Cryptococcus albidus and application of cryptococcus albidus in synthesis of norambrox ether

Abstract

The invention discloses cryptococcus albidus and application of the cryptococcus albidus in synthesis of norambrox ether, and belongs to the technical field of bioengineering. The preservation number of the cryptococcus albidus JMIC881 is CCTCC (China Center for Type Culture Collection) NO: M 20251916, the preservation date is September 1, 2025, and the preservation unit is the China Center for Type Culture Collection. The strain JMIC881 is separated from clary sage flowers, and is a microbial strain obtained by natural breeding and ARTP mutation breeding of wild cryptococcus light white yeast. The invention also provides a method for producing norambrox ether by using sclareol as a substrate through fermentation by using the strain JMIC881, and the method has the advantages of mild reaction conditions, no chemical pollutant emission, high product selectivity, low energy consumption and the like, solves the environmental problem caused by the traditional chemical synthesis method, reduces the equipment cost and energy consumption of industrial production, and is suitable for industrial production. And a new path is provided for green preparation of the ambrogyl ether.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a Cryptococcus albidus and application thereof in synthesis of ambrox. BACKGROUND

[0002] Ambrox (chemical name: (-)-ambrox, molecular formula: C 16 H 28 O) is a sesquiterpenoid compound with extremely persistent, warm, animalistic and woody-succinic aroma. As the most core fragrance component of natural ambergris, ambrox is an indispensable top-grade fixative and aroma booster in the modern high-end perfume industry, which can significantly improve the persistence, level and overall aroma of perfume. Due to the strict restrictions on its natural source (sperm whale digestive material) and the involvement of ethical and protection issues, the market demand for synthetic ambrox is growing.

[0003] The preparation of ambrox has long relied on two paths: one is to extract natural products from sperm whale excretion, but due to the protection of animal regulations, the yield can only meet less than 5% of market demand; the other is chemical synthesis method, taking sclareol as raw material, through strong acid catalysis cyclization, dehydration reaction to generate target product, but there are the following key problems: 1) Environmental risk: liquid acid catalyst (such as sulfuric acid, trifluoroacetic acid) will corrode equipment, produce a large amount of acid wastewater, and the treatment cost accounts for 15%-20% of the production cost; 2) Process limitations: the reaction requires high temperature and high pressure conditions of 120-150℃, the energy consumption accounts for more than 60% of the total energy consumption, and there are many side reactions, so the product purity needs to be refined several times to reach more than 95%; 3) Poor sustainability: the chemical synthesis process cannot realize the recycling of raw materials, which is contrary to the current green manufacturing concept.

[0004] In recent years, biotransformation technology has been concerned due to its mildness and specificity. In existing research, there are two major bottlenecks in microbial catalysis of sclareol conversion: one is the lack of high-efficiency catalytic strains, and the conversion rate of most microorganisms to sclareol is less than 60%; the second is the low solubility of the substrate (solubility in water <0.5g / L), which limits the substrate concentration in the fermentation system and reduces the efficiency of industrial production. Therefore, screening high-activity strains and optimizing the fermentation system to improve the solubility and conversion efficiency of the substrate have become the core breakthrough direction of the biological method for preparing ambrox, and it is of great significance to develop a green and efficient biotransformation method.

[0005] Microbial transformation has become a research hotspot in recent years due to its advantages such as mild reaction conditions, environmental friendliness, and high selectivity. Currently, the main method for obtaining sclareol is extraction from sclareol. However, the complex composition of the plant makes separation and purification difficult, and its cultivation is affected by geographical and climatic factors, which restricts the stable supply of sclareol. Constructing microbial cell factories, by introducing heterologous terpene synthesis pathways and modifying endogenous metabolic pathways, holds promise for achieving efficient synthesis of high-value terpene compounds. Existing literature reports the production of sclareol using metabolically engineered yeast or Escherichia coli. The team led by Zhou Yongjin at the Dalian Institute of Chemical Physics developed a fermentation-based sclareol technology, using Saccharomyces cerevisiae as a cell factory and systematically modifying its central metabolism to achieve efficient synthesis of sclareol from glucose, with a yield of 11.4 g / L (Xuan Cao. et al., Engineering yeast for high-level production of diterpenoidsclareol, Metabolic Engineering, Volume 75, Januray 2023, pages 19-28). The fermentation method for synthesizing perillyl alcohol has the advantages of simple production process, short cycle, environmental friendliness and independence from land and climate factors; however, it still requires perillyl alcohol as a reactant to obtain ambroxol through chemical conversion, and it is difficult to directly synthesize ambroxol by microorganisms. Summary of the Invention

[0006] The main objective of this invention is to provide a Cryptococcus faecium strain and its application in the synthesis of ambroxol. This strain can produce an enzyme that catalyzes the conversion of perillol into ambroxol. Therefore, the production of ambroxol from perillol can be achieved by using this Cryptococcus faecium strain.

