Engineered Saccharomyces cerevisiae and its construction method and application

Abstract

The present invention relates to the field of bioengineering technology, and particularly relates to an engineered Saccharomyces cerevisiae strain, a method for constructing the same, and applications thereof. The Saccharomyces cerevisiae strain with the preservation number of CGMCC No. 26221 provided by the present invention is obtained by screening on the basis of the chassis strain Saccharomyces cerevisiae SyBE_Sc14D18 after site-directed mutagenesis, atmospheric and room temperature plasma mutagenesis, and adaptive laboratory evolution induced by hydrogen peroxide. On the basis of this strain, the expression of geranyl pyrophosphate synthase TmCrtE is increased and the expression of the Ura3 gene is restored to obtain an engineered Saccharomyces cerevisiae strain with high lycopene yield.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to engineered Saccharomyces cerevisiae and its construction method and application. Background Art

[0002] Lycopene is a dark red carotenoid with the molecular formula C 40 H 56 , which is a linear polyunsaturated olefin compound composed of 11 conjugated double bonds and 2 non-conjugated double bonds, and mainly exists in fruits and vegetables such as grapefruit, watermelon, and tomatoes in nature. This covalent polyene structure endows lycopene with strong antioxidant ability. It prevents cells from being damaged by oxidation by quenching singlet oxygen and scavenging free radicals, and can effectively prevent and treat various diseases caused by aging and decreased immunity. Studies have found that lycopene has physiological functions such as anti-cancer, prevention of cardiovascular diseases, and lipid-lowering. Therefore, as a natural and effective lipophilic antioxidant, lycopene is widely used in the fields of food engineering and the cosmetics industry, and has broad application prospects. It is estimated that the global market value of lycopene will increase to 172 million US dollars in 2025, with an annual growth rate of 4.4% during the forecast period from 2020 to 2025.

[0003] According to relevant research reports, in the lycopene biosynthesis pathway, CrtE encoding geranylgeranyl pyrophosphate (GGPP) synthase and CrtI encoding phytoene dehydrogenase are key rate-limiting enzymes. The sufficient supply of biosynthetic precursors plays an important role in microbial production. Increasing the expression level of CrtE is beneficial to increasing the accumulation of the key precursor GGPP, which is an effective way to enhance the lycopene biosynthesis flux. CrtI is responsible for catalyzing the continuous dehydrogenation of phytoene to lycopene, and its structural characteristics significantly affect the dehydrogenation steps and the ratio of each dehydrogenation product.

[0004] Traditional methods for producing lycopene include natural extraction and chemical synthesis, which cannot meet the growing market demand due to resource and cost limitations. The microbial fermentation method has the advantages of short production cycle, being not restricted by climate conditions, low production cost, non-toxic side effects of products, etc., and shows good commercial prospects in the large-scale synthesis of lycopene. Saccharomyces cerevisiae is a generally recognized as safe (GRAS) model microorganism, whose genome has been completely sequenced for convenient genetic manipulation, and it has a short growth cycle and is easy to culture. In recent years, it has become a popular host for heterologous product synthesis.

[0005] Atmospheric and Room Temperature Plasma (ARTP) mutagenesis technology, as an environmentally friendly, highly safe, and simple-to-operate efficient physical mutagenesis method, has been widely used in industrial and laboratory strain breeding due to its characteristics such as uniform discharge, high concentration of active particles, and high mutation diversity. Some studies have shown that based on the antioxidant properties of carotenoids, using hydrogen peroxide as a selection pressure can force strains to enhance the accumulation of carotenoids to resist oxidative stress, thereby increasing the production ceiling of carotenoids. Therefore, ARTP mutagenesis can be further combined with hydrogen peroxide-induced Adaptive Laboratory Evolution (ALE) to give full play to the advantages of both for strain breeding.

