Cryptococcus albidus strain bc96 and its use in the production of humuladiol

Abstract

The present application belongs to the field of biotechnology and microbial fermentation engineering, and particularly relates to a Cryptococcus albidus strain BC96 and application thereof in production of sclareol diol. Cryptococcus albidus The present application is screened from soil planted with sclarea in Yili Kazakh Autonomous Prefecture of Xinjiang Uygur Autonomous Region through systematic wild strain screening, natural breeding and ARTP mutagenesis breeding, and the optimal mutant strain is named BC96 with a preservation number of CCTCC NO:M20251917, a preservation date of September 1, 2025 and a preservation unit of China Center for Type Culture Collection. The present application also provides application of the above strain BC96 in production of sclareol diol. With sclareol as a substrate, the conversion rate of sclareol reaches more than 96% through fermentation, the fermentation liquor has less impurities, sclareol diol is easy to separate and purify, has high purity and other advantages, and a new path is provided for green preparation of sclareol diol.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and microbial fermentation engineering, specifically relating to a Cryptococcus lightifolia strain BC96 and its application in the production of perillaldehyde. Background Technology

[0002] Perillaldehyde, an essential intermediate in the synthesis of the rare fragrance ambergris, possesses significant industrial value. Ambergris is highly regarded for its remarkably similar aroma to the natural animal fragrance ambergris, exhibiting a unique, mild, delicate, and pleasant musky animal scent. It is considered the synthetic fragrance that best reproduces the essential characteristics of ambergris. Given its superior fragrance quality, ambergris has found widespread application as a high-end flavoring and fixative in the perfume manufacturing, tobacco processing, and pharmaceutical industries, largely replacing the increasingly scarce natural ambergris resources.

[0003] In the early stages of exploring biotransformation, researchers discovered that certain microbial strains possessed the ability to convert perillyl alcohol into perillyl glycol or further metabolize it into other products. These findings initially confirmed the feasibility of biological production, but early research had not yet overcome the technical bottleneck of achieving a complete synthetic route starting from simple carbon sources (such as glucose). Only in recent years, through metabolic engineering to modify the cofactor regeneration system and substrate-specific enzyme gene expression of *Cryptococcus faecalis*, has efficient and targeted conversion using glycerol as a carbon source and perillyl alcohol as a precursor been achieved.

[0004] Notably, a research team from Zhejiang University developed a highly innovative co-culture technique. In their published patent (A combination of strains, application, and method for producing perillaldehyde), they first constructed a genetically modified Saccharomyces cerevisiae engineered strain. This strain optimized the endogenous metabolic network by exogenously expressing the LPPS and SCS genes, downregulating the ERG9 gene expression, and knocking out the rox1 gene, significantly improving the synthesis capacity of perillaldehyde. Subsequently, the researchers established a co-culture system with other fungi using this engineered yeast. The engineered yeast was specifically responsible for converting glucose into perillaldehyde, while the fungal cells focused on further converting perillaldehyde into perillaldehyde, thus achieving for the first time the complete biosynthesis of perillaldehyde from a single carbon source, glucose.

[0005] Although significant progress has been made in the biosynthesis of sagebearing diol, there is still considerable room for optimization. Current research focuses on the following aspects: First, the development and utilization of alternative substrates. In addition to glucose, researchers are actively exploring the use of more economical and environmentally friendly carbon sources such as crude glycerol and waste oils.

[0006] Secondly, precise regulation of metabolic pathways is the core strategy for increasing the yield of perillaldehyde. By accurately adjusting the expression levels of key enzymes, introducing heterologous regulatory elements, or optimizing promoter sequences, the distribution of metabolic flux can be effectively balanced, reducing the ineffective accumulation of intermediate products. Furthermore, the rapid development of synthetic biology tools provides more innovative possibilities for the efficient production of perillaldehyde: including using the CRISPR-Cas system to achieve precise genome editing and constructing artificially designed gene regulatory circuits to achieve dynamic metabolic control. The comprehensive application of these cutting-edge technologies will strongly promote the large-scale and industrialized production of perillaldehyde, injecting new impetus into the sustainable development of the fragrance industry. Summary of the Invention

[0007] In view of this, the main objective of this invention is to provide a Cryptococcus lightans strain BC96 and its application in the production of perillaldehyde. This strain BC96 acts as a biocatalyst, enabling an innovative process for converting perillaldehyde into high-value-added perillaldehyde. This invention primarily achieves high-yield production of perillaldehyde through systematic strain selection and fermentation process optimization, resulting in a conversion rate of over 96%.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, this invention provides a Cryptococcus lighthani strain BC96, classified and named as follows: Cryptococcus albidus BC96, accession number CCTCC NO: M20251917, accession date September 1, 2025, deposited by China Center for Type Culture Collection.

