Candida viswanathii and application thereof

Abstract

The application provides a Candida viswanathii CATH2402, the preservation number of which is CCTCC NO: M 20241166, and also provides application of the aforementioned Candida viswanathii in fermentative production of azelaic acid, and a production method of azelaic acid. The Candida viswanathii CATH2402 can utilize various substrates to fermentatively produce azelaic acid, and has high tolerance to the product azelaic acid, thereby significantly improving the yield of azelaic acid by biological method, and having the potential for industrialized scale production.

Description

Technical Field

[0001] This invention belongs to the field of fermentation technology, specifically relating to a Candida viride strain for fermenting and producing azelaic acid and its application. Background Technology

[0002] Azelaic acid, also known as azelaic acid, is a saturated dicarboxylic acid containing 9 carbon atoms, with the molecular formula C9H12O. 16 O4, a medium-chain dicarboxylic acid, is widely used in many industrial fields. For example, azelaic acid can be used to produce dioctyl azelate plasticizer; azelaic acid can be used in the synthesis of resins and synthetic fibers; it can also be used as a raw material for the production of fragrances, lubricants, cosmetics, and food additives.

[0003] The preparation of azelaic acid mainly relies on chemical synthesis methods, such as potassium permanganate oxidation and hydrogen peroxide oxidation. Although microbial synthesis has the advantages of being environmentally friendly and sustainable, there is relatively little research on microbial synthesis for azelaic acid production. Existing engineered strains generally suffer from problems such as low product tolerance and difficulty in regulating metabolic flux. There are almost no reports, both domestically and internationally, on the synthesis of azelaic acid using Candida virescens through a fully bio-fermentation method. Summary of the Invention

[0004] The purpose of this invention is to provide an engineered strain for producing azelaic acid, which can utilize a variety of substrates for fermentation and conversion to produce azelaic acid, significantly increasing the yield of azelaic acid produced by biological methods and possessing the potential for industrial-scale production.

[0005] In a first aspect, this invention provides a Candida viswanathii strain, specifically Candida viswanathii CATH2402, with accession number CCTCC NO: M 20241167. It was deposited on June 13, 2024, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), and classified as Candida viswanathii.

[0006] The present invention obtained Candida viswanathii CATH2402 through adaptive evolution screening. Candida viswanathii 2402 was identified as belonging to Candida viswanathii by ITS (Internal Transcribed Spacer) sequence, and the ITS sequence is referenced from GenBank No. MK394122.1.

[0007] Specifically, this invention employs a laboratory adaptive evolution method, using Candida albicans strain CAES2113 (accession number: CCTCC NO: M2020048, disclosed in patent application CN111748480B) as the starting strain, gradually increasing the nonane concentration in the culture medium to screen out Candida albicans strains with high nonane tolerance, and then conducting fermentation transformation experiments to further screen for Candida albicans strains with higher azelaic acid production.

[0008] The second aspect of this invention is to provide an application of the aforementioned Candida virescens in the fermentation production of azelaic acid.

[0009] A third aspect of the present invention provides a method for producing azelaic acid, comprising: fermenting and culturing Candida vesicularia CATH2402 to obtain a fermentation broth, and extracting azelaic acid from the fermentation broth, or extracting and purifying azelaic acid from the fermentation broth; wherein the fermentation substrate is selected from any one or more combinations of nonanoic acid, nonanoic acid ester, nonanoic acid salt, or nonane.

[0010] In some embodiments, the nonane is more preferably n-nonane. The nonane can be a petroleum-based alkane or a coal-derived alkane, or a bio-based alkane obtained through the processing of vegetable oils.

[0011] In some embodiments, the nonanoate ester is selected from any one or a combination of glycerol nonanoate ester, nonanoate nonyl ester, and hexanoate nonyl ester.

[0012] In some embodiments, the nonanoate is selected from any one or a combination of sodium nonanoate, potassium nonanoate, and calcium nonanoate.

[0013] In some embodiments, the fermentation substrate is selected from any of the following combinations: a combination of nonanoic acid and nonanoic acid ester; a combination of nonanoic acid and nonane; a combination of nonanoic acid ester and nonane; a combination of nonanoic acid salt and nonane; or a combination of nonanoic acid, nonanoic acid ester and nonane.

[0014] In the above combinations consisting of two or three different types of fermentation substrates, the mass ratio of the different types of fermentation substrates is not particularly limited. For example, when the fermentation substrate is a combination of nonanoic acid and nonanoic acid ester, the mass ratio of nonanoic acid and nonanoic acid ester is not particularly limited, and can be, for example, 1:99 to 99:1.