[0007] 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 JMIC881, accession number CCTCC NO: M 20251916, accession date September 1, 2025, deposited at the China Center for Type Culture Collection.

[0008] The wild type Cryptococcus albidus strain JMIC881 is obtained by the following method: first, the wild type Cryptococcus albidus strain capable of converting sclareol to ambrox is isolated from sclareol flowers; then, the wild type Cryptococcus albidus strain is subjected to natural selection and ARTP mutagenic selection to obtain a microbial strain capable of converting sclareol to ambrox with high yield, and the ITS sequence of the microbial strain is shown in SEQ ID NO. 1. The strain is named Cryptococcus albidus JMIC881, which grows well in YPD medium, and the strain is observed to have an oval, amastigote morphology with a diameter of 0.5-1 μm, which is consistent with the morphology of Saccharomycetes. The colony is round, smooth, white, and has a moist surface, which is a gram-positive yeast. The optimal growth temperature of the strain JMIC881 is 25-30 ℃, and the optimal pH is 4.0-6.5. The strain JMIC881 can produce enzymes capable of catalyzing sclareol and converting sclareol to ambrox. The entire conversion process of ambrox does not require the addition of exogenous chemical catalysts, but relies on microbial metabolism to achieve directional conversion, so that ambrox can be synthesized by microbial fermentation using sclareol as a substrate.

[0009] In a second aspect, the present application provides an application of the Cryptococcus albidus strain JMIC881 in synthesis of ambrox.

[0010] In a third aspect, the present application provides a fermentation medium of the Cryptococcus albidus strain JMIC881, which comprises the following raw materials: 20-60 g / L glycerol, 5-20 g / L yeast powder, 5-30 g / L peptone, 5-30 g / L ammonium sulfate, 1-3 g / L disodium hydrogen phosphate, 5-10 g / L potassium dihydrogen phosphate, and 0-2 mL / L trace elements, wherein the trace elements comprise 0.8-1.2 g / L magnesium sulfate, 0.8-1.2 g / L biotin, 0.8-1.2 g / L vitamin B1, 0.04-0.06 g / L nicotinic acid, and 8-12 g / L ferrous sulfate; so as to further catalyze sclareol to produce ambrox.

[0011] Further, the fermentation medium comprises the following raw materials: 40 g / L glycerol, 10 g / L yeast powder, 15 g / L peptone, 15 g / L ammonium sulfate, 1 g / L disodium hydrogen phosphate, 6 g / L potassium dihydrogen phosphate, and 2 mL / L trace elements, wherein the trace elements consist of 1 g / L magnesium sulfate, 1 g / L biotin, 1 g / L vitamin B1, 0.05 g / L nicotinic acid, and 10 g / L ferrous sulfate; in this way, the conversion rate of sclareol is improved to be more than 80%.

[0012] In a fourth aspect, the present application provides a fermentation production method of ambrein, comprising: taking sclareol as a substrate, Cryptococcus albidus JMIC881 as a chassis cell, and performing fermentation culture at 25-30 DEG C to produce ambrein.

[0013] To improve the conversion rate of ambrein, the fermentation production method comprises: inoculating the seed liquid of Cryptococcus albidus JMIC881 into a fermentation medium at an inoculation amount of 8-12%, then adding 5-20 g / L of sclareol, 10%-30% of n-dodecane and 0.5-2 mL / L of IPTG, adjusting the pH to 4.0-6.5, and performing fermentation culture at a temperature of 25-30 DEG C and a rotation speed of 150-250 rpm to produce ambrein. The fermentation medium comprises 20-60 g / L of glycerol, 5-20 g / L of yeast powder, 5-30 g / L of peptone, 5-30 g / L of ammonium sulfate, 1-3 g / L of disodium hydrogen phosphate, 5-10 g / L of potassium dihydrogen phosphate and 0-2 mL / L of trace elements, wherein the trace elements comprise 0.8-1.2 g / L of magnesium sulfate, 0.8-1.2 g / L of biotin, 0.8-1.2 g / L of vitamin B1, 0.04-0.06 g / L of nicotinic acid and 8-12 g / L of ferrous sulfate. The viable cell count of the seed liquid of Cryptococcus albidus JMIC881 is 5x10 7 -2x10 8 CFU / mL.

