# US 10,870,873 B2 · Method of using carbohydrate esters as inducers for gene expression

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- Number: US 10,870,873 B2
- Document ID: US10870873B2
- Status: Granted
- Kind: B2
- Application number: 16/020,429
- Filed: 2018-06-27
- Published: 2018-11-15
- Granted: 2020-12-22
- Earliest claimed: 2012-07-20
- Assignees: Qingdao Viand Biotech Group Co., Ltd.
- Applicants: Qingdao Vland Biotech Group Co., Ltd.
- Inventors: Tom Tao Huang
- Web page: https://patentgrep.com/patent/US10870873B2

## CPC

- C12P 21/005: {Glycopeptides, glycoproteins}
- C07H 13/04: having the esterifying carboxyl radicals attached to acyclic carbon atoms
- C07K 14/475: Growth factors; Growth regulators
- C07K 14/575: Hormones (derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695)
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 1/14: Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor
- C12N 1/38: Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound (C12N1/34 takes precedence)
- C12N 9/2408: {acting on alpha -1,4-glucosidic bonds}
- C12N 9/2411: {Amylases}
- C12N 9/2434: {acting on beta-1,4-glucosidic bonds}
- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12N 15/80: for fungi
- C12P 19/12: Disaccharides
- C12P 19/44: Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- 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)}
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention provides novel carbohydrate esters, in particular disaccharide esters, and the methods of their preparation. These compounds find use as microbial media components for the induction of gene expression in microbial fermentation processes.

## Claims

### Claim 1

A method for producing a protein of interest, comprising:
a) providing a fermentation host; and
b) culturing the fermentation host with a carbon source and an inducer compound of the following formula:
where Ra is H or C(O)CH3; Rb is H or C(O)CH3; Rc is H or C(O)CH3; Rd is H or C(O)CH3; Re is H or C(O)CH3; Rf is H or C(O)CH3; and Rg is H or C(O)CH3, R is a C1-C24 aliphatic moiety, and
wherein the fermentation host is cultured under conditions sufficient to produce the protein of interest.

### Claim 2 (depends on 1)

The method of claim 1, wherein the carbon source comprises one or more of glucose, sucrose, fructose, lactose, cellulose, cellulose hydrolysate, starch, starch hydrolysate, maltose, or maltodextrin.

### Claim 3 (depends on 1)

The method of claim 1, wherein the protein of interest comprises a cellulase or an amylase.

### Claim 4 (depends on 1)

The method of claim 1, wherein the fermentation host comprises a filamentous fungus of the genus Trichoderma or Aspergillus.

### Claim 5 (depends on 4)

The method of claim 4, wherein the fermentation host is Trichoderma reesei, Trichoderma viride, Aspergillus niger, or Aspergillus oryzae.

### Claim 6 (depends on 1)

The method of claim 1, wherein the inducer compound is provided in at least trace amounts in an inducer crude mixture.

### Claim 7 (depends on 6)

The method of claim 6, wherein the inducer crude mixture is added to the culture in an amount of about 0.9 grams of inducer crude mixture per liter of culture.

### Claim 8 (depends on 6)

The method of claim 6, wherein the inducer crude mixture provides a specific induction of greater than or equal to about 17.4 U/mg at day 3.

### Claim 9 (depends on 1)

The method of claim 1, wherein R is selected from the group consisting of unsubstituted C1-C24 alkyl; C1-C24 alkyl substituted with hydroxyl, carboxyl or aromatic groups; unsubstituted C1-C24 alkenyl; and C1-C24 alkenyl substituted with hydroxyl, carboxyl or aromatic groups.

### Claim 10 (depends on 1)

The method of claim 1, wherein R is an aliphatic moiety selected from unsubstituted C1-C24 alkyl, substituted C1-C24 alkyl, unsubstituted C1-C24 alkenyl, and substituted C1-C24 alkenyl.

### Claim 11 (depends on 1)

The method of claim 1, wherein R is an aliphatic moiety selected from C1-C24 alkyl substituted with hydroxyl groups and C1-C24 alkenyl substituted with hydroxyl groups.

### Claim 12 (depends on 1)

The method of claim 1, wherein R is an aliphatic moiety selected from C1-C24 alkyl substituted with carboxyl groups and C1-C24 alkenyl substituted with carboxyl groups.

### Claim 13 (depends on 1)

The method of claim 1, wherein R is an aliphatic moiety selected from C1-C24 alkyl substituted with aromatic groups and C1-C24 alkenyl substituted with aromatic groups.