[0015] In some embodiments, the fermentation culture temperature is 28°C to 35°C, for example 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C.

[0016] In some embodiments, the dissolved oxygen level during fermentation is 10% to 80%, for example 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 80%. Preferably, it is 20% to 80%.

[0017] In some implementations, the air volume for fermentation is 0.2 to 0.8 vvm, for example 0.2 vvm, 0.3 vvm, 0.4 vvm, 0.5 vvm, 0.6 vvm, 0.7 vvm or 0.8 vvm.

[0018] In some implementations, the fermentation culture pressure is 0.05–0.2 MPa, for example 0.05 MPa, 0.08 MPa, 0.11 MPa, 0.12 MPa, 0.15 MPa, or 0.2 MPa.

[0019] In some implementations, the pH of the fermentation culture is 4.5 to 7.5.

[0020] In some embodiments, the fermentation culture is carried out under acidic conditions; preferably, the pH value during fermentation culture is 4.5 to 6.9, and further 4.5 to 6.5, for example 4.5, 4.8, 5.0, 5.5, 5.8, 6.0, 6.4, or 6.5. This can be adjusted by adding sodium hydroxide or dilute sulfuric acid.

[0021] In some embodiments, the fermentation culture is carried out under alkaline conditions; preferably, the pH value during fermentation culture is 7.0 to 7.5, for example, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. This can be adjusted by adding sodium hydroxide or dilute sulfuric acid.

[0022] In some embodiments, during fermentation culture, when the optical density OD620 of the strain diluted 30 times reaches 0.5–1.0, preferably 0.7–1.0, for example 0.7, 0.8, 0.9, or 1.0, the fermentation substrate is added to begin fermentation conversion. The fermentation substrate can be added in a single step, in batches, or continuously fed. Preferably, during fermentation conversion, the concentration of the fermentation substrate is controlled to be below 8% (v / v), and further to 0.1–5% (v / v).

[0023] In some embodiments, when the fermentation substrate is solid, it is first heated to dissolve it before being added to the fermentation broth; wherein, the temperature at which the fermentation substrate is heated can be 40 to 100°C.

[0024] In some implementations, the fermentation time is 120 to 180 hours.

[0025] In some implementations, the culture medium for fermentation contains at least a carbon source, a nitrogen source, and / or inorganic salts.

[0026] In some preferred embodiments, the carbon source is selected from at least one of glucose, sucrose, lactose, maltose, fructose, molasses, glycerol, sorbitol, arabinose, rhamnose, cellobiose, sophorose, and gentiobiose, and more preferably from at least one of glucose, lactose, and sucrose.

[0027] In some preferred embodiments, the nitrogen source is selected from at least one of yeast extract, peptone, corn steep liquor, urea, ammonium salts, and nitrates.

[0028] In some preferred embodiments, the inorganic salt is selected from at least one of sulfates, hydrochlorides, nitrates and phosphates, more preferably from at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, calcium chloride and potassium nitrate.

[0029] In some preferred embodiments, the culture medium comprises a seed culture medium and a fermentation culture medium, both containing the carbon source, the nitrogen source, and the inorganic salts. *Candida virescens* is first cultured in the seed culture medium, and then inoculated into the fermentation culture medium for further culture and fermentation transformation.

[0030] In some specific embodiments, the seed culture medium contains at least: 15-30 g / L sucrose or glucose, 1-3 g / L corn steep liquor, 4-7 g / L yeast extract, 5-8 g / L potassium dihydrogen phosphate, and 1-3 g / L urea.

[0031] In some specific embodiments, the fermentation medium contains at least: 20-35 g / L sucrose or glucose, 3-5 g / L corn steep liquor, 2-5 g / L yeast extract, 5-8 g / L potassium dihydrogen phosphate, 2-4 g / L potassium nitrate, 1-2 g / L sodium chloride, and 0.5-2 g / L urea.

[0032] Preferably, the fermentation medium further contains an antifoaming agent, which includes polyether antifoaming agents, organosilicon antifoaming agents, silicone-ether composite antifoaming agents, mineral oil antifoaming agents, or nonionic surfactants, preferably, for example, polydimethylsiloxane, polyoxypropylene glycerol, or polyoxyethylene oxypropylene glycerol.

[0033] In some embodiments, during fermentation culture, the inoculum size of Candida virescens is 5 to 50% (v / v) of the fermentation medium, that is, the volume ratio between the seed culture and the fermentation medium is (5 to 50): 100.

[0034] A fourth aspect of the present invention is to provide a fermentation broth containing Candida viswanathii CATH2402 and azelaic acid.

