Recombinant pichia pastoris strain with high yield of beta-carotene as well as construction method and application of recombinant pichia pastoris strain

Abstract

The invention discloses a recombinant pichia pastoris strain with high yield of beta-carotene as well as a construction method and application of the recombinant pichia pastoris strain, and belongs to the technical field of biological genetic engineering. In order to improve the yield of beta-carotene, a beta-carotene synthesis route is constructed by a multi-copy method, the expression level of a target gene and the yield of a target product are improved, and meanwhile, an exogenous gene is integrated into a genome instead of free plasmid expression, so that the genetic stability of the strain is improved. In addition, the recombinant strain also has good stability and repeatability, the yield fluctuation range of beta-carotene in the multi-batch fermentation process is less than 10%, and the recombinant strain has significant industrial application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of biological genetic engineering, and in particular relates to a recombinant Pichia pastoris strain with high beta-carotene production, a construction method and an application thereof. Background Art

[0002] Beta-carotene is a common, orange-yellow carotenoid found in nature. It possesses excellent antioxidant properties and can slow aging. It is also a key precursor for vitamin A synthesis, playing an essential role in maintaining the normal metabolism and function of complex organisms. Therefore, it is widely used in aquaculture, food, cosmetics, and pharmaceutical industries.

[0003] Currently, global β-carotene production is primarily carried out through chemical synthesis and natural extraction. Chemical synthesis is the current mainstream method for producing β-carotene, offering high production efficiency and relatively low costs. However, this method also presents certain issues, such as environmental pollution and the generation of byproducts. Long-term use may also have potential toxic side effects on the human body. Another method is natural extraction, where β-carotene is extracted from plants (mostly algae). However, since plant growth is affected by factors such as season and climate, and the β-carotene content is limited, this method results in high production costs and struggles to meet market demand. Therefore, to meet the growing market demand for β-carotene while addressing the challenges of low production efficiency, high costs, and environmental pollution in industrial production, the use of genetic engineering techniques to modify microbial strains for efficient β-carotene production is a viable solution.

[0004] At present, some studies have used CRISPR / Cas9 technology to introduce β-carotene synthesis genes (crtE, crtI, crtYB) from Phaffia rhodozyma into cerevisiae, while replacing xylose as the carbon source, and finally achieving a yield of 772.8 mg / L in a 5L fermenter. There are also studies that exogenously added 2 mM oleic acid and used the IZH1 promoter to dynamically regulate the expression of the ERG9 gene in cerevisiae to balance the relationship between bacterial growth and product synthesis, with a yield of 142 mg / L.

[0005] In addition to brewer's yeast, some unconventional yeasts such as Yarrowia lipolytica and Candida tropicalis are also gradually being developed and utilized by people. Chinese patent CN106987550A discloses a recombinant β-carotene-producing yeast, Yarrowia lipolytica. By introducing a codon-optimized β-carotene synthesis gene, Yarrowia lipolytica is endowed with the ability to produce β-carotene. Subsequently, by overexpressing geranylgeranyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-CoA reductase, the β-carotene yield was successfully increased to 26.03 mg / gDCW. Chinese patent CN114806914A also discloses an unconventional β-carotene-producing yeast. Using Candida tropicalis as a starting strain, the β-carotene synthesis pathway was first constructed in the cytoplasm. The β-carotene anabolic flux was further increased by overexpressing genes related to the MVA pathway and the terpenoid synthesis pathway. Subcellular compartment engineering was then used to construct the β-carotene synthesis pathway in the peroxisome. Ultimately, after 216 hours of fermentation in a 5-L fermentor, the yield reached 6.5 g / L.

[0006] Although some progress has been made in genetically engineering microorganisms to produce β-carotene, its application in industrial production remains elusive. Therefore, further research is needed to develop efficient, genetically stable strains for industrial β-carotene production, thereby reducing production costs.

[0007] Pichia pastoris is a methanol-trophic yeast, capable of growing using methanol as its sole carbon and energy source. It possesses a robust protein expression system and is amenable to high-density fermentation, making it an ideal platform for expressing exogenous genes. Compared to Saccharomyces cerevisiae, Pichia pastoris possesses a more rigorous central carbon metabolism system. Fermentation of glucose does not produce ethanol, resulting in higher biomass yields, making it a promising strain for producing β-carotene. Currently, the maximum β-carotene yield in Pichia pastoris is only 366 mg, but metabolic engineering and other approaches are expected to increase yields further. Summary of the Invention

[0008] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0009] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0010] Therefore, the purpose of the present invention is to overcome the shortcomings of the prior art and provide a recombinant Pichia pastoris strain HL-8 Komagataella phaffii HL-8, which was deposited in the China Center for Type Culture Collection on March 31, 2025, with the deposit number CCTCC No. M 2025667, and the deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0011] Another object of the present invention is to overcome the deficiencies in the prior art and provide the use of a recombinant Pichia pastoris strain in increasing β-carotene production.

[0012] As a preferred embodiment of the use of the recombinant Pichia pastoris strain of the present invention for increasing β-carotene production, the recombinant Pichia pastoris strain increases β-carotene production by expressing genes related to β-carotene synthesis, overexpressing genes related to the endogenous mevalonate pathway, increasing genes related to the supply of acetyl-CoA precursors, and downregulating genes related to squalene synthesis;

[0013] The β-carotene synthesis-related genes include the gene CrtE encoding geranylgeranyl diphosphate synthase, the gene CrtI encoding phytoene dehydrogenase, and the gene CrtYB encoding phytoene synthase / lycopene cyclase;

[0014] The mevalonate pathway-related genes include the gene tHMGR encoding truncated 3-hydroxy-3-methylglutaryl-CoA reductase, the gene GGPPS encoding geranylgeranyl diphosphate synthase, the gene IDI1 encoding isopentenyl pyrophosphate isomerase, the gene ERG10 encoding acetyl-CoA transacetylase, the gene ERG13 encoding hydroxymethylglutaryl-CoA synthase, the gene ERG12 encoding mevalonate kinase, and the gene ERG8 encoding phosphomevalonate kinase;

[0015] The genes related to increasing the supply of acetyl-CoA precursors include the gene ACL1 encoding the large subunit of ATP-citrate lyase, the gene ACL2 encoding the small subunit of ATP-citrate lyase, the gene YHM2 encoding the carboxylate transporter, and the gene IDP2 encoding isocitrate dehydrogenase;

[0016] The squalene synthesis-related genes include the gene ERG9 encoding squalene synthase.

