Recombinant yeast for producing alpha-ionone and its construction method and application

Abstract

This invention discloses a recombinant yeast for producing α-ionone, its construction method, and its applications. The construction method involves using a yeast encoding osmanthus flowers... Osmanthus fragrans The gene for the carotenoid cleavage dioxygenase OfCCD1 was obtained through rational modification and codon optimization. OfCCD1m The gene, with its 5' end fused to the N-terminal membrane-anchored peptide coding sequence of human tyrosine kinase LCK, was transformed into the δ site of Saccharomyces cerevisiae TW3 to obtain recombinant yeast TW4 producing α-ionone. The Saccharomyces cerevisiae strain TW4 producing α-ionone of this invention has the following beneficial effects: it achieves de novo synthesis of α-ionone in Saccharomyces cerevisiae for the first time, filling a technological gap in this field; by using a membrane anchoring strategy to locate the OfCCD1m enzyme in the yeast plasma membrane, the contact efficiency between the enzyme and the substrate ε-carotene is significantly improved, resulting in an α-ionone yield of 1.46 mg / L.

Description

Technical Field

[0001] This invention relates to recombinant yeast for producing α-ionone, its construction method and application, belonging to the fields of genetic engineering and application and microbial technology. Background Technology

[0002] α-ionone (CAS No.: 127-41-3) is a cyclic monoterpenoid compound with a violet aroma, widely found in economic crops such as raspberries, osmanthus, and tea, as well as plants in the Iridaceae family. Due to its extremely low odor threshold and unique aroma characteristics, this compound has become one of the most important commercial fragrance varieties, widely used in the fields of fragrances, cosmetics, pharmaceuticals, and food. Furthermore, α-ionone also possesses various physiological activities, including antibacterial, antioxidant, anti-pest, anti-photoaging, and tumor cell apoptosis-inducing activities, demonstrating broad application prospects.

[0003] Currently, the production of α-ionone mainly relies on plant extraction and chemical synthesis. Plant extraction is limited by the extremely low content of α-ionone in natural raw materials, and its frequent coexistence with β-ionone leads to high separation and purification costs, making it difficult to meet stable commercial demand. Chemical synthesis uses citral and acetone as raw materials, condensing them to produce pseudoionone, which is then cyclized to generate α-ionone. However, this method suffers from high costs, poor safety, and mixed reaction products, and the high cost of key starting materials makes it unsustainable.

[0004] In recent years, the biosynthesis of α-ionone using microbial cell factories has emerged as a highly promising alternative. Currently, heterologous synthesis of α-ionone has been achieved in *E. coli*. However, *E. coli* as a production host faces challenges such as metabolic flux bottlenecks and limitations in industrial production, restricting its application in the food and cosmetic fields. In *Yarrowia lipolytica*, related research has only reached the stage of isotope tracing of added substrates and optimization of fermentation conditions, and de novo biosynthesis of α-ionone has not yet been achieved. Furthermore, *Yarrowia lipolytica* suffers from relatively scarce genetic manipulation tools, low efficiency of multi-gene co-integration, and long gene editing cycles, limiting the efficiency and speed of its metabolic engineering modification.

[0005] Among numerous microbial systems, Saccharomyces cerevisiae (Saccharomyces cerevisiae) Saccharomyces cerevisiaeWith its mature genetic manipulation system, robust industrial fermentation performance, and the advantages of its natural MVA pathway, *Saccharomyces cerevisiae* has been recognized as a Generally Recognized as Safe (GRAS) yeast by the U.S. Food and Drug Administration, making it more suitable for the production of food flavorings and fragrances or cosmetic raw materials. Furthermore, the natural mevalonic acid (MVA) pathway of *Saccharomyces cerevisiae* provides ample isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) precursors for the synthesis of terpenoids, and its endomembrane system and organelle compartments provide a more suitable environment for the synthesis of target products.

[0006] Although *Saccharomyces cerevisiae* has shown great potential in the synthesis of terpenoids, and β-ionone has been biosynthesized in *Saccharomyces cerevisiae*, there are no reports to date of de novo biosynthesis of α-ionone in *Saccharomyces cerevisiae*. Therefore, developing engineered yeast capable of synthesizing α-ionone in *Saccharomyces cerevisiae* is of great significance for achieving the green and sustainable production of α-ionone. Summary of the Invention

[0007] The first objective of this invention is to overcome the shortcomings of the prior art and provide a recombinant yeast for producing α-ionone.

