Method for prolonging service life of rhodosporidium toruloides and application

Abstract

The invention discloses application of proline and / or glucosylglycerol in prolonging the service life of rhodosporidium toruloides in a high-glucose culture medium, a method for prolonging the service life of the rhodosporidium toruloides in the high-glucose culture medium and a microbial culture medium, and the service life of the rhodosporidium toruloides in the high-glucose culture medium is prolonged by adding the proline and / or the glucosylglycerol into the culture medium. The service life of the rhodosporidium toruloides in a high-glucose culture environment can be obviously prolonged.

Description

Technical Field

[0001] The present application relates to a method for prolonging the lifespan of Rhodotorula glutinis and its applications, belonging to the field of microbial engineering. Background Art

[0002] Rhodosporidium toruloides ( Rhodosporidium toruloides , R. toruloides , Rhodotorula glutinis) belongs to the basidiomycete heterothallic fungus and is an extremely important microorganism in the fermentation industry. It can use hexoses and pentoses derived from biomass as raw materials to produce important bio-based products: microbial oils, with intracellular oils accounting for more than 60% of the cell dry weight (Ratledge C, Wynn J P. Adv. Appl. Microbiol. 2002, 51:1-51; Li Y, Zhao Z, Bai F. Enzyme Microb. Technol. 2007, 41(3):312-317); industrial enzymes or enzymes for drug synthesis such as phosphodiesterase, phenylalanine ammonia-lyase (Hodgins D S. J Biol.Chem. 1971,246(9):2977-2985; Gilbert H J, Clarke I N, Gibson R K, et al. J Bacteriol. 1985,161(1):314-320), D-amino acid oxidase (Gadda G, Negri A, Pilone M S. J Biol.Chem. 1994,269(27):17809-17814; Liao G J, Lee Y J, Lee Y H, et al. Biotechnol.Appl.Biochem. 1998,27(Pt1):55-61), etc., as well as β-carotene and exopolysaccharides; and has relatively wide applications in sewage treatment and biopharmaceuticals.

[0003] Aging is a complex biological process that leads to the deterioration of the physical and biochemical state of cells, and further leads to diseases and ultimately cell death. Any intervention that can reduce the relevant environmental and genetic factors leading to aging is considered novel (López-Otín et al., 2013). In addition to the overall significance of aging research in biomedical and medical research, a better understanding of cellular aging in organisms will undoubtedly enhance the production of metabolites in many biotechnology-related cells, such as industrially important microorganisms for lipid production.

[0004] The effect of glucose on the lifespan of organism cells has been widely studied (Olivares-Marin et al., 2018). For example, the unicellular eukaryote, Saccharomyces cerevisiae, shows a shortened replicative and chronological lifespan when exposed to high glucose concentrations (>2%). In contrast, when lower glucose is given in the culture (≤2%), which is called calorie restriction (CR), the lifespan response shows a significant increase. This indicates that glucose's involvement in energy metabolism affects multiple lifespan-promoting mechanisms and pathways (Tello Padilla et al., 2018).

[0005] Excessive glucose, i.e., calorie excess in chronological lifespan (>2%), usually leads to a sharp cell death during aging in laboratory strains (McCleary & Rine, 2017). Since the industrial fermentation of Rhodotorula glutinis usually requires high concentrations of glucose as a carbon source, its lifespan and vitality are also affected or inhibited to a certain extent. Therefore, there is an urgent need for a method to extend the lifespan of Rhodotorula glutinis and improve its vitality in a high-sugar environment. Summary of the Invention

[0006] In the early research on the lifespan of Rhodotorula glutinis by the inventors of this application, it was found that proline and / or glucosylglycerol can extend the lifespan of Rhodotorula glutinis under conventional culture conditions (high glucose concentration ≤2%); through further research, the inventors unexpectedly found that proline and / or glucosylglycerol can also effectively extend the lifespan of Rhodotorula glutinis under high glucose concentration (high glucose concentration >2%), and based on this, this application was completed.

[0007] According to the first aspect of this application, there is provided the use of proline and / or glucosylglycerol to extend the lifespan of Rhodotorula glutinis ( R. toruloides ) in a high-sugar medium, wherein in the high-sugar medium, the concentration of glucose is greater than 20 g / L; preferably 30 - 200 g / L; more preferably 70 - 120 g / L.

[0008] Optionally, the concentration of glucosylglycerol in the medium is 20 - 150 mmol / L; preferably 50 - 120 mmol / L; more preferably 80 - 120 mmol / L.