[0017] As a preferred embodiment of the use of the recombinant Pichia pastoris strain of the present invention in increasing β-carotene production, among the β-carotene synthesis-related genes, CrtE, CrtI and CrtYB are all derived from Xanthophylomyces dendrorhous.

[0018] As a preferred embodiment of the use of the recombinant Pichia pastoris strain of the present invention in increasing β-carotene production, among the mevalonate pathway-related genes, tHMGR, GGPPS, IDI1, ERG10, ERG13, ERG12 and ERG8 are all derived from Pichia pastoris (Komagataelaphaffi).

[0019] As a preferred embodiment of the use of the recombinant Pichia pastoris strain of the present invention in increasing β-carotene production, among the genes related to increasing the supply of acetyl-CoA precursor, ACL is derived from Aspergilus niger, and YHM2 and IDP2 are both derived from Saccharomyces cerevisiae.

[0020] As a preferred embodiment of the use of the recombinant Pichia pastoris strain of the present invention for increasing β-carotene production, the gene expression level of the squalene synthase gene ERG9 is downregulated by replacing the promoter, and the promoter is derived from Pichia pastoris PAS_chr1-4_0582.

[0021] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for constructing a recombinant Pichia pastoris strain.

[0022] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0023] The β-carotene synthesis related genes were constructed into a gene expression cassette "P GAP -crtE-T AOX1 "P GAP -crtI-T AOX1 "P GAP -crtYB-T AOX1 ", electroporation was performed into the rDNA multi-copy site of the original Pichia pastoris strain GS115 genome to obtain the β-carotene synthesis gene multi-copy strain HL-1;

[0024] Construction of gene expression cassette "P GAP -tHMGR-T AOX1 -P GAP -IDI1-T AOX1 -P GAP -GGPPS-T AOX1 ", introduced the neutral site I-5 of the genome of the recombinant strain HL-1 to obtain the recombinant strain HL-2;

[0025] Construction of gene expression cassette "P GAP-crtYB-T AOX1 ", introduced into the neutral site II-7 of the genome of the recombinant strain HL-2 to obtain the recombinant strain HL-3;

[0026] Construction of gene expression cassette "P GAP -anACL-T AOX1 ", introduced the neutral site I-4 of the genome of the recombinant strain HL-3 to obtain the recombinant strain HL-4;

[0027] Construction of gene expression cassette "P GCW14 -ERG10-T AOX1 -P TEF1 -ERG13-T AOX1 ", introduced into the neutral site II-4 of the genome of the recombinant strain HL-4 to obtain the recombinant strain HL-5;

[0028] Construction of gene expression cassette "P GCW14 -ERG12-T AOX1 -P GAP -ERG8-T AOX1 ", introduced into the neutral site IV-4 of the genome of the recombinant strain HL-5 to obtain the recombinant strain HL-6;

[0029] Construction of gene expression cassette "P GAP -ScYHM2-T AOX1 -P GCW14 -ScIDP2-T AOX1 ", introduced the neutral site I-2 of the genome of the recombinant strain HL-6 to obtain the recombinant strain HL-7;

[0030] Based on the recombinant strain HL-7, the expression of squalene synthase was down-regulated using the promoter of the Pichia pastoris PAS_chr1-4_0582 gene to obtain the recombinant strain HL-8, which is the recombinant Pichia pastoris strain.

[0031] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for producing β-carotene.

[0032] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0033] The recombinant Pichia pastoris strain was inoculated into YPD medium at an inoculum rate of 1-5%, and cultured overnight at 28-30° C. and 200-220 rpm to obtain an activated glycerol strain;

[0034] Take the activated glycerol bacteria to start with OD 600nm = 0.1-0.5 was inoculated into YPD medium and cultured at 28-30°C and 200-220 rpm until OD 600nm=10-15, to obtain seed solution;

[0035] The seed liquid was inoculated into YPD medium and cultured at 28-30°C and 200-220 rpm for 72 hours. Samples were taken every 24 hours for microscopic examination and the fermentation products were preserved.

[0036] β-carotene was extracted from the fermentation broth using acetonitrile as the extraction solvent.

[0037] As a preferred embodiment of the method for producing beta-carotene according to the present invention, the YPD culture medium comprises 9.5-10.5 g / L yeast extract, 19-21 g / L peptone, and 19-21 g / L glucose.

[0038] As a preferred embodiment of the method for producing β-carotene according to the present invention, the seed solution is inoculated into YPD culture medium, wherein the initial concentration of the seed solution is OD 600nm =0.5.

[0039] Beneficial effects of the present invention:

[0040] (1) The β-carotene biosynthesis pathway was constructed using a multi-copy method, which improved the expression level of the target gene and the yield of the target product. At the same time, the exogenous gene was integrated into the genome instead of being expressed on a free plasmid, which improved the genetic stability of the strain.