[0008] A second objective of this invention is to provide a method for constructing the aforementioned recombinant yeast.

[0009] A third objective of this invention is to provide the application of the aforementioned recombinant yeast in the fermentation production of α-ionone.

[0010] A fourth objective of this invention is to provide a second recombinant yeast for producing α-ionone.

[0011] The fifth objective of this invention is to provide a method for constructing the second recombinant yeast described above.

[0012] The sixth object of the present invention is to provide the application of the second recombinant yeast described above in the fermentation production of α-ionone.

[0013] The technical solution of this invention is summarized as follows: A method for constructing recombinant yeast for producing α-ionone includes the following steps: The code originates from osmanthus. Osmanthus fragrans The gene for the carotenoid cleavage dioxygenase OfCCD1, according to literature reports, underwent physical modification and codon optimization. OfCCD1m The gene was fused to the 5' end with the N-terminal membrane anchoring peptide coding sequence of human tyrosine kinase LCK, and transformed into the δ site of Saccharomyces cerevisiae TW3 to obtain recombinant yeast TW4 that produces α-ionone. The brewing yeast TW3 is a yeast capable of producing ε-carotene; The managerial modifications and codon optimizations OfCCD1m The nucleotide sequence of the gene is shown in SEQ ID NO.1; The nucleotide sequence encoding the membrane-anchored peptide is shown in SEQ ID NO.12.

[0014] The above-described method was used to construct the recombinant yeast TW4, which produces α-ionone.

[0015] The above-mentioned application of recombinant yeast TW4 fermentation for the production of α-ionone.

[0016] The second method for constructing recombinant yeast for producing α-ionone includes the following steps: The code is derived from Salmonella ( Salmonella enteritidis The gene and encoding of acetyl-CoA synthase SeACS are derived from Pantotheca pineapple. Pantoea ananatis The codons of the GGPP synthase PaCrtE were optimized to obtain the optimized SeACS enzyme encoding gene and the optimized PaCrtE enzyme encoding gene. The optimized SeACS enzyme encoding gene, the optimized PaCrtE enzyme encoding gene, and the endogenous Saccharomyces cerevisiae gene were used. ADH2 Genes and ALD6 Gene expression cassettes were constructed and recombinant yeast TW4 was introduced. ypl062w The site was identified to obtain the recombinant yeast TW6, which produces α-ionone; The ADH2 The nucleotide sequence of the gene is shown in SEQ ID NO.15; The ALD6 The nucleotide sequence of the gene is shown in SEQ ID NO.16; The nucleotide sequence of the gene encoding the optimized SeACS enzyme is shown in SEQ ID NO.14; The nucleotide sequence of the gene encoding the optimized PaCrtE enzyme is shown in SEQ ID NO.7.

[0017] The recombinant yeast TW6 that produces α-ionone was constructed using the second construction method described above.

[0018] The above-mentioned application of recombinant yeast TW6 fermentation for the production of α-ionone.

[0019] The present invention has the following beneficial effects: The *Saccharomyces cerevisiae* strain TW4, which produces α-ionone according to this invention, has the following beneficial effects: (1) For the first time, de novo synthesis of α-ionone was achieved in Saccharomyces cerevisiae, filling a technological gap in this field; (2) By using a membrane anchoring strategy to localize the OfCCD1m enzyme to the yeast plasma membrane, the contact efficiency between the enzyme and the substrate ε-carotene was significantly improved, resulting in an α-ionone yield of 1.46 mg / L.

[0020] The *Saccharomyces cerevisiae* strain TW6, which produces α-ionone according to this invention, has the following beneficial effects: (1) The supply of acetyl-CoA was effectively increased by the precursor enhancement strategy, and the yield of α-ionone in shake flasks reached 2.07 mg / L; (2) A two-stage feeding strategy was adopted in a 5L fermenter, and the yield of α-ionone reached 20.5 mg / L, laying the foundation for industrial production. Attached Figure Description

[0021] Figure 1 The yield of α-ionone synthesized by shake-flask fermentation of recombinant yeasts TW4 and TW6 was determined. All recombinant strains were fermented using YPD liquid medium.