[0006] The microbial metabolic network is extremely complex, and the regulatory mechanisms of many biosyntheses have not been fully elucidated. Random mutation combined with adaptive evolution and high-throughput screening provides a feasible strategy for further exploring the production potential of engineering strains. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide an engineered Saccharomyces cerevisiae strain, its construction method, and its application. The present invention provides an engineered Saccharomyces cerevisiae strain into which a BtCrtI mutant is introduced and subjected to mutagenesis and gene editing, as well as the application of this strain in the production of lycopene.

[0008] The present invention provides a method for constructing an engineered strain, which is obtained by subjecting the CrtI gene in the chassis strain to site-directed mutagenesis and then mutagenesis;

[0009] Among them, the site-directed mutagenesis of the CrtI gene includes mutating alanine at position 355 to valine, tyrosine at position 160 to phenylalanine, and / or asparagine at position 576 to serine in the amino acid sequence encoded by it; specifically, the site of the site-directed mutagenesis of the CrtI gene can be 1, for example, alanine at position 355 is mutated to valine, tyrosine at position 160 is mutated to phenylalanine, or asparagine at position 576 is mutated to serine; it can be 2, for example, alanine at position 355 is mutated to valine and tyrosine at position 160 is mutated to phenylalanine, alanine at position 355 is mutated to valine and asparagine at position 576 is mutated to serine, tyrosine at position 160 is mutated to phenylalanine and asparagine at position 576 is mutated to serine; it can also be 3, for example, alanine at position 355 is mutated to valine, tyrosine at position 160 is mutated to phenylalanine, and asparagine at position 576 is mutated to serine, and the present invention does not limit this.

[0010] The mutagenesis includes atmospheric and room temperature plasma mutagenesis and / or hydrogen peroxide-induced adaptive laboratory evolution. The atmospheric and room temperature plasma mutagenesis randomly generates a large number of mutant libraries, and the hydrogen peroxide-induced adaptive laboratory evolution can enrich a large number of beneficial mutants. The combination of the two is beneficial to improving the mutagenesis efficiency.

[0011] Specifically, in the embodiments of the present invention, through the statistics of the lethality rate and apoptosis number of the strain, it is found that more effective mutations can be obtained when the time of the atmospheric and room temperature plasma mutagenesis is 35 s, and when the number of times of the hydrogen peroxide-induced adaptive laboratory evolution is 15 times, a mutant strain with a higher lycopene yield can be obtained.

[0012] Furthermore, the method for constructing the engineering bacteria of the present invention also includes enhancing the expression of the CrtE gene and / or restoring the expression of the Ura3 gene. The CrtE gene encodes geranylgeranyl pyrophosphate (GGPP) synthase. Increasing the expression level of CrtE is beneficial to increasing the accumulation of the key precursor GGPP, enhancing the lycopene biosynthesis flux, and restoring the expression of the Ura3 gene promotes cell growth and increases the lycopene yield.

[0013] Even further, the chassis bacterium in the construction method provided by the present invention is Saccharomyces cerevisiae SyBE_Sc14D18, which is the strain SyBE_Sc14D18 mentioned in the paper "Design, Construction and Fermentation Process Optimization of High-yield Lycopene-producing Saccharomyces cerevisiae".

[0014] The method for constructing the engineering bacteria provided by the present invention improves the yield and purity of lycopene of the strain through site-directed mutagenesis on the basis of Saccharomyces cerevisiae SyBE_Sc14D18; and a strain yZK006 with high lycopene yield is screened through atmospheric and room temperature plasma mutagenesis and hydrogen peroxide-induced laboratory evolution and is preserved in a preservation institution. On the basis of the strain yZK006, the biosynthesis of lycopene is promoted by enhancing the expression of geranyl pyrophosphate synthase TmCrtE, and the growth of the strain is promoted by restoring the expression of the Ura3 gene, further increasing the lycopene yield, and no additional uracil needs to be added in the fermentation culture, reducing the fermentation cost.

[0015] The present invention provides Saccharomyces cerevisiae with the preservation number of CGMCC No. 26221, which was preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 25, 2022, at the address of No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0016] The present invention also provides a Saccharomyces cerevisiae engineering bacterium, which includes enhancing the expression of the CrtE gene and / or restoring the expression of the Ura3 gene in the Saccharomyces cerevisiae with the preservation number of CGMCC No. 26221.

