Aspergillus niger capable of efficiently utilizing glycerol to produce malic acid as well as construction method and application of aspergillus niger

Abstract

The invention belongs to the technical field of genetic engineering and microorganisms, and discloses an Aspergillus niger LH-S8 capable of efficiently utilizing glycerol to produce malic acid, which is constructed by strengthening glycerol transport and glycerol metabolism and decomposition capabilities through a genetic engineering means. According to the present invention, the strain can produce 100-120 g / L of L-malic acid in 5-7 days in a fermentation tank, the average glycerol consumption rate is 0.5-0.9 g / L / h, and the carbon conversion rate is 0.9-1.2 C-mol / C-mol, which is close to 70-90% of the theoretical maximum value; in the research field of producing malic acid by using glycerol, the raw material conversion rate reaches the highest value reported at present under the condition of keeping higher production concentration and shorter production period. According to the method, the raw material conversion rate is increased, and a new thought is provided for green and sustainable development of bio-based chemicals.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and microbial technology, in particular to an Aspergillus niger strain capable of efficiently utilizing glycerol to produce malic acid, a construction method and application thereof. Background Art

[0002] Aspergillus niger is a filamentous fungus widely found in nature. With its tenacious vitality and excellent adaptability to diverse carbon sources, it plays an important role in the field of organic acid production. Especially in the production of key organic acids such as citric acid and malic acid, Aspergillus niger has not only promoted high fermentation efficiency and large-scale industrial production practices, but also deeply penetrated into many important fields such as food, medicine, and materials, and continued to stimulate strong interest and exploration in scientific research and industry. However, in the face of increasingly fierce competition in the organic acid market and the bottlenecks encountered by industrial fermentation technology, how to find and implement low-cost and high-efficiency production solutions has become an urgent need for the current development of the industry. In this context, crude glycerol, a by-product produced in the process of biodiesel production, is regarded as an ideal alternative carbon source in the field of organic acid fermentation because of its rich resources and huge utilization potential. This strategy can not only effectively alleviate the by-product disposal pressure of the biodiesel industry, but also open up a new cost-saving path for organic acid production, which has far-reaching economic and environmental significance. Despite this, glycerol utilization still faces many challenges, including cost-effectiveness, microbial conversion efficiency, industrial-scale production, product purity and application value. Although genetic engineering methods have been used to try to improve conversion efficiency, it is still a difficult task to achieve high-concentration products, fast production rates and high conversion efficiency in microbial processes. In response to this challenge, based on the previously constructed engineered strain Aspergillus niger LH-11, glycerol transporter, glycerol kinase, glycerol-3-phosphate dehydrogenase, glycerol dehydrogenase and dihydroxyacetone kinase were further expressed in sequence through advanced genetic engineering technology. The present invention successfully developed a malic acid production strain Aspergillus niger LH-S8 that can efficiently utilize glycerol. This innovative achievement not only significantly enhances the strain's ability to utilize glycerol, a cheap carbon source, but also greatly improves the cost-effectiveness and market competitiveness of malic acid production, providing an effective solution to cope with raw material cost pressure. In summary, using genetic engineering to transform Aspergillus niger and giving it the ability to efficiently convert crude glycerol into high-value organic acids is an important strategy to reduce the cost of raw materials for organic acid industrial production and improve the economic efficiency of products. The research results of the present invention not only enrich the raw material selection for organic acid fermentation, improve the flexibility and economy of the production process, but also contribute to the green and sustainable development of bio-based chemicals. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an Aspergillus niger strain capable of efficiently utilizing glycerol to produce malic acid, a construction method and an application thereof.

[0004] The technical solution adopted by the present invention to solve its technical problem is:

[0005] The invention discloses an Aspergillus niger LH-S8 which can efficiently utilize glycerol to produce malic acid. The Aspergillus niger LH-S8 is constructed by enhancing the glycerol transport and glycerol metabolism and decomposition capabilities through genetic engineering means.

[0006] Furthermore, the Aspergillus niger LH-S8 is constructed by genetic engineering in Aspergillus niger LH-11 (the Aspergillus niger LH-11 is an engineered strain created in the patent applied for by the applicant "A strain of Aspergillus niger producing organic acid, method and application" (application number: 2024102531598, application date: March 6, 2024, applicant: Nanjing Haohe Biotechnology Co., Ltd.). It is a strain known in the art.) by sequentially expressing the glycerol transporter gene stl1, the glycerol kinase gene glpK, the glycerol-3-phosphate dehydrogenase gene gpdA, the glycerol dehydrogenase gene gdh and the dihydroxyacetone kinase gene dhaK.

[0007] Furthermore, the nucleotide sequences of the glycerol transporter gene stl1, glycerol kinase gene glpK, glycerol-3-phosphate dehydrogenase gene gpdA, glycerol dehydrogenase gene gdh and dihydroxyacetone kinase gene dhaK are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively, and the corresponding amino acid sequences are SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0008] Furthermore, the Aspergillus niger LH-S8 is suitable for producing malic acid using glycerol or a mixture containing more than 30% glycerol as a raw material.

