Anastazia, a new species of the genus Anastazia (Trichoptera: Leptoceridae) from China

Abstract

The present application first provides an enzyme system for improving astaxanthin yield, including the beta-carotene hydroxylase CrtZ and the beta-carotene ketolase CrtW with significantly improved conversion rate discovered by the inventors. The beta-carotene hydroxylase CrtZ has the amino acid sequence shown in SEQ ID NO: 1, and the beta-carotene ketolase CrtW has the amino acid sequence shown in SEQ ID NO: 2. The present application constructs a high-yield astaxanthin Saccharomyces cerevisiae engineering strain by overexpressing the coding genes of the aforementioned beta-carotene hydroxylase crtZ and beta-carotene ketolase crtW in a beta-carotene-producing base strain. The Saccharomyces cerevisiae engineering strain is used for microbial fermentation to synthesize astaxanthin, and the yield of astaxanthin can reach 64.8 mg / g, which is significantly improved compared with the prior art, and the technical progress is significant. On this basis, the present application provides a Saccharomyces cerevisiae engineering strain capable of growing in a medium containing corn stalk hydrolysate and high-yield astaxanthin, realizing the conversion of low-cost agricultural and forestry waste to high-value astaxanthin, and having the application prospect of green and sustainable development.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and relates to an engineered strain of Saccharomyces cerevisiae, specifically a high-astaxanthin-producing strain of Saccharomyces cerevisiae 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, there are three main methods for producing astaxanthin: natural extraction, chemical synthesis, and microbial fermentation. Natural extraction primarily refers to extraction from crustacean processing waste (such as shrimp and crab) or Haematococcus pluvialis. The former suffers from low astaxanthin content and high levels of impurities such as ash and chitin in the extract; the latter, while providing all-trans astaxanthin, suffers from high production costs due to the long growth cycle of algae, the need for light, low biomass, and difficulty in cell wall disruption, hindering large-scale promotion. Due to the technical problems of natural extraction, chemical synthesis is currently the main source in the market, accounting for over 95% of the total market share. However, the selenium produced by chemical synthesis is mainly a 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 has low safety and antioxidant activity. Furthermore, the synthesis process is complex and cannot meet consumers' demand for natural and safe products. Compared to the previous two methods, the production of astaxanthin by microbial fermentation has advantages such as short production cycle, no impact from climate and season, easy to achieve high-density industrial cultivation, and the product is of natural configuration. It is regarded as the core technology route for the next generation of natural L-astaxanthin.

[0004] Among numerous microbial hosts, Saccharomyces cerevisiae is widely recognized as one of the ideal substrates for constructing astaxanthin cell factories. The main reasons include: (1) Saccharomyces cerevisiae is a recognized GRAS microorganism, free from endotoxins and phage contamination risks, 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 highly active 3S,3'S-L-astaxanthin has been achieved by introducing heterologous CrtZ and CrtW into Saccharomyces cerevisiae, but the yield of the final product astaxanthin is low. This is mainly because 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 number of intermediate products (such as canthaxanthin and zeaxanthin). Even so, the synthesis of astaxanthin by Saccharomyces cerevisiae is the most competitive technical route for the industrialization of natural astaxanthin. Therefore, how to increase the yield of astaxanthin in the process of synthesizing astaxanthin with brewer's yeast is an urgent technical problem to be solved. Summary of the Invention

[0005] To address the problems existing in the synthesis of astaxanthin by *Saccharomyces cerevisiae* in current technologies, this invention provides a high-yield *Saccharomyces cerevisiae* engineered strain, its construction, and its applications. The inventors discovered β-carotene hydroxylase CrtZ and β-carotene ketoylase CrtW, which significantly improve conversion rates compared to existing technologies. Based on this, by overexpressing the encoding genes of the aforementioned β-carotene hydroxylase CrtZ and β-carotene ketoylase CrtW in a basic strain, a high-yield *Saccharomyces cerevisiae* engineered strain was constructed, significantly increasing astaxanthin production.