[0017] The method for constructing the engineered Saccharomyces cerevisiae strain includes enhancing the expression of the CrtE gene and / or restoring the expression of the Ura3 gene in Saccharomyces cerevisiae with the preservation number of CGMCC No.26221. In some specific embodiments, enhancing the expression of the CrtE gene includes inserting P GAL1 -TmCrtE-T GPM1 into the HO locus of the strain, and restoring the expression of Ura3 includes inserting P Ura3 -Ura3-T Ura3 into the DAK2-ZNF1 locus of the strain.

[0018] The present invention provides the use of the Saccharomyces cerevisiae with the preservation number of CGMCC No.26221 or the engineered Saccharomyces cerevisiae strain in the preparation of lycopene.

[0019] The present invention also provides a method for preparing lycopene, which includes fermenting and culturing the Saccharomyces cerevisiae with the preservation number of CGMCC No.26221 or the engineered Saccharomyces cerevisiae strain.

[0020] Specifically, in the embodiments of the present invention, the fermentation temperature for the fermentation culture is 30 °C, the aeration rate is 1.5 vvm, the stirring speed is 200 - 700 rpm, the dissolved oxygen content is not less than 30% (v / v), and the pH value is 6.0. The process of the fermentation culture includes two stages: cell growth and lycopene accumulation.

[0021] After site-directed mutagenesis of the engineered Saccharomyces cerevisiae strain provided by the present invention, both the yield and purity of lycopene are significantly improved. After ARTP mutagenesis and ALE, the lycopene yield of the strain is further significantly improved, and it has stronger oxidative tolerance. After regulating the expression level of CrtE and restoring the expression of the Ura3 gene, the yield of lycopene is further increased, and in the fermentation culture, uracil does not need to be added, further reducing the fermentation cost and increasing the industrial application value.

[0022] Biological deposit description

[0023] Saccharomyces cerevisiae was deposited on December 25, 2022 at the China General Microbiological Culture Collection Center, with the address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.26221. Description of the drawings

[0024] Figure 1 Shows the schematic diagram of BtCrtI site-directed mutagenesis;

[0025] Figure 2 Shows the plasmid map of pZK003;

[0026] Figure 3 Show the effect of the introduction of excellent BtCrtI mutants on the yield and purity of lycopene;

[0027] Figure 4 Show the flow chart of breeding high-yield mutant strains by ARTP mutagenesis combined with hydrogen peroxide-induced ALE;

[0028] Figure 5 Show the relationship between ARTP treatment time and the lethality rate of strain yZK002;

[0029] Figure 6 Show the YPDG plate coating situation after each round of hydrogen peroxide treatment in adaptive evolution;

[0030] Figure 7 Show the lycopene yield of mutant strains in each round of adaptive evolution;

[0031] Figure 8 Show the results of transcriptome analysis of high-yield mutant strain yZK006;

[0032] Figure 9 Show the schematic diagrams of the construction of strains yZK011 and yZK016;

[0033] Figure 10 Show the effect of adjusting TmCrtE expression and Ura3 restoration on lycopene yield;

[0034] Figure 11 Show the comparison of cell growth between strains yZK011 and yZK016;

[0035] Figure 12 Show the fed-batch fermentation results of strain yZK016 in a 7L fermenter. Detailed implementation manners

[0036] The present invention provides an engineered Saccharomyces cerevisiae strain, its construction method and application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0037] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0038] Table 1 List of strains and plasmids used in the present invention

[0039]

[0040] Table 2 List of primers used in the present invention

[0041]

[0042] Table 3 Gene sequences involved in this experiment

[0043]

[0044] The present invention will be further described below in conjunction with embodiments:

[0045] Example 1 Introduction of excellent BtCrtI mutants to increase lycopene production

[0046] Using CRISPR / Cas9 technology to perform site-directed mutagenesis on the Saccharomyces cerevisiae genome, replacing the wild-type BtCrtI with an excellent mutant ( Figure 1 ). Mutation sites are A355V (GCA→GTC), Y160F (TAC→TTC), N576S (AAC→TCT).

