A method for regulating the morphology of brewer's yeast to increase yeast protein content

Abstract

The present invention discloses a method for regulating the morphology of saccharomyces cerevisiae to increase the protein content of yeast, and belongs to the technical field of microbial genetic engineering. The present invention integrates the Gic1 gene at the HO site on the genome to reshape the morphology of saccharomyces cerevisiae, improves the ability of saccharomyces cerevisiae to synthesize proteins globally, increases protein accumulation, and increases the protein content of saccharomyces cerevisiae from 46.16 g / 100 g to 51.04 g / 100 g, providing technical support for low-cost, green single-cell protein production, and having broad application prospects in the field of alternative proteins.

Description

Technical Field

[0001] The invention relates to a method for regulating the morphology of brewer's yeast and increasing the protein content of yeast, and belongs to the technical field of microbial genetic engineering. Background Art

[0002] Existing protein production strategies cannot meet people's demand for protein. Therefore, finding more sustainable, more accessible and healthier protein sources has become the focus of current food research.

[0003] Microbial protein, or single-cell protein (SCP), has become a promising protein source. It can be obtained through fermentation and culture of industrial and agricultural waste materials. Compared with traditional proteins, microbial protein raw materials are widely available, with high production efficiency and short cycle times. Yeast cells, among others, have advantages such as a clear genetic background, a complete molecular operating system, and strong tolerance to environmental stress, and have been widely used in the production of organic acids, bioenergy, biomaterials, biopharmaceuticals, and natural products. Yeast protein is an ideal new alternative protein. Currently, the selection of Saccharomyces cerevisiae to increase protein content is relatively mature, but the operation is cumbersome. Adaptive evolution to increase protein content is limited to increasing strain biomass with a certain decrease in protein content, while mutagenesis breeding is time-consuming and has poor stability. Therefore, how to rationally design Saccharomyces cerevisiae to improve its global protein synthesis capacity has begun to attract attention.

[0004] As an important part of microbial fermentation regulation, bacterial fermentation morphology regulation plays an important role in improving the production level of microorganisms in industrial fermentation and has received widespread attention. For large-volume intracellular products (such as polylactic acid and polyhydroxybutyric acid), small cell volume will affect the accumulation of products, ultimately leading to a decrease in production efficiency. The volume of microbial cells is mainly affected by their own morphology. Therefore, reshaping microbial cell morphology is an effective strategy to increase cell volume. However, some literature shows (Mapping pathways and phenotypes by systematic gene overexpression. Mol Cell 21(3):319-30) that overexpression of some genes affects the growth of bacterial cells, making it difficult to achieve effective accumulation of products. Therefore, screening genes that increase protein content by regulating cell morphology is of great significance for improving global protein synthesis. Summary of the Invention

[0005] Technical issues:

[0006] The purpose of the present invention is to overcome the shortcomings of existing breeding technology and provide a rational design method that can achieve efficient synthesis of single-cell protein in Saccharomyces cerevisiae.

[0007] Technical solution:

[0008] This invention provides a method for regulating Saccharomyces cerevisiae morphology to increase yeast protein content. This method involves overexpressing cytoskeleton and cell cycle-related genes to reshape cell morphology and thereby enhance global protein synthesis in Saccharomyces cerevisiae. This invention has important implications for promoting global protein synthesis in Saccharomyces cerevisiae and for the application of alternative proteins.

[0009] The present invention provides a method for regulating the morphology of saccharomyces cerevisiae and increasing the protein content of yeast, which comprises integrating one or more genes of Gic1, Acf2, Aip1, Cln1 and Cln2 into the genome of saccharomyces cerevisiae.

[0010] In one embodiment, the method is to integrate the Gic1 gene into the genomic HO site.

[0011] In one embodiment, the method comprises the steps of:

[0012] (1) The Gic1, Acf2, Aip1, Cln1, or Cln2 gene was ligated into the plasmid pUMRI-A-△HO to obtain the recombinant plasmids pUMRI-△HO-GIC1, pUMRI-△HO-ACF2, pUMRI-△HO-AIP1, pUMRI-△HO-CLN1, and pUMRI-△HO-CLN2, respectively;

[0013] (2) The recombinant plasmid from step (1) is linearized and integrated into the chromosome of Saccharomyces cerevisiae by homologous recombination.

