Saccharomyces cerevisiae strain with high yield of campesterol as well as construction method and application of saccharomyces cerevisiae strain

Abstract

The invention discloses a saccharomyces cerevisiae strain with high yield of campesterol as well as a construction method and application of the saccharomyces cerevisiae strain. One of the key rate-limiting enzymes, which are heterologously synthesized in the yeast, of the campesterol ((24R)-Ergosta-5-en-3beta-ol) is from a plant C-24 (28) reductase DWF1 mutant (Ar207). In view of the characteristics that the catalytic activity of the enzyme is relatively low and substrates are dispersedly distributed in different compartments of cells, on the basis of the original recombinant saccharomyces cerevisiae for producing campesterol, a rate-limiting enzyme gene copy positioned to a specific subcellular organelle is additionally added, so that the campesterol can be produced. The expression quantity is effectively increased; meanwhile, the contact between the enzyme and a substrate in a corresponding chamber is enhanced; and the yield of campesterol is increased. By utilizing the strategy, Ar207 is positioned to recombinant bacteria of peroxisome and lipid droplets, and the yield of campesterol reaches 3.00 g / l and 3.77 g / l respectively during fermentation in a 5L tank.

Description

Technical Field

[0001] The present invention relates to the technical field of synthetic biology, and in particular to a high-yield campesterol yeast strain and a construction method and application thereof. Background Art

[0002] Phytosterols are a class of steroidal compounds containing a cyclopentane polyhydrogen phenanthrene nucleus and different alkane side chains at the C17 position. They have a similar structure to cholesterol and are widely found in various plants. They have a variety of physiological functions and activities. Due to their many physiological effects and safety and stability, phytosterols are widely used in food, medicine, chemical and other industries. β-Sitosterol, campesterol ((24R)-Ergosta-5-en-3β-ol), stigmasterol and rapeseed sterol are the main components of natural phytosterols.

[0003] Campesterol is one of the most important plant sterols, which has anti-tumor, cholesterol-lowering, anti-cancer and anti-inflammatory, growth-promoting, and hormone level regulating effects. In addition, campesterol may also have special anti-cancer effects. Some literature has statistically analyzed the relationship between the intake of various plant sterols and cancer risk, and found that there is no linear relationship between the intake of mixed plant sterols and cancer risk. Interestingly, the intake of campesterol is significantly negatively correlated with cancer risk (Jiang, et al, Journal of Oncology, 2019, 7479518). This shows that campesterol may have unique advantages in anti-cancer, and may have more applications in health care products and medicine in the future.

[0004] At present, the method of obtaining campesterol is to extract mixed plant sterols from byproducts and waste liquids such as degumming wastewater, bleaching clay and deodorization distillate in the oil production industry, and then separate and purify to obtain campesterol. This method of extracting campesterol from vegetable oil has extremely low campesterol content and complex composition, making separation and purification difficult.

[0005] The ergosterol synthesis pathway exists naturally in yeast, and the biosynthesis of sterols in eukaryotes is highly conserved. The ergosterol synthesis pathway of yeast and the natural synthesis pathway of campesterol in plants share many intermediates. A literature (Qin, et al, J. Agr. Food. Chem, 2024, 72(9), 4814-4824) uses the ergosterol synthesis pathway of Saccharomyces cerevisiae to introduce exogenous C-7 sterol reductase DHCR7 and knock out the yeast C-22 desaturase ERG5 to obtain a strain producing 24-epi-campesterol ((24S)-Ergosta-5-en-3β-ol). At present, most of the technologies for producing rapeseed oil sterol by microorganisms use the yeast's own C-24 (28) sterol reductase ERG4 in the last step of the reaction, and the product is 24-epi-rapeseed oil sterol. As described in the patent (publication number CN112812983A), the yield of 24-epi-rapeseed oil sterol in a 5L fermentation tank reaches 916.88 mg / L. There is a document (Xu, et al, ACS Synth. Biol, 2020, 9, 3157-3170) expressing DWF1, DWF5 (encoding C-7 sterol reductase) and DWF7 (encoding sterol C-5 desaturase) from Arabidopsis thaliana in Saccharomyces cerevisiae, and successfully constructed a yeast strain that produces natural rapeseed oil sterol, but the titer of rapeseed oil sterol in shake flask fermentation is only about 40 mg / L. Part of the reason is that plant-derived enzymes are poorly expressed in yeast and have poor activity on substrates.

