Recombinant Strains for Improving the Production Level and Fermentation Intensity of Organic Acids, and Methods for Constructing and Using the Same

Abstract

The present invention relates to bioengineering technology, and discloses a recombinant strain for improving the production level and fermentation intensity of organic acids, and a construction method and application thereof. The recombinant strain is obtained by genetically engineering a starting strain. Compared with the starting strain, the coding gene of the anchor protein GPI is overexpressed in the recombinant strain. The construction method of the recombinant strain includes: genetically engineering the starting strain so that the coding gene of the anchor protein GPI is overexpressed in the starting strain. The present invention also provides the application of the recombinant strain and / or the recombinant strain obtained by the construction method in the production of organic acids. The production level and fermentation intensity of organic acids of the recombinant strain are significantly improved, the production cost of organic acids is effectively reduced, and it has the prospect of industrial application.

Description

Technical Field

[0001] The present invention relates to bioengineering technology. Specifically, it relates to a recombinant strain for improving the production level and fermentation intensity of organic acids, a construction method for a recombinant strain for improving the production level and fermentation intensity of organic acids, and an application of a recombinant strain for improving the production level and fermentation intensity of organic acids. Background Art

[0002] Four-carbon organic acids are a class of low-molecular-weight organic compounds containing 4 carbon atoms and having one or two acidic functional groups. Four-carbon organic acids represented by fumaric acid, malic acid, and succinic acid have been widely used in fields such as food, medicine, cosmetics, pesticides, detergents, and textiles. They are important platform chemicals and have great market application value.

[0003] Traditional production methods of four-carbon organic acids mainly use chemical synthesis or enzymatic conversion of petrochemical raw materials. However, chemical synthesis and enzymatic conversion have problems such as high temperature, substrate inhibition effect, easy formation of by-products, high substrate cost, depletion of petroleum resources, and greenhouse gas emissions, which hinder the sustainable large-scale production of organic acids. Therefore, under the trend of seeking industrial sustainable solutions, microbial fermentation for the production of organic acids has received extensive attention. Compared with chemical synthesis and enzymatic conversion, using renewable biomass raw materials and relying on biological fermentation to produce four-carbon organic acids has advantages such as high fermentation efficiency and low cost, and is considered a more promising route.

[0004] Through a large number of studies on the biosynthesis of four-carbon organic acids, a production method has been obtained in which filamentous fungi convert carbon sources such as glucose into organic acids through glycolysis and other pathways under stress conditions. However, stress conditions affect the physiological metabolic activities of filamentous fungi to a certain extent, thereby reducing the fermentation performance. Cellular adaptive engineering targeting the stress defense pathway may be a key step in delivering highly efficient microbial biocatalysts to achieve high titer, high yield, and high productivity. However, there is currently no effective method to enhance stress tolerance and increase glycolytic flux using metabolic engineering means to improve the production efficiency of organic acids. Summary of the Invention

[0005] The object of the present invention is to overcome the problem that stress conditions in the prior art affect the physiological metabolic activities of filamentous fungi and reduce the fermentation performance, and to provide a recombinant strain, its construction method, and application. The production level and fermentation intensity of organic acids of this recombinant strain are significantly improved, the production cost of organic acids is effectively reduced, and it has the prospect of industrial application.

[0006] To achieve the above object, a first aspect of the present invention provides a recombinant strain for improving the production level and fermentation intensity of organic acids. This recombinant strain is obtained by genetically engineering a starting strain. Compared with the starting strain, the coding gene of the anchor protein GPI is overexpressed in the recombinant strain.

[0007] Preferably, the organic acid is a four-carbon organic acid; the starting strain is a fungus, preferably a filamentous fungus.

[0008] Preferably, the organic acid is fumaric acid, the starting strain is Rhizopus oryzae, the amino acid sequence of the anchor protein GPI is as shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.2.

[0009] Preferably, the organic acid is malic acid, the starting strain is Aspergillus oryzae, the amino acid sequence of the anchor protein GPI is as shown in SEQ ID NO.3, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.4.

[0010] Preferably, the organic acid is succinic acid, the starting strain is Aspergillus niger, the amino acid sequence of the anchor protein GPI is as shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.2.

[0011] A second aspect of the present invention provides a method for constructing a recombinant strain for improving the production level and fermentation intensity of organic acids. This method includes: genetically engineering a starting strain to overexpress the coding gene of the anchor protein GPI.

[0012] Preferably, the organic acid is a four-carbon organic acid; the starting strain is a fungus, preferably a filamentous fungus.

[0013] Preferably, the organic acid is fumaric acid, the starting strain is Rhizopus oryzae, the amino acid sequence of the anchor protein GPI is as shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.2.

[0014] Preferably, the organic acid is malic acid, the starting strain is Aspergillus oryzae, the amino acid sequence of the anchor protein GPI is as shown in SEQ ID NO.3, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.4.

[0015] Preferably, the organic acid is succinic acid, the starting strain is Aspergillus niger, the amino acid sequence of the anchor protein GPI is as shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.2.

[0016] Preferably, the process of genetically engineering the starting strain includes: constructing an anchor protein GPI recombinant vector and transforming the anchor protein GPI expression vector into the starting strain.

[0017] Preferably, the expression vector of the anchor protein GPI recombinant vector is the pEASY-Blunt Zero plasmid.

[0018] The third aspect of the present invention provides the use of the aforementioned recombinant strain and / or the recombinant strain obtained by the aforementioned construction method in the production of organic acids.

[0019] The fourth aspect of the present invention provides a method for producing organic acids, comprising the following steps: culturing the aforementioned recombinant strain and / or the recombinant strain obtained by the aforementioned construction method.

