A Saccharomyces cerevisiae for improving ovalbumin production by modifying the endoplasmic reticulum

Abstract

The present invention relates to a Saccharomyces cerevisiae for improving the production of ovalbumin by modifying the endoplasmic reticulum, belonging to the technical field of genetic engineering. In the Saccharomyces cerevisiae CEN.PK2-1C of the present invention, the ovalbumin-encoding gene is heterologously expressed, and the INU1 signal peptide is inserted upstream of the N-terminus of ovalbumin to achieve the extracellular secretion of ovalbumin; then the fluorescence protein is used to characterize the unfolded protein response in the endoplasmic reticulum, and molecular chaperones Kar2 and PDI are co-expressed with promoters of different strengths in Saccharomyces cerevisiae to promote the folding of the endoplasmic reticulum; the opi1 gene is knocked out to increase the product secretion amount; the transcription factors INO2 and INO4 are overexpressed to expand the endoplasmic reticulum, increase the protein synthesis ability of Saccharomyces cerevisiae and promote the synthesis of ovalbumin. The yield of ovalbumin produced by the constructed Saccharomyces cerevisiae through fermentation for 72 hours reaches 6.2 mg / L.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to a Saccharomyces cerevisiae for improving the production of ovalbumin by modifying the endoplasmic reticulum. Background Art

[0002] High-quality proteins are essential for maintaining normal physiological functions of the human body. It plays a key role in tissue repair, immune system enhancement, energy supply, etc. With the growth of the global population and the improvement of living standards, the demand for high-quality proteins is also increasing. The land, water and other resources on the earth are limited, which restricts the production capacity of high-quality proteins. Especially for those protein sources that require a large amount of land and water resources, such as animal husbandry. Therefore, how to improve the production efficiency of proteins under limited resources and ensure the sustainability of protein supply is an urgent problem to be solved.

[0003] Eggs are an important source of high-quality proteins, especially the ovalbumin (OVA) in them has high nutritional value and is easily absorbed by the human body. However, the production of eggs is affected by various factors, such as the outbreak of avian influenza, changes in feeding costs, etc., which challenges the stability of eggs as a protein source. Ovalbumin is the most abundant protein in egg white, accounting for about 50%. It has been reported that ovalbumin has been synthesized in microorganisms such as Escherichia coli, Pichia pastoris and Trichoderma reesei, but none of these hosts have been proven to be the best host for expressing OVA.

[0004] Saccharomyces cerevisiae has the advantages of a short growth cycle, strong fermentation ability, easy large-scale culture, and rich nutritional components such as various proteins, amino acids, vitamins, and bioactive substances. It has become an ideal host for protein production. By genetic engineering technology, the target gene is introduced into Saccharomyces cerevisiae, and its efficient protein expression system can be used to produce a large amount of recombinant proteins with specific functions. In addition, Saccharomyces cerevisiae is also considered generally recognized as safe (GRAS), which mainly benefits from its viability, activity, and biosafety. Therefore, it has great application value in the production of edible and medicinal proteins.

[0005] As the first organelle in the secretory pathway of Saccharomyces cerevisiae, the endoplasmic reticulum (ER) plays a crucial role in protein synthesis, folding, and processing. However, when the expression level of heterologous proteins is too high, it will have a negative impact on the function of the endoplasmic reticulum. But when the expression level of heterologous proteins exceeds the processing capacity of the endoplasmic reticulum, a large number of proteins cannot be properly folded and thus accumulate in the endoplasmic reticulum. This accumulation leads to the generation of endoplasmic reticulum stress. Among them, the Unfolded Protein Response (UPR) is an important response to endoplasmic reticulum stress. When the unfolded or misfolded proteins in the endoplasmic reticulum accumulate to a certain extent, the UPR will be activated. This response aims to restore the normal function of the endoplasmic reticulum by increasing the expression of molecular chaperones and proteases involved in protein folding and degradation in the endoplasmic reticulum, as well as reducing the protein synthesis rate. However, this may also lead to a decrease in protein secretion, because when cells respond to endoplasmic reticulum stress, they will prioritize dealing with existing protein problems rather than continuing to synthesize and secrete new proteins, further limiting the yield of heterologous proteins. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a Saccharomyces cerevisiae for improving the yield of ovalbumin by modifying the endoplasmic reticulum. The ovalbumin-encoding gene is heterologously expressed in Saccharomyces cerevisiae CEN.PK2-1C, and the INU1 signal peptide is inserted upstream of the N-terminus of ovalbumin. Molecular chaperones Kar2 and PDI are co-expressed using promoters with different strengths, the opi1 gene is knocked out, and the transcription factors INO2 and INO4 are overexpressed.