[0009] The method for obtaining the *Cryptococcus faecium* strain BC96 includes: firstly, isolating wild-type *Cryptococcus faecium* from soil in which *Perilla frutescens* is planted; then, subjecting the wild-type *Cryptococcus faecium* to natural selection and ARTP mutagenesis to obtain a microbial strain with an ITS sequence as shown in SEQ ID NO.1 that can convert perillyl alcohol to perillyl diol, and naming it... Cryptococcus albidus BC96. This strain BC96 grows well in YPD medium. The observed strains are typically 0.5-1 μm in diameter, elliptical in shape, and non-flagellated, consistent with yeast morphology. Colonies are round, smooth, white, and moist, indicating it is a Gram-positive yeast. The optimal growth temperature for strain BC96 is 25-30 ℃, and the optimal pH is 5.0-6.0. It produces an enzyme that catalyzes the conversion of perillol to perillyl glycol. The entire conversion to perillyl glycol does not require an external chemical catalyst; it relies solely on microbial metabolism for directional conversion, thus enabling the microbial fermentation synthesis of perillol using perillol as a substrate.

[0010] Secondly, this invention provides the application of a Cryptococcus cephalosporin strain BC96 in the production of perillaldehyde.

[0011] Thirdly, the present invention provides a fermentation medium for Cryptococcus lightensis strain BC96, comprising: 0-40 g / L glucose, 20-60 g / L glycerol, 1-10 g / L ammonium nitrate, 0-3 g / L yeast extract, 1-10 g / L diammonium hydrogen phosphate, 1-10 g / L sodium dihydrogen phosphate, and 0-2 mL / L trace elements; thereby achieving further efficient catalysis of perillyl alcohol to perillyl glycol, with a perillyl alcohol conversion rate of over 96%.

[0012] Furthermore, the fermentation medium comprises: 20 g / L glucose, 40 g / L glycerol, 7 g / L ammonium nitrate, 1 g / L yeast extract, 7 g / L ammonium dihydrogen phosphate, 10 g / L disodium hydrogen phosphate, and 1 mL / L trace elements. Experimental verification showed that under optimized conditions, this strain can efficiently convert 40 g / L of perillyl alcohol substrate into perillyl glycol, with a conversion rate exceeding 90%, achieving a final product concentration of 30.60 g / L, while the residual substrate concentration is only 0.96 g / L, demonstrating excellent catalytic efficiency.

[0013] Fourthly, the present invention provides a method for producing perillaldehyde, comprising: using perillaldehyde as a substrate and Cryptococcus baiji strain BC96 as chassis cells, fermenting and culturing at 25-30°C to produce perillaldehyde.

[0014] Furthermore, the above production method includes: inoculating the seed culture of *Cryptococcus faecium* strain BC96 into a fermentation medium at an inoculation rate of 8-12%, then adding 10-40 g / L perillyl alcohol and 20-60 g / L Tween 80, adjusting the pH to 5.0-6.0, and fermenting at 25-30℃ and 200-250 rpm to obtain a fermentation broth producing perillyl glycol. The fermentation medium comprises: 0-40 g / L glucose, 20-60 g / L glycerol, 1-10 g / L ammonium nitrate, 0-3 g / L yeast extract, 1-10 g / L diammonium hydrogen phosphate, 1-10 g / L sodium dihydrogen phosphate, and 0-2 mL / L trace elements; the viable count of the *Cryptococcus faecium* strain BC96 seed culture is 5 × 10⁻⁶. 7 -2×10 8 CFU / mL.

[0015] Preferably, the liquid volume of the fermentation container during the fermentation process is 8%-10% v / v.

[0016] To further improve the activity of the *Cryptococcus faecium* strain BC96 seed culture, the preparation method of the *Cryptococcus faecium* strain BC96 seed culture includes: inoculating a single colony of activated *Cryptococcus faecium* strain BC96 into a seed culture medium and culturing it at 25-30℃ and 200-250 rpm to obtain the *Cryptococcus faecium* strain BC96 seed culture; wherein, the seed culture medium comprises: 10-20 g / L glucose, 3-10 g / L yeast extract, 8-20 g / L peptone, 1-5 g / L ammonium dihydrogen phosphate and 5-10 g / L dipotassium hydrogen phosphate.