[0020] Preferably, the culturing includes: obtaining strain spores after plate activation of the recombinant strain, inoculating the strain spores into a seed medium for seed culture to obtain a seed liquid, and inoculating the seed liquid into a fermentation medium for fermentation culture to obtain a fermentation liquid.

[0021] Preferably, the plate activation uses a PDA medium, and the conditions for the plate activation include at least: a temperature of 25-35°C and a time of 4-5 days.

[0022] Preferably, the seed medium contains: 30-50 g / L glucose, 4-8 g / L beef extract powder, 0.5-1 g / L anhydrous potassium dihydrogen phosphate, 0.5-1 g / L anhydrous dipotassium hydrogen phosphate, 0.05-0.15 g / L magnesium sulfate heptahydrate, 0.05-0.15 g / L calcium chloride dihydrate, 0.001-0.005 g / L trace element salt, wherein the trace element salt contains sodium chloride, ferrous sulfate heptahydrate and anhydrous citric acid;

[0023] The conditions for the seed culture include at least: an inoculum size of 1×10 8 -5×10 8 strain spores / L, a temperature of 25-35°C, a rotation speed of 150-250 rpm, and a time of 20-30 h.

[0024] Preferably, the fermentation medium contains: 80-120 g / L cane molasses, 4-8 g / L beef extract powder, 0.1-0.2 g / L anhydrous potassium dihydrogen phosphate, 0.1-0.2 g / L anhydrous dipotassium hydrogen phosphate, 0.05-0.15 g / L magnesium sulfate heptahydrate, 0.05-0.15 g / L calcium chloride dihydrate;

[0025] The process of fermentation culture includes: inoculating the seed liquid into the fermentation medium at an inoculation amount of 80 - 120 mL / L, and fermenting at a temperature of 28 - 35 °C and a rotation speed of 150 - 250 rpm until the residual sugar content in the fermentation broth is below 10 g / L.

[0026] Through the above technical solution, the beneficial effects of the present invention are as follows:

[0027] In the recombinant strain provided by the present invention, the coding gene of the anchored protein GPI is overexpressed, which can effectively stress - defense against stress conditions, enhance stress tolerance, improve glycolytic flux. When using filamentous fungi for producing four - carbon organic acids as the starting strain, it can significantly improve the production level and fermentation intensity of organic acids, reduce the cost in the process of fermentative production of four - carbon organic acids, provide excellent strains for industrial fermentation production of four - carbon organic acids, and have good industrial application prospects.

[0028] The recombinant strain with enhanced stress resistance obtained by the genetic engineering means of the present invention effectively shortens the fermentation cycle, improves the production efficiency of four - carbon organic acids, reduces the production and fermentation costs. Compared with the current technical solution, it increases the output of fermentation products per unit time, improves the benefits of enterprises producing such products, and also expands the application field of microbial fermentation production of four - carbon organic acids.

[0029] The recombinant strain provided by the present invention overexpresses the anchored protein GPI gene, effectively enhancing the overall ability of the stress - defense system of filamentous fungi, including the enzyme system (catalase and total antioxidant capacity) and non - enzymatic defense (ROS); this indicates that overexpressing the anchored protein GPI gene, which has a potential impact on the integrity of the cell wall and the tolerance to adverse environments by covalently binding to the cell wall network or attaching to the cytoplasmic membrane in filamentous fungal strains, can enhance the ability of the strain's stress - defense system, and thus improve the production level and fermentation intensity of four - carbon organic acids by filamentous fungi.

[0030] In the most preferred embodiment of the present invention, molasses is used as a component of the medium for fermenting and producing organic acids by the recombinant strain, reusing the by - product of sugar production - the sugarcane juice squeezed industrially, improving the added value of the product, greatly reducing resource waste and environmental pollution, and being suitable for large - scale green and efficient industrial production of organic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a schematic diagram of the recombinant vector of the anchored protein GPI in the present invention;

[0032] Figure 2 is a gel electrophoresis diagram of the genomic verification of the transformants in Examples 1 - 3;

[0033] Figure 3It is a graph showing the results of biochemical analysis of the stress resistance of recombinant Rhizopus oryzae strain I and the parental strain in Test Example 2;

[0034] Figure 4 It is a graph showing the results of biochemical analysis of the stress resistance of recombinant Aspergillus oryzae strain I and the parental strain in Test Example 2;

[0035] Figure 5 It is a graph showing the results of biochemical analysis of the stress resistance of recombinant Aspergillus niger strain I and the parental strain in Test Example 2. Detailed implementation manners

[0036] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0037] The first aspect of the present invention provides a recombinant strain for improving the production level and fermentation intensity of organic acids. This recombinant strain is obtained by genetically engineering a parental strain. Compared with the parental strain, the coding gene of the anchored protein GPI is overexpressed in the recombinant strain.

[0038] According to the present invention, preferably, the organic acid is a four-carbon organic acid; the parental strain is a fungus, preferably a filamentous fungus.

[0039] In the recombinant strain provided by the present invention, the overexpression of the coding gene of the anchored protein GPI can effectively stress defense against stress conditions, enhance stress tolerance, and increase the glycolysis flux. When using a filamentous fungus producing four-carbon organic acids as the parental strain, it can significantly improve the production level and fermentation intensity of organic acids, reduce the cost in the process of fermenting and producing four-carbon organic acids, provide an excellent strain for industrial fermentation production of four-carbon organic acids, and have good industrial application prospects.