[0007] The first object of the present invention is to provide a Saccharomyces cerevisiae for improving the yield of ovalbumin by modifying the endoplasmic reticulum. The Saccharomyces cerevisiae heterologously expresses the ovalbumin-encoding gene using the INU1 signal peptide, knocks out the gene opi1, and overexpresses the encoding genes of molecular chaperones Kar2 and PDI.

[0008] Furthermore, the starting strain is Saccharomyces cerevisiae CEN.PK2-1C.

[0009] Furthermore, the Saccharomyces cerevisiae overexpresses the encoding genes of transcription factors INO2 and INO4.

[0010] Furthermore, the P GAP promoter is used to express the Kar2-encoding gene and the INO4-encoding gene, and the P PGK1 promoter is used to express the PDI-encoding gene and the INO2-encoding gene.

[0011] Furthermore, overexpress the Kar2-encoding gene at locus 208a of the Saccharomyces cerevisiae genome, the PDI-encoding gene at locus 720a, the INO2-encoding gene at locus 1309a, and the INO4-encoding gene at locus 416d.

[0012] The second object of the present invention is to provide a microbial preparation comprising the above-mentioned Saccharomyces cerevisiae.

[0013] The third object of the present invention is to provide a construction method of the above-mentioned Saccharomyces cerevisiae, comprising the following steps:

[0014] S1. Heterologously express the ovalbumin-encoding gene in the starting strain Saccharomyces cerevisiae, and insert the INU1 signal peptide upstream of the N-terminal sequence of ovalbumin;

[0015] S2. At locus 208a in the genome of the strain obtained in S1, use the P GAP promoter to express the chaperone Kar2-encoding gene, and at locus 720a in the genome of the strain obtained in S2, use the P PGK1 promoter to express the chaperone PDI-encoding gene;

[0016] S3. Knock out the opi1 gene in the genome of the strain obtained in S3;

[0017] S4. At locus 1309a in the genome of the strain obtained in S4, use the P GAP promoter to express the transcription factor INO2-encoding gene, and at locus 416d in the genome of the strain obtained in S5, use the P PGK1 promoter to express the transcription factor INO4-encoding gene.

[0018] The fourth object of the present invention is to provide the application of the above-mentioned Saccharomyces cerevisiae, the above-mentioned microbial preparation or the above-mentioned method in the preparation of ovalbumin.

[0019] The fifth object of the present invention is to provide a method for preparing ovalbumin. Add the above-mentioned Saccharomyces cerevisiae or the above-mentioned microbial preparation to the fermentation system, purify the fermentation product to obtain ovalbumin.

[0020] Furthermore, the fermentation substrate is glucose.

[0021] Furthermore, the inducer is galactose.

[0022] Furthermore, streak culture the above-mentioned Saccharomyces cerevisiae on YPD medium to obtain single colonies, pick the single colonies into the seed liquid medium to prepare a seed liquid, transfer the seed liquid to YPD medium, and add 2% galactose at 24 hours. Purify the fermentation supernatant using a His-Binding-resin purification column to obtain the product ovalbumin.

[0023] Advantages of the present invention:

[0024] Based on the endoplasmic reticulum remodeling strategy, the present invention improves the expression level of molecular chaperones that promote protein folding, increases the volume of the endoplasmic reticulum lumen, expands the endoplasmic reticulum, increases the protein synthesis ability of Saccharomyces cerevisiae, and promotes the synthesis of ovalbumin. The extracellular secretion amount of ovalbumin of the constructed Saccharomyces cerevisiae reaches 6.2 mg / L in 48 hours, and this endoplasmic reticulum-optimized strain has generality in the expression of target proteins, and can also achieve efficient production of other target proteins besides ovalbumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to the specific embodiments of the present invention in combination with the drawings, where

[0026] Figure 1 is a schematic diagram of the remodeling idea of the present invention;