[0006] The technical solution of the present invention:

[0007] This application first provides an enzyme system for increasing astaxanthin production, comprising β-carotene hydroxylase CrtZ and β-carotene ketolase CrtW; wherein the β-carotene hydroxylase CrtZ has the amino acid sequence shown in SEQ ID NO: 1, and the β-carotene ketolase CrtW has the amino acid sequence shown in SEQ ID NO: 2. The β-carotene hydroxylase CrtZ is derived from *Sphingobacterium griseoflavum*, and the β-carotene ketolase CrtW is derived from *Stigmatella aurantiaca*. It is known to those skilled in the art that the β-carotene hydroxylase and β-carotene ketolase work synergistically to introduce a hydroxyl group (-OH) and a ketone group (=O) onto the β-carotene ring, thereby achieving the conversion of β-carotene to astaxanthin. The β-carotene hydroxylase CrtZ and β-carotene ketoylase CrtW used in the enzyme system described in this application are newly discovered and disclosed by the inventors. Using this enzyme system significantly improves the conversion efficiency of β-carotene to astaxanthin, thereby increasing astaxanthin yield. Therefore, this enzyme system is suitable for β-carotene-producing microorganisms.

[0008] The recombinant nucleic acid is isolated from the enzyme system described above.

[0009] A transformed microorganism comprising the recombinant nucleic acid as described above. The microorganism is a β-carotene-producing strain.

[0010] A brewer's yeast strain for synthesizing astaxanthin is obtained by introducing recombinant nucleic acids as described above, using a β-carotene-producing basic strain as the starting strain.

[0011] Preferably, the gene encoding β-carotene hydroxylase CrtZ in the recombinant nucleic acid is shown in SEQ ID NO:6, and the gene encoding β-carotene ketylase CrtW is shown in SEQ ID NO:7.

[0012] The β-carotene-producing basic strain is a recombinant *Saccharomyces cerevisiae* strain engineered to include and express exogenous β-carotene synthesis genes. These β-carotene synthesis genes include genes encoding geraniol pyrophosphate synthase (CrtE), phytoene desaturase (CrtI), and lycopene cyclase (CrtYB). These geraniol pyrophosphate synthase (CrtE), phytoene desaturase (CrtI), and lycopene cyclase (CrtYB) are all derived from *Xanthophyllomyces dendrorhous*. The integration of the carotene synthase genes (CrtE, CrtI, CrtYB) and astaxanthin synthase genes (CrtZ, CrtW) into the *Saccharomyces cerevisiae* strain DYF01 genome is a stepwise process, but their order can be interchanged.

[0013] More preferably, the encoding gene of the geraniol pyrophosphate synthase CrtE is shown in SEQ ID NO:3, the encoding gene of the phytoene desaturase CrtI is shown in SEQ ID NO:4, and the encoding gene of the lycopene cyclase CrtYB is shown in SEQ ID NO:5.

[0014] This application first overexpresses geraniol pyrophosphate synthase (CrtE), phytopene desaturase (CrtI), and lycopene cyclase (CrtYB) in the genome of *Saccharomyces cerevisiae* to obtain a basic strain producing β-carotene. Further overexpression of β-carotene ketolase (CrtW) and hydroxylase (CrtZ) yields a high-astaxanthin-producing engineered *Saccharomyces cerevisiae* strain, DYF05. The astaxanthin yield reaches 64.8 mg / g DCW, a significant improvement compared to the yield of existing *Saccharomyces cerevisiae* strains (maximum 24.4 mg / g, *Construction of lycopene-overproducing *Saccharomyces cerevisiae* by combining directed evolution and metabolic engineering. *Metabolic Engineering*, 2015, 30, 69-78). This invention does not specifically limit the gene editing method, but CRISPR-Cas9 technology is preferred.

[0015] Preferably, the *Saccharomyces cerevisiae* engineered strain described in this application overexpresses heterologous cellulase (CBH), cellulase (CelA), and β-glucosidase (BglZ), and is localized to the outer side of the cell wall via the C-terminus of the cell wall anchoring protein CWP2 derived from *Saccharomyces cerevisiae*, thereby obtaining an engineered *Saccharomyces cerevisiae* strain capable of growing using corn stalk hydrolysate as the sole carbon source. The encoding gene for the heterologous cellulase (CBH) is shown in SEQ ID NO:8, the encoding gene for the cellulase (CelA) is shown in SEQ ID NO:9, and the encoding gene for the β-glucosidase (BglZ) is shown in SEQ ID NO:10. The nucleotide sequence of the cell wall anchoring protein CWP2 is shown in SEQ ID NO:11. The integration order of the cellulase gene, β-carotene synthase gene, and astaxanthin synthase gene can be arbitrarily interchanged; as long as all are expressed, the conversion of low-cost agricultural and forestry waste, represented by corn stalks, into high-value astaxanthin can be achieved.