[0040] During the research process, the inventors of the present invention unexpectedly found that by overexpressing the anchored protein GPI gene, the overall ability of the stress defense system of filamentous fungi can be effectively enhanced, including the enzyme system (catalase and total antioxidant capacity) and non-enzymatic defense (ROS); this indicates that overexpressing the anchored protein GPI gene, which is potentially covalently bound to the cell wall network or attached to the cytoplasmic membrane in filamentous fungal strains and has an impact on the integrity of the cell wall and the tolerance to adverse environments, can enhance the ability of the strain's stress defense system, thereby improving the production level and fermentation intensity of four-carbon organic acids by filamentous fungi.

[0041] According to the present invention, preferably, the organic acid is fumaric acid, the starting strain is Rhizopus oryzae, the amino acid sequence of the anchor protein GPI is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2. Under this preferred embodiment, the production level and fermentation intensity of the four-carbon organic acid fumaric acid obtained by fermenting the recombinant strain under flask conditions for 5 days are increased by 31%-38% compared with the starting strain Rhizopus oryzae.

[0042] According to the present invention, preferably, the organic acid is malic acid, the starting strain is Aspergillus oryzae, the amino acid sequence of the anchor protein GPI is shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4. Under this preferred embodiment, the production level and fermentation intensity of the four-carbon organic acid malic acid obtained by fermenting the recombinant strain under flask conditions for 5 days are increased by 12.8%-17% compared with the starting strain Aspergillus oryzae.

[0043] According to the present invention, preferably, the organic acid is succinic acid, the starting strain is Aspergillus niger, the amino acid sequence of the anchor protein GPI is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2. Under this preferred embodiment, the production level and fermentation intensity of the four-carbon organic acid succinic acid obtained by fermenting the recombinant strain under flask conditions for 5 days are increased by 38.1%-57.6% compared with the starting strain Aspergillus niger.

[0044] The second aspect of the present invention provides a method for constructing a recombinant strain with improved organic acid production level and fermentation intensity, the method comprising: genetically engineering the starting strain to overexpress the encoding gene of the anchor protein GPI.

[0045] According to the present invention, preferably, the organic acid is a four-carbon organic acid, and the starting strain is a fungus; preferably a filamentous fungus.

[0046] According to the present invention, preferably, the organic acid is fumaric acid, the starting strain is Rhizopus oryzae, the amino acid sequence of the anchor protein GPI is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0047] According to the present invention, preferably, the organic acid is malic acid, the starting strain is Aspergillus oryzae, the amino acid sequence of the anchor protein GPI is shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0048] According to the present invention, preferably, the organic acid is succinic acid, the starting strain is Aspergillus niger, and the amino acid sequence of the anchor protein GPI is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0049] In the present invention, overexpression refers to inserting a gene that already exists in the cell into the strain, so that the gene is up-regulated and the final gene expression product exceeds the normal level. Overexpression uses the method of gene homologous recombination, setting homologous arms above and below at the designated site, and placing the gene expression cassette to be expressed between the two homologous arms.

[0050] In the present invention, the overexpression of the above-mentioned anchor protein GPI can be achieved by conventional genetic engineering modification methods in the art. For example, for the overexpression of the above-mentioned anchor protein GPI, the corresponding recombinant vector can be first constructed and replicated in Trelief 5α Chemically Competent Cell, and then introduced into the competent starting strain in a certain order. Various methods for constructing recombinant vectors are known in the art to ligate the encoding gene of the anchor protein GPI to an expression vector to prepare a recombinant vector. For example, but not limited to, the classical "enzymatic digestion - ligation" method, the Gateway cloning system developed by Invitrogen Corporation, and the ClonExpress cloning system developed by Novozymes Corporation (such as the ClonExpress MultiS OneStep Cloning Kit).

[0051] Exemplarily, the recombinant vector of the present invention can be constructed for the encoding gene of the anchor protein GPI by the recombinase method: based on the genome of the starting strain, the upstream and downstream homologous arm sequences of the targeted insertion site are amplified by the PCR method; the encoding gene expression cassette of the anchor protein GPI to be inserted, the upstream and downstream homologous arm sequences, and the resistance gene expression cassette are connected in series to obtain a recombinant vector, but the present invention is not limited thereto. For example, when the expression vector of the recombinant vector of the anchor protein GPI uses the pEASY - Blunt Zero plasmid, the map of the recombinant vector is as Figure 1 shown.

[0052] Subsequently, this recombinant vector can be introduced into the starting strain (such as Rhizopus oryzae, Aspergillus oryzae, or Aspergillus niger) by conventional methods in the art, such as but not limited to microinjection, gene gun, transformation (such as electroporation). The above microinjection, gene gun, or transformation are all conventional operations in the art. For example, transformation refers to treating cells by using some known methods in molecular biology and genetic engineering, making the treated cells in a competent state, and thus contacting with exogenous DNA, so that the exogenous DNA enters the cells in the competent state. Commonly used transformation methods include protoplast transformation method, chemical transformation method, and electroporation transformation method.

[0053] According to the present invention, preferably, the process of genetically engineering the starting strain includes: constructing an anchored protein GPI recombinant vector and transforming the anchored protein GPI expression vector into the starting strain. More preferably, the expression vector of the anchored protein GPI recombinant vector is the pEASY-Blunt Zero plasmid.

