Saccharomyces cerevisiae engineering bacteria constructed based on CRISPR-Cas9 system and used for producing radix pseudostellariae cyclic peptide HB and construction method
By constructing engineered Saccharomyces cerevisiae using the CRISPR-Cas9 system, the problems of complex and costly methods for obtaining Codonopsis pilosula cyclic peptide HB have been solved, enabling efficient and stable production of Codonopsis pilosula cyclic peptide HB in Saccharomyces cerevisiae, which is applicable to the food and biopharmaceutical industries.
Patent Information
- Application Number
- CN202511227711.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-29
- Publication Date
- 2026-01-09
AI Technical Summary
The current methods for obtaining Codonopsis pilosula cyclic peptide HB mainly rely on chemical extraction, which suffers from low content, complex processes, and high costs. There is an urgent need for an efficient biological preparation method.
A CRISPR-Cas9 system was used to construct an engineered Saccharomyces cerevisiae strain. Through gene editing, the cyclopeptide HB was expressed in the Saccharomyces cerevisiae. Utilizing the gene editing capability of the CRISPR-Cas9 system, an engineered Saccharomyces cerevisiae strain containing the pESC-URA-prePhHB-PhPOP2 plasmid, the gRNA-1 plasmid, and Donor DNA was constructed to achieve efficient production of the cyclopeptide HB.
Stable production of Codonopsis pilosula cyclic peptide HB in Saccharomyces cerevisiae has been achieved, which is suitable for large-scale production. The production process is environmentally friendly and harmless to humans and the environment, and is applicable to the food and biopharmaceutical industries.
Smart Images
Figure CN121294491A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to a brewer's yeast strain for producing heterophyllin B (heterophyllin B) based on the CRISPR-Cas9 system, its construction method, and its application. Background Technology
[0002] The CRISPR system has become an essential tool in almost all aspects of synthetic biology and metabolic engineering, including gene editing, heterologous expression, transcriptional regulation, and genome-wide screening. The CRISPR-Cas9 gene editing system was used to edit genes in Saccharomyces cerevisiae. This system comprises three components: the Cas9 protein, a gRNA plasmid, and donor DNA.
[0003] The Cas9 protein is a key component of the CRISPR-Cas9 gene editing system, which originated from the immune mechanisms of bacteria and archaea to defend against invading viruses. Cas9 is a nuclease capable of cleaving double-stranded DNA. Cas9 activity depends on its paired single-stranded guide RNA (gRNA), which directs Cas9 to a specific DNA sequence. Donor DNA refers to a specific DNA sequence used for homologous recombination (HR) or other gene repair mechanisms. When using a system like CRISPR-Cas for precise gene editing, donor DNA serves as a template, providing the necessary genetic information to guide and facilitate the cell's DNA repair process, thereby achieving the correction or insertion of a specific gene.
[0004] The traditional Chinese medicine *Pseudostellaria heterophylla* (Miq.) Pax exPax et Hoffm., a plant in the Caryophyllaceae family, is known for its dried tuberous root. It is believed to invigorate qi and strengthen the spleen, promote fluid production and moisten the lungs. In traditional Chinese medicine, it is commonly used for spleen and lung deficiency, qi and yin deficiency, spontaneous sweating, and thirst. The main chemical components of *Pseudostellaria heterophylla* include cyclic peptides, saponins, and polysaccharides. Modern pharmacological studies have shown that the cyclic peptide *Hyperophyllin B* exhibits low cytotoxicity, tyrosinase inhibitory activity, anti-melanin production, and improvement of inflammation and oxidative stress, demonstrating good biological activity and medicinal development value. Compared to linear peptides, plant cyclic peptides are more stable in their skeletal structure, exhibiting superior heat resistance, chemical degradation resistance, and enzymatic resistance. In recent years, they have received considerable attention in the pharmaceutical field as drug skeletons. Codonopsis pilosula cyclic peptide HB was once listed as an indicator component of Codonopsis pilosula in the 2010 edition of the Chinese Pharmacopoeia. The main method of obtaining it is through chemical extraction and separation. Due to its low content, the extraction process is complex and costly. Therefore, there is an urgent need for a method to construct a Saccharomyces cerevisiae engineered strain for producing Codonopsis pilosula cyclic peptide HB based on the CRISPR-Cas9 system. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Saccharomyces cerevisiae engineered strain for producing Codonopsis pilosula cyclic peptide HB based on the CRISPR-Cas9 system.
