Aspergillus niger engineering strain utilizing cheap carbon source as well as construction method and application of aspergillus niger engineering strain

Abstract

The invention relates to an aspergillus niger engineering strain using a cheap carbon source and a construction method and application thereof, and the engineering strain is obtained by overexpressing a sucB gene with a nucleotide sequence as shown in SEQ ID NO.1 in an aspergillus niger starting strain. According to the invention, the aspergillus niger engineering bacteria capable of efficiently utilizing the cheap carbon source cane molasses are constructed, and the L-malic acid is efficiently synthesized from the aspergillus niger by utilizing the cheap raw material. Furthermore, the cane molasses pretreated, especially the cane molasses pretreated by a phosphoric acid method, is taken as a carbon source, and a fermentation culture medium and culture conditions are optimized by a response surface method, so that the yield of the L-malic acid is further improved, the cost of raw materials in the fermentation process is reduced, and a good foundation is laid for industrial production of the L-malic acid.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to an Aspergillus niger engineering strain utilizing a cheap carbon source, a construction method and an application thereof. Background Art

[0002] Carbon sources mainly provide microorganisms with cell carbon frameworks, energy required for cell life activities, and carbon frameworks for synthetic products. Glucose is the preferred carbon source for most microorganisms because of the presence of the tricarboxylic acid cycle metabolic pathway in most microorganisms. However, the cost of glucose is relatively high, and most of the substrates used for malic acid microbial fermentation are relatively expensive glucose. In order to increase profit margins, low-cost sugar raw materials are preferred for large-scale fermentation.

[0003] Malic acid, also known as 2-hydroxysuccinic acid, is an important four-carbon organic acid. As an important platform chemical, it is widely used in food, agriculture, chemical industry, pharmaceuticals and biomaterials, with an annual demand of more than 200,000 tons. At present, the synthesis of malic acid mainly includes chemical synthesis, enzymatic method and fermentation method. The direct fermentation production of malic acid using sugar raw materials as substrates has attracted widespread attention due to its mild process and natural raw material sources. Aspergillus niger is the most widely used microorganism in industry. In previous studies, a strain of Aspergillus niger that can efficiently accumulate malic acid was obtained, but it can only use glucose as a substrate, and the raw material cost is relatively high. Summary of the invention

[0004] The first purpose of the present invention is to find a cheap carbon source to replace glucose, and to construct an Aspergillus niger engineered strain that can efficiently utilize the cheap carbon source using a metabolic engineering strategy, so as to enable Aspergillus niger to efficiently synthesize L-malic acid using cheap raw materials.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] An engineered strain of Aspergillus niger utilizing a cheap carbon source is obtained by overexpressing a sucB gene having a nucleotide sequence as shown in SEQ ID NO. 1 in a starting strain of Aspergillus niger.

[0007] As a preferred embodiment, the strong promoter PgpdA with a nucleotide sequence as shown in SEQ ID NO. 2 is used to overexpress the sucB gene.

[0008] As a preferred embodiment, the Aspergillus niger starting strain is Aspergillus niger LH-1, and its preservation number is CGMCC No. 41020.

[0009] The second object of the present invention is to provide a method for constructing the above-mentioned Aspergillus niger engineering strain, comprising: using pEASY plasmid as a gene expression vector, utilizing Cre-lox recombination technology to construct a recombinant expression plasmid, and introducing the recombinant expression plasmid into the Aspergillus niger starting strain to construct the Aspergillus niger engineering strain.

[0010] As a preferred embodiment, the construction method comprises:

[0011] Using pEASY plasmid as a vector, constructing a loxP-hph-loxP recombinant vector pEASY-Cre-loxP-Hph-loxP; connecting PgpdA with the pEASY-Cre-loxP-Hph-loxP to obtain a recombinant plasmid pEASY-Cre-loxP-hph-loxP-Pgpd;

[0012] The target gene sucB was introduced into the recombinant plasmid pEASY-Cre-loxP-hph-loxP-Pgpd to obtain the recombinant expression plasmid pEASY-Cre-loxP-hph-loxP-Pgpd-sucB of sucB;

[0013] The recombinant expression plasmid is transformed into the starting strain to obtain the Aspergillus niger engineered strain.

[0014] The third object of the present invention is to provide the use of the above-mentioned Aspergillus niger engineered strain in producing L-malic acid.

