Salt-tolerant strain of aureobasidium pullulans and its application in poly-malic acid production

Abstract

This invention relates to the field of microbial fermentation technology, and discloses a salt-tolerant melanin-producing *Syngonium brevicorum* strain and its application in the production of polymalic acid. The strain is a melanin-producing *Syngonium brevicorum* (…). Aureobasidium melanogenum The strain, tust8-2, with accession number CGMCC No. 42419, was obtained from the starting strain *Bacillus melanin-producing* 2pe-1 through atmospheric pressure room temperature plasma mutagenesis and adaptive evolution with increasing NaCl concentration. It can grow in a culture system with a NaCl concentration of 6%–7% (w / v). When used for polymalic acid fermentation, this strain achieved a polymalic acid concentration of 72.74 g / L in a 5 L fermenter system at 25°C with sodium hydroxide as a neutralizing agent and an initial glucose concentration of 190 g / L.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to salt-tolerant melanin-producing short-stem mold strains and their application in the production of polymalic acid. Background Technology

[0002] Polymalic acid (PMLA) is a linear polyester with L-malic acid as its sole monomer. Its degradation products are non-toxic and harmless, exhibiting excellent water solubility, bioabsorbability, and chemical derivatization. It demonstrates irreplaceable advantages in areas such as drug delivery carriers, surgical sutures, food preservation packaging, and cosmetic additives. PMLA synthesis methods mainly include microbial synthesis and chemical synthesis. Microbial synthesis, due to its mild reaction, high product purity, low pollution, and environmental friendliness, has become the mainstream method for PMLA synthesis. However, most strains have limited PMLA synthesis capabilities, resulting in generally low yields. Therefore, strain improvement has become a core means to enhance the synthetic capacity of microbial products and overcome industrialization bottlenecks. Traditional high-yield PMLA fermentation processes use calcium carbonate as a neutralizing agent, but the extraction process generates a large amount of calcium sulfate contaminants. Simultaneously, high-yield PMLA strains have limited tolerance to free salt ions, leading to a significantly lower yield of PMLA produced using soluble neutralizing agents compared to fermentation processes using calcium carbonate.

[0003] Traditional mutagenesis techniques, such as ultraviolet mutagenesis and chemical mutagens, suffer from drawbacks such as low mutation efficiency, narrow mutation spectrum, and easy generation of toxic residues, making it difficult to meet the needs of high-efficiency breeding. ARTP, as a novel physical mutagenesis technology, has been widely used in the improvement of strains of various microorganisms such as bacteria, actinomycetes, and yeasts, and has successfully increased the yield of various metabolites.

[0004] Salt tolerance adaptation is achieved through long-term subculturing of microorganisms in media with progressively increasing salt concentrations, followed by targeted screening to identify mutant strains with enhanced salt tolerance. *Brachystomum* is a typical osmotic-tolerant fungus, exhibiting outstanding salt tolerance; some fungi can tolerate high-salt environments of 10–25 g / L NaCl, classifying them as moderately salt-tolerant fungi. Their salt tolerance adaptation mechanisms possess both general microbial characteristics and distinct species specificity. Polymalic acid (PMLA) synthesis is also regulated by salt stress osmotic pressure. Although PMLA synthesized by *Brachystomum* is an extracellular macromolecular metabolite and not a salt-compatible solute, its monomeric malic acid is a small molecule involved in intracellular osmotic regulation, which can assist *Brachystomum* in coping with salt stress by regulating osmotic pressure and ion balance. The synthesis process of PMLA itself is directly affected by salt stress osmotic pressure and is closely related to the salt tolerance metabolic regulation of *Brachystomum*. PMLA synthesis mainly relies on the TCA cycle, glyoxylate cycle, and pyruvate carboxylation pathway. Salt stress can synergistically promote the synthesis of the target product by interfering with these metabolic pathways, making open fermentation of PMLA possible.

[0005] The combination of ARTP mutagenesis and salt tolerance adaptation is essentially a synergistic effect of random mutation and targeted selection, exhibiting excellent complementarity and synergistic effects. The core function of ARTP mutagenesis is to generate extensive genetic variation, disrupting the original metabolic balance of the strain and potentially inducing gene mutations related to salt tolerance and PMLA synthesis. However, these mutations are random and require targeted selection to obtain strains with superior phenotypes. On the other hand, salt tolerance adaptation, through salt stress, conducts targeted selection on the mutation library generated by ARTP mutagenesis, retaining those salt-tolerant mutant strains that can grow normally and synthesize PMLA, while eliminating salt-intolerant strains with low yields.

[0006] From a metabolic regulation perspective, ARTP mutagenesis may lead to mutations in PMLA synthesis-related genes (such as malate dehydrogenase genes and PMLA synthase genes) and salt tolerance-related genes (such as compatibility solute synthesis genes and ion transporter genes) in *Brachys melanin-producing brachys*. Salt tolerance adaptive evolution then positively selects for these mutated genes, allowing the mutant strain to gradually optimize its metabolic pathways under high-salt conditions, achieving a simultaneous increase in salt tolerance and PMLA production. Furthermore, the oxidative stress response induced by salt stress can further induce *Brachys melanin-producing brachys* to produce more reactive oxygen species, thereby promoting DNA repair and mutation. This synergistic effect with the genetic variations generated by ARTP mutagenesis further enhances the strain's improvement.

[0007] However, existing technologies still have the following drawbacks: First, most high-yield PMLA strains are susceptible to high concentrations of salt ions (especially Na+). + First, the strains have poor tolerance and cannot maintain high yields in soluble neutralization systems without calcium carbonate, leading to a heavy reliance on calcium carbonate in traditional fermentation processes. This results in a large amount of calcium sulfate solid waste, increasing post-treatment costs and environmental burden. Second, existing mutation breeding methods (such as using ARTP alone or salt tolerance acclimatization alone) often only improve a single trait, making it difficult to simultaneously improve salt tolerance and PMLA yield in a synergistic way. Moreover, the screening efficiency is low, and the overall performance of the obtained strains is not ideal.

[0008] Therefore, there is an urgent need to develop a breeding method and strain that can simultaneously endow the strain with high salt tolerance and high PMLA yield, and can be efficiently fermented in a calcium carbonate-free system. Summary of the Invention

[0009] This invention provides a salt-tolerant melanin-producing *Synthia spp.* strain and its application in polymalic acid production. The method uses *Synthia spp.* (a type of *Synthia spp.*) as the starting point for this process. Aureobasidium melanogenum Using strain 2pe-1 (CGMCC No. 42418) as the starting strain, a melanin-producing short-stem mold resistant to high concentrations of NaCl and producing high levels of polymalic acid was obtained through screening. Aureobasidium melanogenum)tust8-2 (CGMCC No. 42419). This invention solves the problems of poor salt tolerance and low polymalic acid yield of existing strains, and provides excellent strains and processes for the industrial-scale and efficient production of polymalic acid.

[0010] In a first aspect, the present invention provides a salt-tolerant melanin-producing short-stem mold strain, namely melanin-producing short-stem mold tust8-2, which was deposited on December 30, 2025 at the Beijing Culture Collection Center for Microbial Cultures (address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, 100101, China), with accession number CGMCC No. 42419.