[0017] To further enhance the activity of the activated Cryptococcus lightlyi strain BC96, the method for culturing single colonies of the activated Cryptococcus lightlyi strain BC96 includes: inoculating a suspension of Cryptococcus lightlyi strain BC96 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 BC96; 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 BC96 suspension is 0.5 × 10⁻⁶. 7 -0.8×10 7 CFU / mL.

[0018] Therefore, the *Cryptococcus faecium* strain BC96, which produces high-yield perillaldehyde, was initially isolated from soil used for planting perilla in Ili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region. Through systematic screening of wild-type strains, natural selection, and ARTP mutagenesis breeding, the *Cryptococcus faecium* strain BC96, capable of producing an enzyme that catalyzes the conversion of perillaldehyde to perillaldehyde, was finally obtained. Using this *Cryptococcus faecium* strain BC96, perillaldehyde can be efficiently produced through microbial fermentation without the need for exogenous chemical catalysts, thus achieving efficient biosynthesis of perillaldehyde. Furthermore, experiments showed that the shake-flask fermentation broth using perillaldehyde as a substrate and strain BC96 as the substrate bacteria contained few impurities, and the product was easily separated from the substrate; moreover, the strain had a high conversion efficiency for perillaldehyde, reaching over 96%.

[0019] Furthermore, the fermentation production method and various culture media provided by the present invention can achieve efficient conversion of salidomide as a substrate. For example, shaking flask culture for 72 hours can convert 40 g / L salidomide substrate into 30.60 g / L salidomide, with a conversion rate of 92.72%. Moreover, the fermentation production has few side reactions, and salidomide is easy to purify and separate, resulting in high-purity salidomide.

[0020] Furthermore, the fermentation production method for synthesizing perillaldehyde using Cryptococcus lightans strain BC96 provided by this invention features mild reaction conditions (room 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

[0021] Figure 1 The morphology and microscopic images of Cryptococcus BC96 strain on a plate; Figure 2 Gas chromatogram of shake-flask fermentation broth of Cryptococcus BC96; Figure 3 The graph shows the changes in the content and conversion rate of perillaldehyde in the shake-flask fermentation medium of Cryptococcus lighti strain BC96. Figure 4 The graph shows the changes in the content and conversion rate of perillaldehyde in the shake-flask fermentation of Cryptococcus lightifolius strain BC96.

[0022] In the sequence list of the present invention: SEQ ID NO.1 is the ITS sequence of strain BC96. Detailed Implementation

[0023] 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.

[0024] The main objective of this invention is to develop and improve a biosynthesis process for perilla glycol based on microbial transformation technology, specifically utilizing Cryptococcus lighthani (… Cryptococcus albidus Using strain BC96 as a biocatalyst, a complete biosynthesis system for perillaldehyde was established through systematic strain screening and improvement, culture medium formulation optimization, fermentation process parameter control, and biotransformation condition regulation, thereby achieving the efficient conversion of natural perillaldehyde into high-value perillaldehyde.

[0025] To achieve the above objectives, this invention first systematically isolates and collects microbial samples from soils containing various plants of the genus *Perilla*. 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 is selectively bred. Cryptococcus albidus The mutant strain BC96. This Cryptococcus lighti strain BC96, 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: M20251917.

[0026] Another major objective of this invention is to provide the application of the aforementioned Cryptococcus lightanis strain BC96 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.

[0027] 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.

[0028] Unless otherwise specified, the culture media, culture methods, and detection methods used in the following examples are all existing commercial culture media or existing technologies. For example, YPD solid culture medium includes: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, and 20 g / L agar; YPD liquid culture medium includes: 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract.

[0029] In the following experimental schemes of this invention, the trace elements in the culture medium consist of 1 g / L magnesium sulfate, 1 g / L biotin, 1 g / L vitamin B1, 0.05 g / L niacin and 10 g / L ferrous sulfate.

[0030] The gas chromatograph analysis method and conditions of this invention are as follows: The detection instrument is an Agilent 7890B gas chromatograph with a flame ionization detector; the carrier gas is high-purity nitrogen (99.999%); the chromatographic column is a DB-5 (30m × 0.25mm × 0.25μm); the injection port temperature is 200℃; the detector temperature is 280℃; the column temperature program is: initial temperature 60℃, hold for 1 min, increase to 250℃ at 10℃ / min, hold for 5 min; carrier gas flow rate is 1.0 mL / min; injection volume is 1.0 μL; and the split ratio is 10:1. The peak times for perillaldehyde standard were 14.760 min, perillyl lactone 14.170 min, and perillyl diol standard 14.095 min. Figure 2 As shown in the standard sample.

