Recombinant saccharomyces cerevisiae for producing lycopene and application thereof
By modifying the glucose concentration-inducible promoter of Saccharomyces cerevisiae and replacing it with the mutant HXT1 promoter, the problem of galactose-dependent lycopene fermentation in Saccharomyces cerevisiae was solved, achieving efficient and low-cost lycopene production with a significant increase in yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HEILONGJIANG NHU BIOTECH CO LTD
- Filing Date
- 2026-03-27
- Publication Date
- 2026-06-30
AI Technical Summary
Existing brewing yeast fermentation of lycopene relies on galactose induction, resulting in high costs, and galactose is expensive, making industrial-scale production difficult.
By modifying the glucose concentration-induced promoter and replacing the PGAL80 promoter on the Saccharomyces cerevisiae genome with the mutant HXT1 promoter, glucose was used as an inducer to precisely regulate the expression of key enzymes in the lycopene synthesis pathway. The genes were integrated using the CRISPR-Cas9 technology to construct recombinant Saccharomyces cerevisiae, thereby achieving decoupled regulation of fermentation and production.
The efficient production of lycopene was achieved through glucose induction, which reduced production costs, improved fermentation efficiency, decoupled cell growth and product synthesis, and achieved an OD600 of over 400 and a lycopene yield of over 4.90 g/L after 120 h of fermentation in YPD medium.
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Abstract
Description
Technical Field
[0001] This invention relates to a recombinant brewing yeast for producing lycopene and its application, belonging to the field of microbial technology. Background Technology
[0002] Lycopene is a carotenoid widely found in nature, an isomer of β-carotene, with the molecular formula C6H2O. 40 H 56 Lycopene has a molecular weight of 536.85. It is a highly effective antioxidant that can effectively scavenge free radicals. Studies have shown that lycopene can prevent and treat cardiovascular disease, atherosclerosis, cancer, and neurodegenerative diseases.
[0003] Saccharomyces cerevisiae is an important model microorganism in biological research, serving as a crucial host for the synthesis of many natural compounds. It possesses advantages such as high safety and acid resistance, and currently, there are relatively comprehensive gene manipulation techniques available for Saccharomyces cerevisiae. In recent years, through the modification of Saccharomyces cerevisiae, the efficient synthesis of many substances has been achieved, realizing the transformation of scientific research results and creating significant commercial value. For example, Saccharomyces cerevisiae has been used as a chassis cell for the efficient synthesis of β-carotene, astaxanthin, and vitamin A. Saccharomyces cerevisiae synthesizes lycopene based on the MVA pathway. Currently, most Saccharomyces cerevisiae strains capable of producing lycopene utilize the galactose-inducible system (GAL-inducible system) to regulate protein expression. However, because galactose itself can be utilized by cells, the induction capacity decreases after galactose utilization, and galactose is expensive. Using this induction system for industrial production presents problems such as excessively high costs. Therefore, it is necessary to develop new strains with lycopene production capabilities suitable for industrial fermentation production. Summary of the Invention
[0004] The present invention also provides a mutant HXT1 promoter having any of the nucleotide sequences shown in SEQ ID NO. 7 to SEQ ID NO. 10.
[0005] The present invention also provides a recombinant expression vector containing the mutant HXT1 promoter.
[0006] The present invention also provides a host cell containing the mutant HXT1 promoter.
[0007] In one embodiment, the host cell includes, but is not limited to, brewer's yeast.
[0008] In one embodiment, the host cell is based on Saccharomyces cerevisiae BY4741 with promoter P... GAL80 Replace with the recombinant Saccharomyces cerevisiae with the mutant HXT1 promoter.
[0009] This invention provides a recombinant brewing yeast for producing lycopene, expressing phytorepinephrine synthase crtB, geraniol-geraniol pyrophosphate synthase crtE, phytorepinephrine dehydrogenase crtI, and 3-hydroxy-3-methylglutaryl-CoA reductase HMG1, and expressing the promoter P on the genome. GAL80 Replace with any of the promoters shown in SEQ ID NO.4 to SEQ ID NO.10.
[0010] In one embodiment, the encoding genes for phytoene synthase crtB and phytoene dehydrogenase crtI are integrated into the GAL1 site of the genome.
[0011] In one embodiment, the genes encoding geraniol geraniol pyrophosphate synthase crtE and 3-hydroxy-3-methylglutaryl-CoA reductase HMG1 are integrated into the HO site of the genome.