[0011] In one alternative embodiment, the strain is capable of growing in a culture medium with a NaCl concentration of 6-7% (w / v); and / or, when the strain ferments to produce polymalic acid in a fermentation system with NaOH as a neutralizing agent, the polymalic acid yield is ≥72 g / L.

[0012] Secondly, the present invention also provides a method for breeding the salt-tolerant melanin-producing short-stalked mold strain as described above, comprising the following steps: (1) Using the melanin-producing short-stalked mold as the starting strain, the ARTP mutant library was obtained by mutagenesis using a normal pressure room temperature plasma mutagenesis system. (2) The ARTP mutant library obtained in step (1) was inoculated into a liquid culture medium containing an increasing concentration of NaCl for continuous subculture and targeted screening of mutant strains with salt tolerance. (3) Spread the bacterial solution obtained in step (2) onto a screening plate containing an indicator and select candidate strains with strong acid production ability; (4) The candidate strains selected in step (3) were subjected to shake-flask fermentation, and the polymalic acid yield was measured. The strain with the highest polymalic acid yield was selected from them.

[0013] In an optional implementation, the starting strain in step (1) is *Bacillus melanin-producing* 2pe-1, with accession number CGMCC No. 42418.

[0014] In one optional embodiment, the parameters for atmospheric pressure room temperature plasma mutagenesis in step (1) are: radio frequency power 100-120 W, helium flow rate 10-12 SLM, distance between plasma torch nozzle outlet and substrate 2-5 mm, and mutagenesis time 30-360 s; and / or, the initial concentration of NaCl in step (2) is 0.5% (w / v), the NaCl concentration increases by 0.5% (w / v) after each passage, and the final concentration is 7% (w / v); and / or, the screening plate containing the indicator in step (3) is a bromocresol green indicator PDA plate and / or a trichomoniasis polysaccharide screening plate; and / or, the fermentation temperature of the shake flask fermentation in step (4) is 25℃, the rotation speed is 180-220 r / min, and the fermentation time is 48-72 h.

[0015] Thirdly, the present invention also provides the application of salt-tolerant melanin-producing short-stalked mold strains obtained by means of the salt-tolerant melanin-producing short-stalked mold strains described above, or by means of the microbial agent or the selection method described above, in the production of polymalic acid.

[0016] Fourthly, the present invention also provides a method for producing polymalic acid, comprising fermentation using the salt-tolerant melanin-producing short-stem mold strain, microbial agent, or salt-tolerant melanin-producing short-stem mold strain selected by the above-mentioned selection method.

[0017] In one alternative embodiment, the method for producing polymalic acid includes the following steps: (1) The melanin-producing short-skinned pustulosa tust8-2 was inoculated into seed culture medium for seed culture to obtain seed liquid; (2) The seed liquid obtained in step (1) is inoculated into the fermentation medium and fermented at 25°C. During the fermentation process, NaOH is used as a neutralizing agent to control the pH. (3) After fermentation, polymalic acid is separated and purified from the fermentation broth.

[0018] In one optional embodiment, the NaCl concentration in the fermentation medium is ≤7% (w / v); And / or, the fermentation medium is a high-sugar medium; optionally, the concentration of glucose in the fermentation medium is ≤300g / L.

[0019] The technical solution of this invention has the following advantages: 1. This invention provides a melanin-producing *Short-circuit mold* strain tolerant to high concentrations of NaCl and its breeding method. A polymalic acid-producing melanin-producing *Short-circuit mold* strain screened in our laboratory (…) Aureobasidium melanogenumStarting with strain 2pe-1, a mutant strain exhibiting significant resistance to high-concentration NaCl and high-concentration polymalic acid fermentation characteristics was obtained through ARTP room-temperature plasma mutagenesis combined with salt tolerance adaptation evolution. This mutant strain was named *Synthia spp.* Aureobasidium melanogenum )tust8-2.

[0020] 2. Strain tust8-2 exhibited excellent performance in high-concentration polymalic acid (PMA) fermentation: In conical flasks, at 25℃ and 180-220 r / min, the PMA concentration reached 18.32 g / L; in a 5 L fermenter, at 25℃, the PMA concentration during batch fermentation reached 72.74 g / L, with a production intensity of 1.13 g / L / h and a sugar-acid conversion rate of 38.3%; in a 5 L fermenter, at 25℃, with an initial glucose concentration of 190 g / L, a fed-batch fermentation process, after three additions of 300 ml of 800 g / L glucose solution, resulted in a PMA yield of 83.5 g / L; simultaneously, the addition of sodium hydroxide as a neutralizing agent increased the final fermentation volume by 53.3%, resulting in a PMA yield of 128 g / L based on the initial batch fermentation volume, with a production intensity of 1.07 g / L. g / L / h, sugar-acid conversion rate is 35.9%; furthermore, the strain still has high PMLA production capacity and excellent cell performance under high sugar conditions with initial sugar concentration of 300 g / L, and can utilize glucose for polymalic acid fermentation with relatively high efficiency.

[0021] 3. This invention enriches the resources of excellent strains for polymalic acid production, effectively solves the contradiction between tolerance to high concentrations of salt ions and high polymalic acid production, and significantly improves the synthesis capacity and production intensity of polymalic acid during fermentation. At the same time, it overcomes the problem of calcium contamination in the traditional fermentation process and simplifies the post-extraction process of polymalic acid, which has important practical significance for the efficient production of polymalic acid.

[0022] Biological Preservation This invention provides a salt-tolerant melanin-producing short-stemmed mold strain, namely melanin-producing short-stemmed mold tust8-2, which was deposited on December 30, 2025 at Beijing Culture Collection Center for Microbial Cultures (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China), with accession number CGMCC No. 42419. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 The results show the growth status of different bacterial strains after the bacterial culture, which has completed its evolution, is spread onto a PDA plate containing 7% NaCl and bromocresol green indicator.

[0025] Figure 2 This represents the growth trend of the starting strain under different concentrations of NaCl during the adaptive evolution process.

[0026] Figure 3 This represents the growth trend of mutant strains under different concentrations of NaCl during adaptive evolution.

[0027] Figure 4 This is a comparison of PMLA synthesis capabilities among different numbered strains in the initial screening library.

[0028] Figure 5 This is a comparison of the PMLA synthesis capabilities of high-yielding strains in the initial screening library.

[0029] Figure 6 The comparison of the synthesis capacity of the starting strain 2pe-1 and the mutant strain tust8-2 using 190 g / L total sugar glucosinolate was conducted in a 5 L fermenter.

[0030] Figure 7 The comparison was conducted in a 5 L fermenter between the starting strain 2pe-1 and the mutant strain tust8-2, focusing on their ability to utilize 190 g / L of total sugar in glucose.

[0031] Figure 8 The comparison of cell growth capacity of the starting strain 2pe-1 and the mutant strain tust8-2 using 190 g / L glucose in a 5 L fermenter.

[0032] Figure 9 The study compared the polymalic acid synthesis capabilities of the starting strain 2pe-1 and the mutant strain tust8-2 in a 5 L fermenter using a total sugar content of 430 g / L glucose and calcium carbonate as a neutralizing agent.

[0033] Figure 10 The study compared the polymalic acid synthesis capabilities of the starting strain 2pe-1 and the mutant strain tust8-2 using a total sugar content of 430 g / L glucose and sodium hydroxide as a neutralizing agent in a 5 L fermenter.

