Yarrowia lipolytica with high yield of gamma-decalactone and / or delta-decalactone and application thereof

Abstract

The application discloses a Yarrowia lipolytica strain with high yield of gamma-decalactone and / or delta-decalactone and application thereof, and belongs to the technical field of biotechnology. The name of the strain is Yarrowia lipolytica Y-1, and the preservation number is GDMCC No: 67810. The strain Y-1 can be directly used for biological transformation to cooperatively and efficiently prepare gamma-decalactone and delta-decalactone with castor oil acid as a substrate. The strain has three outstanding advantages: ① yield advantage; ② tolerance advantage; and ③ application advantage. The biological transformation method has the advantages of mild reaction condition, low cost, product meeting natural perfume standards, environmental friendliness and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a strain of Yersinia lipolytica that produces high levels of γ-decanolide and / or δ-decanolide and its applications. Background Technology

[0002] γ-decalactone (GDL) and δ-decalactone (DDL) are high-value flavor compounds with rich peach and creamy / coconut aromas, respectively. Besides being naturally found in various fruits and fermented foods, they are widely used as important components in food flavorings and daily-use fragrances. Although chemical synthesis can achieve large-scale production, the resulting products are racemic mixtures, with aroma quality differing from the natural configuration, and do not conform to international and Chinese regulations that define "flavors obtained through microbial fermentation" as natural flavorings. Therefore, utilizing microbial cell factories to transform natural substrates such as ricinoleic acid into biosynthetic lactones has become a major development direction in the industry due to the "natural" properties of the products, mild reaction conditions, and environmental friendliness.

[0003] In the biosynthetic pathway, microorganisms, represented by *Yarrowia lipolytica*, can lactone-form γ-decanolide and δ-decanolide by performing a specific number of β-oxidations on their substrates. However, when this process is pushed towards industrial production, it has long faced a core bottleneck that is difficult to overcome: existing production strains cannot simultaneously achieve high yield and high environmental tolerance. Although the reported high-yielding strains have yields close to the industrial threshold, they generally suffer from narrow environmental adaptability, being extremely sensitive to fluctuations in fermentation conditions such as temperature and pH, and unable to maintain stable high yields in complex industrial environments; while strains with better tolerance produce lactone yields far below the industrial economic threshold. The contradiction between high yield and high tolerance has become a core technological barrier in the industry.

[0004] To address these challenges, traditional strain improvement methods each have their limitations. While genetic engineering has a clear objective, the constructed genetically engineered bacteria, especially those carrying exogenous genes, pose biosafety and environmental release risks, their application is subject to strict regulations, and their industrialization prospects are unclear. Traditional single random mutagenesis, on the other hand, suffers from uncontrollable mutation sites, low efficiency, and difficulty in systematically modifying complex traits regulated by multiple genes. Currently, there are numerous reports on the biological production of γ-decanolide, with the highest publicly disclosed yield reaching 35.2 g / L (fermenter level, patent CN117229931 A, strain Y. lipolytica OMK-95). However, existing production strains, including this high-yielding strain, generally suffer from the industry-wide problem of limited environmental adaptability. They have limited tolerance to fluctuations in the fermentation environment and are easily affected by slight deviations in temperature and pH from the optimal range, impacting cell growth and yield, and increasing the risk of contamination by other microorganisms. Therefore, this study adopted a strategy of targeted breeding combined with strain mutagenesis to select high-yield mutant strains that can overcome the bottlenecks in lactone tolerance and synthesis efficiency. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a strain of Yersinia lipolytica that produces high levels of γ-decanolide and / or δ-decanolide and its applications.

[0006] This invention addresses the growing market demand for natural flavorings and the key technical bottlenecks in existing biological production processes of γ-decanolide and δ-decanolide, such as low product concentration and lack of high-performance production strains. It provides a high-yield and highly tolerant mutant strain of Yersinia lipolytica, offering a more stable strain guarantee for industrial production.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A high-yielding strain of Yarrowia lipolytica, named Yarrowia lipolytica Y-1, was obtained by using Yarrowia lipolytica Po1f as the starting strain. Through multiple rounds of directional domestication in a medium containing γ-decanolide, an intermediate strain with high tolerance to the product was obtained. Subsequently, a high-yielding strain was obtained by combined mutagenesis screening with ultraviolet (UVB), nitrosoguanidine (NTG), and diethyl sulfate (DES). The high-yielding strain was then screened for acid resistance and high temperature resistance to finally obtain the target strain.