[0014] In the fermentation culture process, the fermentation production system has a pH of 5.5-6.0, a rotation speed of 200-250 rpm and a liquid volume of 10-25 mL / 250 mL.

[0015] To further improve the activity of the seed liquid of Cryptococcus albidus JMIC881, the preparation method of the seed liquid of Cryptococcus albidus JMIC881 comprises: inoculating the activated single colony of Cryptococcus albidus JMIC881 into a seed culture medium, and culturing at 25-30 DEG C and a rotation speed of 200-300 rpm to obtain the seed liquid of Cryptococcus albidus JMIC881. The seed culture medium comprises 10-20 g / L of glucose, 3-10 g / L of yeast powder, 8-20 g / L of peptone, 1-3 g / L of disodium hydrogen phosphate and 5-9 g / L of potassium dihydrogen phosphate.

[0016] Further improve the activity of the activated Cryptococcus albidus JMIC881 strain, the preparation method of the activated Cryptococcus albidus JMIC881 strain comprises the following steps: inoculating Cryptococcus albidus JMIC881 suspension into a slant culture medium, and culturing in a constant temperature incubator at 25-30 DEG C to obtain the activated Cryptococcus albidus JMIC881 single colony; wherein the slant culture medium comprises 10-20 g / L glucose, 3-10 g / L yeast powder, 8-20 g / L peptone and 15-25 g / L agar, and the viable count of the Cryptococcus albidus JMIC881 suspension is 0.5*10 7 CFU / ml-0.8*10 7 CFU / mL.

[0017] Therefore, the Cryptococcus albidus JMIC881 provided by the application can produce enzymes that can catalyze sclareol and convert sclareol into ambrox, so that the biosynthesis of ambrox is realized. In addition, it is found through experiments that the fermentation broth of the strain JMIC881 as the bottom plate fungus has less impurities when using sclareol as the substrate, and the product is easy to separate from the substrate; and the conversion efficiency of the strain to sclareol is high, and the conversion rate can be more than 80%.

[0018] Further, the fermentation production method of the above ambrox and various culture media provided by the application can realize high-efficiency conversion of sclareol as the substrate, for example, 10 g / L of sclareol substrate can be converted into 6.64 g / L of ambrox after 72 h of shake flask culture, and the conversion rate is 83.31%.

[0019] Further, the fermentation production method for synthesizing ambrox by using the strain JMIC881 provided by the application has the advantages of mild fermentation conditions (normal temperature and pressure), renewable source, no need to use toxic reagents, no chemical pollutant emission, environmental friendliness, high product selectivity, low energy consumption and the like, and is suitable for industrial production; at the same time, the environmental problems caused by traditional chemical synthesis method are solved, the equipment cost and energy consumption of industrial production are reduced, and a new path for green preparation of ambrox is provided. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is the morphology of Cryptococcus albidus JMIC881 on the plate and the microscope examination diagram; Figure 2 It is the gas phase detection spectrum of the fermentation broth of the strain JMIC881; Figure 3 It is the content and conversion rate change curve of ambrox in the single factor screening test of the fermentation culture medium of the strain JMIC881; Figure 4The content of ambrox and the conversion rate change curve in the single-factor screening test of the shake flask fermentation conditions of the strain JMIC881. DETAILED DESCRIPTION

[0021] In order to make the personnel in the technical field better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor should belong to the protection scope of the present application.

[0022] The main purpose of the present application is to provide a high-yield strain with catalytic production of ambrox from sclareol. (Cryptococcus albidus ) JMIC01, through natural selection and ARTP iterative mutagenesis, to obtain a Cryptococcus albidus strain Cryptococcus albidus ) JMIC881, the preservation number of which is CCTCC M20251916, the preservation date of which is September 1, 2025, and the preservation unit of which is the China Center for Type Culture Collection.

[0023] Another main purpose of the present application is to provide the application of the above-mentioned Cryptococcus albidus strain Cryptococcus albidus ) JMIC881 in the synthesis of ambrox. The application further includes optimization of the fermentation medium and fermentation conditions to further improve the conversion rate of sclareol.

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

[0025] In the above test schemes, the trace element formula in the culture medium is as follows: 1 g / L magnesium sulfate, 1 g / L biotin, 1 g / L vitamin B1, 0.05 g / L nicotinic acid, and 10 g / L ferrous sulfate.