[0014] In one embodiment, the Saccharomyces cerevisiae includes but is not limited to BY4741.

[0015] The present invention also provides a recombinant Saccharomyces cerevisiae, wherein one or more genes of Gic1, Acf2, Aip1, Cln1 and Cln2 derived from Saccharomyces cerevisiae BY4741 are integrated into the genome of the recombinant Saccharomyces cerevisiae.

[0016] In one embodiment, the nucleotide sequence of the Gic1 gene is shown as SEQ ID NO.1; the nucleotide sequence of the gene Acf2 is shown as SEQ ID NO.2; the nucleotide sequence of the gene Aip1 is shown as SEQ ID NO.3; the nucleotide sequence of the gene Cln1 is shown as SEQ ID NO.4; and the nucleotide sequence of the gene Cln2 is shown as SEQ ID NO.5.

[0017] In one embodiment, the Saccharomyces cerevisiae includes but is not limited to BY4741.

[0018] The present invention also provides the use of the recombinant Saccharomyces cerevisiae in protein production.

[0019] In one embodiment, the use is to ferment the recombinant Saccharomyces cerevisiae in a culture medium for a period of time.

[0020] In one embodiment, the culture medium contains: tryptone (20 g / L), yeast extract (10 g / L), and galactose (20 g / L).

[0021] In one embodiment, the fermentation is carried out at 28-32°C, especially at 30°C.

[0022] In one embodiment, the fermentation is for at least 48 hours.

[0023] In one embodiment, the recombinant Saccharomyces cerevisiae is first activated in a culture medium containing tryptone, yeast extract, and glucose, and then fermented.

[0024] In one embodiment, the engineered yeast is based on the wild-type yeast BY4741 as a starting strain.

[0025] Beneficial effects:

[0026] Using wild-type BY4741 as a starting strain, the present study screened and regulated genes related to the cytoskeleton and cell cycle, reshaping cell morphology. The results, validated by fermentation, revealed that overexpression of Gic1 enhanced global protein synthesis in Saccharomyces cerevisiae. Compared to the original strain, the protein content of the engineered strain increased from 46.16g / 100g to 51.04g / 100g.

[0027] The present invention regulates the cytoskeleton and cell cycle-related genes to reshape cell morphology and achieve efficient protein synthesis in Saccharomyces cerevisiae. This method can provide a reference for large-scale production of yeast proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the morphology of yeast cells under a microscope.

[0029] Figure 2 The morphology of yeast cells under a scanning electron microscope.

[0030] Figure 3 The protein content in recombinant Saccharomyces cerevisiae cells with different genes integrated. DETAILED DESCRIPTION

[0031] The culture medium involved in the following examples is:

[0032] YPD liquid medium: 20 g / L glucose, 20 g / L tryptone, 10 g / L yeast extract, sterilized by high-pressure steam at 115°C for 30 min; when preparing solid plates, add 15 g / L agar powder to this medium.

[0033] YPG liquid medium: 20 g / L galactose, 20 g / L tryptone, 10 g / L yeast extract, sterilized by high-pressure steam at 115°C for 30 min; when preparing solid plates, add 15 g / L agar powder to this medium.

[0034] 20 mg / mL G418 sulfate stock solution: Dissolve 0.4 g of G418 sulfate powder in 20 mL of sterile water. Filter sterilize using a sterile 0.22 μm syringe filter in a laminar flow hood. Aliquot into brown centrifuge tubes and store at -20°C. Add 10 μL of the stock solution per 1 mL of culture medium for a working concentration of 200 μg / mL for preparation of YPD-G418 and YPG-G418 liquid culture media.

[0035] LB liquid medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, sterilized at 120°C for 20 min. When preparing solid plates, add 15 g / L agar powder.

