Cryptococcus albidus strain and application thereof in production of sclareolide

Abstract

The invention belongs to the field of biotechnology and microbial fermentation engineering, and particularly relates to a cryptococcus albidus strain and application thereof in production of sclareolide. A mutant strain with the most excellent performance is screened out through systematic wild strain screening, natural breeding, ARTP mutation breeding and other technical means, the mutant strain is named as Cryptococcus albidus FM1120, the preservation number is CCTCC NO: M 20251915, the preservation date is September 1, 2025, the preservation number is CCTCC NO: M 20251915, the preservation number is CCTCC NO: M 20251915, the preservation number is CCTCC NO: M 20251915, the preservation number is CCTCC NO: M 20251915, the preservation number is CCTCC NO: M 20251915, the preservation number is CCTCC NO: M 20251915, and the preservation number is CCTCC NO: M 20251915, and the preservation number is CCTCC NO: M 20251915. The preservation unit is China Center for Type Culture Collection. The invention also provides an application of the strain FM1120 in the production of sclareolide, sclareol is taken as a substrate, the conversion rate of sclareol reaches more than 90% through fermentation, the fermentation liquor has the advantages of few impurities, easiness in separation and purification of sclareolide, high purity and the like, and a new path is provided for the green preparation of sclareolide.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and microbial fermentation engineering, specifically relating to a Cryptococcus lighti strain and its application in the production of perilla lactone. Background Technology

[0002] Perilla lactone, an important diterpenoid lactone compound, has the molecular formula C2. 15 H 24 O2 exhibits a unique bicyclic system in its chemical structure, consisting of a lactone ring and a bicyclic diterpene skeleton. This unique structure not only endows the molecule with excellent chemical stability but also gives it significant biological activity. This compound is primarily derived from plants of the genus *Salvia* in the Lamiaceae family, with *Salvia splendens* being the most important natural source. It has also been found in other plants with significant medicinal value, such as *Salvia miltiorrhiza*. In the fragrance industry, salvia lactone is highly valued for its long-lasting fixative effect and unique woody amber aroma, and is widely used in high-end perfume formulations. Particularly noteworthy is its role as a key intermediate in the synthesis of ambergris substitutes, playing an irreplaceable role in mimicking the rare aroma of natural ambergris. In addition, salvia lactone is widely used in various industrial fields, including food additives, cosmetic formulations, and pharmaceutical preparations. In pharmaceutical applications, multiple in vitro studies have shown that this compound exhibits significant growth-inhibiting effects on various cancer cell lines, including breast cancer and lung cancer. Its mechanism of action involves inducing tumor cell cycle arrest, promoting apoptosis, and inhibiting tumor cell migration. Regarding anti-inflammatory and antioxidant properties, it effectively inhibits the release of key inflammatory factors such as TNF-α and IL-6, significantly reducing tissue damage caused by oxidative stress, demonstrating promising application prospects in the treatment of chronic inflammatory diseases. In terms of antibacterial and antifungal properties, studies have confirmed its significant inhibitory effects on pathogenic microorganisms such as Staphylococcus aureus, exhibiting broad-spectrum antibacterial activity. These excellent biological properties give perillaldehyde extremely high economic value in the market.

[0003] The industrial production of perilla lactone mainly employs two traditional methods: chemical synthesis and plant extraction. However, the unstable supply of plant raw materials and the low efficiency of extraction processes limit production costs. From a biosynthetic perspective, the formation of perilla lactone primarily relies on the mevalonic acid (MVA) pathway or the methyl erythritol phosphate (MEP) pathway within the plant, undergoing a series of complex enzymatic reactions involving cyclization and oxidation to ultimately form the lactone structure. Therefore, developing an environmentally friendly, efficient, and economical new biotransformation method is of significant practical importance.

[0004] Studies have shown that microbial systems modified through metabolic engineering can replace traditional plant extraction methods. A patent published by Shaanxi University of Science and Technology, titled "A Method for Biosynthesizing Perillaldehyde," demonstrates that using a mutagenic Cryptococcus lightans strain, 60 g / L of perillol can be converted to 35.2 g / L of perillaldehyde after 10 days of fermentation. Microbial transformation has become a hot research topic due to its significant advantages, including mild reaction conditions, environmental friendliness, and high selectivity. However, improving the conversion rate, optimizing fermentation process parameters, and reducing production costs remain major obstacles to the industrial application of this technology. Summary of the Invention

[0005] In view of this, the main objective of this invention is to provide a Cryptococcus faecium strain and its application in the production of perillaldehyde. This strain can produce an enzyme that catalyzes perillol and efficiently converts perillol into perillaldehyde. Therefore, fermentation using this Cryptococcus faecium strain can catalyze the production of high-purity perillaldehyde from perillol, and the conversion rate of perillol can reach over 90%.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a strain of Cryptococcus lighthani (… Cryptococcus albidus FM1120, accession number CCTCC NO: M 20251915, accession date September 1, 2025, deposited by China Center for Type Culture Collection.