[0009] Preservation information of Yarrowia lipolytica Y-1: Depository institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC), deposit date: February 4, 2026, deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, deposit number: GDMCC No: 67810.

[0010] The application of the Yersinia lipolyticis Y-1 in high-yield production of γ-decanolide and / or δ-decanolide.

[0011] The application of the Yersinia lipolyticis Y-1 in the construction of recombinant engineered bacteria that produce high levels of γ-decanolide and / or δ-decanolide.

[0012] A method for preparing natural γ-decanolide and / or δ-decanolide by biotransformation of the aforementioned Yersinia lipolytica Y-1 includes the following steps: bio-fermentation culture of the aforementioned Yersinia lipolytica Y-1 to obtain γ-decanolide and / or δ-decanolide.

[0013] Furthermore, the culture medium used for bio-fermentation is a culture medium containing ricinoleic acid and / or castor oil; preferably, it is a culture medium containing ricinoleic acid, wherein the concentration of ricinoleic acid is 10-60 g / L.

[0014] Furthermore, the culture medium used for bio-fermentation includes: MgSO4·7H2O: 5-10 g / L, K2HPO4: 5-10 g / L, Tween-80: 1-3 g / L, ricinoleic acid: 10-60 g / L, pH 4.0-9.0.

[0015] Furthermore, the culture medium used for bio-fermentation includes: MgSO4·7H2O: 5-10 g / L, K2HPO4: 5-10 g / L, Tween-80: 1-3 g / L, ricinoleic acid: 20-40 g / L, pH 5.0-7.0.

[0016] Furthermore, the conditions for the biological fermentation culture are 20-37℃ and 100-300 rpm for 24-90 h.

[0017] Furthermore, the conditions for the biological fermentation culture are 25-35℃ and 150-250 rpm for 24-90 h.

[0018] As a preferred embodiment of the present invention, ricinoleic acid can be added to the fermentation system once or multiple times during the bio-fermentation process; preferably, ricinoleic acid is added when the fermentation has proceeded for 50-60 h and / or 80-90 h; more preferably, after adding ricinoleic acid, its target concentration in the fermentation system is maintained at 10-60 g / L.

[0019] The present invention has the following advantages and effects compared with the prior art:

[0020] The *Yarrowia lipolytica* Y-1 strain provided by this invention can be directly used in biotransformation to synergistically and efficiently prepare γ-decanolide and δ-decanolide using ricinoleic acid as a substrate. This strain has three major advantages: ① Yield advantage: γ-decanolide yield reaches 38.2 g / L at the fermenter level, and δ-decanolide yield simultaneously reaches 7.6 g / L, achieving high yield of both products synergistically; ② Tolerance advantage: Directed domestication establishes acid and heat tolerance, which is further enhanced by subsequent mutagenesis. It can stably grow and produce lactones within a temperature range of 25-37℃ and a pH range of 4.0-7.0, and exhibits good tolerance to high concentrations of ricinoleic acid (≤60 g / L) and lactone products (≤40 g / L) in the fermentation system, significantly reducing product feedback inhibition and environmental stress; ③ Application advantage: The fermentation process is stable, with low risk of contamination by other microorganisms, making it more suitable for industrial production. This method has advantages such as mild reaction conditions, low cost, products meeting natural fragrance standards, and environmental friendliness. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the adaptive evolution culture of Yersinia lipophila GDL00.

[0022] Figure 2 This is a graph showing the γ-decanolide production of the Yersinia lipolytica GDL00 mutant strain.

[0023] Figure 3 This is a graph showing the yield of γ-decanolide in the retested Yersinia lipolytica GDL00 mutant strain.

[0024] Figure 4 This is a graph showing the yields of γ-decanolide and δ-decanolide in Yersinia lipophila GDL54 after retesting.

