Method for directional screening of excellent strains of candida viswanathii producing long-chain dibasic acid

Abstract

This invention belongs to the field of microbial fermentation engineering technology, specifically relating to a method for targeted screening of superior strains of *Candida virescens* producing long-chain dicarboxylic acids. Targeted screening is achieved through a dual-pressure approach: first, long-chain alkanes are used as the sole carbon source to screen for strains with dominant ω-oxidation activity; then, these strains are transferred to a late-stage dicarboxylic acid conversion fermentation broth containing norlesin, after the removal of alkanes and cell bodies. This fermentation broth simulates a high product concentration, high osmotic pressure, and complex inhibitory environment, allowing for reverse screening of strains with weak β-oxidation capacity and strong production tolerance. This invention utilizes the production fermentation broth as the screening medium, overcoming the technical bias of traditional methods where the screening environment is disconnected from actual production conditions. The screened strains exhibit high yields and strong production adaptability.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation engineering technology, specifically relating to a method for targeted screening of superior strains of Candida virescens that produce long-chain dicarboxylic acids. Background Technology

[0002] Long-chain dicarboxylic acids are important chemical intermediates for the synthesis of high-performance polyamides, hot melt adhesives, advanced lubricants, and coatings. Currently, industrial production mainly uses *Candida* yeast fermentation with n-alkanes as substrates. In practice, strains are prone to degeneration or non-positive mutations during preservation, subculturing, and repeated freeze-thaw cycles, leading to a decrease in acid-producing capacity. Therefore, efficient and targeted strain screening and rejuvenation techniques are crucial for maintaining and improving industrial fermentation efficiency.

[0003] In existing technologies, screening methods for Candida albicans that produce long-chain dicarboxylic acids mainly include: Mutagenesis methods, such as ultraviolet light and chemical mutagenesis, are highly lethal and random, with an extremely low positive mutation rate (usually <1 / 10000). The screening process is labor-intensive and time-consuming, and there is a risk of reversion mutations.

[0004] Microporous membrane filtration method: Screening is based on the morphology (elliptical and filamentous) of Candida albicans. However, the correlation between cell morphology and acid production performance is weak, resulting in poor screening specificity and low efficiency.

[0005] Two-step concentration and photocopying method: This method indirectly selects specific oxidases through physical mutagenesis combined with chemical reagents (such as nystatin and chlorpromazine). This method has significant drawbacks: First, it relies on artificially synthesized culture media with well-defined components and controllable conditions for screening. Strains screened in this "clean" environment are not well-suited to the complex and harsh environment of actual fermenters (high product concentration, high osmotic pressure, and multiple inhibitors), making them vulnerable in actual production. Second, we found that the β-oxidation blocking strains obtained from the first stage of screening grow slowly in actual production, and the accuracy of the chromogenic zone used to determine the strength of acid production is low, leading to a deviation between the screening results and the actual high-yield target.

[0006] In summary, existing screening methods are either inefficient, weakly correlated with the final production target, or produce strains that are difficult to adapt to real production environments. Therefore, there is an urgent need in this field for a new method that can directly target high-yield goals and perform targeted screening under pressure close to the actual production environment, in order to quickly obtain superior strains with vigorous metabolic activity, strong environmental tolerance, and stable production performance. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of existing technologies and provide a method for screening superior strains of *Candida virescens* that produce long-chain dicarboxylic acids, with clearly defined screening pressure, high correlation with the production environment, and high efficiency and directionality. This method can also be directly used to rejuvenate degenerated strains, and can be combined with physical and chemical mutagenesis to significantly improve the screening efficiency and success rate of superior strains.

[0008] The technical solution of the present invention is as follows: To achieve the above objectives, the present invention provides a method for targeted screening of superior strains of *Candida virescens* that produce long-chain dicarboxylic acids, the method comprising the following steps: (1) Seed culture preparation: A seed culture of *Candida viswanathii* 08001 with accession number CCTCC NO: M 20222052 was prepared; *Candida viswanathii* 08001 is a long-chain dicarboxylic acid producing strain that has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, on December 23, 2022. Its Latin scientific name is... Candida viswanathii ; (2) First screening: The seed liquid obtained in step (1) is inoculated into a liquid culture medium with long-chain alkanes as the only carbon source and cultured at 28-32℃ for 20-40h to the logarithmic growth phase to screen out vigorous strains with strong alkanes assimilation ability and ω-oxidation dominance. (3) Second screening: After centrifugation, the bacterial solution obtained in step (2) is transferred to a fermentation broth containing antibiotics that inhibit the Candida viviparus yeast and has removed alkanes and dicarboxylic acid conversion in the late stage. After culturing at 28-32℃ for 2-6 hours, a suitable dilution is selected and plated to screen out strains with weak β-oxidation capacity and strong tolerance to harsh production environments. (4) Verification of acid production performance: Select single colonies that have grown in the plate in step (3) and perform acid production verification in shake flasks to obtain a superior strain of Candida viviparous yeast that produces long-chain dicarboxylic acid.

