Aspergillus unguiculatus strain and use thereof

Abstract

The present application relates to a strain of Aspergillus elapse, which is preserved in China Center for Type Culture Collection on March 30, 2022, and the preservation number is CCTCC NO: M 2022348. The strain of Aspergillus elapse has a good application prospect in cultivating transgenic plants or resistant genetically engineered bacteria with salt tolerance and / or sugar tolerance and / or osmotic pressure tolerance, solving the problem of soil salinization and treating salt-containing high-osmotic-pressure organic wastewater.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Aspergillus oryzae and its applications. Background Technology

[0002] Currently, microorganisms are widely used in food processing, medicine, cosmetics research and development, energy development, and bioelectronics. The application of microorganisms has promoted the technological transformation of traditional industries and the emergence of new industries, which has had a profound impact on human social life and also has huge economic potential.

[0003] To address the problems caused by the impact of high salt, high sugar, and high osmotic pressure environments on normal production and daily life—such as the reduced efficiency of pollutant treatment due to the inhibition of microbial activity caused by the high osmotic pressure of organic wastewater, and the need for strains with physiological characteristics and metabolic mechanisms that are salt-tolerant, sugar-tolerant, and osmotic pressure-tolerant—and to provide genes for these characteristics to cultivate transgenic plants and engineered bacteria, thereby solving these problems. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a strain of Aspergillus buxiflorus and its applications.

[0005] The technical solution of the present invention is: a strain of Aspergillus spp., which was deposited at the China Center for Type Culture Collection on March 30, 2022, with accession number CCTCC NO: M 2022348.

[0006] This invention also discloses the application of the above-mentioned Aspergillus spp. strain in the cultivation of transgenic plants and resistant genetically engineered bacteria.

[0007] Furthermore, the above-mentioned Aspergillus tufts are used in the cultivation of salt- and / or sugar- and / or osmotic pressure-resistant genetically engineered bacteria. The cultivated resistant genetically engineered bacteria can adapt well to high-salt, high-sugar, and high-osmotic-pressure environments and perform better.

[0008] Furthermore, the above-mentioned Aspergillus tufts are used in the cultivation of transgenic plants that are salt-tolerant and / or sugar-tolerant and / or osmotic pressure-tolerant.

[0009] Furthermore, the genetically modified plant is Arabidopsis thaliana, a transgenic plant with salt tolerance that can grow well in saline soils, thereby improving soil salinization problems.

[0010] The present invention also discloses the application of the above-mentioned Aspergillus cereus strain in the treatment of high osmotic pressure organic wastewater with an osmotic pressure between 280 and 700 mmol / L.

[0011] Furthermore, the high osmotic pressure organic wastewater is saline organic wastewater. The salt tolerance of Aspergillus flavomarginata strains can prevent the activity of Aspergillus flavomarginata strains from being inhibited during the treatment of saline high osmotic pressure organic wastewater, thus preventing the wastewater treatment efficiency from being affected.

[0012] The beneficial effects of this invention are:

[0013] (1) The present invention provides a strain of Aspergillus flavus with physiological characteristics and metabolic mechanism of salt tolerance, sugar tolerance and osmotic pressure tolerance. It can produce bioactive substances with physiological characteristics of salt tolerance, sugar tolerance and osmotic pressure tolerance, which can be used to cultivate transgenic plants with salt tolerance and / or sugar tolerance and / or osmotic pressure tolerance, laying the foundation for further cultivation of highly efficient salt-tolerant and high osmotic pressure-tolerant plants and solving the problem of soil salinization.

[0014] (2) The Aspergillus broom strain provided by the present invention can provide excellent resistance genes for salt and / or sugar and / or osmotic pressure resistance for molecular biology research; using these resistance genes, genetically engineered bacteria such as Escherichia coli and yeast can be produced.

[0015] (3) The present invention provides a strain of Aspergillus broom, which can treat organic wastewater with high salinity and high osmotic pressure. Attached Figure Description

[0016] Figure 1 The colony morphology of Aspergillus fusiforme strain cultured for 7 days at 28°C and 60% humidity on MEA medium with an additional 10g NaCl.

