High-yield D-arabitol strain and mutation breeding method thereof

Abstract

The invention discloses a high-yield D-arabitol strain and a mutation breeding method thereof. The bacterial strain is Wickerhamomyces anomalus A37, and is preserved in the Guangdong Microbial Culture Collection Center (GDMCC), and the preservation number is GDMCC 65731. The invention further discloses a preparation method of the Wickerhamomyces anomalus strain. The strain is obtained through improvement of a multi-stage mutagenesis technology, low-energy N + injection mutagenesis and DES chemical mutagenesis are adopted, high-throughput screening based on an INT color development method and an HPLC secondary screening technology are combined, and the screening efficiency is remarkably improved. After fermentation condition optimization, the yield of D-arabitol can reach 113.63 g / L after the mutant strain A37 is fermented for 156 h under the condition of 350 g / L glucose substrate, and the conversion rate is 32.47%. Physical and chemical mutagenesis technologies are superposed and applied to genetic improvement of Wickersenula anomala for the first time, a foundation is laid for efficient production of arabitol with glucose as a raw material, and the method is suitable for large-scale application in the fields of food, medicine and chemical engineering.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a strain with high yield of D-arabitol and a method for its mutagenesis breeding. Background Art

[0002] D-Arabitol is a pentitol (C 5 H 12 O 5 ), and due to its low calorie (0.2 kcal / g), non-cariogenic, insulin-independent, and slow metabolism characteristics in the human body, it is widely used in the fields of food, medicine, and chemical industry. As one of the 12 high-value bio-based chemicals recognized by the US Department of Energy, its potential for industrial production is significant. Compared with traditional sweeteners, D-arabitol has unique physical and chemical advantages: it does not contain a carbonyl structure, avoiding the Maillard reaction and having excellent stability in high-temperature, acidic, or alkaline environments; at the same time, its low glycemic index makes it an ideal sugar substitute candidate for diabetic patients. In addition, D-arabitol is a key precursor for the synthesis of high-value chiral compounds such as arabic acid and xylitol, and plays an important role in the development of pharmaceutical intermediates and green chemicals.

[0003] Currently, the industrial production of D-arabitol still faces challenges. The chemical synthesis method relies on noble metal catalysts, with problems such as cumbersome steps, many by-products, and environmental pollution; while the microbial conversion method uses glucose as a substrate and directly synthesizes through enzymatic reactions, having the advantages of being green and efficient, but its core bottleneck lies in the lack of high-yield strains. The reported D-arabitol yields of wild-type strains are generally lower than 80 g / L, and the high-concentration substrate inhibition effect is significant, making it difficult to meet industrial demands. Traditional mutagenesis techniques (such as ultraviolet and nitrosoguanidine) are difficult to directionally enhance the activity of the target metabolic pathway due to the strong randomness of mutations and low screening efficiency. Therefore, developing high-yield strains and establishing an efficient mutagenesis breeding system are the keys to breaking through the technical bottleneck of producing D-arabitol by the biological method.

[0004] Glucose, as an inexpensive and abundant carbon source, is an ideal substrate for the production of D-arabitol by the microbial method. However, wild strains have limited tolerance to high-concentration glucose, and the activity of key enzymes in their metabolic pathways is insufficient, resulting in low substrate conversion rates and long fermentation cycles. In addition, traditional screening methods have low throughput and long time consumption, making it difficult to quickly lock in high-yield mutant strains. To address the above problems, it is urgent to strengthen the metabolic ability of strains through multi-stage mutagenesis techniques and combine high-throughput screening strategies to achieve the rapid breeding of high-yield strains and provide core strain resources for the large-scale production of D-arabitol by the biological method. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a strain with high yield of D - arabinitol and its mutagenesis breeding method. By combining the screening of natural strains with multi - stage mutagenesis technology, the D - arabinitol synthesis ability of the strain is significantly improved, and the titer and yield are increased.

[0006] The present invention first provides a strain with high yield of D - arabinitol, which is characterized in that the strain is Wickerhamomyces anomalus A37, deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with the deposition date of February 24, 2025 and the deposition number of GDMCC 65731.