[0016] The application of the engineered strain of Saccharomyces cerevisiae as described above in the fermentation production of astaxanthin. The application is for the microbial fermentation production of astaxanthin, specifically including the following steps: (1) inoculating the engineered strain of Saccharomyces cerevisiae as described above into a seed culture medium to obtain a seed liquid; (2) inoculating the seed liquid into a fermentation culture medium for aerobic fermentation to obtain a fermentation broth; (3) centrifuging the fermentation broth to collect the cells, and obtaining the product astaxanthin through cell disruption, extraction and purification.

[0017] Preferably, the seed culture medium is YPD medium, and the fermentation medium is either YPD medium or corn straw medium. The YPD medium consists of: 10 g / L yeast extract, 10 g / L peptone, and 20 g / L glucose; the corn straw medium has a similar composition to the YPD medium, with corn straw hydrolysate replacing the glucose component. Culture conditions: 30℃, 200 rpm constant temperature shaker culture for 48 hours.

[0018] The beneficial effects of this invention are:

[0019] (1) This application first provides an enzyme system for increasing astaxanthin production, including β-carotene hydroxylase CrtZ and β-carotene ketylase CrtW, which were first discovered and disclosed by the inventors; by using the enzyme system, the conversion efficiency of β-carotene to astaxanthin is significantly improved, thereby increasing the production of astaxanthin.

[0020] (2) This application also provides a high-yield Saccharomyces cerevisiae engineered strain. Using the Saccharomyces cerevisiae engineered strain to ferment and produce astaxanthin, the astaxanthin yield can reach 64.8 mg / g, which is significantly higher than the prior art and represents a significant technological advancement. This lays the foundation for the industrial production of astaxanthin and has broad application prospects and huge market value.

[0021] (3) This application also provides a brewer's yeast strain that can grow in a culture medium containing corn straw hydrolysate and produce high astaxanthin, thereby realizing the conversion of low-cost agricultural and forestry waste into high-value astaxanthin. The raw materials are widely available and have green and sustainable application prospects. Attached Figure Description

[0022] Appendix Figure 1 The growth curve of the engineered strain DYF01 constructed in Example 1 in a culture medium containing corn straw hydrolysate.

[0023] Appendix Figure 2 The growth curve and astaxanthin yield of the engineered strain DYF05 constructed in Example 3 in a culture medium containing corn straw hydrolysate are shown.

[0024] Appendix Figure 3 This is an HPLC chromatogram of the synthesis of astaxanthin by the engineered strain DYF05 in Example 4 in a culture medium containing corn straw hydrolysate. Detailed Implementation

[0025] The present invention will be further described below with reference to the embodiments.

[0026] 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.

[0027] Example 1: Construction of Saccharomyces cerevisiae engineered strain DFY01 overexpressing cellulase gene

[0028] 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:

[0029] Preparation of competent cells of Saccharomyces cerevisiae:

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 50% PEG: Weigh 50 g of PEG3350, dilute with deionized water to 100 mL, and autoclave at 121 °C for 20 min.

[0034] 1×LiTE:

[0035] 10×TE 10 mL 10×LiAc 10 mL sterile water 80 mL Total 100 mL

[0036] Each component is added to a sterilized bottle in an ultra-clean aseptic workbench.

[0037] Prepare 1 L of YPD solution using the following method:

[0038] yeast powder 10 g peptone 20 g glucose 20 g

[0039] Autoclave at 115 ℃ for 20 min. If using solid culture medium, add 20 g of agar powder before sterilization.

[0040] The CEN.PK2-1D strain was streaked onto YPD solid plates.

[0041] 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). 600Prepare 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.

[0042] Conversion of brewing yeast:

[0043] 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.

[0044] Screening of Saccharomyces cerevisiae transformants:

[0045] 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.

[0046] The CBH gene from *Bacillus altitudinis*, the CelA gene from *Clostridium thermocellum*, and the BglZ gene from *Bacillus amyloliquefaciens* were optimized using codons preferred by *Saccharomyces cerevisiae*, resulting in nucleotide sequences as shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. The extracellular wall anchoring protein, CWP2 protein from *Saccharomyces cerevisiae* CEN.PK2-1D, has a nucleotide sequence shown in SEQ ID NO: 11. The optimized CBH, CelA, and BglZ genes were ligated to the 3' end of the CWP2 coding gene to obtain fusion gene sequences. These sequences were chemically synthesized by Shanghai Sangon Biotech Co., Ltd., and then ligated into the pUC-57 vector to form pUC-57-CWP2-CBH, pUC-57-CWP2-CelA, and pUC-57-CWP2-BglZ vectors, respectively.