[0031] I. Wild-type Cryptococcus pluvialis strain (Cryptococcus albidus) )BC01 The specific steps for obtaining wild strain BC01 are as follows: S1. Select soil from the Ili Kazakh Autonomous Prefecture of Xinjiang Uygur Autonomous Region for planting Perilla frutescens, grind it 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 liquid 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 fermentation medium contains 10 g / L perillyl alcohol and 40 g / L Tween 80. The culture is carried out at 30°C and 200 rpm for 120 h. The fermentation medium includes 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, 2.4 g / L diammonium hydrogen phosphate, and 9.4 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 BC01, which catalyzes the production of perillaldehyde from perillyl alcohol, was obtained. Identification of S5 and BC01 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 5.0-6.0.

[0032] II. Pale white Cryptococcus strain (Cryptococcus albidus) BC96 Strain BC96 was mainly obtained from wild strain BC01 through natural selection and ARTP mutagenesis. The specific screening method is as follows: (1) Natural selection A1. Pretreatment of bacterial strains: Pick 10 single colonies of BC01 from the slant culture medium and prepare a bacterial suspension with 0.9% physiological saline. The number of strains is 0.5 × 10⁻⁶. 7 - 0.8×10 7 CFU / mL.

[0033] A2. Preparation of the basic culture medium for the strain: This culture medium consists of 10 g / L glucose, 3 g / L yeast extract, 8 g / L peptone, and 18 g / L agar. After thoroughly mixing all components, the medium is autoclaved and then poured under aseptic conditions to prepare plate screening media. Screening reagents are prepared using perillaldehyde, Tween 80, DMF, Pluronic F127, and PEG4000, respectively, and screening plates are prepared accordingly; the volume concentration of the screening reagent in the basic plate culture medium is 1-10 g / L.

[0034] A3. Strain screening: The bacterial suspension obtained in A1 was mixed with the original solution at a ratio of 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 Dilute the sample to different concentrations, spread it on base plates and screening plates, and incubate it in a 30℃ constant temperature incubator for 48-72 hours to obtain single colony plates.

[0035] A4. Transformation Capacity Test: Single colonies grown on a single-colony plate were picked up using a sterile toothpick and inoculated into 48-well microplates containing 0.5 mL of seed culture medium. The microplates were placed in a shaker at 25-30℃ and incubated at 200 rpm for 24 hours to obtain the seed culture. The viable count of the seed culture was 5 × 10⁻⁶. 7 -2×10 8 CFU / mL; the seed culture was then transferred at a 10% inoculum to a 24-well plate containing 1 mL of fermentation medium and cultured at 25-30°C for 240 h.

[0036] A5. Product Testing: After fermentation, 1 mL of ethyl acetate was added to each well of a 24-well plate for extraction. The mixture was then sonicated at 100 kHz for 30 minutes and centrifuged at 4000 rpm for 10 minutes. The supernatant organic phase was transferred to a 96-well microplate, and potassium permanganate solution was added for color development. The reaction of perillol with potassium permanganate causes the solution to decolorize; the degree of color change can be used to preliminarily determine the product content. Based on this principle, samples with significant color changes were selected for subsequent quantitative analysis to accurately determine the content of perillol and perillyldiol.

[0037] A6. Repeated Experiments: The high-yielding strain obtained from the initial screening was repeatedly tested using steps A1-A5 for verification. Simultaneously, a 10% scale-up culture was performed in 250mL Erlenmeyer flasks for further verification. Sample analysis was performed qualitatively using thin-layer chromatography with ethyl acetate:n-hexane (9:1) as the developing solvent and iodine vapor as the colorimetric reagent. The transformation effect was assessed by observing the color spots of the substrate perillyl alcohol and the product perillyl glycol, and the transformation efficiency was precisely determined using gas chromatography. Each experiment was repeated in triplicate to ensure the reliability and reproducibility of the results. Through repeated verification, the influence of random factors was eliminated, and a stable high-yielding strain with high tolerance to perillyl alcohol, strain BC01, was obtained. The results are shown in Table 1.