[0007] The *Cryptococcus faecium* strain FM1120 was obtained primarily through the following methods: First, wild-type *Cryptococcus faecium* strains capable of converting perillyl alcohol to perillyl lactone were isolated from *Perilla frutescens* flowers. Then, the wild-type *Cryptococcus faecium* strain underwent natural selection and ARTP mutagenesis to obtain a microbial strain capable of converting perillyl alcohol to high-yield perillyl lactone, with an ITS sequence as shown in SEQ ID NO. 1. This strain was named *Cryptococcus faecium* strain FM1120. It grows well in YPD medium. The strain is typically 0.5-1 μm in diameter, elliptical in shape, and non-flagellated, consistent with yeast morphology. The colonies are round, smooth, white, and moist, indicating it is a Gram-positive yeast. The optimal growth temperature of strain FM1120 is 25-28℃, and the optimal pH is 5.5-7.5. It can produce an enzyme that can catalyze the conversion of perillol to perillol lactone. The entire conversion process to perillol lactone does not require the addition of exogenous chemical catalysts. It relies solely on microbial metabolism to achieve directional conversion, thus enabling the microbial fermentation synthesis of perillol lactone using perillol as a substrate.

[0008] Secondly, the present invention provides the application of a Cryptococcus faecium strain FM1120 in the production of perilla lactone.

[0009] Thirdly, the present invention provides a fermentation medium for Cryptococcus lightensis strain FM1120, comprising: 20-60 g / L glycerol, 1-10 g / L yeast extract, 5-20 g / L peptone, 5-20 g / L ammonium sulfate, 3-15 g / L disodium hydrogen phosphate, 1-10 g / L potassium dihydrogen phosphate, and 30-60 g / L perillyl alcohol; to achieve further efficient catalysis of perillyl alcohol to perillyl lactone, so that the conversion rate of perillyl alcohol reaches more than 90%.

[0010] Furthermore, the fermentation medium comprises: 40 g / L glycerol, 5 g / L yeast extract, 15 g / L peptone, 15 g / L ammonium sulfate, 10 g / L disodium hydrogen phosphate, 5 g / L potassium dihydrogen phosphate, and 40 g / L perillyl alcohol; this is beneficial to improving the conversion rate of perillyl alcohol, increasing it to over 90%.

[0011] Fourthly, the present invention provides a method for producing perillaldehyde lactone, comprising: using perillol as a substrate and Cryptococcus lightifolius FM1120 as chassis cells, fermenting and culturing at 25-30°C to produce perillol lactone.

[0012] Further, the above production method includes: inoculating the seed culture of *Cryptococcus faecium* strain FM1120 into a fermentation medium at an inoculation rate of 8-12%, then adding 30-60 g / L perillyl alcohol, 10%-30% n-dodecane, and 0.5-2 mL / L IPTG, adjusting the pH to 5.5-7.5, and fermenting at 25-30℃ and 200-250 rpm to obtain a fermentation broth producing perillyl lactone. The fermentation medium comprises: 20-60 g / L glycerol, 1-10 g / L yeast extract, 5-20 g / L peptone, 5-20 g / L ammonium sulfate, 3-15 g / L disodium hydrogen phosphate, and 1-10 g / L potassium dihydrogen phosphate; the viable count of the *Cryptococcus faecium* strain FM1120 seed culture is 8 × 10⁻⁶. 7 -10×10 7 CFU / mL.

[0013] Preferably, during the fermentation process, the volume of liquid in the shake flask is 10-25 mL / 250 mL.

[0014] To further enhance the activity of the *Cryptococcus faecium* strain FM1120 seed culture, the preparation method of the *Cryptococcus faecium* strain FM1120 seed culture includes: inoculating a single colony of activated *Cryptococcus faecium* strain FM1120 into a seed culture medium and culturing it at 25-30℃ and 200-250 rpm to obtain the *Cryptococcus faecium* strain FM1120 seed culture; wherein, the seed culture medium comprises: 10-20 g / L glucose, 3-10 g / L yeast extract, 8-20 g / L peptone, 1-3 g / L disodium hydrogen phosphate and 5-9 g / L potassium dihydrogen phosphate.

[0015] To further enhance the activity of the activated Cryptococcus lightlyi strain FM1120, the method for culturing single colonies of the activated Cryptococcus lightlyi strain FM1120 includes: inoculating a suspension of Cryptococcus lightlyi strain FM1120 onto an agar slant culture medium and activating it in a constant temperature incubator at 25℃-30℃ to obtain activated single colonies of Cryptococcus lightlyi strain FM1120; wherein the agar slant culture medium comprises: 10-20 g / L glucose, 3-10 g / L yeast extract, 8-20 g / L peptone, and 15-25 g / L agar, and the viable count of the Cryptococcus lightlyi strain FM1120 suspension is 0.5 × 10⁻⁶. 7 CFU / ml - 0.8 × 10 7 CFU / mL.