[0015] As a preferred embodiment, the engineered Aspergillus niger strain produces L-malic acid by fermentation in a culture medium with glucose, sucrose, fructose, xylose, maltose and / or sugarcane molasses as carbon sources; preferably sugarcane molasses.

[0016] As a preferred embodiment, the sugarcane molasses is pretreated sugarcane molasses, and the pretreatment method includes: boiling treatment, activated carbon treatment, sulfuric acid treatment, phosphoric acid treatment, potassium ferrocyanide treatment, lime milk treatment, cationic ion resin adsorption treatment and / or anionic ion resin adsorption treatment.

[0017] As a preferred embodiment, the sugarcane molasses is sugarcane molasses pretreated with phosphoric acid, and the processing method is as follows:

[0018] The sugar cane molasses is diluted, boiled, and centrifuged, and the clarified liquid after centrifugation is acidified with phosphoric acid;

[0019] The acidified clarified liquid is centrifuged, the pH is adjusted with lime milk, activated carbon is added, and the liquid is kept warm at 65-70°C on a magnetic stirrer. After cooling, the activated carbon is removed by centrifugation, and the clear liquid is used for fermentation.

[0020] As a preferred embodiment, the fermentation medium of the engineered Aspergillus niger strain contains a carbon source, a nitrogen source and an inorganic salt;

[0021] A response surface experiment was conducted with carbon source, nitrogen source and inorganic salt concentration as experimental factors and L-malic acid as the response value to obtain the optimal fermentation medium.

[0022] The present invention compares the gene expression differences of Aspergillus niger utilizing sucrose and glucose, screens out three genes related to sucrose degradation, constructs recombinant strains of the three genes respectively, determines sucB as a main degradation gene through fermentation comparison, constructs and screens out enhanced strain AS002 through overexpression of the sucB gene, obtains Aspergillus niger engineered bacteria that can efficiently utilize sugarcane molasses, a cheap carbon source, thereby enabling Aspergillus niger to efficiently synthesize L-malic acid using cheap raw materials.

[0023] Furthermore, the present invention uses sugarcane molasses pretreated, especially pretreated by phosphoric acid, as a carbon source, and optimizes the fermentation medium and culture conditions by response surface methodology. The optimal fermentation conditions for the Aspergillus niger engineering strain are: temperature 28°C, inoculum size 2×10 6 The optimal fermentation conditions were as follows: the molasses concentration after phosphoric acid treatment was 161.323 g / L, the peptone concentration was 8.161 g / L, the K2HPO4 addition was 41.32%, and the Mg2SO4 addition was 61.72%. Under the optimal fermentation conditions, the L-malic acid production in shake flask fermentation was 108.12 ± 2.21 g / L, and the L-malic acid production in fermenter fermentation was 110.72 ± 1.21 g / L. The optimized fermentation method further increased the production of L-malic acid and reduced the cost of raw materials in the fermentation process, laying a good foundation for its industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The invention discloses the effects of different carbon sources on the growth and L-malic acid production of the starting strain Aspergillus niger LH-1.

[0025] Figure 2 It is the effect of using sugarcane molasses treated by different pretreatment methods for strain fermentation to produce L-malic acid.

[0026] Figure 3 This paper is an analysis of the production of L-malic acid by fermentation of sugarcane molasses by Aspergillus niger under different pretreatment methods.

[0027] Figure 4 The fermentation validation optimization predicted results for shake flask fermentation and 5 L batch fermentation in a fermenter. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods.

[0029] The various experimental operations involved in the embodiments, unless otherwise specified, are routine techniques in the art, and those of ordinary skill in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of the present invention.

[0030] The nucleotide sequences provided by the present invention can usually be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities by recombination. The obtained nucleotide sequence is usually cloned into a vector, then transferred into genetically engineered bacteria, and then the relevant nucleotide sequence is isolated from the propagated host cells by conventional methods.

[0031] The starting strain used in the examples is Aspergillus niger LH-1, whose deposit number is CGMCC No. 41020.

[0032] The pEASY plasmid (pEASY-Blunt Zero Cloning Vector) used in the examples is a commercial plasmid.

[0033] The culture medium preparation method / formulation involved in the embodiment is as follows:

[0034] PDA medium: 20% (W / V) potato cooking juice. Accurately weigh 200 g of peeled potatoes and cut them into approximately 1 cm 3 Put the potato pieces in a pot, add water, stir and continue boiling for about 30 minutes; after cooling, filter and squeeze through double-layer gauze to remove the mashed potatoes, add 20 g glucose and 20 g agar powder to the filtrate and stir until completely dissolved, and adjust the volume to 1 L. The pH does not need to be adjusted. Add 100 μg / mL of kanamycin and ampicillin as needed.