[0025] Figure 5 These are growth diagrams of Yersinia lipophila GDL253 and GDL00 under different temperature conditions.

[0026] Figure 6 This is a graph showing the yield of Yeast Extract GDL253 and GDL00 under different temperature conditions.

[0027] Figure 7 These are growth diagrams of Yersinia lipophila GDL253 and GDL00 under different pH conditions.

[0028] Figure 8 This is a graph showing the yields of Yersinia lipophila GDL253 and GDL00 under different pH conditions. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0030] The *Yarrowia lipolytica* Po1f used in the examples is disclosed in the literature “MADZAK C, GAILLARDIN C, BECKERICH J M. Heterologous protein expression and secretion in the non-conventional yeast *Yarrowia lipolytica*: a review [J]. Journal of Biotechnology, 2004, 109(1 / 2):63-81.”; in this invention, this strain is named *Yarrowia lipolytica* GDL00.

[0031] Example 1: Acclimation culture of Yersinia lipophila strain to γ-decanolide substrate

[0032] This embodiment aims to construct a high-yield strain library by using γ-decanolide production as the core screening indicator. Although the target strains need to have the ability to synergistically produce γ-decanolide and δ-decanolide, since their metabolic pathways are homologous and γ-decanolide is the main target product, the screening is based on γ-decanolide production.

[0033] 1. Fermentation culture of Yersinia lipophila GDL00

[0034] The key to obtaining a *Yarrowia lipolytica* strain with high production capacity of γ-decanolide and δ-decanolide lies in adaptive subculturing and domestication culture in a fermentation medium containing γ-decanolide, followed by screening for gene mutant strains with high γ-decanolide production capacity. The specific implementation is as follows:

[0035] The slant nutrient agar medium contains: 20 g / L peptone, 20 g / L glucose, 10 g / L yeast extract, 20 g / L agar, pH 7.0.

[0036] The liquid seed culture medium contains: 20 g / L peptone, 20 g / L glucose, 10 g / L yeast extract, pH 6.5.

[0037] The biotransformation medium contains: MgSO4·7H2O: 7 g / L, K2HPO4: 7 g / L, Tween-80: 2 g / L, ricinoleic acid 20 g / L, pH 5.0-9.0.

[0038] The biotransformation medium containing γ-decanolide is a medium obtained by adding different amounts of γ-decanolide to the biotransformation medium.

[0039] After the starting strain Yarrowia lipolytica GDL00 was plated, a single colony was picked and inoculated into a seed bottle containing 10 mL of YPD medium. The culture was then shaken at 30℃ and 250 r / min for 12-24 h to obtain the seed culture solution.

[0040] 2. Adaptive evolution culture of Yersinia lipophila GDL00

[0041] The seed culture medium was subjected to multiple induction cultures, as detailed below:

[0042] The seed culture was inoculated at a rate of 5% (v / v) into a biotransformation medium containing 0.01% γ-decanolide (v / v), and the first induction culture was performed at 30°C and 250 rpm. When the OD of the culture medium... 600 When the concentration reaches 10 or higher, the culture is stopped, and the fermentation broth is obtained; this broth is the first induction fermentation broth. The fermentation broth is then inoculated at a rate of 5% (volume percentage) into a biotransformation medium containing 0.02% γ-decanolide (volume percentage), and induction culture is performed at 30°C and 250 rpm. If the inoculation to OD rate is completed within 24 hours... 600 If the process reaches a value of 10 or higher, then the fermentation broth is considered the second induced fermentation broth.

[0043] The second induced fermentation broth was inoculated at a rate of 5% (volume percentage) into a biotransformation medium containing 0.03% γ-decanolide (volume percentage), and a third induced culture was performed at 30°C and 250 rpm. When the fermentation broth OD... 600 When the concentration reaches 10 or higher, the culture is stopped, and the fermentation broth is obtained. This fermentation broth is the third induction fermentation broth. The fermentation broth is then inoculated at a rate of 5% (volume percentage) into a biotransformation medium containing 0.04% γ-decanolide (volume percentage), and induction culture is performed at 30℃ and 250 rpm. If the OD (Organic Derivative Oxidation) is completed within 24 hours... 600 If the process reaches a value of 10 or higher, then the culture medium is considered the fourth induced fermentation broth.