[0016] Therefore, the Cryptococcus lighthani strain FM1120, which produces high levels of perillaldehyde in this invention, was initially isolated from perillifer flowers in Ili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region. Through systematic screening of wild strains, natural selection, and ARTP mutagenesis breeding, this excellent strain, which can efficiently produce perillaldehyde through microbial fermentation without the need for exogenous chemical catalysts, was finally obtained.

[0017] Furthermore, the fermentation production method for perillaldehyde and various culture media provided by the present invention can achieve efficient conversion of perillol as a substrate. For example, shaking flask culture for 72 hours can convert 40 g / L perillol substrate into 31.28 g / L perillaldehyde, with a conversion rate of 96.36%. Moreover, the fermentation production has few side reactions, and perillaldehyde is easy to purify and separate, resulting in high-purity perillaldehyde.

[0018] Furthermore, the fermentation production method for synthesizing perillaldehyde using Cryptococcus lightans strain FM1120 provided by this invention features mild reaction conditions (ambient temperature and pressure), no chemical pollutant emissions, high product selectivity, and low energy consumption. These characteristics not only effectively solve the environmental problems caused by traditional processes but also significantly reduce equipment investment and energy consumption for industrial production. In addition, the raw materials for this production method are renewable, and no toxic reagents are used throughout the process, fully complying with green chemistry principles. This provides a completely new environmentally friendly path for the industrial production of perillaldehyde, possessing broad market application prospects and significant social value. Attached Figure Description

[0019] Figure 1 The morphology of Cryptococcus lightensis strain FM1120 under a microscope; Figure 2 Gas chromatogram of shake-flask fermentation broth of Cryptococcus lightiformis strain FM1120; Figure 3 The graph shows the changes in the content and molar conversion rate of perilla lactone in the shake-flask fermentation medium of Cryptococcus lighti strain FM1120. Figure 4 The graph shows the changes in the content and molar conversion rate of perillaldehyde in the shake-flask fermentation of Cryptococcus lighti strain FM1120.

[0020] In the sequence list of the present invention: SEQ ID NO.1 is the ITS sequence of strain FM1120. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0022] The core objective of this invention is to develop and improve a biosynthesis process for perilla lactone based on microbial transformation technology, specifically utilizing Cryptococcus lighthani (… Cryptococcus albidus This invention utilizes a strain of *Cryptococcus lightans* as a biocatalyst to efficiently convert natural perillyl alcohol into high-value perillyl lactone. To achieve this, the invention first systematically isolates and collects microbial samples from the floral organs of various *Perillium* species. Through multiple rounds of natural screening combined with advanced ambient pressure room temperature plasma (ARTP) mutagenesis technology, a *Cryptococcus lightans* strain with excellent transformation performance was selectively bred. Cryptococcus albidusThe mutant strain FM1120. This Cryptococcus lightiformis strain FM1120, as the key biological material of this invention, has been formally deposited at the China Center for Type Culture Collection (CCTCC) in accordance with relevant international regulations on microbial deposits, on September 1, 2025, with accession number CCTCC NO: M 20251915.

[0023] Another main objective of this invention is to provide the above-mentioned Cryptococcus lightans (Cryptococcus) Cryptococcus albidus The application of FM1120 in the synthesis of perillaldehyde. Specifically, by optimizing the fermentation medium formulation, including key components such as carbon source, nitrogen source, and trace elements, and systematically studying the effects of key fermentation parameters such as temperature, pH, dissolved oxygen, and liquid volume on the conversion efficiency of perillaldehyde, a complete and industrially scalable biocatalytic production process system for perillaldehyde was finally established, significantly improving the yield and purity of perillaldehyde.

[0024] The present invention will be described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0025] The gas chromatograph analysis method and conditions of this invention are as follows: The detection instrument is an Agilent 8860 gas chromatograph with a flame ionization detector; the carrier gas is high-purity nitrogen (99.999%); the chromatographic column is an HP-5 (30m × 0.32mm × 0.25μm); the injection port temperature is 280℃; the detector temperature is 300℃; the column temperature program is: initial temperature 80℃, hold for 2 min, increase to 250℃ at 10℃ / min, hold for 5 min; carrier gas flow rate is 1.5mL / min; injection volume is 1.0μL; and the split ratio is 10:1. The peak times for the styracil alcohol standard are 8.555 min, the styracil lactone standard is 8.103 min, and the styracil diol standard is 8.008 min. Figure 2 As shown in the (standard product) diagram.

