Recombinant saccharomyces cerevisiae engineering bacteria with high yield of zeaxanthin and construction method and application thereof

Abstract

The application discloses a recombinant Saccharomyces cerevisiae engineering bacterium with high zeaxanthin yield and a construction method and application thereof. The recombinant Saccharomyces cerevisiae engineering bacterium is obtained by performing genome integration of genes RRT7 and URA3 at a genome Int4 site of a Saccharomyces cerevisiae for producing zeaxanthin, wherein the sequence of the Int4 site is shown as SEQ ID No. 34, the nucleotide sequence of the RRT7 is shown as SEQ ID No. 11, and the nucleotide sequence of the URA3 is shown as SEQ ID No. 18. The application is aimed at a bottleneck of biosynthesis of zeaxanthin of the Saccharomyces cerevisiae, and a high-zeaxanthin-yield Saccharomyces cerevisiae engineering bacterium is obtained through transcriptome analysis and metabolic engineering. The yield of the Saccharomyces cerevisiae engineering bacterium is 489.56 mg / L of zeaxanthin in shake flask fermentation.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and metabolic engineering, and in particular to a high-yield recombinant saccharidin-producing engineered yeast strain, its construction method, and its application. Background Technology

[0002] Zeaxanthin is a type of oxygenated carotenoid containing 40 carbon atoms. Due to its unique conjugated polyene structure, it possesses strong antioxidant, anti-inflammatory, and photoprotective capabilities. As a key component of macular pigment in the retina, zeaxanthin can effectively filter blue light, reducing the risk of age-related macular degeneration and cataracts. It has significant applications in the food, feed, and health product industries, and market demand continues to grow.

[0003] Currently, in industry, zeaxanthin is mainly extracted and separated from plants such as marigolds. However, this method has problems such as long growth cycle, great susceptibility to environmental influence, extremely low content of the target product in the raw materials (only 0.01% to 2%), and complicated extraction and purification processes, making it difficult to meet the demand for large-scale supply.

[0004] The de novo synthesis of zeaxanthin using microbial cell factories (such as Saccharomyces cerevisiae and Escherichia coli) constructed through metabolic engineering offers significant advantages, including being green and sustainable, having a short production cycle, and being easily scaled up, making it an important development direction in the field of biomanufacturing. In the microbial zeaxanthin synthesis system using Saccharomyces cerevisiae as a substrate, although previous studies (such as the team led by Xiao Wenhai at Tianjin University) achieved a zeaxanthin yield of 814.6 mg / L in a 5 L fed-batch fermentation, the overall microbial synthesis system still faces bottlenecks such as low yield and conversion rate, hindering its industrial application.

[0005] Most existing metabolic modification strategies focus on intuitive "carbon flux" drivers, such as overexpressing key rate-limiting enzymes in biosynthetic pathways or knocking out competing pathways. However, this non-targeted and non-systematic modification often overlooks the global metabolic burden on host cells caused by the expression of large amounts of heterologous genes and the flow of unnatural metabolites. Under conventional fermentation culture conditions (e.g., 30°C), this deep-seated physiological contradiction is often masked by the decline in overall cell growth and viability, resulting in synthases failing to form sufficient quantities of highly active proteins despite transcription, or the synthetic reaction stalling due to unknown limitations. Traditional rational design methods struggle to directly predict and repair these complex "hidden bottlenecks."

[0006] To address the aforementioned pain points, there is an urgent need to introduce phenotype-driven systems biology strategies. Research has found that by setting specific environmental pressures (such as 20°C low-temperature culture), the overall metabolic rhythm of host cells can be effectively slowed down, thereby exposing underlying shortcomings that are masked at room temperature and limit the efficient synthesis of products. Based on this, transcriptomics sequencing and differential analysis technologies can precisely capture global feedback signals of cells under specific physiological states. By elucidating the intrinsic molecular mechanisms of high yield at low temperatures, we can break through the blind spots of conventional metabolic network maps and truly uncover the core issues leading to systemic metabolic burden. Based on this discovery, introducing these transcriptome-guided key targets into the host through reverse engineering, fundamentally relieving its deep-seated stress, is a crucial approach to further breaking through the upper limit of maize xanthine yield. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies in zeaxanthin synthesis systems, such as deep metabolic burden and the difficulty of addressing the disconnect between enzyme transcription levels and actual catalytic activity in traditional metabolic modifications, the main objective of this invention is to provide a high-yield recombinant Saccharomyces cerevisiae engineered strain, its construction method, and its applications. Based on transcriptomics analysis strategies, by comparing global gene expression maps under different temperatures and pressures, the underlying key response genes limiting product synthesis were precisely identified. By overexpressing these key endogenous targets in the chassis cells, the metabolic network imbalance caused by global stress was effectively repaired, resolving the disconnect between enzyme activity and transcription levels.

