Saccharomyces cerevisiae engineering strain capable of controllably releasing astaxanthin in self-splitting manner and construction and application of saccharomyces cerevisiae engineering strain

Abstract

The invention provides a saccharomyces cerevisiae engineering strain HYB05 capable of releasing astaxanthin in a controllable self-splitting manner. The saccharomyces cerevisiae engineering strain HYB05 is constructed by taking a basic strain for producing astaxanthin as a starting strain, introducing a coding sequence comprising cell wall lyase Lyc, a coding sequence of a signal peptide mutant F8A and a recombinant nucleic acid construct for expressing a regulatory element, and knocking out an SSD1 gene to relieve a translation inhibition effect. The cell wall lyase Lyc is found and disclosed by the inventor for the first time, and the amino acid sequence is shown as SEQ ID NO: 6; the amino acid sequence of the signal peptide mutant F8A is as shown in SEQ ID NO: 8. According to the engineering strain HYB05, by introducing the recombinant nucleic acid construct, the activation of the catalytic activity of cell wall lyase under specific conditions is realized, so that the cell wall of the saccharomyces cerevisiae is broken, and the astaxanthin synthesized by the saccharomyces cerevisiae is released. Compared with the prior art, the astaxanthin is produced through fermentation of the strain, the extraction steps are simplified, extraction of the astaxanthin in the yeast is achieved to the maximum extent, the yield of the astaxanthin is remarkably increased, and the technical effect is remarkable.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and relates to astaxanthin-producing Saccharomyces cerevisiae strains, specifically to an engineered Saccharomyces cerevisiae strain that can controllably release astaxanthin through self-lysis and its construction and application. Background Technology

[0002] Astaxanthin is a ketocarotenoid with a molecular skeleton composed of eight isoprene units linked together, containing 13 conjugated double bonds. It has a β-ionone ring and a hydroxyl and ketone group at each end, giving it a dark reddish-brown crystalline appearance. It has a high melting point, low polarity, and is almost insoluble in water, but readily soluble in oils and most organic solvents. This molecular structure endows it with transmembrane capabilities, allowing it to simultaneously embed in the hydrophobic and hydrophilic interfaces of cell membranes, comprehensively scavenging reactive oxygen species (ROS) such as singlet oxygen and hydroxyl radicals. Its antioxidant activity is 550 times that of vitamin E and more than 50 times that of β-carotene, earning it the title of a "super antioxidant." Studies have shown that astaxanthin possesses various physiological functions, including antioxidant, anti-aging, anti-tumor, immune-enhancing, vision-protecting, and cardiovascular-protective effects. It is currently widely used in medicine, health care, food preservation, and cosmetics.

[0003] Currently, common methods for synthesizing astaxanthin include algal culture extraction, chemical synthesis, and microbial fermentation. Among these, astaxanthin obtained through microalgal culture extraction is of the levorotatory configuration, exhibiting high activity. However, the long growth cycle of algae, the need for light, low biomass, and difficulties in cell wall disruption lead to high production costs and hinder large-scale promotion. Astaxanthin produced through chemical synthesis is primarily the cis isomer, with a configuration different from natural astaxanthin. This makes it difficult for the human body to efficiently absorb and utilize it, and it also results in low safety and antioxidant activity. Furthermore, the synthesis process is complex, failing to meet consumer demand for natural and safe products. Compared to the first two methods, microbial fermentation for astaxanthin production offers advantages such as a short production cycle, independence from climate and seasons, ease of high-density industrial cultivation, and the production of a natural configuration. It is considered the core technological route for the next generation of natural levorotatory astaxanthin.

[0004] Among many microbial hosts, Saccharomyces cerevisiae is recognized as one of the ideal substrates for building astaxanthin cell factories. The main reasons include: (1) Saccharomyces cerevisiae is a recognized GRAS microorganism, with no endotoxins or risk of phage contamination, and its products can be directly used in dietary supplements, cosmetics and high-end feeds. (2) Saccharomyces cerevisiae has a clear genetic background, well-developed genetic engineering tools, and high growth efficiency. The biosynthesis of astaxanthin begins with β-carotene and requires the catalysis of β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ) in sequence. In the existing technology, the synthesis of 3S,3'S-L-astaxanthin can be highly active by introducing heterologous CrtZ and CrtW into Saccharomyces cerevisiae, but the yield of the final product astaxanthin is low. This is mainly because (1) the enzyme activities of these two exogenous enzymes are mismatched, the catalytic efficiency is low, or the substrate channels are blocked, resulting in the accumulation of a large amount of intermediate products. (2) After synthesis in Saccharomyces cerevisiae, astaxanthin is mainly stored in lipid droplets. Existing technologies rely on high-pressure homogenization, repeated freeze-thaw cycles, or extraction with high-concentration organic solvents (such as ethanol and acetone) to disrupt the cell walls. This not only results in high energy consumption (>10 kWh / kg), protein denaturation, and solvent residue, but also low extraction efficiency, further affecting the yield of astaxanthin. Even so, the synthesis of astaxanthin from Saccharomyces cerevisiae remains the most competitive technical route for the industrialization of natural astaxanthin. Therefore, how to increase the yield of astaxanthin in the Saccharomyces cerevisiae synthesis route is an urgent technical problem to be solved.

[0005] It is well known to those skilled in the art that cell wall lysing enzymes are key tool enzymes for the specific degradation of fungal cell walls. Patent application 2020110522573 discloses a "method for extracting astaxanthin from *Phaeophyte rhodotorulata*", which uses a *Bacillus circulans* cell wall lysing enzyme to release astaxanthin from yeast, achieving the goal of maximizing the extraction of astaxanthin from yeast. However, due to the significant differences in the chemical composition and structure of different fungal cell walls, and the highly specific nature of enzyme action, no cell wall lysing enzymes capable of acting on *Saccharomyces cerevisiae* strains have been reported to date. Summary of the Invention

[0006] To address the problems existing in the synthesis of astaxanthin by *Saccharomyces cerevisiae* in current technologies, this invention first provides an engineered *Saccharomyces cerevisiae* strain capable of controllable self-lysis and astaxanthin release. This engineered strain is constructed using a basic astaxanthin-producing strain as the starting strain and incorporates a recombinant nucleic acid construct including the coding sequence of the cell wall lysin Lyc, the coding sequence of the signal peptide mutant F8A, and expression regulatory elements. By introducing the aforementioned recombinant nucleic acid construct, the engineered strain activates the catalytic activity of the cell wall lysin under specific conditions, thereby causing the cell wall of *Saccharomyces cerevisiae* to rupture and release the synthesized astaxanthin. Compared with existing technologies, using this strain for astaxanthin fermentation not only simplifies the extraction steps but also maximizes the extraction of astaxanthin from yeast, significantly increasing astaxanthin yield and demonstrating remarkable technical effectiveness.