[0026] I. Wild-type Cryptococcus pluvialis strain (Cryptococcus albidus) FM01 The specific steps for obtaining the wild-type strain FM01 are as follows: S1. Select fragrant purslane flowers from Ili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region, grind them into powder, take the juice, dilute it with YPD solid medium, spread it, and incubate it at a constant temperature of 25℃-30℃ until single colonies grow. S2. Inoculation: Inoculate the single colony cultured in step S1 into YPD broth medium and incubate at 30°C in a shaker for 24-48 h. S3. Fermentation: The strain cultured in step S2 is inoculated into the fermentation medium at an inoculation rate of 10%. The medium contains 10 g / L perillyl alcohol, 20% n-dodecane and 1 mL / L LPTG. The culture is carried out at 30℃ and 200 rpm for 96 h. The fermentation medium contains 20 g / L glycerol, 5 g / L yeast extract, 10 g / L peptone, 5 g / L ammonium sulfate, 3 g / L disodium hydrogen phosphate and 9 g / L potassium dihydrogen phosphate. S4. Detection: The fermentation broth obtained in step S3 was soaked in an equal volume of ethyl acetate for 10 min, sonicated for 30 min, and the upper phase was centrifuged at 12000 rpm for 1 min and analyzed by gas chromatography. Finally, strain FM01, which catalyzes the production of perillaldehyde from perillyl alcohol, was obtained.

[0027] Identification of S5 and FM01 strains: (1) Colony morphology observation: The colonies are round, smooth, white, and moist. (2) Prepare slides by Gram staining and observe them under a 100× objective lens. The diameter is 0.5-1 μm and the shape is elliptical and without flagella, which is consistent with the shape of yeast. (3) The optimal growth temperature is 25-30 ℃ and the optimal pH is 5.5-7.5.

[0028] II. Cryptococcus pluvialis strain (Cryptococcus albidus) FM1120 Strain FM1120 was mainly obtained from wild strain FM01 through natural selection and ARTP mutagenesis selection. The specific screening method is as follows: (1) Experimental procedure and analysis of natural selection A1. Pretreatment of bacterial strains: Ten single colonies of JMIC01 were picked from the slant culture medium and a bacterial suspension was prepared using 0.9% physiological saline. The number of strains was 0.5 × 10⁻⁶. 7 - 0.8×10 7 CFU / mL.

[0029] A2. Preparation of the basic culture medium for the strain: 10 g / L glucose, 3 g / L yeast extract, 8 g / L peptone, and 18 g / L agar. Five parallel screening plates were prepared, each containing different screening agents: perillyl alcohol, Tween surfactants, glycerol, DMF organic solvent, and Pluronic F-127 block copolymer. Each screening agent was prepared with a concentration gradient of 1-10 g / L to ensure diverse screening conditions.

[0030] A3. Strain screening: The bacterial suspension obtained in A1 was mixed with the original solution at a ratio of 10... -1 10 -2 10 -310 -4 10 -5 10 -6 Different dilutions were performed, and the samples were spread on base plates and screening plates. The plates were then incubated in a 30°C incubator for 48-72 hours to obtain single colony plates, which can be used for subsequent experiments. The growth of the screening plates is shown in Table 2.

[0031] A4. Transformation Capacity Test: Typical colonies were selected from single-colony plates and inoculated into seed culture medium in 48-well deep-well plates using sterile toothpicks (0.5 mL per well). The culture was pre-cultured for 24 hours at 25℃ and 200 rpm in a constant-temperature shaker to obtain a homogeneous seed culture. The seed culture was then transferred at a 10% inoculum to fermentation medium in 24-well plates (1 mL per well) and cultured for an extended period of 120 hours at 25-30℃, maintaining a shaking condition of 200 rpm throughout to ensure adequate oxygen supply. The changes in the content of perillaldehyde in the fermentation broth are shown in Table 3.

[0032] A5. Product Testing: After fermentation, 1 mL of ethyl acetate was added to each well for extraction, followed by ultrasonic extraction at 100 kHz for 30 minutes. After centrifugation, the organic phase was transferred to a new 24-well plate. The hydroxylamine-FeCl3 colorimetric method was used for detection: first, hydroxylamine hydrochloride methanol solution was added for derivatization, then alkalized with NaOH solution and heated at 60°C for 30 minutes, and finally acidified before adding FeCl3 colorimetric reagent. Samples with the deepest color were selected for GC quantitative analysis.

[0033] A6. Repeated Experiments: Repeat steps A1-A5 2-3 times for the high-yielding strains obtained from the initial screening to ensure the reliability of the results. The concentration gradients of each screening agent are set as follows: perillaldehyde 1-10 g / L, Tween 1-10 g / L, glycerol 1-10 g / L, DMF 1-10 g / L, and Pluronic F-127 1-10 g / L. Three parallel experiments are set up for each concentration.