[0008] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: A method for constructing a high-yield recombinant Saccharomyces cerevisiae engineered strain, using Saccharomyces cerevisiae that produces zeaxanthin as the chassis strain, and introducing target genes MME1, RRT7, or PRP21. The nucleotide sequence of MME1 is shown in SEQ ID No. 3, the nucleotide sequence of RRT7 is shown in SEQ ID No. 11, and the nucleotide sequence of PRP21 is shown in SEQ ID No. 15.

[0009] Using *Saccharomyces cerevisiae*, a strain that produces zeaxanthin, as the chassis strain, the genes RRT7 and URA3 were integrated into the genome at the Int4 site of the *Saccharomyces cerevisiae* genome. The sequence of the Int4 site is shown in SEQ ID No. 34, the nucleotide sequence of RRT7 is shown in SEQ ID No. 11, and the nucleotide sequence of URA3 is shown in SEQ ID No. 18.

[0010] The brewer's yeast that produces zeaxanthin is strain YCGH06, with the genotype CEN.PK2-1C,Δgal1Δgal7Δgal10::nat-TERG13-ERG13-PGAL7-TERG19-ERG19-PGAL10-PGAL1-ERG10-TERG10,Δypl062w::DR-TCYC1-BtCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,trp1::TRP1_TCYC1-BtCrtI-PGAL10-PGAL1-PaCrtB-TPGK1. leu2::LEU2_TTDH2-DR-TCYC1-BtCrtI-PGAL3-TACT1-tHMG1-PGAL10-PGAL1-TmCrtE-TGPM1,his3::HIS3-TENO2-TACT1-tHMG1-PGAL10-PGAL1-(BT S1-ERG20)-TFBA1YGLCtau3::hphA-TIDI1-IDI1-PGAl7-TPDC1-SaPMK-PGAL10-PGAl1-SaMK-THXT7,YMRWdelta15::UAS-PGAL1-PaCrtY-TADH1-DR, YNRCdelta9::UAS-PGAL1-PaCrtY-TADH1-DR, HO::PTEF1-AtRFNR1-TGPM1-PTDH3-AtFD3-TPGI1-G418R, TKL2-PGAL10-EuCrtZ(M92L)-TFBA1-TEF2, Int4:-PGAL10-EuCrtZ(M92L)-TFBA1-TEF2, DAK2-PGAL10-HSC82-TFBA1.

[0011] The genome integration was achieved using the CRISPR / Cas9 gene editing system.

[0012] The RRT7 and URA3 genes in the Saccharomyces cerevisiae genome were knocked into at specific sites using the CRISRPR / Cas9 gene editing system. The sgRNA sequence used for knocking into the RRT7 and URA3 genes was sgRNA-Int4, and the nucleotide sequence is shown in SEQ ID No. 25.

[0013] A high-yield zeaxanthin recombinant brewer's yeast engineered strain was constructed using the aforementioned construction method.

