TH17656U - Products for gene expression to produce target proteins or bioproducts outside of heat-resistant yeast cells without induction, and methods for utilizing such products. - Google Patents

Abstract

------16/10/2020------(OCR) This invention describes the development of a gene expression product for the non-induced extracellular production of target proteins or bioproducts from heat-tolerant yeast cells, and a method for utilizing such product. The product consists of a heat-tolerant yeast host cell strain, Ogataea thermomethanoiica, and a plasmid vector suitable for use with this host cell. A key component of gene expression is a non-induced promoter (GAP promoter) isolated from O. thermomethanoiica. This invented gene expression product allows for controlled expression of target genes simultaneously with the growth of the yeast host cell, without requiring changes in culture medium and utilizing inexpensive carbon sources. Furthermore, the promoter functions effectively at high temperatures, making it suitable for applications in large-scale target protein production where high heat accumulation is a problem. The expression system using this heat-tolerant yeast strain combined with a high-temperature active promoter addresses this issue. This can help reduce production costs by eliminating the need for a cooling system. In addition, it offers other advantages, such as the ability to produce recombinant proteins as the primary protein outside the cell with minimal host cell protein contamination, making subsequent purification easier. ------27/09/2019------(OCR) Page 1 of 1. Abstract of the Invention: This invention describes the development of a product for gene expression to produce target proteins or bioproducts outside of thermotolerant yeast cells without induction, and a method for using such product. The product consists of a thermotolerant yeast host cell strain, Ogataea thermomethanolica, and a plasmid vector suitable for use with this host cell. A key component in gene expression is a non-induced promoter (GAP promoter) isolated from O. thermomethanolica. This allows the gene expression product to control the expression of target genes, enabling their production alongside the growth of the yeast host cell without changing the culture medium and utilizing inexpensive carbon sources. Furthermore, the promoter functions effectively at high temperatures. This characteristic is suitable for application in the large-scale production of target proteins, which presents a problem of high heat accumulation. The expression system using heat-tolerant yeast cell strains combined with a promoter capable of operating at high temperatures can reduce production costs by eliminating the need for cooling systems. Furthermore, it offers additional advantages such as the production of recombinant proteins that are primarily exocrinized outside the cell with minimal contamination from the host cell proteins, making subsequent purification easier.

Claims (1)

------16/10/2563------(OCR) 1. Products for gene expression to produce target proteins or bioproducts outside of thermotolerant yeast cells without induction include plasmid vectors for target gene expression containing the following base sequences crucial for gene expression in the 5’ to 3’ direction: (a) a promoter sequence controlling the production of glyceraltehyde 3-phosphate dehydrogenase enzyme, isolated from the thermotolerant O. thermomethanolica strain, with the sequence shown in Seq. ID. No. 1; (b) an alpha-factor secretion signal sequence, a protein exocrine region, isolated from O. thermomethanoica; and (c) a multiple gene cloning site sequence. (d) The transcription terminator sequences of genes producing alcohol oxidase enzymes isolated from O. thermomethanoiica. (e) Neomycin resistance genes for isolating cells with recombinant plasmids controlled by two ligated promoters: the TEF1 promoter isolated from O. thermomethanoiica. and the L-ldh promoter isolated from L. piantarum BCC9546 (f) the transcription terminator of the gene producing the enzyme glyceraldehyde 3-phosphate dehydrogenase isolated from O. thermomethanoiica (g) the origin of replication (pUC origin of replication, pUC ori) for use in replication in E. coli2. Gene expression products for the production of target proteins or bioproducts outside the cell of thermotolerant yeast without induction, according to claim 1, where the target gene used with such gene expression products is a gene isolated from either a prokaryotic or eukaryotic organism.3. Gene expression products for the production of target proteins or bioproducts outside the cell of thermotolerant yeast without induction, according to claim 1 or 2, where the target gene used with such gene expression products is a gene that encodes a protein with medical or industrial potential. 4. A gene expression product for the non-induced production of target proteins or bioproducts outside the cell of thermophilic yeast, according to any one of claims 1 through 3, where the target gene used for such expression product is a gene encoding an enzyme in the lignocellulose or hemicellulose subgroup or the histidine acid phosphatase group, either individually or in combination. 5. A gene expression product for the non-induced production of target proteins or bioproducts outside the cell of thermophilic yeast, according to claim 4, where the gene encoding an enzyme in the lignocellulose or hemicellulose subgroup or the histidine acid phosphatase group can be selected from genes encoding beta-glucosidase, beta-xylosidase, or phytase enzymes, either individually or in combination. 