[0034] Table 1. Growth of strain FM01 on screening plates

[0035] Table 1 shows that the concentration of perillyl alcohol affects the growth of the strains. Through iterative screening, strains that can tolerate high concentrations of perillyl alcohol can be obtained. Tween, glycerol, and Pluronic F-127 have little effect on growth. DMF has the greatest impact on growth.

[0036] A7. Shake-flask verification of the dominant strain naturally selected in step A6: Experiments were conducted in 250mL Erlenmeyer flasks, with seed culture medium and fermentation medium dispensed at 25mL / flask; culture conditions were consistent with microplate culture. The seed culture medium consisted of: 15 g / L glucose, 6.5 g / L yeast extract, 14 g / L peptone, 7 g / L potassium dihydrogen phosphate, and 2 g / L disodium hydrogen phosphate; the fermentation medium consisted of: 40 g / L glycerol, 5 g / L yeast extract, 15 g / L peptone, 15 g / L ammonium sulfate, 10 g / L disodium hydrogen phosphate, 5 g / L potassium dihydrogen phosphate, 20% n-dodecane, 1 mL / L IPTG, and 40 g / L perillyl alcohol. The hydroxylamine-FeCl3 colorimetric method was used for detection: first, hydroxylamine hydrochloride methanol solution was added for derivatization, then alkalized with NaOH solution and heated at 60℃ for 30 minutes, and finally acidified before adding FeCl3 colorimetric reagent. Select samples with darker colors for GC quantitative analysis.

[0037] (2) ARTP mutagenesis screening Superior strains selected through natural selection were mutagenized using ARTP (Atmospheric and Room Temperature Plasma) technology. Initial screening was performed using a series of plates, followed by high-throughput testing and screening of the mutant library using the Tecan Fluent 1080 automated platform and Agilent RapidFire 400. The specific methods for ARTP mutagenesis selection are as follows: B1. Pretreatment of bacterial strain: Wild-type strain FM01 was prepared into a bacterial suspension using physiological saline, with a strain quantity of 0.5 × 10⁻⁶. 7 - 0.8×10 7 CFU / mL.

[0038] B2. Preparation of screening medium: Preparation of screening medium: 10 g / L glucose, 3 g / L yeast extract, 8 g / L peptone, 5 g / L perillol, and 18 g / L agar are used to prepare screening plate medium.

[0039] B3. Strain Mutagenesis and Screening: The bacterial suspension was subjected to ARTP mutagenesis at 0 s, 30 s, 60 s, 90 s, 120 s, 150 s, and 200 s, according to the original solution and 10 -1 10 -2 10 -3 10 - 4, 10 -5 10 -6 Dilute the sample to different concentrations, spread it on screening plates, and incubate it in an incubator at 25-30℃ for 48-72 h to obtain single colony plates.

[0040] B4. Transformation Capacity Test: Using sterile toothpicks, single colonies were inoculated into 48-well deep-well plates containing 0.5 mL of seed culture medium and incubated on a 25°C shaker at 200 rpm for 24 hours. Subsequently, 50 μL of the seed culture was transferred to 24-well plates containing 1 mL of fermentation medium and incubated at 25-30°C for 96 hours. Stable environmental conditions must be maintained during the incubation process to avoid temperature fluctuations affecting the experimental results.

[0041] B5. Product Detection: After fermentation, 1 mL of ethyl acetate was added to each well for extraction, followed by ultrasonic treatment at 100 kHz for 30 minutes to promote dissolution. After centrifugation, the organic phase was transferred to a new 24-well plate to obtain the extract. Hydroxylamine hydrochloride methanol solution and NaOH solution were added sequentially to the extract, and the mixture was heated in a water bath to ensure complete reaction between the lactone and hydroxylamine. After acidification, FeCl3 solution was added dropwise, and the color change was observed. Quantitative analysis was performed by gas chromatography.

[0042] B6. Validation Experiment: The high-yielding strains selected were repeatedly subjected to the AE step validation experiment. During the shake-flask fermentation stage, the concentration of perillaldehyde substrate was increased to 40 g / L to examine the strain's tolerance to high concentrations. Through multiple rounds of screening and validation, a superior mutant strain with significantly increased perillaldehyde yield was finally obtained. Detailed yield changes in each treatment group were recorded during the experiment, as shown in Table 2, providing a reliable basis for subsequent research.

[0043] Table 2. Changes in the content of perilla lactone in the dominant fungal strain.