[0014] The application of the recombinant brewer's yeast engineered strain in the production of zeaxanthin.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention addresses the bottleneck in the biosynthesis of zeaxanthin in Saccharomyces cerevisiae. Through transcriptomics analysis and metabolic engineering, a high-yield engineered Saccharomyces cerevisiae strain was obtained. The yield of zeaxanthin in shake-flask fermentation was measured to be 489.56 mg / L. Attached Figure Description

[0016] Figure 1 This is a volcano plot of differentially expressed genes. A represents the gene expression differences at 24 hours, and B represents the gene expression differences at 72 hours. Figure 2 This is a diagram showing the effects of overexpression of each gene compared to expression with an empty plasmid. Figure 3 This is a diagram showing the results after overexpression and genome integration. Detailed Implementation

[0017] The embodiments described below are exemplary descriptions of key experimental evidence and are not intended to limit the core content and application scope of this invention due to the amount of evidence. It should be noted that all the accompanying drawings and corresponding descriptions merely illustrate the concept, principles, and representative experimental evidence of the disclosed embodiments of this invention. Where the chain of evidence is complete, it is unnecessary to show all the specific details and extended details of the various embodiments listed in this invention.

[0018] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0019] The initial strain used in this invention was the recombinant strain YCGH06 of Saccharomyces cerevisiae. The construction method of YCGH06 has been described in detail in the patent application "A β-carotene hydroxylase mutant, genetically engineered bacteria and its application" (patent application number: 202610469692.7).

[0020] The genotype of strain YCGH06 is CEN.PK2-1C. Δgal1Δgal7Δgal10::nat-TERG13-ERG13-PGAL7-TERG19-ERG19-PGAL10-PGAL1-ERG10-TERG10, Δypl062w::DR-TCYC1-BtCrtI-PGAL10-PGAL1-PaCrtB-TPGK1, trp1::TRP1_TCYC1-BtCrtI-PGAL10-PGAL1-PaCrtB-TPGK1, leu2::LEU2_TTDH2-DR-TCYC1-BtCrtI-PGAL3-TACT1-tHMG1-PGAL10-PGAL1-TmCrtE-TGPM1, his3::HIS3-TENO2-TACT1-tHMG1-PGAL10-PGAL1-(BTS1-ERG20)-TFBA1 YGLCtau3::hphA-TIDI1-IDI1-PGAl7-TPDC1-SaPMK-PGAL10-PGAl1-SaMK-THXT7, YMRWdelta15::UAS-PGAL1-PaCrtY-TADH1-DR, YNRCdelta9::UAS-PGAL1-PaCrtY-TADH1-DR HO::PTEF1-AtRFNR1-TGPM1-PTDH3-AtFD3-TPGI1-G418R,TKL2:PGAL10-EuCrtZ(M92L)-TFBA1-TEF2,Int4:-PGAL10-EuCrtZ(M92L)-TFBA1-TEF2DAK2:P GAL10 -HSC82 -T FBA1 According to the TIG Genetic Nomenclature Guide, "Δ" indicates gene deletion, followed by the name of the deleted gene, such as Δ KU70 indicating the deletion of the KU70 gene; "::" indicates gene insertion, where the gene before "::" is broken by the insertion of the gene after "::", such as Δ A08::RAD52 indicating the insertion of the RAD52 gene at the A08 site.

[0021] Strain YCGH06 was derived from *Saccharomyces cerevisiae* CENPK2-1C (commercial strain) by deleting gal1 and gal7, and inserting ERG13, ERG19, and ERG10 genes at the gal10 site. BtCrtI and PaCrtB genes were inserted at ypl062w; PaCrtB and BtCrtI genes at the trp1 site; leu2, BtCrtI, tHMG1, and TmCrtE genes were inserted at the leu2 site; his3, tHMG1, and BTS1-ERG20 genes were inserted at the his3 site; SaPMK and SaMK genes were inserted at the YGLCtau3 site; PaCrtY genes were inserted at YMRWdelta15 and YNRCdelta9; and RFNR1 and FD3 genes were inserted at the HO site (as is known). EuCrtZ (M92L) was inserted at the TKL2 and INT14 sites, and the HSC82 gene was inserted at the DAK2 site.

[0022] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0023] Example 1: Key Target Discovery Based on Low-Temperature Transcriptomics (1) Transcriptomics analysis The starting strain, YCGH06, is a *Saccharomyces cerevisiae* strain that produces zeaxanthin. It was inoculated into 50 mL of YPDG liquid medium (60 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, 10 g / L D-(+)-galactose) at an initial OD600 of 0.2. Two fermentation temperature gradients, 20℃ and 30℃, were established, and the cells were cultured at 250 rpm. Cells in the logarithmic growth phase were collected at 24 h and 72 h, flash-frozen in liquid nitrogen, and stored at -80℃. Total RNA was extracted, and the collected cells were sent to Qingke Biotechnology Co., Ltd. for total RNA extraction, transcription, library construction, and sequencing. Differentially expressed genes were screened by comparing gene expression levels at 30 h and 72 h, and deep functional analysis was performed using GO, pathway, clustering, protein-protein interactions, and transcription factor prediction.