[0007] The present invention also provides a multi - stage mutagenesis breeding method for a strain with high yield of D - arabinitol, which comprises the following steps:

[0008] (1) Physically mutagenize the wild strain producing D - arabinitol to be selected by using low - energy nitrogen ion implantation technology, and screen the mutant strains with increased D - arabinitol production by the INT method.

[0009] (2) Further perform DES chemical mutagenesis on the mutant strains obtained in step (1), and screen the mutant strains with increased D - arabinitol production by the INT method. According to the preferred embodiment of the present invention, the wild strain producing D - arabinitol is Wickerhamomyces anomalus producing D - arabinitol.

[0010] The DES chemical mutagenesis in step (2) includes: the bacterial cells to be mutagenized with an OD 600 = 2 are centrifuged to discard the supernatant, and then diluted with a phosphate buffer solution with a pH of 7.0 into a single - cell suspension with an OD 600 = 0.2. Add the cell suspension into a sterile conical flask, then add 20% DES solution, and oscillate and mutagenize for a set time in a shaker at 30 °C and 220 rpm. Then add 25% Na 2 S 2 O 3 solution to terminate the reaction. Aspirate the reaction solution and spread it on a screening plate, and culture it at 30 °C for 2 - 3 days.

[0011] The screening of the mutant strains with increased D - arabinitol production by the INT method in step (1) and step (2) includes:

[0012] Pick single colonies into 96 - well plates respectively, with 200 μL of YPD medium in each well, shake at 30 °C and 220 rpm for 24 h. Then take 50 μL from each well and transfer it to a 96 - deep well plate containing 500 μL of fermentation broth with a glucose concentration of 350 g / L, and culture at 30 °C and 220 rpm for 5 d.

[0013] Add the INT colorimetric reaction system to a 96-well plate, and let the reaction solution react at 30°C and 100 rpm for 30 min. Finally, measure the absorbance at a wavelength of 500 nm, and select the wells with high absorbance for HPLC rescreening.

[0014] Compared with the prior art, the D-arabitol yield of the mutant strain Wickhansenella anomala A37 obtained by the present invention reaches 108.69 g / L, and the conversion rate is 31.05%, which are respectively increased by 33.77% and 16.4% compared with the wild type K955.

[0015] The present invention clarifies the optimal culture conditions of abnormal Wickham's yeast A37, wherein the culture temperature is 30°C, the rotation speed is 220rpm, and the components and concentrations of the fermentation culture liquid are 350g / L glucose, 10g / L Aladdin yeast powder, 5g / L ammonium citrate, 0.5g / L K 2 HPO 4 , 0.25 g / L MgSO 4 Under these fermentation conditions, the D-arabinitol production of the mutant strain A37 could reach 113.63 g / L, and the conversion rate was 32.47%.

[0016] The mutagenesis breeding method of the present invention firstly + Ion implantation and DES chemical mutagenesis technology are superimposed on the genetic improvement of abnormal Wickham's yeast, combined with INT high-throughput screening, which significantly improves the mutagenesis efficiency and strain stability. The present invention is a high-yield strain and supporting breeding technology for efficient production of D-arabinitol by biological methods, which has the advantages of low raw material cost, environmentally friendly process, and easy product separation, and is suitable for large-scale production in the fields of food, medicine and chemical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0018] Figure 1 Schematic diagram of colony morphology;

[0019] Figure 2 The selected strains and their corresponding arabinitol production;

[0020] Figure 3 is the arabinitol production of K955 at different substrate concentrations;

[0021] Figure 4 It is a schematic diagram of substrate consumption ratio under different substrate concentrations of K955;

[0022] Figure 5 The cell growth rate and arabinitol production at a substrate concentration of 350 g / L;

[0023] Figure 6 Phylogenetic tree of Wickerhamomyces anomalus K955;

[0024] Figure 7 For N + Schematic diagram of the survival of injected mutagenized colonies (diluted 1000 times);

