Candida versatilis with high yield of sclerolide and application thereof

Abstract

The present application discloses a high-yield sclerolide Cryptococcus albidus and its application, and belongs to the field of microorganisms and fermentation engineering. The present application screens a Cryptococcus albidus (Cryptococcus albidus SA09-Mc18) with the ability to transform sclerol into sclerolide Cryptococcus albidus ), and uses ARTP high-intensity plasma mutagenesis to broaden the gene mutation spectrum of the strain, and then uses sclerol continuous gradient concentration to achieve long-term adaptive evolution domestication, to obtain a high-yield sclerolide strain Cryptococcus albidus SA09-Mc18, which is preserved in the China General Microbiological Culture Collection Center on June 10, 2026, with the preservation number of CGMCC No.39333. The Cryptococcus albidus SA09-Mc18 can tolerate 80 g / L of sclerol, completely removes the growth inhibition of high-concentration substrates, and is suitable for industrial green production.

Description

Technical Field

[0001] This invention relates to Cryptococcus lightifolia, a high-yield strain of perilla lactone, and its applications, belonging to the field of microbiology and fermentation engineering. Background Technology

[0002] Perilla lactone is a high-value-added diterpenoid flavor compound with an elegant woody amber aroma and long-lasting fixative properties. It is a core raw material for high-end perfumes, ambergris substitutes, tobacco, food flavorings, and cosmetics. It also possesses significant anti-tumor, anti-inflammatory, antioxidant, and broad-spectrum antibacterial activities, showing broad prospects in pharmaceutical applications and extremely high market economic value. Current industrial production mainly involves plant extraction and chemical synthesis: plant extraction is limited by season, origin, and raw material content, resulting in low yields and high costs; chemical synthesis uses strong oxidants, involves multiple reaction steps, causes serious waste pollution, has poor product selectivity, and is difficult to purify. Microbial biotransformation, with its mild conditions, environmental friendliness, and high selectivity, is the inevitable path to replace traditional processes.

[0003] Cryptococcus lightensis can be used to synthesize perillyl lactone, but existing Cryptococcus lightensis strains face several key bottlenecks: 1) Perillyl alcohol is cytotoxic, severely inhibiting yeast cell growth, damaging cell membrane integrity, and suppressing intracellular metabolic enzyme activity at high concentrations; 2) Conventional UV and chemical mutagenesis methods have low mutation efficiency, only slightly improving transformation capacity and failing to address the fundamental problem of growth inhibition under high substrate conditions; 3) Single breeding methods cannot simultaneously achieve the simultaneous improvement of multiple traits such as toxin resistance, rapid growth, high enzyme activity, and strong transformation; 4) Existing wild-type and conventionally mutagenic strains of Cryptococcus lightensis have significant defects: high concentrations of perillyl alcohol strongly inhibit cell growth, resulting in low cell biomass, weak transformation activity, and low fermentation yield. Existing domestic literature and patents show shake-flask yields mostly at the level of 30–35 g / L, making it difficult to achieve high-yield production in fermenters, thus hindering the industrialization of the biological method.

