# US 7,745,200 B2 · Formaldehyde dehydrogenase genes from methylotrophic yeasts

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- Number: US 7,745,200 B2
- Document ID: US7745200B2
- Status: Granted
- Kind: B2
- Application number: 11/810,089
- Filed: 2007-06-04
- Published: 2007-12-27
- Granted: 2010-06-29
- Earliest claimed: 1998-07-30
- Assignees: Research Corporation Technologies, Inc.
- Inventors: James M. Cregg
- Web page: https://patentgrep.com/patent/US7745200B2

## CPC

- C12N 9/0008: {acting on the aldehyde or oxo group of donors (1.2)}
- C12N 15/815: {for yeasts other than Saccharomyces}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides formaldehyde dehydrogenase genes (FLD) from methylotrophic yeasts. The FLD structural genes confer resistance to formaldehyde and are therefore useful as a selectable marker in methylotrophic yeasts. The FLD promoter sequences are strongly and independently induced by either methanol as sole carbon source (with ammonium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source). Induction under either methanol, methylamine or both provides levels of heterologous gene expression comparable to those obtained with the commonly used alcohol oxidase I gene promoter (PAOX1). The FLD promoter of Pichia pastoris (PFLD1) is an attractive alternative to PAOX1 for expression of foreign genes in P. pastoris, allowing regulation by carbon (methanol) or nitrogen (methylamine) source within the same expression strain. Yeast strains, expression cassettes, expression vectors, and host cells-comprising an FLD gene promoter and 3′ termination sequence are also provided.

## Claims

### Claim 1

A method for selecting a formaldehyde resistant cell which comprises:
a) transforming cells of a methylotrophic yeast strain which does not have a functional FLD gene, with a vector comprising an FLD nucleic acid which encodes a protein comprising the amino acid sequence as set forth in SEQ ID NO: 2, wherein said FLD nucleic acid is operably linked on its 5′ end to an FLD promoter or a heterologous promoter and is operably linked on its 3′ end to a 3′ termination sequence, wherein said heterologous promoter and said 3′ termination sequence function in said cells,
b) growing said cells in the presence of formaldehyde; and
c) selecting a transformed yeast cell which grows in the presence of formaldehyde.

### Claim 2 (depends on 1)

The method according to claim 1 wherein said strain is a strain of Pichia pastoris.

### Claim 3 (depends on 1)

The method according to claim 1 wherein said promoter is an FLD promoter.

### Claim 4 (depends on 1)

The method according to claim 1, wherein said strain is a strain of Pichia pastoris and said promoter is an FLD promoter comprising the sequence set forth in SEQ ID NO: 3.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said FLD nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 1 or the nucleotide sequence set forth in SEQ ID NO: 5.