[0044] B7. Shake-flask verification was consistent with natural selection, ultimately yielding the high-yielding strain FM1120. The identification tests for strain FM1120 are as follows: (1) Colony morphology observation: The colonies are round, smooth, white, and moist. (2) Microscopic morphological observation, such as Figure 1 As shown, the slides were prepared using Gram staining and observed under a 100× objective lens. The diameter was 0.5-1 μm, and the shape was oval and non-flagellated, consistent with the morphology of yeast. (3) The optimal growth temperature is 25-30 ℃, and the optimal pH is 5.5-7.5; (4) The fungal properties were identified by PCR amplification and sequencing using universal primers 27F and 1492R. The ITS sequence of the fungus is shown in SEQ ID NO. 1.

[0045] Gas chromatography was used to detect the shake-flask fermentation broth of strain FM1120. The results are as follows: Figure 2 As shown in the (fermentation broth). From Figure 2It is clearly visible that the fermentation broth of strain FM1120 exhibits a strong peak at 8.102 min, which is essentially the same as the peak time of the standard perillaldehyde (8.103 min). Therefore, it can be concluded that perillaldehyde can be produced using strain FM1120. Furthermore, Figure 2 In the (standard) sample, except for the presence of a peak of perillaldehyde at 8.545 min (which is basically the same as the peak time of perillaldehyde standard (8.555 min)), it is similar to... Figure 2 The chromatograms of the fermentation broth were basically the same, with virtually no other impurity peaks such as perillaldehyde. This indicates that the fermentation production of perillaldehyde using strain FM1120 also has the advantages of fewer impurities in the fermentation broth and easier separation of the product from the substrate. For example, the fermentation broth was extracted with 1 BV ethyl acetate, and the organic phase was collected to obtain the extract. Impurities were removed using a chromatography column to obtain a crude extract. The crude extract was then decolorized with activated carbon to obtain a decolorized solution, and finally, the product was cooled and crystallized to obtain a pure product with a perillaldehyde content of 99%.

[0046] Therefore, based on the above analysis and identification results, the strain FM1120 screened in this invention is classified and named *Cryptococcus lightans*. Cryptococcus albidus The accession number, FM1120, was deposited on September 1, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251915.

[0047] III. Optimization of Shake Flask Fermentation Process Using perillaldehyde as a substrate and Cryptococcus lightans strain FM1120 as the substrate cells, the substrate cells were inoculated onto the fermentation medium at an inoculum size of 8%-10%, and the pH of the fermentation culture was adjusted to 5.5-7.5. Shake-flask fermentation was carried out at 25-30℃ and 200-250 rpm to produce perillaldehyde. The specific process is as follows: S1. Strain activation: Prepare slant culture medium with a concentration of 0.7 × 10⁻⁶. 7 A CFU / ml suspension of Cryptococcus lightanis strain FM1120 was inoculated onto the slant culture medium and activated in an incubator at 25°C for 72 h to obtain single colonies. The slant culture medium consisted of 15 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, and 18 g / L agar. S2, Primary Seed Culture: Pick one single colony and place it onto the seed culture medium in the primary seed bottle. Incubate at a constant temperature of 30℃, a rotation speed of 200 rpm, and an amplitude of 50 mm for 24 hours to obtain a concentration of 1×10⁻⁶. 8The seed culture medium for Cryptococcus lightifolius strain FM1120, with a concentration of CFU / mL, comprises: 15 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 1.4 g / L disodium hydrogen phosphate, and 9.7 g / L potassium dihydrogen phosphate. S3. Shake flask fermentation: Before inoculation, add perillyl alcohol powder, n-dodecane and IPTG to the fermentation medium to make the concentration of perillyl alcohol 40 g / L, n-dodecane 20% and IPTG 1 mL / L. Inoculate 20 mL of the above Cryptococcus faecium strain FM1120 primary seed liquid into a 250 mL fermentation shake flask at a 10% inoculation rate and ferment at a constant temperature for 72-96 hours. S4. Detection: Add an equal volume of ethyl acetate, sonicate for 30 min, centrifuge and take the organic phase for gas phase detection.

[0048] Fermentation medium formulation screening This invention, through a systematic review of relevant domestic and international literature, determined that the following basic formulation F was the foundation for a detailed shake-flask fermentation experiment to verify the culture medium formulation containing key components such as carbon source, nitrogen source, phosphate, inorganic salts, and trace elements. The basic formulation F comprises: 40 g / L glycerol, 10 g / L yeast extract, 10 g / L peptone, 10 g / L ammonium sulfate, 6.78 g / L disodium hydrogen phosphate, and 3 g / L potassium dihydrogen phosphate.

[0049] 3.1.1 Single-factor screening Referring to step S3, the selected basic formula F was fermented for 72 h under the following conditions: a substrate concentration of perillyl alcohol (40 g / L), a fermentation temperature of 30℃, a fermentation speed of 200 rpm, a rotation speed of 50 mm, and a pH of 6. The variables were designed as follows: 0-60 g / L glycerol, 0-20 g / L yeast extract, 0-20 g / L peptone, 0-20 g / L ammonium sulfate, 0-20 g / L disodium hydrogen phosphate, and 0-15 g / L potassium dihydrogen phosphate. The perillyl lactone content and conversion rate obtained from the single-factor screening are shown below. Figure 3 .