[0007] The technical solution of the present invention:

[0008] This invention first provides a cell wall lysin Lyc suitable for *Saccharomyces cerevisiae* strains, the amino acid sequence of which is shown in SEQ ID NO: 6. This cell wall lysin Lyc is a first-time discovery and disclosure by the inventors of this application, and is applicable to *Saccharomyces cerevisiae* strains, filling a technological gap in the prior art. The cell wall lysin Lyc is derived from *Cellulosimicrobium cellulans*. Using a genome integration method, the cell wall lysin Lyc gene can be integrated into the genome of *Saccharomyces cerevisiae*, enabling the release of intracellular astaxanthin into the extracellular space after cell wall rupture, without the need for cell wall disruption extraction; thus overcoming the problems of high energy consumption and low extraction efficiency associated with cell wall disruption extraction.

[0009] Based on this, this application also provides a recombinant nucleic acid construct comprising the coding sequence of the cell wall lysin Lyc as described above, the coding sequence of the signal peptide mutant F8A, and expression regulatory elements. The amino acid sequence of the signal peptide mutant F8A is shown in SEQ ID NO: 8. Introducing the construct into a host cell and expressing it yields a fusion protein of the fused signal peptide and cell wall lysin Lyc. Although temperature-sensitive propeptide sequences (yeast α-factor signal peptide) are known in the prior art, the signal peptide mutant F8A is a first discovery and disclosure by the inventors of this application. The signal peptide is cleaved by the signal peptidase under any temperature conditions; this causes the cell wall lysin in the fusion protein to remain in the cytoplasm and exert catalytic activity, initiating the hydrolysis of cell wall β-1,3-glucan, thus rupturing the cell wall of *Saccharomyces cerevisiae*. This leads to the cessation of cell growth and metabolism. However, the inventors of this application unexpectedly discovered that when the construct described in this application was introduced into a basic strain that produces astaxanthin for fermentation to produce astaxanthin, (1) when the culture temperature was 34-37℃, astaxanthin could be extracted without cell wall disruption and simply by adding 0.5% n-dodecane to the culture medium, and its yield was 0.55 times higher than that synthesized under conditions of 26-30℃; (2) astaxanthin synthesized under conditions of 26-30℃ could only be extracted by breaking down the cell wall with organic reagents such as acetone, and could not be extracted by adding 0.5% n-dodecane to the culture medium. The inventors speculate that this may be because the signal peptide mutant F8A is stable in α-helix conformation under normal temperature conditions (26-30℃) and cannot be cleaved by signal peptidase; while the one fused with the signal peptide cannot exert its own catalytic activity, so the cell wall of Saccharomyces cerevisiae does not break. When the temperature is raised to a specific temperature (34-37℃), the α-helical conformation of the signal peptide is disrupted, allowing it to be cleaved by signal peptidase. As a result, the fusion protein exists independently and exerts its own catalytic activity, breaking the cell wall of Saccharomyces cerevisiae.

[0010] Preferably, the expression regulatory element includes a GAL1 promoter; the nucleotide sequence of the GAL1 promoter is shown in SEQ ID NO: 10.

[0011] The GAL1 promoter, as a galactose-inducible promoter, is a commonly used tool in yeast genetic engineering to regulate the expression of genes such as SSD1 recombinase. This application utilizes the GAL1 promoter to control the transcription of cell wall-dissolving enzyme genes. On the one hand, during the early stages of fermentation, at room temperature using YPD medium containing an appropriate amount of glucose (2%), the GAL1 promoter is completely inhibited, and the fusion protein is not expressed. On the other hand, during the later stages of fermentation—when the cell density reaches OD... 600After approximately 8 hours (about 44 hours), an appropriate amount of galactose (2%) was added to the fermentation broth and the temperature was raised to 34-37°C to induce the transcription of cell wall lysin. This allowed for controllable expression of its catalytic activity.

[0012] This application also provides a *Saccharomyces cerevisiae* engineered strain capable of controlled self-lysis and astaxanthin release. The engineered strain is constructed using an astaxanthin-producing basic strain as the starting strain and incorporating the recombinant nucleic acid construct described above. The astaxanthin-producing basic strain is a recombinant *Saccharomyces cerevisiae* engineered to include and express exogenous β-carotene synthesis genes and astaxanthin synthesis genes. The β-carotene synthesis genes include genes encoding geraniol pyrophosphate synthase CrtE, phytoene desaturase CrtI, and lycopene cyclase CrtYB; the astaxanthin synthesis genes include genes encoding β-carotene hydroxylase CrtZ and β-carotene ketylase CrtW. The integration of the carotene synthase genes and astaxanthin synthase genes into the *Saccharomyces cerevisiae* strain DYF01 genome is gradual, but their order can be interchanged. The engineered strain also knocks out the SSD1 gene, the nucleotide sequence of which is shown in SEQ ID NO: 11. SSD1 has been shown to play an important role in yeast senescence and translation regulation. Knocking out SSD1 can alleviate the inhibition of protein synthesis and increase the expression level of exogenous proteins.

[0013] Preferably, the β-carotene hydroxylase CrtZ is derived from Haematococcus pluvialis, and its encoding gene is shown in SEQ ID NO:4; the β-carotene ketylase CrtW is derived from Paracoccus sp., and its encoding gene is shown in SEQ ID NO:5.