[0009] The specific steps of constructing the Aspergillus niger LH-S8 described above and sequentially expressing the above genes in Aspergillus nigerLH-11 are as follows:

[0010] The glycerol transporter gene stl1, glycerol kinase gene glpK, glycerol-3-phosphate dehydrogenase gene gpdA, glycerol dehydrogenase gene gdh and dihydroxyacetone kinase gene dhaK were cloned into the multiple cloning site of vector pLH454, respectively, to construct the corresponding expression plasmids; the expression plasmids were successively transferred into Aspergillus niger LH-11 by Agrobacterium-mediated method, and after each transfer, the hygromycin resistance marker hph was lost before the next plasmid was transferred, and finally Aspergillus niger LH-S8 was obtained.

[0011] Furthermore, the specific steps are as follows:

[0012] First, the glycerol transporter gene stl1, the glycerol kinase gene glpK, the glycerol-3-phosphate dehydrogenase gene gpdA, the glycerol dehydrogenase gdh, and the dihydroxyacetone kinase dhaK were cloned into the multiple cloning site of the pLH454 plasmid to construct the following expression plasmid:

[0013] pLH454-stl1 is a glycerol transporter expression plasmid, pLH454-glpK is a glycerol kinase expression plasmid, pLH454-gpdA is a glycerol-3-phosphate dehydrogenase expression plasmid, pLH454-gdh is a glycerol dehydrogenase expression plasmid, and pLH454-dhaK is a dihydroxyacetone kinase expression plasmid;

[0014] Next, these plasmids were sequentially transferred into Aspergillus nigerLH-11 using the Agrobacterium-mediated method:

[0015] pLH454-stl1 was transformed into Aspergillus niger LH-11, and the strain Aspergillus niger LH-11-stl1 overexpressing the glycerol transporter was obtained by screening with 250 μg / ml hygromycin resistance;

[0016] pLH454-glpK was transferred into Aspergillus niger LH-11-stl1, and the strain Aspergillus niger LH-11-stl1-glpK overexpressing glycerol transporter and glycerol kinase was obtained by screening;

[0017] pLH454-gpdA was transferred into Aspergillus niger LH-11-stl1-glpK, and Aspergillus niger LH-11-stl1-glpK-gpdA was obtained by screening;

[0018] pLH454-gdh was transferred into Aspergillus niger LH-11-stl1-glpK-gpdA, and Aspergillus niger LH-11-stl1-glpK-gpdA-gdh was obtained by screening;

[0019] Finally, pLH454-dhaK was transferred into Aspergillus niger LH-11-stl1-glpK-gpdA-gdh, and the final engineered strain Aspergillus niger LH-11-S8 was obtained by screening;

[0020] After each transfer, the strain was screened for hygromycin resistance at 250 μg / ml to ensure that the target gene was successfully expressed, and the resistance marker of the previous plasmid was removed by losing the marker hph before the next plasmid was transferred.

[0021] The use of Aspergillus niger LH-S8 as described above in producing malic acid by glycerol fermentation.

[0022] As described above, in the method for producing malic acid by fermentation of Aspergillus niger LH-S8, the culture medium contains 5-20 g / L of glucose and 60-200 g / L of glycerol.

[0023] Further, the steps are as follows:

[0024] Aspergillus niger LH-S8 was inoculated on a PDA medium solid plate and cultured for 3 to 5 days; then, sterile water was added and the spores were washed off with a cotton swab; the number of spores in the spore solution was counted using a hemocytometer and inoculated into a seed medium with a final spore concentration of 1.0×10 5 ~2.0×10 6 / mL, culture at 28-37°C and 200-300rpm shaking table for 20-40h; then take 50-140mL seed culture solution and inoculate it into a 2L fermenter with a malic acid fermentation medium filling volume of 1.26L, the fermentation temperature is set at 28-37°C, the ventilation volume is 0.5-2.0vvm, the rotation speed is 200-500rpm, and the fermentation cycle is 7 days; on the 7th day of fermentation, the malic acid concentration reaches 100-120g / L, the average glycerol consumption rate is 0.5-0.9g / L / h, and the carbon conversion rate is 0.9-1.2C-mol / C-mol, which is close to 70-90% of the theoretical maximum value.

[0025] Furthermore, the formula of the seed culture medium is as follows: the components of each liter of the seed culture medium are: 40g of glucose, 6g of bacterial peptone, 0.1g of magnesium sulfate heptahydrate, 0.1g of calcium chloride dihydrate, 0.75g of potassium dihydrogen phosphate, 0.75g of dipotassium hydrogen phosphate, 0.005g of ferrous sulfate heptahydrate, 0.005g of sodium chloride, 0.001g of anhydrous citric acid, pH=6.5, and the solvent is water;

[0026] The formula of the malic acid fermentation medium is as follows: per liter of malic acid fermentation medium components include: 20 g of glucose, 81.7 g of glycerol, 6 g of bacterial peptone, 0.1 g of magnesium sulfate heptahydrate, 0.1 g of calcium chloride dihydrate, 0.15 g of potassium dihydrogen phosphate, 0.15 g of dipotassium hydrogen phosphate, 0.005 g of ferrous sulfate heptahydrate, 0.005 g of sodium chloride, 0.001 g of anhydrous citric acid, 80 g of calcium carbonate, pH=6.5, and the solvent is water.