[0038] Table 1. Growth of strain BC01 on screening plates

[0039] 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 80 and PEG4000 have little effect on growth. DMSO and DMF have the greatest impact on growth.

[0040] A7. Shake-flask verification of the dominant strain naturally selected in step A6: Experiments were conducted in 250 mL Erlenmeyer flasks, with seed culture medium and fermentation medium dispensed at 25 mL / 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: 20 g / L glucose, 20 g / L glycerol, 2.4 g / L ammonium dihydrogen phosphate, 9.4 g / L disodium hydrogen phosphate, 10 g / L ammonium nitrate, 10 g / L yeast extract, 40 g / L Tween 80, and 10 g / L perillyl alcohol. Qualitative analysis of the fermentation broth samples was performed using thin-layer chromatography with ethyl acetate and n-hexane (9:1 v / v) as the developing solvent and iodine as the colorimetric reagent. The conversion effect can be determined by observing the color intensity of the chromatographs of the substrate perillyl alcohol. Based on the TLC spotting principle, samples with darker chromatographs are selected for gas chromatography detection, and the conversion rate is determined by gas chromatography.

[0041] (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. Strain pretreatment: Wild-type strain BC01 was diluted with sterile physiological saline to prepare a homogeneous bacterial suspension containing 0.5 × 10⁻⁶ strains. 7 - 0.8×10 7 CFU / mL.

[0042] 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.

[0043] 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.

[0044] B4. Transformation Capacity Test: Morphologically sound single colonies were selected from single-colony plates and inoculated into 48-well plates containing 0.5 mL of seed culture medium. The plates were then incubated in a shaker at 25-30℃ and 200 rpm for 24 h to obtain the seed culture. Subsequently, the seed culture was transferred at a 10% inoculum to 24-well plates containing 1 mL of fermentation medium and incubated at 25-30℃ with shaking for 240 h. Temperature, shaking speed, and other parameters were strictly controlled during the incubation process to ensure the stability of the fermentation environment.

[0045] B5. Product detection: Same as step A5 in “(1) Natural screening” above.

[0046] B6. Verification experiment: Repeat steps B1-B5 with the single colony with high content selected in step B5, and finally screen out the dominant bacterial strain BC96.

[0047] B7. Shake-flask fermentation was conducted to verify the superior strains screened in step B6. The concentration of perillaldehyde was 10 g / L during fermentation. The changes in perillaldehyde content in the ARTP-induced mutagenesis-screened strains are shown in Table 2. Table 2 shows that strain BC96 had the lowest perillaldehyde residue and the highest perillaldehyde yield. Therefore, the shake-flask verification results are consistent with the ARTP-induced mutagenesis selection, and the high-yielding strain BC96 was finally obtained.

[0048] Table 2. Changes in the content of perillaldehyde in the fermentation broth of dominant microbial strains. BC16 8.30 0.45 8.75 5.14% BC23 8.72 0.30 9.02 3.33% BC47 6.27 0.15 6.42 2.34% BC55 8.33 1.12 9.45 11.85% BC69 7.66 0.14 7.80 1.79% BC75 6.61 1.95 8.56 22.79% BC76 7.08 1.23 8.31 14.80% BC88 8.64 0.82 9.46 8.67% BC96 5.81 4.17 9.98 41.80% BC120 8.30 0.79 9.09 8.70% BC121 5.71 2.56 8.27 30.96% BC188 7.13 2.47 9.60 25.73% The identification test for strain BC96 is 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 5.0-6.0; (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.

[0049] Gas chromatography was used to analyze the shake-flask fermentation broth of strain BC96. The results are as follows: Figure 1 As shown in the fermentation broth. From... Figure 1 It is clearly visible that the fermentation broth of strain BC96 exhibits a strong peak at 14.074 min, which is essentially the same as the peak time of the standard perillaldehyde (14.095 min). Therefore, it can be concluded that perillaldehyde can be produced using strain BC96. Furthermore, Figure 2 Although a peak of perillaldehyde was observed at 14.147 min (similar to the peak time of perillaldehyde standard at 14.170 min) and a peak of perillyl alcohol was observed at 14.736 min (similar to the peak time of perillyl alcohol standard at 14.760 min), the peaks were weak, with intensities below 10; and Figure 2 and Figure 1 The chromatograms of the standard products are basically similar, which shows that the production of perilla glycol by fermentation using strain BC96 has the advantages of fewer impurities in the fermentation broth and easy separation of the product from the substrate.