[0035] The fifth aspect of this invention provides an evolutionary screening method for *Candida viride*, which uses *Candida viride* as the starting strain, gradually increases the nonane concentration in the evolutionary medium, screens for *Candida viride* strains with high nonane tolerance, and then conducts fermentation transformation experiments to further screen for *Candida viride* strains with higher azelaic acid production. The evolutionary medium is a conventional medium in the art, such as commercially available YNB medium; the starting strain can be an industrial *Candida viride* strain used for producing long-chain dicarboxylic acids with 10-16 carbon atoms, such as *Candida viride* strain CAES2113 (accession number: CCTCCNO: M2020048, disclosed in invention patent CN111748480B).

[0036] In some embodiments, the evolutionary screening method includes a first step of laboratory adaptive evolutionary screening and a second step of fermentation transformation screening:

[0037] The first step was to culture the starting strain *Candida viviparous* in an evolution medium containing 0.2–0.3 M nonane. When the OD620 of the cells after a 30-fold dilution was greater than 0.5, the cells were transferred to a fresh evolution medium with the same concentration of nonane. When the strain could grow stably and rapidly in the evolution medium with this concentration of nonane, the concentration of nonane in the growth environment of the strain was gradually increased. After 12–15 months of adaptation and acclimatization, mutant strains tolerant to 0.4–0.5 M nonane concentration were screened.

[0038] The second step involves serially diluting the mutant strain obtained in the first step, selecting well-growing single clones, and fermenting them in a fermentation medium containing the fermentation substrate. The strain with the highest azelaic acid yield is then screened to obtain a high-yield azelaic acid bacteria. The fermentation substrate is preferably 1–3% n-nonane. The nonane used in the first step is preferably n-nonane.

[0039] The technical solution provided by this invention has the following beneficial effects: 1. The *Candida viride* CATH2402 strain of this invention can produce azelaic acid using different types of fermentation substrates and exhibits high tolerance to nonane. 2. The *Candida viride* CATH2402 strain of this invention significantly increases the yield of the target azelaic acid and significantly shortens the fermentation time, showing a marked improvement compared to existing strains, and can be used for industrial-scale production. 3. In the production method of *Candida viride* CATH2402 and azelaic acid of this invention, fermentation conversion can be carried out under both acidic and alkaline conditions, broadening the range of options for subsequent extraction processes. Detailed Implementation

[0040] The present invention will be further illustrated below with specific embodiments. These embodiments are merely illustrative and do not limit the scope of the invention. Unless otherwise specified, the raw materials and ingredients used in the following embodiments are commercially available products, and the methods and conditions used are known in the art and are conventional conditions. Raw materials requiring sterilization were sterilized at 121°C for 20 minutes.

[0041] Materials and methods:

[0042] I. Strains

[0043] Initiating strain: Candida viswanathii CAES2113 (accession number: CCTCCNO: M2020048, published in patent application CN111748480B).

[0044] Candida viswanathii CATH2402 (Preservation number: CCTCC NO: M).

[0045] II. Culture Medium

[0046] Evolution medium: YNB medium supplemented with 2% sucrose, which was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0047] YPD medium (w / v): 2% peptone, 2% glucose and 1% yeast extract (OXOID, LP0021). 1.5% agar powder should also be added to the solid medium.

[0048] III. Product Detection Method: Azelaic acid in the fermentation broth was detected by high performance gas chromatography. The product concentration was calculated based on the ratio of the peak area of ​​the product to the peak area of ​​an internal standard of known concentration.

[0049] IV. Obtaining Candida albicans CATH2402

[0050] Step 1: Laboratory adaptive evolution screening: The original strain Candida viswanathii (CAES2113) was inoculated into evolution medium supplemented with 0.22M n-nonane using glycerol tubes. When the optical density OD620 of the cells after a 30-fold dilution was greater than 0.5, the cells were transferred to fresh evolution medium with the same concentration of n-nonane. When the evolved strain could grow stably and rapidly in the evolution medium with this concentration of n-nonane, the concentration of n-nonane in the growth environment of the strain was gradually increased. After 12 months of adaptive acclimatization, mutant strains tolerant to 0.4M n-nonane concentration were screened.