[0027] The advantages and positive effects achieved by the present invention are:

[0028] 1. The present invention successfully constructed a strain Aspergillus niger LH-S8 that can efficiently utilize glycerol to produce malic acid. In a fermenter, the strain can produce 100-120 g / L of L-malic acid within 5-7 days, with an average glycerol consumption rate of 0.5-0.9 g / L / h and a carbon conversion rate of 0.9-1.2 C-mol / C-mol, which is close to 70-90% of the theoretical maximum value. In the research field of utilizing glycerol to produce malic acid, the raw material conversion rate reaches the highest reported so far while maintaining a high production concentration and a short production cycle. The present invention not only improves the raw material conversion rate, but also provides a new idea for the green and sustainable development of bio-based chemicals.

[0029] 2. The present invention further enhances the glycerol metabolism ability of the Aspergillus niger strain by overexpressing key genes in the glycerol metabolic pathway (glycerol transporter stl1, glycerol kinase glpK, glycerol-3-phosphate dehydrogenase gpdA, glycerol dehydrogenase gdh and dihydroxyacetone kinase dhak). The genetic engineering strategy used in the present invention is replicable in Aspergillus niger, providing a reference for Aspergillus niger to transform glycerol to produce other high-value chemicals.

[0030] 3. The present invention successfully achieved efficient bioconversion of glycerol to L-malic acid, demonstrating the great potential of glycerol as a renewable carbon source. These achievements not only promote the progress of biological production technology of L-malic acid, but also provide new ideas and directions for the bioconversion of glycerol-based chemicals.

[0031] 4. The glycerol transport and metabolic decomposition capabilities of the Aspergillus niger strain Aspergillus niger LH-S8 of the present invention are significantly enhanced, which is achieved through a series of precise genetic engineering operations. Aspergillus niger LH-S8 is particularly suitable for the efficient production of malic acid using a mixture with glycerol as the main component (accounting for more than 30%) as a raw material. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the map of the starting plasmid pLH454 in the present invention;

[0033] Figure 2 The diagram is a map (Figure A) and an enzyme digestion verification diagram (Figure B) of the glycerol transporter stl1 overexpression plasmid pLH454-stl1 of the present invention; in Figure B, M is a DNA Marker, and 1 is an enzyme digestion verification diagram (XbaⅠ, 1748bp / 9956bp) of the pLH454-stl1 expression plasmid;

[0034] Figure 3 The diagram is a map (Figure A) and an enzyme cutting verification diagram (Figure B) of the glycerol kinase glpK expression plasmid pLH454-glpK of the present invention; in Figure B, M is a DNA Marker, and 1 and 2 are enzyme cutting verification diagrams (SacⅠ, 1650bp / 10153bp) of the pLH454-glpK expression plasmid;

[0035] Figure 4 The diagram is a map (Figure A) and an enzyme digestion verification diagram (Figure B) of the glycerol-3-phosphate dehydrogenase gpdA expression plasmid pLH454-gpdA of the present invention; in Figure B, M is a DNA Marker, and 1 and 2 are enzyme digestion verification diagrams of the pLH454-gpdA expression plasmid (EcoRⅠ, SnaBI, 1267bp / 1611bp / 8290bp);

[0036] Figure 5 The diagram is a map (Figure A) and an enzyme digestion verification diagram (Figure B) of the glycerol dehydrogenase gdh expression plasmid pLH454-gdh of the present invention; in Figure B, M is a DNA Marker, and 1 and 2 are enzyme digestion verification diagrams (SpeⅠ, SnaBI, 2643bp / 8290bp) of the pLH454-gdh expression plasmid;

[0037] Figure 6 The diagram is a map (Figure A) and an enzyme digestion verification diagram (Figure B) of the glycerol triphosphate dehydrogenase dhaK expression plasmid pLH454-dhaK of the present invention; in Figure B, M is a DNA Marker, and 1 and 2 are enzyme digestion verification diagrams (BamHI, 1623bp / 10138bp) of the pLH454-dhaK expression plasmid;

[0038] Figure 7 This is a shake flask fermentation diagram of the strain LH-S8 of the present invention using glycerol as the main carbon source;

[0039] Figure 8 This is a diagram of malic acid fermentation using glycerol as the main carbon source by strain LH-S8 in a fermenter of the present invention. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0041] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians 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 this invention.

[0042] The invention discloses an Aspergillus niger LH-S8 which can efficiently utilize glycerol to produce malic acid. The Aspergillus niger LH-S8 is constructed by enhancing the glycerol transport and glycerol metabolism and decomposition capabilities through genetic engineering means.

[0043] Preferably, the Aspergillus niger LH-S8 is constructed by genetic engineering in Aspergillus niger LH-11 (the Aspergillus niger LH-11 is an engineered strain created in the patent applied for by the applicant "A strain of Aspergillus niger producing organic acid, method and application" (application number: 2024102531598, application date: March 6, 2024, applicant: Nanjing Haohe Biotechnology Co., Ltd.). It is a strain known in the art.) by sequentially expressing the glycerol transporter gene stl1, the glycerol kinase gene glpK, the glycerol-3-phosphate dehydrogenase gene gpdA, the glycerol dehydrogenase gene gdh and the dihydroxyacetone kinase gene dhaK.