[0012] In one embodiment, the nucleotide sequence of the gene encoding the phytoene synthase crtB is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the geraniol geraniol pyrophosphate synthase crtE is shown in SEQ ID NO.2; the nucleotide sequence of the gene encoding the phytoene dehydrogenase crtI is shown in SEQ ID NO.3; and the gene sequence encoding the 3-hydroxy-3-methylglutaryl-CoA reductase HMG1 is shown in Gene ID: 854900.
[0013] In one embodiment, the recombinant Saccharomyces cerevisiae will activate the promoter P on its genome. GAL80 Replace with any of the promoters shown in SEQ ID NO.4 to SEQ ID NO.6.
[0014] In one embodiment, the recombinant brewer's yeast will activate the promoter P on its genome. GAL80 Replace with any of the promoters shown in SEQ ID NO.7 to SEQ ID NO.10.
[0015] In one embodiment, the brewing yeast includes, but is not limited to, BY4741.
[0016] The present invention also provides a method for increasing the lycopene production of Saccharomyces cerevisiae, by modifying the promoter P on the Saccharomyces cerevisiae genome. GAL80 Replace with any of the promoters shown in SEQ ID NO.6 to SEQ ID NO.10; the brewing yeast has the ability to synthesize lycopene.
[0017] In one embodiment, the brewing yeast expresses phytoene synthase crtB, geraniol-geraniol pyrophosphate synthase crtE, phytoene dehydrogenase crtI, and 3-hydroxy-3-methylglutaryl-CoA reductase HMG1.
[0018] The present invention also provides a method for preparing lycopene, which is to prepare lycopene by fermentation using the recombinant brewing yeast.
[0019] In one embodiment, the method involves fermenting the recombinant brewer's yeast in YPD medium, with glucose added during the fermentation process.
[0020] In one embodiment, glucose, yeast extract, and ethanol are fed during fermentation to control the concentration of glucose in the fermentation system to be ≤10g / L, yeast extract to be ≤5g / L, and ethanol to be ≤2g / L.
[0021] In one embodiment, the fermentation is carried out at 28-30°C.
[0022] In one implementation, the fermentation process controls the pH of the fermentation system to be 6 ± 0.2.
[0023] In one implementation method, fermentation lasts for 96-120 hours.
[0024] The present invention also provides the application of the recombinant brewing yeast or the method in the preparation of lycopene or products containing lycopene.
[0025] Beneficial effects: 1. This invention modifies the glucose concentration-induced promoter and replaces promoter P in situ with the improved promoter. GAL80， This invention solves the problem of galactose-dependent induction in lycopene fermentation using Saccharomyces cerevisiae, achieving high expression of key enzymes in the metabolic pathway through glucose induction alone. It also resolves the technical issue of galactose-dependent lycopene synthesis in existing Saccharomyces cerevisiae fermentation.
[0026] 2. In response to the problem that the modified control system has insufficient sensitivity to glucose concentration recognition and is prone to premature product expression, this invention mutates the HXT1 promoter and screens out highly sensitive mutant promoters to accurately achieve decoupled control of cell growth and product synthesis during fermentation, thereby effectively improving fermentation potency.
[0027] 3. Lycopene synthesis relies on the MVA pathway of Saccharomyces cerevisiae: Acetyl-CoA generates IPP and DMAPP via the MVA pathway, which are then catalyzed by ERG20 to generate FPP, followed by GGPP catalyzed by CrtE enzyme, and finally synthesized into lycopene through the synergistic catalysis of CrtI and CrtB enzymes. However, lycopene, being a fat-soluble substance, is stored in yeast cell oil droplets and cell membranes. Excessive accumulation can severely affect cell activity, leading to significant inhibition of cell growth in the early stages of fermentation. This invention decouples fermentation and production through precise control of glucose concentration, fundamentally alleviating the inhibitory effect of lycopene accumulation on cell growth. The recombinant strain, fermented in YPD medium for 120 h, achieved an OD600 of over 400 and a lycopene yield of over 4.90 g / L, laying a core regulatory foundation for high lycopene production. Attached Figure Description
[0028] Figure 1 This is a schematic diagram illustrating the expression regulation of the HXT1 promoter.
[0029] Figure 2 The fermentation curve of lycopene from engineered strain HXT1-6 is shown. Detailed Implementation
[0030] (a) Culture medium YPD medium: 20 g / L peptone, 10 g / L yeast extract and 20 g / L glucose.