[0051] The second step, fermentation and transformation screening, involved serially diluting the mutant strains. Appropriately diluted bacterial solutions were spread onto solid plates and cultured until single colonies formed. Well-growing single colonies were selected and transferred to well-drained plates containing primary screening medium (fermentation medium containing 3% n-nonane substrate, 30 g / L sucrose, 5 g / L corn steep liquor, 5 g / L yeast extract, 8 g / L potassium dihydrogen phosphate, 4 g / L potassium nitrate, 1.5 g / L sodium chloride, and 0.5 g / L urea). Fermentation was carried out at 29°C and at natural pH for 70 hours. After fermentation, primary screening was performed, and strains with relatively high azelaic acid production were selected. These strains were then re-screened using well plates. The re-screened strains were further screened by 500 ml shake-flask fermentation at 29°C and at natural pH for 120 hours. Finally, stable, high-yielding strains were obtained and stored in glycerol tubes. ITS sequence identification was performed on the purified single colony ITS sequence obtained in the above steps. The DNA sequence was compared with the known ITS sequence of Candida vesicularis (see GenBank No. MK394122.1). It was identified as Candida vesicularis and thus named Candida vesicularis CATH2402.

[0052] V. Stability verification of Candida albicans strain CATH2402

[0053] Continuous subculturing experiments verified that strain CATH2402 exhibited good stability in morphology, growth, and production performance. No significant changes in colony morphology were observed after five subculturings. CATH2402 colonies were smooth, moist, milky white, glossy, round, and had regular edges. These results indicate that *Candida virescens* strain CATH2402 demonstrates good subculturing stability.

[0054] The performance of the first and fifth generation strains obtained above in producing DC9 was verified by shake-flask fermentation. Glycerol tubes (one for the first generation and one for the fifth generation) of *Candida viswanathii* CATH2402 were inoculated into YPD medium and cultured for 24 h. The culture was then transferred to seed culture medium containing: 20 g / L sucrose, 2 g / L corn steep liquor, 6 g / L yeast extract, 8 g / L potassium dihydrogen phosphate, and 2 g / L urea. After culturing at 29°C for 24 h, the OD620 of the seed culture reached 0.7 (30-fold dilution with water). The seed culture was then inoculated into shake flasks containing fermentation medium and 3.5 mL of n-nonane. The fermentation medium contained: 30 g / L sucrose, 5 g / L corn steep liquor, 5 g / L yeast extract, 8 g / L potassium dihydrogen phosphate, 4 g / L potassium nitrate, 1.5 g / L sodium chloride, and 0.5 g / L urea. Fermentation was carried out at 30℃. After fermentation, the yields of DC9 in the first and fifth generations were measured to be 21.86 g / L and 22.04 g / L, respectively.

[0055] Examples 1-3: Fermentation of Candida virescens CATH2402 in shake flasks for the production of azelaic acid.

[0056] One glycerol tube of *Candida viswanathii* CATH2402 was inoculated into YPD medium and cultured for 24 h. Then, it was inoculated into seed culture medium containing: 20 g / L sucrose, 3 g / L corn steep liquor, 5 g / L yeast extract, 8 g / L potassium dihydrogen phosphate, and 3 g / L urea. After culturing at 29℃ for 26 h, the OD620 of the seed culture reached 0.71 (30-fold dilution with water). 3.8 mL of the seed culture was then inoculated into a shake flask containing 15 mL of fermentation medium containing: 30 g / L sucrose, 5 g / L corn steep liquor, 2 g / L yeast extract, 8 g / L potassium dihydrogen phosphate, 4 g / L potassium nitrate, 1.5 g / L sodium chloride, and 0.5 g / L urea. In Examples 1-3, the amount of n-nonane added to the fermentation medium was 3.0 mL, 3.5 mL, and 4.0 mL, respectively. Fermentation was carried out at 30°C, and the fermentation was stopped when the n-nonane content in the fermentation broth was 0. The test results of the obtained fermentation broth are shown in Table 1 below.

[0057] Comparative Example 1: Fermentation of Candida albicans CAES2113 in shake flasks to produce azelaic acid.

[0058] One germinating strain of Candida viswanathii (CAES2113) was taken and fermented in a glycerol tube using the same process as in Example 2. Fermentation was stopped when the n-nonane content in the fermentation broth was 0. The test results of the obtained fermentation broth are shown in Table 1 below.

[0059] Table 1

[0060]

[0061] Examples 4-8: Fermentation of azelaic acid using *Candida viride* CATH2402 in a fermenter.

[0062] (1) Activation culture: 100 mL of 10 Baume malt extract (pH 5.4) was inoculated into a CATH2402 glycerol tube seed of Candida viviparus. The seed culture was activated in a shake flask at 29℃ and 220 rpm. The culture was stopped when the OD620 value of the cells reached 0.75 (30 times dilution) to obtain the shake flask seed.