[0034] Figure 11 The study compared the polymalic acid synthesis capacity of the mutant strain tust8-2 under conditions of 250 g / L glucose and 300 g / L glucose in a 5 L fermenter.

[0035] Figure 12The comparison of the growth capacity of the starting strain 2pe-1 and the mutant strain tust8-2 in a 5 L fermenter under 70 g / L NaCl conditions was conducted.

[0036] Figure 13 It is the mutagenic mortality rate curve.

[0037] Figure 14 Cell morphology of the original strain 2pe-1 (left) and the mutant strain tust8-2 (right) of *Bacillus melaninus* observed under an oil immersion microscope. Detailed Implementation

[0038] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0039] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0040] The culture medium used in the embodiments of the present invention is prepared according to the following method: (1) PDA slant culture medium: potato 200 g / L, glucose 20 g / L, agar powder 20 g / L, pH=6.

[0041] (2) Primary seed culture medium: glucose 80 g / L, NaNO3 2 g / L, KH2PO4 0.1 g / L, MgSO4·7H2O 0.2 g / L, KCl 0.5 g / L, Tryptone (LP0042, Oxoid) 1 g / L, pH=6 (3) Shake flask fermentation medium: glucose 80 g / L, NaNO3 2 g / L, KH2PO4 0.1 g / L, MgSO4·7H2O 0.2 g / L, KCl 0.5 g / L, Tryptone (LP0042, Oxoid) 1 g / L, pH=6 (4) Fermentation tank culture medium: glucose 190 g / L, Tryptone (LP0042, Oxoid) 15 g / L, NaNO3 6 g / L, yeast extract (LP0021, Oxoid) 5 g / L, KH2PO4 (anhydrous) 0.5 g / L, MgSO4.7H2O 0.2 g / L, KCl 0.5 g / L, ZnSO4.7H2O 5ppm, pH=6; In some of the following examples, due to the different amounts of glucose added, it is uniformly summarized as a fermentation tank culture medium with an initial sugar content of x g / L, where x specifically refers to the amount of glucose used.

[0042] (5) Bromocresol green indicator PDA screening plate: potato 200 g / L, glucose 20 g / L, agar 20 g / L, bromocresol green 0.2 g / L, pH=6.

[0043] (6) Tribenzyl blue polysaccharide screening plate: yeast extract 10 g / L, peptone 20 g / L, agar 20 g / L, tribenzyl blue 0.5 g / L, chloramphenicol 10 g / L, glucose 20 g / L, pH=6.

[0044] In the following embodiments, some of the evaluation indicators are calculated using the following formulas: 1. When sodium hydroxide is used as a neutralizing agent, the volume change rate 1 (%) = (V1 - V0) / V0, V1 (L) = V0 + V G +V NaOH ; 2. The volume change rate caused by using calcium carbonate as a neutralizing agent is 2 (%) = (V2 - V0) / V0, V2 (L) = V0 + V G ; 3. Actual final yield P of polymalic acid when sodium hydroxide is used as a neutralizing agent. PMLA (g / L) = P0 V1 / V0; 4. Actual final yield of polymalic acid (P) when calcium carbonate is used as a neutralizing agent. PMLA (g / L) = P0 V2 / V0; 5. Production intensity (g / L / h) = P PMLA / h; 6. Sugar-acid conversion rate (%) = ΔS G / P PMLA ; Where V0 is the initial fermentation volume of 3 L; V G The total volume of glucose added for the fed-batch fermentation process was 900 mL; V NaOHThe volume increase due to pH adjustment during the fed-batch fermentation process was 700 ml; V1 is the volume at the end of fermentation using sodium hydroxide as a neutralizing agent; V2 is the volume at the end of fermentation using calcium carbonate as a neutralizing agent; ΔS G P represents the total glucose consumption during fermentation; h represents the time at the end of fermentation; P0 represents the polymalic acid yield detected in the diluted liquid phase during fermentation; P PMLA The final yield of polymalic acid is calculated based on the volume after fermentation.

[0045] In the following embodiments, the detection method for polymalic acid is as follows: 1. Fermentation broth pretreatment After centrifuging the fermentation broth, take 1 ml of the supernatant and add 1 ml of H2SO4 (2 mol / L). Place the mixture in a reaction vessel and hydrolyze in a 90℃ oven for 12 h. Remove the vessel and add 2 ml of NaOH (2 mol / L) and 4 ml of water. Mix well and transfer to a 10 ml centrifuge tube.

[0046] 2. High-performance liquid chromatography (HPLC) detection The polymalic acid (PMA) content in fermentation broth was accurately determined using high-performance liquid chromatography (HPLC). A mobile phase of 5 mmol sulfuric acid was used, and an ultraviolet detector was employed. After pretreatment, the fermentation broth was injected into the HPLC system for analysis. A standard curve was plotted by comparing the retention time and peak area with those of malic acid standards. The PMA content in the sample was accurately calculated by substituting the sample peak area into the standard curve.

[0047] The calculation formula is: C PMLA =C MAL ×M PMLA / M MAL ×f 稀释 Among them, C PMLA The concentration of polymalic acid in the fermentation broth; C MAL M represents the concentration of malic acid detected by HPLC. PMLA / M MAL The molar mass ratio of polymalic acid to malic acid is given by f. 稀释 This represents the dilution factor of the pretreated sample.

[0048] The UV detector parameters are as follows: column: 87H column, column temperature: 40℃, mobile phase: 5 mmol H2SO4, flow rate: 0.6 ml / min, analysis time: 30 min, 210 nm. Example 1: Isolation and domestication of strains I. Isolation of melanin-producing short-stalked mold ( Aureobasidium melanogenum 2pe-1 1. Sample collection: Soil from Binhai New Area, Tianjin.

[0049] 2. Strain strain isolation: includes the following steps: (1) Take 1 g of soil sample into a shake flask containing 100 mL of sterile physiological saline, shake at 25℃ and 180 r / min for 30 min, then let stand for 2 min, and take the supernatant liquid and serially dilute it by 10. -3 10 -4 10 -5 ; (2) Take 100 μL of the diluted bacterial solution and place it in the center of a PDA culture plate. Spread the solution evenly over the entire plate with a disposable sterile spreader. Invert the plate and incubate at 25°C in the dark for 3-5 days. (3) Observe the plates during the culture period and screen for colonies that meet the characteristics (screening requirements: young colonies on the 3rd to 5th day are round, milky white or light beige, with a smooth and moist surface, a viscous texture, neat edges, and no black spreading pigment; mature colonies on the 7th to 10th day gradually show black spreading pigment and are viscous). Pick the target colonies with a disposable sterile inoculation loop, streak them in three zones onto the PDA plate, and incubate at 25℃ inverted for 3 to 5 days. (4) When a well-isolated single colony appears in the third zone of the above plate, pick a single colony for microscopic examination. It shows that there are only short-stalked yeast cells with septate hyphae and no bacterial rods or cocci.

[0050] II. Melanin Short-stalked Mold ( Aureobasidium melanogenum )tust8-2 Starting with the melanin-producing short-stem mold 2pe-1, the strain was obtained through ARTP room temperature plasma mutagenesis combined with salt tolerance adaptation evolution. It was then screened using a dual-plate method (bromocresol green and trichomoniasis blue) followed by shake-flask fermentation for secondary screening. The specific steps are as follows: (1) Pick melanin-producing short-stem molds from PDA slant culture medium ( Aureobasidium melanogenum ) 2pe-1 single colony was inoculated into primary seed culture medium and cultured at 25℃ and 180 r / min for 48 h.