[0031] YPG medium: 20 g / L peptone, 10 g / L yeast extract and 2 g / L galactose.
[0032] G418 selection medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, 200 mg / L G418 sulfate.
[0033] Fermentation medium: 20 g / L glucose, 10 g / L yeast extract and 20 g / L tryptone.
[0034] Feeding medium 1: 500 g / L glucose, 400 g / L yeast extract.
[0035] Feeding medium 2: anhydrous ethanol.
[0036] (II) Detection Methods Lycopene extraction: Take 2 mL of the well-vibrated fermentation broth and centrifuge at 12000 rpm for 5 min (wash twice with pure water). After draining the water, resuspend the sample in 3 mL of dimethyl sulfoxide (DMSO) (preheated to 60℃) and vortex until homogeneous. Then, place the sample in a 50℃ water bath for 5 min. Add 3 mL of acetone and incubate at 50℃ in the dark for 10-15 min. Centrifuge the sample at 12000 rpm for 5 min. Transfer the supernatant to a new centrifuge tube and store in the dark.
[0037] Quantitative analysis of lycopene: The concentration of lycopene was determined using high-performance liquid chromatography (HPLC). The HPLC system used in this study was an Agilent Technologies 1200 Infinity series; the column was an Acclaim™ 120 C18 column; the UV absorption wavelength was 450 nm; the mobile phase was methanol, acetonitrile, and dichloromethane (42:42:16); the flow rate was controlled at 1.0 mL / min; and the column temperature was 30 °C. Table 1 Primer sequences
[0038] Example 1 Construction of a recombinant strain producing lycopene 1. Gene Fragment Preparation According to the lycopene metabolism pathway genes from three different sources provided on NCBI, PagcrtB ( Pantoea agglomerate ), BacrtE ( Blakeslea trispora ), BtcrtI ( Blakeslea trispora The gene sequences obtained after codon optimization are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3. The MVA pathway was enhanced by overexpressing HMG1 (Gene ID: 854900). The gene fragments pagcrtB, BacrtE, and BtcrtI were amplified using primers pagcrtB-F and pagcrtB-R, BacrtE-F and BacrtE-R, and BtcrtI-F and BtcrtI-R, respectively. The hmg1 gene fragment was amplified from the genome of *Saccharomyces cerevisiae* BY4741 using primers hmg1-F and hmg1-R. The primer sequences are shown in Table 1.
[0039] 2. Construction of recombinant plasmids PLZL-01 and PLZL-02 Using the genome of Saccharomyces cerevisiae BY4741 as a template, and UP GAL7 -F and UP GAL7 -R is a primer that amplifies promoter P. GAL7 Upstream homology arm UP GAL7The fragments and primer sequences are shown in Table 1.
[0040] Using the genome of Saccharomyces cerevisiae BY4741 as a template, and UP GAL1 -F and UP GAL1 -R is a primer that amplifies promoter P. GAL1 Upstream homology arm UP GAL1 The fragments and primer sequences are shown in Table 1.
[0041] Using the genome of Saccharomyces cerevisiae BY4741 as a template, and P GAL1-10 -F and P GAL1-10 -R is a primer used to amplify the GAL1-10 Promoters fragment. Primer sequences are shown in Table 1.
[0042] Using the genome of Saccharomyces cerevisiae BY4741 as a template, and UP HO -F and UP HO -R indicates a primer, which amplifies UP. HO The fragment is used to integrate foreign genes at the HO site; the primer sequences are shown in Table 1.
[0043] Using the genome of Saccharomyces cerevisiae BY4741 as a template, and based on DN HO -F and DN HO -R is a primer, used to amplify DNA. HO The fragment is used to integrate foreign genes at the HO site; the primer sequences are shown in Table 1.
[0044] Using GJ-F and GJ-R as primers and plasmid PUC57 as a template, the plasmid backbone GJ fragment was amplified. The primer sequences are shown in Table 1.
[0045] Recombinant plasmid PLZL-01 is based on plasmid backbone GJ and contains ADH1 terminator, pagcrtB fragment, and bidirectional promoter P. GAL1,10 BtcrtI fragment, CYC1 terminator.
[0046] Recombinant plasmid PLZL-02 is based on plasmid backbone GJ and contains ADH1 terminator, BacrTE, and bidirectional promoter P. GAL1,10 hmg1 fragment, CYC1 terminator.