[0009] In step (1), the seed culture was obtained by culturing the seed culture in YEPD medium at 30°C for 16-18 hours.

[0010] In step (2), the liquid culture medium using long-chain alkanes as the sole carbon source comprises the following components: Long-chain alkanes: 5.0-50 g / L, preferably 10-40 g / L, more preferably 30 g / L; Potassium dihydrogen phosphate or sodium dihydrogen phosphate 0.05-5.0 g / L, preferably 1.0-3.0 g / L, more preferably 2.0 g / L; The concentration of dipotassium hydrogen phosphate or disodium hydrogen phosphate is 0.1-10 g / L, preferably 2.0-6.0 g / L, and more preferably 4.0 g / L; Ammonium sulfate 1.0-10 g / L, preferably 2.0-8.0 g / L, more preferably 5.0 g / L; Urea concentration: 0.5-5 g / L, preferably 1-4.0 g / L, more preferably 2.0 g / L; Sodium chloride is 0.1-3.0 g / L, preferably 0.5-2.0 g / L, and more preferably 1.0 g / L.

[0011] In step (3), the antibiotic includes Norlesmycin, and the concentration of Norlesmycin added is 0.05-0.2 g / L.

[0012] The preparation method of the late-stage fermentation broth for dicarboxylic acid conversion after removing alkanes and bacterial cells is as follows: After fermenting and culturing the broth according to step (2) for 40-120h (preferably 50-100h, more preferably 80h), remove the bacterial cells, solids, alkanes, and chyme in sequence, and then perform aseptic treatment (filtration sterilization, irradiation sterilization) on the resulting clear liquid to obtain the late-stage fermentation broth for dicarboxylic acid conversion after removing alkanes and bacterial cells, which contains 10.0-120.0 g / L of long-chain dicarboxylic acids.

[0013] The long-chain alkane is selected from one or more of n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, and n-hexadecane; the long-chain dicarboxylic acid is a dicarboxylic acid with a corresponding carbon chain length.

[0014] After step (1) and before step (2), the process may include physical or chemical mutagenesis of the Candida vesicatoria 08001; the physical mutagenesis includes ultraviolet irradiation and plasma implantation, and the mutagen used in the chemical mutagenesis includes camphor, ethyl methanesulfonate (EMS), and 5-fluorouracil.

[0015] The core of this invention lies in breaking away from the traditional mindset of screening in "clean" optimized culture media, and instead using "fermentation broth in the mid-to-late stage of diacid conversion with alkanes and cell cells removed" as the core screening medium. This medium is not simply waste, but a "pressure selector" that highly simulates the complex and harsh environment at the end of an actual fermenter. Its technical effect is reflected in the triple synergistic screening pressure: Reverse metabolic pressure: The high concentration of long-chain dicarboxylic acids (as products) in the fermentation broth is used as a "reverse carbon source", which can directly eliminate strains with strong β-oxidation ability (i.e. strong product degradation ability), forcing the selection direction to focus on product accumulation. Environmental acclimatization stress: The fermentation broth contains high salt, high osmotic pressure, a variety of intermediate metabolites and inhibitors such as reactive oxygen species, simulating the harsh working conditions of real production. The strains that survive in this environment have been pre-acclimatized to the production environment, thus significantly shortening the lag period and entering the high-yield state more quickly in subsequent scale-up production. Assisted biological stress: The addition of low doses of Norlesin exerts additional survival stress on strains with weaker metabolic activity, further enhancing the effect of targeted screening.