[0054] In the present invention, when Rhizopus oryzae, Aspergillus oryzae or Aspergillus niger is used as the starting strain, after the recombinant vector is introduced into the starting strain, positive clones can be screened out through a selection marker (such as hygromycin), and verified by genomic PCR or by sequencing the genomic DNA, so as to obtain the recombinant strain with improved organic acid production level and fermentation intensity. Among them, the method for introducing the recombinant vector into the starting strain is PEG-mediated protoplast transformation. Exemplarily, the method for preparing protoplasts is as follows: inoculate 10 7 fresh filamentous fungal spores into 50 mL of PDA medium and culture at 28 °C and 200 rpm for about 14 h; observe under a microscope at intervals until the germinated hyphae grow to a length twice the diameter of the spores, collect the germinated spores by centrifugation (8000 rpm, 4 °C, 10 min), and remove the supernatant; add 10 mL of an enzymolysis solution prepared with OM buffer, and the enzymolysis solution includes 20 mg of Yatalase enzyme (Takara) and 30 mg of Lysing Enzymes (Sigma), filter and sterilize; enzymolyze at 28 °C and 80 rpm for 4-6 h, and detect under a microscope at intervals until a large number of vacuolated protoplasts appear.

[0055] The third aspect of the present invention provides the use of the aforementioned recombinant strain and / or the recombinant strain obtained by the aforementioned construction method in the production of organic acids. Preferably, the organic acid is fumaric acid, malic acid or succinic acid.

[0056] The fourth aspect of the present invention provides a method for producing an organic acid, comprising the following steps: culturing the aforementioned recombinant strain and / or the recombinant strain obtained by the aforementioned construction method.

[0057] According to the present invention, preferably, the culturing includes: obtaining strain spores after plate activation of the recombinant strain, inoculating the strain spores into a seed medium for seed culture to obtain a seed liquid, and inoculating the seed liquid into a fermentation medium for fermentation culture to obtain a fermentation liquid.

[0058] In the present invention, there is no particular limitation on the culture medium used for activating the flat plate, and it can be a solid culture medium conventionally used in the art for activating the strain culture. Preferably, the flat plate activation uses a PDA culture medium. The conditions for the flat plate activation at least include: the temperature is 25-35°C, and the time is 4-5 days.

[0059] In the present invention, there is no particular limitation on the seed culture medium, and it can be a liquid culture medium conventionally used for the corresponding starting strain, generally containing components such as carbon source, nitrogen source, and inorganic salts, as long as it can enable the recombinant strain to grow and proliferate. Preferably, the seed culture medium contains: 30-50 g / L glucose, 4-8 g / L beef extract powder, 0.5-1 g / L anhydrous potassium dihydrogen phosphate, 0.5-1 g / L anhydrous dipotassium hydrogen phosphate, 0.05-0.15 g / L magnesium sulfate heptahydrate, 0.05-0.15 g / L calcium chloride dihydrate, 0.001-0.005 g / L trace element salt, wherein the trace element salt contains sodium chloride, ferrous sulfate heptahydrate, and anhydrous citric acid.

[0060] There is no particular limitation on the method of seed culture in the present invention, as long as the recombinant strain can grow and proliferate through this method. The parameters such as temperature, pH, rotation speed, and time used can be conventional settings in the art. Preferably, the conditions for the seed culture at least include: the inoculum size is 1×10 8 -5×10 8 spores of the strain / L, the temperature is 25-35°C, the rotation speed is 150-250 rpm, and the time is 20-30 h.

[0061] In the present invention, there is no particular limitation on the fermentation culture medium, and it can be a liquid culture medium conventionally used for the corresponding starting strain, generally containing components such as carbon source, nitrogen source, and inorganic salts, as long as it can enable the recombinant strain to proliferate in large quantities. Preferably, the fermentation culture medium contains: 80-120 g / L cane molasses, 4-8 g / L beef extract powder, 0.1-0.2 g / L anhydrous potassium dihydrogen phosphate, 0.1-0.2 g / L anhydrous dipotassium hydrogen phosphate, 0.05-0.15 g / L magnesium sulfate heptahydrate, 0.05-0.15 g / L calcium chloride dihydrate. In this preferred mode, cane molasses is used as a component of the culture medium for the recombinant strain to ferment and produce organic acids, reusing the sugar by-product - the cane juice squeezed out industrially, improving the added value of the product, greatly reducing resource waste and environmental pollution, and being suitable for large-scale green and efficient industrial production of organic acids.

[0062] The present invention has no particular limitation on the fermentation culture method, as long as the recombinant strain can be proliferated in large quantities by this method. The parameters such as temperature, pH, rotation speed, and time adopted can be conventional settings in the art. In the present invention, in order to improve the yield of four-carbon organic acids, preferably, the process of the fermentation culture includes: inoculating the seed liquid into the fermentation medium at an inoculation amount of 80-120 mL / L, and fermenting at a temperature of 28-35 °C and a rotation speed of 150-250 rpm until the residual sugar content in the fermentation broth is below 10 g / L.

[0063] In the present invention, the organic acids in the obtained fermentation broth can be separated by known methods, and the organic acids in the fermentation broth can also be detected by known methods, or the organic acids separated from the fermentation broth can be detected. For example, the organic acids can be detected by high-performance liquid chromatography and such methods.

[0064] The present invention will be described in detail below through examples.

[0065] In the following examples, the method for measuring the residual sugar concentration in the fermentation broth is as follows: Take an appropriate amount of the fermentation broth, centrifuge at 12000×g for 5 min, dilute the supernatant 400 times with deionized water, and then measure it by the anthrone-sulfuric acid method.

[0066] The method for measuring the content of four-carbon organic acids in the fermentation broth is as follows: Mix the fermentation broth thoroughly, quickly take 2 mL and put it into a 10 mL EP tube, slowly add an equal volume of 2 M HCl to fully acidify it; then centrifuge at 12000×g for 5 min, take the supernatant, dilute it 20-50 times, filter it through a 0.22 μm pore size aqueous filter membrane for testing; measure it by high-performance liquid chromatography (HPLC) ultraviolet detection method, use a Bio-Rad Aminex HPX-87H chromatographic column, the mobile phase is 5 mM dilute sulfuric acid, set the column temperature at 65 °C, the flow rate of the mobile phase is 0.8 mL / min, and the wavelength is 210 nm.