[0047] 1. Construction of Cas9-gRNA plasmid

[0048] Using pZK003 ( Figure 2 ) as the basic plasmid to construct a Cas9 plasmid containing gRNA. Select primers designed with the mutation site within the PAM sequence or the first 20 bp before it, then anneal and bond the two primers to form double-stranded DNA, and use Golden Gate assembly to ligate the annealed double-stranded DNA and pZK003 to obtain the Cas9-gRNA plasmid. Transform the reaction system into DH5α Escherichia coli competent cells, and spread them on the LB+Kan R plate and culture overnight at 37°C. Pick non-luminous single colonies under the fluorescence imager for sequencing to verify whether the gRNA has been successfully inserted.

[0049] Table 4 gRNA primers for site-directed mutagenesis of BtCrtI

[0050] Primer Sequence (5′→3′) gRNA_BtCrtI(A355V)-F gactTCTGAAGCATCTTTCTACGT gRNA_BtCrtI(A355V)-R aaacACGTAGAAAGATGCTTCAGA gRNA_BtCrtI(Y160F)-F gactAGTACGTCCCAGAGATTTTT gRNA_BtCrtI(Y160F)-R aaacAAAAATCTCTGGGACGTACT gRNA_BtCrtI(N576S)-F gactGACAGAACTCTAACGATATT gRNA_BtCrtI(N576S)-R aaacAATATCGTTAGAGTTCTGTC

[0051] 2. Transformation of Saccharomyces cerevisiae

[0052] Using the yeast LiOAc transformation method, 700 ng of the Cas9-gRNA plasmid and 3000 ng of the BtCrtI mutant fragment were co-transformed into the chassis strain SyBE_Sc14D18. SC-Ura plates were used to screen for positive single colonies, and strains yZK001 (A355V) and yZK002 (Y160F&NS76S) were obtained. After correct sequencing, the Cas9-gRNA plasmid containing the Ura3 selection tag in strains yZK001 and yZK002 was discarded using 5-FOA plates, and the genome site-directed mutagenesis experiment was completed.

[0053] Flask fermentation conditions:

[0054] Primary seed culture: 30 μL of glycerol bacteria were inoculated into 3 mL of YPD medium and cultured at 30 °C and 220 rpm for 24 h.

[0055] Secondary seed culture: The primary seed was transferred to 5 mL of fresh YPD medium at an initial OD 600 = 0.2 and cultured at 30 °C and 220 rpm for 16 - 20 h.

[0056] Fermentation: The secondary seed was transferred to a 250 mL flask containing 50 mL of YPDG (40 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, and 10 g / L galactose) medium at an initial OD 600 = 0.1 and fermented for 96 h.

[0057] Results: As Figure 3 shown, the lycopene yields of strains yZK001 (A355V) and yZK002 (Y160F&NS76S) were 447 mg / L and 476 mg / L, respectively, and the purities were increased from 79.9% to 84.6% and 85.2%. Among them, lycopene and neurosporene were the main dehydrogenation products, and the ratios of lycopene to neurosporene in the two strains were increased from 5.7:1 to 7.5:1 and 7.8:1, respectively.

[0058] Example 2 ARTP mutagenesis combined with hydrogen peroxide-induced ALE to breed high-yield mutant strains

[0059] ARTP randomly generates a large number of mutant libraries, and hydrogen peroxide-induced ALE enriches a large number of beneficial mutants. The combination of the two is beneficial to improving the mutagenesis efficiency ( Figure 4 ).

[0060] 1. ARTP mutagenesis

[0061] The experimental strain was mutagenized using the ARTP mutagenesis breeding instrument (ARTP-M) produced by TMAXTREE Biotechnology Co., Ltd. This instrument is equipped with a plasma generator, a helium gas source, and a cooling water circulation system, etc.