[0044] Preferably, the nucleotide sequences of the glycerol transporter gene stl1, glycerol kinase gene glpK, glycerol-3-phosphate dehydrogenase gene gpdA, glycerol dehydrogenase gene gdh and dihydroxyacetone kinase gene dhaK are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively, and the corresponding amino acid sequences are SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0045] Preferably, the Aspergillus niger LH-S8 is suitable for producing malic acid using glycerol or a mixture containing more than 30% glycerol as a raw material.

[0046] The specific steps of constructing the Aspergillus niger LH-S8 described above and sequentially expressing the above genes in Aspergillus nigerLH-11 are as follows:

[0047] The glycerol transporter gene stl1, glycerol kinase gene glpK, glycerol-3-phosphate dehydrogenase gene gpdA, glycerol dehydrogenase gene gdh and dihydroxyacetone kinase gene dhaK were cloned into the multiple cloning site of vector pLH454, respectively, to construct the corresponding expression plasmids; the expression plasmids were successively transferred into Aspergillus niger LH-11 by Agrobacterium-mediated method, and after each transfer, the hygromycin resistance marker hph was lost before the next plasmid was transferred, and finally Aspergillus niger LH-S8 was obtained.

[0048] Preferably, the specific steps are as follows:

[0049] First, the glycerol transporter gene stl1, the glycerol kinase gene glpK, the glycerol-3-phosphate dehydrogenase gene gpdA, the glycerol dehydrogenase gdh, and the dihydroxyacetone kinase dhaK were cloned into the multiple cloning site of the pLH454 plasmid to construct the following expression plasmid:

[0050] pLH454-stl1 is a glycerol transporter expression plasmid, pLH454-glpK is a glycerol kinase expression plasmid, pLH454-gpdA is a glycerol-3-phosphate dehydrogenase expression plasmid, pLH454-gdh is a glycerol dehydrogenase expression plasmid, and pLH454-dhaK is a dihydroxyacetone kinase expression plasmid;

[0051] Next, these plasmids were sequentially transferred into Aspergillus nigerLH-11 using the Agrobacterium-mediated method:

[0052] pLH454-stl1 was transformed into Aspergillus niger LH-11, and the strain Aspergillus niger LH-11-stl1 overexpressing the glycerol transporter was obtained by screening with 250 μg / ml hygromycin resistance;

[0053] pLH454-glpK was transferred into Aspergillus niger LH-11-stl1, and the strain Aspergillus niger LH-11-stl1-glpK overexpressing glycerol transporter and glycerol kinase was obtained by screening;

[0054] pLH454-gpdA was transferred into Aspergillus niger LH-11-stl1-glpK, and Aspergillus niger LH-11-stl1-glpK-gpdA was obtained by screening;

[0055] pLH454-gdh was transferred into Aspergillus niger LH-11-stl1-glpK-gpdA, and Aspergillus niger LH-11-stl1-glpK-gpdA-gdh was obtained by screening;

[0056] Finally, pLH454-dhaK was transferred into Aspergillus niger LH-11-stl1-glpK-gpdA-gdh, and the final engineered strain Aspergillus niger LH-11-S8 was obtained by screening;

[0057] After each transfer, the strain was screened for hygromycin resistance at 250 μg / ml to ensure that the target gene was successfully expressed, and the resistance marker of the previous plasmid was removed by losing the marker hph before the next plasmid was transferred.

[0058] The use of Aspergillus niger LH-S8 as described above in producing malic acid by glycerol fermentation.

[0059] As described above, in the method for producing malic acid by fermentation of Aspergillus niger LH-S8, the culture medium contains 5-20 g / L of glucose and 60-200 g / L of glycerol.

[0060] Preferably, the steps are as follows:

[0061] Aspergillus niger LH-S8 was inoculated on a PDA medium solid plate and cultured for 3 to 5 days; then, sterile water was added and the spores were washed off with a cotton swab; the number of spores in the spore solution was counted using a hemocytometer and inoculated into a seed medium with a final spore concentration of 1.0×10 5 ~2.0×10 6 / mL, culture at 28-37°C and 200-300rpm shaking table for 20-40h; then take 50-140mL seed culture solution and inoculate it into a 2L fermenter with a malic acid fermentation medium filling volume of 1.26L, the fermentation temperature is set at 28-37°C, the ventilation volume is 0.5-2.0vvm, the rotation speed is 200-500rpm, and the fermentation cycle is 7 days; on the 7th day of fermentation, the malic acid concentration reaches 100-120g / L, the average glycerol consumption rate is 0.5-0.9g / L / h, and the carbon conversion rate is 0.9-1.2C-mol / C-mol, which is close to 70-90% of the theoretical maximum value.