[0009] Optionally, the concentration of proline in the medium is 0.1 - 10 mmol / L; preferably 1 - 5 mmol / L; more preferably 1 - 3 mmol / L.

[0010] Optionally, the glucosylglycerol is 2-O-α-d-glucosylglycerol.

[0011] According to the second aspect of this application, this application provides a method for extending the lifespan of Rhodotorula glutinis (R. toruloides A method for prolonging the lifespan, which includes adding proline and / or glucosylglycerol to the high-sugar culture medium; in the high-sugar culture medium, the concentration of glucose is greater than 20 g / L; preferably 30 - 200 g / L; more preferably 70 - 120 g / L.

[0012] According to the method described in claim 5, wherein the concentration of glucosylglycerol in the culture medium is 20 - 150 mmol / L; preferably 50 - 120 mmol / L; more preferably 80 - 120 mmol / L.

[0013] Optionally, the concentration of proline in the culture medium is 0.1 - 10 mmol / L; preferably 1 - 5 mmol / L; more preferably 1 - 3 mmol / L.

[0014] Optionally, the glucosylglycerol is 2-O-α-d-glucosylglycerol.

[0015] According to the third aspect of the present application, a microbial culture medium is provided, which includes a carbon source, a nitrogen source, water, and optionally inorganic salts and / or growth factors, wherein the carbon source includes glucose, and the concentration of glucose is greater than 20 g / L; preferably 30 - 200 g / L; more preferably 70 - 120 g / L; The culture medium further contains proline and / or glucosylglycerol; preferably, the concentration of glucosylglycerol is 20 - 150 mmol / L; preferably 50 - 120 mmol / L; more preferably 80 - 120 mmol / L; the concentration of proline is 0.1 - 10 mmol / L; preferably 1 - 5 mmol / L; more preferably 1 - 3 mmol / L; preferably, the glucosylglycerol is 2-O-α-d-glucosylglycerol; Preferably, the microorganism is Rhodosporidium toruloides ( R. toruloides )

[0016] According to the fourth aspect of the present application, there is provided the application of the method provided in the second aspect of the present application and the microbial culture medium provided in the third aspect of the present application in the industrial fermentation and / or biopharmacy of Rhodosporidium toruloides.

[0017] The beneficial effects that the present application can produce include: The inventors unexpectedly found in the research that proline and / or glucosylglycerol can effectively increase the survival percentage of Rhodosporidium toruloides, affect cell growth kinetics, and increase cell survival integral in a high-sugar culture medium, thereby achieving the extension of the lifespan of Rhodosporidium toruloides under high-sugar culture conditions. Description of the Drawings

[0018] Figure 1Schematic diagram of the process of Bioscreen C MBR automatic microbial growth analyzer in yeast CLS analysis.

[0019] Figure 2 Survival rate-time curves of Rhodotorula glutinis under different culture conditions. The CLS was measured using a Bioscreen C MBR automatic microbial growth analyzer, and the growth data was analyzed using SPOCK; "*" or "**" represents significant differences (* P<0.05, ** P<0.01), and the bars represent the standard error (SE); among them, Figure A shows the change in cell survival rate of different doses of GG compared with the control group; Figure B shows the change in cell survival rate of different doses of Pro compared with the control group.

[0020] Figure 3 Growth kinetic curves of Rhodotorula glutinis under different culture conditions. In the figure, Figure A (control group), Figure B (GG-50mM group), Figure C (GG-100mM group), Figure D (Pro-2mM group), and Figure E (Pro-5mM group) respectively show the average growth kinetics of three replicate samples on high-calorie cultures. The optical density (OD, 600nm) of the cultures was measured within 24 hours. The standard deviation of the three replicate experiments is shown by the bars.

[0021] Figure 4 Survival integral result graph of Rhodotorula glutinis under different culture conditions. The data represents the survival integrals of three replicate experiments of control, GG, and proline. The error bars are the standard errors (SE) calculated using SPOCK; statistical significance was determined by comparing the experimental groups with the control (* P<0.05, ** P<0.01); among them, Figure A shows the survival integral of the GG group compared with the control group; Figure B shows the survival integral of the Pro group compared with the control group.

[0022] Figure 5 Average doubling time of Rhodotorula glutinis under different culture conditions. This illustrates the growth kinetics of cultures treated with control, GG, and proline incubated in SC medium for 12 days. The data here represents the average doubling time of three replicate experiments for each treatment. The error bars are the standard errors (SE) calculated using SPOCK. Compared with the control, the statistical significance was determined as * P<0.05, ** P<0.01, and n.s. represents no significant difference; among them, Figure A shows the average doubling time of the GG group compared with the control group; Figure B shows the average doubling time of the Pro group compared with the control group. Detailed implementation mode

[0023] According to one aspect of the present application, proline and / or glucosylglycerol are provided to prolong Rhodotorula toruloides in high-sugar medium ( R. toruloidesUse for lifespan, wherein in the high-glucose medium, the concentration of glucose is greater than 20 g / L; preferably 30 - 200 g / L; more preferably 70 - 120 g / L.