[0022] Figure 2 Feed-and-batch fermentation process curve of recombinant yeast TW6 in a 5-L fermenter. Detailed Implementation

[0023] The present invention will be further described below through specific embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] The construction methods of the starting strains sc027 and TW3 used in this experiment are as follows: Saccharomyces cerevisiae strain sc027 is derived from Saccharomyces cerevisiae ( Saccharomyces cerevisiaeCEN.PK2-1C was constructed. CEN.PK2-1C (commercially available, genotype: MAT a; ura3-52, trp1-289, leu2-3,112, his311, MAL2-8C, SUC2) was constructed based on homologous recombination of CYB5, ERG19, ERG8, ERG12, ERG10, and tHMG1 (SEQ ID NO: 1). NO.5), IDI1, ERG13, ERG20, ALDH1, and CPR1 genes were inserted into the CEN.PK2-1C genome. Specifically, IDI1 and tHMG1 were inserted into the ADE1 site, ERG20 and tHMG1 were inserted into the TRP1 site, ERG13 and tHMG1 were inserted into the URA3 site, ALDH1, ERG10, and ERG12 were inserted into the HIS3 site, CYB5, ERG19, and ERG8 were inserted into the LEU2 site, and CPR1 was inserted into the GAL1 site. The HIS3, ADE1, and TRP1 genes were then added to finally obtain the Saccharomyces cerevisiae sc027 strain.

[0027] (Gene accession numbers are shown in Table 1) (Article source: Metabolic Engineering of Saccharomyces cerevisiae forEnhanced Dihydroartemisinic Acid Production) The TW3 strain of Saccharomyces cerevisiae used in this embodiment is derived from Saccharomyces cerevisiae (… Saccharomyces cerevisiae The sc027 build specifically includes: (1) DPP1 and LPP1 Site modification The ERG20 gene derived from Saccharomyces cerevisiae was mutated by F96C to obtain the mERG20 gene (SEQ ID NO.28). The mERG20 gene was then placed in P... GAL7 Under the control of the starter, with T ADH1 As a terminator, it was integrated into the DPP1 site of Saccharomyces cerevisiae strain sc027 via homologous recombination; The code is derived from Pantothecin pineapple ( Pantoea ananatis The gene encoding the GGPP synthase PaCrtE was codon-optimized, and the optimized PaCrtE enzyme encoding gene (SEQ ID NO.7) was placed in P... GAL7 Under the control of the starter, with T CYC1 As a terminator, it was integrated into the LPP1 site of the aforementioned Saccharomyces cerevisiae strain via homologous recombination.

[0028] (2) MET17 Site integration The codon-optimized encoding is derived from Bacillus clumps (Pantoea agglomerans The gene for phytoene synthase PagCrtB (SEQ ID NO. 6) was reverse-inserted and placed in P... GAL1 Under the control of the starter (in reverse), with T ERG9 (Reverse) is the terminator; the gene encoding the optimized PaCrtE enzyme (SEQ ID NO.7) is placed in P. GAL10 Under the control of the starter, with T ERG1 The terminator was LEU2; using LEU2 as a screening marker, it was integrated into the MET17 site of the Saccharomyces cerevisiae strain obtained in step (1) through homologous recombination.

[0029] (3) RPL25 Site integration Build RPL25 The site expression cassette is connected in sequence to the following components: The RPL25 gene self-promoter P, which is the 5' homologous arm, RPL25 ; With P RPL25 Starter control, with T RPL25 The hygromycin resistance gene Hyg (SEQ ID NO. 8) from the commercially available plasmid pACBSR-hyg was used as a selection marker for the terminator; The autonomously replicating sequence ARS405 (SEQ ID NO.9) is derived from the genome of Saccharomyces cerevisiae. The codon-optimized gene encoding the phytoene synthase PagCrtB from Bacillus clumps (SEQ ID NO. 6) was reverse-inserted and placed in the P... GAL10 Under the control of the starter (in reverse), with T ERG9 (Reverse) is the terminator; The gene encoding the phytoene dehydrogenase BtCrtI from Blakesleatrispora (SEQ ID NO. 11), with its 3' stop codon TAA removed, and the gene encoding the phytoene ε-cyclase LsLCYE from lettuce (SEQ ID NO. 2), with its nucleotide optimized form, were fused via a linker peptide (coding sequence shown in SEQ ID NO. 3) to obtain the gene encoding the CrtI-linker-LCYE fusion protein (nucleotide sequence shown in SEQ ID NO. 4). This fusion gene was then placed in P... GAL1 Under the control of the starter, with T ENO2 For termination; P derived from the genome of Saccharomyces cerevisiae BTS1 promoter; And a partial fragment of the RPL25 gene as the 3' homologous arm (SEQ ID NO.10); The above RPL25 The site expression cassette was integrated into the genome of the strain obtained in step (2) via homologous recombination. RPL25 The transformed organism was subjected to a series of screenings on YPD plates containing 300 μg / mL, 500 μg / mL, 800 μg / mL, and 1000 μg / mL hygromycin. The resulting highly resistant clone, *Saccharomyces cerevisiae* TW3, was obtained and capable of growing on 1000 μg / mL hygromycin plates. *Saccharomyces cerevisiae* TW3 is a yeast capable of producing ε-carotene.