[0014] Preferably, the encoding gene for the geraniol pyrophosphate synthase CrtE is shown in SEQ ID NO:1, the encoding gene for the phytoene desaturase CrtI is shown in SEQ ID NO:2, and the encoding gene for the lycopene cyclase CrtYB is shown in SEQ ID NO:3; the geraniol pyrophosphate synthase CrtE, the phytoene desaturase CrtI, and the lycopene cyclase CrtYB are all derived from *Xanthophyllomyces dendrorhous* (Red Paffia yeast).

[0015] This invention achieves a controllable self-cleavage and astaxanthin release strain HYB05 by: (1) overexpressing the β-carotene synthesis gene and the astaxanthin synthesis gene on the genome of *Saccharomyces cerevisiae* CEN.PK2-1D; (2) overexpressing Lyc, a cell wall lysin controlled by the GAL1 promoter and fused to the N-terminus of the *Saccharomyces cerevisiae* α-factor signal peptide mutant F8A; and (3) simultaneously knocking out the SSD1 gene to relieve translational repression. During glucose culture (0-44 h), the GAL1 promoter suppresses the transcription of the cell wall lysin Lyc gene to a baseline level, eliminating growth inhibition. Meanwhile, the temperature-sensitive signal peptide undergoes a conformational change during the 37 ℃ induction period, exposing the active site of the cell wall lysin Lyc, while the GAL1 promoter increases enzyme expression. Thus, the GAL1 promoter and the temperature-sensitive signal peptide work synergistically to achieve precise control with zero leakage during fermentation and high efficiency during induction. Astaxanthin was produced using a fermentation-pyrolysis coupled process, achieving a yield of 44.18 mg / g DCW, which represents a significant improvement compared to existing technologies.

[0016] The present invention does not specifically limit the gene editing method, but CRISPR-Cas9 technology is preferred.

[0017] This application also provides the application of the engineered strains of Saccharomyces cerevisiae as described above in the fermentation production of astaxanthin.

[0018] This application provides a method for producing astaxanthin by fermentation using engineered Saccharomyces cerevisiae as described above, comprising the following steps:

[0019] (1) The engineered strain of Saccharomyces cerevisiae as described above is inoculated into a seed culture medium and cultured to obtain a seed liquid; the seed culture medium is YPD medium.

[0020] (2) The seed liquid is inoculated into the fermentation medium and cultured in a constant temperature shaker at 26-30℃ for aerobic fermentation to obtain the fermentation broth; the fermentation medium is YPD medium containing 0.5% (v / v) n-dodecane.

[0021] (3) Cell density reaches OD 600 After approximately 8 hours (about 44 hours), 2% galactose was added to the fermentation broth, and the temperature was raised to 34-37℃ for induction for 4-6 hours. At this time, most of the cell walls (>80%) were destroyed, and intracellular astaxanthin was released into the extracellular environment, entering the n-dodecane organic phase to form the upper oil phase. The oil phase was collected and centrifuged to obtain the product astaxanthin.

[0022] This method eliminates the need for high-pressure homogenization, simplifying the operation and significantly reducing the amount of extraction solvent used (approximately 60%). The above method has the following advantages: (1) High lysis efficiency: After 4 hours of induction at high temperature, cells self-lyse and astaxanthin is released. The operation is simple and requires no special equipment. (2) Reduced cost: Eliminating the investment in cell-wall breaking equipment reduces energy consumption in the extraction stage and reduces solvent usage. (3) Quality maintenance: Mild lysis conditions (37 ℃, pH 6.5) result in a higher astaxanthin degradation rate than chemical cell-wall breaking methods. (4) Simplified process: Cleavage and phase separation are completed in one step in the fermenter. Downstream, only centrifugation is required, shortening the production cycle. (5) Safe and environmentally friendly: No exogenous chemical cell-wall breaking agents are used, and the fermentation waste liquid meets green manufacturing standards.

[0023] The beneficial effects of this invention are:

[0024] (1) This application firstly provides the cell wall dissolving enzyme Lyc, which was first discovered and disclosed by the inventor and is applicable to the strain of Saccharomyces cerevisiae. By using the genome integration method, it is integrated into the genome of Saccharomyces cerevisiae, which is expected to enable the release of intracellular astaxanthin to the extracellular space after cell wall rupture, thus overcoming the problems of cell wall rupture extraction.

[0025] (2) This application also provides a recombinant nucleic acid construct comprising the coding sequence of the cell wall dissolving enzyme Lyc as described above, the coding sequence of the signal peptide mutant F8A, and the expression regulatory element. Introducing and expressing the construct into a host cell allows the cell wall dissolving enzyme to exert its catalytic activity at a specific temperature, thereby rupturing the cell wall of *Saccharomyces cerevisiae*.

[0026] (3) This invention provides an engineered strain HYB05 that can controllably release astaxanthin through self-lysis, and a method for synthesizing astaxanthin using the microbial fermentation method of the strain. The method not only achieves a significant increase in astaxanthin yield (reaching 44.18 mg / g DCW), but also simplifies the operation steps, reduces costs, and is safe and environmentally friendly, thus possessing broad application prospects. Attached Figure Description

[0027] Appendix Figure 1 The growth curve and astaxanthin production yield of the engineered strain HYB01 constructed in Example 1 are shown.

[0028] Appendix Figure 2 The growth curve of the engineered strain HYB05 constructed in Example 3 shows the astaxanthin production yield.

[0029] Figure 3 This is an HPLC chromatogram of astaxanthin synthesis by the engineered strain HYB05 constructed for Example 3. Detailed Implementation

[0030] The present invention will be further described below with reference to the embodiments.

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and all materials and reagents used can be purchased from biological or chemical reagent companies. The *Saccharomyces cerevisiae* strain CEN.PK2-1D was purchased from Zhili Zhongte (Wuhan) Biotechnology Co., Ltd.

[0032] Example 1: Construction of the engineered Saccharomyces cerevisiae strain HYB01 (astaxanthin-producing basic strain)

[0033] The Cirsper-CAS9 technology was used for genome integration in *Saccharomyces cerevisiae*. First, the *Saccharomyces cerevisiae* strain CEN.PK2-1D was transformed into the p414-TEF1p-Cas9-CYC1t plasmid (purchased from Shanghai Qincheng Biotechnology Co., Ltd., product code QCP1810) to enable it to express the exogenous Cas9 nuclease. The specific procedures were as follows:

[0034] Preparation of competent cells of Saccharomyces cerevisiae:

[0035] ssDNA (5 mg / mL): Add 50 mg of chopped salmon extract to deionized water, boil to fully dissolve the solid, dilute to 10 mL, aliquot 200-300 μL into 0.5 mL EP tubes, place in a PCR instrument at 99 ℃ for 10 min, immediately place on ice to cool, and then transfer to a -20 ℃ freezer.