[0062] Experimental parameters: 1) The output power is 120 W; 2) The helium gas flow rate is 10 SLM (Standard Liter per Minute); 3) The distance between the plasma emitter and the metal plate is 2 mm; 4) The ARTP treatment times are 0, 15, 20, 25, 30, 35, and 40 s respectively

[0063] Experimental procedure: Inoculate a single colony after activating the yZK002 glycerol bacteria plate into 3 mL of YPD medium and culture it overnight at 30 °C and 220 rpm. Transfer 200 μL of the primary seed to 5 mL of fresh YPD medium and culture it at 30 °C and 220 rpm for 6 - 8 h until the logarithmic growth phase. Take 1 mL of the bacterial solution into a 1.5 mL centrifuge tube, centrifuge it at 5000 rpm for 1 min, wash the cells twice with 1 mL of physiological saline, and then dilute it with an appropriate amount of sterile water to OD 600 = 1. Take 10 μL of the bacterial suspension and evenly spread it on a sterile metal plate, and start ARTP irradiation.

[0064] Calculation method of lethality:

[0065] Lethality = (total number of control bacteria - total number of colonies after mutagenesis treatment) / total number of control bacteria × 100%

[0066] Appropriate mutagenesis conditions are beneficial to increasing the possibility of obtaining favorable mutant strains. In the ARTP mutagenesis breeding system, the ARTP treatment time is a key parameter affecting the lethality of experimental strains. After plate colony counting, the lethality rates of strain yZK002 at treatment times of 0, 15, 20, 25, 30, 35, and 40 s were calculated to be 0%, 27.8%, 38.9%, 47.2%, 72.2%, 88.9%, and 100% respectively ( Figure 5 ). According to relevant reports, when the lethality rate is between 80% and 90%, it may cause greater DNA damage and more effective mutations. Therefore, 35 s was determined as the optimal treatment time.

[0067] 2. Adaptive laboratory evolution induced by hydrogen peroxide

[0068] Experimental procedure: After ARTP irradiation, immediately transfer the bacterial solution to 5 mL of YPDG medium and culture it until the logarithmic growth phase. Then take 500 μL of the bacterial solution into a sterile 2 mL centrifuge tube, add 30% hydrogen peroxide to a certain concentration, and culture it on a shaker at 30 °C and 220 rpm for 30 min. Wash the cells twice with 1 mL of sterile water and resuspend them in 1 mL of sterile water. Take 200 μL of the bacterial suspension and add it to 5 mL of YPDG medium and culture it for 24 h. Take an appropriate amount of the bacterial solution, dilute it by an appropriate multiple, and spread it on a YPDG plate. This is one round of hydrogen peroxide treatment process.

[0069] Take 500 μL of the bacterial solution again and perform the same hydrogen peroxide treatment process. Repeat this cycle until cell apoptosis occurs as the hydrogen peroxide concentration gradually increases. The hydrogen peroxide treatment was carried out for 15 rounds in total.

[0070] 3. Screening of high-yield mutant strains

[0071] Since the color of each dehydrogenation product is different, ranging from colorless phytoene to dark red lycopene, the color gets darker and darker. Therefore, visually screening can effectively identify mutant strains with higher lycopene production. To study the cumulative effect of iterative hydrogen peroxide stimulation on lycopene production, select 3 single colonies with darker colors from each round of YPDG plates as candidate strains for lycopene production determination in 24-well plates ( Figure 6 ). The results are shown as Figure 7 shown in A below. The lycopene production of the three mutant strains (yZK004, yZK005, and yZK006) was significantly higher than that of the control strain yZK002, increasing from 476 mg / L to 528 mg / L, 540 mg / L, and 548 mg / L respectively. The final hydrogen peroxide treatment concentrations were 377 mM, 725 mM, and 1049 mM respectively. Strains yZK004, yZK005, and yZK006 are highlighted in green, blue, and red respectively. The purple line represents the treatment concentration of H2O2 in each round. In the subsequent retest of the fermentation yield in shake flasks ( Figure 7 shown in B below), only the lycopene production of yZK006 (the sixth round, treated with 1049 mM H2O2) increased significantly, with a yield of 530 mg / L.