[0004] Currently, there are reports on technologies for producing perillaldehyde using microbial conversion of perillyl alcohol. For example, CN121652945 A discloses the use of Cryptococcus lighthani (… Cryptococcus albidusThe technology for producing perillaldehyde from perillyl alcohol catalyzed by strain FM1120 was developed. The optimal shake-flask fermentation medium for this technology is 40 g / L glycerol, 5 g / L yeast extract, 15 g / L peptone, 15 g / L ammonium sulfate, 10 g / L disodium hydrogen phosphate, and 5 g / L potassium dihydrogen phosphate. While the conversion rate can reach over 90% when the perillyl alcohol concentration reaches 40 g / L, the reaction cycle is 72-96 hours. A study published in *Biotechnology and Biological Transformations* in 2024, which achieved de novo synthesis of perillaldehyde through a co-culture system of *Saccharomyces cerevisiae* and *Cryptococcus albidus* ATCC 20918, showed that the actual conversion concentration of perillyl alcohol in this system was only around 536.2 mg / L (approximately 0.5 g / L), and the final product yield was only 626.3 mg / L. A 2023 study (Filobasidium magnum JD1025) achieved good transformation results by screening new strains and optimizing the fermentation process. This literature reported that with an initial substrate concentration of 30 g / L, the product concentration in shake flasks reached 21.62 g / L after 72 hours of transformation. However, the substrate concentration (30 g / L) in the literature is still relatively low, limiting the yield per unit volume. Low concentrations imply large fermentation broth volumes and high downstream extraction costs. US Patent 4970163 discloses transformation using Cryptococcus lightanis ATCC 20918. While this strain can effectively catalyze the reaction, converting 60 g / L of salinomyol to 43 g / L of salinomylide in a fermenter within 120 hours, its substrate tolerance is limited. A fed-batch substrate process is used in the fermenter culture, and the transformation cycle is relatively long (120 hours in the fermenter and 72-264 hours in shake flasks). Other related studies have reported that high concentrations of substrates have an inhibitory effect on bacterial cell activity.

[0005] Therefore, developing a mutant strain with stronger substrate tolerance and faster transformation rate to reduce production costs and increase production intensity has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] Technical issues Perillyl alcohol is cytotoxic. At high concentrations, it inhibits the growth of Cryptococcus lightlingus, damages the cell membrane, and inhibits the activity of intracellular metabolic enzymes, resulting in low biomass and weak transformation capacity of Cryptococcus lightlingus. It is also intolerant to high concentrations of substrate (>40 g / L perillyl alcohol), which cannot meet the high-yield production requirements of industrial fermentation.

[0007] Technical solution This invention utilizes a strain of Cryptococcus lightifolius isolated from soil in Phoenix Mountain, Shenzhen, Guangdong Province, which possesses the ability to convert perillyl alcohol to perillyl lactone. Cryptococcus albidus Using SM18 as the starting strain, the gene mutation spectrum of the strain was broadened by ARTP high-intensity plasma mutagenesis, and then a high-yielding strain of Cryptococcus lightlyi was obtained through long-term adaptive evolution and domestication using a continuous gradient increase of perillyl alcohol concentration. Cryptococcus albidus SA09-Mc18.

[0008] The Cryptococcus syriacus ( Cryptococcus albidus SA09-Mc18 was deposited at the China General Microbiological Culture Collection Center on June 10, 2026, with accession number CGMCC No. 39333. The described Cryptococcus lightifolius SA09-Mc18 can tolerate 80 g / L perillyl alcohol, completely relieving growth inhibition from high concentrations of the substrate.

[0009] This invention provides a method for producing perillaldehyde using the aforementioned Cryptococcus lightifolia SA09-Mc18, comprising the following steps: activating the Cryptococcus lightifolia SA09-Mc18 on plates, performing two-stage seed culture amplification, then inoculating the seed culture into a fermenter, controlling the fermentation conditions for liquid submerged fermentation; after fermentation, separating the solid and liquid components by centrifugation / pressure filtration, collecting the perillaldehyde-containing cell filter cake; extracting the cell filter cake with an organic solvent, and after desolventizing and concentrating the resulting extract, purifying it by recrystallization to obtain pure perillaldehyde.

[0010] In one embodiment of the present invention, the plate activation is performed using YM solid culture medium, with a culture temperature of 28 °C and a culture time of 72 h.

[0011] In one embodiment of the present invention, the first-stage seed culture for the two-stage seed culture is cultured at 28 ℃, 200~220 rpm, and for 24~36 h; the first-stage seed culture is transferred at a volume ratio of 1% and cultured until the logarithmic growth phase of the cells to obtain the second-stage seed culture.