[0025] Figure 8 Color change diagram of INT screening high-yield arabinitol strains at different reaction times;

[0026] Figure 9 For HPLC re-screening N + Results diagram of mutagenized high-yield strains;

[0027] Figure 10 Survival of DES mutagenized colonies (diluted 1000 times);

[0028] Figure 11 Schematic diagram of DES mutagenesis time and lethality;

[0029] Figure 12 Results diagram of HPLC re-screening DES mutagenized high-yield strains;

[0030] Figure 13 Results diagram of re-screening of DES mutagenized forward mutant strains. Specific implementation manners

[0031] The present invention will be further described and explained below in conjunction with specific implementation manners. The described embodiments are only examples of the disclosed content and do not delimit the scope of limitation. Without conflict, the technical features of each embodiment of the present invention can be combined accordingly.

[0032] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0033] The materials, reagents, etc. used in the following embodiments are commercially available reagents and materials unless otherwise specified.

[0034] Example 1: Screening and identification of wild strains with high yield of D-arabinitol

[0035] 1. Strain screening

[0036] Take a small amount of fresh honey, honeycombs, and soil samples from the outer Tongwu beekeeping farm in Xihu District, Hangzhou City, Zhejiang Province, and place them separately in enrichment media. Incubate them in a shaker at 30°C and 220 rpm for 3 days. After the medium becomes turbid, take an appropriate amount of diluted bacterial solution and spread it on a plate containing screening medium, and incubate it in a constant temperature incubator at 30°C for 4 days. The colony morphology on the YPD medium is shown in Figure 1 , pick out the strains with larger colonies, more rounded morphology, and protrusions, which are more similar to the yeast morphology, and inoculate them into a fermentation medium with 200 g / L glucose as the substrate. Ferment at 30°C and 220 rpm for 96 h, and use high-performance liquid chromatography to detect the supernatant of the fermentation broth. A total of 6 strains with higher D-arabitol in the fermentation products were screened, as shown in Figure 2 . Among them, strain K955 produced approximately 42 g / L of arabitol in 96 h.

[0037] The specific medium formulations and liquid phase detection conditions involved in the above experimental process are as follows:

[0038] Enrichment medium: 250 g / L glucose, 10 g / L yeast extract, 50 mg / L ampicillin, 50 mg / L kanamycin, 20 g / L agar powder.

[0039] Screening medium: 250 g / L glucose, 10 g / L yeast extract, 25 mg / L TTC, and 60 mg / L iodoacetic acid, 20 g / L agar powder.

[0040] YPD medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract. For solid medium, add an additional 20 g / L agar powder.

[0041] Fermentation medium: 200 g / L glucose, 10 g / L yeast extract.

[0042] Liquid phase detection conditions: Use the Dionex UltiMate 3000 high-performance liquid system to quantitatively detect arabitol. Detector: Corona Charged Aerosol Detector (CAD), analytical column: Aminex HPX-87H (Φ7.8 mm × 300 mm), mobile phase: 5 mM H 2 SO 4 , flow rate: 0.6 mL / min, temperature: 65°C.

[0043] 2. Determination of the ability to produce D-arabitol

[0044] Subsequently, further identification of the D-arabitol production capacity of strain K955 was carried out. First, K955 was streaked and activated, and single colonies were picked and inoculated into YPD liquid test tubes, cultured at 30 °C for 24 h, and then the seed liquid was inoculated into fermentation broths with different substrate concentrations for fermentation for 144 h to explore the optimal substrate concentration of this strain.