[0006] Previously, our research team (patent publication number CN117070550A) successfully achieved the synthesis of natural campesterol by introducing DHCR7 from zebrafish and DWF1 mutant (Ar207) from Arabidopsis into brewer's yeast with ERG4 and ERG5 knocked out, and achieved the yield of 1.52g / l in a 2L fermenter through lipid droplet regulation and other means. On this basis, further improving the biosynthesis yield of natural campesterol is of great significance for industrialization. Summary of the invention

[0007] The invention provides a high-producing campesterol brewing yeast strain and a construction method and application thereof, which comprises the following steps: in the campesterol-producing campesterol brewing yeast strain, adding a gene copy of Ar207 relocated to peroxisomes or lipid droplets.

[0008] According to a preferred embodiment of the present invention, the campesterol-producing Saccharomyces cerevisiae is Saccharomyces cerevisiae into which exogenous C-7 sterol reductase (DHCR7) is introduced.

[0009] According to a further preferred embodiment of the present invention, the campesterol-producing Saccharomyces cerevisiae is a recombinant strain in which Saccharomyces cerevisiae CICC1746 is used as a host cell, Erg5 and ERG4 are knocked out, and a DWF1 mutant (Ar207) from Arabidopsis thaliana and DHCR7 from zebrafish are introduced. The campesterol-producing recombinant strain is the natural campesterol-producing strain YQC427 (patent publication number CN117070550A) originally constructed by the research team. The preferred embodiment is based on the strain YQC427, the key rate-limiting enzyme Ar207 is relocated to the peroxisome using the localization signal ePTS1, or is relocated to the lipid droplet using the localization signal PLN1, to obtain the campesterol-producing Saccharomyces cerevisiae strain of the present invention.

[0010] According to a preferred embodiment of the present invention, the Ar207 nucleotide sequence encodes the amino acid sequence shown in SEQ ID NO.1.

[0011] In the recombinant strain provided by the present invention, the nucleotide sequence of the localization signal ePTS1 encodes the amino acid sequence shown in SEQ ID NO.2.

[0012] In the recombinant strain provided by the present invention, the nucleotide sequence of the localization signal PLN1 encodes the amino acid sequence shown in SEQ ID NO.3.

[0013] The present invention also provides a high-yield campesterol brewing yeast strain constructed by the above method.

[0014] The present invention also provides application of the high-campesterol-producing yeast strain in producing campesterol.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides a method for constructing a fungal strain for synthesizing high-yield campesterol. This study found that although Ar207 increased the activity of wild-type DWF1, it is still the key rate-limiting enzyme in the campesterol synthesis pathway, and enhancing its expression can strengthen the entire synthesis pathway. This study chose to use the strategy of relocation to subcellular organelles, on the one hand to increase the copy number of Ar207, on the other hand to improve the distribution of Ar207 and reduce the difficulty of contact between the enzyme and the substrate. Peroxisomes are the only place where fatty acid β-oxidation occurs, and the large amount of acetyl-CoA generated can provide sufficient carbon metabolic flow for the synthesis pathway. Lipid droplets are the storage place of yeast sterols, in which a large amount of sterol precursors are stored that cannot be catalyzed by heterologous enzymes. Therefore, the present invention uses synthetic biology technology to connect Ar207 to a specific localization signal, and then recombinantly express it in fungal cells, further improving the biosynthetic yield of natural configuration campesterol.

[0017] The present invention relocates the key rate-limiting enzyme Ar207 to peroxisomes and lipid droplets, respectively, to obtain recombinant strains LP01 and LP02, respectively; the constructed strains LP01 and LP02 are fermented at high density in a 5L tank, and the yields of campesterol can reach 3.00 g / l and 3.77 g / l, respectively, which are 1.97 times and 2.48 times higher than previous studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :Plasmid map of V013-EAr.

[0019] Figure 2 : Plasmid map of V013-PAr.

[0020] Figure 3 :HPLC / MS analysis of campesterol produced by recombinant bacteria fermentation.

[0021] Figure 4 : Test tube fermentation results of YQC427, LP01 and LP02.

[0022] Figure 5 : Results of fed-batch fermentation of Saccharomyces cerevisiae engineered strain LP01 in a 5L fermenter.