[0047] The PCR enzyme used for amplification was Phanta Max Super from Nanjing Novizan Biotechnology Co., Ltd. Fidelity DNA Polymerase. The PCR reaction system is shown in Table 2.
[0048] Table 2 PCR reaction system
[0049] The amplified fragments were recovered and purified by agarose gel electrophoresis.
[0050] The seamless cloning master mix from Nanjing Novizan Biotechnology Co., Ltd. was used. The one-step cloning reaction system is shown in Table 3.
[0051] Table 3 One-step cloning reaction system
[0052] The amount of linearized vector (x) and inserted fragment (y) used can be calculated using the following formula: The optimal amount of fragment A (μL) = 0.04 × number of fragment base pairs (ng) / Y; where Y is the concentration of the insert fragment (ng / μL). The optimal amount of vector used, B~J (μL), is calculated as follows: [0.02 × number of fragment base pairs ng / X; where X is the concentration of the linearized vector (ng / μL).] The circular recombinant vector was transformed into E. coli Top 10 competent cells. Positive recombinant plasmids PLZL-01 and PLZL-02 were obtained through ampicillin-resistant plate selection and verification by colony PCR and sequencing. The plasmid structures are shown in Table 4.
[0053] 3. Construction of recombinant strains integrating and expressing genes for the lycopene synthesis pathway Using CRISPR-CAS9 technology, the pagcrtB and BtcrtI expression frames (ADH1 terminator-pagcrtB fragment-bidirectional promoter P) were first... GAL1,10 The BtcrtI fragment-CYC1 terminator was integrated into the GAL1 site of Saccharomyces cerevisiae BY4741 to obtain JZ-1. Then, the BacrtE and hmg1 expression cassettes (ADH1 terminator-BacrtE-bidirectional promoter P) were integrated into the CYC1 terminator. GAL1,10 The -hmg1-CYC1 terminator was integrated into the HO site of strain JZ-1 to obtain recombinant strain JZ-2, which can regulate lycopene expression. The specific method is as follows: The recombinant plasmids PLZL-01 and PLZL-02 prepared in step 2 above were digested with not I enzyme to obtain the corresponding Donor fragments.
[0054] Competent cells of *Saccharomyces cerevisiae* BY4741 were prepared by overnight culture in YPD liquid medium at 30°C using a shaker. Homologous recombination was then performed by introducing the PLZL-01 Donor fragment and Cas9 plasmid into the competent *Saccharomyces cerevisiae* cells via electroporation. Selection was conducted using G418 selection medium, and single colonies grew after 3-4 days of culture at 30°C. Positive clones identified by PCR were named JZ-1.
[0055] Recombinant strain JZ-1 was cultured overnight in YPD liquid medium at 30°C using a shaker to prepare competent cells. The PLZL-02 Donor fragment and Cas9 plasmid were introduced into the competent Saccharomyces cerevisiae cells via electroporation for homologous recombination. Selection was performed using G418 selection medium, and single colonies grew after 3-4 days of culture at 30°C. Positive clones identified by PCR were named strain JZ-2.
[0056] The recombinant strain JZ-2 was cultured in YPG medium at 220 rpm and 30℃ for 120 h. The lycopene content in the fermentation broth was then measured. The results showed that the lycopene titer in the fermentation broth of strain JZ-2 was 70 mg / L.
[0057] Since the addition of galactose in shake flasks significantly increases fermentation costs, glucose will be used as a regulator to replace galactose in subsequent embodiments to reduce costs.
[0058] Table 4. Inserted sequences in each recombinant plasmid
[0059] Example 2: Using different lengths P HXT Construction of recombinant strains regulating gene expression in the lycopene synthesis pathway The HXT1 promoter originates from the hexose transporter gene family of *Saccharomyces cerevisiae*. It is essentially a sugar-inducible promoter, and its activity is finely regulated by the concentration of hexoses such as glucose in the environment. This promoter replaces the natural promoter P of the core gene gal80 in the galactose regulatory system. gal80 This allows glucose to be used as a fermentation inducer. However, the sequence range within the HXT1 promoter that regulates the gene is still unclear. Therefore, HXT1 promoters of different lengths were selected to regulate lycopene expression. The specific steps are as follows: (1) Preparation of target fragment and recombinant plasmid Using the genome of Saccharomyces cerevisiae BY4741 as a template, UPP was used GAL80 -F and UPP GAL80 -R amplifies UPP GAL80 Fragment; using tP HXT1 -(1~3)-F and tP HXT1-(1~3)-R amplification tP HXT1 - (1~3) segments; using DNP GAL80 -F and DNP GAL80 -R amplification of DNP GAL80 Fragment. Using GJ-F and GJ-R as primers and plasmid PUC57 as a template, the plasmid backbone GJ fragment was amplified.