[0027] Figure 2 Corresponding to Example 1 of the present invention, where A is a schematic diagram of the construction of a signal peptide screening plasmid, B is the result of signal peptide screening, and C is the ovalbumin yield of the recombinant strain WT-INU1-OVA (corresponding plasmid is pESC-P GAL1 -INU1-OVA);

[0028] Figure 3 Corresponding to Example 2 of the present invention, where A is a schematic diagram of the characterization of the unfolded protein response in the endoplasmic reticulum of the recombinant strain using the mScarlet-I fluorescent protein, B is the characterization result using the P HAC1 promoter, and C is the characterization result using the P ERO1 promoter;

[0029] Figure 4 Corresponding to Example 3 of the present invention, where A is a graph of the ovalbumin yields of the recombinant strains SK1-SK3 and SP1-SP3, B is the ovalbumin yield and strain growth of the recombinant strain SA1, and C is the characterization result of the unfolded protein response in the recombinant strain SA1;

[0030] Figure 5 Corresponding to Example 4 of the present invention, where A is a schematic diagram of the functions of transcription factors INO2 and INO4, B is the ovalbumin yields of the recombinant strains SI1, SN1-SN3, and SO1-SO3, C is the ovalbumin yield and growth of the recombinant strain SA2, and D is the characterization results of the unfolded protein response in the recombinant strains SI1 and SA2;

[0031] Figure 6Corresponding to Example 5 of the present invention, where A is the ovalbumin production and strain growth of recombinant strains SA3 and SA4, and B is the result of the unfolded protein response characterization in recombinant strain SA4. Detailed implementation manners

[0032] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited are not intended to limit the present invention.

[0033] Flask fermentation for producing ovalbumin:

[0034] (1) Streak the recombinant strains capable of producing ovalbumin prepared in the following examples on YPD medium to obtain single colonies. First, pick single colonies into 5 mL of seed medium and culture at 30 °C and 220 rpm for 16 hours to prepare the seed solution.

[0035] (2) Transfer 5 mL of the seed solution prepared in step (1) to 45 mL of YPD medium and ferment at 30 °C and 220 rpm for 72 hours. And at 24 hours, add 2% galactose. Purify the 72-hour fermentation supernatant using a His-Binding-resin purification column, and detect the OVA content after SDS-PAGE.

[0036] Example 1: Screening of signal peptides

[0037] The ovalbumin (OVA) gene (NCBI accession number NP_990483.2) was codon-optimized and synthesized by Nanjing Genscript Biotech Co., Ltd. (Nanjing, China), and the nucleotide sequence is shown in SEQ ID NO.1. In order to screen suitable signal peptides for extracellular secretion of OVA, OVA plasmids with different signal peptides were constructed respectively. The ovalbumin gene was cloned into plasmid pESC-URA, and the signal peptides were inserted upstream of the N-terminal sequence of ovalbumin. Details of the signal peptides, signal peptide sequences and constructed plasmids are shown in the following table.

[0038] Table 1 Signal peptides

[0039]

[0040] The constructed signal peptide plasmids were transformed into the parental strain Saccharomyces cerevisiae CEN.PK2-1C to obtain a series of corresponding recombinant strains ( Figure 2 A). According to SDS-PAGE and Western blot analysis, under the guidance of the INU1 signal peptide, the OVA content in the supernatant was the highest ( Figure 2B). When the signal peptide is INU1, the OVA production in the supernatant of the fermentation broth is 3.4 mg / L( Figure 2 C). This strain was named WT-INU1-OVA.

[0041] Example 2: Characterization of the unfolded protein response UPR

[0042] Select the promoters P HAC1 and P ERO1 (the nucleotide sequences are shown in SEQ ID NO.8 - 9 respectively), connect them with the reporter fluorescent protein mScarlet-I (the nucleotide sequence is shown in SEQ ID NO.10), and integrate them into the genome of the recombinant strain obtained in Example 1 to characterize whether UPR occurs in the endoplasmic reticulum of the strain expressing heterologous OVA Figure 3 A). The inducer galactose was added at 0 h, and the UPR of the endoplasmic reticulum was monitored at 24 h. At 48 h, compared with the strain CEN.PK2-1C expressing the pESC-URA empty plasmid, the fluorescence value of the recombinant strain expressing OVA increased significantly, and the UPR was activated 1.8-fold (P HAC1 ) and 3.5-fold (P ERO1 ), respectively, indicating that under the influence of heterologous expression of ovalbumin in the recombinant strain, a certain amount of misfolded or unfolded proteins accumulated in the endoplasmic reticulum Figure 3 B and Figure 3 C).