[0017] Figure 2 The colony morphology of Aspergillus fusiforme strain cultured for 7 days at 28°C and 60% humidity on MEA medium with an additional 60g of glucose.

[0018] Figure 3 The growth curves of colonies in each NaCl experimental group during the salt tolerance test of Aspergillus fusiforme strains are shown after 7 days.

[0019] Figure 4 The 7-day growth curves of colonies in each glucose experimental group were used to determine the sugar tolerance of Aspergillus fusiforme strains.

[0020] Figure 5 The 7-day growth curves of colonies in each NaCl+glucose experimental group were used in the determination of the salt-sugar tolerance characteristics of Aspergillus broomii strains.

[0021] Figure 6 The growth curves of salt-tolerant and salt-intolerant yeasts on yeast limit solid medium supplemented with an additional 10g NaCl are shown in the figures. Detailed Implementation

[0022] Example 1

[0023] (1) Cultivation of Aspergillus broomii strains

[0024] Either MEA or YCA medium can be used to culture Aspergillus flavus strains. The following uses MEA medium to culture Aspergillus flavus strains.

[0025] Using MEA as the basal medium, according to the formula and instructions, 5g, 10g, and 15g of NaCl were added to three 100mL MEA basal mediums respectively. The medium was then autoclaved at 121℃ for 15min, and the culture dishes were poured out to prepare salt-tolerant medium for cooling and later use. An equal amount of Aspergillus flavus strain was inoculated onto the salt-tolerant medium plate and cultured at 28℃ and 60% humidity for 7 days to obtain salt-tolerant and osmotic pressure-resistant Aspergillus flavus strains with colony diameters of 1-2cm.

[0026] (2) Determination of salt and osmotic pressure tolerance of Aspergillus oryzae strains

[0027] The growth of the three salt-tolerant and osmotic pressure-tolerant Aspergillus strains obtained in (1) above was observed. As a control, Aspergillus strains inoculated on 100 mL MEA medium plates without additional NaCl were cultured using the same method as in (1). The growth of the Aspergillus strains was observed, and the colony diameter of each group was measured every other day (since the colony diameter after initial inoculation is negligible, it is counted as 0). The measured data are shown in Table 1.

[0028] Table 1. Record of colony diameter in each NaCl experimental group

[0029]

[0030] From Table 1 and Figure 3 Analysis showed that *Aspergillus flabellulatum* strains did not grow on MEA medium without additional NaCl. They grew on MEA media containing 5g, 10g, and 15g NaCl, but grew best on MEA medium containing 10g NaCl, while growth was slower on MEA media containing 5g and 15g NaCl. Therefore, *Aspergillus flabellulatum* strains can grow on MEA media with different NaCl concentrations, but the growth rate varies, with the highest efficiency observed on MEA medium containing 10g NaCl.

[0031] Sabouraud broth was aliquoted into four 50ml Erlenmeyer flasks. Following the Sabouraud broth formula and instructions, 5g, 10g, 15g, and 20g of NaCl were added to each flask respectively. The flasks were then autoclaved at 121℃ for 15 minutes. An equal volume of Sabouraud broth without added NaCl was added to another 50ml Erlenmeyer flask as a control. Equal volumes of Aspergillus flavus strain were inoculated into each flask. After culturing at 28℃ and 60% humidity for 10 days, the coverage rate of the Aspergillus flavus strain on the surface of the flasks was measured (since the initial colony coverage rate was negligible, it was recorded as 0). The results are shown in Table 2.

[0032] Table 2. Coverage of the Erlenmeyer flask surface by Aspergillus strains grown in each NaCl experimental group.

[0033]

[0034] As shown in Table 2, Aspergillus flavus strains did not grow in Sabouraud broth without additional NaCl. They grew in Sabouraud broth containing 5g, 10g, 15g, and 20g NaCl, but grew faster in Sabouraud broth containing 10g NaCl, slower in Sabouraud broth containing 5g and 15g NaCl, and extremely slowly in Sabouraud broth containing 20g NaCl.