[0050] Therefore, based on the above analysis and identification results, the strain BC96 screened in this invention is classified and named... Cryptococcus albidusBC96 was deposited on September 1, 2025 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20251917. III. Optimization of Shake Flask Fermentation Process

[0051] Using perillaldehyde as a substrate and Cryptococcus lightani BC96 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 5.0-6.0. Shake-flask fermentation was carried out at 25-30℃ and 200-250 rpm. The specific process for producing perillaldehyde diol is described below: S1, Strain Activation Stage: The concentration is 0.7 × 10⁻⁶. 7 A CFU / mL suspension of Cryptococcus lighti BC96 was inoculated onto an agar slant and activated at 25°C for 72 h to obtain single colonies. The agar slant consisted of 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, and 20 g / L agar. S2. Primary Seed Culture: Using a sterile inoculation loop, accurately pick one typical single colony from the activated plate and inoculate it into a primary seed bottle containing 25 mL of liquid culture medium. Place the seed bottle in a constant temperature shaker and incubate for 24 h at 25-30℃, 200 rpm, and an amplitude of 50 mm to obtain a concentration of 2×10⁻⁶. 8 The primary seed culture medium consists of CFU / mL of glucose, yeast extract, peptone, disodium hydrogen phosphate, and potassium dihydrogen phosphate. S3. Fermentation process: Before inoculation, add perillyl alcohol as the substrate and Tween-80 as the co-solvent for perillyl alcohol to a 250 mL fermentation shake flask containing fermentation medium, so that the concentration of perillyl alcohol is 40 g / L and the concentration of Tween-80 is 40 g / L; accurately inoculate 2.5 mL of the primary seed liquid at an inoculation ratio of 10% and carry out shake flask fermentation for 72 h, during which the state of fermentation broth is observed regularly; S4. Product detection and analysis: After fermentation, an equal volume of ethyl acetate extraction solvent was added to the fermentation broth, and the mixture was ultrasonically treated in an ultrasonic homogenizer for 30 min to promote product extraction. Then, the mixture was centrifuged at 4000 rpm for 10 min, and the upper organic phase was collected. The target product was quantitatively detected and analyzed using a gas chromatograph. 3.1 Screening of Fermentation Medium Formulation

[0052] 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 includes: 20 g / L glucose, 20 g / L glycerol, 2.4 g / L ammonium dihydrogen phosphate, 9.4 g / L disodium hydrogen phosphate, 10 g / L ammonium nitrate, 10 g / L yeast extract, and 1 mL / L trace elements. During the screening of the fermentation culture medium formulation, the fermentation method followed the aforementioned step S3, and the fermentation conditions were: fermentation temperature 30℃, rotation speed 200 rpm, 50 mm, pH 6, and shake flask volume 25 mL.

[0053] 3.1.1 Single-factor screening The variables were designed as follows: 0-40 g / L glucose, 0-60 g / L glycerol, 0-10 g / L ammonium nitrate, 0-10 g / L yeast extract, 0-10 g / L disodium hydrogen phosphate, 0-10 g / L ammonium dihydrogen phosphate, and 0-2 mL / L trace elements. The results of the single-factor screening are as follows: Figure 3 The content and conversion rate of perillaldehyde are shown.

[0054] from Figure 3 The results show that under optimal conditions, the potency of perillaldehyde is as follows: 20.22 g / L at 20 g / L glucose; 23.65 g / L at 40 g / L glycerol; 16.01 g / L at 7.0 g / L ammonium nitrate; 22.55 g / L at 5 g / L yeast extract; 20.15 g / L at 7 g / L ammonium dihydrogen phosphate; 20.98 g / L at 3 g / L disodium hydrogen phosphate; and 23.56 g / L at 1 mL / L trace element addition.