6. Cells containing a gene expression product for the non-induced production of target proteins or bioproducts outside the cell of thermophilic yeast. According to claim 1, the cell consists of (1) a host cell, the thermotolerant yeast Ogataea thermomethanolica, and (2) a plasmid vector for target gene expression containing the following sequences of gene expression-important components in the 5’ to 3’ direction: (a) a promoter sequence controlling the production of glyceraldehyde 3-phosphate dehydrogenase, isolated from the thermotolerant O. thermomethanolica, with the sequences shown in Seq. ID. No. 1; (b) an alpha-factor secretion signal sequence, an exocrine protein sequence, isolated from O. thermomethanolica; (c) a multiple clone sequence containing restriction enzymes EcoRI, Kpnl, SacI, Nsil, Apal, Sall, Pmel, BamHI, NheI; and (d) a transcription terminator sequence of an alcohol oxidase gene. (c) A neomycin resistance gene isolated from O. thermomethanolica for the isolation of cells containing a recombinant plasmid regulated by two junctional promoters: the TEF1 promoter isolated from O. thermomethanolica and the L-ldh promoter isolated from L. plantarum BCC9546. (f) A transcription terminator of a gene producing the enzyme glyceraldehyde 3-phosphate dehydrogenase isolated from O. thermomethanolica. (g) A pUC origin of replication (pUC ori) for use in replication in E. coli. 7. Cells containing products for gene expression according to claim 6 where the thermotolerant yeast host O. thermomethanolica grows well at temperatures between 30 and 40 °C and is not resistant to the antibiotic neomycin. 8. Cells containing products for gene expression according to claim 6 or 7 where the host yeast O. thermomethanolica... The suitable thermotolerant strain is the yeast O. thermomethanolica strain TBRC6569. A non-induced gene expression production method for the target protein under any of the claims 1 through 5, where such method comprises the following steps: a. Construction of a recombinant plasmid for the expression of the desired target gene by ligating the target gene to a plasmid vector at a suitable multiple cloning site for the target gene and amplified in E. coli cells. b. Preparation of the recombinant plasmid from (a) to be suitable for transfer and insertion into the chromosome upon inclusion in the thermotolerant yeast host cell O. thermomethanolica strain by extracting the recombinant plasmid from E. coli and cleaving it with restriction enzymes at the beginning of the promoter sequence controlling the production of glyceraldehyde 3-phosphate dehydrogenase, isolated from O. thermomethanoica. c. Construction of recombinant yeast cells O. thermomethanotica for the expression of the target gene by transfer. The recombinant plasmid prepared from (b) is introduced into the host cell of the heat-tolerant yeast strain O. thermomethanotica, and the recombinant yeast cells O. thermomethanotica are selected on solid culture medium containing the antibiotic neomycin. d. Production of the desired target protein by culturing the recombinant yeast cells O. thermomethanotica for non-induced expression of the target gene from (c) under the appropriate culture conditions as follows: First, the recombinant yeast cells are cultured in liquid culture medium at 25 - 40 °C under aeration conditions. Then, the culture medium with appropriate turbidity is transferred to the liquid culture medium again and cultured at 25 - 40 °C under aeration conditions. Then, the culture medium is separated from the cells by centrifugation for analysis to find the desired protein.10. Method for non-induced production of target protein using products for gene expression to produce the target protein according to claim 9, where the optimal conditions for producing the desired target protein by culturing the recombinant yeast cells O. thermomethanotica for non-induced expression of the target gene are: cell culture. Recombinant yeast was cultured in YPD liquid medium at 30°C under aeration for 18-24 hours. The culture, with turbidity of 0.01, was then transferred to 20 ml of YPD liquid medium and cultured at 30°C under aeration for approximately 72 hours. The culture medium was then separated from the cells by centrifugation for analysis to identify the desired proteins. ------27/09/2019------(OCR) Page 1 of 3. Claim 1. The product for gene expression to produce target proteins or bioproducts outside of thermotolerant yeast cells without induction is a plasmid vector for target gene expression containing the following base sequences important for gene expression in the 5’ to 3’ direction: (a) a promoter sequence controlling the production of glyceraldehyde 3-phosphate dehydrogenase enzyme isolated from a thermotolerant O. thermomethanolica strain. (b) The base sequence of the alpha-factor secretion signal, which is the part that carries proteins out of the cell, isolated from O. thermomethanolica. (c) The base sequence that is a multiple clone gene site containing restriction enzyme sites, namely fcoRI, Kpnl, Sacl, Nsil, Apal, Sail, PmeI, BamHI, Nhel. (d) The base sequence that is the transcription terminator of the gene that produces the alcohol oxidase enzyme, isolated from O. thermomethanolica. (e) The neomycin resistance gene for use in isolating cells with recombinant plasmids that are controlled by two fused promoters, namely the TEF1 promoter isolated from O. thermomethanolica. and the L-ldh promoter isolated from L. plantarum BCC9546 (f) the transcription terminator of the gene producing the enzyme glyceraldehyde 3-phosphate dehydrogenase isolated from O. thermomethanolica (g) the origin of replication (pUC origin of replication, pUC ori) for use in replication in E. coli2. Gene expression products for the production of target proteins or bioproducts outside the thermotolerant yeast cell without induction, according to Patent 1, where the target gene used with such expression products is a gene isolated from either a prokaryotic or eukaryotic organism.3. Gene expression products for the production of target proteins or bioproducts outside the thermotolerant yeast cell without induction, according to Patent 1 or 2, where the target gene used with such expression products is a gene that encodes a protein with medical or industrial potential. 