[0044] This process is repeated, with each round of targeted acclimatization involving increasing the concentration of γ-decanolide by 0.01% (volume percentage) compared to the previous round, until the concentration of γ-decanolide in the culture medium reaches 0.05% (volume percentage). The acclimatized bacterial culture is then obtained, and the adaptive acclimatization is as follows: Figure 1 As shown.

[0045] Dilute the acclimatized bacterial culture by 10%. 7 The diluted bacterial solution was spread onto a plate medium containing 0.05% γ-decanolide and incubated at 30°C until a single clone grew, thus obtaining a library of domesticated strains.

[0046] 3. Detection of γ-decanolide

[0047] One hundred domesticated strains were selected from the domesticated strain bank and inoculated into seed bottles containing fermentation medium. After culturing at 30℃ and 250 rpm for 1 day, they were inoculated into biotransformation medium at an inoculum rate of 5% and fermented at 30℃ and 250 rpm for 2 days to obtain fermentation broth. The broth was centrifuged at 8000×g for 5 min, and 100 samples of fermentation supernatant from the domesticated strains were collected.

[0048] Using Yeast Extract GDL00 as a control, fermentation was carried out in the same manner, and the supernatant of GDL00 fermentation was collected.

[0049] 300 μL of the upper organic phase was taken from the fermentation system and placed in a 1.5 mL centrifuge tube. An equal volume of ethyl acetate and a small amount of anhydrous sodium sulfate were added as a desiccant. The mixture was thoroughly shaken and centrifuged at 10,000 rpm for 1 min. The liquid in the 1 mL syringe centrifuge tube was filtered through a 0.22 μm nylon filter into a chromatographic vial for gas chromatography analysis.

[0050] The GC chromatographic conditions were as follows: Agilent HP-5 column, injection port temperature 250℃; detector temperature 285℃; injection volume 0.2 μL; constant flow rate injection, nitrogen as carrier gas, split ratio 1:20; programmed temperature ramping: 120℃ held for 2 min, ramped to 205℃ at 30℃ / min, ramped to 215℃ at 4℃ / min, ramped to 280℃ at 35℃ / min, and equilibrated for 2 min.

[0051] The measurement results are as follows Figure 2As shown, the yield of γ-decanolide in the fermentation supernatant of *Yersinia lipolytica* GDL00 (670.26 mg / L supernatant) was used as the baseline. Strains with yields above this baseline were considered positive mutant strains, while those below were considered negative mutant strains. The results showed that among the 100 domesticated strains, 79 were positive mutants. Of these, 57 strains had γ-decanolide yields more than 1.5 times that of *Yersinia lipolytica* GDL00, 4 strains had yields more than 2 times that of GDL00, and the highest yield was 3.49 times that of GDL00. These results indicate that the mutant strain library constructed using the stress-directed domestication method of this invention has a high positive mutation rate and can improve screening efficiency.

[0052] 4. Identification

[0053] Of the 79 positive mutant domesticated strains obtained from the initial screening, strains 25, 54, 67, 70, 83, and 97, which had high γ-decanolide content in their fermentation broths, were retested using GC, with the detection method being the same as the GC detection in step 3 above. The results are as follows: Figure 3 As shown, the γ-decyl lactone contents of the positive mutant domesticated strains No. 25, 54, 67, 70, 83 and 97 were 1714.2 mg / L, 2121.1 mg / L, 1790.9 mg / L, 1808.1 mg / L, 1340.7 mg / L and 1349.8 mg / L, respectively, all of which were higher than the γ-decyl lactone content in the fermentation broth of Yersinia lipolytica GDL00.