[0067] In the following examples, cane molasses was purchased from Fuyilong Chemical Technology Co., Ltd., model 11-22-24; Rhizopus oryzae was purchased and preserved by the laboratory of the School of Food and Pharmaceutical Engineering, Nanjing Normal University, with the number ATCC20344; Aspergillus oryzae was purchased from the Guangdong Provincial Culture Collection of Microorganisms, with the number ATCC56747; Aspergillus niger was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., with the number ATCC1015; the vector pEASY-Blunt Zero was purchased from Nanjing Novoprotein Biotechnology Co., Ltd.

[0068] Unless otherwise specified, other reagents are all conventional commercially available products, and other methods are all conventional methods; unless otherwise specified, the room temperature is 25 ± 5 °C.

[0069] In the following examples, the preparation process of the seed medium is as follows: Prepared according to 40 g / L glucose, 6 g / L beef extract powder, 0.75 g / L anhydrous potassium dihydrogen phosphate, 0.75 g / L anhydrous dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, 0.1 g / L calcium chloride dihydrate, 0.003 g / L trace element salt I (the components of trace element salt I are: 0.005 g / L sodium chloride, 0.005 g / L ferrous sulfate heptahydrate, 0.001 g / L anhydrous citric acid), and autoclaved at 115 °C for 20 min;

[0070] The preparation process of the fermentation medium is as follows: Prepared according to 100 g / L cane molasses, 6 g / L beef extract powder, 0.15 g / L anhydrous potassium dihydrogen phosphate, 0.15 g / L anhydrous dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, 0.1 g / L calcium chloride dihydrate, and autoclaved at 115 °C for 20 min;

[0071] The preparation process of the PDA solid medium is as follows: Accurately weigh 200 g of peeled potatoes, cut them into small pieces about 1 cm 3 in size, add 1 L of distilled water and continuously stir and boil for 30 min, filter with double-layer gauze to collect the filtrate, add 20 g of glucose and agar with a final mass concentration of 2%, stir until completely dissolved, make up the volume to 1 L with distilled water and dispense into wide-mouth bottles, and autoclave at 121 °C for 20 min;

[0072] The preparation process of the LB solid medium containing kanamycin resistance is as follows: Prepare the medium according to 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder, sterilize at 121 °C for 20 min, and add kanamycin to a final concentration of 100 μg / mL when the sterilized medium is cooled to about 50 °C;

[0073] The preparation process of the transformation medium is as follows: Dissolve 10 g of glucose, 1 ml of trace element salt II (containing sodium chloride, ferrous sulfate heptahydrate, anhydrous citric acid), 20×Salts, 1 g of yeast extract, 218.6 g of sorbitol, and 1.7 g of ammonium tartrate in 1 L of distilled water, and autoclave at 121 °C for 20 min;

[0074] The components of trace element salt II (100 mL): 2.20 g of ZnSO4·7H2O, 1.10 g of H3BO3, 0.50 g of MnCl2·4H2O, 0.16 g of FeSO4·7H2O, 0.16 g of CoCl2·5H2O, 0.16 g of CuSO4·5H2O, 0.11 g of (NH4)6Mo7O 24· 4H2O, 5.00 g of Na4EDTA;

[0075] Composition of 20×Salts (1 L): 10.4 g of KCl, 10.4 g of MgSO4·7H2O, 30.4 g of KH2PO4.

[0076] Example 1

[0077] 1. Construction of the recombinant vector of the ankyrin GPI gene

[0078] Using the cDNA obtained by reverse transcription of the RNA extracted from the starting strain, the filamentous fungus Rhizopus oryzae, as a template, the amplification primer pair root-GPI-F (nucleotide sequence shown in SEQ ID NO.5, see Table 1) and root-GPI-R (nucleotide sequence shown in SEQ ID NO.6, see Table 1) was designed. The coding gene of ankyrin GPI - the root-GPI gene sequence fragment (amino acid sequence shown in SEQ ID NO.1, nucleotide sequence of the coding gene shown in SEQ ID NO.2) was obtained by PCR amplification and recovery. After gel recovery, it was ligated with the vector pEASY-Blunt Zero using the Clon Express MultiS One Step Cloning Kit (purchased from Nanjing Novoprotein Biological Technology Co., Ltd.). The ligation product was transformed into Trelief 5α Chemically Competent Cell (purchased from Beijing Tsingke Biological Technology Co., Ltd.), and evenly spread on the LB solid medium containing 100 μg / mL kanamycin resistance, and cultured overnight at 37°C in an inverted position. Single colonies were picked and verified by colony PCR to obtain the recombinant vector - root-GPI-WW18 that was successfully ligated with the root-GPI gene sequence fragment.

