rDNA NTS-BASED GENE MULTIPLE INSERTION CASSETTE SET AND GRAS-GRADE RECOMBINANT YEAST STRAIN

• US 20170268011A1
• Filed: 12/30/2014
• Published: 09/21/2017
• Est. Priority Date: 08/21/2014
• Status: Active Grant

• Current Assignee: Industry-University Cooperation Foundation Hanyang University
• Original Assignee: Industry-University Cooperation Foundation Hanyang University
• Inventors: KANG, Hyun Ah, MOON, Hye Yun, KIM, Hong Jin
• Granted Patent: US 10,457,949 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2039/523 expressing foreign proteins
• CPC Class Codes: A61K 2039/54 characterised by the route of administration
• CPC Class Codes: A61K 2039/542 oral/gastrointestinal
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 9/0053 Mouth and digestive tract, i.e. intraoral and peroral administration rectal administration A61K9/0031
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 1/185 Saccharomyces isolates
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 2770/30022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2770/30034 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2770/30071 Demonstrated in vivo effect
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0073 with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
• CPC Class Codes: C12P 17/02 Oxygen as only ring hetero atoms
• CPC Class Codes: C12R 2001/865 Saccharomyces cerevisiae
• CPC Class Codes: C12Y 101/01088 Hydroxymethylglutaryl-CoA reductase (1.1.1.88)
• CPC Class Codes: C12Y 114/13132 Squalene monooxygenase (1.14.13.132)

Abstract

Disclosed is a gene multiple insertion cassette set including rDNA NTS fragments and an auxotrophic selection marker having an incomplete promoter is developed, and a safe oral recombinant strain having no antibiotic resistant marker is constructed by multiple insertion of an optimum number of the developed gene multiple insertion cassette sets into chromosomes of a Saccharomyces cerevisiae strain, a vaccine composition including, as an active ingredient, the above strain, a culture product thereof, a cell lysate, or nodavirus capsid protein (NNVcp) isolated and purified therefrom, and a composition for feed addition including, as an active ingredient, the above strain, a culture product thereof, a cell lysate, or squalene or oxidosqualene isolated and purified therefrom.

Claims

1. A gene multiple insertion cassette comprising:

an N-terminal fragment of Sacchoromyces cerevisiae ribosomal DNA nontranscribed spacer (rDNA NTS);

a target insertion gene;

an auxotrophic selection marker gene comprising a promoter region; and

a C-terminal fragment of S. cerevisiae rDNA NTS, in the stated order.

2. The gene multiple insertion cassette of claim 1, wherein the N-terminal fragment of S. cerevisiae rDNA NTS is represented by SEQ ID NO:

1, and the C-terminal fragment of S. cerevisiae rDNA NTS is represented by SEQ ID NO;

2.

3. The gene multiple insertion cassette of claim 1, wherein the auxotrophic selection marker gene is an URA3 gene comprising a promoter region represented by SEQ ID NO:

3 or an LEU2 gene comprising a promoter region represented by SEQ ID NO;

4.

4. The gene multiple insertion cassette of claim 1, wherein the target insertion gene is a nodavirus capsid protein gene (NNV), a HMG-CoA reductase gene (HMG1), or a squalene epoxidase gene (ERG1).

5. A recombinant vector comprising the gene multiple insertion cassette of claim 1.

6. The recombinant vector of claim 5, wherein the recombinant vector has one of cleavage maps selected from the group consisting of FIGS. 1(A), 2(A), 5(A), and 6(A).

7. A recombinant microorganism transformed with the recombinant vector of claim 5.

8. The recombinant microorganism of claim 7, wherein the recombinant microorganism is Escherichia coli.

9. The recombinant microorganism of claim 8, wherein the recombinant microorganism is one selected from NTS-16U-NNV (Accession No:

KCTC 12608BP), NTS-50L-NNV (Accession No;

KCTC 12610BP), NTS-16U-tHMG1 (Accession No;

KCTC 12609BP), and NTS-16U-ERG1 (Accession No;

KCTC 12607BP).

10. A recombinant Sacchoromyces cerevisiae strain in which the gene multiple insertion cassette of claim 1 is multiple-inserted into S. cerevisiae ribosomal DNA (rDNA).

11. The recombinant S. cerevisiae strain of claim 10, wherein the strain is a recombinant S. cerevisiae Y2805/NTS-16U-NNV strain (Accession No:

KCTC 12611BP) or a recombinant S. cerevisiae Y2806/NTS-50L-NNV strain (Accession No;

KCTC 12613BP), wherein each of the strains comprises a nodavirus capsid protein gene (NNVcp) multiple-inserted thereto.

12. The recombinant S. cerevisiae strain of claim 10, wherein the strain is a recombinant S. cerevisiae Y2805/NTS-16U-tHMG strain (Accession No:

KCTC 12612BP), wherein the strain comprises a HMG-CoA reductase gene (HMG1) multiple-inserted thereto.

13. The recombinant S. cerevisiae strain of claim 10, wherein the strain is a recombinant S. cerevisiae CEN.PK-1C/NTS -16U-tHMG/NTS-16U-ERG1 strain, wherein the strain comprises HMG1 and a squalene epoxidase gene (ERG1) multiple-inserted thereto.

14. A vaccine composition comprising, as an active ingredient, the strain of claim 11, a culture product thereof, a cell lysate, or a nodavirus capsid protein (NNVcp) isolated and purified therefrom.

15. The vaccine composition of claim 14, wherein the vaccine is an oral vaccine.

16. A composition for feed additive, the composition comprising, as an active ingredient, the strain of claim 12, a culture product thereof, a cell lysate, or squalene or squalene isolated and purified therefrom.

17. A composition for feed additive, the composition comprising, as an active ingredient, the strain of claim 13, a culture product thereof, a cell lysate, or squalene or squalene isolated and purified therefrom.