[0062] Preferably, the formula of the seed culture medium is: the components of each liter of seed culture medium are: 40g of glucose, 6g of bacterial peptone, 0.1g of magnesium sulfate heptahydrate, 0.1g of calcium chloride dihydrate, 0.75g of potassium dihydrogen phosphate, 0.75g of dipotassium hydrogen phosphate, 0.005g of ferrous sulfate heptahydrate, 0.005g of sodium chloride, 0.001g of anhydrous citric acid, pH=6.5, and the solvent is water;

[0063] The formula of the malic acid fermentation medium is as follows: per liter of malic acid fermentation medium components include: 20 g of glucose, 81.7 g of glycerol, 6 g of bacterial peptone, 0.1 g of magnesium sulfate heptahydrate, 0.1 g of calcium chloride dihydrate, 0.15 g of potassium dihydrogen phosphate, 0.15 g of dipotassium hydrogen phosphate, 0.005 g of ferrous sulfate heptahydrate, 0.005 g of sodium chloride, 0.001 g of anhydrous citric acid, 80 g of calcium carbonate, pH=6.5, and the solvent is water.

[0064] The organic acid liquid phase detection methods mentioned below all used HPLC (Agilent 1260 Infinity II Prime) for detection, using an organic acid column (amino HPX-87H, 300 mm×7.8 mm), the column temperature was controlled at 65°C, the mobile phase was 5 mM sulfuric acid, the flow rate was 0.6 mL / min, and the UV detection wavelength was set to 210 nm.

[0065] The pLH454 plasmid used in the following may be referred to: Development of a Cre-loxP-based genetic system in Aspergillus niger ATCC1015 and its application to construction of efficient organic acid-producing cell factories. Appl Microbiol Biotechnol. 2019, 103(19): 8105-8114. doi: 10.1007 / s00253-019-10054-3. The pLH454 plasmid is also disclosed in the patent publication CN111218408A.

[0066] Aspergillus niger LH-11 itself is a strain of Aspergillus niger with the ability to produce organic acids, created and described by Nanjing Haohe Biotechnology Co., Ltd. in a previous patent (application number: 2024102531598, application date: March 6, 2024).

[0067] Specifically, the relevant preparation and detection are as follows:

[0068] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians 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 this invention.

[0069] Example 1: Construction of strain Aspergillus niger LH-S8

[0070] (1) Construction of pLH454-stl1 (glycerol transporter expression plasmid)

[0071] The synthetic codon-optimized stl1 sequence (SEQ NO 1) was used as a template and primers stl1-F / stl1-R were used to amplify the gene stl1 sequence fragment by PCR reaction. The pLH454 ( Figure 1 ) for ligation, and the ligation product was transformed into E. coli JM109 competent cells, evenly coated in LB solid medium containing 100 μg / mL kanamycin resistance, and inverted overnight culture at 37°C. Single clones were picked for colony PCR verification and plasmid double enzyme digestion verification to obtain pLH454-stl1. Its map and plasmid enzyme digestion verification are shown in Figure 2 shown.

[0072] (2) Construction of pLH454-glpK (glycerol kinase expression plasmid)

[0073] Glycerol kinase glpK was amplified by PCR reaction using cDNA of Aspergillus niger LH-11 as template and primers glpK-F / glpK-R to obtain the gene glpK sequence fragment (sequence number 2). The One Step Clone Kit was used to connect with pLH454 double-digested with Sac I and pst I, and the connection product was transformed into E. coli JM109 competent cells, evenly spread in LB solid medium containing 100 μg / mL kanamycin resistance, and inverted overnight culture at 37°C. Single clones were picked and verified by colony PCR and plasmid enzyme digestion to obtain pLH454-glpK. Its map and plasmid enzyme digestion verification are shown in Figure 3 shown.

[0074] (3) Construction of pLH454-gpdA (glycerol-3-phosphate dehydrogenase expression plasmid)

[0075] Glycerol-3-phosphate dehydrogenase gpdA was amplified by PCR reaction using cDNA of Aspergillus niger LH-11 as template and primers gpdA-F / gpdA-R to obtain the gene gpdA sequence fragment (sequence number SEQ NO 3). The OneStep Clone Kit was used to connect with pLH454 double-digested with EcoRI and SacI, and the connection product was transferred to E.coli JM109 competent cells, evenly spread on LB solid medium containing 100 μg / mL kanamycin resistance, and inverted overnight culture at 37°C. Single clones were picked and verified by colony PCR and plasmid enzyme digestion to obtain pLH454-gpdA. Its map and plasmid enzyme digestion verification are shown in Figure 4 shown.

[0076] (4) Construction of pLH454-gdh (glycerol dehydrogenase expression plasmid)

[0077] Glycerol dehydrogenase gdh was amplified by PCR reaction using cDNA of Aspergillus niger LH-11 as template and primers gdh-F / gdh-R to obtain the gene gdh sequence fragment (sequence number SEQ NO 4). The One Step CloneKit kit was used to connect with pLH454 double-digested with EcoRI and SacI, and the connection product was transferred to E. coli JM109 competent cells, evenly spread in LB solid medium containing 100 μg / mL kanamycin resistance, and inverted overnight culture at 37°C. Single clones were picked and verified by colony PCR and plasmid enzyme digestion to obtain pLH454-gdh. Its map and plasmid enzyme digestion verification are shown in Figure 5 shown.