[0047] 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 fusion gene of cell wall anchoring protein and three cellulose degrading enzymes, and the termination sequence ADH1 was added to the 3' end.

[0048] Using pUC-57-CWP2-CBH, pUC-57-CWP2-CelA, and pUC-57-CWP2-BglZ vectors as templates, and CWP2-F and CBH-R, CWP2-F and CelA-R, and CWP2-F and BglZ-R as primers, respectively, the corresponding fusion genes were amplified by PCR. The PCR reaction system is as follows:

[0049] 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

[0050] PCR amplification program: 95 Pre-denaturation at ℃ for 3 min; denaturation at 95 ℃ for 15 s; denaturation at 65 ℃ Annealing at ℃ for 15 s, 72 ℃ extension for 1 min; 32 cycles, 72 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).

[0051] The primer sequences are shown below:

[0052] CWP2-F: 5'-AACACACATAAACAAACAAAATGGAATCAGCTGCCGCTATTAG-3';

[0053] CBH-R: 5'-ATAAATCATAAGAAATTCGGTTACCTATGATAGGTAAAGC-3';

[0054] CelA-R: 5'-ATAAATCATAAGAAATTCGGTTAGTAAGGCAAATGTGGGA-3';

[0055] BglZ-R: 5'-CTACTAACGGAGAGTCCTTACTACTAACGGAGAGTCCTTA-3'.

[0056] Using the pYQ01 vector as a template, and CWP2-R and CBH-F, CWP2-R and CelA-F, and CWP2-R and BglZ-F as primers, the linearized vector gene was amplified by PCR. The PCR reaction system is as follows:

[0057] 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

[0058] PCR amplification program: 95 Pre-denaturation at ℃ for 3 min; 95 Denaturation at ℃ for 15 s; 65 Annealing at ℃ for 15 s, 72 ℃ extension for 1 min; 32 cycles, 72 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).

[0059] The primer sequences are shown below:

[0060] CWP2-R: 5'-CTAATAGCGGCAGCTGATTCCATTTTGTTTGTTTATGTGTGTT-3';

[0061] CBH-F: 5'-GCTTTACCTATCATAGGTAACCGAATTTCTTATGATTTAT-3';

[0062] CelA-F: 5'-TCCCACATTTGCCTTACTAACCGAATTTCTTATGATTTAT-3';

[0063] BglZ-F: 5'-TAAGGACTCTCCGTTAGTAGTAAGGACTCTCCGTTAGTAG-3'.

[0064] The pYQ01 linearized vector and the cellulase gene fragment were fused using a seamless cloning kit (Vazyme, catalog number C112-01), and the resulting fusions 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.

[0065] 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 CBH gene was integrated into the ho gene site, the CelA gene into the ygr250c gene site, and the BglZ gene into the gal80 gene site. The amplification system is shown below:

[0066] 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

[0067] The PCR program was as follows: 95 ℃ for 3 min; 30 cycles × (95 ℃ for 15 s, 55 ℃ for 15 s, 72 ℃ for 2 min); 72 ℃ for 5 min; 16 ℃ for 10 min. PCR products were purified using a gel extraction kit (Vazyme, catalog number DC301-01).

[0068] The primer sequences are shown below:

[0069] CBH-F1:

[0070] 5'-TTTCTATTACAACTATTAGCTCTAAATCCATATCCTCATAAGCAGCAATCAATTCTATCTATACTTTAAATCATTATCAATACTGCCATT-3';

[0071] CBH-R1:

[0072] 5'-TGGTTTTTTTCATCCAAAATATTAAATTTTACTTTTATTACATACAACTTTTTAAAACTAATATACACATTCCGGTAGAGGTGTGGTCAAT-3';

[0073] CelA-F1:

[0074] 5'-AGCACCGAAACAGGTCAAACAGATACTCATCATTAATGGCGGACCCATAATTTTCAGAAGGTTAGTACCTTCATTATCAATACTGCCATT-3';

[0075] CelA-R1:

[0076] 5'-GCAGAAGGCTTATATAAAAAAAAAAAGAAATATTTCTTCACTTTCGAGCATTGAACTATTGTCATTACCTCCGGTAGAGGTGTGGTCAAT-3';

[0077] BglZ-F1:

[0078] 5'-TCACTGCTGGTCCTTGCCGACCAGCGTATACAATCTCGATAGTTGGTTTCCCGTTCTTTCCACTCCCGTCTCATTATCAATACTGCCATT-3';

[0079] BglZ-R1:

[0080] 5'-TAGATATATACTCAGTATTCGTTTTTATAACGTTCGCTGCACTGGGGGCCAAGCACAGGGCAAGATGCTTCCGGTAGAGGTGTGGTCAAT-3'.