[0006] The second objective of this invention is to provide a method for constructing an engineered Saccharomyces cerevisiae strain that produces Codonopsis pilosula cyclic peptide HB based on a CRISPR-Cas9 system.
[0007] The third objective of this invention is to provide an application of a CRISPR-Cas9 system-based engineered Saccharomyces cerevisiae strain for producing Codonopsis pilosula cyclic peptide HB.
[0008] The technical solution of this invention is summarized as follows:
[0009] A method for constructing an engineered Saccharomyces cerevisiae strain for producing Codonopsis pilosula cyclic peptide HB based on the CRISPR-Cas9 system includes the following steps:
[0010] 1) Construction of pESC-URA-prePhHB-PhPOP2 plasmid: The prePhHB gene was synthesized into the pESC-URA vector to obtain the pESC-URA-prePhHB vector; the nucleotide sequence of the prePhHB gene is shown in SEQ ID NO.1; the PhPOP2 gene was synthesized into the pESC-HIS vector to obtain the pESC-HIS-PhPOP2 vector; the nucleotide sequence of the PhPOP2 gene is shown in SEQ ID NO.2; the pESC-URA-prePhHB vector was linearized by double digestion at both BamHI and XhoI restriction sites; the linearized pESC-URA-prePhHB vector was obtained; using the pESC-HIS-PhPOP2 vector as a template, and with PhPOP2-F and PhPOP2-R as primers, the PhPOP2 gene fragment was obtained; the nucleotide sequences of PhPOP2-F and PhPOP2-R are shown in SEQ ID NO.1. As shown in NO.3 and SEQ ID NO.4, the PhPOP2 gene fragment and the linearized pESC-URA-prePhHB vector were seamlessly cloned to obtain the pESC-URA-prePhHB-PhPOP2 plasmid.
[0011] 2) Construction of gRNA-1 plasmid: Using the gRNA plasmid as a template, and ROX1-gRNA-73.4-F and ROX1-gRNA-73.4-R as primers, reverse PCR was performed to eliminate the original plasmid template. E. coli transformation was then performed to obtain the gRNA-1 plasmid. The nucleotide sequence of the gRNA plasmid is shown in SEQ ID NO.5, the nucleotide sequence of ROX1-gRNA-73.4-F is shown in SEQ ID NO.6, and the nucleotide sequence of ROX1-gRNA-73.4-R is shown in SEQ ID NO.7.
[0012] 3) Construction of Donor DNA: Using a single colony of *Saccharomyces cerevisiae* as a template, the upstream homologous arm ROX-455 was amplified using primers ROX-repeat500F-F and ROX-repeat500F-R. The nucleotide sequence of ROX-repeat500F-F is shown in SEQ ID NO. 8; the nucleotide sequence of ROX-repeat500F-R is shown in SEQ ID NO. 9. Using a single colony of *Saccharomyces cerevisiae* as a template, the downstream homologous arm ROX-467 was amplified using primers ROX-repeat500R-F and ROX-repeat500R-R. The nucleotide sequence of ROX-repeat500R-F is shown in SEQ ID NO. 10; the nucleotide sequence of ROX-repeat500R-R is shown in SEQ ID NO. 9. As shown in NO.11, fragment 1 was amplified using the pESC-URA-prePhHB-PhPOP2 plasmid as a template and ROX-repeat-F and ROX-repeat-R as primers. The nucleotide sequence of ROX-repeat-F is shown in SEQ ID NO.12, and the nucleotide sequence of ROX-repeat-R is shown in SEQ ID NO.13. Using the upstream homologous arm ROX-455, fragment 1, and the downstream homologous arm ROX-467 as templates and ROX-repeat500F-F and ROX-repeat500R-R as primers, overlap extension PCR ligation was performed to obtain Donor DNA.
[0013] 4) The cas9 plasmid, gRNA-1 plasmid and Donor DNA were co-transformed into Saccharomyces cerevisiae. Transformants were selected for colony PCR verification to obtain an engineered Saccharomyces cerevisiae strain that produces Codonopsis pilosula cyclic peptide HB based on the CRISPR-Cas9 system. The nucleotide sequence of the cas9 plasmid is shown in SEQ ID NO.14.
[0014] The above-described method was used to construct engineered Saccharomyces cerevisiae.