[0054] To verify the ability of the aforementioned high-yielding strains to synergistically synthesize δ-decanolide, the δ-decanolide content in the same batch of fermentation samples was detected. Qualitative and quantitative analyses were performed using δ-decanolide standards. The results showed that all mutant strains could simultaneously synthesize δ-decanolide, and its yield was positively correlated with γ-decanolide yield. Among them, strain 54 (later named GDL54) achieved the highest γ-decanolide yield of 2121.4 mg / L, and its δ-decanolide yield also reached the highest level of 586.4 mg / L. Figure 4 Further verification of the acid and heat resistance characteristics of the six high-yielding strains (No. 25, No. 54, etc.) obtained from screening was conducted: the strains were inoculated into acidic medium at pH 4.0 and high-temperature medium at 37℃ and cultured for 24 h, respectively, and the OD was measured. 600 Value. The results showed that the OD values ​​of the six high-yielding domesticated strains under the above-mentioned stress conditions were... 600 All values ​​were ≥6.0, and the cells could still grow normally. Among them, strain GDL54 showed the best performance, with OD values ​​under acidic conditions. 600 =11.2, OD under high temperature conditions 600 =10.7, proving that the strain obtained through targeted domestication is not only high-yielding, but also has significant acid and high-temperature resistance, laying an environmental adaptability foundation for subsequent mutation breeding and industrial application.

[0055] Example 2: Ultraviolet (UVB) mutagenesis screening of Yarrowia lipolytica strain

[0056] In this embodiment, the culture medium formulation and screening method described in Example 1 were used to further subject the high-yielding strain obtained through directional domestication to ultraviolet (UVB) mutagenesis treatment in order to select mutant strains with better γ-decanolide synthesis ability.

[0057] 1. Preparation of GDL54 strain for mutagenesis

[0058] The GDL54 domesticated strain obtained in Example 1 was inoculated onto a fresh nutrient agar slant and incubated statically at 30°C for 32 hours. Subsequently, a single colony was picked and inoculated into a shake flask containing 10 mL of seed culture medium and cultured with shaking at 30°C and 250 rpm until mid-log growth, which served as the starting seed solution for UV mutagenesis.

[0059] 2. Ultraviolet mutagenesis treatment

[0060] Take 0.5 mL of logarithmic-phase bacterial culture and serially dilute it with sterile deionized water. Spread 200 µL of the appropriately diluted culture onto nutrient agar plates. Place the plates under a 20 W UV lamp at a distance of 20 cm and irradiate for 1 minute and 2 minutes respectively. Immediately after mutagenesis treatment, wrap the plates with aluminum foil and incubate in the dark at 30°C for 3–4 days.

[0061] 3. Screening and identification of mutant strains

[0062] After clear single colonies appeared on the plates, a total of 60 single colonies were picked from the different irradiation time treatment groups for initial screening. The initial screening method was the same as in Example 1, using shake-flask fermentation and GC detection to screen for mutant strains with increased γ-decanolide production. After the initial screening, the 5 mutant strains with the highest yield were selected for the first round of secondary screening, with 3 parallel shake-flask experiments for each strain.

[0063] 4. Screening Results

[0064] After multiple rounds of screening, a UV mutant strain with the highest γ-decanolide yield was obtained and named GDL137. The yield of γ-decanolide in its shake-flask fermentation broth reached 2621.1 mg / L, and the yield of δ-decanolide was 620.5 mg / L, which was a further improvement over the mutagenic starting strain.

[0065] Example 3: Screening for mutagenesis of Yersinia lipophilia using nitrosoguanidine (NTG)

[0066] This embodiment uses the same culture medium formulation and screening method as Example 1, and takes the high-yielding strain obtained in Example 2 as the starting strain, and further uses NTG for chemical mutagenesis.

[0067] 1. Preparation of the starting strain for mutagenesis

[0068] The strain GDL137 obtained in Example 2 was inoculated onto fresh nutrient agar slant and incubated statically at 30°C for 32 h. An appropriate amount of bacterial cells was transferred to a shake flask containing 10 mL of seed culture medium and cultured with shaking at 30°C and 250 rpm until mid-log growth, which served as the starting seed solution for NTG mutagenesis.

[0069] 2. NTG mutagenesis treatment

[0070] Collect the above seed culture by centrifugation at 5000 rpm for 5 min, and wash twice with pH 7.0 phosphate buffer. Then, resuspend the cells in 20 mL of the same buffer, transfer them to a light-protected glass Erlenmeyer flask, and shake to fully disperse the cells.