[0045] The final fermentation results are as Figure 3 shown. When the glucose concentration was 100 g / L, strain K955 reached the highest yield of D-arabitol (18.8 g / L) on the 4th day of fermentation, and then the yield decreased sharply, probably because the strain began to absorb and utilize D-arabitol after the substrate was exhausted. When the glucose concentration was 350 g / L, strain K955 produced the highest level of arabitol on the 6th day of fermentation, and the highest yield reached 81.25 g / L. As can be seen in Figure 4 , at lower substrate concentrations (100, 200 g / L), the substrate of strain K955 was completely consumed on the 2nd day, the substrate was almost completely consumed on the 6th day at a substrate concentration of 300 g / L, about 87% of the substrate was consumed on the 6th day at a concentration of 350 g / L, and only 50% of the substrate was consumed at a concentration of 400 g / L. Combining the D-arabitol yield and glucose utilization rate, the optimal substrate concentration for strain K955 to produce D-arabitol is 350 g / L, and under this condition, the D-arabitol titer can reach 81.25 g / L ( Figure 5 ).

[0046] 3. Strain identification

[0047] Subsequently, the strain K955 was identified by 18S rDNA determination. The 18S conserved region reflects the phylogenetic relationship between biological species, while the variable region can reflect the differences between species and is suitable as a classification criterion at the species level and above. The sequencing results were subjected to BLAST alignment, and the alignment results are as Figure 6As shown, this bacterium belongs to the domain Eukaryota - kingdom Fungi - subkingdom Dikarya - phylum Ascomycota - subphylum Saccharomycotina - class Saccharomycetes - order Saccharomycetales - family Phaffomycetales - genus Wickerhamomyces - Wickerhamomyces anomalus. Wickerhamomyces anomalus is a species of the genus Ascomycota. Its cells are mostly round or oval, and it can reproduce asexually by budding and sexually by forming cap - shaped or spherical ascospores. It is widely present on the surfaces of fruits such as grapes, mulberries, and citrus fruits, and can also be isolated from silage such as grains and corn, high - sugar foods, and traditional koji.

[0048] To deeply analyze the D - arabinitol metabolic mechanism of K955, whole - genome sequencing of K955 was carried out on the PacBio platform to obtain its whole - genome data. The SMRT Link v5.0.1 software was used to perform genome reassembly on the reads, and a preliminary reassembly result that can reflect the basic situation of the sample genome was obtained: the original data volume was 3,084,833,071 bp, 411,935 reads were sequenced, the average sequencing read length was 7,488.6 bp, the N50 length of the sequencing read length was 10,974 bp, a total of 32 contigs were obtained, with a total sequence information of 13,834,765 bp. Among them, the N50 size for judging the quality of genome splicing was 935,510 bp, and the GC content was 32.04%. Starting from the final reassembly result (≥500 bp) of the sample, de novo and homologous prediction were used, and the total number of genes predicted to encode regions in K955 was 5,306, accounting for 57.73% of the entire genome sequence size. Due to the GC content and sequencing depth of the strain genes, the GC content after splicing and recombination was mainly distributed between 30% and 50%, and the sequencing depth was mostly distributed in the range of 30 - 300×; the Unigene length distribution was concentrated in the range of ≥2,500 bp, reaching 721, accounting for about 13.59% of the total.

[0049] Example 2: Low - energy N + Injection mutagenesis and breeding

[0050] Different combinations of the charge number, mass number, energy, and dose of ions injected by low - energy ion implantation can provide more mutagenesis conditions. Low - energy ion implantation mutagenesis has the characteristics of a wide mutation spectrum and a high mutation rate, so mutant strains that meet the production requirements and have increased yields can be screened.

[0051] 1. Mutagenesis method

[0052] (1) Preparation of bacterial suspension: The K955 strain was activated in YPD medium at 30 °C for 12 h, inoculated into the seed medium at an inoculation amount of 5% and cultured for 18 h to obtain a seed solution, and the seed solution was diluted with fresh YPD medium.

[0053] (2) Preparation of bacterial film: 100 μL of the bacterial suspension was aspirated with a pipette and evenly spread on a sterile plate, and dried in a laminar flow hood to form a bacterial film.

[0054] (3) Injection of N + ions: The bacterial film was placed in a sterile vacuum target chamber, and ion implantation was carried out in a vacuum environment of 10 -3 Pa with ion beams of different doses and energies.

[0055] (4) Incubation: After completion, the mutagenized bacterial film was quickly soaked in 1 mL of sterile water in a 1.5 mL EP tube and cultured at a constant temperature of 30 °C. At the same time, a control bacterial film without ion implantation was cultured.