[0036] 10×TE: Tris 1.211 g, EDTA (disodium EDTA dihydrate) 0.372 g, dissolve all components in deionized water, adjust pH to 7.5 with glacial acetic acid, bring to a final volume of 100 mL, and filter through a 0.22 μm membrane for sterilization.

[0037] 10×LiAc (1 M): Weigh 6.6 g of LiAc, dilute to 100 mL with deionized water, and autoclave at 121 °C for 20 min.

[0038] 50% PEG: Weigh 50 g of PEG3350, dilute with deionized water to 100 mL, and autoclave at 121 °C for 20 min.

[0039] 1×LiTE:

[0040] Element content 10×TE 10 mL 10×LiAc 10 mL sterile water 80 mL Total 100 mL

[0041] Each component is added to a sterilized bottle in an ultra-clean aseptic workbench.

[0042] Prepare 1 L of YPD solution using the following method:

[0043] drug Weigh yeast powder 10 g peptone 20 g glucose 20 g

[0044] Autoclave at 115 ℃ for 20 min. If using solid culture medium, add 20 g of agar powder before sterilization.

[0045] The CEN.PK2-1D strain was streaked onto YPD solid plates.

[0046] Single colonies of *Saccharomyces cerevisiae* were picked from plates and activated overnight on YPD medium. The cells were then cultured at 30°C with a shaker at 200 rpm. The next day, the overnight colonies were transferred and allowed to grow to OD (October Observation Time). 600 Prepare competent cells at a density of approximately 0.6. Pre-chill at 4 °C for 10 min, centrifuge at 3000 g for 2 min at 4 °C, and discard the supernatant. Resuspend in 15 mL of pre-chilled ddH₂O, centrifuge at 3000 g for 2 min at 4 °C, and discard the supernatant. Repeat once. Resuspend in 5 mL of pre-chilled 100 mM LiTE, centrifuge at 3000 g for 2 min at 4 °C, and discard the supernatant. Resuspend in 500 μL of 100 mM LiTE into a 1.5 mL centrifuge tube and place on ice before transformation.

[0047] Conversion of brewing yeast:

[0048] Transfer 100 μL of competent cells to a new 1.5 mL centrifuge tube and centrifuge at 8000 g for 2 min, discarding the supernatant. In a new 1.5 mL centrifuge tube, add 240 μL of 50% (m / v) PEG3350, 36 μL of 1M LiAc, 20 μL of 5 mg / ml ssDNA (denatured salmon sperm DNA), and 1000 ng of plasmid p414-TEF1p-Cas9-CYC1t. Bring the volume to 360 μL with ddH2O and mix thoroughly. Resuspend the competent cells in 360 μL of the mixture using a pipette. Heat shock at 42 ℃ for 30 min. Add 700 μL of YPD medium and incubate at 30 ℃ for 2 hours. Centrifuge at 6000 g for 2 min and discard 900 μL of supernatant. The remaining supernatant was resuspended and spread onto G418 resistant (final concentration 300 μg / mL) YPD solid plates, and then incubated statically at 30 °C.

[0049] Screening of Saccharomyces cerevisiae transformants:

[0050] Single colonies were picked from plates and cultured in YPD medium containing antibiotic G418. The culture was then stored for later use, resulting in the Saccharomyces cerevisiae strain CEN.PK2-1D-Cas9, which can express exogenous Cas9 nuclease.

[0051] The CrtE, CrtI, and CrtYB gene sequences from *Xanthophyllomyces dendrorhous* were optimized using codons preferred by *Saccharomyces cerevisiae*, resulting in the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. These sequences were then chemically synthesized by Shanghai Sangon Biotech Co., Ltd., and ligated into the pUC-57 vector to form pUC-57-CrtE, pUC-57-CrtI, and pUC-57-CrtYB vectors, respectively.

[0052] The CrtZ gene sequences from *Haematococcus pluvialis* and the CrtW gene sequences from *Paracoccus* sp. were optimized using codons preferred by *Saccharomyces cerevisiae*, resulting in nucleotide sequences as shown in SEQ ID NO: 4 and SEQ ID NO: 5. These sequences were then chemically synthesized by Shanghai Sangon Biotech Co., Ltd., with the C-terminus of CrtW linked to CrtZ using a GS linker to obtain a fusion gene. This fusion gene was then ligated into the pUC-57 vector to form the pUC-57-CrtWZ vector.

[0053] Using the pYQ01 vector (nucleotide sequence as shown in SEQ ID NO: 12), the promoter sequence TDH3 was added to the 5' end of the above genes and the termination sequence ADH1 was added to the 3' end, respectively.

[0054] Using pUC-57-CrtE, pUC-57-CrtI, pUC-57-CrtYB, and pUC-57-CrtZW vectors as templates, and CrtE-F and CrtE-R, CrtI-F and CrtI-R, CrtYB-F and CrtYB-R, and CrtZW-F and CrtZW-R as primers, respectively, the corresponding genes were amplified by PCR. The PCR reaction system is as follows:

[0055] 2 x Phanta Max Buffer 25 μL dNTP Mix (10 mM each) 1 μL F 2 μL R 2 μL Phanta Max Super-Fidelity DNA Polymerase 1 μL plasmid 1 μL <![CDATA[ddH2O]]> Up to 50 μL

[0056] PCR amplification program: 95 Pre-denaturation at ℃ for 3 min; denaturation at 95 ℃ for 15 s; denaturation at 65 ℃ Annealing at ℃ for 15 s, 48 ℃ extension for 1 min; 32 cycles, 48 Extend the PCR product at ℃ for 5 min. The PCR product was then purified using a gel extraction and purification kit (Vazyme, catalog number DC301-01).