[0030] Table 1. Gene Accession Numbers (NCBI)

[0031] Example 1: Construction of recombinant yeast TW4 for producing α-ionone This embodiment constructs a recombinant yeast TW4 with OfCCD1m enzyme anchored to the yeast plasma membrane.

[0032] (1) Synthesis and codon optimization of the OfCCD1m gene The code originates from osmanthus ( Osmanthus fragrans The gene encoding the carotenoid cleavage dioxygenase OfCCD1 was rationally modified and codon-optimized according to the literature (Integrating Enzyme and Metabolic Engineering Tools for Enhanced α-Ionone Production) to obtain the OfCCD1m gene, which was synthesized by Genewiz Biotechnology Co., Ltd. and integrated into the pESC-URA plasmid to obtain pESC-OfCCD1m. The nucleotide sequence of the rationally modified and codon-optimized OfCCD1m gene is shown in SEQ ID NO.1.

[0033] (2) Construction of δ site integration expression cassette Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PCR amplification was performed using primers δup-F (SEQ ID NO.17) and δup-PGAL10-R (SEQ ID NO.18) to obtain the δup fragment, the homologous arm upstream of the δ site.

[0034] Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, PCR amplification was performed using primers PGAL10-δup-F (SEQ ID NO.19) and PGAL10-LCK-R (SEQ ID NO.20) to obtain P... GAL10 Promoter fragment; Using plasmid pESC-OfCCD1m as a template, PCR amplification was performed using LCK-PGAL10-F (SEQ ID NO.21) and OfCCD1m-TENO2-R (SEQ ID NO.22) to obtain a gene fragment encoding the N-terminal membrane anchoring peptide of human tyrosine kinase LCK fused to the 5' end of OfCCD1m. The nucleotide sequence of the membrane anchoring peptide encoding sequence is shown in SEQ ID NO.12. Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, PCR amplification was performed using primers TENO2-OfCCD1m-F (SEQ ID NO. 23) and TENO2-TCYC1-R (SEQ ID NO. 24) to obtain T... ENO2 Termination of sub-fragment.

[0035] Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, PCR amplification was performed using primers TCYC1-TENO2-F (SEQ ID NO.25) and TCYC1-AurR-R (SEQ ID NO.26) to obtain T... CYC1 Termination of sub-fragment.

[0036] Using the commercially available pAbAi plasmid containing the aurin resistance selection marker AurR as a template, PCR amplification was performed using primers AurR-TCYC1-F (SEQ ID NO.27) and AurR-PHIS3-R (SEQ ID NO.29) to obtain the aurin resistance selection marker AurR fragment, the nucleotide sequence of which is shown in SEQ ID NO.13.

[0037] Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, PCR amplification was performed using primers PHIS3-AurR-F (SEQ ID NO.30) and PHIS3-δR-R (SEQ ID NO.31) to obtain P... HIS3 Promoter segment.

[0038] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PCR amplification was performed using primers δdown-PHIS3-F (SEQ ID NO.32) and δdown-R (SEQ ID NO.33) to obtain the δdown fragment, the homologous arm downstream of the δ site. The amplified δup, PGAL10, the 5'-fused N-terminal membrane anchor peptide encoding sequence of human tyrosine kinase LCK OfCCD1m fragment, TENO2, TCYC1, AurR, PHIS3, and δdown were fused by overlap extension PCR. Using δup-F (SEQ ID NO.17) and δdown-R (SEQ ID NO.33) as primers, the δ site integration expression cassette fragment was obtained.