[0036] The final working concentration of ampicillin was 100 μg / mL; the final working concentration of kanamycin sulfate was 50 μg / mL; and the final working concentration of G418 was 200 μg / mL.

[0037] The biochemical materials involved in the following embodiments are as follows:

[0038] The wild-type Saccharomyces cerevisiae strain BY4741 was preserved in our laboratory, and the competent Escherichia coli TOP10 was purchased from Novagen, USA.

[0039] The yeast integrative plasmid pUMRI-A-ΔHO was disclosed in the paper "De novo production of hydroxytyrosol by metabolic engineering of Saccharomyces cerevisiae".

[0040] The methods involved in the following embodiments are:

[0041] Determination of protein content: The bacterial cells in the fermentation broth were collected, washed and dried to constant weight, and the protein quality was determined by Kjeldahl method, referring to GB 5009.5-2016 "National Food Safety Standard for Determination of Protein in Food". Unless otherwise specified, the "protein content" mentioned in the examples refers to the amount of protein per unit cell (g 蛋白 / 100g 菌体细胞 ).

[0042] The information of the strains, genes, plasmids and primers involved in the following examples are shown in Tables 1 to 4.

[0043] Table 1 Cytoskeleton and cell cycle related genes and their functions

[0044]

[0045] Table 2: Primer list

[0046]

[0047] Table 3: Plasmid list

[0048]

[0049]

[0050] Table 4: List of strains

[0051]

[0052] Basic operations in molecular biology:

[0053] (1) E. coli plasmid extraction, genome extraction, PCR product purification, and gel recovery were performed according to the instructions of the corresponding kit unless otherwise specified.

[0054] (2) Amplify the DNA fragment using PCR technology, using different reaction systems and procedures according to experimental requirements. See Table 4 for the reaction system and Table 5 for the reaction procedure.

[0055] Table 5: PCR reaction system

[0056]

[0057] Table 6: PCR reaction program

[0058]

[0059]

[0060] The pUMRI-A-△HO involved in the following embodiments is disclosed in the following paper: Liu YJ, Liu H, Hu HT, et al. De Novo Production of Hydroxytyrosol by Metabolic Engineering of Saccharomyces cerevisiae[J]. Journal of Agricultural and Food Chemistry, 2022, 70(24): 7490-7499.

[0061] Example 1: Construction of yeast strain PUMRI-ΔHO-GIC1

[0062] The Gic1 fragment (nucleotide sequence shown in SEQ ID NO. 1) was amplified from the genome of wild-type yeast BY4741 using PCR using primers Gic1-F (BamH1) and Gic1-R (Sal1). The Gic1 fragment was ligated with the plasmid pUMRI-A-△HO to generate the recombinant plasmid pUMRI-△HO-GIC1. The recombinant plasmid pUMRI-△HO-GIC1 was digested with SfiI, linearized, and then integrated into the HO site of BY4741 to generate the engineered yeast strain PUMRI-△HO-GIC1. Primer sequences are shown in Table 2, and the genotype of PUMRI-△HO-GIC1 is shown in Table 4. The PCR reaction system is shown in Table 5, and the PCR procedure is shown in Table 6.

[0063] Example 2: Construction of yeast strain PUMRI-ΔHO-ACF2

[0064] The Acf2 fragment (nucleotide sequence shown in SEQ ID NO. 2) was amplified from the genome of wild-type yeast BY4741 using PCR using primers Acf2-F (BamH1) and Acf2-R (Sal1). The Acf2 fragment was ligated with the plasmid pUMRI-A-△HO to generate the recombinant plasmid pUMRI-△HO-ACF2. The recombinant plasmid pUMRI-△HO-ACF2 was digested with SfiI, linearized, and then integrated into the HO site of BY4741 to generate the engineered yeast strain PUMRI-△HO-ACF2. Primer sequences are shown in Table 2, and the genotype of PUMRI-△HO-ACF2 is shown in Table 4. The PCR reaction system is shown in Table 5, and the PCR procedure is shown in Table 6.

