Rhodotorula mucilaginosa for producing high-yield grease by using acetic acid as well as preparation method and application of

Abstract

The invention discloses a yeast mutant strain capable of efficiently utilizing acetic acid to generate grease. The strain is named as rhodotorula mucilaginosa in taxonomy. The strain is adaptively domesticated for 211 days, and the obtained mutant strain can grow in a culture medium containing at most 1.4 M of sodium acetate. Under the condition of sodium acetate stress, compared with a wild strain, the hysteresis period of the mutant strain is reduced by at least 24 hours, and the grease yield is increased by 197%. According to the strategy, the glucose and sodium acetate metabolism rate and the grease generation efficiency are improved, and a technical basis is provided for establishment of a cell factory for producing lipid-based chemicals.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a red yeast for high-yield oil by utilizing acetic acid, and a preparation method and application thereof. Background Art

[0002] Lipids are a class of water-insoluble organic compounds. In addition to being stored as carbon and energy in cells, they also play an important role in the formation of cell membrane structures and signal transduction. A large part of the common oil products on the market come from animals and plants, but due to climate, season and site restrictions, the output of animal and plant oils is difficult to meet the needs of biofuel and other chemical production. Some oil-producing yeasts can use a variety of carbon sources to produce oils, and are not affected by seasons, regions and climate, and have received widespread attention. Among them, Rhodotorula mucilaginosa is a promising class of industrial application microorganisms that can use different carbon sources to reproduce and produce lipids for use in cosmetics, oleochemicals and biofuel processing. Improving the utilization rate of some renewable carbon sources by Rhodotorula mucilaginosa can not only reduce the production cost of oils, but also realize waste utilization and promote sustainable development.

[0003] Acetic acid can be prepared greenly by utilizing renewable electricity and carbon dioxide, and has become a renewable resource that has attracted much attention. At the same time, acetic acid is also a high-quality carbon source for yeast to produce oils. After entering the yeast cell, it can be directly converted into acetyl-CoA, becoming a direct precursor for fatty acid synthesis. Through a series of enzymatic reactions, the carbon chain is gradually extended to form longer fatty acid molecules. Compared with the synthesis of fatty acids by the β-oxidation pathway, its synthesis pathway is shorter and more efficient. However, acetic acid is toxic to yeast when it exists in a protonated form (pH≈4.75) and an acetic acid anion form (pH≈5-6), inhibiting strain energy consumption and metabolic enzyme activity, resulting in strain growth stagnation and productivity loss. Therefore, how to overcome the above-mentioned defects when acetic acid is used as a carbon source, enrich the strains of beneficial mutations produced by Rhodotorula glutinosae, obtain stable mutant strains that can efficiently utilize acetic acid, and realize the green and sustainable production of single-cell oils is a technical problem to be solved in this field. Summary of the invention

[0004] The invention aims to solve the technical problem that red yeast is inhibited by acetic acid and is difficult to efficiently utilize acetic acid to synthesize fatty acids, and provides a mutant red yeast that tolerates sodium acetate and efficiently utilizes sodium acetate to synthesize oil and fat, and application thereof.

[0005] The first aspect of the present invention provides a red yeast for high oil production using acetic acid, wherein the red yeast is classified as Rhodotorula mucilaginosa, named as red yeast A2, and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No.31121, a deposit date of July 1, 2024, and a deposit address of Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0006] The second aspect of the present invention provides the use of the above-mentioned red yeast in oil preparation;

[0007] Furthermore, the red yeast is grown in an environment containing 0.6-1.4M sodium acetate to prepare oil;

[0008] Further, the oil includes oleic acid, palmitic acid and stearic acid;

[0009] Furthermore, the oil yield produced by fermentation of the red yeast is at least 90% higher than that of ordinary red yeast.

[0010] The third aspect of the present invention provides a method for preparing the above-mentioned Rhodotorula glutinosus, comprising the following steps:

[0011] The wild-type Rhodotorula glutinosus WT was cultured in a medium containing sodium acetate, and the sodium acetate concentration in the medium was gradually increased to a maximum value of 1.4 M by an adaptive acclimation method, and the wild-type strain was continuously cultured for at least 5 generations, thereby obtaining a Rhodotorula glutinosus mutant strain A2 with high sodium acetate concentration tolerance;

[0012] Further, the culture medium is YPD culture medium;

[0013] Beneficial Effects

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] 1. Non-GMO adaptive domestication was used to improve the strain's tolerance to sodium acetate, further achieving efficient utilization of cheap renewable carbon sources.

[0016] 2. Compared with the wild-type strain, the mutant strain obtained by the present invention has greatly improved lipid synthesis efficiency, providing a technical basis for the establishment of a cell factory for the production of lipid-based chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the tolerance of the starting strain to sodium acetate

[0018] Figure 2 Figure 2 shows the (A) glucose consumption, (B) sodium acetate consumption, and (C) biomass of the starting strain and mutant strain in 0.6 M furfural medium.