[0057] The primer sequences are shown below:

[0058] CrtE-F: 5'-AACACACATAAACAAACAAAATGGACTATGCTAATATATT-3';

[0059] CrtE-R: 5'-ATAAATCATAAGAAATTCGGTTATAGCGGAATATCCGCCA-3';

[0060] CrtI-F: 5'-AACACACATAAACAAACAAAATGGGAAAAGAAAAGGATCA-3';

[0061] CrtI-R: 5'-ATAAATCATAAGAAATTCGGTTAAAAGGCCAAAACACCAA-3'.

[0062] CrtYB-F: 5'-AACACACATAAACAAACAAAATGTCACCTTACTTATTCTT-3';

[0063] CrtYB-R: 5'-ATAAATCATAAGAAATTCGGTTATTGGCCTTCCCAACCAC-3'.

[0064] CrtWZ-F: 5'-AACACACATAAACAAACAAAATGTCTGCTCACGCACTGCC-3';

[0065] CrtWZ-R: 5'-ATAAATCATAAGAAATTCGGTTATCTCTTGGACCAGTCCA-3'.

[0066] Using pYQ01 vector as a template, and CrtE-F1 and CrtE-R1, CrtI-F1 and CrtI-R1, CrtYB-F1 and CrtYB-R1, and CrtWZ-F1 and CrtWZ-R1 as primers, the linearized vector gene was obtained by PCR amplification. The PCR reaction system is as follows:

[0067] 2 x Phanta Max Buffer 25 μL dNTP Mix (10 mM each) 1 μL F 2 μL R 2 μL Phanta Max Super-Fidelity DNA Polymerase 1 μL plasmid 1 μL <![CDATA[ddH2O]]> Up to 50 μL

[0068] PCR amplification program: 95 Pre-denaturation at ℃ for 3 min; 95 Denaturation at ℃ for 15 s; 65 Annealing at ℃ for 15 s, 48 ℃ extension for 1 min; 32 cycles, 48 Extend the PCR product at ℃ for 5 min. The PCR product was then purified using a gel extraction and purification kit (Vazyme, catalog number DC301-01).

[0069] The primer sequences are shown below:

[0070] CrtE-F1: 5'-TGGCGGATATTCCGCTATAACCGAATTTCTTATGATTTAT-3';

[0071] CrtE-R1: 5'-AATATATTAGCATAGTCCATTTTGTTTGTTTATGTGTGTT-3';

[0072] CrtI-F1: 5'-TTGGTGTTTTGGCCTTTTAACCGAATTTCTTATGATTTAT-3';

[0073] CrtI-R1: 5'-TGATCCTTTCTTTTCCCATTTTGTTTGTTTATGTGTGTT-3'.

[0074] CrtYB-F1: 5'-GTGGTTGGGAAGGCCAATAACCGAATTTCTTATGATTTAT-3'.

[0075] CrtYB-R1: 5'-AAGAATAAGTAAGGTGACATTTTGTTTGTTTATGTGTGTT-3'.

[0076] CrtWZ-F1: 5'-TGGACTGGTCCAAGAGATAACCGAATTTCTTATGATTTAT-3'.

[0077] CrtWZ-R1: 5'-GGCAGTGCGTGAGCAGACATTTTGTTTGTTTATGTGTGTT-3'.

[0078] The pYQ01 linearized vector and the astaxanthin synthase gene fragment were fused using a seamless cloning kit (Vazyme, catalog number C112-01), and the resulting fragments were transformed into *E. coli* DH5, following the manufacturer's instructions. Positive transformants were obtained and their sequences were verified by sequencing by Qingdao Qingke Biotechnology Co., Ltd., and then stored for future use.

[0079] Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1D as a template, 70 bp each of the upstream and downstream homologous arms of the genomic integration site were amplified. The CrtE gene was integrated into the ndt80 gene locus, the CrtI gene into the hxt3 gene locus, the CrtYB gene into the gpp1 gene locus, and the CrtZW gene into the dit1 gene locus. The amplification system is shown below:

[0080] template 100 ng Primer 1 0.5 μM Primer 2 0.5 μM Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Cat. No. P505-D1) 1 μL 2×Phanta Max Buffer 25 μL dNTP 4 μL <![CDATA[ddH2O]]> Add to 50 μL

[0081] The PCR program was as follows: 95 °C for 3 min; 30 cycles × (95 °C for 15 s, 55 °C for 15 s, 48 ​​°C for 2 min); 48 °C for 5 min; 16 °C for 10 min. PCR products were purified using a gel extraction kit (Vazyme, catalog number DC301-01).

[0082] The primer sequences are shown in the table below:

[0083] CrtE-F2:

[0084] 5'-TAAGCAAAAAATTGAAAGTTTACTAACCTTTCATTAAAGAGAAATAACAATATTATAAAAAGCGCTTAAATCATTATCAATACTGCCATT-3';

[0085] CrtE-R2:

[0086] 5'-AAGTTATCTGGAGGTCCTGTGTTCGATCCACAGAATTCGCATATTTTTTTAACGATTTAAAATCATTAGTCCGGTAGAGGTGTGGTCAAT-3';

[0087] CrtI-F2:

[0088] 5'-ATAATTTTACTTAATAGCTTTTCATAAAATAATAGAATCACAAACAAAATTTACATCTGAGTTAAACAATCTCATTATCAATACTGCCATT-3';

[0089] CrtI-R2:

[0090] 5'-TTTATCATTATTGACTAGCACATCGAATCTTAAAATACACTATTATTCAGCACTACGGTTTAGCGTGAAACCGGTAGAGGTGTGGTCAAT-3';

[0091] CrtYB-F2:

[0092] 5'-TAAATCTTTCGTAAGTATCTCTTGATTGCCATTTTTTTCTTTCCAAGTTTCCTTGTTATGAAACGTTTCATCATTATCAATACTGCCATT-3';

[0093] CrtYB-R2:

[0094] 5'-AAATGGAGGGAAATCATACATTTTTATTTTATTTTTAGCGTAGTAGTTTTATCAAAAAAATAAAAGAAAACCGGTAGAGGTGTGGTCAAT-3';

[0095] CrtWZ-F2:

[0096] 5'-AAAGTCTTGACTAAATAAACAATTTGTTAATATCCTAATTCGGTAAAGCTTTGTCGAGACATTAACAAAATCATTATCAATACTGCCATT-3';

[0097] CrtWZ-R2:

[0098] 5'-AAAGAAACGAACTAACTAATGTTTAAGTAAAAGAACAAAAAGGTAGACCAATGTAGCGCTCTTACTTTACCGGTAGAGGTGTGGTCAAT-3'.

