TH17655Y - A product for gene expression to produce target proteins or bioproducts outside of thermo-resistant yeast cells via methanol induction; cells containing such product; and a method for methanol-induced production of target proteins using such product. - Google Patents

Abstract

------October 22, 2020------(OCR) This invention describes the development of a gene expression product for the production of target proteins or bioproducts outside of thermotolerant yeast cells via methanol induction. The product consists of cells containing the product and a methanol-induced production method for target proteins using the product. The product comprises a thermotolerant yeast strain, Ogataea thermomethanolica, and a plasmid vector suitable for use with this cell. A key component of the gene expression product is a methanol-induced promoter isolated from O. thermomethanolica. This gene expression product can control the expression of target genes, as the AOX promoter from O. thermomethanolica is functional at high temperatures. Furthermore, the induction of recombinant protein production under the action of the AOX promoter begins while the yeast cells are cultured in glycerol, during the cell growth phase before the culture medium is changed to methanol. Subsequently, the yeast cells are cultured in methanol, which is the induction condition. Recombinant proteins are synthesized at high levels, allowing the use of O. thermomethanolica as a host cell in combination with the AOX promoter from O. thermomethanolica to produce recombinant proteins from the glycerol step to the methanol step, resulting in increased protein yield in a shorter time. Furthermore, other advantages include the ability to produce recombinant proteins outside the cell as the primary protein with little or no host protein contamination, facilitating subsequent purification. In cases where the target protein is an enzyme, the resulting gene expression product exhibits high enzyme activity.

Claims (1)