[0035] Based on the above experiments, it can be shown that this Aspergillus broomi strain has the characteristics of salt resistance and osmotic pressure resistance.

[0036] Example 2

[0037] (1) Cultivation of Aspergillus broomii strains

[0038] Using MEA as the basal medium, according to the formula and instructions, 20g, 40g, 60g, 80g, 100g, 120g, 140g, 160g, and 180g of glucose were added to nine 100mL MEA basal mediums respectively. The medium was then autoclaved at 121℃ for 15min, and the culture dishes were poured out to prepare glucose-tolerant medium for cooling and later use. An equal amount of Aspergillus flavus strain was inoculated onto the glucose-tolerant medium plate and cultured at 28℃ and 60% humidity for 7 days to obtain glucose-tolerant and osmotic pressure-resistant Aspergillus flavus strains with colony diameters of 1-2cm.

[0039] (2) Determination of sugar tolerance and osmotic pressure tolerance of Aspergillus oryzae strains

[0040] The culture and growth of the nine sugar-tolerant and osmotic pressure-tolerant Aspergillus strains obtained in (1) above were observed. As a control, Aspergillus strains inoculated on 100 mL MEA medium plates without additional glucose were cultured using the culture method described in (1) above. The culture and growth of the Aspergillus strains were observed. The colony diameter of each group was measured every other day (since the colony diameter after initial inoculation is negligible, it is counted as 0). The measured data are shown in Table 3.

[0041] Table 3. Record of Colony Diameter in Each Glucose Experimental Group

[0042]

[0043]

[0044] From Table 3 and Figure 4 Analysis showed that *Aspergillus flavomarginata* strains did not grow on MEA medium without added glucose. They grew on MEA medium containing 20g, 40g, 60g, 80g, 100g, 120g, 140g, 160g, and 180g of glucose, but grew best on MEA medium containing 60g of glucose. Growth was slower on MEA medium containing 20g, 40g, 80g, 100g, 120g, 140g, and 160g of glucose, and extremely slow on MEA medium containing 180g of glucose. Therefore, *Aspergillus flavomarginata* strains can grow on various MEA mediums containing glucose, but the growth rate differs, with the highest efficiency observed on MEA medium containing 60g of glucose.

[0045] Sabouraud broth was aliquoted into five 50ml Erlenmeyer flasks. Following the Sabouraud broth formula and instructions, 40g, 60g, 80g, 100g, and 120g of glucose were added to each flask respectively. The flasks were then autoclaved at 121℃ for 15 minutes. An equal volume of Sabouraud broth without added glucose was added to another 50ml Erlenmeyer flask as a control. Aspergillus flavus strain was inoculated into each flask in equal amounts. After culturing at 28℃ and 60% humidity for 7 days, the coverage rate of the Aspergillus flavus strain on the surface of the flasks was measured (since the initial colony coverage rate was negligible, it was recorded as 0). The results are shown in Table 4.

[0046] Table 4. Coverage of the Erlenmeyer flask surface by Aspergillus strains grown in each glucose experimental group.

[0047]

[0048] As shown in Table 4, Aspergillus flavus strains did not grow in Sabouraud broth without added glucose. They could grow in Sabouraud broth containing 40g, 60g, 80g, 100g, and 120g of glucose, but the growth rate was fastest in Sabouraud broth containing 60g of glucose. Growth was slower in Sabouraud broth containing 40g, 80g, and 100g of glucose, and extremely slow in Sabouraud broth containing 120g of glucose.

[0049] Based on the above experiments, it can be shown that this Aspergillus broomi strain has the characteristics of sugar resistance and high osmotic pressure resistance.