[0099] Preparation of competent cells of Saccharomyces cerevisiae:

[0100] ssDNA (5 mg / mL): Add 50 mg of chopped salmon extract to deionized water, boil to fully dissolve the solid, dilute to 10 mL, aliquot 200-300 μL into 0.5 mL EP tubes, place in a PCR instrument at 99 ℃ for 10 min, immediately place on ice to cool, and then transfer to a -20 ℃ freezer.

[0101] 10×TE: Tris 1.211 g, EDTA (disodium EDTA dihydrate) 0.348 g, dissolve all components in deionized water, adjust pH to 7.5 with glacial acetic acid, dilute to 100 mL, and filter sterilize using a 0.22 μm filter membrane.

[0102] 10×LiAc (1 M): Weigh 6.6 g of LiAc, dilute to 100 mL with deionized water, and autoclave at 121 °C for 20 min.

[0103] 50% PEG: Weigh 50 g of PEG3350, dilute with deionized water to 100 mL, and autoclave at 121 °C for 20 min.

[0104] 1×LiTE:

[0105] Element content 10×TE 10 mL 10×LiAc 10 mL sterile water 80 mL Total 100 mL

[0106] Each component is added to a sterilized bottle in an ultra-clean aseptic workbench.

[0107] Prepare 1 L of YPD solution using the following method:

[0108] drug Weigh yeast powder 10 g peptone 20 g glucose 20 g

[0109] Autoclave at 115 ℃ for 20 min. If using solid culture medium, add 20 g of agar powder before sterilization.

[0110] The Saccharomyces cerevisiae strain CEN.PK2-1D-Cas9 was streaked onto YPD solid plates.

[0111] Single colonies of *Saccharomyces cerevisiae* were picked from plates and activated overnight on YPD medium. The cells were then cultured at 30°C with a shaker at 200 rpm. The next day, the overnight colonies were transferred and allowed to grow to OD (October Observation Time). 600 Prepare competent cells at a density of approximately 0.6. Pre-chill at 4 °C for 10 min, centrifuge at 3000 g for 2 min at 4 °C, and discard the supernatant. Resuspend in 15 mL of pre-chilled ddH₂O, centrifuge at 3000 g for 2 min at 4 °C, and discard the supernatant. Repeat once. Resuspend in 5 mL of pre-chilled 100 mM LiTE, centrifuge at 3000 g for 2 min at 4 °C, and discard the supernatant. Resuspend in 500 μL of 100 mM LiTE into a 1.5 mL centrifuge tube and place on ice before transformation.

[0112] Conversion of brewing yeast:

[0113] Transfer 100 μL of competent cells to a new 1.5 mL centrifuge tube and centrifuge at 8000 g for 2 min, discarding the supernatant. In a new 1.5 mL centrifuge tube, add 240 μL of 50% (m / v) PEG3350, 36 μL of 1M LiAc, 20 μL of 5 mg / ml ssDNA (denatured salmon sperm DNA), 1000 ng of plasmid sgRNA (plasmid construction according to Biotechnology for Biofuels and Bioproducts, 2023, 16:124), and 2000 ng of DNA fragment (the CrtE gene containing the integration site homologous arm obtained by PCR). Add ddH2O to a final volume of 360 μL and mix thoroughly. Resuspend the competent cells in 360 μL of the mixture using a pipette. Heat shock at 42 ℃ for 30 min. Add 700 μL of YPD medium and incubate at 30 ℃ for 2 hours. Centrifuge at 6000 g for 2 min and remove 900 μL of supernatant. Resuspend the supernatant in the remaining supernatant and spread it on YPD solid plates containing G418 and HygB resistance (final concentration 300 μg / mL). Incubate statically at 30 °C.

[0114] Screening of Saccharomyces cerevisiae transformants:

[0115] Single colonies were picked from a plate and cultured in YPD medium containing antibiotic G418 + HygB. After preservation, the genome was extracted from the remaining bacterial culture using the following method: overnight cultured yeast was centrifuged at 5000 rpm for 3 min to remove the medium; resuspended in 100 μL of 200 mM LiAc and 1% SDS; incubated at 70 ℃ for 15 min; 300 μL of anhydrous ethanol was added and vortexed; centrifuged at 13000 g for 5 min to remove the supernatant; 300 μL of 70% ethanol was added, gently shaken, and then centrifuged at 13000 g for 1 min to discard the supernatant, and the liquid was aspirated with a pipette; the bottom precipitate was dried in an oven for 15-30 min, as residual ethanol would affect DNA dissolution; the genome was dissolved in 40 μL of ddH2O solution.

[0116] Validation of yeast transformants:

[0117] Primers were designed to validate the transformant genome. One upstream and one downstream primer were located inside the integrative gene, and the other was located in the homologous arm (one inside and one outside) to ensure the rigor of the experiment. Sequencing validation was performed subsequently.

[0118] In summary, the integration of CrtE, CrtI, CrtYB, and CrtWZ into the genome of the Saccharomyces cerevisiae CEN.PK2-1D-Cas9 strain in this embodiment was carried out stepwise. Specifically, the CrtE gene was first integrated into the genome of the CEN.PK2-1D-Cas9 strain, then the CrtI gene was integrated into the genome of an engineered strain containing the CrtE gene, then the CrtYB gene was integrated into the genome of an engineered strain containing both the CrtE and CrtI genes, then the CrtWZ gene was integrated into the genome of an engineered strain containing the CrtE, CrtI, and CrtYB genes, and finally the astaxanthin-producing basic strain—the Saccharomyces cerevisiae engineered strain HYB01—was obtained.