[0024] Proline (Pro) and glucosylglycerol (GG) can effectively extend the lifespan of Rhodosporidium toruloides under conventional culture conditions (i.e., when the glucose concentration is 2% or 20 g / L or less). However, due to the fact that high concentrations of glucose (above 2%) can cause severe cell death, it is thus impossible to determine whether substances that can extend cell lifespan under conventional culture conditions also have the effect of extending lifespan in a high-glucose environment. For example, certain polyphenolic compounds that have been reported currently, such as resveratrol; NAD precursors such as NMN; and some isoprenyl flavonoid compounds that act through glucose or nutrient-dependent mechanisms and other compounds that can extend cell lifespan, have been proven to have no effect on extending cell lifespan in a high-glucose environment. The inventors of the present application unexpectedly found during the research that proline (Pro) and glucosylglycerol (GG) also have a significant effect on extending the chronological lifespan (CLS) of Rhodosporidium toruloides cells in a high-glucose environment.

[0025] In some embodiments, the concentration of glucosylglycerol in the medium is 20 - 150 mmol / L; in some embodiments, preferably 50 - 120 mmol / L; more preferably 80 - 120 mmol / L.

[0026] In some embodiments, the concentration of proline in the medium is 0.1 - 10 mmol / L; preferably 1 - 5 mmol / L; more preferably 1 - 3 mmol / L.

[0027] In some embodiments, the glucosylglycerol (GG) includes 6 stereostructures, including 2-αGG, 2S-1-αGG, 2R-1-αGG, 2-βGG, 2S-1-βGG, and 2R-1-βGG. In some embodiments, the glucosylglycerol is its natural configuration 2-αGG or 2-βGG. In some specific embodiments, the glucosylglycerol is 2-O-α-d-glucosylglycerol.

[0028] According to another aspect of the present application, the present application provides a method for extending the lifespan of Rhodosporidium toruloides ( R. toruloides in a high-glucose medium, which includes adding proline and / or glucosylglycerol to the high-glucose medium; in the high-glucose medium, the concentration of glucose is greater than 20 g / L; preferably 30 - 200 g / L; more preferably 70 - 120 g / L.

[0029] In some embodiments, the concentration of glucosylglycerol in the medium is 20 - 150 mmol / L; preferably 50 - 120 mmol / L; more preferably 80 - 120 mmol / L.

[0030] In some embodiments, the concentration of proline in the medium is 0.1 - 10 mmol / L; preferably 1 - 5 mmol / L; more preferably 1 - 3 mmol / L.

[0031] In some embodiments, the glucosylglycerol is 2 - O - α - d - glucosylglycerol.

[0032] In some embodiments, the high - sugar medium can be obtained by adding extra glucose to any Rhodosporidium toruloides medium, such as adding extra glucose to yeast extract peptone dextrose medium (YPD / YEPD medium), synthetic complete medium (SC medium), nitrogen - limited medium, inorganic salt medium, etc., so as to obtain the high - sugar medium.

[0033] According to another aspect of the present application, a microbial medium is provided, which includes a carbon source, a nitrogen source, water, and optionally inorganic salts and / or growth factors, wherein the carbon source includes glucose, and the concentration of the glucose is greater than 20 g / L; preferably 30 - 200 g / L; more preferably 70 - 120 g / L; The medium further contains proline and / or glucosylglycerol; preferably, the concentration of the glucosylglycerol is 20 - 150 mmol / L; preferably 50 - 120 mmol / L; more preferably 80 - 120 mmol / L; the concentration of the proline is 0.1 - 10 mmol / L; preferably 1 - 5 mmol / L; more preferably 1 - 3 mmol / L; preferably, the glucosylglycerol is 2 - O - α - d - glucosylglycerol; Preferably, the microorganism is Rhodosporidium toruloides ( R. toruloides )

[0034] In some embodiments, the carbon source may further include at least one of fructose, maltose, sucrose, ethanol, and ethyl fatty acid.

[0035] In some embodiments, the nitrogen source is selected from at least one of peptone, yeast powder, ammonium sulfate, ammonium chloride, and nitrate.