[0081] Preparation of competent cells of Saccharomyces cerevisiae:

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 50% PEG: Weigh 50 g of PEG3350, dilute with deionized water to 100 mL, and autoclave at 121 °C for 20 min.

[0086] 1×LiTE:

[0087] 10×TE 10 mL 10×LiAc 10 mL sterile water 80 mL Total 100 mL

[0088] Each component is added to a sterilized bottle in an ultra-clean aseptic workbench.

[0089] Prepare 1 L of YPD solution using the following method:

[0090] yeast powder 10 g peptone 20 g glucose 20 g

[0091] Autoclave at 115 ℃ for 20 min. If using solid culture medium, add 20 g of agar powder before sterilization.

[0092] The CEN.PK2-1D-Cas9 strain was streaked onto YPD solid plates.

[0093] 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.

[0094] Conversion of brewing yeast:

[0095] 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 (the CBH gene fragment containing the integration site homologous arm obtained by PCR). 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 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.

[0096] Screening of Saccharomyces cerevisiae transformants:

[0097] 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.

[0098] Validation of yeast transformants:

[0099] 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.

[0100] The integration of cellulase exonuclease (CBH), cellulase endonuclease (CelA), and β-glucosidase (BglZ) into the genome of *Saccharomyces cerevisiae* CEN.PK2-1D was carried out stepwise. Specifically, the CBH gene was first integrated into the CEN.PK2-1D-Cas9 genome, then the CelA gene was integrated into the genome of an engineered strain containing the CBH gene, and then the BglZ gene was integrated into the genome of an engineered strain containing both the CBH and CelA genes. Finally, the *Saccharomyces cerevisiae* engineered strain DYF01, which overexpressed the cellulase gene, was obtained.

[0101] Example 2: Verification of the growth of strain DYF01 in a culture medium containing corn straw hydrolysate.

[0102] The corn stalk pretreatment solution was obtained by reacting corn stalks with a eutectic solvent (DES). The specific method was as follows: Prepare the DES reagent by weighing 13.9 g of choline chloride, 63 g of diethanolamine, and 6.2 g of ethylene glycol into a round-bottom flask and mixing in an oil bath at 80 °C until clear. Weigh 4 g of corn stalks and 60 g of DES reagent into a round-bottom flask and incubate in an oil bath at 120 °C for 60 min. Filter the mixture under vacuum. Wash the solid fraction repeatedly with 100 mL of 75% ethanol, then with deionized water at 80 °C, and dry in an oven at 60 °C to obtain a solid fraction containing carbohydrates such as cellulose. To remove inhibitors from this fraction, add Ca(OH)₂ solution to adjust the pH to 10.0, react at 50 °C for 30 min, and then adjust the pH back to 6.0 to precipitate phenols and furfural. Finally, adjust the pH to 5.5-6.0 with 5 M NaOH or ammonia to obtain the corn stalk hydrolysate.

[0103] Single colonies of the DYF01 strain constructed in Example 1 were picked from YPD solid plates and activated overnight in YPD liquid medium, then cultured at 30 ℃ on a shaker at 200 rpm. The next day, the cells were transferred and cultured in a medium containing 10 g / L corn straw hydrolysate (YPD medium does not contain glucose) at 30 ℃ on a shaker at 200 rpm. OD was measured at regular intervals. 600 Values ​​were used to observe the growth status of the strains, and the results were as follows: Figure 1 As shown. By Figure 1 It can be seen that strain DYF01 grows well when corn straw hydrolysate is the sole carbon source, with an OD of 26 hours. 600 =4.21, 65-hour OD 600 =6.62, after which it entered a stable period. This indicates that the engineered Saccharomyces cerevisiae strain DYF01 constructed in Example 1 can grow normally in a culture medium containing corn straw hydrolysate.

[0104] Example 3: Construction of Saccharomyces cerevisiae DYF01 genome overexpressing astaxanthin synthase gene strain DFY05

[0105] 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: 3, SEQ ID NO: 4, and SEQ ID NO: 5. 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.