[0039] (3) Conversion of brewing yeast The above-mentioned δ-site integrated expression cassette fragment was introduced into the δ-site of Saccharomyces cerevisiae TW3 using the lithium acetate conversion method.

[0040] Specifically, the pre-prepared TW3 competent cells of *Saccharomyces cerevisiae* were removed from a -80°C freezer and thawed on ice. The yeast transformation system was prepared as follows: 120 μL 50% PEG-3350, 18 μL 1 M lithium acetate (water) solution, and 5 μL 10 mg / mL salmon sperm DNA. The thawed competent cells were centrifuged at 5000 rpm for 3 min, the supernatant was discarded, and the cells were thoroughly mixed with 37 μL of sterile aqueous solution containing 2000 ng of the δ-site integration expression cassette fragment. This mixture was then added to the yeast transformation system and vortexed for 30 s to ensure homogeneity. The mixture was then heat-shocked in a 42°C water bath for 30 min, centrifuged at 5000 rpm for 3 min, the supernatant was discarded, 1 mL of YPD medium was added, and the mixture was thoroughly mixed. The mixture was then incubated at 30°C and 220 rpm for 4 h in a shaker. After incubation, the cells were centrifuged at 5000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in sterile water. The cells were then centrifuged again at 5000 rpm for 3 min, the supernatant was discarded, and the washing was repeated once to remove as much residual YPD medium as possible. The cells were then evenly aspirated with residual sterile water and spread onto YPD plates containing 300 ng / mL adenosine monophosphate. The plates were incubated at 30°C for 3 days. Positive clones were selected and sequentially transferred to YPD plates containing 600 ng / mL adenosine monophosphate for selection to obtain positive yeast clones. Single colonies were selected for colony PCR verification, and the correctly verified positive clone was named TW4.

[0041] Example 2: Fermentation of recombinant yeast TW4 and determination of α-ionone yield (1) Fermentation of recombinant yeast TW4 Recombinant yeast TW4 was inoculated into a test tube containing 3 mL of YPD liquid medium and cultured overnight at 30°C in a shaker for 16 h. When the yeast reached the logarithmic growth phase, it was transferred to a 250 mL shake flask containing 50 mL of YPD liquid medium. The inoculation amount was controlled so that the initial OD600 was 0.05. The yeast was cultured and fermented at 30°C in a shaker at 220 rpm for 96 h. After 30 h of fermentation, 5% galactose was added to induce gene expression, and 10% dodecane was added as an extraction solvent.

[0042] (2) Preparation of α-ionone standard curve Weigh out α-ionone standard (purchased from Sigma-Aldrich, purity ≥95%), dissolve and dilute it with dodecane to different concentrations (0.1 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 2.5 mg / L), and analyze it by GC-MS. Plot a standard curve with concentration as the x-axis and peak area as the y-axis for quantitative analysis of α-ionone in the sample.

[0043] (3) GC-MS detection conditions After fermentation, 35 mL of fermentation broth was transferred to a 50 mL centrifuge tube and centrifuged at 5000 rpm for 3 min. 1 mL of the upper organic layer was transferred to a 2 mL EP tube, dried with a small amount of anhydrous sodium sulfate, filtered through a 0.22 μm nylon 66 filter membrane, and then placed into a sample vial for GC-MS detection.

[0044] The GC detection conditions were as follows: HP-5 column (30 m × 0.250 mm, 0.25 μm), injection volume 1 μL, split ratio 1:10, initial temperature 80℃ held for 1 min, temperature increased to 120℃ at 10℃ / min held for 1 min, and then increased to 300℃ at 10℃ / min held for 2 min.

[0045] (4) Measurement results The α-ionone yield of recombinant yeast TW4 was 1.46 mg / L, see [link to relevant documentation]. Figure 1 .