[0024] (2) Key target discovery: Transcriptome sequencing data of the strain were obtained under culture conditions of 20℃ and 30℃, and differential expression analysis was performed. A volcano plot of differentially expressed genes was then constructed. Figure 1 The significance threshold was set as p-value < 0.05 and |log2(FoldChange)| > 1. Combined with KEGG metabolic pathway enrichment analysis ( Figure 2The results showed that under low-temperature induced high-yield stress, gene clusters related to cofactor metabolism and RNA / protein quality control exhibited a significant upregulation trend. Based on the above transcriptomic analysis results, this invention ultimately screened and identified three key endogenous target genes: the MME1 gene involved in NADPH cofactor supply, the PRP21 gene involved in mRNA splicing and quality control, and the RRT7 gene involved in protein translation system optimization. The MME1, PRP21, and RRT7 genes are derived from *Saccharomyces cerevisiae*.

[0025] Example 2: Construction of target gene expression cassette and transformation of recombinant strains (1) Construction of expression box Using the genome of Saccharomyces cerevisiae YCGH06 as a template, and MME1-F (SEQ ID No. 1) and MME1-R (SEQ ID No. 2) as primers, the full-length fragment of the endogenous gene MME1 (GenBank: 855208) was obtained by PCR amplification. The nucleotide sequence of MME1 is shown in SEQ ID No. 3.

[0026] The empty plasmid Pcgh01:pRS416-T was cleaved using NotI. TDH2 -P GAL1 / 10 -T FBA1 (SEQ ID No. 4) (can be directly synthesized) to obtain a linearized vector cleaved between a promoter and a terminator, terminator T TDH2 The nucleotide sequence is shown in SEQ ID No. 5, and the promoter P GAL10 The nucleotide sequence is shown in SEQ ID No. 6. Terminator T FBA1 The nucleotide sequence is shown in SEQ ID No. 7. The fragment MME1 and the linearized vector were ligated in vitro using a seamless cloning method to obtain the fragment pRS416-T. TDH2 -MME1-P GAL1 / 10 - T FBA1 (SEQ ID No.8).

[0027] The fragment pRS416-T TDH2 -MME1-P GAL1 / 10 The TOP10 competent cells of *E. coli* were imported, transformants were selected and sequenced, and after preliminary screening by plating with plates containing the corresponding antibiotics, single-clone transformants were picked, plasmids were extracted and sequenced for verification. The obtained plasmid was Pcgh02: pRS416-T. TDH2 -MME1-P GAL1 / 10 (SEQ ID No.26).

[0028] Using the genome of Saccharomyces cerevisiae YCGH06 as a template, and RRT7-F (SEQ ID No. 9) and RRT7-R (SEQ ID No. 10) as primers, the full-length fragment of the endogenous gene RRT7 (GenBank: 850629) was obtained by PCR amplification. The nucleotide sequence of RRT7 is shown in SEQ ID No. 11.

[0029] Empty plasmid pRS416-T was cleaved using NotI. TDH2 -P GAL1 / 10 A linearized vector was obtained by cutting between the promoter and terminator, and the promoter was ligated in vitro using a seamless cloning method to obtain the pRS416-T fragment. TDH2 - RRT7-P GAL1 / 10。 (SEQ ID No. 12) The fragment pRS416-T TDH2 - RRT7-P GAL1 / 10 The TOP10 competent cells of *E. coli* were imported, transformants were selected and sequenced, and after initial screening by plating with appropriate antibiotics, single-clone transformants were picked, plasmids were extracted and sequenced for verification. The obtained plasmid was Pcgh03: pRS416-T. TDH2 - RRT7-P GAL1 / 10 (SEQ ID No.27).