[0106] The CrtZ and CrtW gene sequences from *Sphingobacterium griseoflavum* and *Stigmatella aurantiaca*, respectively, were optimized using codons preferred by *Saccharomyces cerevisiae*, resulting in nucleotide sequences as shown in SEQ ID NO: 6 and SEQ ID NO: 7. These sequences were then chemically synthesized by Shanghai Sangon Biotech Co., Ltd., and the CrtZ and CrtW gene sequences were ligated using a GS linker to obtain a fusion gene, which was then inserted into the pUC-57 vector to form the pUC-57-CrtZW vector.

[0107] Using the pYQ01 vector, 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.

[0108] 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, the corresponding genes were amplified by PCR. The PCR reaction system and procedure were the same as in Example 1.

[0109] The primer sequences are shown below:

[0110] CrtE-F: 5'-AACACACATAAACAAACAAAATGGACTATGCTAATATATT-3';

[0111] CrtE-R: 5'-ATAAATCATAAGAAATTCGGTTATAGCGGAATATCCGCCA-3';

[0112] CrtI-F: 5'-AACACACATAAACAAACAAAATGGGAAAAGAAAAGGATCA-3';

[0113] CrtI-R: 5'-ATAAATCATAAGAAATTCGGTTAAAAGGCCAAAACACCAA-3'.

[0114] CrtYB-F: 5'-AACACACATAAACAAACAAAATGTCACCTTACTTATTCTT-3';

[0115] CrtYB-R: 5'-ATAAATCATAAGAAATTCGGTTATTGGCCTTCCCAACCAC-3'.

[0116] CrtZW-F: 5'-AACACACATAAACAAACAAAATGATGCAAATCTTGATTAA-3';

[0117] CrtZW-R: 5'-ATAAATCATAAGAAATTCGGTTATGCCGCCGGACGGGCAT-3'.

[0118] Using the pYQ01 vector as a template, and CrtE-F1 and CrtE-R1, CrtI-F1 and CrtI-R1, CrtYB-F1 and CrtYB-R1, and CrtZW-F1 and CrtZW-R1 as primers, the linearized vector gene was obtained by PCR amplification. The PCR reaction system and procedure were the same as in Example 1.

[0119] The primer sequences are shown below:

[0120] CrtE-F1: 5'-TGGCGGATATTCCGCTATAACCGAATTTCTTATGATTTAT-3';

[0121] CrtE-R1: 5'-AATATATTAGCATAGTCCATTTTGTTTGTTTATGTGTGTT-3';

[0122] CrtI-F1: 5'-TTGGTGTTTTGGCCTTTTAACCGAATTTCTTATGATTTAT-3';

[0123] CrtI-R1: 5'-TGATCCTTTCTTTTCCCATTTTGTTTGTTTATGTGTGTT-3'.

[0124] CrtYB-F1: 5'-GTGGTTGGGAAGGCCAATAACCGAATTTCTTATGATTTAT-3'.

[0125] CrtYB-R1: 5'-AAGAATAAGTAAGGTGACATTTTGTTTGTTTATGTGTGTT-3'.

[0126] CrtZW-F1: 5'-ATGCCCGTCCGGCGGCATAACCGAATTTCTTATGATTTAT-3'.

[0127] CrtZW-R1: 5'-TTAATCAAGATTTGCATCATTTTGTTTGTTTATGTGTGTT-3'.

[0128] 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.

[0129] Using the genome of Saccharomyces cerevisiae CEN.PK2-1D as a template, 70 bp each of the upstream and downstream homologous arms of the genome integration site were amplified. The CrtE gene was integrated into the ndt80 gene site, the CrtI gene was integrated into the hxt3 gene site, the CrtYB gene was integrated into the gpp1 gene site, and the CrtZW gene was integrated into the dit1 gene site. The PCR amplification system and procedure were the same as in Example 1.

[0130] The primer sequences are shown below:

[0131] CrtE-F2:

[0132] 5'-TAAGCAAAAAATTGAAAGTTTACTAACCTTTCATTAAAGAGAAATAACAATATTATAAAAAGCGCTTAAATCATTATCAATACTGCCATT-3';

[0133] CrtE-R2:

[0134] 5'-AAGTTATCTGGAGGTCCTGTGTTCGATCCACAGAATTCGCATATTTTTTTAACGATTTAAAATCATTAGTCCGGTAGAGGTGTGGTCAAT-3';

[0135] CrtI-F2:

[0136] 5'-ATAATTTTACTTAATAGCTTTTCATAAAATAATAGAATCACAAACAAAATTTACATCTGAGTTAAACAATCTCATTATCAATACTGCCATT-3';

[0137] CrtI-R2:

