Candida viswanathii and application thereof

Abstract

The application provides a Candida viswanathii CATH2401, the preservation number of which is CCTCC NO: M 20241166, and also provides application of the aforementioned Candida viswanathii in fermentation production of suberic acid and a production method of suberic acid. The Candida viswanathii CATH2401 has high tolerance to a substrate octane, and significantly improves the yield of suberic acid produced by a biological method.

Description

Technical Field

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

[0002] Octyl succinic acid, with the general formula HOOC(CH2)6COOH, is a medium-chain dicarboxylic acid widely used in various industrial sectors. It is a key raw material for the synthesis of polyesters, polyurethanes, and polyamides, and can be used to manufacture plastics, coatings, and fiber products. In the food industry, octyl succinic acid is used as a food additive to adjust the acidity and taste of foods such as carbonated beverages, fruit juices, and baked goods. In the pharmaceutical field, octyl succinic acid is used as an intermediate in the synthesis of antibiotics, anticancer drugs, and antiviral drugs. It can also be used in the production of cosmetics.

[0003] The preparation of octanediic acid mainly relies on chemical synthesis methods, such as the oxidation of castor oil or cyclooctane. Although microbial synthesis has the advantages of being environmentally friendly and sustainable, there is relatively little research on the microbial synthesis of octanediic acid. Existing engineered strains generally suffer from problems such as low product tolerance and difficulty in regulating metabolic flux. There are almost no reports on the synthesis of octanediic acid by the whole bio-fermentation method, both domestically and internationally. Summary of the Invention

[0004] The purpose of this invention is to provide an engineered strain for producing octanoic acid, which has high tolerance to the substrate octane and significantly improves the yield of octanoic acid produced by biological methods.

[0005] In a first aspect, this invention provides a Candida viswanathii strain, specifically Candida viswanathii CATH2401, with accession number CCTCC NO: M20241166. It was deposited on June 6, 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 CATH2401 through adaptive evolution screening. Candida viswanathii CATH2401 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 vesicatoria CAES2113 strain (accession number: CCTCC NO: M2020048, disclosed in patent application CN111748480B) as the starting strain, gradually increasing the octane concentration in the culture medium to screen out Candida vesicatoria strains with high octane tolerance, and then conducting fermentation transformation experiments to further screen for Candida vesicatoria strains with higher succinic acid production.

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

[0009] A third aspect of the present invention is to provide a method for producing octanoic acid, comprising: fermenting and culturing Candida viswanathii CATH2401 to obtain a fermentation broth, and extracting octanoic acid from the fermentation broth, or extracting and purifying octanoic acid from the fermentation broth; wherein the fermentation substrate is selected from any one or more combinations of octanoic acid, octanoic acid ester, octanoic acid salt, or octane.

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

[0011] In some embodiments, the octanoate is selected from any one or a combination of glyceryl octanoate, octanoate, and hexyl octanoate.

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

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

[0014] In the above combinations consisting of two or three different types of fermentation substrates, there is no particular limitation on the mass ratio of the different types of fermentation substrates. For example, when the fermentation substrate is a combination of octanoic acid and caprylate, there is no particular limitation on the mass ratio of octanoic acid and caprylate, for example, it can be 1:99 to 99:1.

[0015] 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 for heating the fermentation substrate can be 45–65°C.

[0016] In some implementations, the pH of the fermentation culture is 4.5–7.5. This can be adjusted by adding sodium hydroxide or dilute sulfuric acid.

[0017] 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.

[0018] 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 6.5.

[0019] 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.

[0020] In some embodiments, the fermentation substrate is added intermittently or all at once during fermentation culture, preferably intermittently. All-at-one addition is preferably done during inoculation with *Candida virescens*.

[0021] In some implementations, the interval between intermittent replenishments is 24 to 90 hours, or even 24 to 65 hours, for example, 24 hours, 48 ​​hours, or 65 hours.

[0022] In some implementations, the fermentation cycle is 120 to 220 hours.