[0012] In one embodiment of the present invention, the fermenter is a 50 L mechanically stirred fermenter with a culture medium volume of 25 L. The fermentation culture medium contains 0.5-2 g / L disodium hydrogen phosphate, 0.5-3 g / L ammonium chloride, 0.5-2% (v / v) malt extract, and 86 g / L perillyl alcohol.

[0013] In one embodiment of the present invention, the inoculation amount of the seed liquid into the fermentation tank is 10% (v / v).

[0014] In one embodiment of the present invention, the fermentation conditions for the liquid deep fermentation are: temperature 28 ℃, stirring speed 200 rpm, aeration ratio 1 vvm, and total fermentation cycle 96 h.

[0015] Beneficial effects This invention addresses the shortcomings of existing Cryptococcus lightensis strains, including poor tolerance to perillaldehyde, high substrate inhibition of cell growth, low conversion efficiency, and difficulty in industrializing fermentation yields. First, the genetic variation of the strain is enriched through ARTP mutagenesis. Then, superior traits are fixed through substrate gradient stress acclimation. The strain's performance is simultaneously improved from multiple dimensions, including cellular stress resistance, cell growth, and catalytic metabolism, resulting in a high-perillaldehyde-producing strain, SA09-Mc18. The perillaldehyde tolerance limit of SA09-Mc18 is increased from 20 g / L in the wild-type strain to 80 g / L, a fourfold increase. Under substrate conditions of 40 g / L and 80 g / L, the growth rate of SA09-Mc18 is approximately 10 times higher than that of the pre-acclimation strain SA09, completely eliminating the growth inhibition caused by high substrate concentrations. When 40 g / L of substrate is added to shake-flask fermentation and cultured for 48 h, strain SA09-Mc18 can transform 32 g / L of perillyl lactone with a conversion rate of over 98%; when 80 g / L of substrate is added and cultured in shake-flask for 96 h, 64.552 g / L of perillyl lactone is transformed with a conversion rate of over 98%.

[0016] This invention establishes a 50 L fermenter production process to achieve high-yield production of perillaldehyde. The 50 L fermenter is fed with 86 g / L perillol, with a fermentation cycle of 96 h. After 48-72 h of cultivation, the perillaldehyde yield can reach a maximum of 70 g / L, with a substrate molar conversion rate ≥97%. Compared to the unacclimated strain SA09 (yield 32 g / L) induced by mutagenesis alone, the yield is increased by 118.75%, and the fermentation cycle is shortened by 48 h. The fermentation system produces few byproducts, and the purified product has a purity exceeding 99%, making it suitable for industrial-scale green production.

[0017] Preservation of biological materials Cryptococcus syriacus ( Cryptococcus albidus SA09-Mc18 was deposited on June 10, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39333. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0018] Figure 1 Growth curves of wild strain SM18 in different concentrations of perillaldehyde system.

[0019] Figure 2 : Observation diagram of bacterial strain morphology. Among them, a is the colony morphology and b is the cell micromorphology.

[0020] Figure 3 Lethality curves of strains corresponding to different mutagenesis times of ARTP.

[0021] Figure 4 Results of the detection of perilla lactone produced by the fermentation of the initial screening strain.

[0022] Figure 5 Results of genetic stability testing of strain SA09 after continuous passage.

[0023] Figure 6 : Growth change curves of strains during gradient adaptive evolution.

[0024] Figure 7 Comparison of growth curves of SA09 and SA09-Mc18 under different concentrations of styraxol. Where a represents SA09 and b represents SA09-Mc18.

[0025] Figure 8 Results of genetic stability assay for strain SA09-Mc18 after continuous passage.

[0026] Figure 9 The curves showing the changes in substrate and product content during the fermentation process of SA09 and SA09-Mc18 in a 50 L fermenter. Where a represents SA09 and b represents SA09-Mc18.