[0063] (2) Seed culture: The shake flask seeds obtained in step (1) were inoculated into a seed tank containing seed culture medium containing 20 g / L sucrose, 3 g / L corn steep liquor, 5 g / L yeast extract, 8 g / L potassium dihydrogen phosphate, 3 g / L urea, and an inoculum of 1.8% (v / v). The culture was carried out at a temperature of 29℃, an air flow of 0.3 vvm, a pressure of 0.12 MPa, and a dissolved oxygen of 15%. The culture was stopped when the OD620 value of the seed solution reached 0.8 (30 times dilution) to obtain the seed solution.

[0064] (3) Fermentation culture: The seed culture obtained in step (2) was inoculated into a fermenter containing fermentation culture medium containing 30 g / L sucrose, 5 g / L corn steep liquor, 2 g / L yeast extract, 8 g / L potassium dihydrogen phosphate, 4 g / L potassium nitrate, 1.5 g / L sodium chloride, and 0.5 g / L urea. The inoculum size was 20% (v / v). Fermentation culture was carried out at a temperature of 29℃, an air volume of 0.31 vvm, a pressure of 0.12 MPa, and a dissolved oxygen content of 45%. When the OD620 value of the fermentation broth reached 0.8 (30 times dilution), the fermentation substrate was added to the fermenter in batches for fermentation conversion. The substrate concentration in the fermentation broth was controlled to not exceed 2.0% (v / v). After 100 h of fermentation, the addition of substrate was stopped, and the remaining substrate in the fermentation system was used for fermentation. Fermentation was stopped when the substrate concentration in the fermentation broth was detected to be 0. The total fermentation time was calculated. The fermentation substrate, fermentation pH, total fermentation time, and azelaic acid yield are shown in Table 2 below.

[0065] Comparative Example 2: Fermentation of Candida albicans CAES2113 in a fermenter to produce azelaic acid.

[0066] The method for preparing azelaic acid was the same as in Example 6, except that the strain used was *Candida viswanathii* CAES2113. The results are shown in Table 2 below.

[0067] Table 2

[0068]

[0069] As shown in Table 2, under the same fermentation conditions, the difference between Example 6 and Comparative Example 2 lies in the different *Candida viviparous* strains. Example 6 shows that the DC9 yield of the strain CATH2402 of this invention is significantly increased, and the fermentation time is significantly shortened. Examples 4, 5, and 8 show that the strain CATH2402 of this invention can ferment at lower pH levels, with acid production all exceeding 55 g / L, and Example 4 even reaching over 66 g / L. Example 8 shows that the strain of this invention can also ferment a mixture of alkanes and fatty acids to produce DC9.

[0070] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A type of Candida virescens, characterized in that, The *Candida viswanathii* strain mentioned is *Candida viswanathii* CATH2402, with accession number CCTCC NO: M 20241166.

2. The application of the Candida virescens strain as described in claim 1 in the fermentation production of azelaic acid.

3. A method for producing azelaic acid, characterized in that, It includes: Fermentation broth was obtained by fermenting and culturing the Candida viswanathii CATH2402 as described in claim 1; as well as, Azelaic acid is extracted from the fermentation broth, or azelaic acid is extracted from and purified from the fermentation broth; The fermentation substrate is selected from any one or more combinations of nonanoic acid, nonanoic acid ester, nonanoic acid salt, or nonane.

4. The production method according to claim 3, characterized in that, The fermentation substrate is selected from any of the following combinations: a combination of nonanoic acid and nonanoic acid ester; a combination of nonanoic acid and nonane; a combination of nonanoic acid ester and nonane; a combination of nonanoic acid salt and nonane; or a combination of nonanoic acid, nonanoic acid ester and nonane.

5. The production method according to claim 3, characterized in that, The fermentation culture temperature is 28℃~35℃, and / or, dissolved oxygen is 10%~80%, and / or, air volume is 0.2~0.8vvm, and / or, pressure is 0.05~0.2Mpa.

6. The production method according to claim 3, characterized in that, The pH value for fermentation culture is 4.5–7.

5.

7. The production method according to claim 3, characterized in that, Fermentation time is 120–180 hours.

8. The production method according to claim 3, characterized in that, The culture medium for fermentation contains at least a carbon source, a nitrogen source, and / or inorganic salts.

9. A fermentation broth, characterized in that, It contains Candida viswanathii CATH2402 and azelaic acid.

10. A method for evolutionary screening of Candida virescens as described in claim 1, characterized in that, Using *Candida viride* as the starting strain, the concentration of nonane in the evolutionary medium was gradually increased to screen out *Candida viride* strains with high nonane tolerance. Then, fermentation transformation experiments were conducted to further screen for *Candida viride* strains with higher azelaic acid production. The starting strain is an industrial strain of *Candida viride* strain used to produce long-chain dicarboxylic acids with 10 to 16 carbon atoms.

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