[0072] Example 3 Transcriptome analysis of the high-yield mutant strain yZK006

[0073] Experimental procedure: Take 5 mL of the fermentation broth of the mutant strain yZK006 and the control strain yZK002 at 30 h and 60 h respectively, centrifuge at 4000 rpm for 5 min at 4°C, wash the cells twice with sterile water and collect the cell pellets, quickly freeze them in liquid nitrogen, and store them at -80°C. All samples were sent to BGI for further RNA sequencing detection and analysis through the DNBSEQ platform. To gain a deeper understanding of the changes in the strain phenotype, pathway analysis of differentially expressed genes was performed based on the KEGG database. And the Dr.Tom network platform of BGI was used for data mining and graph presentation. The RNA sequencing data was calculated from three biological replicates. The genomic information of the reference species was derived from Saccharomyces cerevisiae S288C.

[0074] Result analysis:

[0075] Differential gene expression analysis was performed using a selection criterion of |Log2(Fold Change)| ≥ 1.5 and Q-value ≤ 0.05. Compared with the parental strain yZK002, 155 genes were up-regulated and 172 genes were down-regulated in the mutant strain yZK006 at 60 h ( Figure 8 in A). Then, we used GO biological process enrichment analysis and KEGG Mapper analysis to screen gene functions and found that the differentially expressed genes were mainly distributed in the processes of hydrogen peroxide catabolism, ergosterol biosynthesis, fatty acid oxidation, and central carbon metabolism ( Figure 8 in B). As Figure 8 shown in C, the expression levels of several key enzymes in the antioxidant system of yZK006 were significantly up-regulated at the later stage. Among them, catalase A (CTA1) in the peroxisomal matrix and catalase T (CTT1) in the cytoplasm are mainly responsible for decomposing hydrogen peroxide entering the cell to protect it from oxidative damage. Superoxide dismutase encoded by SOD1 is another important antioxidant enzyme that can convert superoxide anion radicals into hydrogen peroxide, which is then scavenged under the catalysis of catalase. In addition, the transcription factors YAP1 and MSN2 that regulate the expression of a series of antioxidant genes were also significantly up-regulated. MSN2 is a zinc finger transcription factor that can enhance the expression of catalase CTT1 located in the cytoplasm. These results indicate that the yZK006 strain may have evolved stronger oxidative tolerance when resisting oxidative attacks from the external environment.