[0015] The application of the above-mentioned engineered Saccharomyces cerevisiae in the fermentation production of Codonopsis pilosula cyclic peptide HB.
[0016] Advantages of this invention:
[0017] The method of this invention constructs a CRISPR-Cas9-based engineered Saccharomyces cerevisiae strain that produces Codonopsis pilosula cyclic peptide HB. This strain is based on a recombinant expression system of Saccharomyces cerevisiae. Codonopsis pilosula cyclic peptide HB is generated in the engineered Saccharomyces cerevisiae strain. The gene-edited yeast can be used to construct an industrial strain that can produce antibiotic-free fermentation with long-term stable production, which is suitable for large-scale production. The production process is environmentally friendly, and Saccharomyces cerevisiae is harmless to humans and the environment. It can also be used in the food and biopharmaceutical industries. Attached Figure Description
[0018] Figure 1 Schematic diagram of pESC-URA-prePhHB-PhPOP2 plasmid;
[0019] Figure 2 For transformant validation, the agarose gel electrophoresis results are shown (M: 5000bp DNA Marker; 1-10: transformants);
[0020] Figure 3 This is a diagram showing the construction of the gRNA-1 plasmid;
[0021] Figure 4 A diagram of the Donor DNA construction;
[0022] Figure 5 Agarose gel electrophoresis diagram for constructing Donor DNA (M: 5000bp DNA Marker; 4, 5: Construction of Donor DNA);
[0023] Figure 6 The agarose gel electrophoresis results after co-transformation are shown in the image (M: 2000bp DNA Marker; 1-6: transformants). Detailed Implementation
[0024] The present invention will be further described below with reference to specific embodiments in order to better understand the technical solution.
[0025] Example 1: Construction of pSEC-URA-prePhHB-PhPOP2 plasmid:
[0026] The prePhHB gene (SEQ ID NO.1) was synthesized into the pESC-URA vector to obtain the pESC-URA-prePhHB vector; the PhPOP2 gene (SEQ ID NO.2) was synthesized into the pESC-HIS vector to obtain the pESC-HIS-PhPOP2 vector; the pESC-URA-prePhHB vector was linearized by double digestion at both BamHI and XhoI restriction sites; the linearized pESC-URA-prePhHB vector was obtained; using the pESC-HIS-PhPOP2 vector as a template, the PhPOP2 gene fragment was obtained using PhPOP2-F (SEQ ID NO.3) and PhPOP2-R (SEQ ID NO.4) as primers; the PhPOP2 gene fragment and the linearized pESC-URA-prePhHB vector were seamlessly cloned to obtain the pESC-URA-prePhHB-PhPOP2 plasmid;
[0027] Extract the plasmid and send it for analysis. If the sequence matches, the pESC-URA-prePhHB-PhPOP2 plasmid has been successfully constructed. See pESC-URA-prePhHB-PhPOP2 plasmid [link missing]. Figure 1 The pESC-URA-prePhHB-PhPOP2 plasmid was successfully constructed, and the transformants were validated. Agarose gel electrophoresis results are as follows: Figure 2 As shown.
[0028] Example 2: Construction of gRNA-1 plasmid
[0029] The ROX1 genome targeting sequence was selected using the website http: / / www.rgenome.net / cas-designer / . The sequence of out-of-frame socre73.4 was selected to obtain ROX1-gRNA-73.4-F and ROX1-gRNA-73.4-R. Using the gRNA plasmid (SEQ ID NO.5) as a template, and using ROX1-gRNA-73.4-F (SEQ ID NO.6) and ROX1-gRNA-73.4-R (SEQ ID NO.7) as primers, reverse PCR was performed to eliminate the original plasmid template. The PCR product was recovered according to the kit instructions and transformed into E. coli to obtain the gRNA-1 plasmid.
[0030] The build process is as follows Figure 3 As shown.
[0031] The PCR reaction procedure is shown in Table 1, and the PCR system is shown in Table 2.
[0032] Table 1: PCR reaction procedure
[0033]
[0034] Table 2: PCR reaction system
[0035]
[0036] Conversion method:
[0037] Add 5 μL of gRNA-1 plasmid to each 50 μL tube of E. coli DH5α competent cells (without removing them from the ice surface), gently mix with a pipette tip, incubate on ice for 30 min, heat shock at 42.5℃ for 90 s, and incubate on ice for 2 min. Add 400 μL of LB medium and activate on a shaker at 37℃ for 1 hour. Spread 100 μL of the bacterial culture onto an AmpR selection plate and incubate upside down at 37℃ overnight. Extract plasmid from transformants and sequence to verify; correct sequences are identified as gRNA-1 plasmids.