[0026] The gas chromatograph analysis method and conditions of the present application are as follows: the detection instrument is Agilent 8860 gas chromatograph-hydrogen flame ionization detector, the carrier gas is high-purity nitrogen (99.999%), the chromatographic column is HP-5 (30 m x 0.25 mm x 0.25 μm), the injection port temperature is 200℃, the detector temperature is 280℃, the column temperature rising program is: the initial temperature is 120℃, maintained for 1 min, increased to 250℃ at 20℃ / min, maintained for 2 min, the carrier gas flow rate is 1.0 mL / min, the injection amount is 1.0 μL, and the split ratio is 10:1. It is detected that the peak time of the sclareol standard is 8.511 min, and the peak time of the ambrox standard is 7.043 min, as shown in Figure 2 (above).

[0027] I. Wild-type Cryptococcus albidus (Cryptococcus albidus ) JMIC01 The specific steps for obtaining the wild-type strain JMIC01 are as follows: S1, select a Salvia farinacea flower from Yili Kazakh Autonomous Prefecture of Xinjiang Uygur Autonomous Region, grind to powder, take the juice and dilute and coat with YPD solid culture medium, and incubate at 25-30°C constant temperature to grow single colonies; S2, inoculation: inoculate the single colonies cultured in step S1 into YPD broth medium and incubate at 30°C constant temperature shaker for 24-48 h; S3, fermentation: inoculate the strain cultured in step S2 into fermentation medium at a inoculation amount of 10%, add 10 g / L salviol to the medium, incubate at 30°C and 200 rpm for 120 h, wherein the fermentation medium comprises 40 g / L glycerol, 10 g / L yeast powder, 10 g / L peptone, 10 g / L ammonium sulfate, 3 g / L disodium hydrogen phosphate, 9 g / L potassium dihydrogen phosphate, 2 mL / L trace elements, 20% n-dodecane and 1 mL / L IPTG; S4, detection: soak the fermentation broth obtained in step S3 in equal volume of ethyl acetate for 10 min, ultrasonic for 30 min, take the upper phase and centrifuge at 12000 rpm for 1 min, analyze by gas chromatograph, and finally obtain the strain JMIC01 with catalytic production of ambrethiol from salviol; S5, JMIC01 strain identification: (1) colony morphology observation: the colony is round, smooth, white, and the surface texture is moist; (2) use gram staining to make a film, and observe under objective 100x, diameter, 0.5-1 μm, and the morphology is oval, non-flagellated, consistent with the morphology of yeast; (3) the optimum growth temperature is 25-30°C, and the optimum pH is 4.0-6.5.

[0028] II. Cryptococcus albidus (Cryptococcus albidus ) JMIC881 Wild-type Cryptococcus albidus Cryptococcus albidus ) JMIC881 is mainly obtained by natural selection and ARTP mutagenesis selection of wild-type strain JMIC01, and the specific screening method is as follows: (1) natural selection A1, strain pretreatment: pick 10 JMIC01 single colonies from the slope medium, prepare a bacterial suspension with 0.9% normal saline, and the strain number is 0.5×10 7 - 0.8×10 7CFU / mL.

[0029] A2, strain basic medium preparation: 10 g / L glucose, 3 g / L yeast powder, 8 g / L peptone, 18 g / L agar are prepared to prepare the basic flat plate culture medium, and the screening agent is prepared by using vetivazulene, Tween 80, DMF, Pluronic F 127, PEG4000 respectively and the screening plate is prepared; wherein the volume concentration of the screening agent in the basic flat plate culture medium is 5-20 g / L.

[0030] A3, strain screening: the bacterial suspension obtained in A1 is diluted according to the stock solution, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Different dilutions are diluted, coated on the basic flat plate and the screening plate, and placed in a 30°C constant temperature incubator for culture for 48-72h to obtain a single colony plate; A4, strain transformation ability test: the single colony on the single colony plate is inoculated into a 48-well plate seed culture medium, and is shaken in a 25°C shaking bottle cabinet for 24h to obtain a single colony seed liquid; then the single colony seed liquid is inoculated into a 24-well plate fermentation culture medium, and is shaken in a 25-30°C shaking bottle cabinet for 240h to obtain a single colony fermentation product; the liquid volume of the 48-well plate is 0.5mL, and the liquid volume of the 24-well plate is 1mL.

[0031] A5, product detection: 1mL of ethyl acetate is added to each well of the 24-well plate, and is ultrasonically treated at a frequency of 100kHz for 30min, and is centrifuged, and is spotted on the plate by using a spotting needle array, and according to the principle of TLC spotting, the strains with deep color are selected for gas phase quantitative detection of ambrox content.