[0071] Add 0.5 mL of NTG solution to the bacterial suspension to achieve a final concentration of 0.25 mg / mL, and treat with constant temperature shaking at 30℃ and 250 rpm for 30 min. After the reaction is complete, immediately add 0.1 mL of the treated solution to 100 mL of sterile physiological saline to terminate the reaction.

[0072] 3. Isolation and initial screening of mutant strains

[0073] The terminated bacterial culture was serially diluted and spread onto nutrient agar plates, then incubated at 30°C in the dark for 3–4 days. After colonies grew, 60 single colonies were selected for initial screening via shake-flask fermentation.

[0074] 4. Screening and validation of high-yielding strains

[0075] After initial screening, strains with high γ-decanolide production were selected for secondary screening, using the same method as in Example 2. A UV mutant strain with the highest γ-decanolide production was obtained, named GDL216, whose shake-flask fermentation broth yielded 2843.7 mg / L of γ-decanolide and 634.1 mg / L of δ-decanolide.

[0076] Example 4: Screening of Yersinia lipolyticis by diethyl sulfate (DES) mutagenesis

[0077] This embodiment uses the same culture medium and screening process as in Example 2. The high-yielding strain obtained in Example 3 is used as the starting strain, and DES is used for chemical mutagenesis to select mutant strains with further enhanced γ-decanolide synthesis ability.

[0078] 1. Preparation of the starting strain for mutagenesis

[0079] The strain GDL216 obtained in Example 3 was inoculated onto fresh nutrient agar slant and incubated statically at 30°C for 32 h. An appropriate amount of bacterial cells was transferred to a shake flask containing 10 mL of seed culture medium and cultured with shaking at 30°C and 250 rpm until mid-log growth, which served as the starting seed solution for DES mutagenesis.

[0080] 2. DES-induced mutagenesis treatment

[0081] Collect the above seed culture, centrifuge at 5000 rpm for 5 min to collect the bacterial cells, and wash twice with pH 7.0 phosphate buffer. Resuspend the bacterial cells with an appropriate amount of the same buffer, and add DES solution to make a final concentration of 2% (v / v). Incubate at 30°C with shaking for 30 min.

[0082] After the treatment was completed, 1 mL of the treated solution was added to 0.5 mL of 25% sodium thiosulfate (Na2S2O3) solution to terminate the reaction.

[0083] 3. Isolation and screening of mutant strains

[0084] The bacterial suspension after the reaction was terminated was serially diluted and spread onto nutrient agar plates, and incubated at 30°C in the dark for 3–4 days. After colony formation, a total of 60 single colonies were selected for initial screening via shake-flask fermentation.

[0085] 4. Screening and identification of high-yielding strains

[0086] After initial screening, strains with high γ-decanolide production were selected for secondary screening, using the same method as in Example 2. A mutant strain with the highest γ-decanolide production was obtained, named GDL253, with a γ-decanolide concentration of 3013.6 mg / L and a δ-decanolide production of 652.3 mg / L in its shake-flask fermentation broth.

[0087] Example 5: Efficient synthesis of γ-decanolide and δ-decanolide by mutant strain GDL253 under fed-batch fermentation mode

[0088] This embodiment relates to a method for preparing γ-decanolide and δ-decanolide by biotransformation of ricinoleic acid using the mutant strain GDL253 obtained in Example 4 above.

[0089] The culture medium formulation used in this embodiment is as follows:

[0090] The slant agar medium consisted of 20 g / L peptone, 20 g / L glucose, 10 g / L yeast extract, and 20 g / L agar. The initial pH of the medium was 7.0, and it was sterilized at 115°C for 20 min.

[0091] The liquid seed culture medium consisted of 20 g / L peptone, 20 g / L glucose, and 10 g / L yeast extract. The initial pH of the medium was 6.5, and it was sterilized at 115°C for 20 min.