[0079] 2. Obtaining the recombinant strain overexpressing the ankyrin gene GPI

[0080] Preparation of the protoplasts of the starting strain: Inoculate 10 7 spores of the fresh starting strain Rhizopus oryzae (ATCC 20344) into 50 mL of PDA medium, and culture at 28°C and 200 rpm for about 14 h; observe under the microscope at intervals until the germ tubes of the young spores grow to a length of 2 times the spore diameter. After centrifugation (8000 rpm, 4°C, 10 min), collect the germinated spores and remove the supernatant; add 10 mL of the enzyme solution prepared with OM buffer. The enzyme solution includes 20 mg of Yatalase enzyme (Takara) and 30 mg of Lysing Enzymes (Sigma), and filter sterilize; enzymolyze at 28°C and 80 rpm for 4 - 6 h, and detect under the microscope at intervals until a large number of vacuolated protoplasts appear;

[0081] The vector Root - GPI - WW18 was transferred into the protoplasts of filamentous fungi by PEG - mediated protoplast transformation. The protoplasts were mixed with the correctly extracted plasmid and incubated on ice for 50 min. Then, 1.5 mL of PEG 4000 solution was added to induce transformation at room temperature for 15 min. A thin layer of the pre - warmed transformation medium was first poured, and then the transformation medium was mixed with the induced filamentous fungal protoplasts and poured onto the solidified lower - layer medium. The mixture was cultured in an incubator at 28 °C for 3 - 4 d until transformants grew out. The transformants were screened. The phenotype of the transformants was insensitive to hygromycin resistance. The genomic DNA of such transformants was extracted for verification. Verification primers Root - GPI - yz - F (nucleotide sequence is shown in SEQ ID NO.7, see Table 1) and Root - GPI - yz - R (nucleotide sequence is shown in SEQ ID NO.8, see Table 1) were designed. The amplification result showed that the amplification of the primer pair Root - GPI - yz - F and Root - GPI - yz - R was positive (the gel electrophoresis pattern is shown in Figure 2 ). Such transformants were recombinant Rhizopus oryzae strains that successfully overexpressed the anchored protein gene GPI. Two strains with the clearest gel electrophoresis bands were selected as recombinant Rhizopus oryzae strain I and recombinant Rhizopus oryzae strain II.

[0082] Table 1 Primer Sequences

[0083]

[0084]

[0085] Example 2

[0086] 1. Construction of the recombinant vector of the anchored protein GPI gene

[0087] Using the cDNA obtained by reverse transcription of the RNA extracted from the starting strain Aspergillus oryzae, a filamentous fungus, as a template, amplification primers were designed, namely, Mi-GPI-F (nucleotide sequence as shown in SEQ ID NO.9, see Table 1) and Mi-GPI-R (nucleotide sequence as shown in SEQ ID NO.10, see Table 1). The coding gene of the anchor protein GPI, the Mi-GPI gene sequence fragment (amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence of the coding gene as shown in SEQ ID NO.4), was obtained by PCR amplification and recovery. After gel recovery, it was ligated with the vector pEASY-Blunt Zero using the Clon Express MultiS One Step Cloning Kit (purchased from Nanjing Novozymes Biotech Co., Ltd.). The ligation product was transformed into Trelief 5α Chemically Competent Cells (purchased from Beijing Tsingke Biotechnology Co., Ltd.), evenly spread on an LB solid medium containing 100 μg / mL kanamycin resistance, and cultured overnight at 37 °C in an inverted position. Monoclonal colonies were picked and verified by colony PCR to obtain the recombinant vector Mi-GPI-WW18 successfully ligated with the Mi-GPI gene sequence fragment.

[0088] 2. Obtaining a recombinant strain overexpressing the anchor protein gene GPI

[0089] Preparing protoplasts of the starting strain: Inoculate 10 7 spores of the fresh starting strain Aspergillus oryzae (ATCC 56747) into 50 mL of PDA medium and culture at 28 °C and 200 rpm for about 14 h; observe under a microscope at intervals until the germ tubes of the young spores grow to a length twice the diameter of the spores. After centrifugation (8000 rpm, 4 °C, 10 min), collect the germinated spores and remove the supernatant; add 10 mL of an enzymatic hydrolysis solution prepared with OM buffer. The enzymatic hydrolysis solution includes 20 mg of Yatalase enzyme (Takara) and 30 mg of Lysing Enzymes (Sigma), and is filtered and sterilized; perform enzymatic hydrolysis at 28 °C and 80 rpm for 4 - 6 h, and detect under a microscope at intervals until a large number of vacuolated protoplasts appear;

[0090] The vector rice - GPI - WW18 was transferred into the protoplasts of filamentous fungi by PEG - mediated protoplast transformation method. The protoplasts were mixed with the correctly extracted plasmid and incubated on ice for 50 min, then 1.5 mL of PEG 4000 solution was added to induce transformation at room temperature for 15 min. First, pour a thin layer of the transformed medium, then mix the transformed medium with the induced filamentous fungal protoplasts and pour it onto the solidified lower - layer medium, and culture it in an incubator at 28 °C for 3 - 4 d until transformants grow out. Screen the transformants. The phenotype of the transformants is insensitive to hygromycin resistance. Extract the genome of such transformants for verification. Design verification primers rice - GPI - yz - F (the nucleotide sequence is as shown in SEQ ID NO.11, see Table 1) and rice - GPI - yz - R (the nucleotide sequence is as shown in SEQ ID NO.8, see Table 1). The amplification result shows that the primer pair rice - GPI - yz - F and rice - GPI - yz - R amplify positively (the gel electrophoresis pattern is shown in Figure 2 ), and such transformants are recombinant Aspergillus oryzae strains that have successfully overexpressed the anchored protein gene GPI. Select two strains with the clearest gel electrophoresis bands as recombinant Aspergillus oryzae strain I and recombinant Aspergillus oryzae strain II.

[0091] Example 3

[0092] According to the method of Example 1, replace the starting strain Rhizopus oryzae with Aspergillus niger (ATCC 1015) to construct recombinant Aspergillus niger strains overexpressing the anchored protein gene GPI. Select two strains with the clearest gel electrophoresis bands as recombinant Aspergillus niger strain I and recombinant Aspergillus niger strain II.