[0032] A6, repeated test: the single colony with high content selected in step A5 is repeated 2-3 times of steps A1-A5 to obtain natural breeding to improve the tolerance of JMIC01 to vetivazulene, and the results are shown in Table 1.

[0033] Table 1 JMIC01 screening plate growth

[0034] It can be seen from Table 1 that the concentration of vetivazulene affects the growth of the strain, and through iterative screening, a strain tolerant to high concentration of vetivazulene can be obtained; Tween, PEG4000 and Pluronic F 127 basically do not affect the growth; DMF has the greatest influence on the growth.

[0035] A7. Shake flask verification of the dominant strain selected by natural selection in step A6: a 250 mL triangular flask is used for the experiment, and the seed culture medium and fermentation culture medium are loaded with 25 mL / flask; the culture conditions are consistent with those of the microplate culture. The seed culture medium comprises 15 g / L glucose, 6.5 g / L yeast extract powder, 14 g / L peptone, 7 g / L potassium dihydrogen phosphate, and 2 g / L sodium hydrogen phosphate. The fermentation culture medium comprises 40 g / L glycerol, 10 g / L yeast powder, 10 g / L peptone, 10 g / L ammonium sulfate, 3 g / L sodium hydrogen phosphate, 9 g / L potassium dihydrogen phosphate, 2 mL / L trace elements, 20% n-dodecane, 1 mL / L IPTG, and 10 g / L bomeol. Fermentation broth samples can be qualitatively detected by thin layer chromatography, using ethyl acetate and n-hexane in a volume ratio of 9:1 as the developing agent, and iodine coloration. The conversion effect can be determined by observing the color intensity of the color group of the substrate bomeol. According to the TLC point plate principle, samples with deep color groups are selected for gas phase detection to determine the conversion rate.

[0036] (2) ARTP mutagenesis selection After the dominant strain selected by natural selection is subjected to ARTP (Atmospheric and Room Temperature Plasma) mutagenesis, preliminary screening is performed through serial plates, and then high-throughput testing and screening of the mutant library are performed through a Tecan Fluent 1080 automatic platform and an Agilent RapidFire 400. The specific method of ARTP mutagenesis selection is as follows: B1. Strain pretreatment: wild-type strain JMIC01 is prepared into a bacterial suspension with physiological saline, and the strain number is 0.5×10 7 - 0.8×10 7 CFU / mL.

[0037] B2. Preparation of strain screening medium: 10 g / L glucose, 3 g / L yeast powder, 8 g / L peptone, 5 g / L bomeol, and 18 g / L agar are used to prepare a screening plate medium.

[0038] B3. Strain screening: the bacterial suspension is subjected to ARTP mutagenesis for 0 s, 30 s, 60 s, 90 s, 120 s, 150 s, and 200 s, and is diluted according to the original solution, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 different dilutions, spread on the screening plate, and placed in a 25-30°C incubator for 48-72 h to obtain a single colony plate.

[0039] B4, strain transformation ability test: single colony on single colony plate was inoculated into 48-well plate seed culture medium, and was shaken in a 25°C shaker bottle cabinet for 24h to obtain seed liquid; then the single colony seed liquid was inoculated into 24-well plate fermentation medium, and was shaken in a 25-30°C shaker bottle cabinet for 240h to obtain single colony fermentation product; the liquid volume of 48-well plate was 0.5mL, and the liquid volume of 24-well plate was 1mL.

[0040] B5, product detection: 1mL ethyl acetate was added to each well of 24-well plate, and was ultrasonicated at 100kHz for 30min, and then was centrifuged, and then was spotted on TLC plate by using a spotting needle array, and then strains with deep color were selected for gas phase quantitative detection of ambrox content.

[0041] B6, repeated test: the single colony with high content selected in step B5 was repeated steps B1-B5, and finally the dominant strain JMIC881 was screened out.

[0042] B7, the dominant strain screened in step B6 was verified by shake flask fermentation, and the concentration of sclavatol in fermentation was 10g / L, and the change of ambrox content of the strain screened by ARTP mutagenesis was as shown in Table 2. Table 2 shows that the strain JMIC881 has the least sclavatol residue and the highest ambrox yield, so the shake flask verification result is consistent with the ARTP mutagenesis breeding, and finally the high-yield strain JMIC881 is obtained.