[0035] Potato dextrose water (PDB) medium (liquid PDA): potato extract powder 6 g / L, glucose 20 g.

[0036] All the above culture media were sterilized by high pressure steam at 121°C for 20 min; relevant antibiotics were added before use.

[0037] Transformation medium: 10 g glucose, 1 mL trace elements, 20×Salts, 1 g yeast extract, 218.6 g sorbitol, 1.7 g ammonium tartrate, and add corresponding drugs as needed.

[0038] Trace elements (100 mL) composition: 2.20 g ZnSO4·7H2O, 1.10 g H3BO3, 0.50 g MnCl2·4H2O, 0.16 g FeSO4·7H2O, 0.16 g CoCl2·5H2O, 0.16 g CuSO4·5H2O, 0.11 g (NH4)6Mo7O 24 4H2O, 5.00 g Na4EDTA.

[0039] 20×Salts (1L): 10.4 g KCl, 10.4 g MgSO4·7H2O, 30.4 g KH2PO4.

[0040] Screening medium (PDA+screening agent): PDA medium, hygromycin 250 μg / mL.

[0041] DNA extraction buffer (1L): sodium chloride 8.766 g, disodium ethylenediaminetetraacetic acid (EDTA·Na2) 22.33 g, tris (hydroxymethylaminomethane) (Tris) 48.456 g, sodium dodecyl sulfate (SDS) 10 g.

[0042] Seed culture medium preparation: 4% glucose, 0.6% bacterial peptone, 0.075% anhydrous potassium dihydrogen phosphate, 0.075% anhydrous dipotassium hydrogen phosphate, 0.01% magnesium sulfate heptahydrate, 0.01% calcium chloride dihydrate, 0.0001-0.0005% trace elements (sodium chloride, ferrous sulfate heptahydrate, anhydrous citric acid), sterilized at 115 ℃ for 20 min.

[0043] Fermentation medium preparation: 10% sucrose, 0.6% bacterial peptone, 0.015% anhydrous potassium dihydrogen phosphate, 0.015% anhydrous dipotassium hydrogen phosphate, 0.01% calcium chloride dihydrate, 0.01% magnesium sulfate heptahydrate, 0.00025% trace elements (sodium chloride, ferrous sulfate heptahydrate, anhydrous citric acid), sterilized at 115 °C for 20 min.

[0044] Example 1

[0045] The genes suc1, sucB (nucleotide sequence as shown in SEQ ID NO. 1) and sucC were amplified from the genome of Aspergillus niger LH-1 (CGMCC No. 41020), cloned using the primers shown in Table 1, and gene integration and efficient expression were performed using a vector pEASY-Cre-loxP-hph-loxP-Pgpd containing a 3-phosphoglyceraldehyde dehydrogenase promoter (PgpdA, nucleotide sequence as shown in SEQ ID NO. 2) and loxP-hph-loxP. The specific construction method can refer to the inventor's previous patent application CN118028124A, including:

[0046] (1) Construction of loxP-hph-loxP vector pEASY-Cre-loxP-hph-loxP-Pgpd:

[0047] The loxP-hph-loxP element sequence was connected to the pEASY plasmid to obtain the recombinant plasmid pEASY-Cre-loxP-Hph-loxP; the three sets of primer pairs shown in Table 1 were used to obtain the vector zero-Cre-loxP-hph-loxP with pEASY-Cre-loxP-Hph-loxP as a template, and PgpdA was connected to zero-cre-loxP-hph-loxP to obtain the recombinant plasmid pEASY-Cre-loxP-hph-loxP-Pgpd, which contains the hygromycin resistance gene hph and the 3-phosphoglycerate dehydrogenase gene promoter PgpdA;

[0048] (2) Construction of target gene (suc1, sucB, sucC) high expression plasmids: Using the genome of Aspergillus niger LH-1 as a template, the gene fragment of the target gene was obtained by PCR amplification. Using the plasmid pEASY-Cre-loxP-hph-loxP-Pgpd as a template, the linearized plasmid vector zero-cre-loxP-hph-loxP-Pgpd was obtained by PCR amplification. The linearized plasmid vector zero-cre-loxP-hph-loxP-Pgpd and the gene fragment of the target gene were then ligated to obtain the target gene high expression plasmids, namely the recombinant vectors pEASY-Cre-loxP-hph-loxP-Pgpd-suc1, pEASY-Cre-loxP-hph-loxP-Pgpd-sucB and pEASY-Cre-loxP-hph-loxP-Pgpd-sucC.

