Method for the Targeted Integration of Multiple Copies of a Gene of Interest in a Yarrowia Strain

a technology of yarrowia and gene, applied in the field of targeted integration of multiple copies of a gene of interest in the genome of a yarrowia strain, can solve the problems of large yield and great stability, none of these technologies have completely satisfied the requirements of industrial production, and the majority of yarrowia lipolytica /i>strains transformed by lip2 had mediocre genetic stability, and the effect of reducing the number of copies

Inactive Publication Date: 2012-02-09
INST NAT DE RECH POUR LAGRICULTURE LALIMENTATION & LENVIRONNEMENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0002]Yarrowia lipolytica yeast is now increasingly used as expression host for genes of interest. This yeast has the specific characteristic of producing and secreting high molecular weight proteins such as alkaline proteases, acid proteases or RNase with great effectiveness in the culture medium, these proteins then being easily recovered from the culture medium. In order to permit the expression of these genes of interest, various technologies were developed early such as those described more particularly in patent EP 0 138 508 B1 or EP 0 220 864 B1. These different technologies have in common that they use “integrative” vectors that allow the insertion of a DNA segment carrying the gene of interest in the chromosomal DNA; this gene of interest is then expressed. Nevertheless, none of these technologies have completely satisfied the requirements of industrial production, namely a large yield and great stability over time.
[0079]The method according to the invention thus makes it possible to reuse a same selection marker, particularly an auxotrophic marker, for another targeted integration during a new transformation step.

Problems solved by technology

Nevertheless, none of these technologies have completely satisfied the requirements of industrial production, namely a large yield and great stability over time.
However, it appeared that the majority of the Yarrowia lipolytica strains transformed by LIP2 had mediocre genetic stability, generally below 50%.
Furthermore, since these transformed strains are obtained as a result of random integration events, monitoring the genetic stability of the strains via sequencing of the insertion sites is extremely tedious.
Added to the problems relating to the stability of the obtained strains and the difficulty in identifying the precise integration site of the exogenous sequence are the additional problems associated with the absence of Yarrowia lipolytica strains lacking multiple selection markers due to the instability of these strains.
However these vectors are dispersed and random.
This is a lengthy, inefficient method that also requires the use of a counter-selection marker.

Method used

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  • Method for the Targeted Integration of Multiple Copies of a Gene of Interest in a Yarrowia Strain
  • Method for the Targeted Integration of Multiple Copies of a Gene of Interest in a Yarrowia Strain
  • Method for the Targeted Integration of Multiple Copies of a Gene of Interest in a Yarrowia Strain

Examples

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example 1

Strains Used in the Examples

[0107]The strains used in the method according to the invention are derived from a wild-type strain of Yarrowia lipolytica, in particular the wild-type strain Yarrowia lipolytica W29 (atcc 20460, indexed under CLIB89 in the Collection of the CIRM (Centre International de Ressources Microbiennes).

[0108]Thus, we can use new mutant strains derived from the Yarrowia lipolytica strain ATCC 20460 through the intermediary of the strain Po1d [auxotrophic strain got leucine (Leu−) and uracil (Ura−)], described in the publication of G. Barth et al.: Yarrowia lipolytica. in Nonconventional Yeasts in Biotechnology: A Handbook (Wolf, K., Ed.), Vol. 1, 1996, pp. 313-388. Springer-Verlag. It is indexed under CLBI139 in the CIRM.

[0109]Obtaining these new receiver strains (FF-lu, FF-lug (CNCM I-3911), FF-lua (CNCM I-3912), FF-luga (CNCM I-3913)) will be described later.

[0110]Obtaining the Strains

[0111]The mutant receiver strains usable in the method according to the inven...

example 2

Construction and Use of an Integration / Expression Cassette for the Targeted Insertion of a Gene Encoding for a Therapeutic Protein of Interest not Produced by Y. lipolytica

[0196]We took as example the L-asparaginase protein of Erwinia chyrsanthemi. The gene encoding for this protein was introduced into the vector pVC-pPOX2 at the HindIII / AvrII site. To do this, a fusion PCR was performed between the preproLIP2 sequence and the portion of the sequence of the gene encoding for the mature form of the L-asparaginase protein. PCR was performed with the primers preproLip2 and Lip2KR to amplify the pre-pro-LIP2 sequence and with the Laspens and Lasprev primers to amplify the sequence encoding for the mature protein. Then, by mixing the 2 PCR products obtained, the fusion with the preproLIP2 and pLasprev primers was carried out.

[0197]The preproLip2 primer contains the HindIII site and the Lasprev primer the AvrII site for cloning the fusion product in the pCV-pPOX2. The vector pVC-pPOX2-pr...

example 3

Transformation of the Integration / Expression Cassettes of the L-Asparaginase Protein in Y. lipolytica

[0199]It is possible, for example, to introduce successively 4 copies of the expression cassette of L-asparaginase in the 4 loci mentioned earlier in the FF-luga strain.

[0200]1 Insertion of the First Copy at the ADE2 Locus.

[0201]The first expression cassette was introduced by transformation of the FF-luga strain at the ADE2 locus using the integration / expression cassette PTAde2-Gut2-1.0Ex-LAsp from the vector JME941. The transformants are selected on selective YNBcasa medium supplemented with uracil and adenine and containing 1% glycerol as sole carbon source. The transformants obtained are isolated on this same selective medium and integration at the locus is verified by PCR using the primers Ver1ade2 and Ver2ade2 located upstream and downstream of the integration sequences.

[0202]The transformation rate obtained is 8.2 103 transformants per μg of DNA transformed.

[0203]Verification ...

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Abstract

This invention concerns a method for the targeted integration of at least three copies of a gene of interest in the genome of a Yarrowia strain including the steps of: (a) cultivating a Yarrowia strain, said strain including a deletion among at least three genes, the phenotype associated with each of these deletions corresponding to an auxotrophy or to a dominant character for this strain; (b) transforming said Yarrowia strain thus obtained with at least three recombinant vectors that include selection markers allowing, for this strain, the complementation of auxotrophy and, potentially, of the dominant character resulting from each of these deletions; and (c) selecting, on a minimum medium, the yeasts having integrated said at least three recombinant vectors. This invention also includes a method for producing a polypeptide of interest using this method as well as a method for obtaining a modified Yarrowia strain including a deletion among at least three genes, the phenotype associated with each of these deletions corresponding to an auxotrophy or to a dominant character for this strain.

Description

[0001]This invention concerns a method for the targeted integration of at least three copies of a gene of interest in the genome of a Yarrowia strain that includes the steps of (a) cultivating a Yarrowia strain, said strain including a deletion among at least three genes, the phenotype associated with each of said deletions corresponding to an auxotrophy or to a dominant character for said strain; (b) transforming the Yarrowia strain thus obtained using at least three recombinant vectors, including selection markers enabling, for this Yarrowia strain, the complementation of auxotrophy and, potentially, the dominant character resulting from each of the deletions; and (c) selecting, on a minimum medium, yeasts having integrated these at least three recombinant vectors. This invention also includes a method for producing a polypeptide of interest using said method as well as a method for obtaining a modified Yarrowia strain including a deletion among at least three genes, the phenotype...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/02C12N1/15C12N15/80
CPCC12N15/815
Inventor NICAUD, JEAN-MARCFUDALEJ, FRANCKNEUVEGLISE, CECILEBECKERICH, JEAN-MARIE
Owner INST NAT DE RECH POUR LAGRICULTURE LALIMENTATION & LENVIRONNEMENT
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