[0043] Example 3: Using molecular chaperones to promote endoplasmic reticulum folding

[0044] Insert the kar2 gene expression cassette controlled by different promoters at the 208a site of the genome of the recombinant Saccharomyces cerevisiae WT-INU1-OVA, and insert the pdi gene expression cassette controlled by different promoters at the 720a site.

[0045] Select three promoters P TDH2 、P GAP and P PGK1 (the nucleotide sequences are shown in SEQ ID NO.11 - 13), overexpress the molecular chaperone Kar2 and the PDI coding genes (the nucleotide sequences are shown in SEQ ID NO.14 - 15) respectively to support the efficient folding of heterologous proteins. All expression cassettes are designed to be integrated as an additional copy in the genome, while the endogenous expression cassette remains unchanged. The results show that among the 6 strains co-constructed with molecular chaperones controlled by three different strengths of promoters, 5 strains showed favorable OVA secretion. Molecular chaperones controlled by three different strengths of promoters were all beneficial to OVA secretion, and there was no significant difference. Strain SK2 (P GAPThe OVA production of ( -kar2) was increased to 3.8 mg / L, while that of the SP3 strain (P PGK1 -pdi) was increased to 4.5 mg / L( Figure 4 A). Using the pACYCDuet-1 plasmid as the backbone, P GAP -kar2 and P PGK1 -pdi were co-expressed in the recombinant Saccharomyces cerevisiae WT-INU1-OVA to obtain the SA1 strain. The OVA production of the SA1 strain was increased to 5.1 mg / L, and a slight improvement in strain growth could be observed( Figure 4 B). However, compared with the control strain, the UPR of the SA1 strain was increased instead, activating 1.3-fold (P HAC1 ) and 1.4-fold (P ERO1 )( Figure 4 C). This may be because the additional proteins in the endoplasmic reticulum occupy the narrow space of the endoplasmic reticulum lumen, resulting in increased endoplasmic reticulum stress.

[0046] Example 4: Expanding the endoplasmic reticulum membrane of recombinant strains

[0047] Using the CRISPR gene editing system, with the recombinant Saccharomyces cerevisiae WT-INU1-OVA as the starting strain, the gene opi1 (nucleotide sequence as shown in SEQ ID NO.16) was knocked out to obtain the recombinant strain SI1. The genes ino2 and ino4 (nucleotide sequences as shown in SEQ ID NOs.17-18 respectively) were overexpressed using promoters with different strengths (P TDH2 , P GAP and P PGK1 ) to obtain the SN1-SN3 and SO1-SO3 strains respectively. The results showed that the OVA secretion of the SI1 strain was increased to 5.5 mg / L. Among the strains overexpressing the INO2-encoding gene, the secretion of SN3 was the highest, reaching 5.3 mg / L. Among the engineered strains with increased expression levels of the INO4-encoding gene, the secretion of SO2 reached 4.7 mg / L, while the SO3 strain overexpressing the INO4-encoding gene using the strong promoter P PGK1 was not conducive to the improvement of protein secretion( Figure 5 B). Further combining the modifications of the expression levels of these three genes, the SA2 strain was obtained. The extracellular secretion of OVA was approximately 5.2 mg / L, and the growth decreased slightly( Figure 5 C). Monitoring the unfolded protein response (UPR) of the SI1 and SA2 strains with significantly increased protein secretion, at 48 hours, the UPR intensity of SI1 was 0.9-fold (P HAC1 ) and 0.7-fold (P ERO1 ) of the control strain respectively, while the UPR intensity of SA2 did not change significantly( Figure 5 D).

[0048] The expansion of the endoplasmic reticulum membrane in SI1 strain alleviates the unfolded protein response, probably because the enlarged endoplasmic reticulum lumen reduces the concentration of incompletely folded intermediates, decreases the formation of aggregates, and provides more time for protein folding. In addition, the expanded endoplasmic reticulum can accommodate more misfolded proteins. Although the endoplasmic reticulum membrane of SA2 strain is expanded relative to the control strain, its unfolded protein response is not significantly reduced, which may be due to the over-engineering of the transcription factor regulating lipid synthesis genes.