[0030] Using the genome of Saccharomyces cerevisiae YCGH06 as a template, and with PRP21-F (SEQ ID No. 13) and PRP21-R (SEQ ID No. 14) as primers, the full-length fragment of the endogenous gene PRP21 (GenBank: 850629) was obtained by PCR amplification. The nucleotide sequence of PRP21 is shown in SEQ ID No. 15.

[0031] Empty plasmid pRS416-T was cleaved using NotI. TDH2 -P GAL1 / 10 A linearized vector, cut between the promoter and terminator, was obtained and ligated in vitro using a seamless cloning method to obtain the pRS416-T fragment. TDH2 - PRP21-P GAL1 / 10。

[0032] The fragment pRS416-T TDH2 - PRP21-P GAL1 / 10 The TOP10 competent cells of *E. coli* were imported, transformants were selected and sequenced, and after initial screening by plating with appropriate antibiotics, single-clone transformants were picked, plasmids were extracted and sequenced for verification. The obtained plasmid was Pcgh04: pRS416-T. TDH2 - PRP21-PGAL1 / 10 (SEQ ID No. 28) (2) Transformation and screening of target strains: Empty plasmid Pcgh01 and the aforementioned plasmids Pcgh02, Pcgh03, and Pcgh04 were transformed into YCGH06 using the lithium acetate method, respectively, to construct strains SC-CGH01, SC-CGH02, SC-CGH03, and SC-CGH04.

[0033] After transformation, the transformants were screened on SC-URA solid medium (synthetic yeast nitrogen source YNB 6.7 g / L, glucose 20 g / L, mixed amino acid powder lacking tryptophan, leucine, histidine and uracil 2 g / L, 0.1 g / L leucine, 0.02 g / L tryptophan, 0.02 g / L histidine). The selected transformants were verified by boiling in 20 mM NaOH solution and then PCR. The verified single colonies were isolated, purified and then tested for fermentation.

[0034] (3) Fermentation method of zeaxanthin The zeaxanthin-producing strain was activated by two stages of seed culture and then cultured in shake flasks for two-phase fermentation. 1. Primary seed: Select a single colony of the transformant containing the empty plasmid pRS416 and incubate it overnight at 30 ℃ and 250 rpm in 3 mL SC-URA liquid medium.

[0035] 2. Secondary seed: Transfer the primary seed to 5 mL of SC-URA liquid medium with an initial OD600 of 0.2 and incubate overnight at 30 °C and 250 rpm until the OD600 is approximately 6.

[0036] 3. Shake flask fermentation: The secondary seed was inoculated into a 250mL shake flask containing 50mL of liquid culture at an initial OD600=0.2 and cultured at 20 ℃ and 250 rpm for 120 h.

[0037] 4. Detection methods for zeaxanthin After fermentation, 100 μl of the sample was transferred to a clean EP tube and centrifuged at 12000 rpm for 2 min. The supernatant was aspirated using a syringe, and 500 μl of DMSO (dimethyl sulfoxide) was added. The mixture was shaken at 45°C and 1600 rpm for 20 min in the enzyme reactor. Then, 1 mL of acetone was added, and the mixture was shaken at 45°C and 1600 rpm for 20 min. After centrifugation, the sample was centrifuged at 12000 rpm for 4 min. The supernatant organic phase was aspirated into a 2 mL EP tube, filtered through a 0.22 μm organic filter, and transferred to a clean vial for HPLC analysis. The sample yield was determined using an HPLC system. The column was a BDS Hypersil C18 (150 mm × 4.6 mm, 5 μm, Thermo Scientific). HPLC conditions: Mobile phase A was acetonitrile-water containing 2% formic acid (7:3, v / v), and mobile phase B was methanol-isopropanol (3:2, v / v). The gradient conditions were: 100% A for 5 minutes, 100-10% A for 5-10 minutes, 10% A for 15 minutes, then back to 100% A after 5 minutes, and equilibrate for 10 minutes before injection. The column temperature was set to 40℃, and the flow rate was 1 mL / min. Zeaxanthin substrate was observed at a wavelength of 450 nm.