[0038] Example 3: Construction of Donor DNA
[0039] Using a single colony of Saccharomyces cerevisiae BY4741 (commercial) as a template, the upstream homologous arm ROX-455 was amplified using ROX-repeat500F-F (SEQ ID NO.8) and ROX-repeat500F-R (SEQ ID NO.9) as primers;
[0040] Using a single colony of Saccharomyces cerevisiae BY4741 (the strain is a commercial product) as a template, the downstream homologous arm ROX-467 was amplified using ROX-repeat500R-F (SEQ ID NO.10) and ROX-repeat500R-R (SEQ ID NO.11) as primers;
[0041] Fragment 1 was amplified using the pESC-URA-prePhHB-PhPOP2 plasmid as a template and ROX-repeat-F (SEQ ID NO.12) and ROX-repeat-R (SEQ ID NO.13) as primers;
[0042] Using purified upstream homologous arm ROX-455, fragment 1, and downstream homologous arm ROX-467 as templates, and ROX-repeat500F-F and ROX-repeat500R-R as primers, overlap extension PCR ligation was performed to obtain Donor DNA.
[0043] The PCR reaction system is shown in Table 3. The construction of the donor DNA is as follows: Figure 4 As shown, the agarose gel electrophoresis image is as follows: Figure 5 As shown in the figure. The primer sequences are shown in Table 4.
[0044] Table 3: PCR reaction system
[0045]
[0046] The ligation product was validated by PCR and used in subsequent experiments.
[0047] Example 4: Co-conversion
[0048] The Cas9 plasmid (SEQ ID NO.14), gRNA-1 plasmid, and Donor DNA were co-transformed into *Saccharomyces cerevisiae* BY4741. Transformants were selected for colony PCR verification, yielding an engineered *Saccharomyces cerevisiae* strain capable of producing *Codonopsis pilosula* cyclic peptide HB based on the CRISPR-Cas9 system. The specific transformation method is as follows:
[0049] 1) Take a sterile 1.5ml EP tube and add 1μg of Cas9 plasmid, 1μg of gRNA-1 plasmid, 2μg of Donor DNA, 10μL of Carrier DNA, 100μL of Saccharomyces cerevisiae BY4741 competent cells, and 500μL of PEG / LIAC yeast transformation medium in sequence. Mix well by pipetting. Incubate at 30℃ for 30min, and invert 6-8 times after 15min.
[0050] 2) Transfer the bacteria obtained in step 1) to a 42°C water bath for 15 minutes, and invert it 6-8 times at 7.5 minutes.
[0051] 3) Centrifuge at 5000 rpm for 40 s, discard the supernatant, resuspend in 400 μL of ddH2O, centrifuge for 30 s, and discard the supernatant.
[0052] 4) Resuspend in 50 μL of ddH2O, plate, and incubate at 28°C for 2-3 days. Select transformants for validation.
[0053] Pick colonies and add them to 20 μL of 20 mM NaOH aqueous solution. Incubate at 95 °C for 5 min to use as a template for PCR reaction.
[0054] After co-transformation, the agarose gel electrophoresis results are as follows: Figure 6 As shown.
[0055] Colony PCR verification, primers are as follows:
[0056] Gene editing validation -F (SEQ ID NO.15)
[0057] Gene editing validation - R: (SEQ ID NO.16)
[0058] The PCR reaction system is shown in Table 4.
[0059]
[0060] The successfully verified single colony is the Saccharomyces cerevisiae engineered strain that produces Codonopsis pilosula cyclic peptide HB based on the CRISPR-Cas9 system.
[0061] Example 5: Fermentation
[0062] A single colony of the Saccharomyces cerevisiae engineered strain that produces Codonopsis pilosula cyclic peptide HB based on the CRISPR-Cas9 system constructed in Example 4 was inoculated into 5mLYPDA medium and cultured to obtain seed culture.
[0063] 5 ml of seed culture was inoculated into 1 L of YPDA medium and fermented at 30 °C and 150 rpm until OD600 = 0.8 (approximately 24-36 h). The cells were collected by centrifugation and transferred to induction medium. The cells were induced at 30 °C and 150 rpm (or 200 rpm) for 36 h to obtain Codonopsis pilosula cyclic peptide HB, with a yield of 1.01 mg / L.