[0019] Figure 3 Lipid accumulation of the starting strain and mutant strain Specific implementation plan

[0020] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods, but these embodiments shall not be used to interpret the limitation of the present invention; the experimental methods in the following embodiments are conventional methods unless otherwise specified, and the test materials used in the following embodiments are commercially available unless otherwise specified; after reading the description of the present invention, various equivalent changes, modifications and modifications made by technical personnel in this field all fall within the scope defined by the claims of the present invention.

[0021] In a specific embodiment, the present invention is implemented by the following technical solution:

[0022] The present invention is achieved through the following technical solutions:

[0023] 1. Cultivation of Mutant Rhodotorula A2

[0024] The wild-type Rhodotorula was named WT, and the concentration of sodium acetate in the culture medium was gradually increased by adaptive domestication technology. The wild-type strain was continuously cultured to obtain a mutant strain A2 that was tolerant to high concentrations of sodium acetate.

[0025] 2. Identification of Mutant Rhodotorula A2

[0026] ① Morphological characteristics of strain A2: When grown on YPD agar medium, the colonies are orange-red, with a bright surface, soft and sticky texture, no obvious change in the color of the culture medium, and no exudate.

[0027] ② Molecular identification: After morphological, physiological characteristics and ITS rDNA gene sequence identification, the similarity with the NCBI GenBank Rhodotorula was as high as 99.8%. Finally, the strain was identified as Rhodotorula.

[0028] 3. By measuring the biomass, glucose and sodium acetate consumption of the starting strain and the mutant strain in sodium acetate medium, it was found that the mutant strain metabolized sodium acetate faster.

[0029] 4. The lipid production of the starting strain WT and the mutant strain A2 was detected by GC-MS; the results showed that the bacteria had a good ability to produce lipids using sodium acetate as a substrate;

[0030] Example 1: Acetate tolerance and identification of bacterial strains

[0031] Take an appropriate amount of sodium acetate and dissolve it in YPD medium with a pH of 5.0 to prepare 0.1, 0.2, 0.3, 0.4, and 0.5 M solutions. Use a 0.22 μm microporous filter membrane to sterilize the solution and store it at 4°C.

[0032] The frozen wild-type Rhodotorula glutinosus (WT) was activated on a YPD agar plate in advance, and a single colony was picked from the cultured plate and inoculated into a YPD liquid medium, and cultured at 30°C and 210 rpm for 72 h. After centrifugation to collect the cells, the cells were washed twice with sterile water, and then resuspended in a sodium acetate medium with a concentration of 0-0.5 M. The initial OD 600 The wild type red yeast of 0.6 was used as the starting point for acclimatization, with the concentration of the original strain in the hysteresis phase and the concentration in YPD medium that was significantly different. 600 The inoculum size was 1.0, and the YPD medium without inoculation of the strain was used as a blank. 200 μL of the bacterial suspension was transferred to a 96-well plate with a transparent cover and placed in an automatic microbial growth characteristic instrument for culturing at 30°C and 500 rpm for 180 h. The instrument measured the OD value of the bacterial suspension every 20 min. 600 The original strain was tested to determine its tolerance to different sodium acetates. Figure 1 As shown in the figure, the strain significantly increased in the growth lag phase in the 0.2 M sodium acetate medium, with the maximum OD 600 It decreased significantly, indicating that 0.2M sodium acetate had caused obvious inhibition on the strain, so 0.2M sodium acetate was used as the starting point for domestication.

[0033] Pick a single colony from the pre-cultured YPD plate and pre-culture the strain in a YPD liquid medium with a pH of 5.0 at 30°C and 210rpm. After 72 hours, collect and wash the bacteria, and inoculate the yeast in a 0.2M sodium acetate medium with an inoculation amount of OD600 1.0. Observe the changes in the strain OD600 every 24 hours. When the strain enters the stable phase, subculture to a medium with an increased sodium acetate concentration of 0.05M, and the initial OD600 is 1.0. If the strain hysteresis period increases significantly during the period, reduce the sodium acetate concentration by 0.05M and repeat the subculture process. Repeat the above steps until the strain reaches the biological limit, that is, the strain growth rate and hysteresis period can no longer be restored after repeated cultivation in a certain concentration of sodium acetate medium for more than 5 generations. Culture the domesticated strain in the maximum domestication concentration (1.4M) sodium acetate medium for 5 generations to obtain the mutant strain A2 and preserve the strain.

[0034] Identification of the strain: When grown on YPD agar medium, the colonies were orange-red, with a bright surface, soft and sticky texture, no obvious change in the color of the medium, and no exudate. ITS rDNA gene sequence identification showed a similarity of up to 99.8% with Rhodotorula glutinosus in NCBI GenBank. The mutant strain was finally identified as Rhodotorula glutinosus.

[0035] Example 2: Determination of growth characteristics of the original strain and mutant strain under sodium acetate stress

[0036] A single colony was picked from the cultured single colony plate and inoculated into YPD medium, and cultured at 30°C and 210rpm for 72h. After the culture, the bacteria were washed twice with sterile water, the bacterial sludge was collected, and resuspended in the maximum concentration of sodium acetate (0.6M) medium that the original strain could grow, and the initial OD600 of the inoculation was adjusted to 0.6. The bacterial biomass, glucose content, and sodium acetate content in the fermentation broth were sampled every 24h to monitor.