[0023] In some implementations, the culture medium for fermentation contains at least a carbon source, a nitrogen source, and / or inorganic salts.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] In some preferred embodiments, the culture medium includes a seed culture medium and a fermentation culture medium, both containing the carbon source, the nitrogen source, and the inorganic salts. Specifically, *Candida virescens* is first cultured in the seed culture medium, and then inoculated into the fermentation culture medium for further culture and fermentation transformation.

[0028] 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.

[0029] A fourth aspect of the present invention is to provide a fermentation broth containing Candida viswanathii CATH2401 and succinic acid.

[0030] The fifth aspect of this invention provides an evolutionary screening method for *Candida virescens*, which uses *Candida virescens* as the starting strain, gradually increases the octane concentration in the evolutionary medium, screens for *Candida virescens* strains with high octane tolerance, and then conducts fermentation transformation experiments to further screen for *Candida virescens* strains with higher succinic 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 virescens* strain used for producing long-chain dicarboxylic acids with 10-16 carbon atoms, such as *Candida virescens* strain CAES2113 (accession number: CCTCC NO: M2020048, disclosed in patent application CN111748480B).

[0031] In some embodiments, the evolutionary screening method includes a first step of laboratory adaptive evolutionary screening and a second step of fermentation transformation screening:

[0032] The first step was to culture the starting strain, *Candida virescens*, in an evolution medium containing 0.05–0.1 M octane. When the OD620 of the cells after a 30-fold dilution was greater than 0.5, the strain was transferred to a fresh evolution medium with the same concentration of octane. When the strain could grow stably and rapidly in the evolution medium with this concentration of octane, the octane concentration in the growth environment of the strain was gradually increased. After 30–36 months of adaptive acclimatization screening, mutant strains tolerant to 1.0–1.5 M octane concentrations were obtained.

[0033] 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 succinic acid yield is then screened to obtain a high-succinic acid-producing bacterium. The fermentation substrate is preferably 5–10% n-octane. The octane used in the first step is preferably n-octane.

[0034] The technical solution provided by this invention has the following beneficial effects: the production of succinic acid by the *Candida virescens* CATH2401 strain of this invention is significantly improved; for example, compared with the starting strain, the succinic acid production is significantly improved compared with existing strains. The *Candida virescens* CATH2401 strain of this invention can use bio-based fermentation substrates, increasing the bio-based content of the succinic acid product, which is of great significance for the sustainable development of industrial processes. Detailed Implementation

[0035] 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.

[0036] Materials and methods:

[0037] I. Strains

[0038] Initiating strain: Candida viswanathii CAES2113 (accession number: CCTCC NO: M2020048, published in patent application CN111748480B).

[0039] Candida viswanathii CATH2401 (Preservation number: CCTCC NO: M20241166).

[0040] II. Culture Medium

[0041] Evolution medium: YNB medium supplemented with 2% sucrose, which was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0042] 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.

[0043] III. Product Detection Method: High performance gas chromatography was used to detect octanoic acid in the fermentation broth. 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.

[0044] IV. Obtaining Candida albicans CATH2401

[0045] Step 1: Laboratory adaptive evolution screening: The original strain Candida viswanathii (CAES2113) was inoculated into evolution medium supplemented with 0.1M n-octane 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-octane. When the evolved strain could grow stably and rapidly in the evolution medium with this concentration of n-octane, the concentration of n-octane in the growth environment of the strain was gradually increased. After 34 months of adaptive acclimatization, mutant strains tolerant to 1.2M n-octane concentration were screened.

[0046] 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 activated in seed culture medium, then transferred to shake-flask primary screening medium (a fermentation medium containing n-octane, 30 g / L sucrose, 8 g / L corn steep liquor, 5 g / L yeast extract, 8 g / L potassium dihydrogen phosphate, 7 g / L potassium nitrate, 1.5 g / L sodium chloride, and 0.5 g / L urea). Fermentation was carried out at 29°C and natural pH, with intermittent addition of n-octane, totaling 6.6 M n-octane, for a fermentation period of 144 hours. After fermentation, strains producing relatively high levels of succinic acid were selected. These strains were then subjected to a second screening in 500 ml shake flasks at 29°C and natural pH for a fermentation period of 192 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 CATH2401.