[0038] 5. Experimental Results: Further testing and phenotypic analysis confirmed that, Figure 3 As shown, overexpression of the candidate gene significantly promotes product synthesis. Specifically, this was confirmed by high-performance liquid chromatography (HPLC). The sample pretreatment steps are as follows: the reaction solution is first concentrated by vacuum centrifugation to remove the solvent, the residue is dissolved in methanol and then filtered through a syringe filter (13mm 0.22μm) for injection.

[0039] The mobile phase system consisted of phase A (acetonitrile:water, v / v 9:1) and phase B (methanol:isopropanol, v / v 3:2). The gradient elution program was set as follows: within 0–10 min, the proportion of phase B linearly increased from 0% to 90%; from 10–25 min, the proportion of phase B was maintained at 90%; from 25–35 min, the proportion of phase B linearly decreased from 90% to 0%; and then equilibrated under the initial conditions for 5 min. The column temperature was kept constant at 40℃, the flow rate was 1 mL / min, and zeaxanthin was detected at a wavelength of 450 nm.

[0040] Compared with the starting strain, overexpression of genes RRT7, PRP21, and MME1 effectively increased the synthesis and accumulation of zeaxanthin in recombinant cells. Notably, among the tested genes, SC-CGH02 achieved a yield of 465.821 mg / L, SC-CGH04 achieved a yield of 461.842 mg / L, and overexpression of the RRT7 gene showed the most significant promoting effect on the zeaxanthin synthesis pathway, with the largest increase in yield. SC-CGH03 achieved a yield of 480.502 mg / L, demonstrating extremely high potential for industrial application.

[0041] (5) Genomic integration of expression elements and construction of recombinant engineered strains Based on the screening results of the aforementioned advantageous genes, this invention further implemented the stable integration and expression modification of the target fragment in chassis cells using CRISPR / Cas9 gene editing technology. The specific construction steps of the recombinant integration plasmid are as follows: First, using plasmid Pcgh03 as a template and primers URA3-F (SEQ ID No. 16) and URA3-R (SEQ ID No. 17), the auxotrophic gene fragment (P) was obtained as a selection marker. URA -URA3-T URA Simultaneously, the template was linearized by digestion with the restriction endonuclease EcoRI at 37°C overnight to obtain fragment T. TDH2 -RRT7-P GAL1 / 10 -T FBA1 Using overlap extension PCR (OE-PCR) technology, fragment T was... TDH2 -RRT7-P GAL1 / 10 -T FBA1 P URA -URA3-T URA3 Fragment and linearized fragment T DH2 -RRT7-P GAL1 / 10 -T FBA In vitro assembly was performed, and the assembly product was transformed into E. coli competent cells TOP10. After preliminary screening by plating with appropriate antibiotics, single-clone transformants were picked, plasmids were extracted, and sequencing verification was performed. The recombinant plasmid with correct sequencing results was named pcgh05.

[0042] Pcgh05: pRS416-T DH2 -RRT7-P GAL1 / 10 -T FBA -P URA3 -URA3-T URA3 (SEQ ID No. 29) URA3 (SEQ ID No. 18) is derived from Saccharomyces cerevisiae and is a key gene involved in the biosynthesis of uracil nucleotides in yeast, providing the strain with uracil nutrient synthesis function.

[0043] Subsequently, the plasmid Pcgh06:pRS416K-Int4-L-Int4-R (SEQ ID No. 30) was linearized by single digestion with the restriction endonuclease BamHI to obtain the linearized vector pRS416-Int4-L-Int4-R. The sequence of the Int4 site is shown in SEQ ID No. 34. Using plasmid pcgh05 as a template, and Int4-TDH-F (SEQ ID No. 19) and Int4-URA3-R (SEQ ID No. 20) as primers with 25bp homologous arms added to both ends of each primer, fragment T was obtained by PCR amplification. DH2 -RRT7-P GAL1 / 10 -T FBA -P URA3 -URA3-T URA3 The fragment T was ligated using a seamless cloning method in vitro. DH2 -RRT7-P GAL1 / 10 -T FBA -P URA3 -URA3-T URA3 The plasmid pRS416-Int4-L-Int4-R was combined with linearized vector pRS416-Int4-L and introduced into E. coli competent cells TOP10. Transformants were selected and sequenced to obtain plasmid pcgh07: pRS416-Int-LT. DH2 -RRT7-P GAL1 / 10 -T FBA -P URA3 -URA3-T URA3 -Int-R (SEQ ID No. 31) Using pcgh07 as a template and Int4-F (SEQ ID No. 21) and Int4-R (SEQ ID No. 22) as primers, the fragment Int4-L-TDH2-RRT7-P was obtained. GAL1 / 10 -T FBA -P URA3 -URA3-T URA3-Int4-R, and then used an online tool (http: / / crispor.tefor.net / crispor.py) to find and identify the target gRNA sequence. Using primers: GRNA-Int4-F (SEQ ID No. 23), GRNA-Int4-R (SEQ ID No. 24) at both ends of sgRNA-Int4 (SEQ ID No. 25), the synthesized upstream and downstream primers were diluted with double-distilled water (ddH2O) to a final concentration of 100 μM.