[0041] (2) Overexpression of the genes tHMGR, IDI1, and GGPPS can better balance the MVA pathway, allowing more carbon metabolic flow to the β-carotene synthesis pathway, increasing yield by approximately 99%;

[0042] (3) By overexpressing crtYB, a key rate-limiting enzyme in the β-carotene biosynthesis pathway, the accumulation of lycopene during β-carotene biosynthesis was reduced, its toxic effects on cells were reduced, and the yield was increased by approximately 37%;

[0043] (4) Heterologous expression of anACL, YHM2, and IDP2 can increase the intracellular supply of acetyl-CoA. Simultaneous overexpression of ERG10, ERG13, ERG12, and ERG8 can pull more acetyl-CoA metabolic flux into the mevalonate pathway, providing sufficient precursors for the synthesis of β-carotene, increasing production by approximately 70%;

[0044] (5) By downregulating the expression of the ERG9 gene, the endogenous farnesyl pyrophosphate competition pathway can be weakened, increasing the supply of geranylgeranyl diphosphate, a precursor for the synthesis of tetraterpenoid compounds in the cell, and increasing the yield by about 21%;

[0045] (6) The recombinant strain of the present invention achieved a maximum yield of 1.38 g / L of β-carotene in shake flask fermentation, the highest yield reported to date for Pichia pastoris. Furthermore, the recombinant strain exhibited excellent stability and reproducibility, with β-carotene yield fluctuations of less than 10% across multiple fermentation batches, demonstrating significant potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0047] Figure 1 Schematic diagram of the structure of plasmid pGAPZB in an embodiment of the present invention.

[0048] Figure 2 Schematic diagram of the structure of plasmid pTEF1ZB in an embodiment of the present invention.

[0049] Figure 3 Schematic diagram of the structure of plasmid pGCW14ZB in an embodiment of the present invention.

[0050] Figure 4 Schematic diagram of the structure of plasmid G-PNSI-5-tHMGR-GGPPS-IDI1 in Example 2 of the present invention.

[0051] Figure 5 Schematic diagram of the structure of plasmid G-PNSII-7-crtYB in Example 3 of the present invention.

[0052] Figure 6 Schematic diagram of the structure of plasmid G-PNSI-4-anACL1-anACL2 in Example 4 of the present invention.

[0053] Figure 7 Schematic diagram of the structure of plasmid G-PNSII-4-ERG10-ERG13 in Example 5 of the present invention.

[0054] Figure 8 Schematic diagram of the structure of plasmid G-PNSIV-4-ERG12-ERG8 in Example 6 of the present invention.

[0055] Figure 9 Schematic diagram of the structure of plasmid G-PNSI-2-YHM2-IDP2 in Example 7 of the present invention.

[0056] Figure 10This is a graph showing the changes in β-carotene production by different recombinant bacteria during shake flask fermentation in Example 9 of the present invention.

[0057] Figure 11 Schematic diagram of the production pathway of β-carotene in Pichia pastoris according to Example 9 of the present invention. DETAILED DESCRIPTION

[0058] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0059] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0060] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0061] Unless otherwise specified, the raw materials used in the present invention are commercially available.

[0062] The sequence of geranylgeranyl diphosphate synthase (CrtE) from Xanthophylomyces dendrorhous used in the present invention can be found at:

[0063] Found at https: / / www.ncbi.nlm.nih.gov / protein / QSV51901.1?report=genbank&log$=prottop&blast_rank=1&RID=ZBXNW80Y016.

[0064] The sequence of phytoene dehydrogenase (CrtI) from Xanthophylomyces dendrorhous used in the present invention can be found at:

[0065] Found at https: / / www.ncbi.nlm.nih.gov / protein / CED83513.1?report=genbank&log$=prottop&blast_rank=3&RID=ZBY75KMZ016.

[0066] The sequence of the phytoene synthase / lycopene cyclase (CrtYB) from Xanthophylomyces dendrorhous used in the present invention can be found at:

[0067] The protein was obtained by searching https: / / www.ncbi.nlm.nih.gov / protein / Q7Z859.1?report=genbank&log$=prottop&blast_rank=1&RID=ZBYM9NDD013.

[0068] The sequence of 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) from Pichia pastoris (Komagataelaphaffi) used in the present invention can be found at:

[0069] Available from https: / / www.ncbi.nlm.nih.gov / gene / 8198637.

[0070] The sequence of the geranylgeranyl diphosphate synthase (GGPPS) from Pichia pastoris used in the present invention can be found at:

[0071] Available from https: / / www.ncbi.nlm.nih.gov / gene / 8199751.

[0072] The sequence of isopentenyl pyrophosphate isomerase (IDI1) from Pichia pastoris used in the present invention can be found at:

[0073] Available from https: / / www.ncbi.nlm.nih.gov / gene / 8197017.

[0074] The sequence of acetyl-CoA transacetylase (ERG10) from Pichia pastoris (Komagataelaphaffi) used in the present invention can be found at:

[0075] Available from https: / / www.ncbi.nlm.nih.gov / gene / 8197878.

[0076] The sequence of the hydroxymethylglutaryl-CoA synthase (ERG13) from Pichia pastoris used in the present invention can be found at:

[0077] Available from https: / / www.ncbi.nlm.nih.gov / gene / 8198573.

[0078] The sequence of mevalonate kinase (ERG12) from Pichia pastoris used in the present invention can be found at:

[0079] Available from https: / / www.ncbi.nlm.nih.gov / gene / 8197654.

[0080] The sequence of phosphomevalonate kinase (ERG8) from Pichia pastoris (Komagataelaphaffi) used in the present invention can be found at:

[0081] Obtained from https: / / www.ncbi.nlm.nih.gov / gene / 8198218.

[0082] The sequence of the large subunit of ATP-citrate lyase (ACL1) from Aspergillus niger used in the present invention can be found at:

[0083] The protein was obtained by searching https: / / www.ncbi.nlm.nih.gov / protein / XP_001394055.1?report=genbank&log$=prottop&blast_rank=1&RID=ZBYW1HFY013.

[0084] The sequence of the ATP-citrate lyase small subunit (ACL2) from Aspergillus niger used in the present invention can be found at:

[0085] The protein was obtained by searching https: / / www.ncbi.nlm.nih.gov / protein / XP_001394057.1?report=genbank&log$=prottop&blast_rank=1&RID=ZBZ45CMD013.

[0086] The sequence of the carboxylate transporter (YHM2) from Saccharomyces cerevisiae used in the present invention can be found at:

[0087] Obtained from https: / / www.ncbi.nlm.nih.gov / gene / 855282.