[0065] Example 3: Construction of yeast strain PUMRI-ΔHO-AIP1

[0066] The Aip1 fragment (nucleotide sequence shown in SEQ ID NO. 3) was amplified from the genome of wild-type yeast BY4741 using PCR using primers Aip1-F (BamH1) and Aip1-R (Sal1). The Aip1 fragment was ligated with the plasmid pUMRI-A-△HO to obtain the recombinant plasmid pUMRI-△HO-AIP1. The recombinant plasmid pUMRI-△HO-AIP1 was digested with SfiI, linearized, and then integrated into the HO site of BY4741 to obtain the engineered yeast strain PUMRI-△HO-AIP1. The primer nucleotide sequences are shown in Table 2, and the genotype of PUMRI-△HO-AIP1 is shown in Table 4. The PCR reaction system is shown in Table 5, and the PCR reaction procedure is shown in Table 6.

[0067] Example 4: Construction of yeast strain PUMRI-ΔHO-CLN1

[0068] The Cln1 fragment (nucleotide sequence shown in SEQ ID NO. 4) was amplified from the genome of wild-type yeast BY4741 using PCR using primers Cln1-F (BamH1) and Cln1-R (Sal1). The Cln1 fragment was ligated with plasmid pUMRI-A-△HO to generate recombinant plasmid pUMRI-△HO-CLN1. The recombinant plasmid pUMRI-△HO-CLN1 was digested with SfiI, linearized, and then integrated into the HO site of BY4741 to generate the engineered yeast strain PUMRI-△HO-CLN1. Primer nucleotide sequences are shown in Table 2, and the genotype of PUMRI-△HO-CLN1 is shown in Table 4. The PCR reaction system is shown in Table 5, and the PCR reaction procedure is shown in Table 6.

[0069] Example 5: Construction of yeast strain PUMRI-ΔHO-CLN2

[0070] The Cln2 fragment (nucleotide sequence shown in SEQ ID NO. 5) was amplified from the genome of wild-type yeast BY4741 using PCR using primers Cln2-F (BamH1) and Cln2-R (Sal1). The Cln2 fragment was ligated with plasmid pUMRI-A-△HO to generate the recombinant plasmid pUMRI-△HO-CLN2. The recombinant plasmid pUMRI-△HO-CLN2 was digested with SfiI, linearized, and then integrated into the HO site of BY4741 to generate the engineered yeast strain PUMRI-△HO-CLN2. The primer sequences are shown in Table 2, and the genotype of PUMRI-△HO-CLN2 is shown in Table 4. The PCR reaction system is shown in Table 5, and the PCR reaction procedure is shown in Table 6.

[0071] Example 6: Construction of yeast strain PUMRI-A-ΔHO

[0072] The plasmid pUMRI-A-△HO was transformed into the Saccharomyces cerevisiae BY4741 strain to obtain the yeast engineered strain PUMRI-A-△HO.

[0073] Example 7: Yeast cell culture

[0074] The above-constructed strains were inoculated into YPD liquid medium and cultured at 30°C and 250 r·min -1 The cells were activated and then inoculated into 50 mL YPG liquid medium at a ratio of 2% (v / v) and incubated at 30°C and 250 r·min -1 Culture for 48h.

[0075] Microscopic observation of yeast cell morphology:

[0076] The microscopic morphology of bacterial cells in shake flasks was observed using a digital optical microscope equipped with a built-in 3-megapixel digital camera (Ted Pella, Inc., Redding, CA, USA) and Motic Images Plus 2.0 image analysis software. One drop of fermentation broth was placed on a clean, transparent glass slide, and the cells were observed using an optical microscope with a maximum magnification of ×1000 using an oil immersion lens.