[0051] (2) Under sterile conditions, take 5 mL of bacterial culture from step (1), centrifuge at 10000 rpm for 8 min to collect cells, wash twice with sterile PBS, resuspend again and take 20 μL on ARTP mutagenesis slide.

[0052] (3) The cells from step (2) were subjected to ambient temperature and ambient pressure plasma mutagenesis in an ARTP mutagenesis system. The parameters were set as follows: 1) Set the RF power input to 100 W and the helium flow rate to 10 SLM; 2) Set the distance between the plasma torch nozzle outlet and the substrate to 2~5 mm (preferably 2 mm in this embodiment).

[0053] 3) Select different mutagenesis times: 30 s, 60 s, 120 s, 180 s, 240 s, 360 s. After mutagenesis, wash the slides with PBS to generate an ARTP mutant library.

[0054] (4) After washing the mutant library from step three with PBS, take 100 μL and spread it on a PDA selection plate containing bromocresol green. Culture for 2-3 days and observe the cell growth status and number. Figure 13 The mutation curve is shown. From... Figure 13 The results showed that the mutagenesis mortality rate was between 80% and 90% at 240 s. Within a suitable lethal range, the mortality rate approached 96%-97% when the mutagenesis time was increased to 360 s. Too few surviving bacteria limited screening, and excessively long mutagenesis times easily led to multiple DNA damage. The 240 s mutagenesis time had not yet reached the plateau phase; this time provided a suitable number of surviving bacteria without causing significant DNA damage. Therefore, the optimal mutagenesis time was determined to be 240 s.

[0055] (5) Using the optimal mutagenesis time obtained in step (4) as the standard, repeat steps (1), (2) and (3) again.

[0056] (6) The mutant sample from step (5) was revived by adding 5 mL of PBS at pH 6.8, and then transferred to a shake flask fermentation medium and cultured at 25°C and 180 r / min for 48 h.

[0057] (7) Transfer the bacterial culture obtained in step (6) to an adaptive evolutionary incubator (Tianmu Biotechnology and Tsinghua University Wuxi Institute of Applied Technology; model: EVOL cell). The parameters and facilities of the evolutionary incubator are as follows: 1) The temperature was set to 25℃, the inoculum size was set to 6% (v / v), the OD detection wavelength was set to 600 nm, the detection cycle was set to 480 min, and the passage time was set to 48 h. 2) Chemical factor A concentration setting: NaCl concentration (mother liquor) set to 30% (w / v); 3) Subculture gradient setting: Increase the NaCl concentration by 0.5% (w / v) after each subculture. (Increasing the NaCl concentration too much will cause the strain to die directly, and due to the limited precision of the instrument, the liquid pumping volume cannot be accurate to a decimal point. During subculture, the NaCl concentration needs to be increased in small increments within the precision range of the instrument.) (8) After the evolutionary culture is completed, transfer the culture chamber in step (7) to the clean bench and keep all the fermentation broth inside in a glycerol tube containing 20-50% glycerol.

[0058] (9) Take 10-100 μL of the glycerol tube from step (8), dilute it, and then take 100 μL to spread on a PDA screening plate containing 7% NaCl bromocresol green indicator. Incubate for 2-3 days and observe the size of the clear zone of the colony.

[0059] (10) such as Figure 1 As shown, select strains with larger discoloration zones from the plates in step (9), transfer them to PDA slant to form a preliminary screening cell bank, and number them (e.g., ...). Figure 4 As shown, the strains in the initial screening library were numbered sequentially as 7-A~7-K, 7-1~7-6, 8-1~8-3, J1~J8, and then stored at 4℃. At the same time, strains with larger discoloration zones were selected again and transferred to bromocresol green plates and trebengan blue polysaccharide plates according to the initial screening library numbers (the selection requirement for the target strain is that it retains a relatively obvious discoloration zone on the bromocresol green plate and a smaller discoloration zone on the trebengan blue polysaccharide plate), and cultured at 25℃.

[0060] (11) The strains in the initial screening library described in step (10) are sequentially introduced into the shake flask fermentation medium according to the numbering order, and cultured for 5 days at 25℃, 180 r / min, with 65 g / L calcium carbonate as the neutralizing agent (65 g / L calcium carbonate powder and shake flask fermentation medium are sterilized separately and then the powder is added to the fermentation liquid at one time, and the pH is controlled within the range of 6 to 7).

[0061] like Figure 4 and Figure 5 As shown, the strain 8-2, which exhibited the highest polymalic acid concentration (18.32 g / L), the highest fermentation efficiency, and good stability, was ultimately obtained and named *Brocadobromis melanogaster*. Aureobasidium melanogenum )tust8-2.

[0062] Example 2: Identification and Preservation of Strains I. Melanin Short-Stemmed Pulsatilla ( Aureobasidium melanogenum Identification of 2pe-1 1. Morphological identification (1) Absorbing melanin from short-stemmed mold ( Aureobasidium melanogenu 20 μL of m)2pe-1 glycerol tube bacterial suspension was diluted, spread onto PDA plates, and incubated at 25℃ in the dark for 3-5 days to observe colony growth. (2) Macroscopic colony morphology observation: young colonies on days 3-5 are round, milky white or light beige, with smooth and moist surfaces, viscous texture, neat edges, and no black diffusion pigment; mature colonies on days 7-10 gradually show black diffusion pigment and become viscous.

[0063] (3) Microscopic observation of cell morphology: Take a small amount of colony and place it on a glass slide. Observe the cells under 10×, 40× and oil immersion respectively. The cells are short rod-shaped, budding and multiplying, with septate branching transparent hyphae. Figure 14 The left image shows the cell morphology of *Syngonium melanogaster* 2pe-1 as observed under an oil immersion microscope.

[0064] 2. Molecular biological identification (1) Extraction of melanin from *Syngonium brevicornu* ( Aureobasidium melanogenum 2pe-1 glycerol tube bacterial suspension was inoculated into YPD medium and cultured for 24-48 h; (2) Take 4 mL of the above bacterial culture, centrifuge at 8000 r / min for 5 min and discard the supernatant. Grind the bacterial precipitate with liquid nitrogen and extract whole genome DNA from the bacterial cells using a plant extraction kit. (3) The whole genome DNA was sent for sequencing, and the sequence was compared with NCBI BLAST and found to match *Synthia spp.* (a type of spp.). Aureobasidium melanogenum Homology ≥ 99%.

[0065] II. Melanin Short-stalked Mold ( Aureobasidium melanogenum Identification of tust8-2 1. Morphological identification (1) Absorbing melanin from short-stemmed mold ( Aureobasidium melanogenu 20 μL of m)tust8-2 glycerol tube bacterial suspension was diluted, spread onto PDA plates, and incubated at 25℃ in the dark for 3-5 days to observe colony growth. (2) Macroscopic colony morphology observation: Young colonies on days 3-5 are round, milky white or light beige, with a smooth, moist surface, a viscous texture, and neat edges without black diffusion pigment; mature colonies on days 7-10 gradually show black diffusion pigment and become viscous. Compared with the original strain 2pe-1, the colonies of the mutant strain tust8-2 are relatively smaller, and the melanin diffusion in mature colonies is relatively faster.