------October 22, 2020------(OCR) 1. Products for gene expression to produce target proteins or bioproducts outside of thermoresistant yeast cells via methanol induction include plasmid vectors for target gene expression containing the following base sequences crucial for gene expression in the 5’ to 3’ direction: a. Promoter sequence controlling alcohol oxidase enzyme production, isolated from a thermoresistant strain of O. thermomethanolica. b. Alpha-factor secretion signal sequence, a protein exocrine region, isolated from O. thermomethanolica. c. Multiple gene cloning site sequence. These include: 1. Restriction enzyme sites: EcoRI, KpnI, SacI, NsiI, ApaI, SalI, PmeI, BamHI, NheI. 2. Transcription terminator sequences of genes producing alcohol oxidase enzyme isolated from O. thermomethanolica. 3. Neomycin resistance genes for isolating cells with recombinant plasmids controlled by two ligated promoters: promoter TEF1 isolated from O. thermomethanolica and promoter L-ldh isolated from L. plantarum BCC9546. 4. Transcription terminator of genes producing glyceraldehyde 3-phosphate dehydrogenase enzyme isolated from O. thermomethanolica. 5. pUC origin of replication (pUC ori) for use in E. coli replication. 6. Products for gene expression to produce target proteins or bioproducts outside of thermotolerant yeast cells. Methanol-induced gene expression product for the production of target proteins or bioproducts outside of heat-resistant yeast cells, as per Relief 1, where the target gene used with the expression product is a gene isolated from either a prokaryotic or eukaryotic organism. 3. Methanol-induced gene expression product for the production of target proteins or bioproducts outside of heat-resistant yeast cells, as per Relief 1 or 2, where the target gene used with the expression product is a gene that encodes a protein with potential for medical or industrial applications, either individually or in combination. 4. Methanol-induced gene expression product for the production of target proteins or bioproducts outside of heat-resistant yeast cells, as per Reliefs 1 to 3, where the target gene used with the expression product is a gene that encodes an enzyme in the lignocellulose or hemicellulose subgroup or the histidine acid phosphatase group, either individually or in combination. 5. Methanol-induced gene expression product for the production of target proteins or bioproducts outside of heat-resistant yeast cells. Methanol-induced thermotolerance yeast, according to claim 4, where the gene encoding an enzyme in the group of lignocellulose or hemicellulose or histidine acid phosphatase enzymes is selected from genes encoding either beta-glucosidase, beta-xylosidase, or phytase enzymes, either individually or in combination.6. Cells containing products for gene expression to produce target proteins or bioproducts outside the cell. Methanol-induced thermotolerance yeast, according to claim 1, where such cells consist of (1) a host cell of the thermotolerant yeast strain Ogataea thermomethanolica; (2) a plasmid vector for target gene expression containing the following sequences of base sequences important for gene expression in the 5' to 3' direction: a. a promoter sequence controlling the production of alcohol oxidase enzyme isolated from the thermotolerant O. thermomethanolica; b. an alpha-factor secretion signal sequence. These include: a. Protein transport sequences isolated from O. thermomethanolica; c. Multiple clone sequences containing specific enzymes: EcoRI, KpnI, SacI, NsiI, ApaI, SalI, PmeI, SomHI, NheI; d. Transcription terminator sequences of genes producing alcohol oxidase enzyme, isolated from O. thermomethanolica; e. Neomycin resistance genes for isolating cells with recombinant plasmids controlled by two ligated promoters: promoter TEF1 isolated from O. thermomethanolica. f. The L-ldh promoter isolated from L. plantarum BCC9546. g. The transcription terminator of the gene producing glyceraldehyde 3-phosphate dehydrogenase isolated from O. thermomethanolica. h. The pUC origin of replication (pUC ori) for use in E. coli replication. 7. Cells containing the gene expression product according to claim 6, where the thermotolerant yeast host strain O. thermomethanolica grows well at temperatures between 30 and 40 °C and is not resistant to the antibiotic neomycin. 8. Cells containing the gene expression product according to claim 6 or 7, where the thermotolerant yeast host strain O. thermomethanolica is TBRC656. 9. A methanol-induced method for producing target proteins using the gene expression product. According to any one of claims 1-5, the procedure consists of the following steps: a. Fabrication of recombinant plasmids for the expression of the desired target gene by ligating the target gene to a plasmid vector at a suitable multiple cloning site and amplified in E. coli cells. b. Preparation of the recombinant plasmid from step a to be suitable for transfer and insertion into the chromosome once inoculated into a thermotolerant yeast host cell strain O. thermomethanolica. This is achieved by extracting the recombinant plasmid from E. coli and cleaving it with restriction enzymes at the region of the base sequence that controls the production of alcohol oxidase enzyme isolated from O. thermomethanolica. c. Construction of O. thermomethanolica recombinant yeast cells for the expression of the target gene by transferring the recombinant plasmid prepared in step b into a thermotolerant yeast strain O. thermomethanolica and selecting O. thermomethanolica recombinant yeast cells that received the recombinant plasmid on the medium. 1. Culturing a solid culture containing the antibiotic neomycin. 2. Production of the desired target protein by culturing recombinant yeast cells O. thermomethanolica for the expression of the target gene, as in point C, under the following optimal cultivation conditions: Begin by culturing recombinant yeast cells in liquid medium at 25-40°C under aeration. Transfer the culture to the liquid medium and continue culturing at 25-40°C under aeration. Concentrate the cells by precipitating the cultured cells and dissolving them in a liquid medium containing 1-3% methanol. Then, produce the target protein by further culturing the cells in the aforementioned liquid medium at 25-40°C under aeration, adding 1-3% methanol daily. 10. Methanol-induced target protein production method using the gene expression product for target protein production according to claim 9, where the optimal conditions for production are... To produce the target protein using recombinant yeast cell culture (O. thermomethanolica), the process involves culturing the recombinant yeast cells in YPD liquid medium at 30°C under aeration for 18-24 hours. The cells are then transferred to BMMY liquid medium and cultured at 30°C under aeration for another 18-24 hours, or until the absorbance at 600 nm is measured at 8-12. Cell concentration is then achieved by precipitating the cultured cells and dissolving them in BMMY liquid medium containing 2% methanol, using a tenfold reduction in volume compared to the previous BMMY medium. The target protein is then produced by further culturing the cells in the same BMMY liquid medium at 30°C under aeration. For a total of 48 hours, 2% methanol was added daily. ------------ Revised 26/10/2018 Claims 1. Products for gene expression to produce target proteins or bioproducts outside of thermo-resistant yeast cells via methanol induction include plasmid vehicles for target gene expression containing the following base sequences crucial for gene expression in the 5' to 3' direction: (a) a promoter sequence controlling alcohol oxidase enzyme production, isolated from the thermo-resistant O. thermomethanolica strain; (b) an alpha-factor secretion signal sequence, a protein export region, isolated from O. thermomethanolica; (c) a multiple clone sequence containing restriction enzymes such as EcoRl, Kpnl, Sacl, Nsil, Apal, Sall, Pmel, Bamhl, and Nhel; (d) a transcription terminator sequence of an alcohol oxidase enzyme gene, isolated from O. thermomethanolica; and (e) a neomycin resistance gene for isolating cells containing recombinant plasmids. (f) Transcription terminer of the glyceraldehyde 3-phosphate dehydrogenase enzyme gene isolated from O. thermomethanolica. (g) pUC origin of replication (pUC ori) for use in E. coli replication.