[0056] (5) Plate culture: 100 μL of the eluate from the previous step was aspirated in a laminar flow hood, spread on a YPD plate, cultured at 30 °C for 24 h, and the colonies were observed and the survival rate was calculated.

[0057] The survival situation of the mutagenized plate is shown in Figure 7 , the injection dose of plate No. 11 is appropriate and the lethality rate is relatively high. The ion implantation dose is 1031 keV·10 13 ions / cm 2 , and the mutagenized plate with this injection dose was selected for screening.

[0058] 2. High-throughput screening method

[0059] Iodonitrotetrazolium violet, namely INT, can be used as an artificial hydrogen acceptor to accept hydrogen atoms and be reduced to show color, causing a change in the color depth of the reaction solution before and after. Since glucose enters the pentose phosphate pathway through glycolysis during the synthesis of D-arabitol, which involves multiple dehydrogenase reactions, free hydrogen ions are generated during the reaction, and INT is a strong artificial hydrogen acceptor, so the color of the fermentation broth containing INT will change from light yellow to dark purple. Based on the above principle, high-yield mutant strains of D-arabitol can be screened by the INT method. Since INT has an absorption value at a wavelength of 500 nm after accepting hydrogen atoms, the ability of the mutant strains in the fermentation broth to produce D-arabitol is characterized by detecting the absorbance value of the fermentation broth by a microplate reader per unit time, thereby realizing high-throughput screening.

[0060] Pick single colonies from different plates into a 96-well plate, add 200 μL of YPD medium to each well, shake at 220 rpm at 30 °C for 24 h. Then, take 50 μL from each well and transfer it to a 96-deep well plate containing 500 μL of fermentation broth with a glucose concentration of 350 g / L, and culture at 30 °C and 220 rpm for 5 d for INT high-throughput screening.

[0061] Add 5 μL of fermentation broth, 50 μL of glucose solution and 145 μL of INT solution (100 mg of INT dissolved in 100 mL of 50 mM Tris-HCl) to the 96-well plate. The INT reaction solutions at different reaction times are as Figure 8 shown. Finally, select the reaction solution after 30 min of reaction to measure the absorbance. The reaction solution reacts at 100 rpm at 30 °C for 30 min, and finally measure the absorbance at a wavelength of 500 nm. Select the wells with high absorbance for HPLC re-screening.

[0062] As Figure 9 shown, after high-throughput screening of nearly 2000 strains of bacteria, 42 positive mutant strains were re-screened by HPLC, and a strain ③-3-8C, hereinafter referred to as 8C for short, was screened out. The yield was 90.98 g / L, which was 11.98% higher than that of the wild type K955; the conversion rate was 25.99%, an increase of 11.99%.

[0063] Example 3: N + Physical mutagenesis combined with DES chemical mutagenesis screening

[0064] On the basis of N + injection mutagenesis, the positive strain 8C screened out was mutagenized with diethyl sulfate (DES). The cells grown to OD 600 = 2 were centrifuged at 4000 rpm for 4 min. After discarding the supernatant, they were diluted with a phosphate buffer solution with a pH of 7.0 to a single-cell suspension with OD 600 = 0.2. Add 5 mL of the cell suspension to a sterile conical flask, and then add 0.25 mL of a dilute solution of 20% DES. When starting to count, shake in a shaker at 30 °C and 220 rpm for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, and 35 min respectively, and immediately add 0.75 mL of 25% Na 2 S 2 O 3 solution to terminate the reaction. Pipette 50 μL of the reaction solution and spread it on the screening plate, and culture at 30 °C for 2 - 3 days. Use the untreated bacterial strain spread as a control, count the number of colonies on the plate ( Figure 10 ), and calculate the mutagenic lethality rate ( Figure 11) According to the lethality rate and mutagenesis time, 25 min was selected as the mutagenesis time. At 25 min, the lethality rate had reached 76.7%, and viable colonies were retained while having a relatively high lethality rate.