[0037] The biomass of the strain was determined by the gravimetric method. After each sampling, the bacteria were washed twice with sterile water, and the bacterial sludge was collected and dried at 60°C to a constant weight (about 24 h) and then weighed.

[0038] The content of glucose and sodium acetate in the fermentation broth was analyzed by HPLC equipped with a differential refractive index detector and an Aminex HPX-87H column at 60°C. The mobile phase was 5 mM sulfuric acid, eluted at 0.6 mL / min for 50 min, and the injection volume was 20 μL. Glucose and sodium acetate standards were used for qualitative and quantitative analysis.

[0039] The growth, glucose and sodium acetate consumption of the original strain and mutant strain under sodium acetate stress were as follows: Figure 2 As shown in A, the mutant strain entered the exponential phase within 24 hours, while the original bacteria entered the exponential phase after 48 hours. At the end of the fermentation, the biomass of the mutant strain was significantly higher than that of the wild strain, indicating that the mutant strain had a certain resistance to sodium acetate. Figure 2 B and C reflect that the mutant strain consumes glucose and sodium acetate significantly faster than the original strain, indicating that the mutant strain consumes carbon sources faster and has a higher substrate utilization rate than the original strain.

[0040] Example 3: Determination of lipid accumulation in strains

[0041] At the end of fermentation (144h), the fermentation broth was collected, and the cells were collected by centrifugation at 2772×g for 5min. After washing the bacteria twice with sterile water, the bacterial sludge was freeze-dried to obtain bacterial powder, which was stored at -40°C in the dark. The freeze-dried bacterial powder was suspended in methanol, and 0.15mg / mL lauric acid was used as the internal standard. 0.12mm zirconium oxide beads were added and crushed in a freeze grinder. An appropriate amount of chloroform was added to the crushed cells, so that the volume ratio of chloroform / methanol / water was 2 / 1 / 0.8, vortexed for 3min, centrifuged at 1728×g for 10min, the chloroform layer was recovered, transferred to a glass test tube, and dried under N2. Subsequently, 2% H2SO4 methanol solution was added to the above extract, and methylated at 70°C for 3h. An equal amount of saturated NaCl solution was added to the sample, vortexed for 15s, and then an equal amount of n-hexane was added, vortexed for 3min, and centrifuged at 1728×g for 10min. The n-hexane layer was recovered, a small amount of anhydrous sodium sulfate was added to remove the water in the sample, and centrifuged at 1728×g for 10 minutes. Finally, the supernatant was filtered through a 0.22μm filter for further analysis and measurement of strain lipid accumulation. A gas chromatograph-mass spectrometer equipped with an FID and an HP-88 column was used for measurement, with an inlet temperature of 250°C, an injection volume of 1μL, helium as a carrier gas, a flow rate of 0.6mL / min, and a split ratio of 15:1. Chromatographic conditions: initial temperature 180 degrees Celsius, maintained for 2min; increased to 210 degrees Celsius at 2°C / min, maintained for 5min; then increased to 220°C at 2.5°C / min, maintained for 4min, and a total running time of 30min. Mass spectrometry conditions: the ion source was an electron ionization source, the temperature was 230°C, and the electron energy was 70eV. A full scan was performed in the mass range of 50 to 550amu.

[0042] The lipid accumulation results of the strain in 0.6 M sodium acetate are shown in Figure 3 As shown, oleic acid (C18:1n9c), palmitic acid (C16:0) and stearic acid (C18:0) are the main fatty acids. The accumulated lipid content of the mutant strain is 596.5±10.0 mg / g cell dry weight, which is significantly higher than that of the original strain (200.7±10.4 mg / g cell dry weight). After long-term sodium acetate stress, the total lipid accumulation of the mutant strain can be increased by 197% compared with the original strain.

[0043] The basic principles, main features and advantages of the present invention are shown and described above, but the present invention is not limited to the embodiments. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for producing high-yield oil using acetic acid. It is characterized in that The taxonomic name of the red yeast Rhodotorula mucilaginosa is named as red yeast A2, and it is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.31121.

2. Use of the red yeast rice according to claim 1 in oil preparation.

3. The use according to claim 2, It is characterized in that The red yeast is grown in an environment containing 0.6-1.4M sodium acetate to prepare oil.

4. The use according to claim 2, It is characterized in that The oils include oleic acid, palmitic acid and stearic acid.

5. The use according to claim 2, It is characterized in that The oil yield produced by fermentation of the red yeast is at least 90% higher than that of common red yeast.

6. A method for preparing the red yeast rice according to claim 1, It is characterized in that The following steps are involved: The wild-type Rhodotorula glutinosus was cultured in a medium containing sodium acetate, and the sodium acetate concentration in the medium was gradually increased to a maximum value of 1.4 M using an adaptive domestication method. The wild-type strain was then cultured continuously for at least 5 generations to obtain a Rhodotorula glutinosus mutant strain with high concentration sodium acetate tolerance.

7. The preparation method according to claim 6, It is characterized in that The culture medium is YPD culture medium.

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