[0050] Example 3

[0051] (1) Cultivation of Aspergillus broomii strains

[0052] Using MEA as the basal medium, according to the formula and instructions, add 5g NaCl + 20g glucose, 10g NaCl + 60g glucose, and 15g NaCl + 80g glucose to three 100mL MEA basal mediums respectively. Autoclave at 121℃ for 15min, pour out the culture dishes, and prepare salt-tolerant and sugar-tolerant medium for cooling and later use. Inoculate an equal amount of Aspergillus flavus strain onto salt-tolerant and sugar-tolerant medium plates and culture at 28℃ and 60% humidity for 7 days to obtain salt-tolerant and sugar-tolerant Aspergillus flavus strains with colony diameters of 1-2cm.

[0053] (2) Determination of salt-sugar tolerance characteristics of Aspergillus fusiforme strains

[0054] The growth of the three salt-tolerant and sugar-tolerant Aspergillus strains obtained in (1) above was observed. Aspergillus strains inoculated on MEA medium plates without additional NaCl and glucose were cultured as controls using the culture method in (1) above. The growth of the Aspergillus strains was observed. The colony diameter of each group was measured every other day (since the colony diameter after initial inoculation can be ignored, it is counted as 0). The measured data are shown in Table 5.

[0055] Table 5. Record of colony diameter in each NaCl+ glucose experimental group.

[0056]

[0057] From Table 5 and Figure 5Analysis showed that *Aspergillus flabellulatum* strains did not grow on MEA medium without additional NaCl and glucose. They grew on MEA media containing 5g NaCl + 20g glucose, 10g NaCl + 60g glucose, and 15g NaCl + 80g glucose, but grew best on MEA medium containing 10g NaCl + 60g glucose. Growth was slower on MEA media containing 5g NaCl + 20g glucose and 15g NaCl + 80g glucose. Therefore, *Aspergillus flabellulatum* strains can grow on various MEA media containing NaCl and glucose, but the growth rate differs, with the highest efficiency observed on MEA medium containing 10g NaCl + 60g glucose.

[0058] Based on the above experiments, it can be concluded that this Aspergillus broomi strain has salt- and sugar-tolerant characteristics.

[0059] Example 4

[0060] Isolation and purification of Aspergillus broom-shaped strains

[0061] Using one of the following media: MEA medium with additional 5g, 10g, or 15g NaCl; MEA medium with additional 20g, 40g, 60g, 80g, 100g, 120g, 140g, 160g, or 180g glucose; or MEA medium with additional 5g NaCl + 20g glucose, 10g NaCl + 60g glucose, or 15g NaCl + 80g glucose, single colonies can be isolated by streaking or stab inoculation. These single colonies can be purified by re-streaking or stab inoculating into the aforementioned media. Repeated purification is performed until no contaminants are detected under a microscope. Because this bacterium produces conidia, it should be preserved on test tube slant agar to prevent contamination.

[0062] Example 5

[0063] Identification of Aspergillus broom-shaped strains

[0064] (1) Morphological observation of Aspergillus broom strains

[0065] On salt-only plates, the colonies of this fungus are circular on the front, with an uneven surface, a raised center, wrinkled edges, a dense and dry texture, no exudate, and a black or gray color, with a darker color in the raised center; the back is circular, black in the center, and grayish-white around the edges. The colony morphology of *Aspergillus buxiflorus* strain cultured for 7 days at 28°C and 60% humidity on MEA medium supplemented with 10g NaCl is shown below. Figure 1 .

[0066] On sugar-only plates, the colonies of this bacterium are circular with a raised center. Initially, the center of the colony is light green with a grayish-white periphery, and the surface is uneven and velvety. With prolonged incubation, the colony color gradually turns dark green to black, the center collapses, and wrinkles form on the surface. When gently lifted with an inoculation needle, it detaches from the plate in flakes. The reverse side is circular with a light black center and a light gray periphery with a white periphery. The colony morphology of *Aspergillus buxiflorus* strain cultured for 7 days on MEA medium supplemented with 60g of glucose at 28°C and 60% humidity is shown below. Figure 2 .