[0016] This dual-pressure strategy of "alkane forward screening activity + fermentation broth reverse screening tolerance and product accumulation ability" applies a squeeze-style selection from both ends of the metabolic pathway, ensuring that the selected strains possess excellent traits of efficient substrate utilization and efficient product accumulation, and can quickly adapt to the production environment.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. Highly targeted and efficient: This invention employs targeted pressure screening from two dimensions: reverse metabolism pathways and adaptability to the production environment. The objectives are clear, avoiding the blindness of traditional mutagenesis. 2. Close to production and highly adaptable: Using real fermentation broth as the screening medium allows the strains to undergo harsh environmental testing close to production during the screening stage. When the selected strains are scaled up to fermenter production, they have a short lag period, strong adaptability, and stable performance. 3. Flexible application and wide compatibility: This invention can be used independently as a means of rejuvenating degenerated strains, or it can be used in conjunction with various physical and chemical mutagenesis methods as an efficient and targeted screening tool after mutagenesis, significantly improving the success rate of traditional breeding methods; 4. Low cost and easy operation: The screening medium mainly utilizes the fermentation broth produced by the manufacturer, without the need for special expensive reagents or equipment, making it easy to promote and implement in industrial production. Detailed Implementation

[0018] To make the objectives and technical solutions of this invention clearer, this invention will be further described in detail. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; where specific techniques or conditions are not specified in the experiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions; unless otherwise specified, the reagents and materials mentioned are all commercially available.

[0019] Example 1 1. Seed culture preparation: Take 1 mL of bacterial culture from the glycerol cryopreservation tube of Candida viviparus 08001 with accession number CCTCC NO: M 20222052, inoculate it into 20 mL of YEPD liquid medium (10 g / L glucose, 10 g / L peptone, 5 g / L yeast extract), and incubate at 30℃ and 240 rpm for 18 h to the logarithmic phase.

[0020] 2. First screening: The seed culture obtained in step (1) was inoculated into the first screening medium at a 20% inoculum and cultured at 30℃ for 30h. First screening medium: with n-dodecane as the sole carbon source, the formula is: n-dodecane 30 g / L, potassium dihydrogen phosphate 2.0 g / L, disodium hydrogen phosphate 4.0 g / L, ammonium sulfate 5.0 g / L, sodium chloride 1.0 g / L, pH 7.3-7.4. After sterilization at 121℃ for 20 minutes, aseptically added filtered and sterilized urea to a final concentration of 2.0 g / L.

[0021] 3. Second screening: Centrifuge the bacterial culture obtained in step (2), and transfer the centrifuged cells to the second screening medium, and incubate at 30°C for 4 hours. Second screening medium: Remove the bacterial cells, solids, alkanes, and chyme from the fermentation broth that has been fermented for 80 hours according to step (2) in sequence, and then filter the resulting clear liquid to remove bacteria. Before use, add Norilskin with a final concentration of 0.1 g / L after filtration and sterilization.

[0022] 4. Acid production verification: After diluting the bacterial culture to a suitable concentration, select 10... -4 10 -5 10 -6 Three gradients of YEPD solid plates were spread, with three replicates for each plate. The plates were incubated at 30°C for 3 days, and plates with 40-80 single colonies were selected. Healthy single colonies from these plates were then selected for shake-flask fermentation verification, using the following method: Single colonies were inoculated into malt extract seed culture medium and cultured on a shaker for 18 hours. Then, a 1% inoculum was added to primary seed culture medium and cultured on a shaker for 30 hours. Finally, a 20% inoculum was added to shake-flask fermentation medium and cultured on a shaker for 110 hours. All shake-flask culture conditions were 30℃ and 240 rpm. The fermentation broth after shake-flask culture was analyzed using standard methods for acid production determination: alkaline boiling to break the emulsion, acid precipitation, ether extraction, drying, re-dissolving in alcohol, and titration with 0.2M NaOH standard solution to obtain the crude acid content. Acid production = crude acid concentration × shake-flask fermentation broth volume.

[0023] Among them, the malt extract seed culture medium has a sugar content of 10-15, is filled in a 250mL shake flask with a volume of 20mL, and is used after being sterilized at 121℃ for 20min. Primary seed culture medium: sucrose 15g / L, KH2PO4 6g / L, yeast extract 3g / L, corn flour 0.2 g / L, urea 0.3%, 30mL in a 500mL shake flask, sterilized at 121℃ for 20min before use; Shake flask fermentation medium: 20 g / L sucrose, 6 g / L KH2PO4, 5 g / L KNO3, 1.2 g / L urea, 1 g / L NaCl, 3 g / L yeast extract, 0.2 g / L corn flour, 10.0 g / L long-chain alkanes corresponding to the screening, 20 mL in a 500 mL shake flask, sterilized at 121 °C for 20 min before use.