[0055] 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 seven factors with three levels, L... 18 (3 7 An orthogonal array was used to develop 18 experimental schemes. 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 A glucose 5 20 (best single-factor analysis) 40 B glycerin 5 40 (best single-factor result) 60 C ammonium nitrate 1 7 (Single-factor optimal) 10 D yeast powder 1 5 (Single-factor optimal) 10 E disodium hydrogen phosphate 1 3 (Single-factor optimal) 7 F Ammonium dihydrogen phosphate 1 7 (Single-factor optimal) 10 G Trace elements 0 1 (Single-factor optimal) 2 Table 4 L 18 (3 7 Orthogonal Experimental Design Table 1 5 5 1 1 1 1 0 1.78 2 5 5 7 5 3 7 1 8.73 3 5 5 10 10 10 10 2 9.45 4 5 40 1 5 10 10 1 14.22 5 5 40 7.0 10 1 0 2 13.97 6 5 40 10 1 3 7 0 2.25 7 5 60 1 10 3 10 2 16.90 8 5 60 7.0 1 10 1 0 3.77 9 5 60 10 5 1 7 1 16.85 10 20 5 7.0 10 1 10 1 15.43 11 20 5 10 1 3 1 2 17.39 12 20 5 1 5 10 7 0 2.93 13 20 40 10 1 10 7 1 27.87 14 20 40 1 5 1 10 2 25.44 15 20 40 7.0 10 3 7 0 3.47 16 60 5 10 5 3 1 0 4.38 17 60 40 1 10 3 7 2 23.64 18 60 60 7.0 1 1 10 1 19.65 Therefore, the preferred formulation of the shake flask fermentation medium includes: glucose 5-20 g / L, glycerol 20-60 g / L, ammonium nitrate 3-7 g / L, yeast extract 3-7 g / L, ammonium dihydrogen phosphate 1-10 g / L, disodium hydrogen phosphate 1-10 g / L, and trace elements 0.2-2 mL / L; most preferably, the shake flask fermentation medium includes: 20 g / L glucose, 40 g / L glycerol, 7 g / L ammonium nitrate, 1 g / L yeast extract, 7 g / L ammonium dihydrogen phosphate, 10 g / L disodium hydrogen phosphate, and 1 mL / L trace elements. 3.2 Optimization of fermentation conditions

[0056] Based on the optimal shake-flask fermentation medium obtained from the above orthogonal experiment, a systematic single-factor screening experiment for fermentation conditions was conducted. The specific fermentation method was as described in step S3 above, and the basic fermentation conditions were: fermentation temperature 30℃, rotation speed 200 rpm, 50 mm, pH value 6, and shake flask volume 25 mL (10% v / v).

[0057] The effects of four key parameters on the bioconversion of perillaldehyde were investigated through single-factor experiments: the temperature range was set at 20-45℃ to cover the optimal growth range of microorganisms; the pH range covered different acidity and alkalinity conditions from 3 to 8; the shaking speed was adjusted between 150-250 rpm; and the liquid volume levels ranged from 4% to 20% v / v (10-50 mL / 250 mL). Detailed data on the yield and conversion efficiency of perillaldehyde under different fermentation conditions can be found in [link to relevant data]. Figure 4 As shown.

[0058] from Figure 4It can be seen that the culture temperature of strain BC96 should be maintained within a suitable range of 25-28℃, the pH value should be stably controlled in a weakly acidic environment of 5.0-6.0, and the stirring speed should be maintained at 200-250 rpm to ensure sufficient dissolved oxygen. When culturing in shake flasks, the liquid volume should be controlled within the range of 8%-10% v / v (20-25 mL / 250 mL) to ensure good aeration. In this way, the conversion rate of perillaldehyde after fermentation culture of strain BC96 can reach more than 96%. Furthermore, using 40 g / L perillyl alcohol as the substrate concentration, the optimal parameters and fermentation conditions for single-factor screening in shake-flask fermentation yielded the following optimal potency of perillyl alcohol: 24.12 g / L at 25℃; 21.77 g / L at 200 rpm; 21.87 g / L at an optimized liquid volume of 10% v / v; and 16.98 g / L at pH 6.0. These data provide important reference for subsequent process scale-up. The single-colony stability of strain BC96 under optimal fermentation medium and conditions with 40 g / L perillyl alcohol is shown in Table 5.

[0059] Table 5. Changes in the content of perillaldehyde in single colony screening. Single colony 1 0.88 29.23 30.11 97.08% Single colony 2 0.33 30.17 30.50 98.92% Single colony 3 0.21 28.76 28.97 99.28% Single colony 4 0.34 29.22 29.56 98.85% Single colony 5 0.56 29.50 30.06 98.14% Single colony 6 0.38 29.51 29.89 98.73% Single colony 7 0.44 28.99 29.43 98.50% Single colony 8 0.76 30.22 30.98 97.55% Single colony 9 0.42 28.22 28.64 98.53% Single colony 10 0.11 30.15 30.26 99.64% Table 5 shows that the conversion rate error among the individual colonies of strain BC96 is small, and it has good conversion ability for perillaldehyde. Therefore, strain BC96 has good stability and can maintain stable genetic characteristics and physiological activity during continuous subculturing, laying a solid foundation for subsequent industrial-scale production.