[0044] Prepare 10× Annealing Buffer, which is a mixture of 0.5M Tris-HCl (pH 7.4) and 0.1 M MgCl2; or use 30 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid (HEPES)) buffer with a pH of 7.8 instead. Prepare a 20 μL annealing reaction system in a PCR tube. This system contains: 9 μL of diluted upstream primer and 2 μL of 10× Annealing Buffer (Note: the remaining volume is usually made up to 20 μL with downstream primer and double-distilled water). Place the above system in a PCR instrument for annealing. The reaction conditions are set as follows: first, incubate at 95℃ for 5 min; then begin a cooling program, starting from 94℃ and decreasing the temperature by 1℃ every 6 s until the temperature drops to 14℃ (this can also be set to 40 cycles, with the temperature decreasing by 2℃ per cycle); finally, incubate at 4℃.

[0045] Prepare a 20 μL Golden Gate assembly reaction system in a sterile PCR tube. The specific reaction components and amounts are as follows: target plasmid vector pcgh08:pZK003 (SEQ ID No. 32): 1 μL; annealed gRNA product: 3 μL; 10×T4 DN ligase buffer: 2 μL; T4 DNA ligase: 1 μL; restriction endonuclease BsaI-v2: 1 μL; double-distilled water (ddH2O): 12 μL. Place the prepared reaction system in a PCR instrument and perform the Golden Gate assembly reaction according to the following temperature cycling program: Cyclic reaction: Incubate at 37°C for 10 minutes, then at 22°C for 10 minutes. This process is repeated 9 times. Enzyme inactivation and stability: After the cycle is completed, incubate at 65 °C for 10 minutes, followed by incubation at 80 °C for 20 minutes (to completely inactivate the endonuclease and ligase). Storage: Finally, keep warm at 4℃. After the assembly reaction, an appropriate amount of the assembly product was transformed into TOP10 *E. coli* competent cells for subsequent positive clone screening and expansion culture. The day after transformation, the colonies on the culture medium were validated by fluorescence. Green excitation fluorescence was used for fluorescence imaging, and colonies showing no fluorescence were selected as positive colonies correctly ligated to the target fragment. Two positive colonies that passed the initial fluorescence screening were picked from the plate and submitted for Sanger sequencing verification. The correctly sequenced transformants were cultured at 37°C for 12 hours, and the plasmid was extracted to obtain plasmid pcgh09:Int4-CRISPR / Cas9 (SEQ ID No. 33). Finally, using the conventional lithium acetate conversion method in this field, the plasmid pcgh07 containing the target gene RRT7 and the DNA expression framework marked with URA3 and pcgh09 targeting the Int4 site were introduced into strain YCGH06, and chromosome integration was achieved through homologous recombination.

[0046] After transformation, the bacterial cells were revived and plated on uracil-deficient auxoselective medium (SC-Ura) for initial screening. Single colonies of well-grown transformants were selected for further verification by colony PCR and gene sequencing to confirm the target expression cassette T. TDH2 -RRT7-P GAL1 / 10 -T FBA1 -P URA3 -URA3-T URA3 The recombinant high-yield yeast strain was successfully and stably integrated into the Int4 site of the Saccharomyces cerevisiae genome. The strain that ultimately achieved positive validation was named YC01, with a yield reaching 489.56 mg / L. Figure 3 This strain can serve as an excellent fermentation strain for the subsequent large-scale fermentation production of zeaxanthin.