[0046] Example 3: Construction of recombinant yeast TW6 for producing α-ionone This embodiment constructs a recombinant yeast TW6 with enhanced supply of precursor acetyl-CoA. (1) Genes encoding SeACS and PaCrtE of Gene synthesis and codon optimization The code is derived from Salmonella ( Salmonella enteritidis Acetyl-CoA synthase SeACS The gene encodes a gene from Pantotheca pineapple ( Pantoea ananatis GGPP synthase PaCrtEThe gene was developed by Genewiz Biotechnology Co., Ltd. using Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae Codon optimization was performed on the host to obtain optimized SeACS enzyme coding genes and optimized PaCrtE enzyme coding genes. The optimized SeACS enzyme coding genes and optimized PaCrtE enzyme coding genes were integrated into the pESC-URA plasmid to obtain pESC-SeACS and pESC-PaCrtE, respectively. The nucleotide sequence of the optimized SeACS enzyme coding gene is shown in SEQ ID NO.14, and the nucleotide sequence of the optimized PaCrtE enzyme coding gene is shown in SEQ ID NO.7.

[0047] (2) Construction of the ypl062w site integration expression cassette Using the genome of Saccharomyces cerevisiae CEN.PK2-1C as a template, PCR amplification was performed using primers LF (SEQ ID NO.34) and L-PGAL2-R (SEQ ID NO.35) to obtain the homologous arm fragment at the ypl062w site; Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, PCR amplification was performed using primers PGAL2-LF (SEQ ID NO.36) and PGAL2-ALD6-R (SEQ ID NO.37) to obtain the PGAL2 promoter P. GAL2 Fragment; Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, PCR amplification was performed using primers ALD6-PGAL2-F (SEQ ID NO.38) and ALD6-TACS1-R (SEQ ID NO.39) to obtain a genome containing ALD6 and its corresponding terminator T. ALD6 The fragment, the nucleotide sequence of the ALD6 gene is shown in SEQ ID NO.16; Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, PCR amplification was performed using primers TACS1-ALD6-F (SEQ ID NO.40) and TACS1-CrtE-R (SEQ ID NO.41) to obtain T... ACS1 Fragment; Using the plasmid pESC-PaCrtE containing the PaCrtE gene as a template, PCR amplification was performed using primers CrtE-TACS1-F (SEQ ID NO. 42) and PGAL1-SeACS-R (SEQ ID NO. 43) to obtain a plasmid containing PaCrtE and the promoter P. GAL10-GAL1 A fragment.

[0048] Using the plasmid pESC-SeACS containing the SeACS gene as a template, PCR amplification was performed using primers SeACS-PGAL1-F (SEQ ID NO.44) and SeACS-TADH3-R (SEQ ID NO.45) to obtain the SeACS gene fragment.

[0049] Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, PCR amplification was performed using primers TADH3-SeACS-F (SEQ ID NO.46) and TADH3-ADH2-R (SEQ ID NO.47) to obtain T... ADH3 Fragment; Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, PCR amplification was performed using primers ADH2-TADH3-F (SEQ ID NO.48) and ADH2-PGAL7-R (SEQ ID NO.49) to obtain the gene containing the ADH2 gene and its corresponding terminator T. ADH2 The fragment, the nucleotide sequence of the ADH2 gene is shown in SEQ ID NO.15; Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, PCR amplification was performed using primers PGAL7-ADH2-F (SEQ ID NO.50) and PGAL7-URA-R (SEQ ID NO.51) to obtain P... GAL7 Promoter segment.

[0050] Using the expression vector pESC-URA empty plasmid (commercially available) as a template, PCR amplification was performed using primers URA-PGAL7-F (SEQ ID NO. 52) and URA-RR (SEQ ID NO. 53) to obtain the URA3 gene fragment.

[0051] PCR amplification was performed using primers R-URA-F (SEQ ID NO.54) and RR (SEQ ID NO.55) to obtain the homologous arm fragment at the ypl062w site.

[0052] The fragments amplified above were subjected to overlap extension PCR (OE-PCR) using LF (SEQ ID NO.34) and RR (SEQ ID NO.55) as primers to obtain the ypl062w site integration expression cassette.

[0053] (3) Conversion of brewing yeast Following the transformation method in step (3) of Example 1, the above-mentioned ypl062w site integration expression cassette was transformed into recombinant bacteria TW4. The recombinant bacteria were plated on SD medium plates lacking uracil for screening. Single colonies were picked for colony PCR verification, and the positive clones that were verified correctly were named TW6.

[0054] Example 4: Fermentation process and α-ionone content detection of recombinant yeast TW6 (1) Shake flask fermentation Shake-flask fermentation of recombinant yeast TW6 was carried out according to the method in Example 2. The results showed that the α-ionone yield of recombinant yeast TW6 was 2.07 mg / L. Figure 1 .