[0043] Table 2 Change of sclavatol residue and ambrox content in fermentation product of dominant strain

[0044] Strain JMIC881 identification: (1) colony morphology observation: the colony was round, smooth, white, and the surface texture was moist; (2) microscope morphology observation as shown in Figure 1 , the slide was prepared by using gram staining, and was observed under an objective lens of 100x, the diameter was 0.5-1μm, and the morphology was oval and non-flagellated, which was consistent with the morphology of yeast; (3) the optimum growth temperature was 25-30°C, and the optimum pH was 4.0-6.5; (4) the universal primers 27F and 1492R were used for PCR amplification and sequencing identification of fungal properties, and the ITS sequence of the fungus was as shown in SEQ ID NO.1.

[0045] The shake flask fermentation liquid of the strain JMIC881 was detected by gas chromatography, and the detection result was as shown in Figure 2 (lower). Figure 2It is clearly visible that the fermentation broth of strain JMIC881 exhibits a strong peak at 7.039 min, which is essentially the same as the peak time of the standard ambroxol (7.043 min). Therefore, it can be concluded that ambroxol can be produced using strain JMIC881. Furthermore, Figure 2 (Below) Except for the peak of perillaldehyde at 8.497 min (which is basically the same as the peak time of perillaldehyde standard (8.511 min)), it is similar to... Figure 2 The spectra (above) are basically the same, with virtually no other impurities. This indicates that the fermentation production of ambroxol using strain JMIC881 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. Finally, the product was cooled and crystallized to obtain a pure ambroxol with a content of 99%.

[0046] Therefore, based on the above analysis and identification results, the strain JMIC881 screened in this invention is classified and named *Cryptococcus faecium*. Cryptococcus albidus) The accession number, JMIC881, 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 20251916.

[0047] III. Optimization of Shake Flask Fermentation Process Using perillyl alcohol as a substrate and Cryptococcus lightaniae JMIC881 as chassis cells, the chassis cells were inoculated onto the fermentation medium at an inoculum size of 8%-9%, and the pH of the fermentation culture was adjusted to 4.0-6.5. Shake-flask fermentation was carried out at 25-30℃ and 150-250 rpm. The specific process for producing ambroxol 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 JMIC881 was inoculated onto the slant culture medium and activated in an incubator at 28°C for 56 h to obtain single colonies; wherein the slant culture medium comprises: 15 g / L glucose, 6.5 g / L yeast extract, 14 g / L peptone, and 20 g / L agar. S2, Primary Seed Culture: Select 3 single colonies and place them 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 h to obtain a concentration of 2×10⁻⁶. 8The seed culture medium comprises 15 g / L glucose, 6.5 g / L yeast powder, 14 g / L peptone, 2 g / L sodium phosphate dibasic, 7 g / L potassium dihydrogen phosphate; S3, shake flask fermentation: adding sclerol powder to the fermentation medium before inoculation, inoculating 20 mL of the first-stage seed liquid of the Cryptococcus albidus JMIC881 into a 250 mL fermentation shake flask at a 10% inoculation amount, and performing constant-temperature fermentation culture; S4, detection: adding an equal volume of ethyl acetate, ultrasonic treatment for 30 min, centrifuging to obtain an organic phase, and performing gas chromatography detection.

[0048] Fermentation medium formula screening A large number of relevant information is consulted to verify the shake flask fermentation of the formula containing a carbon source, a nitrogen source, a phosphate, an inorganic salt and trace elements.

[0049] The basic formula F is 40 g / L glycerol, 10 g / L yeast powder, 10 g / L peptone, 10 g / L ammonium sulfate, 6.78 g / L sodium phosphate dibasic, 3 g / L potassium dihydrogen phosphate, and 1 mL / L trace elements; wherein the trace element formula is 1 g / L magnesium sulfate, 1 g / L biotin, 1 g / L vitamin B1, 0.05 g / L nicotinic acid, and 10 g / L ferrous sulfate. Moreover, 20% n-dodecane and 1 mL / L IPTG are added to each screening formula of the basic formula F.

[0050] (1) Single-factor screening Referring to step S3, the basic formula F screened is subjected to single-factor screening under the conditions of an initial concentration of the fermentation substrate sclerol of 10 g / L, a fermentation temperature of 30°C, a rotation speed of 200 rpm, a 50 mm, a pH value of 6 and a fermentation time of 72 h. The variable design is 0-60 g / L glycerol, 0-20 g / L yeast powder, 0-20 g / L peptone, 0-20 g / L ammonium sulfate, 0-10 g / L sodium phosphate dibasic, 0-15 g / L potassium dihydrogen phosphate, and 0-5 mL / L trace elements. The content of ambrethether and the conversion rate in single-factor screening are shown in Table 1. Figure 3 .