[0064] The YPDA culture medium formula is: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, 0.03 g / L adenine sulfate; the balance is distilled water.
[0065] The induction medium consisted of 20 g / L peptone, 10 g / L yeast extract, 20 g / L galactose, and 0.03 g / L adenine sulfate; the remainder was distilled water.
Claims
1. A method for constructing an engineered Saccharomyces cerevisiae strain for producing Codonopsis pilosula cyclic peptide HB based on a CRISPR-Cas9 system, characterized in that... Includes the following steps: 1) Construction of pESC-URA-prePhHB-PhPOP2 plasmid: The prePhHB gene was synthesized into the pESC-URA vector to obtain the pESC-URA-prePhHB vector; the nucleotide sequence of the prePhHB gene is shown in SEQ ID NO.1; the PhPOP2 gene was synthesized into the pESC-HIS vector to obtain the pESC-HIS-PhPOP2 vector; the nucleotide sequence of the PhPOP2 gene is shown in SEQ ID NO.2; the pESC-URA-prePhHB vector was linearized by double digestion at both BamHI and XhoI restriction sites. A linearized pESC-URA-prePhHB vector was obtained; using the pESC-HIS-PhPOP2 vector as a template and PhPOP2-F and PhPOP2-R as primers, the PhPOP2 gene fragment was obtained; the nucleotide sequences of PhPOP2-F and PhPOP2-R are shown in SEQ ID NO.3 and SEQ ID NO.4; the PhPOP2 gene fragment and the linearized pESC-URA-prePhHB vector were seamlessly cloned to obtain the pESC-URA-prePhHB-PhPOP2 plasmid; 2) Construction of gRNA-1 plasmid: Using the gRNA plasmid as a template, and ROX1-gRNA-73.4-F and ROX1-gRNA-73.4-R as primers, reverse PCR was performed to eliminate the original plasmid template. E. coli transformation was then performed to obtain the gRNA-1 plasmid. The nucleotide sequence of the gRNA plasmid is shown in SEQ ID NO.5, the nucleotide sequence of ROX1-gRNA-73.4-F is shown in SEQ ID NO.6, and the nucleotide sequence of ROX1-gRNA-73.4-R is shown in SEQ ID NO.
7. 3) Construction of Donor DNA: Using a single colony of *Saccharomyces cerevisiae* as a template, the upstream homologous arm ROX-455 was amplified using primers ROX-repeat500F-F and ROX-repeat500F-R. The nucleotide sequence of ROX-repeat500F-F is shown in SEQ ID NO. 8; the nucleotide sequence of ROX-repeat500F-R is shown in SEQ ID NO.
9. Using a single colony of *Saccharomyces cerevisiae* as a template, the downstream homologous arm ROX-467 was amplified using primers ROX-repeat500R-F and ROX-repeat500R-R. The nucleotide sequence of ROX-repeat500R-F is shown in SEQ ID NO. 10; the nucleotide sequence of ROX-repeat500R-R is shown in SEQ ID NO.
9. As shown in NO.11, fragment 1 was amplified using the pESC-URA-prePhHB-PhPOP2 plasmid as a template and ROX-repeat-F and ROX-repeat-R as primers. The nucleotide sequence of ROX-repeat-F is shown in SEQ ID NO.12, and the nucleotide sequence of ROX-repeat-R is shown in SEQ ID NO.
13. Using the upstream homologous arm ROX-455, fragment 1, and the downstream homologous arm ROX-467 as templates and ROX-repeat500F-F and ROX-repeat500R-R as primers, overlap extension PCR ligation was performed to obtain Donor DNA. 4) The cas9 plasmid, gRNA-1 plasmid and Donor DNA were co-transformed into Saccharomyces cerevisiae. Transformants were selected for colony PCR verification to obtain an engineered Saccharomyces cerevisiae strain that produces Codonopsis pilosula cyclic peptide HB based on the CRISPR-Cas9 system. The nucleotide sequence of the cas9 plasmid is shown in SEQ ID NO.
14.
2. The engineered Saccharomyces cerevisiae obtained by the construction method of claim 1.
3. The application of the engineered Saccharomyces cerevisiae of claim 2 in the fermentation production of Codonopsis pilosula cyclic peptide HB.