# US 10,767,207 B2 · Trichoderma reesei host cells expressing a glucoamylase from Aspergillus fumigatus and methods of use thereof

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- Number: US 10,767,207 B2
- Document ID: US10767207B2
- Status: Granted
- Kind: B2
- Application number: 16/281,989
- Filed: 2019-02-21
- Published: 2019-06-13
- Granted: 2020-09-08
- Earliest claimed: 2012-12-11
- Assignees: Danisco US Inc.
- Applicants: Danisco US Inc
- Inventors: Jing Ge; Ling Hua; Sung Ho Lee; Jalsen Li; Jayarama K Shetty; Zhongmei Tang; Bo Zhang; Kun Zhong
- Web page: https://patentgrep.com/patent/US10767207B2

## CPC

- C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase}
- C12N 9/2428: {Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase}
- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- C12Y 302/01003: Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
- Y02E 50/10: Biofuels, e.g. bio-diesel
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

Fungal glucoamylases from Aspergillus fumigatus—expressed in Trichoderma reesei host cells (AfGATR) are provided. Trichoderma reesei host cells express AfGATRs at higher, or at least comparable, levels to natively expressed AfGA Aspergillus fumigatus. AfGATRs, including AfGA1TR and AfGA2TR, exhibit high activity at elevated temperatures and at low pH, so AfGATRs can be used efficiently in a process of saccharification in the presence of alpha-amylase, such as Aspergillus kawachii alpha-amylase (AkAA). AfGATRs advantageously catalyze starch saccharification to an oligosaccharide composition significantly enriched in DP1 (i.e., glucose) compared to the products of saccharification catalyzed by Aspergillus niger glucoamylase (AnGA) or native AfGA expressed in Aspergillus fumigatus. AfGATRs such as AfGA1TR, AfGA2TR or a variant thereof can be used at a lower dosage than AnGA and natively expressed AfGAs to produce comparable levels of glucose.

## Claims

### Claim 1

A recombinant Trichoderma reesei (T. reesei) host cell expressing an AfGATR having at least 90% sequence identity to SEQ ID NO: 12 or 13, wherein the AfGATR is more thermostable than an AfGA having the same amino acid sequence of AfGATR and wherein the AfGA is expressed in an A. fumigatus host cell.

### Claim 2 (depends on 1)

A recombinant AfGATR produced by culturing the host cell of claim 1 in a fermentation medium.

### Claim 3 (depends on 2)

The recombinant AfGATR of claim 2, wherein said AfGATR has at least 70% activity at 74° C. at pH 5.0 over 10 min.

### Claim 4 (depends on 3)

The recombinant AfGATR of claim 3, wherein said AfGATR is an A. fumigatus glucoamylase 1 polypeptide expressed in T. reesei (AfGA1TR).

### Claim 5 (depends on 4)

The recombinant AfGA1TR of claim 4, wherein said AfGA1TR has at least 70% activity over a temperature range of 55° to 74° C. at pH 5.0 over 10 min.

### Claim 6 (depends on 5)

The recombinant AfGA1TR of claim 5, wherein said AfGAT1R has an optimum temperature of about 68° C.

### Claim 7 (depends on 3)

The recombinant AfGATR of claim 3, wherein said AfGATR is an A. fumigatus glucoamylase 2 polypeptide expressed in T. reesei (AfGA2TR).

### Claim 8 (depends on 7)

The recombinant AfGA2TR of claim 7, wherein said AfGA2TR has at least 70% activity over a temperature range of 61° to 74° C. at pH 5.0 over 10 min.

### Claim 9 (depends on 8)

The recombinant AfGA2TR of claim 8, wherein said AfGA2TR has an optimum temperature of about 69° C.

### Claim 10 (depends on 3)

The recombinant AfGATR of claim 3, wherein said AfGATR comprises an amino acid sequence with at least 95% or 99% amino acid sequence identity to SEQ ID NO: 12.

### Claim 11 (depends on 10)

The recombinant AfGATR of claim 10, wherein said AfGATR comprises SEQ ID NO: 12.

### Claim 12 (depends on 3)

The recombinant AfGATR claim 3, wherein said AfGATR consists of an amino acid sequence with at least 99% amino acid sequence identity to SEQ ID NO: 12.

### Claim 13 (depends on 3)

The recombinant AfGATR of claim 3, wherein said AfGATR comprises an amino acid sequence with at least 95% or 99% amino acid sequence identity to SEQ ID NO: 13.

### Claim 14 (depends on 13)

The recombinant AfGATR of claim 13, wherein said AfGATR comprises SEQ ID NO: 13.

### Claim 15 (depends on 3)

The recombinant AfGATR of claim 3, wherein said AfGATR consists of an amino acid sequence with at least 99% amino acid sequence identity to SEQ ID NO: 13.

### Claim 16 (depends on 3)

A method of saccharifying a composition comprising starch to produce a composition comprising glucose, wherein said method comprises:
(i) contacting a starch composition with the AfGATR of claim 3; and
(ii) saccharifying the starch composition to produce said glucose composition;
wherein said AfGATR catalyzes the saccharification of the composition comprising starch to a composition comprising glucose.

### Claim 17 (depends on 16)

The method of claim 16, wherein said composition comprising glucose is more significantly enriched in DP1 compared to a second composition comprising DP1 produced by Aspergillus niger glucoamylase (AnGA) after 24 hours of saccharification under the same conditions.

### Claim 18 (depends on 16)

The method of claim 16, wherein said composition comprising glucose is enriched in DP1 compared to a second composition comprising DP1 produced by a wild-type AfGA under the same conditions.

### Claim 19 (depends on 16)

The method of claim 16, wherein said AfGATR is AfGATR2 and wherein said composition comprising glucose is enriched in DP1 compared to a second composition comprising DP1 produced by AfGA1TR under the same conditions.

### Claim 20 (depends on 16)

The method of claim 16, wherein the AfGATR is present at an amount about 40%-50% the amount of AnGA, to produce the same DP1 yield after 24 hours of saccharification under the same conditions.

### Claim 21 (depends on 16)

The method of claim 16, wherein the method further comprises contacting a starch composition with an alpha-amylase.

### Claim 22 (depends on 21)

The method of claim 21, wherein the alpha-amylase is AkAA.

### Claim 23 (depends on 16)

The method of claim 16, wherein the method further comprises contacting a starch composition with a pullulanase.

### Claim 24 (depends on 16)

The method of claim 16, further comprising fermenting the glucose composition to produce an End of Fermentation (EOF) product.

### Claim 25 (depends on 24)

The method of claim 24, wherein the EOF product comprises a metabolite.

### Claim 26 (depends on 16)

The method of claim 16, wherein said AfGATR is secreted by said Trichoderma reesei host cell.

### Claim 27 (depends on 26)

The method of claim 26, wherein said host cell further expresses and secretes an alpha-amylase.

### Claim 28 (depends on 27)

The method of claim 27, wherein said host cell further expresses and secretes a pullulanase.

### Claim 29 (depends on 26)

The method of claim 26, wherein said composition comprising starch is contacted with said host cell.