[0066] (3) Microscopic observation of cell morphology: Take a small amount of colony and place it on a glass slide. Observe the cells under 10×, 40× and oil immersion respectively. The cells are short rod-shaped, budding and multiplying, with septate branching transparent hyphae. Figure 14 The right image shows the cell morphology of *Tust8-2* melaninus observed under an oil immersion microscope.

[0067] 2. Molecular biological identification (1) Absorbing melanin from short-stemmed mold ( Aureobasidium melanogenum Inoculate the bacterial culture from tust8-2 glycerol tubes into YPD medium and incubate for 24-48 h; (2) Take 4 mL of the above bacterial culture, centrifuge at 8000 r / min for 5 min and discard the supernatant. Grind the bacterial precipitate with liquid nitrogen and extract whole genome DNA from the bacterial cells using a plant extraction kit. (3) The whole genome DNA was sent for sequencing, and the sequence was compared with NCBI BLAST and found to match *Synthia spp.* (a type of spp.). Aureobasidium melanogenum Homology ≥ 99%.

[0068] III. Biological Preservation: Melanin Short-Stemmed Pulmonaria ( Aureobasidium melanogenum 2pe-1 was deposited on December 30, 2025 at Beijing Culture Collection Center for Microbial Cultures (Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postcode: 100101), with accession number: CGMCC No. 42418, and classified as melanin-producing short-stemmed sclerotium.

[0069] Melanin Short-Stemmed Pulmonaria ( Aureobasidium melanogenum )tust8-2 was deposited on December 30, 2025 at Beijing Culture Collection Center for Microbial Cultures (Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China), with accession number CGMCC 42419.

[0070] Example 3: Effect of different concentrations of NaCl on the growth of the starting strain 2pe-1 This invention aims to investigate the effects of different NaCl concentrations on the growth of *Bretschneidera melanin-producing* 2pe-1 strain. Firstly, the starting strain *Bretschneidera melanin-producing* (… Aureobasidium melanogenum 2pe-1 culture was cultured to the logarithmic growth phase, and then the NaCl concentration was gradually increased in an adaptive evolutionary apparatus, with each increase being 0.5% (w / v). OD was measured every 8 hours during the growth process. The specific operating steps are as follows: (1) Pick melanin-producing short-stalked dermatophyte 2pe-1 from PDA slant culture medium and inoculate it into primary seed culture medium; (2) Cultured on a shaker at 25℃ and 180~220 r / min for 48~64 h, and inoculated into an adaptive evolution incubator containing fresh shake flask fermentation medium at an inoculation rate of 6% (v / v) under aseptic conditions; (3) Set the parameters of the adaptive evolution instrument: the temperature is set to 25℃, the inoculum size is set to 6% (v / v), the OD detection wavelength is set to 600 nm, and 0.5% (w / v) NaCl is added after each passage; (4) Samples were taken and OD was tested every 480 min during the growth process.

[0071] The above results are as follows Figure 2 As shown, the melanin-producing short-stem mold ( Aureobasidium melanogenumAfter one subculture of strain 2pe-1, the first addition of 0.5% (w / v) NaCl resulted in normal growth of the strain. However, when the NaCl concentration was increased to 3% (w / v), *Brachys melanocarpa* (…)… Aureobasidium melanogenum The growth of strain 2pe-1 was significantly slow, and it essentially ceased to grow under a 3.5% (w / v) NaCl concentration, indicating that it is a melanin-producing short-stemmed fungus. Aureobasidium melanogenum 2pe-1 can only grow normally under 2.5% (w / v) NaCl conditions.

[0072] Example 4: Effect of different concentrations of NaCl on the growth of mutant strains This invention aims to investigate the effects of different NaCl concentrations on the growth of *Tust8-2*, a melanin-producing fungus. Firstly, *Tust8-2* was subjected to... Aureobasidium melanogenum The tust8-2 culture was cultured to the logarithmic growth phase, and then the NaCl concentration was gradually increased in an adaptive evolutionary apparatus, with each increase being 0.5% (w / v). OD was measured every 8 hours during the growth process. The specific operating steps are as follows: (1) Pick out *Syngonium melanogaster* from the slant culture medium ( Aureobasidium melanogenum )tust8-2 was inoculated into primary seed culture medium; (2) The mixture was cultured in a shaker at 25℃ and 180 r / min for 48 h. Under aseptic conditions, the melanin short-stem mold ( Aureobasidium melanogenum ) tust8-2, inoculated at a rate of 6% (v / v) into an adaptive evolution incubator containing fresh shake-flask fermentation medium; (3) Set the parameters of the adaptive evolution instrument: the temperature is set to 25℃, the inoculum size is set to 6% (v / v), the OD detection wavelength is set to 600 nm, and 0.5% (w / v) NaCl is added after each passage; (4) Samples were taken and OD was tested every 480 min during the growth process.

[0073] The above results are as follows Figure 3As shown, the mutagenized strain was cultured with a NaCl concentration of 2.5% as the initial concentration. After one subculture, the NaCl concentration was increased by 0.5% (w / v). As the NaCl concentration increased, the growth of the strain slowed down. However, when the NaCl concentration was between 4% and 6% (w / v), the OD of *B. melanin-producing* continuously changed and gradually increased with increasing NaCl concentration, indicating that *B. melanin-producing* could gradually adapt to an environment containing NaCl. When the NaCl concentration increased to 6% to 7% (w / v), the OD of the strain increased significantly, but the growth showed a continuous downward trend, at which point adaptive evolution ceased. This demonstrates that *B. melanin-producing* tust8-2, obtained by mutagenesis and screening using the *B. melanin-producing* strain 2pe-1 as the starting point, possesses salt tolerance and can grow in a high-concentration NaCl environment.

[0074] Example 5: Melanin Short-Stemmed Pulsatilla ( Aureobasidium melanogenum Application of 2pe-1 in polymalic acid fermentation The fermentation conditions in this embodiment are shake flask fermentation with 80 g / L glucose or a 5 L fermenter with 190 g / L initial glucose. The specific operation steps are as follows: Fermentation Method 1: Shake Flask Fermentation (1) Under aseptic conditions, pick melanin-producing short-stem molds from PDA slant culture medium ( Aureobasidium melanogenum ) 2pe-1 single colony was inoculated into primary seed culture medium and cultured at 25℃ and 180 r / min for 48 h; (2) Under aseptic conditions, the seed culture medium from step (1) was inoculated into the shake flask fermentation medium at an inoculation rate of 10% (v / v). Shake flask fermentation was carried out at 25°C, 180 r / min, and with 65 g / L calcium carbonate as a neutralizing agent (65 g / L calcium carbonate powder and shake flask fermentation medium were sterilized separately and then the powder was added to the fermentation liquid at once, and the pH was controlled within the range of 6 to 7).