[0077] Morphological observation of yeast cells under scanning electron microscope:

[0078] Yeast morphology was primarily characterized using cold-field emission scanning electron microscopy (CFE-SEM). The CFE-SEM imaging steps are briefly described as follows: First, yeast cells were collected by centrifugation (12,000 rpm for 1 minute) and washed three times with phosphate buffered saline. The cells were then fixed overnight with 2.5% glutaraldehyde. After washing three times with phosphate buffered saline, the cells were collected and dehydrated in successive steps using 30%, 50%, 70%, 90%, 95%, and finally anhydrous ethanol. Finally, the yeast powder was dried at low temperature for imaging.

[0079] The results show Figure 1 and Figure 2After 48 hours of fermentation, cells were collected and observed under a light microscope. Compared to the control strain BY4741, overexpression of the Aip1, Acf2, and Cln1 genes did not significantly affect cell morphology. However, overexpression of the Cln2 and Gic1 genes significantly altered Saccharomyces cerevisiae morphology. Cln2 overexpression resulted in the development of numerous elongated buds. This morphological defect is hypothesized to be due to the prolonged growth of apical buds caused by abnormally high levels of Cln2-CDK, which is normally localized to the bud tip and promotes its growth. These elongated buds may explain the increase in cell size. Overexpression of the Gic1 gene resulted in a significant increase in the size of the Saccharomyces cerevisiae cells, with the cell surface exhibiting ruptures. It is hypothesized that Gic1 regulates the cytoskeletal component, the septin ring, thereby controlling polar growth and mitosis in Saccharomyces cerevisiae cells.

[0080] Example 8: Effect of morphological manipulation on protein content of Saccharomyces cerevisiae

[0081] The strains constructed in Examples 1 to 6 were inoculated into YPD liquid medium and cultured at 30°C and 250 r·min. -1 The cells were activated and then inoculated into 50 mL YPG liquid medium at a ratio of 2% (v / v) and incubated at 30°C and 250 r·min -1 Take 4 mL of fermentation broth and culture at 8000 r·min -1 Centrifuge for 10 minutes and discard the culture medium. Resuspend the precipitate in sterile water and centrifuge again. Wash twice and then centrifuge and discard the supernatant. Place in a 105°C oven and dry to constant weight to measure the dry weight (DCW) of the bacteria. -1 The remaining fermentation liquid was collected by centrifugation for 10 minutes, washed twice with distilled water and then dried. The protein content (g / 100g) was determined by the Kjeldahl method to screen out strains with excellent protein content and production capacity.

[0082] Fermentation results such as Figure 3 As shown, the protein content of strain PUMRI-△HO-GIC1 (51.04g / 100g) was significantly higher than that of the control strain BY4741 (46.16g / 100g). The Gic1 gene, a cytoskeleton-regulating gene, causes morphological changes in Saccharomyces cerevisiae. The increased size of Saccharomyces cerevisiae leads to increased protein accumulation and cellular protein content.

[0083] Therefore, by regulating the morphology of Saccharomyces cerevisiae, the cell morphology can be reshaped, the efficient synthesis of Saccharomyces cerevisiae protein can be achieved, and the production efficiency of Saccharomyces cerevisiae protein can be greatly improved.

[0084] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for regulating the morphology of brewer's yeast to increase the protein content of yeast, characterized in that: The Gic1 gene is integrated into the HO site of the Saccharomyces cerevisiae genome; the nucleotide sequence of the Gic1 gene is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that The cerevisiae yeast is BY4741.

3. The use of recombinant Saccharomyces cerevisiae in improving protein production of yeast, characterized in that: The recombinant Saccharomyces cerevisiae is obtained by overexpressing the Gic1 gene in Saccharomyces cerevisiae BY4741; the Gic1 gene is integrated into the genomic HO site; the nucleotide sequence of the Gic1 gene is shown in SEQ ID NO.

1.

4. The use according to claim 3, characterized in that: The recombinant Saccharomyces cerevisiae is fermented in the culture medium for a period of time.

5. The use according to claim 4, characterized in that: The recombinant brewer's yeast is first activated in a culture medium containing tryptone, yeast extract and glucose, and then fermented.

6. The use according to any one of claims 4 to 5, characterized in that: The fermentation is carried out at 28-32° C. for at least 48 hours.

Images