[0060] In summary, this invention systematically isolates and collects microbial samples from soils containing various plants of the genus *Perilla*, and through multiple rounds of natural screening combined with advanced ambient pressure room temperature plasma (ARTP) mutagenesis technology, ultimately selects the highest-performing *Cryptococcus lightans*. Cryptococcus albidusThe mutant strain BC96, 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 BC96 strain provided by this invention in the production of perillaldehyde diol, the conversion rate of perillaldehyde can reach over 90%, with few impurities in the fermentation broth, easy separation and purification of perillaldehyde diol, and 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 diol. 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 diol, and providing a new path for the green preparation of perillaldehyde diol.

[0061] 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 lighthani BC96, classified and named as follows: Cryptococcus albidus BC96, accession number CCTCC NO: M20251917, accession date September 1, 2025, deposited by China Center for Type Culture Collection.

2. A method for obtaining the Cryptococcus lighthanii strain BC96 as described in claim 1, comprising: First, wild-type Cryptococcus lightensis was isolated from the soil in which Perilla frutescens was planted. Then, the wild-type Cryptococcus lightensis was subjected to natural selection and ARTP mutagenesis to obtain a microbial strain with an ITS sequence as shown in SEQ ID NO.1 that can convert perillyl alcohol to perillyl diol.

3. The use of the Cryptococcus cephalosporin strain BC96 as described in claim 1 in the production of perillaldehyde.

4. A fermentation medium for the Cryptococcus lightanis strain BC96 according to claim 1, characterized in that, include: 0-40 g / L glucose, 20-60 g / L glycerol, 1-10 g / L ammonium nitrate, 0-3 g / L yeast extract, 1-10 g / L diammonium hydrogen phosphate, 1-10 g / L sodium dihydrogen phosphate, 0-2 mL / L trace elements.

5. A method for producing perillaldehyde, comprising: Using perillyl alcohol as a substrate and Cryptococcus baiji strain BC96 as the chassis cell as described in claim 1, perillyl alcohol was produced by fermentation culture at 25-30℃.

6. The production method according to claim 5, characterized in that, include: The seed culture of *Cryptococcus faecium* strain BC96 was inoculated into the fermentation medium at an inoculum rate of 8-12%, followed by the addition of 10-40 g / L perillyl alcohol and 20-60 g / L Tween 80. The pH was adjusted to 5.0-6.0, and fermentation was carried out at a fermentation temperature of 25-30℃ and a fermentation speed of 200-250 rpm to obtain a fermentation broth producing perillyl glycol. The fermentation medium consisted of: 0-40 g / L glucose, 20-60 g / L glycerol, 1-10 g / L ammonium nitrate, 0-3 g / L yeast extract, 1-10 g / L diammonium hydrogen phosphate, 1-10 g / L sodium dihydrogen phosphate, and 0-2 mL / L trace elements. The viable count of the *Cryptococcus faecium* strain BC96 seed culture was 5 × 10⁻⁶. 7 -2×10 8 CFU / mL.

7. The production method according to claim 6, characterized in that, The fermentation temperature is 25-28℃.

8. The production method according to claim 6 or 7, characterized in that, The liquid volume of the fermentation vessel during the fermentation process is 8%-10% v / v.

9. The production method according to claim 8, characterized in that, The method for preparing the seed culture of Cryptococcus BC96 strain includes: inoculating a single colony of activated Cryptococcus BC96 strain into a seed culture medium and culturing it at 25-30℃ and 200-250 rpm to obtain the seed culture of Cryptococcus BC96 strain; wherein the seed culture medium comprises: 10-20 g / L glucose, 3-10 g / L yeast extract, 8-20 g / L peptone, 1-5 g / L ammonium dihydrogen phosphate and 5-10 g / L dipotassium hydrogen phosphate.

10. The production method according to claim 9, characterized in that, The method for culturing the activated Cryptococcus lightiformis strain BC96 single colonies includes: inoculating a suspension of Cryptococcus lightiformis strain BC96 onto an agar slant culture medium and activating it in a constant temperature incubator at 25℃-30℃ to obtain the activated Cryptococcus lightiformis strain BC96 single colonies; 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 lightiformis strain BC96 suspension is 0.5 × 10⁻⁶. 7 -0.8×10 7 CFU / mL.

Images