[0092] The biotransformation medium consisted of: MgSO4·7H2O: 7 g / L, K2HPO4: 7 g / L, Tween-80: 2 g / L, and ricinoleic acid: 20 g / L. The initial pH of the medium was 7.0, and it was sterilized at 115℃ for 20 min.

[0093] The fermentation system used in this embodiment is 100 mL, and the fermentation flask is a 500 mL Erlenmeyer flask; the detailed steps involved in its preparation are as follows:

[0094] (1) Slant culture: The mutant strain GDL253 was inoculated onto nutrient agar medium and cultured statically at 30℃ for 24 h;

[0095] (2) Preparation of seed culture: Using the slant culture obtained in step (1), pick a single colony and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL of liquid seed culture medium. Incubate at 30°C and shake at 250 rpm for 24 h to obtain a mature seed culture.

[0096] (3) Biotransformation: Using the seed culture medium obtained in step (2), transfer it into a 500 mL Erlenmeyer flask containing 100 mL of biotransformation medium at an inoculation rate of 5% (v / v). Under the condition of 30℃, shake at 250 rpm for 24 h. During the transformation process, add 40 g / L of ricinoleic acid and transform for 48 h.

[0097] (4) Detection of γ-decyl lactone and δ-decyl lactone concentrations: After extraction with analytical grade butyl acetate, the concentrations of γ-decyl lactone and δ-decyl lactone were detected by gas chromatography. The final concentration of γ-decyl lactone reached 7054.2 mg / L, and the yield of δ-decyl lactone was 1402.5 mg / L.

[0098] Example 6: Screening and verification experiments on the acid and high temperature resistance of mutant strain GDL253

[0099] This embodiment focuses on screening and verifying the acid and high-temperature resistance of the high-yielding mutant strain GDL253 obtained by composite mutagenesis. The specific steps are as follows:

[0100] (1) Temperature tolerance test: GDL253 and control strain GDL00 were inoculated into liquid seed culture medium and cultured at 250 rpm for 48 h at 25℃, 30℃, 35℃, 37℃ and 40℃ respectively. OD was measured. 600 Values ​​and γ-decyl lactone yields, results are as follows Figure 5-6 As shown. GDL253 has an OD value in the range of 25-35℃. 600 The values ​​were all above 10.4, and the yield of γ-decanolide remained stable above 5.8 g / L; at 37℃, both growth and yield decreased significantly, and OD... 600 The OD value dropped to 6.5, and the yield dropped to 3.5 g / L; it barely grew at 40℃, and no effective yield was detected; the control strain GDL00 had an OD value of 30℃. 600 The control strain showed a growth rate of 12.9, indicating good growth but a yield of only 0.7 g / L. Growth was poor at both below 30℃ and above 30℃, with a yield of only 0.5 g / L. No growth was observed at 37℃ and above. This demonstrates that GDL253 has a wider temperature tolerance range, and its high-temperature tolerance and high-yield stability are significantly superior to the control strain.

[0101] (2) pH tolerance test: GDL253 and control strain GDL00 were inoculated into liquid seed culture medium at pH 3.0, 4.0, 5.0, 6.0 and 7.0, respectively, and cultured at 30℃ and 250 rpm for 48 h with shaking. OD was then measured. 600 Values ​​and γ-decyl lactone yields, results are as follows Figure 7-8 As shown, GDL253 can grow normally within the pH range of 5.0-7.0, with a stable γ-decanolide yield of no less than 5.8 g / L; while the control strain GDL00 only showed an OD of [missing value] under optimal conditions of pH 6.0-7.0. 600 The pH is approximately 11.0, with a yield of only 0.7 g / L, and the OD value is below pH 5.0. 600 When the pH value dropped below 5.9, the yield was less than 0.4 g / L. This indicates that GDL253 has stronger acid tolerance and better fermentation stability.