[0036] In some embodiments, the growth factor is selected from at least one of vitamins, amino acids, purines, and pyrimidines.

[0037] In some embodiments, the microbial culture medium can be obtained by adding glucose, proline, and / or GG to any culture medium known in the art, particularly the culture medium of Rhodotorula glutinis, especially the fermentation medium of Rhodotorula glutinis; in some embodiments, the culture medium is selected from YPD medium, YEPD medium, SC medium, nitrogen-limited medium, and inorganic salt medium.

[0038] According to another aspect of the present application, there is provided the application of the method provided in the second aspect of the present application and the microbial culture medium provided in the third aspect of the present application in the industrial fermentation and / or biopharmaceuticals of Rhodotorula glutinis.

[0039] The present application will be described in detail below with reference to examples, but the present application is not limited to these examples.

[0040] Unless otherwise specified, the materials and reagents in the examples of the present application are purchased through commercial channels.

[0041] Example 1 R. toruloides NP11 grows in YEPD agar plates (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, and 20 g / L agar), grows at 30 °C for 3 days, collects yeast cells, and inoculates them onto 5 mL of YEPD liquid medium (2% bacteriological peptone, 1% yeast extract, and 2% glucose), and incubates them in a shaker at 30 °C and 200 rpm for 24 hours. Thereafter, the culture is inoculated onto freshly prepared SC medium containing 10% glucose (containing amino acids, adenine, yeast nitrogen base (1.8 g / L), glucose (100 g / L), ammonium sulfate (5 g / L), and four-fold excess of histidine, leucine, tryptophan, and uracil (Hu et al., 2013)).

[0042] The SC medium is divided into three groups. The control group (control) does not add Pro or GG. The proline group adds 2 mM proline (99%, Sigma-Aldrich) (Pro-2 mM) and 5 mM proline (Pro-5 mM) respectively; the glucosylglycerol group adds 50 mM glucosylglycerol (2-O-α-d-glucosylglycerol, 2-αGG, 99.3%, Zhongke SmartCyano Co., Ltd. (Qingdao, China)) (GG-50 mM) and 100 mM glucosylglycerol (GG-100 mM) respectively; three parallel experimental groups are set for each group.

[0043] In a shaker (30 oIncubation began after 48 hours in C and 200 rpm. Every 48 hours, 5 μL of yeast culture was collected and transferred to a 100-well honeycomb plate of the Bioscreen C MBR automatic microbial growth analyzer containing 145 μL of YEPD, and recorded every 15 minutes at 600 nm. At 30 o C and constant stirring for 24 hours to obtain the growth data of the cells. Finally, the R language survival analysis tool Survival Percentages and Outgrowth Collection Kit (SPOCK) was used to calculate the survival percentage, growth kinetics, survival integral, and doubling time of yeast cells in each experimental group (Smallet al., 2020). The schematic diagram of the analysis process is as Figure 1 shown.

[0044] (A) Glucose glycerol and proline can extend the lifespan of Rhodotorula glutinis After culturing in SC medium for 96 hours, cell viability analysis was performed, indicating that GG and proline can eliminate the so-called toxicity through the pro-aging effect of high glucose on yeast cells. The results are shown in Table 1.

[0045] Table 1. Cell viability of R. toruloides treated with different concentrations of GG and proline after 96 hours Number Experimental group Concentration (mM) Cell viability (%) Number of replicates P value 1 Control - 41.9 ± 6.5 4 - 2 GG 50 49 ± 9.5 4 ns 3 GG 100 67.2 ± 9.1 4 *P<0.05 4 Pro 2 76.9 ± 12.6 4 *P<0.05 5 Pro 5 63.2 ± 5.1 4 *P<0.05 During the chronological life cycle, calorie excess, that is, glucose concentration > 2%, usually leads to severe cell death in laboratory strains during aging (McCleary & Rine, 2017). The results of this experiment show that GG and proline can weaken these negative effects after administration.

[0046] Figure 2 showed that GG and proline significantly increased the survival rate of Rhodotorula glutinis on high glucose medium. Although both concentrations of GG (50 mM and 100 mM) produced good results, the cells exposed to the 100 mM concentration showed more obvious effects (**P < 0.01) ( Figure 2 A). Proline showed better effects at lower concentrations (2 mM) (** < P0.01) ( Figure 2 B).