[0065] Among them, 20% DES solution: Diethyl sulfate and absolute ethanol are mixed at a volume ratio of 1:4. 25% Na 2 S 2 O 3 : 5 g Na 2 S 2 O 3 is dissolved in 20 mL ddH 2 O and used after filtration with a sterile filter membrane.

[0066] Similarly, the INT method was used for high-throughput screening, and 21 positive mutant strains were selected for HPLC re-screening. The results of the re-screening are shown in Figure 12 . Six mutagenic strains with relatively high yield increases were screened out, namely ①-D34, ②-2-B22, ②-2-C5, ②-4-H40, ②-4-A2, and ②-5-A37. Subsequently, these 6 DES mutagenic positive mutant strains, the wild type K955, and 8C were re-screened by shake flask fermentation again, and the results are as Figure 13 shown. Among them, the strain ②-5-A37 (hereinafter referred to as A37 for short) had the highest yield, which was 108.69 g / L, a 33.77% increase compared to the wild type K955; the conversion rate was 31.05%, an increase of 16.4%.

[0067] The obtained high-yield mutant strain Wickerhamomyces anomalus A37 was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC). The deposit date was February 24, 2025, and the deposit number was GDMCC 65731.

[0068] Example 4: Exploration of A37 fermentation conditions

[0069] To improve the D-arabitol production ability of the mutant strain Wickerhamomyces anomalus A37 (deposit number: GDMCC 65731), based on the optimal substrate concentration (350 g / L glucose) of the wild strain K955 in Example 1, the fermentation conditions of A37 were further systematically optimized, including the type of nitrogen source, the ratio of inorganic salts, the culture temperature, and the stirring speed. The specific experimental methods and results are as follows:

[0070] Screening and concentration optimization of nitrogen source

[0071] Commercially available OXOID, Aladdin and Angel brand yeast powder were selected as nitrogen sources, and the final concentrations were set to 5, 10, 15, and 20 g / L for single-factor experiments. The carbon source of the fermentation medium was 350 g / L glucose. The results showed that when the final concentration of Aladdin yeast powder was 10 g / L, the D-arabinitol production reached 112.9 g / L, which was significantly higher than other brands and concentration conditions (P<0.05).

[0072] Inorganic salt ratio verification

[0073] Based on previous research, 5g / L ammonium citrate and 0.5g / L K 2 HPO 4 , 0.25 g / L MgSO 4 The initial concentration was adjusted within the range of ±20% to conduct verification experiments. The results showed that the above inorganic salt concentration changes had no significant effect on bacterial growth and D-arabinitol production (P>0.05), so the initial formula was maintained.

[0074] Cultivation temperature optimization

[0075] The temperature gradient was set at 26, 28, 30, 32, and 34°C to investigate its effect on bacterial metabolism. The results showed that the bacterial growth rate was the highest at 30°C, and the D-arabinitol production reached 113.63 g / L, which was significantly better than other temperature groups (P<0.05). When the temperature was lower than 28°C, the bacterial metabolic rate decreased; when it was higher than 32°C, the product synthesis was inhibited by heat stress.

[0076] Stirring speed optimization

[0077] The effects of rotation speed (180, 200, 220, 240 rpm) on dissolved oxygen and shear force were investigated. The experiment showed that the bacterial biomass and D-arabinitol production reached peak values ​​at 220 rpm. When the rotation speed was lower than 200 rpm, insufficient dissolved oxygen led to uneven dispersion of the bacteria and a decrease in the specific growth rate; when the rotation speed was higher than 220 rpm, high shear force inhibited the metabolic activity of the bacteria and the yield was reduced to 105.4 g / L.

[0078] Comprehensive optimization results

[0079] Based on the above experiments, the optimal fermentation conditions for mutant A37 were determined to be: 10 g / L Aladdin yeast powder, 5 g / L ammonium citrate, 0.5 g / L K 2 HPO 4 , 0.25 g / L MgSO 4 , culture temperature 30℃, stirring speed 220rpm. Under these conditions, the yield of D-arabinitol increased to 113.63g / L, which was 4.5% higher than that of A37 before optimization, and the fermentation period was stable at 156h.