[0138] 5'-TTTATCATTATTGACTAGCACATCGAATCTTAAAATACACTATTATTCAGCACTACGGTTTAGCGTGAAACCGGTAGAGGTGTGGTCAAT-3';

[0139] CrtYB-F2:

[0140] 5'-TAAATCTTTCGTAAGTATCTCTTGATTGCCATTTTTTTCTTTCCAAGTTTCCTTGTTATGAAACGTTTCATCATTATCAATACTGCCATT-3';

[0141] CrtYB-R2:

[0142] 5'-AAATGGAGGGAAATCATACATTTTTATTTTATTTTTAGCGTAGTAGTTTTATCAAAAAAATAAAAGAAAACCGGTAGAGGTGTGGTCAAT-3';

[0143] CrtZW-F2:

[0144] 5'-AAAGTCTTGACTAAATAAACAATTTGTTAATATCCTAATTCGGTAAAGCTTTGTCGAGACATTAACAAAATCATTATCAATACTGCCATT-3';

[0145] CrtZW-R2:

[0146] 5'-AAAGAAACGAACTAACTAATGTTTAAGTAAAAGAACAAAAAGGTAGACCAATGTAGCGCTCTTACTTTACCGGTAGAGGTGTGGTCAAT-3'.

[0147] The steps for preparing competent yeast cells, yeast transformation, and screening and verifying transformants are the same as in Example 1.

[0148] The integration of carotene synthase genes (CrtE, CrtI, CrtYB) and astaxanthin synthase genes (CrtZ, CrtW) into the genome of *Saccharomyces cerevisiae* strain DYF01 is a stepwise process, but the order of integration can be interchanged. In this embodiment, the integration is carried out using the following steps: First, the CrtE gene is integrated into the genome of strain DYF01. Then, the CrtI gene is integrated into the genome of an engineered strain containing the CrtE gene. Next, the CrtYB gene is integrated into the genome of an engineered strain containing both the CrtE and CrtI genes. Finally, the CrtZ and CrtW genes are integrated into the genome of an engineered strain containing the CrtE, CrtI, and CrtYB genes. Finally, *Saccharomyces cerevisiae* engineered strain DYF05, which overexpresses the astaxanthin synthase gene, is obtained.

[0149] In addition, it should be noted that (1) the Saccharomyces cerevisiae engineered strain DYF05 constructed in this embodiment is constructed based on the DYF01 strain constructed in Example 1. Therefore, the Saccharomyces cerevisiae engineered strain DYF05 can grow in a culture medium containing corn straw hydrolysate and further realize its conversion from β-carotene to astaxanthin. (2) In the construction of the Saccharomyces cerevisiae engineered strain DYF05 constructed in this embodiment, the integration order of the cellulose degradation enzyme gene, carotene synthase gene and astaxanthin synthase gene can be arbitrarily changed. As long as all of them are expressed, the conversion of low-cost agricultural and forestry waste represented by corn straw into high-value astaxanthin can be realized.

[0150] Example 4: Construction of Saccharomyces cerevisiae engineered strain DFY05-2 overexpressing astaxanthin synthase gene

[0151] Unlike Example 3, the basic strain was Saccharomyces cerevisiae CEN.PK2-1D-Cas9.

[0152] Similarly, the integration of carotene synthase genes (CrtE, CrtI, CrtYB) and astaxanthin synthase genes (CrtZ, CrtW) into the genome of *Saccharomyces cerevisiae* strain CEN.PK2-1D-Cas9 is carried out stepwise, but the order can be interchanged. In this embodiment, the integration is performed using the following steps: First, the CrtE gene is integrated into the genome of *Saccharomyces cerevisiae* strain CEN.PK2-1D-Cas9; then, the CrtI gene is integrated into the genome of an engineered strain containing the CrtE gene; next, the CrtYB gene is integrated into the genome of an engineered strain containing both the CrtE and CrtI genes; then, the CrtZ and CrtW genes are integrated into the genome of an engineered strain containing the CrtE, CrtI, and CrtYB genes; finally, *Saccharomyces cerevisiae* engineered strain DYF05-2, overexpressing the astaxanthin synthase gene, is obtained. This *Saccharomyces cerevisiae* engineered strain DYF05-2 can achieve the conversion of β-carotene to astaxanthin, but it cannot grow in a culture medium containing corn straw hydrolysate.

[0153] Example 5: Synthesis of astaxanthin using engineered strains of *Saccharomyces cerevisiae* constructed in Example 3 or Example 4.