[0047] (B) Glucose glycerol and proline support the growth kinetics of R. toruloides Cell growth provides important information on how organisms respond to different substances or growth conditions. Examine the 24-hour growth data to evaluate the effects of GG and Pro on Rhodotorula glutinis. The experimental results are as Figure 3 shown, compared with the control group (Figure 3 (Figure A), the growth kinetic curves of cells in each experimental group at different times showed impressive growth increases ( Figure 3 as shown in Figures B, C, D, and E), indicating a significant effect on cell lifespan extension in each experimental group. The control group showed more right-shifted retardation, and both treatments (Figures B - D) showed positive kinetic rises relative to the control (Figure A). Without being limited to any theory, the inventors believe this may be due to the induction of gene expression by the two substances that control cell metabolism, stress response, and extend the lifespan of Rhodotorula glutinis under hypernutritional conditions. Among all experimental groups, 100 mM GG showed better effects than 50 mM GG, especially on the 10th and 12th days, indicating that high-concentration GG has a longer lifespan effect. In addition, growth kinetic analysis showed the universality of cell viability within the population (Molon et al., 2016).

[0048] (C) Glucose, glycerol, and proline increase R. toruloides the survival score and doubling time Figure 4 shows the yeast cells cultured in SC medium for 12 days R. toruloides and the survival scores in different experimental groups. The results showed that GG and proline could improve the survival rate. The survival score, also known as the area under the curve, represents the overall cell performance of each treatment. When comparing the treatments with the control, 100 mM GG and 2 mM proline had more significant (**P < 0.01) growth responses, and this finding confirmed the geroprotective efficacy of GG and Pro and their ability to extend lifespan at the provided concentrations.

[0049] Figure 5 shows the doubling times of Rhodotorula glutinis in different experimental groups cultured in SC medium for 12 days. Compared with the control, there was no significant change in the doubling times of the strains in each experimental group, which may be due to the stress caused by high glucose in the medium.

[0050] As described above, these are only several embodiments of the present application and do not impose any form of limitation on the present application. Although the present application is disclosed with preferred embodiments as above, it is not intended to limit the present application. Any person skilled in the art, without departing from the scope of the technical solution of the present application, making some changes or modifications using the disclosed technical content is equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

Claims

1. Use of proline and / or glucosylglycerol for prolonging the lifespan of Rhodotorula glutinis in a high-sugar culture medium, wherein, R. toruloides ), In the high-sugar medium, the concentration of glucose is greater than 20 g / L; preferably 30 - 200 g / L; more preferably 70 - 120 g / L.

2. The use according to claim 1, wherein, The concentration of glucosylglycerol in the medium is 20 - 150 mmol / L; preferably 50 - 120 mmol / L; more preferably 80 - 120 mmol / L.

3. The use according to claim 1, wherein The concentration of proline in the medium is 0.1 - 10 mmol / L; preferably 1 - 5 mmol / L; more preferably 1 - 3 mmol / L.

4. Use according to any one of claims 1 - 3, wherein, The glucosylglycerol is 2-O-α-d-glucosylglycerol.

5. A method for prolonging the lifespan of Rhodosporidium toruloides in a high-sugar medium, which comprises adding proline and / or glucosylglycerol to the high-sugar medium; in the high-sugar medium, the concentration of glucose is greater than 20 g / L; preferably 30 - 200 g / L; more preferably 70 - 120 g / L. R. toruloides ​ 6. The method according to claim 5, wherein The concentration of glucosylglycerol in the medium is 20 - 150 mmol / L; preferably 50 - 120 mmol / L; more preferably 80 - 120 mmol / L.

7. The method according to claim 4, wherein, The concentration of proline in the medium is 0.1 - 10 mmol / L; preferably 1 - 5 mmol / L; more preferably 1 - 3 mmol / L.

8. The method according to any one of claims 5-7, wherein The glucosylglycerol is 2-O-α-d-glucosylglycerol.

9. A microbial medium comprising a carbon source, a nitrogen source, water, and optionally inorganic salts and / or growth factors, wherein the carbon source comprises glucose, and the concentration of glucose is greater than 20 g / L; preferably 30 - 200 g / L; more preferably 70 - 120 g / L; The medium further comprises proline and / or glucosylglycerol; preferably, the concentration of glucosylglycerol is 20 - 150 mmol / L; preferably 50 - 120 mmol / L; more preferably 80 - 120 mmol / L; the concentration of proline is 0.1 - 10 mmol / L; preferably 1 - 5 mmol / L; more preferably 1 - 3 mmol / L; preferably, the glucosylglycerol is 2-O-α-d-glucosylglycerol; Preferably, the microorganism is Rhodosporidium toruloides ( R. toruloides ).

10. Use of the method according to any one of claims 5 - 8 and the medium according to claim 9 in the industrial fermentation and / or biopharmacy of Rhodosporidium toruloides.

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