[0119] Example 2: Synthesis of astaxanthin using the engineered Saccharomyces cerevisiae strain HYB01 constructed in Example 1

[0120] (1) Synthesis and extraction of astaxanthin

[0121] Single colonies of HYB01 strain were picked from YPD solid plates and activated overnight in YPD liquid medium, then cultured at 30 ℃ with a shaker at 200 rpm. The next day, the cells were transferred and cultured in YPD liquid medium at 30 ℃ with a shaker at 200 rpm. Odulescence (OD) was measured at regular intervals. 600 Values ​​and astaxanthin. See details for results. Figure 1 .Depend on Figure 1 It can be seen that strain HYB01 is growing well, with an OD of 26 hours. 600 =2.9, 72-hour OD 600 =6.31, after which it entered a stable period.

[0122] Astaxanthin was extracted from the bacterial cells using organic reagents. The method was as follows: After centrifuging 0.2 mL of bacterial culture, the culture medium was removed, 1 mL of acetone was added to the bacterial cells, and the mixture was incubated in a water bath at 55°C for 15 minutes. The astaxanthin content in the organic phase was then detected.

[0123] (2) Detection of astaxanthin production

[0124] Astaxanthin yield was determined by HPLC. Detection conditions: Column: C18 (4.6 × 250 mm, 5 μm); Column temperature: 30℃; Flow rate: 0.5 mL / min; Injection volume: 5 μL; Detection wavelength: 476 nm; Mobile phase A: methanol: acetonitrile: dichloromethane = 21:21:8; Mobile phase B: methanol: water = 1:9. The gradient elution program was: 0–18 min (phase A increases from 80% to 100%, phase B decreases from 20% to 0%), 18–40 min (phase A 100%, phase B 0%), 40–45 min (phase A decreases from 100% to 80%, phase B increases from 0% to 20%), 45–50 min (phase A 80%, phase B 20%).

[0125] For details on astaxanthin production, please refer to [link / reference]. Figure 1 .Depend on Figure 1 It can be seen that the astaxanthin production of the engineered strain HYB01 constructed in Example 2 increased with the extension of time, and tended to stabilize after 48 hours, with the highest production being 28.52 mg / g DCW.

[0126] Example 3: Astaxanthin Synthesis by the Engineered Saccharomyces cerevisiae strain HYB05 with Controllable Self-Destruction and Astaxanthin Release

[0127] The coding sequence of the cell wall lysin Lyc gene from *Cellulosimicrobium cellulans* was optimized using codons preferred by *Saccharomyces cerevisiae*, resulting in the nucleotide sequence shown in SEQ ID NO: 7. The α-factor signal peptide mutant F8A and GAL1 promoter sequences from the *Saccharomyces cerevisiae* genome are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The optimized Lyc coding gene was ligated to the 3' end of the α-factor signal peptide mutant F8A coding gene, and the GAL1 promoter sequence was added to the 5' end of the fusion gene to obtain the fusion gene sequence. Shanghai Sangon Biotech Co., Ltd. chemically synthesized the fusion gene and ligated it into the pUC-57 vector to form the pUC-57-Lyc vector.

[0128] The termination sequence ADH1 was added to the 3' end of the above fusion gene using the pYQ01 vector (nucleotide sequence as shown in SEQ ID NO: 12).

[0129] Using pUC-57-Lyc vector as a template and Lyc-F and Lyc-R as primers, the PCR reaction system and procedure are as shown in Example 1.

[0130] The primer sequences are shown in the table below:

[0131] Lyc-F: 5'-AACACACATAAACAACAAAGATTAGAAGCCGCCGAGCGG-3';

[0132] Lyc-R: 5'-ATAAATCATAAGAAATTCGGTTAAGCTCTTAGCCAGACAG-3';

[0133] Using the pYQ01 vector as a template and Lyc-F1 and Lyc-R1 as primers, the linearized vector gene was obtained by PCR amplification. The PCR reaction system and procedure are shown in Example 1.

[0134] The primer sequences are shown in the table below:

[0135] Lyc-F1: 5'-CTGTTCTGGCTAAGAGCTTAACCGAATTTCTTATGATTTAT-3';

[0136] Lyc-R1: 5'-GCTTTACCTATCATAGGTAACCGAATTTCTTATGATTTAT-3';

[0137] The pYQ01 linearized vector and gene fragment were fused using a seamless cloning kit (Vazyme, catalog number C112-01), and transformed into *E. coli* DH5, following the manufacturer's instructions. Positive transformants were obtained and their sequences were verified for correctness by sequencing by Qingdao Qingke Biotechnology Co., Ltd., and then stored for future use.

[0138] Using the Saccharomyces cerevisiae CEN.PK2-1D genome as a template, 70 bp each of the upstream and downstream homologous arms of the genomic integration site were amplified. Lyc-F2 and Lyc-R2 were used as primers, and the above genes were integrated into the SSD1 gene site. The PCR reaction system and procedure are as shown in Example 1.

[0139] The primer sequences are shown below:

[0140] Lyc-F2:

[0141] 5'-TATCGCAAAACAGAACACCAAAAACCTTTCAGCGCAAAGATTTGGCCCAATTATTCCATCTTTATACACTGATTAGAAGCCGCCGAGCGG-3';

[0142] Lyc-R2:

[0143] 5'-CGGTGAAGCACAATCCAAAGAACGAAAGTGAAAAACAAGAAAAACAGCAATGACGATATTGGTAGAAGAGACGGTAGAGGTGTGGTCAAT-3';

[0144] Preparation of sgRNA plasmids:

[0145] Design the Saccharomyces cerevisiae Guide-RNA on the website (http: / / www.e-crisp.org / ). Since unwanted genes need to be completely eliminated, the chosen gRNA sequence should be located as centrally as possible within the gene to improve the efficiency of homologous recombination.

[0146] The primer sequences are as follows:

[0147] gRNA-F: 5'-CTAAATAACTGACCAGGGATGATCATTTATCTTTCACTGC-3'

[0148] gRNA-R: 5'-ATCCCTGGTCAGTTATTTAGGTTTTAGAGCTAGAAATAGC-3'

[0149] PCR amplification was performed using pRS42H plasmid as a template, following the system and procedure described previously. The PCR products were fused using a seamless cloning kit (Vazyme, catalog number C112-01) and transformed into *E. coli* DH5, following the manufacturer's instructions. Positive transformants were obtained and their sequences were verified for correctness by sequencing by Qingdao Qingke Biotechnology Co., Ltd., and then stored for future use.