[0024] Example 2 The method is the same as in Example 1, except that n-tetradecane is replaced by n-tetradecane for the long-chain alkanes. When verifying acid production in single-strain shake flasks, n-tetradecane is also used for the long-chain alkanes in the shake flask fermentation medium, and Tween 80 is added at a final concentration of 0.15%.

[0025] Example 3 The method is the same as in Example 1, except that in step (1), when preparing the seed solution, UV mutagenesis is performed 16 hours after inoculation during the logarithmic growth phase. Specifically, the seed solution is centrifuged, the supernatant is discarded, diluted 10 times with physiological saline, resuspended, and placed in a sterile culture dish with magnetic stirring. Magnetic stirring is turned on, and the solution is irradiated for 60 seconds with a 15W UV lamp at a distance of 30cm in a darkroom laminar flow hood to complete the mutagenesis. The mutagenized seed suspension is then centrifuged again, concentrated to one-tenth of its volume with physiological saline, and then subjected to subsequent directional screening.

[0026] Example 4 The method is the same as in Example 1, except that in step (1), when preparing the seed culture, chromosome doubling is performed 6 hours after inoculation. Specifically, camphor powder ethanol solution with a final concentration of 0.2 g / L, which has been filtered and sterilized, is added, and the seed culture time is extended to 30 hours. After the seed culture is completed, when more than 30% of the individuals show significant enlargement under microscopic examination or significant enlargement of the cell nucleus region after staining, subsequent targeted screening is carried out.

[0027] Example 5 The method is the same as in Example 1, except that in step (1), when preparing the seed solution, chemical mutagenesis is performed 18 hours after inoculation during the logarithmic growth phase. Specifically, EMS solution with a final concentration of 20 μL / mL, filtered and sterilized, is added. After mutagenesis for 30 minutes, an equal volume of 5% sodium thiosulfate solution is added to terminate the mutagenesis. Subsequent targeted screening is then carried out after the seed mutagenesis is completed.

[0028] Example 6 The method is the same as in Example 1, except that n-decane is replaced by n-decane for the long-chain alkanes. When verifying acid production in single-strain shake flasks, n-decane is also used for the long-chain alkanes in the shake flask fermentation medium. The screening temperature and acid production detection temperature are both adjusted to 29°C.

[0029] Comparative Example 1 This comparative example uses the traditional UV mutagenesis screening method. Seed culture that has grown to the logarithmic growth stage was prepared according to the method in Example 1. 10 mL of seed suspension was placed in a sterile petri dish with magnetic stirring. The magnetic stirring was turned on, and a 15W UV lamp was used to irradiate the culture for 60 s at a distance of 30 cm. An appropriate amount of bacterial culture was diluted and spread on a conventional YEPD solid plate. The plate was incubated at 30°C for 3 days. Single colonies grew on the plate, and shake-flask fermentation with n-dodecane as the substrate was performed for verification.

[0030] Comparative Example 2 This comparative example uses a simple EMS mutagenesis screening method. Seed culture grown to the logarithmic growth phase was prepared according to the method in Example 1. Cells were collected by centrifugation, washed twice with sterile sodium phosphate buffer, and resuspended. A filtered, sterile EMS solution with a final concentration of 20 μL / mL was added, and mutagenesis was induced for 30 min. An equal volume of 5% sodium thiosulfate solution was added to terminate the mutagenesis. The mutagenized seed culture was centrifuged, washed twice with sterile water, and resuspended. An appropriate amount of bacterial culture was diluted and plated onto a standard YEPD agar plate. The plates were incubated at 30°C for 3 days. Single colonies grew on the plates, and shake-flask fermentation using n-dodecane as a substrate was performed for verification.

[0031] Comparative Example 3 This comparative example demonstrates a two-step concentration-printing method for screening high-yield dodecanoic acid (DOA)-producing strains. Seed culture grown to the logarithmic growth stage was prepared according to the method in Example 1. Then, following the method described in "Two-Step Concentration and Printing Techniques for Screening Mutagenic Strains of Candida Tropicalis" (Jiao Peng et al., 2000), *Candida virescens* 08001 was first mutagenized with diethyl sulfate, followed by two-step concentration using nystatin and chlorpromazine. Finally, the culture was printed on SEL1 and SEL2 plates, and the strain with the highest R-value was selected based on visually observed acid-producing zones. This selected strain was used as the target strain for final β-oxidation inhibition and enhanced α and ω-oxidation performance, and shake-flask fermentation with n-dodecane as the substrate was conducted for verification.