[0049] Table 1 Primers used

[0050]

[0051] Inoculate fresh Aspergillus niger LH-1 (CGMCC No. 41020) spores into 100 mL liquid PDA medium and culture at 28°C and 180 rpm for 12 h; aspirate 10 mL of the bacteria into a 50 mL centrifuge tube and centrifuge at 8000 rpm for 10 min, remove the supernatant, add the enzyme solution after membrane (the preparation process of the enzyme solution is: dissolve 20 mg Yatalase and 30 mg Lysing Enzymes in 10 mL Osmotic medium) into the centrifuge tube, resuspend, and transfer to a 50 mL conical flask for enzyme hydrolysis for 3-4 h. In the middle, blow once to make the bottom plate strain fully hydrolyzed, and examine the protoplasts under an optical microscope;

[0052] Transfer the enzymatic hydrolyzed product to a 50 mL centrifuge tube, slowly add 10 mL of Trapping Buffer close to the tube wall, centrifuge at 4°C and 5000 rpm for 5 min, pipette the protoplasts at both liquid levels into another 50 mL centrifuge tube, dilute the protoplast suspension with one volume of STC buffer, centrifuge at 4°C and 6000 rpm for 5 min, remove the supernatant, resuspend the protoplasts with 1 mL of STC buffer to obtain a protoplast solution, and place on ice;

[0053] Take a sterile 1.5 mL centrifuge tube, mix the recombinant vectors pEASY-Cre-loxP-hph-loxP-Pgpd-suc1, pEASY-Cre-loxP-hph-loxP-Pgpd-sucB and pEASY-Cre-loxP-hph-loxP-Pgpd-sucC obtained in Example 2 with 70 μL of the protoplast solution, and bathe on ice for 50 min to obtain a mixture; take a sterile 15 mL centrifuge tube, add the mixture to the 15 mL centrifuge tube, and then add 1.5 mL PEG4000 solution was gently mixed, and after standing at room temperature for 20 min, 10 mL of transformation medium was added, mixed, and plated to obtain transformants, including the recombinant strain AS001 transferred into the recombinant vector pEASY-Cre-loxP-hph-loxP-Pgpd-suc1, the recombinant strain AS002 transferred into the recombinant vector pEASY-Cre-loxP-hph-loxP-Pgpd-sucB, and the recombinant strain AS003 transferred into the recombinant vector pEASY-Cre-loxP-hph-loxP-Pgpd-sucC.

[0054] Example 2

[0055] The original strain of Aspergillus niger LH-1 (CGMCC No. 41020) was streaked onto PDA medium and cultured at 28°C for 5 days. The strain in the culture dish was inoculated into an eggplant bottle containing seed medium and cultured once at 28°C for 5 days to obtain a primary seed solution to obtain sufficient spores. The primary seed solution was diluted with 1×10 6 The spores were inoculated into the seed culture medium at a concentration of spores / mL, and the seed solution was obtained by secondary seed culture at 28°C for 20 hours.

[0056] The seed liquid was inoculated into 50 mL of fermentation medium at a concentration of 10% (v / v), and cultured at a temperature of 28°C and a rotation speed of 200 rpm for 4 days to obtain the fermentation liquid. During the fermentation process, fermentation samples were taken regularly every day, and the glucose content was measured by SBA-40E biosensor analyzer, and the production of organic acids was measured by high performance liquid chromatography, and the cell dry weight was measured by suction filtration. The results are as follows Figure 2 As shown in the figure, at 5 days of fermentation, the L-malic acid yield of the experimental group with sucrose as the carbon source was the highest at 86.01 ± 1.7 g / L, which was 11.7% higher than the L-malic acid yield of 77.11 ± 2.40 g / L when glucose was used as the carbon source. After adding fructose, xylose, maltose, and lactose, the L-malic acid yields were 71.04 g / L, 42.59 g / L, 63.01 g / L, and 11.87 g / L, respectively, which were significantly lower than glucose fermentation. The results show that sucrose is more suitable for Aspergillus niger to ferment and synthesize L-malic acid.