[0027] Figure 10 Gas phase detection spectrum of fermentation broth of strain SA09-Mc18. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0029] The culture media involved in the following examples are as follows, wherein perillyl alcohol is added as a substrate, and the concentration is adjusted according to experimental requirements: YM medium: 10% malt extract (v / v), 10 g / L yeast extract; YM solid medium: 10% malt extract (v / v), 10 g / L yeast extract, 20 g / L agar; Seed culture medium: 5% malt extract (v / v), 5-10 g / L yeast extract, 1-3 g / L disodium hydrogen phosphate; Shake flask fermentation medium: 0.5–2 g / L disodium hydrogen phosphate, 0.5–3 g / L sodium chloride, 0.5–3 g / L ammonium chloride, 1–5% wort (v / v); Fermentation medium for fermenters: 0.5–2 g / L disodium hydrogen phosphate, 0.5–3 g / L ammonium chloride, 0.5–2% (v / v) wort.

[0030] Example 1: Detailed Breeding Process of ARTP Mutagenesis and Gradient Adaptive Evolution in Cryptococcus lightbait I. Screening for superior strains using ARTP mutagenesis 1. Starting strain: wild Cryptococcus faecium Cryptococcus albidus SM18 was isolated from soil from Phoenix Mountain in Shenzhen, Guangdong. The specific steps are as follows: (1) Weigh 10 g of soil, add 90 mL of sterile physiological saline, shake for 30 min (180 rpm), and prepare 10 g of soil. - ¹Suspension. Serially dilute to 10⁻⁶. - ²、10 - ³、10 -4 Take 10 - ²–10 -4 Dilute 0.1 mL of each solution and spread it onto YM solid medium. Before use, add (filter sterile): chloramphenicol 50 mg / L, streptomycin 50 mg / L, and nystatin 10 mg / L. Incubate at 28℃ for 24-72 h. Select strains with different colony morphologies and streak them three times on a plate to ensure purification, obtaining the isolated strain SM18.

[0031] (2) Identification of the above SM18 strain (a) Colony morphology observation: such as Figure 2 The colonies are round, smooth, white, and moist.

[0032] (b) Observation was performed using a motorized inverted fluorescence microscope (ECLIPSE Ti2-E), such as... Figure 2 When observed under a 40x objective lens, the shape is round or oval, without flagella, consistent with the morphology of yeast.

[0033] (c) The optimal growth temperature is 28-30℃ and the optimal pH is 5-8.

[0034] (d) Molecular biological identification (ITS rRNA gene sequencing): Genomic DNA was extracted from strain SM18. The ITS rRNA gene region was amplified by PCR using universal fungal primers F: TCCGTAGGTGAACCTGCGG, R: TCCTCCGCTTATTGATATGC. The amplified product was sequenced, and the gene sequence is shown in SEQ ID NO:1 of the sequence listing. BLAST homology comparison of the sequenced sequence was performed in the NCBI GenBank database. The results showed that the sequence had ≥99.5% homology with the ITS sequence of the Cryptococcus lighti type strain. A phylogenetic tree was constructed, and the target strain clustered with Cryptococcus lighti in one clade.

[0035] Based on morphological, physiological, biochemical, and molecular identification results, this strain was identified as *Cryptococcus lightans*. Cryptococcus albidus It was named SM18.

[0036] The ITS rRNA sequence is shown in SEQ ID NO:1, and is detailed below: .

[0037] (3) Activation: The isolated SM18 strain was diluted and spread on YM solid medium and incubated at 28℃ for 72 h to grow single colonies. (4) Inoculation: Inoculate the single colony cultured in step (3) into YM medium and culture in a constant temperature shaker at 28℃ for 24-48h; (5) Determination of bacterial growth curve: Using a 48-well plate, the seed culture from step (4) was inoculated into the fermentation medium containing 0-20 g / L of perillyl alcohol, and cultured at 700-800 r / min at 28℃. OD was measured every 1 h. 600 Record the data and plot the curve. For example... Figure 1As shown, the overall growth of the strain was relatively slow under the fermentation medium containing perillyl alcohol. The growth of the strain was significantly inhibited at 19 g / L and 20 g / L perillyl alcohol, and the strain activity was also poor.