[0088] The sequence of isocitrate dehydrogenase (IDP2) from Saccharomyces cerevisiae used in the present invention can be found at:

[0089] Obtained from https: / / www.ncbi.nlm.nih.gov / gene / 850871.

[0090] The components of the YPD liquid culture medium used in the present invention include: 10 g / L yeast extract, 20 g / L peptone and 20 g / L glucose.

[0091] The components of the YPD plate used in the present invention include: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose and 20 g / L agar.

[0092] The components of the YPDZ plate used in the present invention include: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 20 g / L agar and 100 μg / mL bleomycin.

[0093] The components of the MDZ plate used in the present invention include: 13.4 g / L YNB culture medium, 20 g / L glucose, 20 g / L agar and 100 μg / mL bleomycin.

[0094] The primer sequences used in the examples of the present invention are shown in Table 1 below.

[0095] Table 1 Primer sequences

[0096]

[0097]

[0098]

[0099]

[0100] Example 1 Construction of recombinant Pichia pastoris strain HL-1

[0101] (1) Three missing (Leu - 、Arg - 、His - ) Pichia pastoris GS115 derivative strains were cultured overnight in YPD liquid medium to prepare competent cells.

[0102] (2) Entrust a biotechnology company to synthesize gene fragments crtE, crtI, and crtYB containing EcoRI and SalI restriction sites at both ends, and use the endonucleases EcoRI and SalI to digest at 37°C for 1 hour. The reaction solution is purified and recovered using the Novozyme gel recovery kit. Separately, take the vector pGAPZB (structure as Figure 1 The resulting mixture was treated with endonucleases EcoRI and SalI at 37°C for 1 h, and the reaction solution was purified and recovered using a Novozyme gel recovery kit.

[0103] (3) The crtE, crtI, and crtYB gene fragments were mixed with the pGAPZB vector, respectively, and reacted with DNA ligase at 25°C for 1 h to obtain plasmids pGAPZB-crtE, pGAPZB-crtI, and pGAPZB-crtYB.

[0104] (4) Using plasmids pGAPZB-crtE, pGAPZB-crtI, and pGAPZB-crtYB as templates, PCR amplification was performed using primers “rDNA-Promoter-F2” and “rDNA-Terminator-R2” to obtain the gene expression cassette “P GAP -crtE-T AOX1 ”, “P GAP -crtI-T AOX1 ” and “P GAP -crtYB-T AOX1 ”.

[0105] (5) Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primers “LEU2-P20-F” and “LEU2-T250-R”, primers “ARG4-P20-F” and “ARG4-T250-R”, and primers “HIS4-P20-F” and “HIS4-T250-R” to obtain the gene expression cassette “P Leu2-20 -Leu2-T Leu2 ”, “P Arg4-20 -Arg4-T Arg4 ” and “P His4-20 -His4-T His4 ”.

[0106] (6) Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primers “rDNA-500-F” and “rDNA-AUXO-R” and primers “rDNA-T-F2” and “rDNA-500-R”, respectively, to obtain the NTS sequence of the Pichia pastoris rDNA locus (500 bp each).

[0107] (7) After mixing the DNA fragments obtained above, fusion PCR was performed using primers "rDNA-500-F" and "rDNA-500-R" to obtain the gene expression cassette "NTS-P Leu2-20 -Leu2-T Leu2 -P GAP -crtE-T AOX1 -NTS" "NTS-P Arg4-20 -Arg4-T Arg4 -P GAP -crtI-T AOX1 -NTS" and "NTS-P His4-20 -His4-T His4 -P GAP -crtYB-T AOX1 -NTS". The three gene expression cassettes were simultaneously transformed into competent cells by electroporation.

[0108] (8) Screening was performed using MDZ plates. Single colonies grew in 3–4 days. Single colonies with color were selected and positive clones were identified by PCR. These clones were named recombinant Pichia pastoris strain HL-1.

[0109] Example 2 Construction of recombinant Pichia pastoris strain HL-2

[0110] (1) The recombinant Pichia pastoris strain HL-1 prepared in Example 1 was cultured overnight in YPD liquid medium to prepare competent cells.

[0111] (2) Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primers "tHMG1-530-F" and "tHMG1-R", primers "IDI1-KpnI-F" and "IDI1-SalI-R", and primers "GGPPS-KpnI-F" and "GGPPS-SalI-R" to obtain genes tHMGR (about 1370 bp), IDI1 (about 880 bp), and GGPPS (about 1020 bp). The tHMGR gene fragment, IDI1 gene fragment, and GGPPS gene fragment were treated with nucleases EcoRI and SalI, and nucleases KpnI and SalI, respectively, at 37°C for 1 hour. The reaction solution was purified and recovered using a Novagen gel recovery kit.

[0112] (3) Take another vector pGAPZB, pTEF1ZB (structure as Figure 2 ) and pGCW14ZB (structure as shown Figure 3 The cells were treated with endonucleases EcoRI, SalI, KpnI, and SalI at 37°C for 1 h, respectively, and the reaction solution was purified and recovered using a Novozymes gel recovery kit.

[0113] (4) The tHMGR gene fragment was mixed with the pGAPZB vector, the IDI1 gene fragment was mixed with the pTEF1ZB vector, and the GGPPS gene fragment was mixed with the pGCW14ZB vector. DNA ligase was used to react at 25°C for 1 h to obtain plasmids pGAPZB-tHMGR, pTEF1ZB-IDI1, and pGCW14ZB-GGPPS.

[0114] (5) Using plasmids pGAPZB-tHMGR, pGCW14ZB-GGPPS, and pTEF1ZB-IDI1 as templates, PCR amplification was performed using primers “PZB-A1*-F” and “PZB-A2*-R”, primers “PZB-A2*-F” and “PZB-A3*-R”, and primers “PZB-A3*-F” and “PZB-A4*-R”, respectively, to obtain the gene expression cassette “P GAP -tHMGR-T AOX1 ”, “P GCW14 -GGPPS-T AOX1 ” and “P TEF1 -IDI1-T AOX1 ”.

