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1. WO2020156903 - CO-EXPRESSION DE FOLDASE PARENTE

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CLAIMS

1. A nucleic acid construct comprising:

a) a first heterologous promoter operably linked to at least one polynucleotide encoding a foldase; and

b) a second heterologous promotor operably linked to at least one polynucleotide encoding a polypeptide of interest;

wherein the foldase and the polypeptide of interest are from the same Gram-positive species.

2. The nucleic acid construct according to claim 1 , wherein the first heterologous promotor and the second heterologous promoter are identical copies of the same heterologous promoter.

3. The nucleic acid construct according to any of the preceding claims, wherein the polypeptide of interest comprises an enzyme; preferably the enzyme is selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase; more preferably an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, nuclease, oxidase, pectinolytic enzyme, peroxidase, phosphodiesterase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, and beta-xylosidase; most preferably the polypeptide of interest is an amylase.

4. The nucleic acid construct according to any of the preceding claims, wherein the foldase and the polypeptide of interest are selected from the same Bacillus species; preferably the Bacillus species 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 sp. NSP9.1 , Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus safensis, Bacillus sonorensis L12, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells; most preferably Bacillus species is selected from the group consisting of B. amyloliquefaciens, B. licheniformis, B. sp. NSP9.1 , B. sonorensis L12, and B. subtilis.

5. The nucleic acid construct according to any of the preceding claims, wherein the foldase has a sequence identity of at least 80% to SED ID NO:3, and the polypeptide of interest is from B. amyloliquefaciens ; preferably the polypeptide of interest is an amylase; more preferably the

polypeptide of interest has a sequence identity of at least 80% to SEQ ID NO:6; most preferably, the polypeptide of interest comprises or consists of SEQ ID NO:6.

6. The nucleic acid construct according to any of claims 1-4, wherein the foldase has a sequence identity of at least 80% to SED ID NO:9, and the polypeptide of interest is from B. licheniformis ; preferably the polypeptide of interest is an amylase; more preferably the polypeptide of interest has a sequence identity of at least 80% to SEQ ID NO: 12; most preferably, the polypeptide of interest comprises or consists of SEQ ID NO: 12.

7. The nucleic acid construct according to any claims 1-4, wherein the foldase has a sequence identity of at least 80% to SED ID NO: 15, and the polypeptide of interest is from B. sp. NSP9.1 ; preferably the polypeptide of interest is an amylase; more preferably the polypeptide of interest has a sequence identity of at least 80% to SEQ ID NO: 18; most preferably, the polypeptide of interest comprises or consists of SEQ ID NO: 18.

8. The nucleic acid construct according to any of claims 1-4, wherein the foldase has a sequence identity of at least 80% to SED I D NO:21 , and the polypeptide of interest is from B. sonorensis L12; preferably the polypeptide of interest is an amylase; more preferably the polypeptide of interest has a sequence identity of at least 80% to SEQ ID NO:24; most preferably, the polypeptide of interest comprises or consists of SEQ ID NO:24.

9. The nucleic acid construct according to any of claims 1-4, wherein the foldase has a sequence identity of at least 80% to SED ID NO:27, and the polypeptide of interest is from B. subtilis ; preferably the polypeptide of interest is an amylase; more preferably the polypeptide of interest has a sequence identity of at least 80% to SEQ ID NO:30; most preferably, the polypeptide of interest comprises or consists of SEQ ID NO:30.

10. An expression vector comprising a nucleic acid construct according to any of the preceding claims.

1 1. A Gram-positive host cell comprising in its genome:

1) a nucleic acid construct comprising:

a) a first heterologous promoter operably linked to at least one polynucleotide encoding a foldase; and

b) a second heterologous promotor operably linked to at least one polynucleotide encoding a polypeptide of interest;

and/or

2) an expression vector comprising said nucleic acid construct;

wherein the foldase and the polypeptide of interest are from the same Gram-positive species and are heterologous to the Gram-positive host cell.

12. The Gram-positive host cell according to claim 11 , which is a Bacillus host cell; preferably the Bacillus 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 sp. NSP9.1 , Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus safensis, Bacillus sonorensis L12, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells; most preferably Bacillus species is selected from the group consisting of B. amyloliquefaciens, B. licheniformis, B. sp. NSP9.1 , B. sonorensis L12, and B. subtilis.

13. A method for producing a polypeptide of interest, the method comprising:

I) providing a Gram-positive host cell comprising in its genome:

1) a nucleic acid construct comprising:

a) a first heterologous promoter operably linked to at least one polynucleotide encoding a foldase; and

b) a second heterologous promotor operably linked to at least one polynucleotide encoding the polypeptide of interest;

and/or

2) an expression vector comprising said nucleic acid construct;

wherein the foldase and the polypeptide of interest are from the same Gram positive species and are heterologous to the Gram-positive host cell;

II) cultivating said host cell under conditions conducive for expression of the foldase and the polypeptide of interest; and, optionally

III) recovering the polypeptide of interest.

14. The method according to claim 13 wherein the Gram-positive host cell is a Bacillus host cell; preferably the Bacillus 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 sp. NSP9.1 , Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacillus pumilus, Bacillus safensis, Bacillus sonorensis L12, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells; most preferably Bacillus

species is selected from the group consisting of B. amyloliquefaciens, B. licheniformis , B. sp. NSP9.1 , B. sonorensis L12, and B. subtilis.

15. The method according to any of claims 13-14, wherein the polypeptide of interest comprises an enzyme; preferably the enzyme is selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase; more preferably an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, nuclease, oxidase, pectinolytic enzyme, peroxidase, phosphodiesterase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, and beta-xylosidase; most preferably the polypeptide of interest is an amylase.

16. The method according to any of claims 13-15, wherein the secretion stress experienced by the Gram-positive host cell is on par or reduced; preferably the secretion stress experienced by the Gram-positive host cell is reduced; most preferably the secretion stress is reduced by at least 1 %, e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, or more.