[0038] 2. The starting strain was mutagenized using the ARTP system, and the specific steps are as follows: (1) Activation of bacterial strain and preparation of bacterial suspension: Wild-type strain SM18 was inoculated into YM solid medium and cultured at 28 ℃ for 72 h. Single colonies were picked and cultured in YM medium at 28 ℃ and 200 rpm / min until the logarithmic phase. The bacterial suspension was washed 2-3 times with 0.9% physiological saline solution, and then the OD of the bacterial suspension was adjusted with physiological saline. 600 Dilute to approximately 0.6–0.8 to obtain a bacterial suspension.

[0039] (2) Preparation of strain screening medium and well plate fermentation medium: Screening medium: 0.5-2 g / L disodium hydrogen phosphate, 0.5-3 g / L sodium chloride, 0.5-3 g / L dipotassium hydrogen phosphate, 5-8 g / L glucose, 5-10 g / L PEG40 hydrogenated castor oil, 1-15 g / L perillyl alcohol, 20 g / L agar. Three parallel samples were set up for each bacterial culture gradient.

[0040] Fermentation medium for perforated plates: 0.5–2 g / L disodium hydrogen phosphate, 0.5–3 g / L sodium chloride, 15 g / L perillyl alcohol, 5–8% malt extract.

[0041] (3) Detailed parameters of ARTP mutagenesis: Plasma generator power 110 W, high-purity helium flow rate 12 L / min, irradiation distance 2 mm, mutagenesis time 0 s, 60-120 s; after mutagenesis, the bacterial culture was serially diluted to 10. -2 10 -3 10 -4 The bacteria were coated onto screening plates and incubated upside down at 28 °C for 72 h. After colony counting, the bacterial suspension treated at 0 s (i.e. before mutagenesis) was used as the control group. The lethality rate was calculated according to the following formula: lethality rate % = (number of colonies in the control group - number of colonies in the mutagenesis group) / number of colonies in the control group / 100. The lethality rate curve was plotted (Figure 3).

[0042] (4) Transformation rate test of the mutant strain Larger single colonies from the screening plate were picked and transferred to 96-well plates containing 0.6 mL of seed culture medium. The plates were incubated at 28°C and 350 rpm for 24 hours to obtain the seed culture. Then, the OD... 600A high concentration of seed culture was inoculated at a rate of 5% into 48-well plates containing 1 mL of fermentation medium. Fermentation was carried out at 28°C and 800 rpm for 120 h to obtain the fermentation broth. An equal volume of ethyl acetate was added to the fermentation broth, followed by sonication for 30 min to promote dissolution. The broth was centrifuged at 4000 rpm for 10 min, and the supernatant was filtered. The content of perillaldehyde in the fermentation broth was determined by GC. The results are as follows: Figure 4 Five mutant strains were able to completely transform the substrate, namely SA09, 20, 52, 63, and 88.

[0043] (5) Shaking flask and sieving a. Strain activation and fermentation: Strains with high perillol content and wild-type strains in the well plates were inoculated into screening medium and incubated statically at 28℃ for 72h. Three larger single colonies of each strain were picked and transferred to Erlenmeyer flasks containing 10mL of seed medium. The flasks were incubated at 28℃ and 200-220r / min for 24h-36h to obtain seed liquid. The seed liquid was then inoculated at a rate of 5% into Erlenmeyer flasks containing 20mL of shake-flask fermentation medium (capacity 100mL). The perillol content in the shake-flask fermentation medium was 30g / L and 40g / L. The fermentation was carried out at 28℃ and 200-220r / min for 120h to obtain fermentation broth.