[0078] (5) Construction of pLH454-dhaK (dihydroxyacetone kinase expression plasmid)

[0079] Dihydroxyacetone kinase dhaK was amplified by PCR reaction using cDNA of Aspergillus niger LH-11 as template and primers dhaK-F / dhak-R to obtain the gene dhaK sequence fragment (sequence number 5). The One Step Clone Kit was used to connect with pLH454 double-digested with EcoRI and SacI, and the connection product was transformed into E. coli JM109 competent cells, evenly spread in LB solid medium containing 100 μg / mL kanamycin resistance, and inverted overnight culture at 37°C. Single clones were picked and verified by colony PCR and plasmid enzyme digestion to obtain pLH454-dhaK. Its map and plasmid enzyme digestion verification are shown in Figure 6 shown.

[0080] (6) Construction of strain Aspergillus niger LH-S8

[0081] Agrobacterium containing plasmid pLH454-stl1 was co-cultured with spores of Aspergillus niger LH-11 on a solid plate of IM medium (for specific methods, see Development of a Cre-loxP-based genetic system in Aspergillus niger ATCC1015 and its application to construction of efficient organic acid-producing cell factories. Appl Microbiol Biotechnol. 2019, 103(19): 8105-8114. doi: 10.1007 / s00253-019-10054-3.), and then the co-culture was transferred to a solid plate of CM medium containing 200 μM cefotaxime, 100 μg / mL ampicillin, 100 μg / mL streptomycin, and 250 μg / mL hygromycin B and cultured at 28°C until a single clone was formed. Single clones were picked and transferred to potato dextrose agar plates and potato dextrose agar plates containing hygromycin B (250 μg / mL), and transformants with hygromycin B resistance were selected for genome extraction and verification to obtain hygromycin-resistant expression strains containing the stl1 gene. One of the correct clones was picked for induced recombination of the resistance marker hygromycin to obtain the stl1 gene overexpression strain Aspergillus niger LH-11-stl1 without hygromycin resistance.

[0082] After resistance marker-induced recombination of Aspergillus niger LH-11-stl1, pLH454-glpK was transferred into Aspergillus niger LH-11-stl1 using the same method as above, and the strain Aspergillus niger LH-11-stl1-glpK overexpressing glycerol transporter and glycerol kinase was obtained by screening.

[0083] According to the above method, the following steps were performed: pLH454-gpdA was transferred into Aspergillus niger LH-11-stl1-glpK, and Aspergillus niger LH-11-stl1-glpK-gpdA was obtained by screening. pLH454-gdh was transferred into Aspergillus niger LH-11-stl1-glpK-gpdA, and Aspergillus niger LH-11-stl1-glpK-gpdA-gdh was obtained by screening. Finally, pLH454-dhaK was transferred into Aspergillus niger LH-11-stl1-glpK-gpdA-gdh, and the final engineered strain Aspergillus niger LH-11-S8 was obtained by screening.

[0084] The Agrobacterium involved in the co-cultivation method is the AGL-1 strain, and the electroporation conditions are: Capacitnce: 25uF, Voltage: 2.5kV, Resistance: 200Ω, Pulse: 5msec. The IM culture medium preparation method is: 15g agar is added with water to a volume of 905.7mL, sterilized at 121℃ for 20min, and the prepared sterile Kbuffer 0.8mL, MN buffer 20mL, 1% calcium chloride solution 1mL, 0.01% ferrous sulfate solution 10mL, IM Trace elements 5mL, 20% ammonium nitrate solution 2.5mL, 50% glycerol 10mL, 1M MES 40mL, 20% glucose 5mL are added, and when the temperature is cooled to about 50℃, kanamycin is added to a final concentration of 100μg / mL, and acetosyringone is added to a final concentration of 200μM; wherein the above percentages are all mass percentages. MNbuffer ingredients: 3 g magnesium sulfate, 1.5 g sodium chloride, dilute to 100 mL with water, filter sterilize. IMTrace elements ingredients: 10 mg zinc sulfate, 10 mg copper sulfate, 10 mg boric acid, 10 mg manganese sulfate, 10 mg sodium molybdate, dilute to 100 mL with water, filter sterilize.

[0085] The CM culture medium is prepared as follows: 20 g agar is diluted with water to 897 mL, sterilized at 121° C. for 20 min, and 20 mL of sterile ASPN solution (containing 26.1 g / L potassium chloride, 74.8 g / L potassium dihydrogen phosphate, 297.5 g / L sodium nitrate, with water as solvent and pH adjusted to 5.5) prepared in advance, 20 mL of 50% glucose, 2 mL of 1 M manganese sulfate, 1 mL of CM Trace elements (containing 2.1 g zinc sulfate, 1.1 g boric acid, 0.5 g manganese chloride, 0.5 g ferrous sulfate, 0.17 g cobalt chloride, 0.16 g copper sulfate, 0.15 g sodium molybdate, 5.1 g EDTA per 100 mL) is added. 10% casein hydrolysate, 50mL 10% yeast extract, when the temperature is cooled to about 50°C, add hygromycin to make the final concentration of 250μg / mL, add streptomycin to make the final concentration of 100μg / mL, add ceftriaxone to make the final concentration of 100μg / mL, add ampicillin to make the final concentration of 100μg / mL; wherein the above percentages are all mass percentages.