[0067] Microscopic examination: In the early stages of growth, with sufficient nutrients, conidiophores branch to produce sterigmata. During the vigorous growth period, conidiophores form spherical heads at the distal ends of the sterigmata. The conidia are cord-like, borne at the apical sacs of the conidiophores, arranged radially. Under a scanning electron microscope, the conidiophores are 40-60 micrometers long, some even longer than 40-60 micrometers, and 2-3 micrometers wider, with a smooth surface. The conidia are cylindrical, flat at both ends and slightly bulging in the middle, 2-3 micrometers long and 1-2 micrometers wide, with an uneven, wrinkled surface.

[0068] (2) Molecular biological identification of Aspergillus broom strains

[0069] Genomic DNA was extracted from Aspergillus penicillioides using a genomic DNA extraction kit. The genomic DNA was amplified, and the amplified products were detected by electrophoresis. The samples were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO.1. Based on the results report, the strain was preliminarily identified as Aspergillus, and presumed to be Aspergillus penicillioides. The preservation classification name is Aspergillus penicillioidesstcustom01.

[0070] DNA sequencing, combined with the morphological characteristics of the strain, identified the strain as Aspergillus fusiformis. This strain was deposited on March 30, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M2022348. The nucleotide sequence is shown in SEQ ID No. 1.

[0071] SEQ ID No. 1:

[0072] ggatgccctt ctcggggtgt ccgtcctccc atccgtgtct atttgtaccc tgttgcttcggcgggcccgc ccttcgtggc cgccgggggg cttccctgcc cccgggcccg tgcccgccgg agacctcaaccatgaacact gtctgaaggt tgcagtctga gtaccgatat aaaaaatcgt taaaactttc aacaacggatctcttggttc cggcatcgat gaagaacgca gcgaaatgcg atacgtaatg tgaattgcag aattcagtgaatcatcgagt ctttgaacgc acattgcgcc ccctggtatt ccggggggca tgcctgtccg agcgtcattgctgccctcaa gcacggcttg tgtgttgggc ccccgtcccc ctcccaggaa ggggacgggc ccgaaaggcagcggcggcac cgtgtccggt cctcgagcgt atgggaagca acttttttgt cacccgctcc tgtaggtccggccggcggcc tgcccaaccc caaccttctt tttttaacca ggtgacctcggatc

[0073] Example 6

[0074] A salt-tolerant Aspergillus strain was cultured according to the culture method in Example 1. The salt-tolerant resistance gene of the salt-tolerant Aspergillus strain was transferred into Arabidopsis thaliana via Agrobacterium tumefaciens-mediated transformation to obtain transgenic Arabidopsis thaliana. The following experiments aim to demonstrate that the transgenic Arabidopsis thaliana cultivated using the salt-tolerant Aspergillus strain has salt-tolerant characteristics compared to non-transgenic Arabidopsis thaliana.

[0075] Transgenic Arabidopsis thaliana was selected, with non-transgenic Arabidopsis thaliana as a control. Transgenic Arabidopsis thaliana seeds and non-transgenic Arabidopsis thaliana seeds were planted in soil with similar fertility from the same area of ​​a farmland in this city. They were cultivated according to conventional methods, and were also irrigated daily with 400 ml of a 10% NaCl aqueous solution for 30 days. The germination rate of transgenic Arabidopsis thaliana seeds and control Arabidopsis thaliana seeds after 10 days of planting and the seedling survival rate after 30 days were recorded. The results are shown in Table 6.

[0076] Table 6. Records of germination rate and seedling survival rate

[0077] Transgenic Arabidopsis 85.1 81.3 Non-GMO Arabidopsis thaliana 20.3 11.6

[0078] Through observation and recording, it was found that when transgenic Arabidopsis seeds and non-transgenic Arabidopsis seeds were watered with 400ml of a 10% NaCl aqueous solution every day for 30 days, the non-transgenic Arabidopsis seeds developed slowly or even stopped developing, with only a few seeds germinating and a low survival rate after germination. In contrast, most transgenic Arabidopsis seeds developed normally and germinated, with a high survival rate after germination. Therefore, it can be proven that transgenic Arabidopsis has significantly higher salt tolerance than the non-transgenic control group and can be planted in soil with high salt content.