[0032] In Examples 1-6 and Comparative Examples 1-3, 100 single strains were selected for shake-flask acid production verification. The growth time of single colonies and the total number of single strains with acid production greater than 0.7, 0.9, and 1.1 were counted. The results are shown in Tables 1-4.

[0033] Table 1. Time for single colony growth

[0034] Table 2. Total number of single bacteria with acid production > 0.7

[0035] Table 3. Total number of single bacteria with acid production > 0.9

[0036] Table 4. Total number of single bacteria with acid production > 1.1

[0037] The data in Tables 1-4 show that the single colonies screened using the method of this invention have a shorter growth time, and the number of high-yielding strains grown on plates with appropriate dilutions after treatment with a mutagen is significantly better than that of using it alone. The screening efficiency of positive mutation is significantly improved after targeted pressure using this method, which proves the superiority and practicality of the dual-pressure targeted screening method of this invention.

Claims

1. A method for targeted screening of superior strains of *Candida virescens* that produce long-chain dicarboxylic acids, characterized in that, Includes the following steps: (1) Prepare seed culture of Candida viviparous 08001 with preservation number CCTCC NO: M 20222052; (2) The seed liquid obtained in step (1) is inoculated into a liquid culture medium with long-chain alkanes as the only carbon source and cultured to carry out the first screening to obtain strains with vigorous growth. (3) After centrifugation, the bacterial culture obtained in step (2) is collected and transferred to a fermentation broth containing antibiotics that inhibit the Candida viviparous yeast and in which alkane and dicarboxylic acid conversion of the cells has been removed, for a second screening. (4) Spread the bacterial culture after step (3) onto a plate, pick a single colony for acid production performance verification, and obtain an excellent strain of Candida viviparous yeast that produces long-chain dicarboxylic acid.

2. The method for targeted screening of superior strains of Candida virescens producing long-chain dicarboxylic acids according to claim 1, characterized in that, In step (2), the liquid culture medium with long-chain alkanes as the sole carbon source comprises the following components: long-chain alkanes 5.0-50 g / L, potassium dihydrogen phosphate or sodium dihydrogen phosphate 0.05-5.0 g / L, dipotassium hydrogen phosphate or disodium hydrogen phosphate 0.1-10 g / L, ammonium sulfate 1.0-10 g / L, urea 0.5-5 g / L, and sodium chloride 0.1-3.0 g / L.

3. The method for targeted screening of superior strains of Candida virescens producing long-chain dicarboxylic acids according to claim 2, characterized in that, The preparation method of the late-stage fermentation broth for dicarboxylic acid conversion after removing alkanes and bacteria is as follows: After fermentation culture for 40-120 hours in step (2), remove bacteria, solids, alkanes, and chyme in sequence, and then sterilize the resulting clear liquid to obtain the late-stage fermentation broth for dicarboxylic acid conversion after removing alkanes and bacteria, which contains 10.0-120.0 g / L of long-chain dicarboxylic acid.

4. The method for targeted screening of superior strains of *Candida virescens* producing long-chain dicarboxylic acids according to claim 3, characterized in that, The long-chain alkane is selected from one or more of n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, and n-hexadecane; the long-chain dicarboxylic acid is a dicarboxylic acid with a corresponding carbon chain length.

5. The method for targeted screening of superior strains of *Candida virescens* producing long-chain dicarboxylic acids according to claim 4, characterized in that, The antibiotics include Norlesmycin.

6. The method for targeted screening of superior strains of *Candida virescens* producing long-chain dicarboxylic acids according to claim 5, characterized in that, The concentration of Norlesmycin added is 0.05-0.2 g / L.

7. The method for targeted screening of superior strains of Candida virescens producing long-chain dicarboxylic acids according to claim 1, characterized in that, After step (1) and before step (2), the method further includes a step of physical or chemical mutagenesis of the Candida vesicatoria 08001.

8. The method for targeted screening of superior strains of *Candida virescens* producing long-chain dicarboxylic acids according to claim 7, characterized in that, The physical mutagenesis includes ultraviolet irradiation and plasma implantation.

9. The method for targeted screening of superior strains of *Candida virescens* producing long-chain dicarboxylic acids according to claim 7, characterized in that, The mutagen used in the chemical mutagenesis includes camphor, ethyl methanesulfonate, and 5-fluorouracil.

10. The method for targeted screening of superior strains of *Candida virescens* producing long-chain dicarboxylic acids according to claim 1, characterized in that, In step (2), the culture temperature is 28-32℃ and the culture time is 20-40h; in step (3), the culture temperature is 28-32℃ and the culture time is 2-6h.

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