[0115] (6) Plasmid G-PNSI-5 was circularized by PCR using primers "PNSI-5-DO-A4-F" and "PNSI-5-UP-A1-R" to obtain a DNA vector fragment containing the PNSI-5 homology arms. After the above gene expression cassette and vector fragment were evenly mixed, the plasmid G-PNSI-5-tHMGR-GGPPS-IDI1 was obtained by Gibson assembly. The structure is as follows Figure 4 shown.

[0116] (7) Using plasmid G-PNSI-5-tHMGR-GGPPS-IDI1 as a template, the gene expression cassette "homologous arm (up)-P" was obtained by PCR using primers "PNSI-5-UP-A0-F" and "PNSI-5-DO-AR-R". GAP -tHMGR-T AOX1 -P GCW14 -GGPSS-T AOX1 -P TEF1 -IDI1-T AOX1- The gene expression cassette was transformed into competent cells by electroporation.

[0117] (8) Screening was performed using YPDZ plates, and single colonies grew in 3–4 days. Positive clones were identified by PCR and named the recombinant Pichia pastoris strain HL-2.

[0118] Example 3 Construction of recombinant Pichia pastoris strain HL-3

[0119] (1) The recombinant Pichia pastoris strain HL-2 prepared in Example 2 was cultured overnight in YPD liquid medium to prepare competent cells.

[0120] (2) A biotechnology company was commissioned to synthesize a gene fragment crtYB containing EcoRI and SalI restriction sites at both ends. The fragment was digested with EcoRI and SalI at 37°C for 1 h, and the reaction solution was purified and recovered using a Novagen gel recovery kit.

[0121] (3) Separately, the vector pGAPZB was treated with the endonucleases EcoRI and SalI at 37°C for 1 h. The reaction solution was purified and recovered using the Novozyme Gel Recovery Kit.

[0122] (4) The crtYB gene fragment was mixed with the pGAPZB vector and reacted with DNA ligase at 25°C for 1 h to obtain the plasmid pGAPZB-crtYB.

[0123] (5) Using plasmid pGAPZB-crtYB as a template, PCR amplification was performed using primers “PNSII-7-CrtYB-F” and “PNSII-7-CrtYB-R” to obtain the gene expression cassette “P GAP -crtYB-T AOX1 ”.

[0124] (6) Plasmid G-PNSII-7 was circularized by PCR using primers "PNSII-7-DO-CrtYB-F" and "PNSII-7-UP-CrtYB-R" to obtain a DNA vector fragment containing the PNSII-7 homology arms. After the above gene expression cassette and vector fragment were evenly mixed, plasmid G-PNSII-7-crtYB was obtained by Gibson assembly. The structure is as follows Figure 5 shown.

[0125] (7) Using plasmid G-PNSII-7-crtYB as a template, the gene expression cassette "homologous arm (up)-P" was obtained by PCR using primers "PNSII-7-UP-A0-F" and "PNSII-7-DW-AR-R". GAP -crtYB-T AOX1 - homology arm (down)". The gene expression cassette was transformed into competent cells by electroporation.

[0126] (8) Screening was performed using YPDZ plates, and single colonies grew in 3–4 days. Positive clones were identified by PCR and named the recombinant Pichia pastoris strain HL-3.

[0127] Example 4 Construction of recombinant Pichia pastoris strain HL-4

[0128] (1) The recombinant Pichia pastoris strain HL-3 prepared in Example 3 was cultured overnight in YPD liquid medium to prepare competent cells.

[0129] (2) A biotechnology company was commissioned to synthesize gene fragments anACL1 and anACL2 containing KpnI and SalI restriction sites at both ends. The fragments were digested with KpnI and SalI endonucleases at 37°C for 1 h, and the reaction solution was purified and recovered using a Novozymes gel recovery kit.

[0130] (3) Separately, vectors pGAPZB and pGCW14ZB were treated with endonucleases KpnI and SalI at 37°C for 1 h. The reaction solution was purified and recovered using a Novozyme gel recovery kit.

[0131] (4) The anACL1 gene fragment was mixed with the pGAPZB vector, and the anACL2 gene fragment was mixed with the pGCW14ZB vector. DNA ligase was used to react at 25°C for 1 hour to obtain plasmids pGAPZB-anACL1 and pGCW14-anACL2.

[0132] (5) Using plasmid pGAPZB-anACL1 and plasmid pGCW14-anACL2 as templates, PCR amplification was performed using primers “PZB-A1*-F” and “PZB-A2*-R” and primers “PZB-A2*-F” and “PZB-A3*-R”, respectively, to obtain the gene expression cassette “P GAP -anACL1-T AOX1 ” and “P GCW14 -anACL2-T AOX1 ”.

[0133] (6) Plasmid G-PNSI-4 was circularized by PCR using primers "A3-F" and "A1-R" to obtain a DNA vector fragment containing the PNSI-4 homology arms. The above gene expression cassette and vector fragment were evenly mixed and assembled by Gibson to obtain plasmid G-PNSI-4-anACL1-anACL2, with the structure shown in FIG. Figure 6 shown.

[0134] (7) Using plasmid G-PNSI-4-anACL1-anACL2 as a template, the gene expression cassette "homologous arm (up)-P" was obtained by PCR using primers "PNSI-4-UP-A0-F" and "PNSI-4-DW-AR-R". GAP -anACL1-T AOX1 -P GCW14 -anACL2-T AOX1 - homology arm (down)". The gene expression cassette was transformed into competent cells by electroporation.

[0135] (8) Screening was performed using YPDZ plates, and single colonies grew in 3–4 days. Positive clones were identified by PCR and named the recombinant Pichia pastoris strain HL-4.

[0136] Example 5 Construction of recombinant Pichia pastoris strain HL-5

[0137] (1) The recombinant Pichia pastoris strain HL-4 prepared in Example 4 was cultured overnight in YPD liquid medium to prepare competent cells.