[0057] Example 3

[0058] Transcriptomics was used to analyze the mechanism of changes in L-malic acid production rate when sucrose and glucose were used as carbon sources, and the expression profile changes of Aspergillus niger were compared when the acid production processes of sucrose and glucose were most different.

[0059] Three genes related to sucrose hydrolase (suc1, sucB, sucC) were overexpressed in Aspergillus niger, and the resulting engineered strains (AS001, AS002, AS003) showed different degrees of improvement in the L-malic acid production rate. In particular, the strain overexpressing the sucB gene achieved an L-malic acid titer of 95.05 ± 2.15 g / L within 120 h, with a production rate of 0.79 g / (L·h) ( Figure 2 ), the L-malic acid production increased by 11.6% compared with the initial strain when fermented with sucrose as the carbon source. However, there was no obvious advantage in fermentation time and acid production after overexpression of the other two sucrose hydrolase genes suc1 and sucC ( Figure 2). It further proves that enhancing the gene expression of enzymes that decompose sucrose can enhance the efficiency of metabolic pathways and promote the synthesis of target products.

[0060] Example 4

[0061] In this example, AS002 was used as a fermentation strain to study the effect of using sugarcane molasses treated with different pretreatment methods for strain fermentation to produce L-malic acid.

[0062] The original sugarcane molasses was diluted with pure water in a ratio of 1:1 (referred to as diluted sugarcane molasses), and then treated by the following methods:

[0063] (1) Boiling method: Weigh an appropriate amount of diluted sugarcane molasses into a large beaker, boil at 100°C for 8 min, centrifuge at 8000 r / min for 15 min, remove the precipitate, and clear for later use;

[0064] (2) Activated carbon method: Take an appropriate amount of diluted sugarcane molasses in a large beaker, add activated carbon at a concentration of 80 g / L, mix well, heat on a magnetic stirrer at 45 °C for 5 h, centrifuge at 8000 r / min for 20 min, remove the activated carbon, and adjust the pH of the clear solution to 6.0 with 5 M NaOH for later use;

[0065] (3) Sulfuric acid method: Take an appropriate amount of diluted sugarcane molasses and adjust the pH to 3.5 with 1 M H2SO4. After standing overnight, centrifuge at 8000 r / min for 10 min to remove the precipitate. The clarified liquid is then adjusted to pH 6.0 with 5 M NaOH and set aside.

[0066] (4) Phosphoric acid method: In the first step, take an appropriate amount of diluted sugarcane molasses in a large beaker, boil it at 100℃ for 8 min, centrifuge it at 8000 r / min for 15 min, adjust the pH of the clarified liquid to 2.5 with concentrated phosphoric acid, let it stand for 12 h after acidification, and centrifuge it at 8000 r / min for 10 min; In the second step, adjust the pH of the clear liquid to about 6.0 with newly prepared 15% lime milk, add 1% activated carbon and place it on a magnetic stirrer at 65-70℃ for 30 min, let it stand and cool, then centrifuge it at 8000 r / min for 15 min to remove the activated carbon, and set the clear liquid aside;

[0067] (5) Potassium ferrocyanide method: Take an appropriate amount of diluted sugarcane molasses in a large beaker, adjust the pH to 6.0 with low concentration of potassium ferrocyanide, boil at 100°C for 20 min, let it stand and cool to room temperature, centrifuge at 8000 r / min for 15 min to remove the precipitate, and keep the clear solution for later use;

[0068] (6) Lime milk method (CaO): Take an appropriate amount of diluted sugarcane molasses in a large beaker, adjust the pH to 8.0-9.0 with 20% CaO, heat and boil for 25 min, let it stand and cool, filter it with four layers of gauze, and then adjust the pH to 6.0 with 10% H2SO4. Centrifuge at 8000 r / min for 15 min to remove the precipitate and keep the clear solution for later use;

[0069] (7) Cationic ion resin adsorption method: Take an appropriate amount of diluted sugarcane molasses in a large beaker, add 20% treated cationic resin, place it on a magnetic stirrer at 30°C and 200 r / min for 24 h, then filter with four layers of gauze to remove the resin, adjust the filtrate to pH 6.0 with 5 M NaOH and set aside;

[0070] (8) Negative ion resin adsorption method: Take an appropriate amount of diluted sugarcane molasses in a large beaker, add 20% treated anion resin, place it on a magnetic stirrer at 30°C and 200 r / min for 24 h, then filter with four layers of gauze to remove the resin, adjust the filtrate to pH 6.0 with 5 M NaOH and set aside.