[0102] Example 7: Experiment on high-yield production of γ-decanolide and δ-decanolide by mutant strain GDL253 in a fermenter

[0103] To verify the industrial application potential of the mutant strain GDL253, this embodiment uses a 50 L fermenter with a 30 L fermentation system for scale-up experiments. The specific steps are as follows:

[0104] (1) Seed liquid preparation: Same as steps (1)-(2) in Example 5, to obtain mature seed liquid;

[0105] (2) Preparation of fermentation tank culture medium: MgSO4·7H2O: 7 g / L, K2HPO4: 7 g / L, Tween-80: 2 g / L, ricinoleic acid 20 g / L, pH 7.0, sterilized at 115℃ for 20 min;

[0106] (3) Inoculation and fermentation: The seed liquid was inoculated into the fermenter at a rate of 5% (v / v). The fermentation parameters were set as follows: temperature 30℃, stirring speed 300-500 rpm, aeration rate 1.5-2.0 vvm, and fermentation cycle 90 h.

[0107] (4) Feeding strategy: ricinoleic acid was added at 55 h and 85 h of fermentation, respectively, and the amount added each time was such that the final concentration of ricinoleic acid in the system reached 40 g / L;

[0108] (5) Product detection: Samples were taken every 12 hours, and the concentrations of γ-decanolide and δ-decanolide were detected using the GC method described in Example 1. At 72 hours of fermentation, the yield of γ-decanolide reached a peak of 38.2 g / L, while the yield of δ-decanolide simultaneously reached 7.6 g / L and remained stable until the end of fermentation. Based on the product yield, γ-decanolide accounted for 83.4% of the total product, and δ-decanolide accounted for 16.6%. This indicates that the ratio of γ-decanolide to δ-decanolide in the total product was approximately 5:1, demonstrating that the mutant strain GDL253 can efficiently convert ricinoleic acid into the target lactone product.

[0109] In this invention, the mutant strain GDL253 was renamed Yarrowia lipolytica Y-1. The preservation information of Yarrowia lipolytica Y-1 is as follows: depositary institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC), deposit date: February 4, 2026, deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, deposit number: GDMCC No: 67810.

[0110] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of *Yersinia lipolytica* that produces high levels of γ-decanolide and δ-decanolide, characterized by: The name of the *Yersinia lipolytica* is *Yersinia lipolytica* (… Yarrowia lipolytica Y-1 was deposited on February 4, 2026 at the Guangdong Provincial Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, accession number: GDMCC No: 67810.

2. The application of the *Yersinia lipolytica* as described in claim 1 in the high production of γ-decanolide and δ-decanolide.

3. The application of the *Yersinia lipophila* strain according to claim 1 in the construction of recombinant engineered bacteria that produce high levels of γ-decanolide and δ-decanolide.

4. A method for preparing natural γ-decanolide and δ-decanolide by biotransformation of *Yarrowia lipolytica* as described in claim 1, characterized in that, The process includes the following steps: using the *Yersinia lipolytica* yeast of claim 1 for bio-fermentation to obtain γ-decanolide and δ-decanolide.

5. The method according to claim 4, characterized in that: The culture medium used for bio-fermentation is a medium containing ricinoleic acid and / or castor oil; The concentration of ricinoleic acid is 10–60 g / L.

6. The method according to claim 4, characterized in that: The culture medium used for bio-fermentation includes: MgSO4·7H2O: 5-10 g / L, K2HPO4: 5-10 g / L, Tween-80: 1-3 g / L, ricinoleic acid: 10-60 g / L, pH 4.0-9.0; And / or, the conditions for the biological fermentation culture are 20-37℃, 100-300 rpm for 24-90 h.

7. The method according to claim 6, characterized in that: The culture medium used for bio-fermentation includes: MgSO4·7H2O: 5-10 g / L, K2HPO4: 5-10 g / L, Tween-80: 1-3 g / L, ricinoleic acid: 20-40 g / L, pH 5.0-7.0; And / or, the conditions for the biological fermentation culture are 25-35℃, 150-250 rpm for 24-90 h.

8. The method according to claim 4, characterized in that: During the bio-fermentation process, ricinoleic acid is added to the fermentation system once or multiple times.

9. The method according to claim 8, characterized in that: Add ricinoleic acid when fermentation reaches 50-60 h and 80-90 h.

10. The method according to claim 8 or 9, characterized in that: After adding ricinoleic acid, its target concentration in the fermentation system is maintained at 10-60 g / L.

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