[0047] V. Stability verification of Candida albicans strain CATH2401

[0048] Continuous subculturing experiments verified that the *Candida viscera* strain CATH2401 exhibited good stability in morphology, growth, and production performance. No significant changes in colony morphology were observed after five subculturings. *Candida viscera* CATH2401 colonies were smooth, moist, milky-white, glossy, round, and had regular edges. These results indicate that *Candida viscera* CATH2401 demonstrates good subculturing stability.

[0049] The performance of the first and sixth generation strains obtained above in producing succinic acid was verified by shake-flask fermentation. One tube each of *Candida viviparus* CATH2401 glycerol was 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 0.6 (30-fold dilution with water). The seed culture was then inoculated into shake flasks containing 15 mL of fermentation medium and 10 mL of n-octane. 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 DC8 from the first and sixth generation strains were measured to be 3.65 g / L and 3.88 g / L, respectively.

[0050] Examples 1-3: Production of succinic acid from *Candida viride* CATH2401 in shake flasks

[0051] One tube of Candida viswanathii CATH2401 glycerol 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. The culture was maintained at 29°C for 26 h, and the OD620 of the seed culture reached 0.65 (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, 7 g / L corn steep liquor, 5 g / L yeast extract, 8 g / L potassium dihydrogen phosphate, 7 g / L potassium nitrate, 1.5 g / L sodium chloride, and 0.5 g / L urea.

[0052] In Examples 1 and 3, the n-octane was added intermittently, specifically as follows:

[0053] In Example 1, 2.1 mL of n-octane was added at the time of inoculation, and 2.1 mL of n-octane was added every 48 hours.

[0054] In Example 3, 3.7 mL of n-octane was added at the time of inoculation, and 2.1 mL of n-octane was added after 65 hours, followed by 2.1 mL of n-octane every 48 hours thereafter.

[0055] Example 2: The method of adding n-octane was to add 10 mL of n-octane at the time of inoculation.

[0056] Fermentation was carried out at 30℃, and the fermentation was stopped when the n-octane 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

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

[0059] Comparative Example 2

[0060] 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-octane content in the fermentation broth was 0. The test results of the obtained fermentation broth are shown in Table 1 below.

[0061] Table 1

[0062]

[0063] As shown in Table 1, under the same fermentation conditions, the difference between Example 1 and Comparative Example 1, and between Example 2 and Comparative Example 2, lies in the different *Candida virescens* strains. Examples 1 and 2 show that the succinic acid production of the strain CATH2401 of the present invention is significantly increased, and the average acid production rate is also significantly increased. Example 3 further demonstrates that the intermittent addition of n-octane as a substrate can enable strain CATH2401 to ferment and produce more succinic acid.

[0064] 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* CATH2401, 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 succinic acid.

3. A method for producing octanoic acid, characterized in that, It includes: Fermentation broth was obtained by fermenting and culturing the Candida viswanathii CATH2401 as described in claim 1; as well as, Succinic acid is extracted from the fermentation broth, or succinic acid is extracted from and purified from the fermentation broth; The fermentation substrate is selected from any one or more combinations of octanoic acid, octanoic acid ester, octanoic acid salt, or octane.

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

5.

5. The production method according to claim 3, characterized in that, During fermentation culture, the fermentation substrate can be added intermittently or all at once.

6. 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.

7. The production method according to claim 3, characterized in that, The fermentation culture temperature is 28℃~35℃.

8. The production method according to claim 3, characterized in that, During fermentation culture, the inoculum size of Candida virescens is 5-50% of the fermentation medium.

9. A fermentation broth, characterized in that, It contains Candida viswanathii CATH2401 and succinic acid.

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

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