# US 2014/0154738 A1 · Protein glycosylation modification in methylotrophic yeast

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2014/0154738 A1
- Document ID: US20140154738A1
- Status: Published application
- Kind: A1
- Application number: 14/152,329
- Filed: 2014-01-10
- Published: 2014-06-05
- Earliest claimed: 2000-06-30
- Assignees: Vib Vzw; Universiteit Gent; Research Corporation Technologies, Inc.
- Applicants: VIB, vzw; Universiteit Gent; Research Corporation Technologies, Inc.
- Inventors: Roland Contreras; Nico L.M. Callewaert; Steven C.J. Geysens
- Web page: https://patentgrep.com/patent/US20140154738A1

## CPC

- C12P 21/005: {Glycopeptides, glycoproteins}
- C12N 1/165: Yeast isolates
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12N 9/2488: {Mannanases}
- C12N 15/81: for yeasts
- C12N 15/815: {for yeasts other than Saccharomyces}
- C12Y 302/0102: Alpha-glucosidase (3.2.1.20)
- C12Y 302/01024: Alpha-mannosidase (3.2.1.24)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C12R 2001/84: Pichia
- Y02P 20/52: using catalysts, e.g. selective catalysts
- 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
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The present invention provides genetically engineered strains of Pichia capable of producing proteins with reduced glycosylation. In particular, the genetically engineered strains of the present invention are capable of expressing either or both of an α-1,2-mannosidase and glucosidase II. The genetically engineered strains of the present invention can be further modified such that the OCH1 gene is disrupted. Methods of producing glycoproteins with reduced glycosylation using such genetically engineered stains of Pichia are also provided.

## Claims

### Claim 1

A genetically engineered methylotrophic yeast strain, which lacks a functional enzyme involved in production of high mannose structures, and expresses at least one exogenous enzyme for production of Man5GlcNAc2, wherein said enzyme involved in production of high mannose structures is α-1,6-mannosyltransferase encoded by the OCH1 gene, said at least one enzyme for production of Man5GlcNAc2 is α-1,2-mannosidase or an enzymatically active fragment thereof.

### Claim 2 (depends on 1)

The strain of claim 1, wherein said enzyme is of a fungal origin or a mammalian origin.

### Claim 3 (depends on 1)

The strain of claim 1, wherein said enzyme for production of Man5GlcNAc2 is targeted to the ER.

### Claim 4 (depends on 3)

The strain of claim 3, wherein the targeting of said enzyme to the ER is achieved by engineering said enzyme to include the ER retention signal as set forth in SEQ ID NO: 1.

### Claim 5 (depends on 1)

The strain of claim 1, wherein said strain produces Man5GlcNAc2 as a predominant N-glycan structure or a predominant intermediate N-glycan structure.

### Claim 6 (depends on 1)

The strain of claim 1, wherein said methylotrophic yeast is of the genera Candida, Hansenula, Torulopsis, or Pichia.

### Claim 7

A method for producing a glycoprotein with reduced hyperglycosylation in methylotrophic yeast, comprising providing a methylotrophic yeast strain, which lacks a functional enzyme involved in production of high mannose structures, and expresses at least one exogenous enzyme for production of Man5GlcNAc2, wherein said enzyme involved in production of high mannose structures is α-1,6-mannosyltransferase encoded by the OCH1 gene, said at least one enzyme for production of Man5GlcNAc2 is α-1,2-mannosidase or an enzymatically active fragment thereof.

### Claim 8 (depends on 7)

The method of claim 7, wherein said enzyme is of a fungal origin or a mammalian origin.

### Claim 9 (depends on 8)

The method of claim 8, wherein said enzyme for production of Man5GlcNAc2 is targeted to the ER.

### Claim 10 (depends on 9)

The method of claim 9, wherein the targeting of said enzyme to the ER is achieved by engineering said enzyme to include the ER retention signal as set forth in SEQ ID NO: 1.

### Claim 11 (depends on 7)

The method of claim 7, wherein said strain produces Man5GlcNAc2 as a predominant N-glycan structure or a predominant intermediate N-glycan structure.

### Claim 12 (depends on 7)

The method of claim 7, wherein said methylotrophic yeast is of the genera Candida, Hansenula, Torulopsis, or Pichia.