[0044] b. Detection: 10 mL of fermentation broth was added to an equal volume of ethyl acetate, followed by sonication for 30 min to promote dissolution. The upper phase was centrifuged at 12000 rpm for 10 min and analyzed by gas chromatography. Five dominant strains (SA09, 20, 52, 63, and 88) were obtained by mutagenesis screening of the wild-type strain in well plates. After shake-flask screening, two strains showed positive conversion in shake-flask fermentation medium containing 30 g / L perillyl alcohol. The strain with the highest conversion rate was SA09, with an average conversion rate of 93.6%, followed by SA52, with an average conversion rate of 91.6%, which was 45% higher than the starting strain. In shake-flask fermentation medium containing 40 g / L perillyl alcohol, the strain with the highest conversion rate was SA09. The results are shown in Tables 1 and 2 below.

[0045] Table 1. Conversion results of 30 g / L perillyl alcohol

[0046] Table 2. Conversion results of 40 g / L perillyl alcohol

[0047] (6) Genetic stability test of strain SA09 Strains SA09 were cultured on selection medium at 28°C for 72 h (first subculture). Then, they were inoculated onto seed medium and cultured at 28°C for 24 h to obtain seed culture. The seed culture was then inoculated at a rate of 5% into shake-flask fermentation medium (containing 30 g / L perillyl alcohol) and cultured at 28°C for 120 h. Five replicates were set up for the experiment. Single colonies of SA09 from the first subculture plates were picked and spread onto fresh selection medium for the second subculture, yielding seed culture and fermentation broth. The same method was used for a total of 6 subcultures, with fermentation culture performed in each generation. The perillyl lactone content in the fermentation broth was measured to investigate the genetic stability of perillyl lactone transformation by strain SA09. The results are as follows: Figure 5 The results showed that the transformation rate remained stable at around 93-94% in each generation, indicating genetic stability. Only ARTP mutagenesis was used to screen for superior strains, and the transformation rate of perilla lactone during shake-flask fermentation was 45% higher than that of wild-type strains.

[0048] II. Gradient Adaptive Evolutionary Breeding Process 1. Preparation of adaptive evolution medium: Evolution medium: 0.5-2 g / L disodium hydrogen phosphate, 0.5-3 g / L sodium chloride, 5% malt extract, 5-10 g / L PEG40 hydrogenated castor oil, 5-10 g / L HP-β-cyclodextrin, 40 g / L perillyl alcohol (concentration gradually increased).

[0049] 2. Detailed domestication process of gradient adaptive evolution of perillaldehyde: According to the results in section 1 (5) above, the strain showed some inhibition at 40 g / L perillaldehyde, so a concentration gradient was set: 40 g / L → 50 g / L → 60 g / L → 70 g / L → 80 g / L. Using SA09 as the starting strain, the corresponding concentration of perillaldehyde was added to the evolutionary medium. The culture was carried out at 28 ℃ using a fully automated high-throughput microbial droplet culture instrument. A total of 65 droplets were generated for subculturing, with each generation cultured for 24-48 h. Each concentration was subcultured 4-5 times before moving to the next concentration, and the entire strain was continuously subcultured for 29 generations. Figure 6 As shown, after gradient evolution and domestication, strains SA09-Mc18 and SA09-Mc55 grew well and were able to fully adapt to the high concentration of perillaldehyde environment, thus improving their substrate metabolism capacity.

[0050] 3. Shake flask screening a. Strain activation and fermentation: SA09-Mc18 and SA09-Mc55 strains, derived from the evolution of SA09, were inoculated into selection medium and statically cultured at 28℃ for 48 h. Five larger single colonies from each strain were then transferred to Erlenmeyer flasks containing 10 mL of seed culture medium and cultured at 28℃ and 200-220 rpm for 24 h to obtain the seed culture. This seed culture was then inoculated at a rate of 5% into Erlenmeyer flasks containing 40 mL of shake-flask fermentation medium (250 mL capacity), with a perillyl alcohol content of 80 g / L. Fermentation was carried out at 28℃ and 200-220 rpm for 72-96 h to obtain the fermentation broth. The results are shown in Table 3 below. Compared to SA09-Mc55, SA09-Mc18 exhibited a higher molar conversion rate.