[0051] From Figure 3It can be seen that: under the optimal conditions of each factor, the ambrin titer is 6.05 g / L when the concentration of glycerol is 40 g / L, the ambrin titer is 5.54 g / L when the concentration of yeast powder is 5 g / L, the ambrin titer is 6.55 g / L when the concentration of protein peptone is 10 g / L, the ambrin titer is 6.01 g / L when the concentration of ammonium sulfate is 15 g / L, the ambrin titer is 6.12 g / L when the concentration of potassium dihydrogen phosphate is 9 g / L, the ambrin titer is 5.87 g / L when the concentration of sodium phosphate dibasic is 3 g / L, and the ambrin titer is 6.56 g / L when the concentration of trace elements is 2 g / L.

[0052] (2) Optimal concentration range combination experiment In order to optimize the combined effect of multiple factors, the ambrin titer was used as the evaluation index, and the orthogonal experiment design was used to screen the optimal nutritional formula. Each factor was set to 3 levels (low concentration, medium concentration / optimal concentration of single factor, high concentration), and the levels were set as shown in Table 3. For the 7 factors with 3 levels, L 18 (3 7 ) orthogonal table was used to develop 18 experimental schemes. At the same time, 20% n-dodecane, 1 mL / L IPTG and 10 g / L basilthujone were added to each formula. The specific scheme is shown in Table 4: Table 3 Orthogonal experiment factor and level table

[0053] Table 4 L 18 (3 7 ) orthogonal experiment scheme table

[0054] As can be seen, the optimal formula of the shake flask fermentation medium includes: 20-60 g / L glycerol, 5-20 g / L yeast powder, 5-30 g / L protein peptone, 5-30 g / L ammonium sulfate, 1-3 g / L sodium phosphate dibasic, 5-10 g / L potassium dihydrogen phosphate, 0-2 mL / L trace elements, and 5-20 g / L basilthujone. Most preferably, the shake flask fermentation medium includes: 40 g / L glycerol, 10 g / L yeast powder, 15 g / L protein peptone, 15 g / L ammonium sulfate, 1 g / L sodium phosphate dibasic, 6 g / L potassium dihydrogen phosphate, and 2 mL / L trace elements.

[0055] 3.2 Fermentation conditions The fermentation of strain JMIC881 requires high dissolved oxygen. On the basis of the optimal shake flask fermentation medium in the above orthogonal experiment, single-factor screening was performed on the fermentation conditions. The temperature concentration range is 20-45℃, the pH concentration range is 3-8, and the rotation speed is 150-250 rpm.

[0056] Single-factor screening was performed using fermentation with a substrate of perillyl alcohol at 10 g / L, and 20% n-dodecane and 1 mL / L IPTG were added to the formulation; ambroxol content and conversion rate are shown in [reference needed]. Figure 4 .

[0057] from Figure 4 It can be seen that the optimal control range for shake flask fermentation parameters is: fermentation temperature 25-30 ℃, pH 5.5-6.0, and rotation speed 200-250 rpm. The optimal parameters and ambroxol titers obtained through single-factor screening for shake flask fermentation are as follows: ambroxol titer 6.37 g / L at 30 ℃; 6.57 g / L at 220 rpm; and 3.82 g / L at pH 6.5.

[0058] Table 5 shows the single-colony stability of strain JMIC881 under optimal fermentation formulation and conditions, with the addition of 10 g / L perillyl alcohol, 20% n-dodecane, and 1 mL / L IPTG. Table 5 indicates that the conversion rate error among the single colonies of strain JMIC881 is small, demonstrating good conversion ability for perillyl alcohol. Therefore, strain JMIC881 exhibits good stability.

[0059] Table 5. Changes in ambroxol content during single colony screening

[0060] In summary, the Cryptococcus lighthani strain provided by this invention ( Cryptococcus albidus The JMIC881 microbial strain, possessing specific catalytic activity, achieves biotransformation reactions using perillyl alcohol as a substrate in an optimized fermentation system. The JMIC881 strain provided by this invention offers advantages in the production of ambroxol, including mild fermentation conditions (ambient temperature and pressure), no chemical pollutant emissions, high product selectivity, and low energy consumption. It solves the environmental problems associated with traditional chemical synthesis methods while reducing equipment costs and energy consumption for industrial production, providing a new pathway for the green preparation of ambroxol.

[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 palea ( Cryptococcus albidus JMIC881, characterized in that, The accession number is CCTCC NO: M 20251916, the accession date is September 1, 2025, and the depositary institution is the China Center for Type Culture Collection.