[0071] The AS002 strain was fermented using the method of Example 2, and the carbon source in the fermentation medium was replaced with an equal amount of pretreated cane molasses (10% cane molasses). The fermentation results are as follows: Figure 3 shown.

[0072] from Figure 3 As shown in Figure A, during the entire fermentation period using sugarcane molasses after different pretreatment methods as the carbon source, the change trend of L-malic acid concentration is basically the same, but the changes in the final L-malic acid concentrations are quite different.

[0073] At 120 h of fermentation, the concentrations of L-malic acid in the boiling, activated carbon, sulfuric acid, phosphoric acid, potassium ferrocyanide, calcium oxide, cationic resin adsorption, and anionic resin adsorption treatments reached 82.44 g / L, 78.75 g / L, 81.15 g / L, 84.45 g / L, 79.05 g / L, 77.35 g / L, 78.75 g / L, and 79.15 g / L, respectively; compared with the L-malic acid yield of 75.45 g / L in the control group when fermented with untreated molasses as the carbon source, the L-malic acid concentration in the phosphoric acid treatment group was the highest at 84.05 ± 1.1 g / L, which was 12% higher than the L-malic acid yield of untreated raw molasses fermentation, and the generation rate was 9.7%. The L-malic acid concentrations at the end of the experimental groups with other pretreatment methods were also greater than those of the control group. The colloid, ash and metal contents in sugarcane molasses after treatment with eight pretreatment methods were analyzed. It was found that the phosphoric acid method was the best for removing colloid, ash and metal contents. After treatment, the colloid content in molasses dropped from 6.23% to 0.57%, removing 91% of the colloids in the molasses; the ash content dropped from 13.84% to 5.27%, removing 62% of the ash in the molasses; and the removal rate of the detected metal ions also reached more than 50%.

[0074] Depend on Figure 1 From Figure B, we can see that the maximum generation rate of L-malic acid exists between 48 and 72 h. In this period, the maximum generation rates of L-malic acid in the untreated, boiling, activated carbon, sulfuric acid, phosphoric acid, potassium ferrocyanide, calcium oxide, cation resin adsorption, and anion resin adsorption treatment methods are 1.27 g / (L·h), 1.34 g / (L·h), 1.19 g / (L·h), 1.35 g / (L·h), 1.37 g / (L·h), 1.27 g / (L·h), 1.28 g / (L·h), 1.30 g / (L·h), and 1.31 g / (L·h). In addition, it was found that in the first 72 h of fermentation, the generation rate of L-malic acid in each test group was higher than that in the untreated control group. The above results show that after pretreatment, the harmful substances that affect cell growth and fermentation are removed from molasses, which is more conducive to the growth and reproduction of Aspergillus niger cells and the production capacity of organic acid L-malic acid.

[0075] Example 5

[0076] Based on the research results in Example 4, the fermentation was carried out using sugarcane molasses pretreated with phosphate group as the carbon source. In this example, the fermentation medium was further optimized by single factor and response surface methodology.

[0077] Molasses concentration (A), peptone concentration (B), K2HPO4 concentration (C), and MgSO4·7H2O concentration (D) were selected as experimental factors, and L-malic acid yield (Y) was selected as the response value for response surface experiment. The results were analyzed by method. The response surface experiment design and results are shown in Table 2, and the variance analysis is shown in Table 3. Design-Expert software was used to perform RSM experimental design and analysis, and the regression equation between Y (L-malic acid production) and four factors A, B, C, and D was obtained: L-malic acid production = 111.956 +0.996667 A + 1.24667 B + 1.07083 C + 1.35583 D - 0.27 AB + 0.07 AC + 1.33 AD- 0.17 BC - 0.12 BD - 0.1775 CD - 12.3159 A2 - 7.48842 B2 - 7.86967 C2 -8.08217 D2. When the coefficient of the independent variable in the linear model is positive, it means that the dependent variable increases with the increase of the independent variable, showing a promoting effect; when the coefficient is negative, it indicates an inhibitory effect. Among them, the coefficients of A, B, C, and D are all positive, indicating that these four factors play a promoting role within the research scope, which is beneficial to increasing the production of L-malic acid.

[0078] Table 2 Response surface experimental design and results

[0079]

[0080] Table 3 Analysis of variance of response surface test results

[0081]

[0082] Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).

