease that has at least 80% sequence identity to SEQ ID NO:5.
8:
- The prokaryotic host cell according to claim 1, wherein the propeptide has an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:4;
- OR wherein the propeptide has an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:8.
9:
- The prokaryotic host cell according to claim 1, wherein the polynucleotide encoding the propeptide has at least 80% sequence identity to SEQ ID NO:3;
- OR wherein the polynucleotide encoding the propeptide has at least 80% sequence identity to SEQ ID NO:7.
10:
- The prokaryotic host cell according to claim 1, wherein the mature protease comprises or consists of PfuS or a variant thereof;
- OR wherein the mature protease comprises or consists of BH-S8A or a variant thereof.
11:
- The prokaryotic host cell according to claim 1, wherein the propeptide comprises or consists of the PfuS propeptide or a variant thereof;
- OR wherein the propeptide comprises or consists of the S8A propeptide or a variant thereof.
12:
- The prokaryotic host cell according to claim 1, wherein the at least one polynucleotide encoding the protease proenzyme and the at least one polynucleotide encoding the propeptide have been integrated into same locus or different loci in the genome of the prokaryotic host cell.
13:
- The prokaryotic host cell according to claim 1, wherein the at least one polynucleotide encoding the protease proenzyme and the at least one polynucleotide encoding the propeptide are operably linked with a promotor in an operon.
14:
- The prokaryotic host cell according to claim 1, wherein the at least one polynucleotide encoding the protease proenzyme and the at least one polynucleotide encoding the propeptide are each operably linked with a separate promoter;
- preferably, the separate promoters are identical copies of the same promoter.
15:
- A method for producing a mature protease, the method comprising:
- a. providing a prokaryotic host cell according to claim 1;
- b. cultivating said prokaryotic host cell under conditions conducive for expression of the mature protease;
- and, optionally,
- c. recovering the mature protease.
2 more elements