[0138] (2) Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primers "ERG10-XbaI-F" and "ERG10-SalI-R" and primers "ERG13-EcoRI-F" and "ERG13-XbaI-R" to obtain genes ERG10 (approximately 1220 bp) and ERG13 (approximately 1360 bp). The ERG10 gene fragment and the ERG13 gene fragment were treated with nucleases SalI and EcoRI and endonucleases EcoRI and XbaI at 37°C for 1 hour, and the reaction solution was purified and recovered using the Novozyme gel recovery kit.

[0139] (3) Separately, the vectors pGCW14ZB and pTEF1ZB were treated with endonucleases SalI, EcoRI and EcoRI, XbaI at 37°C for 1 h, and the reaction solution was purified and recovered using a Novozymes gel recovery kit.

[0140] (4) The recovered ERG10 gene fragment was mixed with the pGCW14ZB vector, and the ERG13 gene fragment was mixed with the pTEF1ZB vector. DNA ligase was used to react at 25°C for 1 hour to obtain plasmid pGCW14-ERG10 and plasmid pTEF1-ERG13.

[0141] (5) Using plasmid pGCW14ZB-ERG10 and plasmid pTEF1ZB-ERG13 as templates, PCR amplification was performed using primers “PZB-A1*-F” and “PZB-A2*-R” and primers “PZB-A2*-F” and “PZB-A3*-R”, respectively, to obtain the gene expression cassette “P GCW14 -ERG10-T AOX1 ” and “P TEF1 -ERG13-T AOX1 ”.

[0142] (6) Plasmid G-PNSII-4 was circularized by PCR using primers "A3-F" and "A1-R" to obtain a DNA vector fragment containing the PNSII-4 homology arms. After the above gene expression cassette and vector fragment were evenly mixed, plasmid G-PNSII-4-ERG10-ERG13 was obtained by Gibson assembly, with the structure shown in FIG. Figure 7 shown.

[0143] (7) Using plasmid G-PNSII-4-ERG10-ERG13 as a template, the gene expression cassette "homologous arm (up)-P" was obtained by PCR using primers "PNSII-4-UP-A0-F" and "PNSII-4-DW-AR-R". GCW14 -ERG10-T AOX1 -P TEF1 -ERG13-T AOX1 - homology arm (down)". The gene expression cassette was transformed into competent cells by electroporation.

[0144] (8) Screening was performed using YPDZ plates, and single colonies grew in 3–4 days. Positive clones were identified by PCR and named the recombinant Pichia pastoris strain HL-5.

[0145] Example 6 Construction of recombinant Pichia pastoris strain HL-6

[0146] (1) The recombinant Pichia pastoris strain HL-5 prepared in Example 5 was cultured overnight in YPD liquid medium to prepare competent cells.

[0147] (2) Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primers "pGCW14-ERG12-F" and "pGCW14-ERG12-R" and primers "pGAP-ERG8-F" and "pGAP-ERG8-R" to obtain genes ERG12 (about 1300 bp) and ERG8 (about 1360 bp). Separately, vectors pGCW14ZB and pGAPZB were obtained and circularized using primers "C-GCW14ZB-F" and "C-GCW14ZB-R" and primers "C-GAPZB-F" and "C-GAPZB-R", respectively.

[0148] (3) The circularized vectors pGCW14ZB and pGAPZB were mixed evenly with the ERG12 gene fragment and the ERG8 gene fragment, respectively, and the plasmids pGCW14ZB-ERG12 and pGAPZB-ERG8 were obtained by Gibson assembly.

[0149] (4) Using plasmid pGCW14ZB-ERG12 and plasmid pGAPZB-ERG8 as templates, PCR amplification was performed using primers “PZB-A1*-F” and “PZB-A2*-R” and primers “PZB-A2*-F” and “PZB-A3*-R”, respectively, to obtain the gene expression cassette “P GCW14 -ERG12-T AOX1 ” and “P GAP -ERG8-T AOX1 ”.

[0150] (5) Plasmid G-PNSIV-4 was circularized by PCR using primers "A3-F" and "A1-R" to obtain a DNA vector fragment containing the PNSIV-4 homology arms. The above gene expression cassette and vector fragment were evenly mixed and assembled by Gibson to obtain plasmid G-PNSIV-4-ERG12-ERG8, with the structure shown in FIG. Figure 8 shown.

[0151] (6) Using plasmid G-PNSIV-4-ERG12-ERG8 as a template, the gene expression cassette "homologous arm (up)-P" was obtained by PCR using primers "PNSIV-4-UP-A0-F" and "PNSIV-4-DW-AR-R". GCW14 -ERG12-T AOX1 -P GAP -ERG8-T AOX1 - homology arm (down)". The gene expression cassette was transformed into competent cells by electroporation.

[0152] (7) Screening was performed using YPDZ plates, and single colonies grew in 3–4 days. Positive clones were identified by PCR and named the recombinant Pichia pastoris strain HL-6.

[0153] Example 7 Construction of recombinant Pichia pastoris strain HL-7

[0154] (1) The recombinant Pichia pastoris strain HL-6 prepared in Example 6 was cultured overnight in YPD liquid medium to prepare competent cells.

[0155] (2) Using the Saccharomyces cerevisiae genome as a template, PCR amplification was performed using primers "scYHM2-KpnI-F" and "scYHM2-SalI-R" and primers "scIDP2-KpnI-F" and "scIDP2-SalI-R" to obtain genes YHM2 (approximately 950 bp) and IDP2 (approximately 1250 bp). The two gene fragments were treated with nucleases KpnI and SalI at 37°C for 1 hour, and the reaction solution was purified and recovered using a Novozymes gel recovery kit.

[0156] (3) Separately, vectors pGAPZB and pGCW14ZB were treated with endonucleases KpnI and SalI at 37°C for 1 h. The reaction solution was purified and recovered using a Novozyme gel recovery kit.

[0157] (4) The recovered YHM2 gene fragment was mixed with the pGAPZB vector, and the IDP2 gene fragment was mixed with the pGCW14ZB vector. DNA ligase was used to react at 25°C for 1 hour to obtain plasmids pGAPZB-YHM2 and pGCW14ZB-IDP2.

