Rasamsonia transformants

Patent metadata

Field	Value
Publication number	US-9631197-B2
Application number	US-201314382768-A
Country	US
Kind code	B2
Filing date	Mar 12, 2013
Priority date	Mar 12, 2012
Publication date	Apr 25, 2017
Grant date	Apr 25, 2017

Abstract

The invention relates to a method for carrying out recombination at a target locus in a Rasamsonia cell. The invention also relates to Rasamsonia cells, for example Rasamsonia cells produced by such a process. The invention further relates to processes in which such Rasamsonia cells are used and to the resulting enzyme compositions. The invention further relates to nucleic acid and amino acid sequences.

First claim

The invention claimed is:

1. A method for carrying out recombination at a target locus in a Rasamsonia cell, which method comprises:
(a) providing a first nucleic acid comprising, in order:
(i) a region capable of homologous recombination with a 5′ region of the target locus,
(ii) a first site-specific recombination site,
(iii) a first promoter, and
(iv) a sequence encoding a first non-functional portion of a marker gene;

(b) providing a second nucleic acid comprising, in order:
(v) a sequence encoding a second non-functional portion of the marker gene of (a) and capable of homologous recombination with the first non-functional portion,
(vi) a first terminator sequence,
(vii) a second promoter,
(viii) a sequence encoding a site-specific recombinase,
(ix) a second terminator sequence,
(x) a second site-specific recombination site, and
(xi) a region capable of homologous recombination with a 3′ region of the target locus,

(c) recombining the sequence encoding a first non-functional portion of a marker gene with the sequence encoding a second non-functional portion of the marker gene to give rise to a single nucleic acid comprising, in order, the region capable of homologous recombination with the 5′ region of the target locus, the first site-specific recombination site, the first promoter, a sequence encoding the functional marker gene, the first terminator sequence, the second promoter, the sequence encoding the site-specific recombinase, the second terminator sequence, the second site-specific recombination site, and the region capable of homologous recombination with the 3′ region of the target locus; and
(d) recombining the single nucleic acid of (c) with the homologous 5′ region and 3′ region of the target locus in the Rasamsonia cell,
thereby to carry out recombination at the target locus in the Rasamsonia cell.

2. A method according to claim 1 , which further comprises expressing the site-specific recombinase so that the sequence located between the site-specific recombination sites is out-recombined.

3. A method according to claim 2 , wherein expression of the site-specific recombinase is controlled by an inducible promoter.

4. A method according to claim 1 , wherein recombination of the first and second nucleic acids with each other and with the homologous 5′ region and 3′ region flanking the target locus is carried out in vivo.

5. A method according to claim 2 , wherein out-recombination of a nucleic acid sequence between the site-specific recombination sites is carried out in vivo.

6. A method according to claim 1 , wherein the in vivo recombination is carried out in a Rasamsonia emersonii cell.

7. A method according to claim 1 , wherein the host cell is a variant of a parent host cell, the parent host cell having a preference for non-homologous recombination, wherein the ratio of non-homologous recombination/homologous recombination is decreased in the variant as compared to said ratio in said parent organism measured under the same conditions.

8. A method according to claim 1 , wherein: the site-specific recombination sites are lox sites and the site-specific recombinase is Cre; the site-specific recombination sites are FRT sites and the site-specific recombinase is Flp; the recombination sites are Vlox sites and the site-specific recombinase is VCre; or the recombination sites are Slox and the site-specific recombinase is SCre.

9. A method according to claim 2 , wherein the site-specific recombination sites are such that out-recombination following site-specific recombinase expression gives rise to a single mutant site-specific recombination site at the target locus which is not recognized by the site-specific recombinase.

10. A method according to claim 1 , wherein the target locus comprises a coding sequence which is disrupted and/or partially or fully deleted.

11. The method according to claim 1 , wherein the first nucleic acid further comprises one or more expression cassettes and each expression cassette comprising, in order, a promoter, a coding sequence encoding a polypeptide of interest, and a terminator sequence; wherein the one or more expression cassettes are positioned between (i) the region capable of homologous recombination with the 5′ region of the target locus and (ii) the first site-specific recombination site.

12. The method according to claim 1 , wherein the second nucleic acid further comprises one or more expression cassettes and each expression cassette comprising, in order, a promoter, a coding sequence encoding a polypeptide of interest, and a terminator sequence; wherein the one or more expression cassettes are positioned between (x) the second site-specific recombination site and (xi) the region capable of homologous recombination with a 3′ region of the target locus.

Assignees

• Dsm Ip Assets Bv

Inventors

• Los Alrik Pieter
• Arendsen Yvonne Johannes Odilia
• Vollebregt Adrianus Wilhelmus Hermanus

Classifications

• C12N9/2437

  Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150) · CPC title

• C12N15/905

  in yeast · CPC title

• C12N15/80 Primary

  for fungi · CPC title

• C12Y304/23

  Aspartic endopeptidases (3.4.23) · CPC title

• C12N9/58

  derived from fungi · CPC title