[0083] The response surface design regression equation showed that when the molasses concentration was 161.323 g / L, the peptone concentration was 8.161 g / L, the K2HPO4 addition amount was 41.32%, and the Mg2SO4 addition amount was 61.72%, the predicted value of L-malic acid production was up to 112.12 g / L. In order to verify the accuracy of the result, the optimal fermentation conditions after optimization (temperature 28℃, seed inoculation amount 2×10 6 The fermentation experiment was carried out according to the simulated optimal medium component addition amount at 250 rpm. Figure 3 As shown, according to the optimization strategy, after 120 h of fermentation in shake flasks ( Figure 4The maximum bacterial biomass can reach 15.92 ± 0.81 g / L, the residual sugar content of molasses is about 28 g, and the average L-malic acid yield is 108.12 ± 2.21 g / L. After fermentation in a 5 L fermenter for 120 h ( Figure 4 In the middle (B), the residual sugar content of molasses was about 20 g / L, and the average L-malic acid yield was 110.72 ± 1.21 g / L.

Claims

1. An engineered strain of Aspergillus niger utilizing a cheap carbon source, characterized in that: The engineered strain is obtained by overexpressing the sucB gene having a nucleotide sequence as shown in SEQ ID NO. 1 in a starting strain of Aspergillus niger.

2. The engineered Aspergillus niger strain according to claim 1, characterized in that The strong promoter PgpdA with the nucleotide sequence shown in SEQ ID NO. 2 was used to overexpress the sucB gene.

3. The Aspergillus niger engineered strain according to claim 1, characterized in that The Aspergillus niger starting strain is Aspergillus niger LH-1, and its preservation number is CGMCC No. 41020.

4. The method for constructing the Aspergillus niger engineered strain according to any one of claims 1 to 3, characterized in that: The pEASY plasmid is used as a gene expression vector, the Cre-lox recombination technology is used to construct a recombinant expression plasmid, and the recombinant expression plasmid is introduced into the Aspergillus niger starting strain to construct the Aspergillus niger engineering strain.

5. The construction method according to claim 4, characterized in that: include: Using pEASY plasmid as a vector, constructing a loxP-hph-loxP recombinant vector pEASY-Cre-loxP-Hph-loxP; connecting PgpdA with the pEASY-Cre-loxP-Hph-loxP to obtain a recombinant plasmid pEASY-Cre-loxP-hph-loxP-Pgpd; The target gene sucB was introduced into the recombinant plasmid pEASY-Cre-loxP-hph-loxP-Pgpd to obtain the recombinant expression plasmid pEASY-Cre-loxP-hph-loxP-Pgpd-sucB of sucB; The recombinant expression plasmid is transformed into the starting strain to obtain the Aspergillus niger engineered strain.

6. Use of the engineered Aspergillus niger strain according to any one of claims 1 to 3 in producing L-malic acid.

7. The use according to claim 6, characterized in that: The engineered Aspergillus niger strain produces L-malic acid by fermentation in a culture medium with glucose, sucrose, fructose, xylose, maltose and / or sugarcane molasses as carbon sources; sugarcane molasses is preferred.

8. The use according to claim 7, characterized in that: The sugar cane molasses is pretreated sugar cane molasses, and the pretreatment method includes: boiling treatment, activated carbon treatment, sulfuric acid treatment, phosphoric acid treatment, potassium ferrocyanide treatment, lime milk treatment, cationic ion resin adsorption treatment and / or anionic ion resin adsorption treatment.

9. The use according to claim 8, characterized in that: The sugarcane molasses is sugarcane molasses pretreated with phosphoric acid, and the processing method is as follows: The sugar cane molasses is diluted, boiled, and centrifuged, and the clarified liquid after centrifugation is acidified with phosphoric acid; The acidified clarified liquid is centrifuged, the pH is adjusted with lime milk, activated carbon is added, and the liquid is kept warm at 65-70°C on a magnetic stirrer. After cooling, the activated carbon is removed by centrifugation, and the clear liquid is used for fermentation.

10. The use according to claim 6, characterized in that: The fermentation medium of the Aspergillus niger engineered strain contains a carbon source, a nitrogen source and an inorganic salt; A response surface experiment was conducted with carbon source, nitrogen source and inorganic salt concentration as experimental factors and L-malic acid as the response value to obtain the optimal fermentation medium.

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