[0050] from Figure 3 It can be seen that, through systematic single-factor optimization experiments, the potency of perillaldehyde content under the optimal conditions of each factor is as follows: 23.8 g / L for perillaldehyde fermented with 40 g / L glycerol; 20.7 g / L for perillaldehyde fermented with 5 g / L yeast powder; 25.2 g / L for perillaldehyde fermented with 10 g / L peptone; 25.8 g / L for perillaldehyde fermented with 10 g / L ammonium sulfate; 27.2 g / L for perillaldehyde fermented with 1 g / L potassium dihydrogen phosphate; and 18.5 g / L for perillaldehyde fermented with 10 g / L disodium hydrogen phosphate.

[0051] 3.1.2 Optimal Concentration Range Combination Experiment To optimize the combined effect of multiple factors, using perillaldehyde as the evaluation index, an orthogonal experimental design was employed to screen the optimal nutritional formula. Each factor had three levels (low concentration, single-factor optimal concentration, and high concentration), as shown in Table 3 below. For the six factors with three levels, L... 18 (3 7 An orthogonal array was used (with the remaining column designated as the error term), and 18 experimental schemes were developed. Each formulation was supplemented with 20% n-dodecane, 1 mL / L IPTG, and 40 g / L perillyl alcohol. The specific schemes are shown in Table 4 below. Table 3. Factors and Levels in the Orthogonal Experiment

[0052] Table 4 L 18 (3 7 Orthogonal Experimental Design Table

[0053] Therefore, the preferred formulation of the shake flask fermentation medium includes: 20-60 g / L glycerol, 1-10 g / L yeast extract, 5-20 g / L peptone, 5-20 g / L ammonium sulfate, 3-15 g / L disodium hydrogen phosphate and 1-10 g / L potassium dihydrogen phosphate. Most preferably, the shake flask fermentation medium includes: 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.

[0054] Fermentation conditions Based on the optimal shake-flask fermentation medium obtained from the above orthogonal experiments, a systematic single-factor screening experiment of fermentation conditions was conducted. The experiment investigated the effects of four key parameters on the biotransformation of perilla lactone: the temperature test range was set at 20-45℃ to cover the optimal growth range of microorganisms; the pH gradient was set at 3-10, spanning the acidic to alkaline range; the stirring speed was controlled in the range of 0-250 rpm; and the dissolved oxygen volume test gradient was 10-100 mL / 250 mL to investigate the effect of dissolved oxygen.

[0055] from Figure 4It can be seen that, using 40 g / L perillyl alcohol as the substrate concentration, the optimal control range for shake-flask fermentation parameters is: fermentation temperature 25-30℃, pH 5.5-7.5, rotation speed 200-250 rpm, and liquid volume 20-25 mL / 250 mL. The optimal parameters and perillyl lactone potency obtained by single-factor screening in shake-flask fermentation are as follows: the highest potency of 28.87 g / L was obtained under fermentation conditions of 25℃; the potency reached 27.52 g / L at a rotation speed of 250 rpm; the potency reached 23.99 g / L under fermentation conditions of pH 6.5; and the potency reached 27.43 g / L under fermentation conditions of 25 mL liquid volume.

[0056] After a series of optimizations and improvements, strain FM1120 was shake-flask fermented under the optimal fermentation medium and optimal fermentation conditions, and the stability of single colonies was significantly improved. The relevant experimental data are shown in Table 5.

[0057] Table 5. Changes in the content of perilla lactone in single colony screening.

[0058] Table 5 shows that strain FM1120 can maintain stable genetic characteristics and physiological activity during continuous subculture, laying a solid foundation for subsequent industrial-scale production.

[0059] In summary, this invention systematically isolates and collects microbial samples from the floral organs of various *Perilla* species, and through multiple rounds of natural screening combined with advanced ambient pressure room temperature plasma (ARTP) mutagenesis technology, ultimately screens out the *Cryptococcus lightans* strain with the best performance. Cryptococcus albidus The mutant strain FM1120, a microbial strain with specific catalytic activity, achieves a biotransformation reaction using perillaldehyde as a substrate in an optimized fermentation system. In the application of the FM1120 strain provided by this invention in the production of perillaldehyde lactone, the conversion rate of perillaldehyde can reach over 90%, with few impurities in the fermentation broth, and the perillaldehyde lactone is easily separated and purified with high purity. It has advantages such as mild fermentation conditions (room temperature and pressure), no chemical pollutant emissions, high product selectivity, and low energy consumption, solving the environmental problems caused by traditional chemical synthesis methods, while reducing equipment costs and energy consumption in industrial production, providing a new path for the green preparation of perillaldehyde lactone. Therefore, this invention achieves full-process optimization from natural raw material screening to high-efficiency strain breeding, and then to the establishment of the optimal fermentation process, significantly improving the yield and purity of perillaldehyde lactone.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A strain of Cryptococcus lightensis ( Cryptococcus albidus FM1120, accession number CCTCC NO: M20251915, accession date September 1, 2025, deposited by China Center for Type Culture Collection.