[0086] The resistance marker induced recombination method comprises the following steps: spores of about 300 pyc gene expression clones are evenly spread on an MM medium containing 30 μg / mL tetracycline, cultured at 28°C until a single clone grows out, 200 single clones are randomly selected and transferred to a potato dextrose agar medium and cultured at 28°C for 24 hours, then the clones are transferred one by one to a potato dextrose agar medium containing hygromycin (250 μg / mL) and cultured at 28°C for 24 hours, and finally phenotype observation is performed to screen transformants of resistance marker induced recombination, that is, transformants that grow normally on the potato dextrose agar medium and cannot grow on the potato dextrose agar medium containing hygromycin are transformants of successfully induced recombination.

[0087] Primer sequences used:

[0088] Primers Sequence(5'→3’) stl1-F CACATCTAAACAATGGAATTCATGAAGGATCTGAAGCTGTC stl1-F TCCCTGCAGGGTACCGAGCTCTTAGCCCTCGAAGTTGGCCT glpK-F TAAACAATGGAATTCGAGCTCATGCAGAAATCACCCACCAT glpK-R ACGTTAAGTGGATCCCTGCAGCTATGCACCGTTCTGTCCTT gpdA-F TTCGAGCTCGGTACCCTGCAGATGGCCTCCCGCCAGTCTCG gpdA-R TCAGTAACGTTAAGTGGATCCCTATTTGTTTGCCGGGGACT gdh-F CACATCTAAACAATGGAATTCGAATTCGCTCCTCCCACTCA gdh-R TCCCTGCAGGGTACCGAGCTCGGATCCTTACTGCGACTTCA dhaK-F CACATCTAAACAATGGAATTCCAGACCAAACACTTCTTCTC dhaK-R TCCCTGCAGGGTACCGAGCTCTTACAACGCCTCAGACAGAC

[0089] Example 2: Application of strain Aspergillus niger LH-S8 to efficiently utilize glycerol to produce malic acid

[0090] The method for producing malic acid by fermenting the strain Aspergillus niger LH-S8 constructed by the present invention in a shake flask and a fermenter, wherein the carbon source conditions are 20 g / L glucose and 81.7 g / L glycerol, and the specific steps are as follows:

[0091] Shake flask fermentation: Inoculate Aspergillus niger LH-S8 on a PDA medium solid plate and culture for 5 days. Then, add an appropriate amount of sterile water and wash off the spores with a cotton swab. Use a hemocytometer to count the number of spores in the spore solution and calculate the number of spores at 2×10 6 The spores / mL were inoculated into malic acid fermentation medium and cultured in a shaker at 28°C, 200rpm, and the fermentation time was 5 days. Finally, the fermentation products were collected and test samples were prepared. The malic acid content of the samples was determined by high performance liquid chromatography. After 10 days of shake flask fermentation, the malic acid concentration of Aspergillus niger LH-S8 reached 104.5 g / L. The results are shown in Figure 7 shown.

[0092] The malic acid fermentation medium comprises the following components per liter: 20 g of glucose, 81.7 g of glycerol, 6 g of bacterial peptone, 0.1 g of magnesium sulfate heptahydrate, 0.1 g of calcium chloride dihydrate, 0.15 g of potassium dihydrogen phosphate, 0.15 g of dipotassium hydrogen phosphate, 0.005 g of ferrous sulfate heptahydrate, 0.005 g of sodium chloride, 0.001 g of anhydrous citric acid, 80 g of calcium carbonate, pH=6.5, and the solvent is water.

[0093] Fermentation in a fermenter: Inoculate Aspergillus niger LH-S8 on a PDA medium solid plate and culture for 5 days. Then, add an appropriate amount of sterile water and wash off the spores with a cotton swab. Count the number of spores in the spore solution using a hemocytometer and inoculate it into a 250 mL triangular flask containing 50 mL of seed culture medium. The final spore concentration is 2.0 × 10 6 / mL, and cultured at 28℃, 200rpm shaking for 20h; then 140mL of seed culture was inoculated into a 2L fermenter with 1.26L of malic acid fermentation medium, and the fermentation temperature was set at 30℃, the ventilation volume was 1vvm, and the rotation speed was 300rpm. Figure 8 As shown, on the 7th day of fermentation, the malic acid concentration reached 108.7 g / L, the malic acid production rate reached 0.65 g / L / h, and the carbon conversion rate was as high as 0.97 C-mol / C-mol, reaching 74% of the maximum theoretical conversion rate (1.33 mol / mol).

[0094] The seed culture medium comprises per liter: 40 g of glucose, 6 g of bacterial peptone, 0.1 g of magnesium sulfate heptahydrate, 0.1 g of calcium chloride dihydrate, 0.75 g of potassium dihydrogen phosphate, 0.75 g of dipotassium hydrogen phosphate, 0.005 g of ferrous sulfate heptahydrate, 0.005 g of sodium chloride, 0.001 g of anhydrous citric acid, pH=6.5, and the solvent is water. The malic acid fermentation medium used in the fermentation tank is the same as the malic acid fermentation medium used in the shake flask fermentation.