[0055] (2) 5 L fermenter feeding and batch fermentation ① Seed liquid preparation Recombinant yeast TW6 was inoculated onto YPD plates and cultured at 30°C for 2 days. A single colony was picked and inoculated into 10 mL of YPD medium and cultured for 24 h. The inoculum was then transferred to 200 mL of YPD medium at a 1% inoculation rate and cultured until the OD600 reached 6-8, which was used as the secondary seed culture.

[0056] ② Fermentation tank cultivation 1.5 L of fermentation medium (20 g / L glucose, 2.5 g / L ammonium sulfate, 8 g / L potassium dihydrogen phosphate, 6.15 g / L magnesium sulfate heptahydrate, 1 g / L methionine, 1 g / L leucine, 1 mL / L defoamer (purchased from Yantai Hengxin Chemical Technology Co., Ltd.), 10 mL / L trace metal solution, 12 mL / L vitamin solution, 20 g / L peptone, and 10 g / L yeast extract) was loaded into a 5 L fermenter. The secondary seed culture was then inoculated into the fermenter. The culture temperature was controlled at 30℃, the pH at 5.5, the dissolved oxygen at above 35%, and the rotation speed was adjusted between 300-900 rpm.

[0057] When OD600 reached 40, 5% galactose was added to induce gene expression, and 300 mL of dodecane was added as the organic phase to collect α-ionone. A two-stage feeding strategy was adopted: glucose was added starting at 19 h, maintaining a glucose concentration not higher than 3 g / L; ethanol was added starting when OD600 reached approximately 90, maintaining an ethanol concentration not higher than 9 g / L.

[0058] ③Product testing After fermentation for 168 h, the fermentation broth was extracted, and the α-ionone content was detected by GC-MS. The results showed that the recombinant yeast TW6 produced 20.5 mg / L of α-ionone, with an OD600 of approximately 100. Figure 2 .

[0059] Table 2 Formulas for Trace Metal Solutions Used in Fermentation Tanks

[0060] Table 3. Vitamin solution formula for fermentation tanks

Claims

1. A method for the construction of a recombinant yeast producing α-ionone, characterized in that Includes the following steps: The code originates from osmanthus. Osmanthus fragrans The gene for the carotenoid cleaving dioxygenase OfCCD1 was modified and its codons optimized. OfCCD1m The gene was fused to the 5' end with the N-terminal membrane anchoring peptide coding sequence of human tyrosine kinase LCK, and transformed into the δ site of Saccharomyces cerevisiae TW3 to obtain recombinant yeast TW4 that produces α-ionone. The managerial modifications and codon optimizations OfCCD1m The nucleotide sequence of the gene is shown in SEQ ID NO.1; The nucleotide sequence encoding the membrane-anchored peptide is shown in SEQ ID NO.

12.

2. The recombinant yeast TW4 for producing α-ionone constructed by the construction method of claim 1.

3. The application of the recombinant yeast TW4 as described in claim 2 for the fermentation production of α-ionone.

4. A method for constructing recombinant yeast for producing α-ionone, characterized in that... Includes the following steps: The code is derived from Salmonella ( Salmonella enteritidis The gene and encoding of acetyl-CoA synthase SeACS are derived from Pantotheca pineapple. Pantoea ananatis The codons of the GGPP synthase PaCrtE gene were optimized to obtain the optimized SeACS enzyme encoding gene and the optimized PaCrtE enzyme encoding gene. The optimized SeACS enzyme encoding gene, the optimized PaCrtE enzyme encoding gene, and the endogenous Saccharomyces cerevisiae gene were used. ADH2 Genes and ALD6 Gene expression cassette was constructed and the recombinant yeast TW4 of claim 2 was introduced. ypl062w The site was identified to obtain the recombinant yeast TW6, which produces α-ionone; The ADH2 The nucleotide sequence of the gene is shown in SEQ ID NO.15; The ALD6 The nucleotide sequence of the gene is shown in SEQ ID NO.16; The nucleotide sequence of the gene encoding the optimized SeACS enzyme is shown in SEQ ID NO.14; The nucleotide sequence of the gene encoding the optimized PaCrtE enzyme is shown in SEQ ID NO.

7.

5. The recombinant yeast TW6 for producing α-ionone constructed by the construction method of claim 4.

6. The application of the recombinant yeast TW6 as described in claim 5 for the fermentation production of α-ionone.

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