[0080] The embodiments described above merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent of the present invention. For those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

1. A strain with high yield of D-arabinitol, characterized in that: The strain is Wickerhamomyces anomalus A37, which is deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on February 24, 2025, and the deposit number is GDMCC 65731.

2. A multi-stage mutagenesis breeding method for a strain with high yield of D-arabinitol, characterized in that: The following steps are involved: (1) Using low-energy nitrogen ions (N + ) injection technology to physically induce mutagenesis on the selected wild strains producing D-arabinitol, and screen the mutants with improved D-arabinitol production by the INT method. (2) The mutant obtained in step (1) is further subjected to DES chemical mutagenesis, and mutants with increased D-arabinitol production are screened by the INT method.

3. The multi-stage mutation breeding method according to claim 2, wherein: The wild strain producing D-arabitol is an abnormal Wilcoxonella producing D-arabitol.

4. The multi-stage mutation breeding method according to claim 2, wherein: The low energy nitrogen ion implantation dose is 1031keV·10 13 ion / cm 2 .

5. The multi-stage mutation breeding method according to claim 2, characterized in that: The DES chemical mutagenesis described in step (2) comprises: Grow to OD 600 =2, the supernatant was discarded after centrifugation, and then diluted with a phosphate buffer with a pH of 7.0 to an OD 600 =0.2 single cell bacterial suspension, add the cell bacterial suspension into a sterile conical flask, then add 20% DES solution, shake in a shaker at 30°C and 220rpm for the set mutagenesis time, then add 25% Na2S2O3 solution to terminate the reaction, aspirate the reaction solution and spread it on the screening plate, and culture it at 30°C for 2 to 3 days.

6. The multi-stage mutation breeding method according to claim 5, characterized in that: The preparation method of 20% DES solution is as follows: diethyl sulfate and anhydrous ethanol are mixed in a volume ratio of 1:4; The preparation method of 25% Na2S2O3 solution is as follows: dissolve Na2S2O3 in double distilled water (ddH2O) and filter through a sterile filter membrane to obtain the solution.

7. The multi-stage mutation breeding method according to claim 5, characterized in that: The oscillation mutagenesis setting time was selected as 25 min.

8. The multi-stage mutation breeding method according to claim 2, wherein: The steps (1) and (2) of screening mutants with improved D-arabinitol production by the INT method include: Single colonies were picked and placed in 96-well plates, with 200 μL YPD medium per well, shaken at 30°C and 220 rpm for 24 h, and then 50 μL per well was placed in a 96-deep-well plate containing 500 μL fermentation broth with a glucose concentration of 350 g / L, and cultured at 30°C and 220 rpm for 5 days; The INT colorimetric reaction system was added to a 96-well plate, and the reaction solution was reacted at 30°C and 100 rpm for 30 min. Finally, the absorbance was measured at a wavelength of 500 nm, and the wells with high absorbance were selected for HPLC rescreening.

9. The multi-stage mutation breeding method according to claim 8, characterized in that: The INT colorimetric reaction system is: 5 μL of yeast fermentation broth, 50 μL of 350 g / L glucose solution, and 145 μL of 1 g / L INT solution, wherein the 1 g / L INT solution is prepared by dissolving 100 mg of INT in 100 mL of 50 mM Tris-HCl buffer.

10. A method for producing D-arabitol by fermentation of the strain according to claim 1, characterized in that: The steps include: The abnormal Wickerhamomyces anomalus A37 was activated in YPD medium, and the activated bacterial solution was inoculated into a shake flask for fermentation and culture to produce D-arabitol, wherein the culture temperature was 30°C, the rotation speed was 220 rpm, and the components and concentrations of the fermentation culture solution were: 350 g / L glucose, 10 g / L Aladdin yeast powder, 5 g / L ammonium citrate, 0.5 g / L K2HPO4, and 0.25 g / L MgSO4.

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