[0051] Table 3

[0052] 4. Growth curve determination of strain SA09-Mc18 after mutagenesis and evolution (1) Activation: Dilute and spread the culture medium using screening medium, and incubate at 28℃ for 72 h to grow single colonies; (2) Inoculation: The single colony cultured in step (1) was inoculated into the seed culture medium and cultured in a constant temperature shaker at 28°C for 24 h; (3) Determination of bacterial growth curve: Using a 48-well plate, the seed culture from step (2) was inoculated into the fermentation medium containing 30 g / L, 40 g / L and 80 g / L of perillyl alcohol, respectively. With strain SA09 as the control, the culture was carried out at 700-800 r / min and 28℃. OD was measured every 1 h. 600 Record the data and plot the curve. For example... Figure 7 As shown, the growth of strain SA09 was initially inhibited at a concentration of 40 g / L perillyl alcohol, resulting in slow growth and reduced biomass. The mutagenic strain SA09-Mc18 showed increased growth rate, significantly faster cell accumulation, and greatly improved tolerance to substrate inhibition at concentrations of 40 g / L and 80 g / L perillyl alcohol. The average growth rate of the mutagenic strain SA09-Mc18 was approximately 10 times higher.

[0053] 5. Genetic stability test of strain SA09-Mc18 (1) Genetic stability The strain SA09-Mc18, obtained through ARTP mutagenesis and adaptive evolution, was cultured on selection medium. It was incubated at 28°C for 48-72 hours (first subculture), then inoculated into seed medium and cultured at 28°C for 24 hours to obtain seed culture. The seed culture was then inoculated at a rate of 5% into shake-flask fermentation medium (containing 80 g / L perillyl alcohol) and cultured at 28°C for 72-96 hours. Five replicates were set up. Single colonies of SA09-Mc18 from the first subculture plates were picked and spread onto fresh selection medium for the second subculture, yielding seed culture and fermentation broth. This process was repeated for 10 generations, with fermentation culture performed after each generation. The perillyl lactone content in the fermentation broth was measured to investigate the genetic stability of perillyl lactone transformation by strain SA09-Mc18. Results are as follows: Figure 8 As shown, the results indicate that the transformation rate remained stable at over 96% in each generation, demonstrating genetic stability.

[0054] (2) Single colony stability Strain SA09-Mc18 was cultured on YM solid medium at 28°C for 48 h. Single colonies from the plates were transferred to shake tubes containing 3 mL of seed culture medium and cultured at 28°C and a rotation speed of 200-220 r / min for 24 h to obtain seed culture. The seed culture was then inoculated at a rate of 5% into Erlenmeyer flasks containing 25 mL of shake-flask fermentation medium (250 mL). Fermentation was carried out at 28°C, pH 6.5, and a rotation speed of 250 r / min for 48-96 h to obtain fermentation broth. Strain SA09-Mc18, obtained through ARTP mutagenesis combined with adaptive evolution screening, exhibited excellent single colony stability under high concentrations of perillaldehyde. Specifically, the fermentation time was 48 h at 40 g / L perillaldehyde and 96 h at 80 g / L perillaldehyde. Specific experimental data are shown in Tables 4 and 5.

[0055] Table 4. Changes in the content of single colonies at 80 g / L perillol during screening.

[0056] Table 5. Changes in single colony screening at 40 g / L perillol content.

[0057] Example 2: Production of perillaldehyde by strain SA09-Mc18 after mutagenesis and evolution. 1. Strain activation: The high-yielding strain SA09-Mc18, after mutagenesis and evolution, was inoculated into YM solid medium and activated at 28 ℃ for 72 h.