[0076] In Saccharomyces cerevisiae, sterols and phospholipids are important components of the cell membrane, regulating the fluidity and permeability of the cell membrane structure. Exogenous hydrogen peroxide acts as a signaling molecule to induce and regulate changes in the cell membrane of Saccharomyces cerevisiae. As can be seen from Figure 8 E, most genes involved in ergosterol biosynthesis (ERG1, ERG11, ERG25, ERG26, ERG6, ERG3, and ERG4) were down-regulated during the lycopene accumulation stage, indicating that yZK006 may improve its viability under oxidative stress by reducing intracellular sterol levels. In addition, the inhibition of the ergosterol synthesis pathway will redirect the FPP flux to the biosynthesis of lycopene. Most genes related to fatty acid oxidation (FAA2, POX1, POT1, SPS19, PXA1, ECI1, DCI1, and CAT2) were transcriptionally up-regulated at 60 h ( Figure 8In D). Fatty acid oxidation in peroxisomes is an important source of acetyl-CoA. POX1 (encoding acyl-CoA oxidase) and SPS19 (encoding peroxisomal 2,4-dienoyl-CoA reductase) are the main rate-limiting enzymes for the β-oxidation of saturated and unsaturated fatty acids, respectively. Under oxidative stress, the high-level expression of these two genes has a positive effect on the β-oxidation of long-chain fatty acids, providing sufficient energy and intermediate metabolites for cell growth. In addition, genes related to the γ-oxidation of fatty acids (ECI1, DCI1, and CAT2) are also upregulated. Acetyl-CoA is an important precursor for carotenoid biosynthesis, mainly derived from the cytoplasmic pyruvate dehydrogenase (PDH) bypass pathway. As Figure 8 shown in D, during the alcohol consumption stage, significant changes occurred in the transcriptional levels of many key genes in the central carbon metabolism pathway. When glucose is depleted, cell growth enters the ethanol consumption stage and starts to produce lycopene. ADH2, which catalyzes the oxidation of ethanol to acetaldehyde, is significantly upregulated in strain yZK006. At the same time, the expression of the ADH6 gene involved in the reverse reaction is downregulated. PDC1 / 5, which is related to the conversion of pyruvate to acetaldehyde, is also significantly downregulated. These results will inhibit the reduction of acetaldehyde to ethanol, which is beneficial for the production of lycopene using ethanol as a carbon source. Ethanol metabolism in Saccharomyces cerevisiae mainly depends on alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALD). Among them, ALD6, which encodes cytoplasmic aldehyde dehydrogenase, is significantly downregulated in the later stage, while ALD4, which encodes mitochondrial aldehyde dehydrogenase, is upregulated, which may be to balance the decrease in acetate metabolism flux caused by the downregulation of ALD6. In addition, the upregulation of ACS1, which encodes acetyl-CoA synthetase, and the downregulation of the glyoxylate cycle-related gene ICL1 are beneficial for increasing the level of acetyl-CoA. The upregulation of genes involved in glycolysis (TDH3, ERR1, and ERR3) and the TCA cycle (CIT3, IDP3, SHH3, and SHH4) can promote the synthesis of lycopene by increasing the supply of reducing power and energy. Combining the above-mentioned weakening of ergosterol biosynthesis and enhancement of fatty acid oxidation, this may be the reason for the increase in target metabolic flux. In summary, we systematically analyzed the differentially expressed genes of strain yZK006 and found that the increase in lycopene production may come from redox processes, the synthesis of cell membrane components, acetyl-CoA synthesis, and energy metabolism.

[0077] Example 4 Regulating the Expression Level of CrtE to Promote Lycopene Biosynthesis and Restoring the Ura3 Gene to Promote Strain Growth

[0078] 1. Using the CRISPR / Cas9 technology, insert P GAL1 [[ID=IO]]-TmCrtE-T GPM1 into the HO locus of strain yZK006 ( Figure 9 in A)

[0079] Experimental procedures: The construction process of the HO-Cas9-gRNA plasmid was the same as above. The gRNA sequence at the HO site was GTAAGGCTTCATTATGGAGA. The fragment P to be inserted GAL1 -TmCrtE-T GPM1 Homologous arms, each 500 bp long, were designed at both the left and right ends, and the three linear fragments were assembled using Gibson assembly to obtain HO Left Arm -P GAL1 -TmCrtE-T GPM1 -HO Right Arm Using the yeast LiOAc transformation method, 700 ng of the HO-Cas9-gRNA plasmid and 3000 ng of the HO Left Arm -P GAL1 -TmCrtE-T GPM1 -HO Right Arm fragment were co-transformed into the strain yZK006, and the SC-Ura plate was spread to screen for positive single colonies. After correct sequencing, the Cas9-gRNA plasmid containing the Ura3 selection tag in the strain yZK011 was discarded using the 5-FOA plate.

[0080] Results: The strain yZK011 was obtained, and the lycopene production was increased to 608 mg / L( Figure 10 ).

[0081] 2. Insert P Ura3 -Ura3-T Ura3 into the DAK2-ZNF1 locus of the strain yZK011( Figure 9 in B)

[0082] Experimental procedures: The fragment P Ura3 -Ura3-T Ura3 Homologous arms, each 500 bp long, were designed at both the left and right ends, and the three linear fragments were assembled using Gibson assembly to obtain DAK2-ZNF1 Left Arm -P Ura3 -Ura3-T Ura3 -DAK2-ZNF1 Right Arm Using the yeast LiOAc transformation method, 3000 ng of the DAK2-ZNF1 Left Arm -P Ura3 -Ura3-T Ura3 -DAK2-ZNF1 Right Arm fragment was transformed into the strain yZK011, and the SC-Ura plate was spread to screen for positive single colonies.