2. The Cryptococcus lightanis strain according to claim 1 ( Cryptococcus albidus JMIC881, characterized in that, The strain was obtained primarily through the following methods: first, wild-type Cryptococcus syringae was isolated from Perilla frutescens flowers to obtain a strain capable of converting perilla frutescens alcohol into ambroxol; then, the wild-type Cryptococcus syringae was subjected to natural selection and ARTP mutagenesis to obtain a microbial strain capable of converting perilla frutescens alcohol into high-yield ambroxol, with an ITS sequence as shown in SEQ ID NO.

1.

3. A Cryptococcus lighthanii strain according to claim 1 or 2 ( Cryptococcus albidus Application of JMIC881 in the synthesis of ambroxol.

4. A Cryptococcus lightans strain as described in claim 1 or 2 ( Cryptococcus albidus The fermentation medium of JMIC881 is characterized by, The product comprises the following ingredients: 20-60 g / L glycerol, 5-20 g / L yeast extract, 5-30 g / L peptone, 5-30 g / L ammonium sulfate, 1-3 g / L disodium hydrogen phosphate, 5-10 g / L potassium dihydrogen phosphate, and 0-2 mL / L trace elements, wherein the trace elements include: 0.8-1.2 g / L magnesium sulfate, 0.8-1.2 g / L biotin, 0.8-1.2 g / L vitamin B1, 0.04-0.06 g / L niacin, and 8-12 g / L ferrous sulfate.

5. The fermentation medium according to claim 4, characterized in that, Including the following raw materials: The formula contains 40 g / L glycerol, 10 g / L yeast extract, 15 g / L peptone, 15 g / L ammonium sulfate, 1 g / L disodium hydrogen phosphate, 6 g / L potassium dihydrogen phosphate, and 2 mL / L trace elements, wherein the trace elements 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.

6. A fermentation method for producing ambroxol, comprising: Using perillyl alcohol as a substrate, the Cryptococcus lighthani strain as described in claim 1 or 2 ( Cryptococcus albidus JMIC881 is a chassis cell that is fermented at 25-30℃ to produce ambroxol.

7. The fermentation production method according to claim 6, characterized in that, include: Cryptococcus lightenicus ( Cryptococcus albidus The JMIC881 seed culture was inoculated into the fermentation medium at an inoculum size of 8%-12%, followed by the addition of 5-20 g / L perillyl alcohol, 10%-30% n-dodecane, and 0.5-2 mL / L IPTG. The pH was adjusted to 4.0-6.5, and fermentation was carried out at a fermentation temperature of 25-30℃ and a fermentation speed of 150-250 rpm to produce ambroxol; wherein, the *Cryptococcus faecium* strain (… Cryptococcus albidus The viable count of JMIC881 seed culture was 5 × 10⁻⁶. 7 -2×10 8 The fermentation medium, at CFU / mL, comprises: 20-60 g / L glycerol, 5-20 g / L yeast extract, 5-30 g / L peptone, 5-30 g / L ammonium sulfate, 1-3 g / L disodium hydrogen phosphate, 5-10 g / L potassium dihydrogen phosphate, and 0-2 mL / L trace elements, wherein the trace elements include: 0.8-1.2 g / L magnesium sulfate, 0.8-1.2 g / L biotin, 0.8-1.2 g / L vitamin B1, 0.04-0.06 g / L niacin, and 8-12 g / L ferrous sulfate.

8. The fermentation production method according to claim 7, characterized in that, During the fermentation process, the pH was 5.5-6.0, the rotation speed was 200-250 rpm, and the volume of liquid in the shake flask was 10-25 mL / 250 mL.

9. The fermentation production method according to claim 7 or 8, characterized in that, The method for preparing the *Cryptococcus lightlyi* JMIC881 seed culture includes: inoculating a single colony of activated *Cryptococcus lightlyi* JMIC881 into a seed culture medium and culturing it at 25-30℃ and 200-300 rpm to obtain the *Cryptococcus lightlyi* JMIC881 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 fermentation production method according to claim 9, characterized in that, The method for preparing the activated Cryptococcus lightensis JMIC881 strain includes: inoculating a suspension of Cryptococcus lightensis JMIC881 onto a slant culture medium and activating it in a constant temperature incubator at 25℃-30℃ to obtain single colonies of the activated Cryptococcus lightensis JMIC881; 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 viable count of the Cryptococcus lightensis JMIC881 suspension is 0.5 × 10⁻⁶. 7 - 0.8×10 7 CFU / mL.

Images