[0060] The PCR enzyme used for amplification was Phanta Max Super from Nanjing Novizan Biotechnology Co., Ltd. Fidelity DNA Polymerase. The amplified fragments were purified and recovered by agarose gel electrophoresis.
[0061] One-step cloning was performed using the seamless cloning master mix from Nanjing Novizan Biotechnology Co., Ltd., and the reaction system is shown in Table 5.
[0062] Table 5 One-step cloning system
[0063] The obtained recombinant vector was transformed into E. coli Top 10 competent cells, and the cells were screened using ampicillin-resistant plates and verified by colony PCR and sequencing to obtain cells containing the promoter tP. HXT1 tP HXT2 tP HXT3 The positive recombinant plasmids PLZL-03~PLZL-05.
[0064] (2) Construction of recombinant strains The strain JZ-2, which is capable of producing lycopene and was constructed in Example 1, was modified. Using CRISPR-Cas9 technology, "P" was... GAL80 Upstream homologous arm -tP HXT1 (1~3)-P GAL80 The downstream homologous arm expression cassette was integrated into the site of the gal80 strain of JZ-2, enabling the strain to regulate lycopene expression. The specific method is as follows: The recombinant plasmids PLZL-03 and PLZL-05 prepared in step (1) of this embodiment were digested with not I enzyme to obtain the corresponding Donor fragments.
[0065] The recombinant strain JZ-2 constructed in Example 1 was cultured overnight in YPD-containing liquid medium to prepare competent cells. The tPHXT1-1~tPHXT1-3 Donor fragments and the Cas9 plasmid were then co-introduced into the competent Saccharomyces cerevisiae cells via electroporation for homologous recombination.
[0066] Transformants were cultured in G418 selection medium and cultured at 28℃-30℃ for 3-4 days to grow single colonies. The positive clones that were correctly identified by PCR were named HXT1-1, HXT1-2, and HXT1-3, respectively.
[0067] Table 6. Promoters of different lengths and their corresponding bacterial strains
[0068] Recombinant strains HXT1-1, HXT1-2, and HXT1-3 were fermented in 100 mL / 250 mL shake flasks of YPD medium at 200 rpm and 30 °C for 120 h. The lycopene content in the fermentation broth was then measured. The results showed that, without the addition of galactose, the lycopene titers in the fermentation broths of strains HXT1-1, HXT1-2, and HXT1-3 were 70 mg / L, 76.5 mg / L, and 81.8 mg / L, respectively.
[0069] Example 3: Mutation at the core site of promoter HXT1 (1) Selection of potential sites Four promoter core regions were obtained through promoter prediction software analysis, with nucleotide sequences TTACAT, TTTCCC, TTGCCG, and TTTCCT, respectively. Base mutation screening was performed using error-prone PCR.
[0070] 2. Construction of recombinant plasmids Error-prone PCR was performed on a 100bp range, consisting of 50bp before and after the core site. The QuickMutation™ Random Mutation Kit (Beyotime) was used as the error-prone PCR kit, and the PCR system is shown in Table 7.
[0071] Table 7 Commonly Misunderstood PCR Systems
[0072] The DNA fragment obtained by error-prone PCR amplification was seamlessly cloned with the plasmid backbone of recombinant plasmid PLZL-05 constructed in Example 2. The circular recombinant vector was transformed into Escherichia coli Top 10 competent cells. Positive recombinant plasmids PLZL-YC001~PLZL-YC100 were obtained by ampicillin-resistant plate screening and colony PCR and sequencing verification.
[0073] The recombinant strain JZ-2 constructed in Example 2 was cultured overnight in YPD liquid medium at 30°C using a shaker to prepare competent cells. Homologous recombination was then performed by electroporation of the Donor fragments containing mutations YCZL-YC001~YCZL-YC100 and the Cas9 plasmid into the Saccharomyces cerevisiae JZ-2 competent cells. The recombinant strains were cultured in G418 selection medium at 30°C for 3-4 days until single colonies grew. Positive clones identified by PCR were named strains YC-1 to YC-100.