[0093] Test Example 1: Test on the ability of recombinant strains to ferment and produce four - carbon organic acids in shake flasks

[0094] The recombinant Rhizopus oryzae strain I and recombinant Rhizopus oryzae strain II obtained in Example 1 and their starting strain Rhizopus oryzae, the recombinant Aspergillus oryzae strain I and recombinant Aspergillus oryzae strain II obtained in Example 2 and their starting strain Aspergillus oryzae, the recombinant Aspergillus niger strain I and recombinant Aspergillus niger strain II obtained in Example 3 and their starting strain Aspergillus niger were respectively inoculated on PDA medium plates and cultured at 28 °C for 4 - 5 days until conidia were produced. Collect the spores and inoculate the spore suspension into the seed medium so that the final added concentration of spores is 1×10 7 spores / 50 mL, and culture it at 28 °C with constant temperature and a stirring rate of 200 rpm for 20 - 23 h to obtain mature seed culture solutions;

[0095] The mature seed culture solution was inoculated into the fermentation medium at an inoculation amount of 10% (v / v), and fermentation culture was carried out at a temperature of 28 - 35 °C and a stirring rate of 200 rpm. Fermentation was terminated when the residual sugar content in the fermentation broth reached below 10 g / L to obtain the organic acid fermentation broth.

[0096] The contents of four-carbon organic acids in the fermentation broths of each recombinant strain and the starting strain were detected, and the results are shown in Tables 2 - 4.

[0097] Table 2

[0098]

[0099] As shown in Table 2, under the condition that the culture medium and culture conditions were exactly the same, the fumaric acid production of the starting strain Rhizopus oryzae was 33.5 g / L of the fermentation broth. While for the recombinant Rhizopus oryzae strains I and II obtained by overexpressing the coding gene of the anchoring protein GPI on the basis of the starting strain Rhizopus oryzae, the fumaric acid productions were 43.87 g / L of the fermentation broth and 46.23 g / L of the fermentation broth respectively. The fumaric acid production increased by 31% and 38% respectively, and the production efficiency increased by 46% and 50% respectively.

[0100] Table 3

[0101]

[0102]

[0103] As shown in Table 3, under the condition that the culture medium and culture conditions were exactly the same, the malic acid production of the starting strain Aspergillus oryzae was 73.5 g / L of the fermentation broth. While for the recombinant Aspergillus oryzae strains I and II obtained by overexpressing the coding gene of the anchoring protein GPI on the basis of Aspergillus oryzae, the malic acid productions were 82.88 g / L of the fermentation broth and 86.1 g / L of the fermentation broth respectively. The malic acid production increased by 12.8% and 17% respectively, and the production efficiency increased by 23% and 28% respectively.

[0104] Table 4

[0105]

[0106] As shown in Table 4, under the condition that the culture medium and culture conditions were exactly the same, the succinic acid production of the starting strain Aspergillus niger was 22.34 g / L of the fermentation broth. While for the recombinant Aspergillus niger strains I and II obtained by overexpressing the coding gene of the anchoring protein GPI on the basis of Aspergillus niger, the succinic acid productions were 30.85 g / L of the fermentation broth and 35.21 g / L of the fermentation broth respectively. The succinic acid production increased by 38.1% and 57.6% respectively, and the production efficiency increased by 53% and 74% respectively.

[0107] Test Example 2

[0108] Biochemical analysis of the stress resistance of each recombinant strain and the parental strain. Among them, the detection process of total antioxidant capacity (T-AOC) is as follows: a. Collect the cells or bacteria of each recombinant strain and the parental strain into centrifuge tubes respectively. According to the ratio of the number of cells or bacteria (10 4 ): the volume of the extraction solution (mL) is 500-1000:1, add 1.0 mL of pre-cooled extraction solution, ultrasonically disrupt the cells (power 200 W, ultrasound on for 3 s, off for 9 s, total time 3 min), then centrifuge at 10000 rpm and 4 °C for 10 min, and take the supernatant and place it on ice for later measurement; b. Preheat the spectrophotometer or microplate reader for more than 30 min, adjust the wavelength to 593 nm, zero the spectrophotometer with distilled water, and measure the absorbance value of the supernatant.

[0109] The detection process of hydrogen peroxide (H2O2) content is as follows: a. Collect the bacteria or cells of each recombinant strain and the parental strain into centrifuge tubes respectively, discard the supernatant after centrifugation; add 1 mL of acetone for every 5 million bacteria or cells, ultrasonically disrupt the bacteria or cells (power 20%, ultrasound for 3 s, interval 10 s, repeat 30 times); centrifuge at 8000 g and 4 °C for 10 min, take the supernatant and place it on ice for later measurement; b. Preheat the spectrophotometer or microplate reader for more than 30 min, adjust the wavelength to 415 nm, zero with distilled water, and measure the absorbance value of the supernatant.

[0110] The detection process of ROS reactive oxygen species includes: a. Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to make the final concentration 10 μmol / L. Remove the supernatant from the culture solutions of each recombinant strain and the parental strain, add an appropriate volume of diluted DCFH-DA. The added volume should be sufficient to cover the cells. Usually, for a six-well plate, add no less than 1 mL of diluted DCFH-DA, incubate in a 37 °C cell culture incubator for 20 min, wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells, and obtain the sample with the probe loaded in situ (usually, the positive control of reactive oxygen species can significantly increase the level of reactive oxygen species after stimulating the cells for 20-30 min); b. Observe the sample with the probe loaded in situ directly with a laser confocal microscope.