[0058] 2. Preparation of Fermentation Generation 1 Seed Culture: Select a single colony from the cultured plate and inoculate it into an Erlenmeyer flask containing 50 mL of seed culture medium. Incubate at 28℃ and 200-220 r / min in a shaker for 24-36 h to obtain generation 1 seed culture.

[0059] 3. Preparation of second-generation fermented seed liquid: The first-generation seed liquid was prepared at 1% ( v / v The inoculum was transferred to a 2.5L Erlenmeyer flask containing seed culture medium for second-generation seed culture and cultured at 28℃ in a shaker at 200-220r / min until the logarithmic growth phase. 4. Fermentation Parameters and Results: 25 L of the prepared fermentation medium (containing 86 g / L perillyl alcohol) was transferred into a 50 L fermenter and sterilized at 115℃ for 30 min. Then, 2.5 L of the cultured second-generation seed culture was transferred into the 50 L fermenter at an inoculation rate of 10% (v / v) for fermentation. The fermentation temperature was 28℃, the stirring speed was 150 rpm-220 rpm, the aeration ratio was 0.5 vvm-1 vvm, and the fermentation period was 96 h. The residual sugar was controlled above 1 g / L during fermentation. The yield was measured every 24 h during fermentation.

[0060] The results are as follows Figure 9 , Figure 10 At 48h, the yield of perillaldehyde from strain SA09-Mc18 reached about 50 g / L. At 72h, the substrate was basically consumed. The high-yielding strain SA09-Mc18 after mutagenesis achieved a perillaldehyde yield of 70.08 g / L, with a substrate molar conversion rate of over 97%, which is 1.18 times higher than the perillaldehyde yield of 32 g / L from strain SA09 alone.

[0061] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A type of Cryptococcus palea ( Cryptococcus albidus Strain SA09-Mc18 was deposited on June 10, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39333. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.

2. The application of the Cryptococcus lighthani as described in claim 1 ( Cryptococcus albidus A method for producing perilla lactone from strain SA09-Mc18, characterized in that, The Cryptococcus lightifolius strain SA09-Mc18 was activated to prepare a seed culture, which was then inoculated into a fermentation medium containing perillyl alcohol for fermentation.

3. The method according to claim 2, characterized in that, The seed culture was obtained through two-stage cultivation. The first-stage seed culture was cultivated at 28 ℃ and 200~220 rpm for 24~36 h. The first-stage seed culture was then transferred to a seed culture medium and cultured until the logarithmic growth phase of the cells to obtain the second-stage seed culture.

4. The method according to claim 2, characterized in that, The fermentation is carried out in a 50 L mechanically stirred fermenter with a culture medium volume of 25 L. The fermentation medium contains 0.5–2 g / L disodium hydrogen phosphate, 0.5–3 g / L ammonium chloride, 0.5–2% (v / v) malt extract, and 86 g / L perillyl alcohol.

5. The method according to claim 4, characterized in that, Fermentation temperature 28 ℃, stirring speed 200 rpm, aeration ratio 1 vvm, fermentation cycle 96 h.

6. The method according to claim 2, characterized in that, After fermentation, the fermentation broth was centrifuged or filtered to separate the solid and liquid components, and the mycelial filter cake containing perillaldehyde was collected. The mycelial filter cake was extracted with an organic solvent, and the resulting extract was desolventized, concentrated, and then purified by recrystallization to obtain pure perillaldehyde.

7. The Cryptococcus lighthanii yeast of claim 1 ( Cryptococcus albidus Application of strain SA09-Mc18 in the production of products containing perilla lactone.

8. The application according to claim 7, characterized in that, The products include fragrances, perfumes, ambergris substitutes, tobacco, cosmetics, and pharmaceuticals.

9. The Cryptococcus lighthanii yeast of claim 1 ( Cryptococcus albidus Application of strain SA09-Mc18 in the production of perilla lactone.

10. The application according to claim 9, characterized in that, The aforementioned Cryptococcus lightifolia strain SA09-Mc18 was genetically modified to produce perilla lactone.

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