[0154] (1) Synthesis of astaxanthin

[0155] The preparation process of corn stalk hydrolysate is the same as in Example 2.

[0156] Single colonies of strains DYF05 and DYF05-2 were picked from YPD solid plates and activated overnight in YPD liquid medium. They were then cultured at 30 °C on a shaker at 200 rpm and transferred the next day. Specifically, the DYF05 strain constructed in Example 3 was cultured in corn straw hydrolysate medium, and the DYF05-2 strain constructed in Example 4 was cultured in YPD medium. The YPD medium consisted of 10 g / L yeast extract, 10 g / L peptone, and 20 g / L glucose. The corn straw medium had a similar composition to the YPD medium, but corn straw hydrolysate replaced the glucose. The culture conditions were: 30 °C on a shaker at 200 rpm, with OD measured at regular intervals. 600 Value and astaxanthin.

[0157] Astaxanthin was extracted from the bacterial cells using organic reagents. The method is as follows: After centrifuging 0.2 mL of bacterial culture and removing the culture medium, 1 mL of acetone was added to the bacterial cells, and the mixture was incubated at 55°C for 15 minutes. The astaxanthin content in the organic phase was then detected. The results for strains DYF05 and DYF05-2 were consistent. The results for strain DYF05 are described below as an example. Figure 2 As shown. By Figure 2 It can be seen that strain DYF05 grows well in the culture medium containing corn straw hydrolysate, with an OD of 26 hours. 600 =2.9, 72-hour OD 600 =6.31, after which it entered a stable period.

[0158] (2) Detection of astaxanthin production

[0159] 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%). Detailed HPLC results can be found [link to HPLC 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 engineered strain DYF05. Combined with... Figure 2It can be seen that the astaxanthin content in the fermentation broth increases with the extension of time, and tends to stabilize at 72 hours, with the highest yield being 64.8 mg / g DCW.

[0160] In summary, the engineered Saccharomyces cerevisiae strains DYF05 and DYF05-2 constructed in this application can produce astaxanthin with a yield of 64.8 mg / g, which is significantly higher than that of existing technologies, demonstrating remarkable technological progress. This lays the foundation for the industrial production of astaxanthin and has broad application prospects and significant market value. Moreover, the engineered strain DYF05 can grow in a culture medium containing corn straw hydrolysate, realizing the conversion of low-cost agricultural and forestry waste into high-value astaxanthin. The raw material sources are wide-ranging, indicating promising green and sustainable application prospects.

[0161] 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 brewer's yeast strain for synthesizing astaxanthin, characterized in that: The engineered strain was constructed by introducing recombinant nucleic acids encoding β-carotene hydroxylase CrtZ and β-carotene ketolase CrtW as the starting strain; the amino acid sequence of β-carotene hydroxylase CrtZ is shown in SEQ ID NO: 1, and the amino acid sequence of β-carotene ketolase CrtW is shown in SEQ ID NO:

2.

2. The engineered strain of *Saccharomyces cerevisiae* for synthesizing astaxanthin according to claim 1, characterized in that: The gene encoding the β-carotene hydroxylase CrtZ is shown in SEQ ID NO:6, and the gene encoding the β-carotene ketylase CrtW is shown in SEQ ID NO:

7.

3. The engineered strain of *Saccharomyces cerevisiae* for synthesizing astaxanthin according to claim 1 or 2, characterized in that: The basic strain producing β-carotene is a recombinant Saccharomyces cerevisiae that has been engineered to include and express exogenous β-carotene synthesis genes; the β-carotene synthesis genes include genes encoding geraniol pyrophosphate synthase CrtE, phytoene desaturase CrtI, and lycopene cyclase CrtYB.

4. The engineered strain of *Saccharomyces cerevisiae* for synthesizing astaxanthin according to claim 3, characterized in that: The gene encoding the geraniol pyrophosphate synthase CrtE is shown in SEQ ID NO:3, the gene encoding the phytocyanin desaturase CrtI is shown in SEQ ID NO:4, and the gene encoding the lycopene cyclase CrtYB is shown in SEQ ID NO:

5.

5. The engineered strain of *Saccharomyces cerevisiae* for synthesizing astaxanthin according to claim 3, characterized in that: The β-carotene-producing basic strain also contains and expresses exogenous cellulase genes; the cellulase genes include cellulase exonuclease, cellulase endonuclease and β-glucosidase.

6. The application of the engineered strain of Saccharomyces cerevisiae as described in any one of claims 1-5 in the fermentation production of astaxanthin.

Images