[0075] Fermentation Method Two: Fermentation in a fermenter using sodium hydroxide as a neutralizing agent. (1) Under aseptic conditions, pick melanin-producing short-stem molds from PDA slant culture medium ( Aureobasidium melanogenum ) 2pe-1 single colony was inoculated into primary seed culture medium and cultured at 25℃ and 180 r / min for 48 h; (2) Under aseptic conditions, the seed culture from step (1) was inoculated at 10% (v / v) into a fermenter culture medium with an initial sugar content of 190 g / L, and the culture was kept at 25°C, 800 r / min, and an aeration rate of 0.3 m³ / min. 3Fermentation was carried out in a 5 L fermenter at a rate of 20 g / L using 4 M sodium hydroxide as a neutralizing agent (4 M sodium hydroxide was prepared as a reagent for the alkali pump; when the pH of the fermentation broth was below 6, 4 M sodium hydroxide was added at a rate of 20 g / L to raise the pH to 5.95–6.05).

[0076] Fermentation Method 3: Fermentation in a fermentation tank, using calcium carbonate as a neutralizing agent. (1) Under aseptic conditions, pick melanin-producing short-stem molds from PDA slant culture medium ( Aureobasidium melanogenum ) 2pe-1 single colony was inoculated into seed culture medium and cultured at 25℃ and 180 r / min for 48 h; (2) Under aseptic conditions, the seed liquid described in step (1) was inoculated into a fermenter culture medium with an initial sugar content of 190 g / L at 10% (v / v). Fermentation was carried out in a 5 L fermenter at 25°C, 800 r / min, aeration rate of 0.3 m3 / h, and with 65 g / L calcium carbonate as a neutralizing agent (65 g / L calcium carbonate powder and fermentation culture medium were sterilized separately and then the powder was added to the fermentation liquid at one time, and the pH was controlled within the range of 6 to 7).

[0077] Example 6: Melanin Short-Stemmed Pulsatilla ( Aureobasidium melanogenum Application of tust 8-2 in polymalic acid fermentation The fermentation conditions in this embodiment are shake flask fermentation with 80 g / L glucose or a 5 L fermenter with 190 g / L initial glucose. The specific operating steps are as follows: Fermentation Method 1: Shake Flask Fermentation (1) Under aseptic conditions, pick melanin-producing short-stem molds from the slant culture medium ( Aureobasidium melanogenum )tust 8-2 single colony inoculated into seed culture medium and cultured at 25℃ and 180 r / min for 48 h; (2) Under aseptic conditions, the seed culture medium from step (1) was inoculated into the shake flask fermentation medium at an inoculation rate of 10% (v / v). Shake flask fermentation was carried out at 25°C, 180 r / min, and with 65 g / L calcium carbonate as a neutralizing agent (65 g / L calcium carbonate powder and shake flask fermentation medium were sterilized separately and then the powder was added to the fermentation liquid at one time, and the pH was controlled within the range of 6 to 7). Fermentation Method Two: Fermentation in a fermenter using sodium hydroxide as a neutralizing agent. (1) Under aseptic conditions, pick melanin-producing short-stem molds from the slant culture medium ( Aureobasidium melanogenum )tust 8-2 single colony inoculated into seed culture medium and cultured at 25℃ and 180 r / min for 48 h; (2) Under aseptic conditions, the seed liquid described in step (1) was inoculated at 10% (v / v) into a fermenter culture medium with an initial sugar content of 190 g / L. Fermentation was carried out in a 5 L fermenter at 25°C, 800 r / min, aeration rate of 0.3 m3 / h, and with 4 M sodium hydroxide as a neutralizing agent (4 M sodium hydroxide was prepared as an alkali pump replenishment reagent, and when the pH of the fermentation liquid was lower than 6, 4 M sodium hydroxide was added at a rate of 20 g / L to raise the pH to 5.95-6.05).

[0078] Fermentation Method 3: Fermentation in a fermentation tank using calcium carbonate as a neutralizing agent. (1) Under aseptic conditions, pick melanin-producing short-stem molds from the slant culture medium ( Aureobasidium melanogenum )tust 8-2 single colony inoculated into seed culture medium and cultured at 25℃ and 180 r / min for 48 h; (2) Under aseptic conditions, the seed liquid described in step (1) was inoculated into a fermenter culture medium with an initial sugar content of 190 g / L at 10% (v / v). Fermentation was carried out in a 5 L fermenter at 25°C, 800 r / min, aeration rate of 0.3 m3 / h, and with 65 g / L calcium carbonate as a neutralizing agent (65 g / L calcium carbonate powder and fermentation culture medium were sterilized separately and then the powder was added to the fermentation liquid at one time, and the pH was controlled within the range of 6 to 7).

[0079] The results of the 2pe-1 shake flask fermentation in Example 5 above are as follows: Figure 4 As shown in Figure 2pe-1, the shake-flask fermentation results of the mutant strain tust 8-2 from Example 6 above are as follows. Figure 4 As shown in the figure, mutant strain tust 8-2 outperformed the original strain 2pe-1 in polymalic acid fermentation under high glucose concentration conditions in terms of total sugar consumption and cell growth. Analysis of the entire fermentation process revealed that in shake-flask fermentation medium, the mutant strain tust 8-2 produced 18.32 g / L of polymalic acid, while the original strain 2pe-1 produced 6.98 g / L, representing a 1.6-fold increase in fermentation efficiency. In summary, the analysis indicates that through ARTP room temperature plasma mutagenesis combined with salt tolerance adaptation evolution, the mutant strain tust 8-2 exhibits higher glucose utilization efficiency and better conversion of glucose into polymalic acid during fermentation.

[0080] The fermentation results of the above fermenters are as follows: Figure 6 to Figure 8When 4M sodium hydroxide was used as a neutralizing agent, the mutant strain tust8-2 exhibited the highest polymalic acid (PMA) production at 64 h, with a PMA yield of 72.74 g / L, a production intensity of 1.13 g / L / h, and a sugar-acid conversion rate of 38.3%. Compared with the original strain 2pe-1, the PMA yield increased by 39.9%, and the sugar-acid conversion rate was effectively improved. In conclusion, the mutant strain tust8-2 possesses excellent cell performance and can utilize glucose more efficiently for PMA fermentation.

[0081] Example 7 Application of melanin-producing short-stem mold 2pe-1 polymalic acid fed-batch fermentation The fermentation conditions used in this embodiment are as follows: 190 g / L glucose as the initial sugar, 800 g / L glucose mother liquor as the feed, and sodium hydroxide or calcium carbonate as the neutralizing agent. The specific operating steps are as follows: Fermentation Method 1: Using sodium hydroxide as a neutralizing agent (1) Under aseptic conditions, pick melanin-producing short-stem molds from PDA slant culture medium ( Aureobasidium melanogenum ) 2pe-1 single colony was inoculated into seed culture medium and cultured at 25℃ and 180 r / min for 48 h; (2) Under aseptic conditions, the seed culture from step (1) was inoculated at 10% (v / v) into a fermenter culture medium containing 190 g / L glucose at 25°C, 800 r / min, and an aeration rate of 0.3 m³ / min. 3 Fermentation was carried out in a 5 L fermenter under the following conditions: 4 M sodium hydroxide was used as a neutralizing agent (4 M sodium hydroxide was prepared as a replenishing agent for the alkali pump; when the pH of the fermentation broth was lower than 6, 4 M sodium hydroxide was added at a rate of 20 g / L to raise the pH to 5.95–6.05). When the residual sugar was lower than 50 g / L, 300 mL of 800 g / L glucose mother liquor was added, and this was done three times in total. Fermentation was terminated after 120 h.