[0083] Result: Strain yZK016 was obtained, with a lycopene production of 703 mg / L( Figure 10 ). Then we compared the cell growth of strains yZK011 and yZK016 and found that the restoration of the Ura3 gene was beneficial to cell growth, thus obtaining the maximum lycopene production( Figure 11 ). In addition, uracil does not need to be added to the fermentation medium, further reducing the fermentation cost and increasing the industrial application value.

[0084] Example 5 Fed-batch fermentation of strain yZK016 in a 7-L fermenter

[0085] Fermentation conditions and strategies:

[0086] Primary seed culture: 50 μL of glycerol bacteria of yZK016 was inoculated into 5 mL of YPD medium and cultured overnight at 30 °C and 220 rpm.

[0087] Secondary seed culture: The primary seed was transferred to a shake flask containing 50 mL of fresh YPD medium at an inoculation amount of 4% (v / v) and cultured at 30 °C and 250 rpm until the logarithmic growth phase.

[0088] Fermenter inoculation: The secondary seed was transferred to a 7-L fermenter containing 3.6 L of YPD (20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract) fermentation medium at an inoculation amount of 10% (v / v) to start fermentation.

[0089] Fermenter control: The fermentation temperature was 30 °C, the aeration rate was 1.5 vvm, the stirring speed was 200 - 700 rpm, the dissolved oxygen (DO) was ≥ 30%, and 10 M sodium hydroxide was automatically fed to control the pH value at 6.0. Galactose was used to induce the expression of heterologous genes in the lycopene biosynthesis pathway. The fermentation process was divided into two stages: cell growth and lycopene accumulation. In the first stage, when the initial 20 g / L glucose was exhausted, 600 g / L of glucose was continuously fed to keep the glucose concentration below 2 g / L. The nitrogen source was fed with 300 g / L of yeast extract at a rate of 1 - 2 g / (L·h). When cell growth was stable, galactose with a final concentration of 25 g / L was added to activate the biosynthesis of lycopene. In the second stage, an appropriate amount of ethanol was continuously fed to keep the ethanol concentration below 5 g / L until the end of fermentation.

[0090] Result: Under the above fermentation conditions, the lycopene production of strain yZK016 reached 8.15 g / L at 138 h, which was 10.6 times higher than that of shake flask fermentation( Figure 12 ).

[0091] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Saccharomyces cerevisiae with the preservation number of CGMCC No.26221, and its preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms.

2. An engineered Saccharomyces cerevisiae strain, characterized in that, Enhancement of the expression of the CrtE gene and / or restoration of the expression of the Ura3 gene in the Saccharomyces cerevisiae with the preservation number of CGMCC No.26221 as claimed in claim 1.

3. The construction method of the engineered Saccharomyces cerevisiae strain according to claim 2, characterized in that It includes enhancing the expression of the CrtE gene and / or restoring the expression of the Ura3 gene in the Saccharomyces cerevisiae with the preservation number of CGMCC No.26221 as claimed in claim 1.

4. The construction method according to claim 3, characterized in that The enhanced expression of the CrtE gene includes inserting P GAL1 -TmCrtE-T GPM1 into the HO locus of the strain; The restoration of Ura3 expression includes inserting P Ura3 -Ura3-T Ura3 into the DAK2-ZNF1 locus of the strain.

5. Use of the Saccharomyces cerevisiae with the preservation number of CGMCC No.26221 as claimed in claim 1 or the engineered strain of Saccharomyces cerevisiae as claimed in claim 2 in the preparation of lycopene.

6. A method for preparing lycopene, characterized in that, It includes fermenting and culturing the Saccharomyces cerevisiae with the preservation number of CGMCC No.26221 as claimed in claim 1 or the engineered strain of Saccharomyces cerevisiae as claimed in claim 2.

7. The preparation method according to claim 6, characterized in that, The fermentation temperature of the said fermentation and culture is 30 °C, the ventilation rate is 1.5 vvm, the stirring speed is 200 - 700 rpm, the dissolved oxygen content is not less than 30% (v / v), and the pH value is 6.0.

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