[0049] Example 5: Combining two strategies to improve the secretion of ovalbumin

[0050] By combining chaperone co-expression and endoplasmic reticulum membrane expansion, further exploration was carried out on how to improve the secretion amount of ovalbumin. Among the strains with modified endoplasmic reticulum membranes, the protein secretion amount of SI1 strain with only the opi1 gene knocked out was higher than that of SA2. Therefore, the PDI and Kar2 chaperones were co-expressed on SI1 and SA2 strains to obtain SA3 and SA4 strains. When the fermentation was carried out for 48 hours, the extracellular secretion amounts of ovalbumin in SA3 and SA4 strains increased to 5.8 mg / L and 6.2 mg / L ( Figure 6 A). The activation intensity of the unfolded protein response in SA4 strain was 1.4 times that of the control strain (P HAC1 ) and 1.2 times (P ERO1 )( Figure 6 B). Compared with the engineered strains (SA1 and SA2), SA4 strain increased the secretion of ovalbumin, which may be because increasing only the size of the endoplasmic reticulum can provide more space for heterologous proteins. However, when the level of chaperones in the endoplasmic reticulum lumen increases simultaneously, the protein folding environment is more favorable.

[0051] Table 2 Strains and modifications involved in Examples 1-5

[0052]

[0053]

[0054] Obviously, the above examples are only for illustration and are not limitations on the implementation methods. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A Saccharomyces cerevisiae for improving ovalbumin production by modifying the endoplasmic reticulum, characterized in that: The Saccharomyces cerevisiae heterologously expresses the ovalbumin-encoding gene using the INU1 signal peptide, and the gene opi1 is knocked out, and the encoding genes of the molecular chaperones Kar2 and PDI are overexpressed; among them, the starting strain is Saccharomyces cerevisiae CEN.PK2-1C; The nucleotide sequence of the ovalbumin-encoding gene is shown in SEQ ID NO.1; The nucleotide sequence of the chaperone Kar2-encoding gene is shown in SEQ ID NO.14; The nucleotide sequence of the chaperone PDI-encoding gene is shown in SEQ ID NO.

15.

2. The Saccharomyces cerevisiae according to claim 1, wherein: The Saccharomyces cerevisiae overexpresses the encoding genes of transcription factors INO2 and INO4; Among them, the nucleotide sequence of the transcription factor INO2-encoding gene is shown in SEQ ID NO.17; The nucleotide sequence of the transcription factor INO4-encoding gene is shown in SEQ ID NO.

18.

3. The Saccharomyces cerevisiae according to claim 2, characterized in that: Use P GAP promoter to express Kar2-encoding gene and INO4-encoding gene, use P PGK1 promoter to express PDI-encoding gene and INO2-encoding gene.

4. A microbial preparation comprising the Saccharomyces cerevisiae according to any one of claims 1-3.

5. The construction method of Saccharomyces cerevisiae according to any one of claims 1-3, characterized in that, Comprising the following steps: S1. Heterologously express the ovalbumin-encoding gene in the starting strain Saccharomyces cerevisiae, and insert the INU1 signal peptide upstream of the N-terminal sequence of ovalbumin; S2. At the 208a locus in the genome of the strain obtained in S1, use the P GAP promoter to express the gene encoding the molecular chaperone Kar2, and at the 720a locus use the P PGK1 promoter to express the gene encoding the molecular chaperone PDI; S3. Knock out the opi1 gene in the genome of the strain obtained by knocking out S2; S4. At the 1309a locus in the genome of the strain obtained in S3, use the P GAP promoter to express the transcription factor INO2-encoding gene, and at the 416d locus, use the P PGK1 promoter to express the transcription factor INO4-encoding gene.

6. Use of the Saccharomyces cerevisiae according to any one of claims 1-3 or the microbial preparation according to claim 4 in the preparation of ovalbumin.

7. A method for preparing ovalbumin, characterized in that: Add the Saccharomyces cerevisiae according to any one of claims 1-3 or the microbial preparation according to claim 4 to the fermentation system, and purify the fermentation product to obtain the ovalbumin.

8. The method according to claim 7, wherein: The fermentation substrate is glucose.

Images