[0082] Fermentation Method Two: Using calcium carbonate as a neutralizing agent (1) Under aseptic conditions, pick melanin-producing short-stem molds from PDA slant culture medium ( Aureobasidium melanogenum ) 2pe-1 single colony was inoculated into seed culture medium and cultured at 25℃ and 180 r / min for 48 h; (2) Under aseptic conditions, the seed culture from step (1) was inoculated at 10% (v / v) into a fermenter culture medium containing 190 g / L glucose at 25°C, 800 r / min, and an aeration rate of 0.3 m³ / min. 3Fermentation was carried out in a 5 L fermenter under the following conditions: 65 g / L calcium carbonate was used as a neutralizing agent (65 g / L calcium carbonate powder and fermenter culture medium were sterilized separately, and the powder was added to the fermentation broth all at once, with the pH controlled within the range of 6-7); when the residual sugar was below 50 g / L, 300 mL of 800 g / L glucose mother liquor was added, and this was repeated three times. Fermentation was terminated after 120 h.

[0083] The above-mentioned residual sugar detection method is as follows: take 1 ml of fermentation broth, centrifuge, take the supernatant and dilute it 100 times, take the Yanhe Bio standard solution and calibrate it in the Yanhe Bio SBA-40E biosensor analyzer, and then directly detect the diluted sample to obtain the residual sugar content.

[0084] Example 8: Application of high-concentration polymalic acid fed-batch fermentation of melanin-producing short-stem mold tust 8-2. The fermentation conditions used in this embodiment are as follows: 190 g / L glucose as the initial sugar, 800 g / L glucose mother liquor as the feed, and sodium hydroxide or calcium carbonate as the neutralizing agent. The specific operating steps are as follows: Fermentation Method 1: Using sodium hydroxide as a neutralizing agent (1) Under aseptic conditions, pick melanin-producing short-stem molds from the slant culture medium ( Aureobasidium melanogenum )tust 8-2 single colony inoculated into seed culture medium and cultured at 25℃ and 180 r / min for 48 h; (2) Under aseptic conditions, the seed liquid described in step (1) was inoculated into the fermenter culture medium at 10% (v / v). Fermentation was carried out in a 5 L fermenter at 25℃, 800 r / min, aeration rate of 0.3 m3 / h, and 4 M sodium hydroxide as a neutralizing agent (4 M sodium hydroxide was prepared as an alkali pump replenishment reagent. When the pH of the fermentation liquid was lower than 6, 4 M sodium hydroxide was added at a rate of 20 g / L to raise the pH to 5.95-6.05). When the residual sugar was lower than 50 g / L, 300 mL of 800 g / L glucose mother liquor was added. This was done three times in total. Fermentation was stopped after 120 h.

[0085] Fermentation Method Two: Using calcium carbonate as a neutralizing agent (1) Under aseptic conditions, pick melanin-producing short-stem molds from the slant culture medium ( Aureobasidium melanogenum )tust 8-2 single colony inoculated into seed culture medium and cultured at 25℃ and 180 r / min for 48 h; (2) Under aseptic conditions, the seed liquid described in step (1) was inoculated into the fermenter culture medium at 10% (v / v). Fermentation was carried out in a 5 L fermenter at 25℃, 800 r / min, aeration rate of 0.3 m3 / h, and 65 g / L calcium carbonate as a neutralizing agent (65 g / L calcium carbonate powder and fermenter culture medium were sterilized separately and then the powder was added to the fermentation liquid at one time, and the pH was controlled within the range of 6 to 7). When the residual sugar was less than 50 g / L, 300 mL of 800 g / L glucose mother liquor was added. This was done three times in total. Fermentation was stopped after 120 h.

[0086] The results of the four batch feed fermentation processes in the four 5L fermenters described in Examples 7 and 8 above are as follows: Figure 9 to Figure 10 When 4M sodium hydroxide was used as a neutralizing agent, the mutant strain tust8-2 was replenished with 300 mL of 800 g / L glucose solution three times after 120 h of fermentation. The polymalic acid yield in the fermentation broth was detected to reach 83.5 g / L (the polymalic acid yield detected by liquid chromatography at this time was the volume dilution and not converted to yield). The use of sodium hydroxide as a neutralizing agent during the fermentation process increased the final fermentation volume by 53.3%. When converted to the initial volume of batch fermentation, the actual polymalic acid yield reached 128 g / L, the production intensity was 1.07 g / L / h, and the sugar-acid conversion rate was 35.9%. Under the same conditions, the polymalic acid yield was increased by 45% compared with the starting strain 2pe-1.

[0087] When calcium carbonate was used as a neutralizing agent, the mutant strain tust 8-2, after 120 h of fermentation, was supplemented with 300 mL of 800 g / L glucose solution three times. The polymalic acid (PMLA) yield in the fermentation broth was detected to be 94.5 g / L. Simultaneously, the addition of feed increased the final fermentation volume by 30%. Converted to the initial volume of the batch fermentation, the actual PMLA yield reached 122.8 g / L, with a production intensity of 1.02 g / L / h and a sugar-acid conversion rate of 34%. Under the same conditions, compared with the starting strain 2pe-1, the PMLA yield was increased by 33%. In conclusion, the mutant strain tust8-2, even with 4M sodium hydroxide as a neutralizing agent, still possesses high PMLA production capacity and excellent cell performance, and can utilize glucose more efficiently for PMLA fermentation.

[0088] Example 9: Application of Aureobasidium melanogenum tust 8-2 in polymalic acid fermentation The fermentation conditions in this embodiment are: 250 g / L initial sugar, glucose in a 5 L fermenter. The specific operating steps are as follows: (1) Under aseptic conditions, pick a single colony of Aureobasidium melanogenum tust 8-2 from the slant culture medium and inoculate it into the seed culture medium. Culture at 25℃ and 180 r / min for 48 h. (2) Under aseptic conditions, the seed liquid described in step (1) was inoculated at 10% (v / v) into a fermenter culture medium with an initial sugar content of 250 g / L glucose. Fermentation was carried out in a 5 L fermenter at 25°C, 800 r / min, aeration rate of 0.3 m3 / h, and 4 M sodium hydroxide as a neutralizing agent (4 M sodium hydroxide was prepared as a reagent for alkali pump replenishment; when the pH of the fermentation liquid was lower than 6, 4 M sodium hydroxide was added at a rate of 20 g / L to raise the pH to 5.95–6.05).

[0089] Example 10: Application of Aureobasidium melanogenum tust 8-2 in polymalic acid fermentation The fermentation conditions in this embodiment are: 300 g / L initial sugar, 5 L glucose fermenter, and the specific operating steps are as follows: (1) Under aseptic conditions, pick a single colony of Aureobasidium melanogenum tust 8-2 from the slant culture medium and inoculate it into the seed culture medium. Culture at 25℃ and 180 r / min for 48 h. (2) Under aseptic conditions, the seed liquid described in step (1) was inoculated into a fermenter culture medium with an initial sugar content of 300 g / L glucose at 10% (v / v). Fermentation was carried out in a 5 L fermenter at 25°C, 800 r / min, aeration rate of 0.3 m3 / h, and 4 M sodium hydroxide as a neutralizing agent (4 M sodium hydroxide was prepared as a reagent for alkali pump replenishment; when the pH of the fermentation liquid was lower than 6, 4 M sodium hydroxide was added at a rate of 20 g / L to raise the pH to 5.95-6.05).

