# US 11,306,319 B2 · Enhanced protein expression and methods thereof

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- Number: US 11,306,319 B2
- Document ID: US11306319B2
- Status: Granted
- Kind: B2
- Application number: 15/771,618
- Filed: 2016-10-27
- Published: 2018-11-15
- Granted: 2022-04-19
- Earliest claimed: 2015-10-30
- Assignees: Danisco US Inc.
- Applicants: Danisco US Inc
- Inventors: Cristina Bongiorni; Marguerite A. Cervin; George England; Chao Zhu; Frank Wouter Koopman
- Web page: https://patentgrep.com/patent/US11306319B2

## CPC

- C12N 15/75: for Bacillus
- C12N 1/205: Bacterial isolates
- C12N 9/1247: {DNA-directed RNA polymerase (2.7.7.6)}
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12N 2320/50: Methods for regulating/modulating their activity
- C12N 2330/51: Specially adapted vectors
- C12N 2800/101: for bacteria
- C12R 2001/01: Bacteria or Actinomycetales {; using bacteria or Actinomycetales}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

The present disclosure is generally related to modified Gram positive bacterial cells producing increased amounts of one or more protein(s) of interest and modified Gram positive bacterial cells having increased genetic competency. Thus, certain embodiments of the disclosure are directed to modified Gram positive bacterial cells expressing an increased amount of a protein of interest, relative to an unmodified (parental) Gram positive bacterial cell expressing the same protein of interest, wherein the modified bacterial cell comprises at least one mutation in a rpoC gene encoding a variant RNA-polymerase (RNAP) β′-subunit polypeptide. In certain embodiments, the rpoC gene encoding the variant β′-subunit polypeptide is integrated into the chromosome of the modified cell. In other embodiments, the rpoC gene encoding the variant β′-subunit polypeptide is comprised on an extrachromosomal plasmid introduced into the modified cell. In other embodiments, the disclosure is directed to competent Bacillus host cells comprising at least one copy of a nucleic acid construct encoding a modified rpoC polypeptide comprising 90% sequence identity to SEQ ID NO: 8 and an aspartic acid to glycine substitution at position 796 of SEQ ID NO: 8, wherein the polynucleotide encoding the rpoC polypeptide is foreign to the Bacillus host cell that was non-competent prior to the introduction of the first nucleic acid construct.

## Claims

### Claim 1

A method for increasing expression of a protein of interest (POI) in a Bacillus cell comprising:
(a) obtaining a modified Bacillus cell expressing an increased amount of a POI, wherein the modified Bacillus cell comprises at least one mutation in a rpoC gene encoding a variant RNA-polymerase (RNAP) β′-subunit polypeptide, wherein the variant β′-subunit polypeptide has at least 90% sequence identity to SEQ ID NO: 6 or 8 and comprises an aspartic acid to glycine substitution at amino acid residue 796 (D796G), or at the equivalent position in any Bacillus RNAP β′-subunit family member and
(b) culturing the modified cell under conditions such that the POI is expressed, wherein the modified Bacillus cell expresses an increased amount of a POI relative to the expression of the POI in an unmodified Bacillus control cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the rpoC gene encoding the variant β′-subunit polypeptide comprises at least 80% sequence identity to SEQ ID NO: 5 or 7.

### Claim 3 (depends on 1)

The method of claim 1, further comprising at least one mutation in a rpoB gene encoding a variant RNAP β-subunit polypeptide.

### Claim 4 (depends on 1)

The method of claim 1, wherein the increased amount of an expressed POI relative to the unmodified Bacillus control cell is at least 5%.

### Claim 5 (depends on 1)

The method of claim 1, wherein the rpoC gene encoding the variant β″-subunit polypeptide is integrated into the chromosome of the modified cell.

### Claim 6 (depends on 1)

The method of claim 1, wherein the rpoC gene encoding the variant β″-subunit polypeptide is comprised on an extrachromosomal plasmid of the modified cell.

### Claim 7 (depends on 1)

The method of claim 1, wherein the variant β″-subunit polypeptide further comprises at least one mutation at an amino acid residue position selected from the group consisting of 751, 784, 797 and 1018-1020.

### Claim 8 (depends on 7)

The method of claim 7, wherein the at least one mutation is selected from the group consisting of: a methionine to isoleucine (M751 I) substitution at amino acid residue position 751, an arginine to histidine (R784H) substitution at amino acid residue position 784, a serine to phenylalanine (S797F) substitution at amino acid residue position 797, a deletion of amino acid residues 1018, 1019 and 1020 (ΔI1018-R1020) at amino acid residue position 1018-1020, of SEQ ID NO: 6 or 8, or a combination thereof.

### Claim 9 (depends on 1)

The method of claim 1, wherein the Bacillus cell is selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. sonorensis, B. halodurans, B. pumilus, B. lautus, B. pabuli, B. cereus, B. agaradhaerens, B akibai, B. clarkii, B. pseudofirmus, B. lehensis, B. megaterium, B. coagulans, B. circulans, B. gibsonii and B. thuringiensis.

### Claim 10 (depends on 1)

The method of claim 1, wherein the POI is selected from the group consisting of acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carbonic anhydrases, carboxypeptidases, catalases, cellulases, chitinases, chymosins, cutinases, deoxyribonucleases, epimerases, esterases, α-galactosidases, β-galactosidases, α-glucanases, glucan lysases, endo-β-glucanases, glucoamylases, glucose oxidases, α-glucosidases, β-glucosidases, glucuronidases, glycosyl hydrolases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, peptidases, rhamno-galacturonases, ribonucleases, transferases, transport proteins, transglutaminases, xylanases, hexose oxidases, and combinations thereof.