Plasmid vectors for continuous, inactivation-free production of target proteins; recombinant heat-tolerant yeast cells of the genus Ogataea for continuous, inactivation-free production of target proteins; and methods for constructing such recombinant yeast cells.

Claims

OCR09WP16 / 07 / 25681. A plasmid vector for continuous production of target proteins without an activator, comprising a promoter, alpha-factor secretion signal, multiple gene cloning sites, transcription terminators of genes producing methanol oxidase, antibiotic resistance genes, transcription terminators of genes producing glyceraldehyde 3-phosphate dehydrogenase, and a plasmid vector replication origin with unique characteristics: • The continuous protein-producing gene promoter is derived from the yeast genus Ogataea; • The alpha-factor secretion signal, which is the target protein exit transport component, is derived from Ogateapolymorpha; and • The transcription terminator of the methanol oxidase gene is derived from Ogateapolymorpha.2.A plasmid vector for the continuous production of target proteins without an activator, according to claim 1, which includes a multiple clone sequence containing a restriction enzyme site selectable from EcoRI, HindiIII, PstI, ApaI, MluI, SalI, NspV, BamHI, and NheI, or a combination of these; an antibiotic resistance gene for isolating cells containing recombinant plasmids regulated by two ligated promoters: the TEF1 promoter from O. polymorpha and the L-ldh promoter from L. plantarum; a transcription terminator of a glyceraldehyde 3-phosphate dehydrogenase gene from O. polymorpha; and a self-replication origin of a bacterial plasmid vector selectable from pUC, pMB1, pBR322, and ColE1.3.A plasmid vector for continuous, inactivation-free production of target proteins under claim 1, where the promoter of the continuous protein-producing gene is selectable from glyceraldehyde-3-phosphate dehydrogenase (GAP), translational longevity factor EF-1alpha (TEF1), glycosylphosphatidylinositolanchored protein (GCW14), alcohol dehydrogenase (ADH1), and pyluvate D.

4. A plasmid vector for continuous, inactivation-free production of target proteins under claim 1 or 2, where the antibiotic resistance gene is selectable from hygromycin, neomycin, and zeosin, or a combination of these.

5. A plasmid vector for continuous, inactivation-free production of target proteins under claim 1, where the yeast genus Ogataea is selectable from O. polymorpha, O. parapolymorpha, or O. thermomethanolica. 6.A recombinant yeast cell for continuous, activator-free production of a target protein containing a plasmid vector for continuous, activator-free production of a target protein according to one of the claims 1-5, which includes: a. a yeast host cell and b. a plasmid vector for continuous target protein production containing an alpha-factor secretion signal promoter, multiple transcription terminator of a methanol oxidase gene, an antibiotic resistance gene, and a transcription terminator of a drug. The plasmid vectors that produce the enzyme glyceraldehyde-3-phosphate dehydrogenase and the origin of self-replication of plasmid vectors in bacteria are characterized by a promoter of a gene that continuously produces a protein (a base sequence derived from the yeast genus Ogataea), an alpha-factor secretion signal which transports the target protein out of the cell (derived from O. polymorpha), and a transcription terminator of a gene that produces the enzyme methanol oxidase (also derived from O. polymorpha). These plasmid vectors are introduced into the host cell to induce gene expression and produce the target protein.7Recombinant yeast cells for continuous production of target proteins without an activator according to claim 6, where such recombinant yeast cells are cultured by fermentation under conditions with sugar as a carbon source.

8. Recombinant yeast cells for continuous production of target proteins without an activator according to claim 7, where the sugar carbon source can be selected from glucose, fructose, maltose, sucrose, and molasses, either individually or in combination.

9. The method for constructing recombinant yeast cells for continuous production of target proteins using a plasmid vector for continuous production of target proteins consists of the following steps: a.The continuous transfer of a recombinant plasmid for target protein production into a yeast host cell yields a recombinant yeast cell containing an alpha-factor secretion signal promoter, multiple transcription terminator of a methanol oxidase gene, an antibiotic resistance gene, a transcription terminator of a glyceraldehyde 3-phosphate dehydrogenase gene, and a characteristic plasmid vector replication origin: the continuous protein-producing gene promoter in a yeast sequence. Ogataea, alpha-factor secretion signaling region for target protein transport derived from O. polymorpha and transcript terminator of methanol oxidase gene derived from O. polymorpha; and b. Selection of recombinant yeast cells for continuous production of target proteins by culturing on solid medium containing specified antibiotics.

10. Method for generating recombinant yeast cells for continuous production of target proteins according to claim 9, where specified antibiotics are selectable from hygromycin, neomycin, and zeosin, either individually or in combination;