2. The Cryptococcus lightanis strain according to claim 1 ( Cryptococcus albidus FM1120, characterized in that, The main method used to obtain it is as follows: first, wild-type Cryptococcus syringa was isolated from Perilla frutescens flowers to obtain a wild-type Cryptococcus syringa that can convert perilla alcohol into perilla lactone; then, the wild-type Cryptococcus syringa was subjected to natural selection and ARTP mutagenesis to obtain a microbial strain with high perilla lactone production and an ITS sequence as shown in SEQ ID NO.

1.

3. A strain of Cryptococcus lightensis as described in claim 1 ( Cryptococcus albidus Application of FM1120 in the production of perilla lactone.

4. A Cryptococcus lighthani strain according to claim 1 ( Cryptococcus albidus The fermentation medium for FM1120 includes: 20-60 g / L glycerol, 1-10 g / L yeast extract, 5-20 g / L peptone, 5-20 g / L ammonium sulfate, 3-15 g / L disodium hydrogen phosphate, 1-10 g / L potassium dihydrogen phosphate, and 30-60 g / L perillyl alcohol.

5. The fermentation medium according to claim 4, characterized in that, include: 40 g / L glycerol, 5 g / L yeast extract, 15 g / L peptone, 15 g / L ammonium sulfate, 10 g / L disodium hydrogen phosphate, 5 g / L potassium dihydrogen phosphate, and 40 g / L perillyl alcohol.

6. A method for producing perillaldehyde, comprising: Using perillyl alcohol as a substrate, the Cryptococcus faecium strain of claim 1 ( Cryptococcus albidus FM1120 is used as the chassis cell, and fermentation culture is carried out at 25-30℃ to produce perillyl lactone.

7. The production method according to claim 6, characterized in that, Cryptococcus lightenis strain ( Cryptococcus albidus The FM1120 seed culture was inoculated into the fermentation medium at an inoculum size of 8-12%, followed by the addition of 30-60 g / L perillyl alcohol, 10%-30% n-dodecane, and 0.5-2 mL / L IPTG. The pH was adjusted to 5.5-7.5, and fermentation was carried out at 25-30℃ and 200-250 rpm. The *Cryptococcus faecium* strain (…) Cryptococcus albidus The viable count of FM1120 seed culture is 8 × 10⁻⁶. 7 -10×10 7 CFU / mL; the fermentation medium comprises: 20-60 g / L glycerol, 1-10 g / L yeast extract, 5-20 g / L peptone, 5-20 g / L ammonium sulfate, 3-15 g / L disodium hydrogen phosphate and 1-10 g / L potassium dihydrogen phosphate.

8. The production method according to claim 7, characterized in that, During the fermentation process, the volume of liquid in the shake flask is 20-25 mL / 250 mL.

9. The production method according to claim 7 or 8, characterized in that, The Cryptococcus syriacus strain ( Cryptococcus albidus The preparation method of FM1120 seed culture includes: activating the Cryptococcus lighthani strain (… Cryptococcus albidus A single colony of FM1120 was inoculated into seed culture medium and cultured at 25-30℃ and 200-250 rpm to obtain the described Cryptococcus lighthani strain ( Cryptococcus albidus FM1120 seed culture; wherein the seed culture medium comprises: 10-20 g / L glucose, 3-10 g / L yeast extract, 8-20 g / L peptone, 1-3 g / L disodium hydrogen phosphate and 5-9 g / L potassium dihydrogen phosphate.

10. The production method according to claim 9, characterized in that, The activated Cryptococcus lightenis strain ( Cryptococcus albidus The culture method for a single colony of FM1120 includes: cultured with the Cryptococcus lightans strain (…). Cryptococcus albidus FM1120 bacterial suspension was inoculated onto slant culture medium and activated in an incubator at 25℃-30℃ to obtain the activated Cryptococcus lightanis strain ( Cryptococcus albidus FM1120 single colony; wherein, the slant culture medium comprises: 10-20 g / L glucose, 3-10 g / L yeast extract, 8-20 g / L peptone and 15-25 g / L agar, and the Cryptococcus lighthani strain ( Cryptococcus albidus The viable count of FM1120 bacterial suspension is 0.5 × 10⁻⁶. 7 CFU / ml - 0.8 × 10 7 CFU / mL.

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