[0158] (5) Using plasmid pGAPZB-YHM2 and plasmid pGCW14ZB-IDP2 as templates, PCR amplification was performed using primers “PZB-A1*-F” and “PZB-A2*-R” and primers “PZB-A2*-F” and “PZB-A3*-R”, respectively, to obtain the gene expression cassette “P GAP -ScYHM2-T AOX1 ” and “P GCW14 -ScIDP2-T AOX1 ”.

[0159] (6) Plasmid G-PNSI-2 was circularized by PCR using primers "A3-F" and "A1-R" to obtain a DNA vector fragment containing the PNSI-2 homology arms. The above gene expression cassette and vector fragment were evenly mixed and assembled by Gibson to obtain plasmid G-PNSI-2-YHM2-IDP2, with the structure shown in FIG. Figure 9 shown.

[0160] (7) Using plasmid G-PNSI-2-YHM2-IDP2 as a template, the gene expression cassette "homologous arm (up)-P" was obtained by PCR using primers "PNSI-2-UP-A0-F" and "PNSI-2-DW-AR-R". GAP -ScYHM2-T AOX1 -P GCW14 -ScIDP2-T AOX1 - homology arm (down)". The gene expression cassette was transformed into competent cells by electroporation.

[0161] (8) Screening was performed using YPDZ plates, and single colonies grew in 3–4 days. Positive clones were identified by PCR and named the recombinant Pichia pastoris strain HL-7.

[0162] Example 8 Construction of recombinant Pichia pastoris strain HL-8

[0163] (1) The recombinant Pichia pastoris strain HL-7 prepared in Example 7 was cultured overnight in YPD liquid medium to prepare competent cells.

[0164] (2) Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primers "XP_002490719.1-F" and "XP_002490719.1-R". The amplified product (about 1000 bp) was purified and recovered using the Novozyme gel recovery kit to obtain the promoter DNA fragment 1 to be replaced.

[0165] (3) Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primers “ERG9-UP-F” and “ERG9-UP-R”. The amplified product (about 770 bp) was purified and recovered using the Norvegian gel recovery kit to obtain DNA fragment 2.

[0166] (4) Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primers “DAS1TT-HA-F” and “DAS1TT-A2-R”. The amplified product (about 250 bp) was purified and recovered using the Novozyme gel recovery kit to obtain DNA fragment 3.

[0167] (5) Using the Pichia pastoris GS115 genome as a template, PCR amplification was performed using primers “ERG9-F” and “ERG9-R”. The amplified product (about 1000 bp) was purified and recovered using the Novozyme gel recovery kit to obtain DNA fragment 4.

[0168] (6) The DNA fragments obtained above were evenly mixed and fusion PCR was performed using primers "ERG9-UP-F" and "ERG9-R". The amplified product (about 3150 bp) was purified and recovered using the Novozyme gel recovery kit to obtain the recombinant DNA fragment to be transformed. The recombinant DNA fragment was transformed into competent cells by electroporation.

[0169] (7) Screening was performed using YPDZ plates, and single colonies grew in 3–4 days. Positive clones were identified by PCR and named the recombinant Pichia pastoris strain HL-8.

[0170] Example 9 Preparation of β-carotene by shake flask fermentation of recombinant Pichia pastoris strains HL-1 to HL-8

[0171] (1) Activation of glycerol bacteria: Thaw the glycerol bacteria stored at -80℃ at room temperature, streak a YPD plate, and culture it in a 28-30℃ incubator for 48-72 hours.

[0172] (2) Seed liquid preparation: Pick a single colony of appropriate size from the streaked plate and inoculate it into 3 mL of YPD liquid medium. Cultivate overnight at 28-30°C and 220 rpm until the OD 600nm When it reaches 10-15, you will get seed liquid.

[0173] (3) Fermentation culture: the seed liquid was heated to the initial OD 600nm =0.5 was inoculated into 20 mL YPD medium and cultured at 28-30°C and 220 rpm for 72 h, during which samples were taken for microscopic examination every 24 h.

[0174] (4) Fermentation broth treatment: 1 mL of 72-h fermentation broth was placed in a disruptor tube and centrifuged at 12,000 rpm for 3 min to collect the cells. The cells were washed twice with 2 mL of sterile ultrapure water, then glass beads and acetonitrile were added. The cells were disrupted and extracted using a high-throughput disruptor until the cells turned white and the intracellular products were essentially dissolved in acetonitrile. The cells were centrifuged at 12,000 rpm for 3 min, and the supernatant was aspirated and passed through a membrane for determination of the intracellular products.

[0175] (5) Detection of β-carotene production by spectrophotometry: Use a pipette to draw 200 μl of the test solution, measure the absorbance at 453 nm using an enzyme marker, and calculate the β-carotene production based on the standard curve. The results are as follows: Figure 10 shown.

[0176] (6) Measure cell dry weight: Take a clean 1.5 mL centrifuge tube, accurately weigh its empty mass using a balance, and record the data. Use a pipette to accurately draw 1 mL of fermentation broth and transfer it to a 1.5 mL centrifuge tube of known mass. Centrifuge at 12,000 rpm for 3 minutes to collect the cells. Resuspend the cells in 1 mL of sterile ultrapure water, wash twice, and transfer them to an 80°C metal bath for heating and drying. After a period of time, weigh the mass using a balance to obtain the cell dry weight.

[0177] Figure 11 Schematic diagram of the production pathway of β-carotene in Pichia pastoris.

[0178] In summary, the present invention provides a recombinant Pichia pastoris strain that produces high β-carotene production, as well as its construction method and application. A multi-copy approach is used to construct the β-carotene biosynthesis pathway, improving target gene expression and β-carotene production. Furthermore, integrating the exogenous gene into the genome rather than expressing it as an episomal plasmid enhances the strain's genetic stability. Furthermore, the recombinant strain exhibits excellent stability and reproducibility. Across multiple fermentation batches, the β-carotene production fluctuation range remains below 10%, demonstrating significant potential for industrial application.