[0079] Example 7

[0080] Salt-tolerant Aspergillus strains were cultured according to the culture method in Example 1. The salt tolerance gene of the salt-tolerant Aspergillus strains was obtained by genetic engineering to produce salt-tolerant yeast with high-efficiency expression of the salt tolerance gene. The following experiments aim to clarify that salt-tolerant yeast has salt tolerance characteristics compared with non-salt-tolerant yeast.

[0081] Salt-tolerant yeasts were selected, with salt-intolerant yeasts serving as controls. Both types of yeasts were inoculated into yeast limit liquid medium and incubated at 30°C for 24 hours. The bacterial solutions were then diluted and inoculated onto yeast limit solid medium supplemented with 10g NaCl, and incubated at 30°C for 24 hours. Every 4 hours, the bacterial solutions were aspirated and the OD values ​​of the salt-tolerant and salt-intolerant yeasts were measured at 600nm.

[0082] like Figure 6 As shown, yeast strains with salt-resistance genes can grow normally on yeast limit solid medium supplemented with 10%, while yeast strains without salt-resistance genes do not grow. These experimental results indicate that yeast strains with salt-resistance genes produced using Aspergillus rufiformis strains possess salt tolerance.

[0083] Meanwhile, using the *Aspergillus frenulum* strains with sugar tolerance and salt-to-sugar tolerance characteristics obtained from Experiments 2 and 3, respectively, to produce sugar-tolerant yeast and salt-to-sugar-tolerant yeast, the results were essentially the same as the above conclusions. This demonstrates that sugar-tolerant yeast exhibits sugar tolerance compared to non-sugar-tolerant yeast, and salt-to-sugar-tolerant yeast exhibits salt-to-sugar tolerance compared to non-salt-to-sugar-tolerant yeast.

[0084] Example 8

[0085] Salt-tolerant Aspergillus strains can effectively treat high-salt organic wastewater. The following experiment aims to clarify the treatment effect of this Aspergillus strain on high-salt organic wastewater.

[0086] Sample 1 (700 mmol / L sodium chloride high osmotic pressure organic wastewater) and sample 2 (580 mmol / L sodium chloride high osmotic pressure organic wastewater) of high salinity organic wastewater discharged during the production process of a chemical plant in this city were selected. Both samples were evenly divided into two parts. The relevant parameters of the samples are shown in Table 7.

[0087] Table 7 Sample-related parameters

[0088] Sample 1 (700 mmol / L) 8000 Sample 2 (580 mmol / L) 8000

[0089] Following the cultivation method described in Example 1, salt-tolerant Aspergillus flavus strains were cultured to the late logarithmic growth stage. Salt-tolerant Bacillus strains cultured to the late logarithmic growth stage were used as controls. Both the cultured Aspergillus flavus and salt-tolerant Bacillus strains were inoculated into four samples at a ratio of 20% of the sample volume. The culture temperature was controlled at 30℃, and after static incubation for 24 hours, the COD degradation in the high-salt organic wastewater was measured. The results of the two strains on the sample treatment are shown in Table 8.

[0090] Table 8 Results of sample treatment by the two strains

[0091]

[0092] The test results in Table 8 show that, at 24 hours, the COD removal rate of the samples with added Aspergillus fusiformis strain can reach 96.7%, while the COD removal rate of the samples with added halophilic Bacillus strain is 90.2% and 90.1%, respectively. Therefore, it can be concluded that the Aspergillus fusiformis strain can efficiently treat high-salt organic wastewater.

Claims

1. A strain of Aspergillus penicillioides, characterized in that, It is deposited at the China Center for Type Culture Collection on March 30, 2022, with accession number CCTCC NO: M 2022348.

2. The application of the Aspergillus broom strain as described in claim 1 in treating high osmotic pressure organic wastewater with an osmotic pressure between 280 and 700 mmol / L, thereby degrading COD in high-salt organic wastewater.

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