[0090] The fermentation results of the above 5 L fermenter are as follows: Figure 11 As shown, when the initial sugar concentration was increased to 250 g / L, the mutant strain tust8-2 completely depleted glucose after 72 h of fermentation, achieving a polymalic acid (PMLA) yield of 67 g / L, a production intensity of 0.93 g / L / h, and a sugar-acid conversion rate of 26.8%. When the initial sugar concentration was increased to 300 g / L, the mutant strain tust8-2 completely depleted glucose after 136 h of fermentation, achieving a PMLA yield of 86 g / L, a production intensity of 0.63 g / L / h, and a sugar-acid conversion rate of 28.7%. In conclusion, the mutant strain tust8-2 maintains high PMLA production capacity and excellent cell performance even under high sugar conditions (initial sugar concentration of 300 g / L), and can efficiently utilize glucose for PMLA fermentation.

[0091] Example 11: Application of Aureobasidium melanogenum tust 2pe-1 in polymalic acid fermentation The fermentation conditions in this embodiment involve adding an additional 70 g / L NaCl to a 5 L fermenter. The specific operating steps are as follows: (1) Under aseptic conditions, pick a single colony of Aureobasidium melanogenum tust 8-2 from the slant culture medium and inoculate it into the seed culture medium. Culture at 25℃ and 180 r / min for 48 h. (2) Under aseptic conditions, the seed culture from step (1) was inoculated at 10% (v / v) into a fermenter culture medium containing 190 g / L glucose at 25°C, 800 r / min, and an aeration rate of 0.3 m³ / min. 3 Fermentation was carried out in a 5 L fermenter under the following conditions: 4M sodium hydroxide was used as a neutralizing agent, and 70 g / L NaCl was added (4M sodium hydroxide was prepared as a replenishing agent for the alkali pump; when the pH of the fermentation broth was lower than 6, 4M sodium hydroxide was added at a rate of 20 g / L to raise the pH to 5.95–6.05; in addition, 70 g / L NaCl (final concentration) was added to the fermentation broth in one go and sterilized together with the fermenter culture medium).

[0092] Example 12 Application of Aureobasidium melanogenum tust 8-2 in polymalic acid fermentation The fermentation conditions in this embodiment involve adding an additional 70 g / L NaCl to a 5 L fermenter. The specific operating steps are as follows: (1) Under aseptic conditions, pick a single colony of Aureobasidium melanogenum tust 8-2 from the slant culture medium and inoculate it into the seed culture medium. Culture at 25℃ and 180 r / min for 48 h. (2) Under aseptic conditions, the seed culture from step (1) was inoculated at 10% (v / v) into a fermenter culture medium containing 190 g / L glucose at 25°C, 800 r / min, and an aeration rate of 0.3 m³ / min. 3 Fermentation was carried out in a 5 L fermenter under the following conditions: 4M sodium hydroxide was used as a neutralizing agent, and 70 g / L NaCl was added (4M sodium hydroxide was prepared as a replenishing agent for the alkali pump; when the pH of the fermentation broth was lower than 6, 4M sodium hydroxide was added at a rate of 20 g / L to raise the pH to 5.95–6.05; the remaining 70 g / L NaCl (final concentration) was added to the fermentation broth in one go and sterilized together with the fermenter culture medium).

[0093] The results of the batch fermentation in the above 5 L fermenter are as follows: Figure 12 As shown, when an additional 70 g / L NaCl was added, the biomass of the mutant strain tust8-2 increased significantly after 72 h of fermentation, and the strain was able to grow normally; while the original strain 2pe-1 showed no growth trend after 72 h of fermentation and the strain could not grow normally. In conclusion, the mutant strain tust8-2 possesses salt tolerance.

[0094] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A salt-tolerant, melanin-producing short-stemmed fungus strain, characterized in that, The strain is *Tust8-2*, a melanin-producing short-stalked fungus, with accession number CGMCC No. 42419.

2. The salt-tolerant melanin-producing short-stemmed fungus strain according to claim 1, characterized in that, The strain is capable of growing in a medium with a NaCl concentration of 6-7% (w / v); and / or, when the strain ferments to produce polymalic acid in a fermentation system with NaOH as a neutralizing agent, the polymalic acid yield is ≥72 g / L.

3. A microbial inoculant, characterized in that, It comprises the salt-tolerant melanin-producing short-stem fungus strain as described in claim 1 or 2, its inactivated form, its culture, its ferment, its extract, and / or at least one metabolite.

4. A method for breeding a salt-tolerant, melanin-producing short-stalked spp. strain as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Using the melanin-producing short-stalked mold as the starting strain, the ARTP mutant library was obtained by mutagenesis using a normal pressure room temperature plasma mutagenesis system. (2) The ARTP mutant library obtained in step (1) was inoculated into a liquid culture medium containing an increasing concentration of NaCl for continuous subculture and targeted screening of mutant strains with salt tolerance. (3) Spread the bacterial solution obtained in step (2) onto a screening plate containing an indicator and select candidate strains with strong acid production ability; (4) The candidate strains selected in step (3) were subjected to shake-flask fermentation, and the polymalic acid yield was measured. The strain with the highest polymalic acid yield was selected from them.

5. The breeding method according to claim 4, characterized in that, The starting strain mentioned in step (1) is *Synthia spp.* 2pe-1, with the preservation number CGMCC No. 42418.

6. The breeding method according to claim 4, characterized in that, The parameters for atmospheric pressure room temperature plasma mutagenesis in step (1) are: radio frequency power 100-120 W, helium flow rate 8-12 SLM, distance between plasma torch nozzle outlet and substrate 2-5 mm, and mutagenesis time 30-360 s; And / or, the initial concentration of NaCl in step (2) is 0.5% (w / v), the NaCl concentration increases by 0.5% (w / v) after each subculture, and the final concentration is 7% (w / v); And / or, the screening plate containing the indicator mentioned in step (3) is a bromocresol green indicator PDA plate and / or a treben blue polysaccharide screening plate; And / or, the fermentation temperature of the shake flask fermentation in step (4) is 25-28℃, the rotation speed is 180-220 r / min, and the fermentation time is 48-72h.

7. The application of the salt-tolerant melanin-producing short-stem mold strain according to claim 1 or 2, the microbial agent according to claim 3, or the salt-tolerant melanin-producing short-stem mold strain selected by the selection method according to any one of claims 4-6 in the production of polymalic acid.

8. A method for producing polymalic acid, characterized in that, Fermentation includes using salt-tolerant melanin-producing short-stem mold strains as described in claim 1 or 2, microbial agents as described in claim 3, or the breeding methods described in any one of claims 4-6.

9. The method for producing polymalic acid according to claim 8, characterized in that, Includes the following steps: (1) The melanin-producing short-skinned pustulosa tust8-2 was inoculated into seed culture medium for seed culture to obtain seed liquid; (2) The seed liquid obtained in step (1) is inoculated into the fermentation medium and fermented at 25°C. During the fermentation process, NaOH is used as a neutralizing agent to control the pH. (3) After fermentation, polymalic acid is separated and purified from the fermentation broth.

10. The method for producing polymalic acid according to claim 9, characterized in that, The NaCl concentration in the fermentation medium is ≤7% (w / v); And / or, the fermentation medium is a high-sugar medium; optionally, the concentration of glucose in the fermentation medium is ≤300g / L.

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