[0150] The method for integrating the Saccharomyces cerevisiae genome is the same as in Example 1, thereby obtaining the Saccharomyces cerevisiae engineered strain HYB05 that can controllably release astaxanthin through self-lysis.

[0151] Example 4: Synthesis of astaxanthin using the engineered Saccharomyces cerevisiae strain HYB05 constructed in Example 3

[0152] (1) Synthesis and extraction of astaxanthin

[0153] Single colonies of HYB05 strain were picked from YPD solid plates and activated overnight in YPD liquid medium, then cultured at 30 ℃ with a shaker at 200 rpm. The next day, the cells were transferred to YPD medium (containing 0.5% (v / v) n-dodecane) and cultured at 26-30 ℃ with a shaker at 200 rpm for 44 hours. OD values ​​were measured at regular intervals. 600 Values ​​and results are detailed in the [link / reference]. Figure 2 .Depend on Figure 1 It can be seen that strain HYB05 is growing well, with an OD of 44 hours.600 ≈8. At this point, add 2% galactose and raise the culture temperature to 34-37℃ to induce cell lysis for 4-6 hours. Collect the product astaxanthin from the n-dodecane phase.

[0154] (2) Detection of astaxanthin production

[0155] Astaxanthin yield was determined by HPLC under the same conditions as in Example 2. See attached results. Figure 3 .like Figure 3 As shown, the chromatographic peak with a retention time of 12.6 min is astaxanthin, indicating that astaxanthin was detected in the fermentation broth of the engineered strain HYB05. Combined with... Figure 2 It was found that the astaxanthin content in the fermentation broth increased with the extension of cell lysis time, and tended to stabilize at 50 hours, with the highest yield being 44.18 mg / g DCW. Compared with the results of Example 2 (28.52 mg / g DCW), the yield increased by 55%, indicating that the engineered strain HYB05 constructed in Example 3 significantly improved the yield of synthesized astaxanthin. This shows that the yield of astaxanthin released by cell self-lysis is higher than that extracted using organic reagents, and this technology does not require a cumbersome organic reagent extraction process, making it simple to operate, low in cost, and safe and environmentally friendly.

[0156] In summary, the fermentation method using the *Saccharomyces cerevisiae* strain HYB05 constructed in this application, which allows for controlled self-lysis and astaxanthin release, increases astaxanthin production by 55% (reaching 44.18 mg / g DCW) compared to the control strain HYB01. Furthermore, it simplifies the operation, reduces costs, and is safe and environmentally friendly, demonstrating broad application prospects. This indicates that the engineered strain HYB05, by introducing and expressing a recombinant nucleic acid construct containing the coding sequence Lyc (a cell wall lysin), the coding sequence of the signal peptide mutant F8A, and expression regulatory elements, achieves controlled (at a specific temperature) catalytic activity of the cell wall lysin and release of astaxanthin, filling a gap in existing technologies and representing significant technological progress.

[0157] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A cell wall-dissolving enzyme, Lyc, suitable for Saccharomyces cerevisiae strains, characterized in that: The amino acid sequence of the cell wall lysin Lyc is shown in SEQ ID NO:

6.

2. A recombinant nucleic acid construct, characterized in that: It comprises the coding sequence of the cell wall lysin Lyc as described in claim 1, the coding sequence of the signal peptide mutant F8A, and the expression regulatory element; wherein the amino acid sequence of the signal peptide mutant F8A is shown in SEQ ID NO:

8.

3. The recombinant nucleic acid construct according to claim 2, characterized in that: The expression regulatory element includes the GAL1 promoter, the nucleotide sequence of which is shown in SEQ ID NO:

10.

4. A brewer's yeast strain capable of controllable self-lysis and astaxanthin release, characterized in that: The engineered strain was constructed by introducing the recombinant nucleic acid construct as described in claim 3 or 4, using the astaxanthin-producing basic strain as the starting strain.

5. The engineered strain of *Saccharomyces cerevisiae* according to claim 4, characterized in that: The engineered strain simultaneously knocked out the SSD1 gene, the nucleotide sequence of which is shown in SEQ ID NO:

11.

6. The engineered strain of *Saccharomyces cerevisiae* according to claim 4 or 5, characterized in that: The astaxanthin-producing basic strain is a recombinant Saccharomyces cerevisiae that has been engineered to include and express exogenous β-carotene synthesis genes and astaxanthin synthesis genes.

7. The engineered strain of *Saccharomyces cerevisiae* according to claim 6, characterized in that: The β-carotene synthesis gene includes genes encoding geraniol pyrophosphate synthase CrtE, phytoene desaturase CrtI, and lycopene cyclase CrtYB; the gene encoding geraniol pyrophosphate synthase CrtE is shown in SEQ ID NO:1, the gene encoding phytoene desaturase CrtI is shown in SEQ ID NO:2, and the gene encoding lycopene cyclase CrtYB is shown in SEQ ID NO:

3.

8. The engineered strain of *Saccharomyces cerevisiae* according to claim 6, characterized in that: The astaxanthin synthesis gene includes genes encoding β-carotene hydroxylase CrtZ and β-carotene ketolase CrtW; the gene encoding β-carotene hydroxylase CrtZ is shown in SEQ ID NO:4, and the gene encoding β-carotene ketolase CrtW is shown in SEQ ID NO:

5.

9. The application of the engineered strain of Saccharomyces cerevisiae as described in any one of claims 4-8 in the fermentation production of astaxanthin.

10. A method for producing astaxanthin by fermentation using any one of the engineered strains of *Saccharomyces cerevisiae* described in any one of 4-8, characterized in that: Includes the following steps: (1) The engineered strain of Saccharomyces cerevisiae according to any one of claims 4-8 is inoculated into a seed culture medium and cultured to obtain a seed liquid; (2) The seed liquid is inoculated into a fermentation medium and aerobic fermentation is carried out at 26-30℃ to obtain fermentation liquid; (3) Add an appropriate amount of galactose to the fermentation broth, heat to 34-37℃ and induce for 4-6 hours, collect the oil phase and centrifuge to obtain the product astaxanthin.

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