[0023] Figure 6 : Results of fed-batch fermentation of 5L fermenter with Saccharomyces cerevisiae engineered strain LP02. DETAILED DESCRIPTION

[0024] The present invention is further described and illustrated below in conjunction with specific embodiments. The embodiments are merely exemplary of the present disclosure and do not define the scope of limitation. The technical features of each embodiment of the present invention may be combined accordingly without conflicting with each other.

[0025] The culture medium of the present invention is as follows:

[0026] (1) YPD medium: 1% yeast powder, 2% peptone, 2% glucose; solid medium with 2% agar powder (add 2% glucose after sterilization); sterilize at 115°C.

[0027] (2) YPD (HygB / G418) medium: 1% yeast powder, 2% peptone, 2% glucose, 100 μg / mL HygB, 200 μg / mL G418; solid medium with 2% agar powder (add 2% glucose after sterilization); sterilize at 115°C; used for screening KanMX markers.

[0028] (3) LB medium: 1% sodium chloride, 1% peptone, 0.5% yeast powder; add 2% agar powder to the solid medium and sterilize at 121°C.

[0029] (4) LB (Amp) medium: 1% sodium chloride, 1% peptone, 0.5% yeast powder, 100 μg / mL ampicillin; solid medium with 2% agar powder, sterilized at 121°C; used for culturing Escherichia coli containing plasmids.

[0030] (5) Fermentation tank medium: 10g / L D-glucose, 10g / L (NH4) 2 SO 4 , 8g / L KH 2 PO 4 、3g / L MgSO 4 、0.7g / L ZnSO 4 7H 2 O, 0.25 g / l NaCl, 8 mL / L trace metal solution, and 12 mL / L vitamin solution.

[0031] The detection methods involved in the following embodiments are as follows:

[0032] Glucose content detection: measured using biosensor analyzer SBA-40E;

[0033] Detection of ethanol content: measured using biosensor analyzer SBA-40E;

[0034] Detection method of campesterol content: The metabolites in the sample were filtered through a 0.22 μm membrane and analyzed using Agilent 1160 high performance liquid chromatography. The chromatographic column model was Thermo C-18 column (ODS Hypersil, 4.6×250 mm, 5 μm), the detection wavelength was 210 nm, the column temperature was 30°C, and the mobile phase was: methanol / water = 96 / 4 (V / V).

[0035] Example 1: Obtaining repositionable expression elements

[0036] Construction of the relocated expression plasmid pV013-PAr: The plasmid used was named pV013, based on pRS42H, with the promoter replaced by GAL7 (SEQ ID NO.4) and the terminator replaced by HIS5 (SEQ ID NO.5). Using the yeast genome as a template, primers were designed to amplify PLN1, with the upstream and downstream primers being PLN1-R and PLN1-F, respectively, to obtain the PLN1 fragment. Primers were designed to amplify DWF1, with the upstream and downstream primers being PAr-R and PAr-F, respectively. The two fragments were ligated to the linearized (BamHⅠ and SalⅠ) pV013 plasmid to obtain the relocated expression plasmid pV013-PAr ( Figure 2 ).

[0037] Similarly, the relocated expression plasmid pV013-EAr ( Figure 1 ), the primers for obtaining the gene with the localization signal were EAr-R and EAr-F, the primers for linearizing the plasmid backbone were V013-ER and V013-EF, and the primer sequences used were shown in Table 1

[0038] Table 1 Primer information for construction of repositionable expression elements

[0039]

[0040] Example 2: Obtaining recombinant strains

[0041] Plasmid pV013-EAr was used as a template, and primers PGAL7 / PHIS5 with 50 bp homology arms were used for PCR amplification to obtain the expression cassette of DWF1 with the localization signal ePTS1; the above expression cassette was transformed into YQC427 (a native campesterol-producing strain) using lithium acetate / PEG3350 chemical transformation method, and the strain was named LP01. Plasmid pV013-PAr was transformed into YQC427 using the same method, and the strain was named LP02.

[0042] Table 2 Primer information for strain construction

[0043]

[0044] Example 3: Fermentation of recombinant strain LP01

[0045] (1) Test tube fermentation

[0046] The successfully edited strain was streaked on YPD solid medium and cultured at 30°C for 48 h. A single clone was picked to inoculate 5 mL of YPD liquid medium and cultured at 30°C and 220 rpm for 96 h.