[0111] The results of the biochemical analysis are as Figures 3-5As shown, overexpressing the encoding gene of the anchor protein GPI enhanced the overall ability of the stress defense systems of the starting strains of Rhizopus oryzae, Aspergillus oryzae, and Aspergillus niger, including the enzyme system ability (catalase and total antioxidant capacity) and the non-enzyme defense ability (ROS). The results indicate that overexpressing the GPI gene of the anchor protein, which has potential effects on both the integrity of the cell wall and the tolerance to adverse environments by covalently binding to the cell wall network or attaching to the cytoplasmic membrane in filamentous fungi, can enhance the overall ability of the stress defense system of filamentous fungi, thereby improving the production level and fermentation intensity of four-carbon organic acids by filamentous fungi.

[0112] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including combining each technical feature in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A recombinant strain for improving the production level and fermentation intensity of organic acids, characterized in that, The recombinant strain is obtained by genetically engineering the starting strain. Compared with the starting strain, the coding gene of the anchored protein GPI is overexpressed in the recombinant strain; The organic acid is fumaric acid, the starting strain is Rhizopus oryzae, and the amino acid sequence of the anchored protein GPI is as shown in SEQ ID NO.1; Alternatively, the organic acid is malic acid, the starting strain is Aspergillus oryzae, and the amino acid sequence of the anchored protein GPI is as shown in SEQ ID NO.3; Alternatively, the organic acid is succinic acid, the starting strain is Aspergillus niger, and the amino acid sequence of the anchored protein GPI is as shown in SEQ ID NO.

1.

2. The recombinant strain according to claim 1, wherein When the amino acid sequence of the anchored protein GPI is as shown in SEQ ID NO.1, the nucleotide sequence of its coding gene is as shown in SEQ ID NO.2; When the amino acid sequence of the anchored protein GPI is as shown in SEQ ID NO.3, the nucleotide sequence of its coding gene is as shown in SEQ ID NO.

4.

3. A method for constructing a recombinant strain with improved organic acid production level and fermentation intensity, characterized in that, The method includes: genetically engineering the starting strain to overexpress the coding gene of the anchored protein GPI; The organic acid is fumaric acid, the starting strain is Rhizopus oryzae, and the amino acid sequence of the anchored protein GPI is as shown in SEQ ID NO.1; Alternatively, the organic acid is malic acid, the starting strain is Aspergillus oryzae, and the amino acid sequence of the anchored protein GPI is as shown in SEQ ID NO.3; Alternatively, the organic acid is succinic acid, the starting strain is Aspergillus niger, and the amino acid sequence of the anchored protein GPI is as shown in SEQ ID NO.

1.

4. The construction method according to claim 3, wherein When the amino acid sequence of the anchored protein GPI is as shown in SEQ ID NO.1, the nucleotide sequence of its coding gene is as shown in SEQ ID NO.2; When the amino acid sequence of the anchored protein GPI is as shown in SEQ ID NO.3, the nucleotide sequence of its coding gene is as shown in SEQ ID NO.

4.

5. The construction method according to claim 3 or 4, characterized in that, The process of genetically engineering the starting strain includes: constructing a recombinant vector of the anchored protein GPI and transforming the expression vector of the anchored protein GPI into the starting strain.

6. The construction method according to claim 5, characterized in that The expression vector of the recombinant vector of the anchored protein GPI is the pEASY-Blunt Zero plasmid.

7. Use of the recombinant strain according to claim 1 or 2 and / or the recombinant strain obtained by the construction method according to any one of claims 3 to 6 in the production of organic acids.

8. A method for producing an organic acid, characterized in that, It includes the following steps: culturing the recombinant strain according to claim 1 or 2 and / or the recombinant strain obtained by the construction method according to any one of claims 3 to 6.

9. The method according to claim 8, wherein The culturing includes: obtaining strain spores after plate activation of the recombinant strain, inoculating the strain spores into a seed medium for seed culture to obtain a seed liquid, and inoculating the seed liquid into a fermentation medium for fermentation culture to obtain a fermentation liquid.

10. The method according to claim 9, wherein The plate activation uses a PDA medium, and the conditions for the plate activation include at least: a temperature of 25 - 35°C and a time of 4 - 5 days.

11. The method according to claim 10, wherein The seed culture medium contains: 30 - 50 g / L of glucose, 4 - 8 g / L of beef extract powder, 0.5 - 1 g / L of anhydrous potassium dihydrogen phosphate, 0.5 - 1 g / L of anhydrous dipotassium hydrogen phosphate, 0.05 - 0.15 g / L of magnesium sulfate heptahydrate, 0.05 - 0.15 g / L of calcium chloride dihydrate, 0.001 - 0.005 g / L of trace element salt, wherein the trace element salt contains sodium chloride, ferrous sulfate heptahydrate and anhydrous citric acid; The conditions for seed cultivation include at least: the inoculation amount is 1×10 8 -5×10 8 spores of the strain per liter, the temperature is 25 - 35°C, the rotation speed is 150 - 250 rpm, and the time is 20 - 30 h.

12. The method according to claim 10, wherein The fermentation culture medium contains: 80 - 120 g / L of cane molasses, 4 - 8 g / L of beef extract powder, 0.1 - 0.2 g / L of anhydrous potassium dihydrogen phosphate, 0.1 - 0.2 g / L of anhydrous dipotassium hydrogen phosphate, 0.05 - 0.15 g / L of magnesium sulfate heptahydrate, 0.05 - 0.15 g / L of calcium chloride dihydrate; The process of the fermentation culture includes: inoculating the seed liquid into the fermentation culture medium at an inoculation amount of 80 - 120 mL / L, and fermenting at a temperature of 28 - 35 °C and a rotation speed of 150 - 250 rpm until the residual sugar content of the fermentation broth is below 10 g / L.

Images