[0179] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. The recombinant Pichia pastoris strain HL-8 was deposited in China Center for Type Culture Collection with the deposit number CCTCC No. M 2025667. The deposit address is Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. Use of the recombinant Pichia pastoris strain according to claim 1 in increasing β-carotene production, characterized in that: The recombinant Pichia pastoris strain increases β-carotene production by expressing β-carotene synthesis-related genes, overexpressing endogenous mevalonate pathway-related genes, increasing acetyl-CoA precursor supply-related genes, and downregulating squalene synthesis-related genes; The β-carotene synthesis-related genes include the gene CrtE encoding geranylgeranyl diphosphate synthase, the gene CrtI encoding phytoene dehydrogenase, and the gene CrtYB encoding phytoene synthase / lycopene cyclase; The mevalonate pathway-related genes include the gene tHMGR encoding truncated 3-hydroxy-3-methylglutaryl-CoA reductase, the gene GGPPS encoding geranylgeranyl diphosphate synthase, the gene IDI1 encoding isopentenyl pyrophosphate isomerase, the gene ERG10 encoding acetyl-CoA transacetylase, the gene ERG13 encoding hydroxymethylglutaryl-CoA synthase, the gene ERG12 encoding mevalonate kinase, and the gene ERG8 encoding phosphomevalonate kinase; The genes related to increasing the supply of acetyl-CoA precursors include the gene ACL1 encoding the large subunit of ATP-citrate lyase, the gene ACL2 encoding the small subunit of ATP-citrate lyase, the gene YHM2 encoding the carboxylate transporter, and the gene IDP2 encoding isocitrate dehydrogenase; The squalene synthesis-related genes include the gene ERG9 encoding squalene synthase.

3. Use of the recombinant Pichia pastoris strain according to claim 2 for increasing β-carotene production, characterized in that: Among the β-carotene synthesis-related genes, CrtE, CrtI and CrtYB are all derived from Xanthophylomyces dendrorhous.

4. Use of the recombinant Pichia pastoris strain according to claim 2 for increasing β-carotene production, characterized in that: Among the mevalonate pathway-related genes, tHMGR, GGPPS, IDI1, ERG10, ERG13, ERG12 and ERG8 are all derived from Pichia pastoris (Komagataelaphaffi).

5. Use of the recombinant Pichia pastoris strain according to claim 2 for increasing β-carotene production, characterized in that: Among the genes related to increasing the supply of acetyl-CoA precursor, ACL is derived from Aspergillus niger, and YHM2 and IDP2 are both derived from Saccharomyces cerevisiae.

6. Use of the recombinant Pichia pastoris strain according to claim 2 for increasing β-carotene production, characterized in that: The squalene synthase gene ERG9 is down-regulated in terms of gene expression level by replacing a promoter, wherein the promoter is derived from Pichia pastoris PAS_chr1-4_0582.

7. A method for constructing a recombinant Pichia pastoris strain, characterized in that: include, The gene expression cassette "P GAP -crtE-T AOX1 ”"P GAP -crtI-T AOX1 ”"P GAP -crtYB-T AOX1 ", electroporation was performed into the rDNA multi-copy site of the original Pichia pastoris strain GS115 genome to obtain the β-carotene synthesis gene multi-copy strain HL-1; Construction of gene expression cassette "P GAP -tHMGR-T AOX1 -P GAP -IDI1-T AOX1 -P GAP -GGPPS-T AOX1 ", introduced the neutral site I-5 of the genome of the recombinant strain HL-1 to obtain the recombinant strain HL-2; Construction of gene expression cassette "P GAP -crtYB-T AOX1 ", introduced into the neutral site II-7 of the genome of the recombinant strain HL-2 to obtain the recombinant strain HL-3; Construction of gene expression cassette "P GAP -anACL-T AOX1 ", introduced the neutral site I-4 of the genome of the recombinant strain HL-3 to obtain the recombinant strain HL-4; Construction of gene expression cassette "P GCW14 -ERG10-T AOX1 -P TEF1 -ERG13-T AOX1 ", introduced into the neutral site II-4 of the genome of the recombinant strain HL-4 to obtain the recombinant strain HL-5; Construction of gene expression cassette "P GCW14 -ERG12-T AOX1 -P GAP -ERG8-T AOX1 ", introduced into the neutral site IV-4 of the genome of the recombinant strain HL-5 to obtain the recombinant strain HL-6; Construction of gene expression cassette "P GAP -ScYHM2-T AOX1 -P GCW14 -ScIDP2-T AOX1 ", introduced the neutral site I-2 of the genome of the recombinant strain HL-6 to obtain the recombinant strain HL-7; Based on the recombinant strain HL-7, the expression of squalene synthase was down-regulated using the promoter of the Pichia pastoris PAS_chr1-4_0582 gene to obtain the recombinant strain HL-8, which is the recombinant Pichia pastoris strain.

8. A method for producing β-carotene, characterized in that: include, The recombinant Pichia pastoris strain according to claim 1 is inoculated into YPD medium at an inoculum rate of 1-5%, and cultured overnight at 28-30° C. and 200-220 rpm to obtain an activated glycerol strain; Take the activated glycerol bacteria to start with OD 600nm = 0.1-0.5 was inoculated into YPD medium and cultured at 28-30°C and 200-220 rpm until OD 600nm =10-15, to obtain seed solution; The seed liquid was inoculated into YPD medium and cultured at 28-30°C and 200-220 rpm for 72 hours. Samples were taken every 24 hours for microscopic examination and the fermentation products were preserved. β-carotene was extracted from the fermentation broth using acetonitrile as the extraction solvent.

9. The method for producing beta-carotene according to claim 8, wherein: The YPD culture medium comprises 9.5-10.5 g / L yeast extract, 19-21 g / L peptone, and 19-21 g / L glucose.

10. The method for producing β-carotene according to claim 8, wherein: The seed solution was inoculated into YPD medium, wherein the initial concentration of the seed solution was OD 600nm =0.5.

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