[0047] (2) 5L tank high density fermentation

[0048] A single colony of LP01 was inoculated into 5 mL YPD medium, cultured at 30°C and 220 rpm for 24 h, and inoculated into four shake flasks with 50 mL YPD at a 2% inoculum (v / v). After 24 h of culture, the culture broth in the four shake flasks was inoculated into a 5 L bioreactor (the fermentation tank medium formula was as described above, and the initial fermentation volume after inoculation was 2 L).

[0049] Fermentation tank parameter setting: the temperature is set to 30 ° C, ammonia is added to control the pH to maintain at 5.0, and the dissolved oxygen is maintained at >25% saturation by adjusting the stirring rate (350 rpm to 900 rpm) and the airflow rate (1 vvm to 6 vvm). After the carbon source in the initial culture medium is used up, a feed liquid containing 500 g / L glucose is added to the fermenter according to the pseudo-exponential feeding model; when the glucose feeding is completed and the ethanol content in the tank is lower than 2 mg / l, anhydrous ethanol is first added at a rate of 6 mL / h, and the ethanol feeding rate is increased or decreased according to the consumption of ethanol by the test bacteria until the fermentation is completed. The feed rate FS in the pseudo-exponential feeding stage is determined by the following equation:

[0050]

[0051] In the formula, X 0 、V 0 and S are the initial biomass density (gDCW / L), initial culture volume (L), and glucose concentration in the culture medium (g / L), respectively; is the productivity of cell biomass to glucose (gDCW / g glucose); μ is the specific growth rate (h -1 ), m is the maintenance factor (g glucose / gDCW / L), and t is the time after the start of feeding (h). is set to 0.5, m is set to 0.05, and the specific growth rate is set to 0.105h -1 .

[0052] (3) Treatment of fermentation products

[0053] Collect 500 μL of fermentation broth from test tube fermentation (for high-density fermentation, dilute to an appropriate multiple and take 500 μL of fermentation broth), remove the supernatant by centrifugation, add 600 μL of ethanol-KOH solution (add 25% [w / v] KOH to 50% ethanol) to the cell pellet, vortex thoroughly, saponify in a boiling water bath for 2 h, cool to room temperature, add 400 μL of water and 800 μL of petroleum ether, vortex thoroughly, let stand for stratification, take 500 μL of the upper petroleum ether extract, dry it under vacuum, add 500 μL of anhydrous ethanol to re-dissolve it as the test sample.

[0054] The test tube fermentation results showed that the LP01 campesterol production was 246.4 mg / l ( Figure 4 ). High-density fermentation took 114 h, and the yield of campesterol was 3.00 g / l ( Figure 5 ).

[0055] Example 4: Fermentation of recombinant strain LP02

[0056] According to the operation method of Example 3, the campesterol yield of LP02 obtained in the test tube fermentation was 231.4 mg / l ( Figure 4During high-density fermentation, the specific growth rate was set at 0.1 h because the strain grew more slowly. -1 , and the other parameters remained unchanged. The fermentation lasted for 181 hours, and the yield of campesterol was 3.77 g / l ( Figure 6 ).

[0057] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for constructing a high-yield campesterol yeast strain, characterized in that: In a campesterol-producing Saccharomyces cerevisiae strain, a gene copy of Ar207 was added that relocalized to peroxisomes or lipid droplets.

2. The construction method according to claim 1, characterized in that: The campesterol-producing Saccharomyces cerevisiae is Saccharomyces cerevisiae into which exogenous C-7 sterol reductase (DHCR7) is introduced.

3. The construction method according to claim 1, characterized in that: The campesterol-producing yeast is a recombinant strain in which yeast CICC1746 is used as a host cell, Erg5 and ERG4 are knocked out, and a DWF1 mutant (Ar207) derived from Arabidopsis thaliana and a C-7 sterol reductase (DHCR7) derived from zebrafish are introduced.

4. The construction method according to claim 1, characterized in that: The Ar207 gene is a gene encoding the amino acid sequence shown in SEQ ID NO.

1.

5. The construction method according to claim 1, characterized in that: The localization signal relocated to the peroxisome is ePTS1, which encodes the amino acid sequence shown in SEQ ID NO.

2.

6. The construction method according to claim 1, characterized in that: The localization signal for relocalization to lipid droplets is PLN1, which encodes the amino acid sequence shown in SEQ ID NO.

3.

7. A Saccharomyces cerevisiae strain with high campesterol production obtained by the method according to any one of claims 1 to 6.

8. Use of the high-campesterol producing Saccharomyces cerevisiae strain according to claim 7 in producing campesterol.

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