4. A gene expression product for the non-induced production of target proteins or bioproducts outside the thermotolerant yeast cell, according to any one of claims 1 to 3, where the target gene used for such expression product is a gene encoding an enzyme in the lignocellulose or hemicellulose subgroup or the histidine acid phosphatase group, either individually or in combination. 5. A gene expression product for the non-induced production of target proteins or bioproducts outside the thermotolerant yeast cell, according to claim 4, where the gene encoding an enzyme in the lignocellulose or hemicellulose subgroup or the histidine acid phosphatase group, either individually or in combination, can be selected from the genes encoding 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 thermotolerant yeast cell non-induced, according to claim 1, where such cells consist of (1) a host cell of the thermotolerant yeast strain Ogataea thermomethanolica, and (2) a plasmid vector for target gene expression containing the following sequences of the base sequences important for gene expression in the 5’ to 3’ direction: (a) a promoter sequence controlling the production of glyceraltehyde 3-phosphate dehydrogenase enzyme isolated from the thermotolerant O. thermomethanolica, with the sequence shown in Figure 2; (b) an alpha-factor secretion signal sequence which is an extracellular protein transport region isolated from O. thermomethanolica; and (c) a sequence of multiple gene cloning sites. (d) The transcript terminator sequence of the gene producing alcohol oxidase, isolated from O. thermomethanolica. (e) The neomycin antibiotic gene for isolating cells with recombinant plasmids regulated by two junctional promoters: promoter TEF1 isolated from O. thermomethanolica and promoter L-ldh isolated from L. plantarum BCC9546. (f) The transcript terminator of the gene producing glyceraldehyde 3-phosphate dehydrogenase, isolated from O. thermomethanolica. (g) The pUC origin of replication (pUC ori) for use in increasing the number of cells in E. coli.7. Cells containing products for gene expression. According to Relief 6, whereby the thermotolerant yeast host cell strain O. thermomethanolica grows well at temperatures between 30 and 40 °C and is not resistant to the antibiotic neomycin.8. Cells containing products for gene expression according to Relief 6 or 7, whereby the suitable thermotolerant yeast host cell strain O. thermomethanolica is strain TBRC656.9. A method for the production of target proteins that does not rely on induction with gene expression products to produce any one of the target proteins according to Reliefs 1 through 8, whereby the method consists of the following steps: a. Construction of recombinant plasmids for the expression of the desired target gene by ligating the target gene to a plasmid vector at a multiple cloning site suitable for the target gene and amplified in E. coli cells. b. Preparation of the recombinant plasmid from (a) to be suitable for transfer and insertion into the chromosome once in the O. thermomethanolica yeast host cell. Heat-tolerant strain: A recombinant plasmid was extracted from E. coli and cut with restriction enzymes at the beginning of the promoter sequence on page 3 of 3 pages, controlling the production of glyceraldehyde 3-phosphate dehydrogenase enzyme isolated from O. thermomethanolica. B. Construction of O. thermomethanolica recombinant yeast cells for target gene expression: The recombinant plasmid prepared in (b) was transferred into a heat-tolerant O. thermomethanolica yeast host cell line, and O. thermomethanolica recombinant yeast cells were selected on solid culture medium containing the antibiotic neomycin. C. Production of the desired target protein by culturing O. thermomethanolica recombinant yeast cells for non-induced target gene expression from (c) under the following optimal culture conditions: First, the recombinant yeast cells were cultured in liquid culture medium at 25-40°C under aeration conditions. Then, the cultures with appropriate turbidity were transferred to the liquid culture medium again and cultured at 25-40°C under aeration conditions. The culture medium is then separated from the cells by centrifugation for analysis to identify the desired proteins.10. Method for the non-induced production of target proteins using products for gene expression to produce target proteins according to claim 9, where the optimal conditions for producing the desired target protein by culturing recombinant yeast cells O. thermomethanolica for non-induced target gene expression are: culturing recombinant yeast cells in YPD liquid medium at 30°C under aeration for 18-24 hours; then transferring the culture with turbidity 0.01 to 20 ml of YPD liquid medium and culturing at 30°C under aeration for approximately 72 hours; then separating the culture medium from the cells by centrifugation for analysis to identify the desired proteins. ------------ Revised 26/10/2018 Claim 1. Products for gene expression to produce target proteins or bioproducts outside of thermotolerant yeast cells without induction include plasmid vehicles for target gene expression containing base sequences crucial for gene expression in the 5' to 3' direction as follows: (a) a promoter sequence controlling the production of glyceraldehyde 3-phosphate dehydrogenase enzyme isolated from the thermotolerant O. thermomethanolica; (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, 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 gene producing the enzyme glyceraldehyde 3-phosphate dehydrogenase, isolated from O. thermomethanolica. (g) pUC origin of replication (pUC ori) for use in replication in E. coli.