[0047] Table 1: Strains used in this study

[0048] Table 2: Plasmids used in this invention

[0049] Table 3 Primer sequences used in this invention

[0050] The above embodiments only describe a portion of the specific implementation methods of the present invention in detail, and are not limited to the embodiments disclosed herein. Furthermore, the substantive content protected by the present invention is not limited thereto. Any other modifications, equivalent substitutions, improvements, etc., made based on the principles and techniques of the present invention without departing from its design scope are all within the protection scope of the present invention.

Claims

1. A method for constructing a high-yield, zeaxanthin-recombinant brewer's yeast engineered strain, characterized in that, Using *Saccharomyces cerevisiae*, a yeast that produces zeaxanthin, as the chassis strain, the target gene MME1, RRT7, or PRP21 was introduced. The nucleotide sequence of MME1 is shown in SEQ ID No. 3, the nucleotide sequence of RRT7 is shown in SEQ ID No. 11, and the nucleotide sequence of PRP21 is shown in SEQ ID No.

15.

2. The method for constructing the high-yield zeaxanthin recombinant brewer's yeast engineered strain according to claim 1, characterized in that, Using *Saccharomyces cerevisiae*, a strain that produces zeaxanthin, as the chassis strain, the genes RRT7 and URA3 were integrated into the genome at the Int4 site of the *Saccharomyces cerevisiae* genome. The sequence of the Int4 site is shown in SEQ ID No. 34, the nucleotide sequence of RRT7 is shown in SEQ ID No. 11, and the nucleotide sequence of URA3 is shown in SEQ ID No.

18.

3. The method for constructing the high-yield zeaxanthin recombinant brewer's yeast engineered strain according to claim 2, characterized in that, The brewer's yeast that produces zeaxanthin is strain YCGH06, with the genotype CEN.PK2-1C,Δgal1Δgal7Δgal10::nat-TERG13-ERG13-PGAL7-TERG19-ERG19-PGAL10-PGAL1-ERG10-TERG10,Δypl062w::DR-TCYC1-BtCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,trp1::TRP1_TCYC1-BtCrtI-PGAL10-PGAL1-PaCrtB-TPGK1. leu2::LEU2_TTDH2-DR-TCYC1-BtCrtI-PGAL3-TACT1-tHMG1-PGAL10-PGAL1-TmCrtE-TGPM1,his3::HIS3-TENO2-TACT1-tHMG1-PGAL10-PGAL1-(BT S1-ERG20)-TFBA1YGLCtau3::hphA-TIDI1-IDI1-PGAl7-TPDC1-SaPMK-PGAL10-PGAl1-SaMK-THXT7,YMRWdelta15::UAS-PGAL1-PaCrtY-TADH1-DR, YNRCdelta9::UAS-PGAL1-PaCrtY-TADH1-DR, HO::PTEF1-AtRFNR1-TGPM1-PTDH3-AtFD3-TPGI1-G418R, TKL2-PGAL10-EuCrtZ(M92L)-TFBA1-TEF2, Int4:-PGAL10-EuCrtZ(M92L)-TFBA1-TEF2, DAK2- P GAL10 -HSC82 -T FBA1 。 4. The method for constructing high-yield zeaxanthin recombinant brewer's yeast engineered strain according to claim 1, characterized in that, The genome integration was achieved using the CRISPR / Cas9 gene editing system.

5. The method for constructing the high-yield zeaxanthin recombinant brewer's yeast engineered strain according to claim 4, characterized in that, The RRT7 and URA3 genes in the genome of Saccharomyces cerevisiae were knocked into at specific sites using the CRISRPR / Cas9 gene editing system. The sgRNA sequence used for knocking into the RRT7 and URA3 genes was sgRNA-Int4, and the nucleotide sequence is shown in SEQ ID No.

25.

6. A high-yield recombinant brewer's yeast strain for zeaxanthin, constructed by the construction method described in any one of claims 2-5.

7. The application of the recombinant brewer's yeast engineered strain according to claim 6 in the production of zeaxanthin.

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