Cell modification method using essential genes as markers and optionally recycling these

Patent metadata

Field	Value
Publication number	US-9951339-B2
Application number	US-201314427698-A
Country	US
Kind code	B2
Filing date	Sep 19, 2013
Priority date	Sep 19, 2012
Publication date	Apr 24, 2018
Grant date	Apr 24, 2018

Abstract

The invention relates to a method for modification of a host cell at a target locus, which method comprises: providing a host cell comprising, at a first locus, at least two site-specific recombination sites and a nucleic acid having an essential function or encoding a product having an essential function; introducing into the host cell, at the target locus, a further nucleic acid having the essential function or encoding for a product having the essential function; and carrying out recombination at the first locus via the at least two site-specific recombination sites, so that the nucleic acid having an essential function or encoding a product having an essential function is rendered non-functional, thereby to modify the host cell at the target locus. The invention also relates to a cell obtainable by a method of the invention.

First claim

The invention claimed is:

1. A method for producing a biological compound of interest, which method comprises transforming a host cell with a nucleic acid encoding the biological compound of interest or a compound involved in synthesis of the biological compound of interest and culturing the resulting host cell under conditions conducive to production of the biological compound of interest, wherein the host cell is obtained by a method comprising:
providing a host cell comprising, at a first locus, at least two site-specific recombination sites and a nucleic acid having an essential function or encoding a product having an essential function;
introducing into the host cell, at a second locus, by homologous recombination, a further nucleic acid having the essential function or encoding for a product having the essential function together with the nucleic acid encoding the biological compound of interest or a compound involved in synthesis of the biological compound of interest; and
carrying out recombination at the first locus via the at least two site-specific recombination sites, so that the nucleic acid having an essential function or encoding a product having an essential function, positioned at the first locus, is rendered non-functional,

thereby to modify the host cell at the second locus, wherein the nucleic acid encoding the biological compound of interest or a compound involved in synthesis of the biological compound of interest is introduced at the second locus,
wherein the host cell, when deficient in the nucleic acid having an essential function or encoding a product having an essential function, is non-viable under all conditions and on any medium.

2. The method according to claim 1 , further comprising isolating the biological compound of interest from culture broth.

3. The method according to claim 1 , wherein introduction into the host cell of the further nucleic acid having the essential function or encoding for a product having the essential function at the second locus results in replacement of nucleic acid sequence at the second locus with the further nucleic acid having the essential function or encoding for a product having the essential function.

4. The method according to claim 1 , wherein at least two of the site-specific recombination sites flank the nucleic acid having an essential function or encoding a product having an essential function at the first locus.

5. The method according to claim 1 , wherein at least two of the site-specific recombination sites flank a sequence encoding a recombinase, said recombinase recognizes at least two of the site-specific recombination sites.

6. The method according to claim 5 , wherein the recombinase is tyrosine recombinase.

7. The method according to claim 5 , wherein expression of the recombinase is under control of an inducible promoter.

8. The method according to claim 1 , wherein the essential function is an essential function in fungi.

9. The method according to claim 1 , wherein
introducing into the host cell, at a second locus, a further nucleic acid having the essential function or encoding for a product having the essential function; and
carrying out recombination at the first locus via the at least two site-specific recombination sites, so that the nucleic acid having an essential function or encoding a product having an essential function is rendered non-functional
are carried out simultaneously, separately or sequentially.

10. The method according to claim 1 , wherein a further copy of the essential nucleic acid is introduced at the locus together with at least two site-specific recombination sites.

11. The method according to claim 10 , which further comprises:
introducing into the host cell, at a further second locus, a further copy of the nucleic acid having the essential function or encoding for a product having the essential function; and
carrying out recombination at the second locus via the at least two site-specific recombination sites, so that the nucleic acid having an essential function or encoding a product having an essential function is rendered non-functional.

12. The method according to claim 11 , which comprises one or more further cycles of:
introducing into the host cell, at a further second locus, a further copy of the nucleic acid having the essential function or encoding for a product having the essential function; and
rendering non-functional the nucleic acid having an essential function or encoding a product having an essential function introduced in the previous introduction cycle via recombination of the at least two site-specific recombination sites flanking that essential gene.

13. The method according to claim 1 , wherein the cell is a prokaryotic, archaeal or eukaryotic cell.

14. The method according to claim 1 , wherein the host cell is a fungal cell.

15. The method according to claim 14 , wherein the fungal cell is selected from the group consisting of an Aspergillus cell, a Chrysosporium cell, a Penicillium cell, a Saccharomyces cell, a Rasamsonia cell, a Talaromyces cell, and a Trichoderma cell.

16. The method according to claim 11 , wherein introducing into the host cell, at a further second locus, of a further copy of the nucleic acid having the essential function or encoding for a product having the essential function occurs by introducing said sequence together with at least two site-specific recombination sites.

17. The method according to claim 12 , wherein introducing into the host cell, at a further second locus, of a further copy of the nucleic acid having the essential function or encoding for a product having the essential function occurs by introducing said sequence together with at least two site-specific recombination sites.

18. A method for transformation of a host cell with a nucleic acid of interest, which method comprises:
providing a host cell comprising, at a first locus, at least two site-specific recombination sites flanking a nucleic acid having an essential function or encoding a product having an essential function;
introducing into the host cell, at a second locus, a nucleic acid of interest and a further nucleic acid having the essential function or encoding for a product having the essential function; and
removing the nucleic acid having the essential function or encoding for a product having the essential function at the first locus via recombination of the at least two site-specific recombination sites,

thereby to transform the host cell with a nucleic acid of interest,
wherein the host cell, when deficient in the nucleic acid having an essential function or encoding a product having an essential function, is non-viable under all conditions and on any medium.

Assignees

• Dsm Ip Assets Bv

Inventors

• De Lange Ilse
• Meijrink Bernard
• Roubos Johannes Andries
• Ooi Siew-Loon

Classifications

• C12N15/81

  for yeasts · CPC title

• C12N15/80

  for fungi · CPC title

• C12N15/65 Primary

  using markers (enzymes used as markers C12N15/52) · CPC title