[0074] Recombinant bacteria YC-1 to YC-100 were cultured in YPD medium at 200 rpm and 30°C for 120 h. The lycopene content in the fermentation broth was then measured. The results showed that strains with high lycopene potency were selected without the addition of galactose. These strains possessed the HXT1 mutant sequences of SEQ ID NO.7 to SEQ ID NO.10. The lycopene potencies of the strains HXT1-4, HXT1-5, HXT1-6, and HXT1-7 were 85 mg / L, 90 mg / L, 89.2 mg / L, and 82.2 mg / L, respectively.
[0075] Example 4: Application of recombinant bacteria in lycopene production The recombinant bacteria constructed in Examples 2 and 3 were fermented in 15L fermenters to produce lycopene. The specific steps are as follows: The recombinant strains were streaked onto YPD solid medium and cultured at 30℃ for 24 h. Strains were then picked and inoculated into YPD test tubes, and cultured at 30℃ and 220 rpm for 24 h to obtain the primary seed culture. This primary seed culture was transferred to 300 ml of YPD medium and cultured at 30℃ and 220 rpm for 6 h to obtain the secondary seed culture. The secondary seed culture was inoculated at a rate of 1% into a 15L fermenter containing YPD medium and fermented at 30℃. Twenty h after the start of fermentation, 500 g / L glucose, 400 g / L yeast extract, and anhydrous ethanol were added sequentially to control the glucose concentration in the fermentation system to ≤10 g / L, yeast extract ≤5 g / L, and ethanol ≤2 g / L. The fermentation system was automatically controlled using 6M NaOH at pH 6, aeration rate of 1.5 vvm, dissolved oxygen ≥30, and stirring speed of 400–700 rpm for a total fermentation time of 120 h. The fermentation broth was collected, and the lycopene yield was measured.
[0076] The results showed that after 120 hours of fermentation, the OD600 of the fermentation broth of engineered strain HXT1-6 reached 604, and the final titer reached 6.11 g / L (see...). Figure 2 The efficacy was 19.80% higher than that of strain HXT1-3.
[0077] Table 8 Fermentation effects of different strains
[0078] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A recombinant brewing yeast for producing lycopene, characterized in that, Promoter P located on the genome GAL80 SEQ ID NO. 4 to SEQ ID NO. 10, and expressed phytoene synthase crtB, geranylgeranyl pyrophosphate synthase crtE, phytoene dehydrogenase crtI, and 3-hydroxy-3-methylglutaryl-CoA reductase HMG1.
2. The recombinant brewing yeast according to claim 1, characterized in that, The encoding genes for phytoene synthase crtB and phytoene dehydrogenase crtI are integrated into the GAL1 site of the genome.
3. The recombinant brewing yeast according to claim 2, characterized in that, The genes encoding geraniol geraniol pyrophosphate synthase crtE and 3-hydroxy-3-methylglutaryl-CoA reductase HMG1 are integrated into the HO site of the genome.
4. The recombinant brewing yeast according to any one of claims 1 to 3, characterized in that, The starting strain was Saccharomyces cerevisiae BY4741.
5. A mutant HXT1 promoter, characterized in that, The nucleotide sequence is shown in any of SEQ ID NO. 7 to SEQ ID NO.
10.
6. A biomaterial, characterized in that, include: (a) A recombinant expression vector containing the mutant HXT1 promoter of claim 5; or (b) A host cell containing the mutant HXT1 promoter as described in claim 5.
7. A recombinant brewing yeast, characterized in that, Promoter P located on the genome GAL80 is replaced with any one of the promoters shown in SEQ ID NO. 4 to SEQ ID NO.
10.
8. A method for increasing lycopene yield in brewing yeast, characterized in that, replacing the promoter P GAL80 SEQ ID NO. 6 ~ SEQ ID NO. 10; and the Saccharomyces cerevisiae has the ability of lycopene synthesis.
9. A method for preparing lycopene, characterized in that, Lycopene is prepared by fermentation using the recombinant brewing yeast according to any one of claims 1 to 5.
10. The use of the recombinant brewing yeast according to any one of claims 1 to 4 or the method according to any one of claims 8 to 9 in the preparation of lycopene or products containing lycopene.