[0095] The Aspergillus niger LH-S8 provided by the present invention has the ability to efficiently utilize glycerol to produce malic acid, and can be used as a good strain for producing malic acid based on glycerol. In the production of malic acid using glycerol, LH-S8 obtained 108.7 g / L of malic acid after 7 days of fermentation at a production intensity of 0.69 g / L / h, and the carbon conversion rate reached 0.97 C-mol / C-mol. In order to highlight the advantages of this study, in related studies at home and abroad, Aspergillus niger fermented for 288 h (12 days) at a production intensity of 0.45 g / L / h to obtain a malic acid concentration of 131.5 g / L, and the C conversion rate was 0.97 C-mol / C-mol, while Echinochloa crus-galli fermented for 264 h (11 days) at a production intensity of 0.56 g / L / h to obtain a malic acid concentration of 134 g / L, and the C conversion rate was only 0.37 C-mol / C-mol. On the basis of obtaining a higher malic acid concentration, this study shortened the fermentation cycle. The malic acid production intensity and carbon yield have great advantages over the existing malic acid production strains using glycerol as a carbon source, providing a new solution for reducing the fermentation production cost and improving the economy of microbial fermentation to produce malic acid.

[0096] The sequences used in the present invention are as follows:

[0097] SEQ NO 1 Codon-optimized stl1 nucleotide sequence

[0098] ATGAAGGATCTGAAGCTGTCCAACTTCAAGGGCAAGTTCATCTCCCGCACCTCCACTG

[0099] GGGTCTGACTGGTAAAAAACTGCGCTACTTCATTACCATCGCCTCCATGACCGGCTTCTC

[0100] CCTGTTTGGCTATGATCAGGGCCTGATGGCCTCCCTGATTACCGGTAAACAGTTCAACTA

[0101] CGAGTTCCCCGCCACCAAGGAGAACGGCGATCATGATCGCCATGCCACCGTCGTCCAAG

[0102] GCGCTACTACTTCCTGTTACGAGCTGGGCTGCTTTGCCGGTTCCCTGTTTGTCATGTTCT

[0103] GCGGCGAGCGCATCGGCCGCAAACCTCTGATTCTGATGGGTTCCGTCATCACCATCATCG

[0104] GCGCCGTCATTTCCACCTGCGCTTTCCGCGGTTACTGGGCTCTGGGTCAATTTATTATCGG

[0105] CCGCGTCGTCACTGGCGTCGGCACTGGTTTGAATACTTCCACTATTCCCGTCTGGCAGTC

[0106] CGAGATGTCCAAAGCCGAAAATCGCGGCCTGCTGGTCAACCTGGAAGGTTCCACTATCG

[0107] CCTTTGGCACCATGATCGCCTACTGGATCGATTTCGGCCTGTCCTACACCAACTCCTCCG

[0108] TCCAGTGGCGCTTTCCCGTCTCTATGCAGATTGTCTTCGCCCTGTTCCTGCTGGCCTTCAT

[0109] GATCAAGCTGCCCGAGTCCCCCCGCTGGTTGATTTCCCAATCTAGAACTGAAGAAGCCC

[0110] GCTACCTGGTCGGCACCCTGGATGATGCTGATCCCAATGATGAGGAGGTCATCACCGAG

[0111] GTCGCCATGCTGCATGATGCCGTCAATCGCACTAAGCACGAGAAGCACTCCCTGTCCTC

[0112] CCTGTTCTCCCGCGGTAGATCCCAAAATCTGCAACGCGCTCTGATTGCTGCTTCCACTCA

[0113] ATTTTTTCAGCAGTTCACCGGCTGCAACGCCGCCATTTATTATTCCACCGTCCTGTTCAAC

[0114] AAGACCATCAAGCTGGATTACCGCCTGTCCATGATCATCGGCGGCGTCTTTGCCACCATC

[0115] TACGCCCTGTCTACTATCGGCTCCTTCTTCCTGATCGAGAAGCTGGGCCGCCGCAAACTG

[0116] TTTCTGCTGGGCGCCACCGGTCAAGCTGTCTCTTTTACCATTACTTTCGCCTGCCTGGTC

[0117] AAGGAGAACAAGGAGAACGCCCGCGGCCGGCGCGCGTCGGCTTGTTTCTGTTTATTACTTT

[0118] TTTCGGCCTGTCCCTGCTGTCCCTGCCTGGATTTATCCCCTGAAATTGCTTCCATGAAG

[0119] GTCCGCGCCTCCACCAACGCTTTTCCACCTGTACCAATTGGCTGTGCAACTTCGCCGTC

[0120] GTCATGTTCACCCCCATCTTCATCGGCCAGTCCGGCTGGGGTTGTTACCTGTTTTTCGCC

[0121] GTCATGAACTACCTGTACATCCCCGTCATCTTCTTCTTCTACCCCGAACCGCCGGCCGC

[0122] TCCCTGGAAGAAATTGATATTATCTTCGCCAAGGCCTACGAGGATGGCACCCAGCCCTGG

[0123] CGCGTCGCTAATCATCTGCCTAAACTGTCCCTGCAGGAGGTCGAAGATCACGCCAATGC

[0124] TCTGGGCTCCTACGATGATGAGATGGAGAAGGAGGATTTCGGCGAGGATCGCGTCGAGG

[0125] ATACCTACAACCAGATCAACGGCGATAACTCCTCCTCCTCCTCCAACATCAAGAACGAG

[0126] GATACCGTCAACGATAAGGCCAACTTCGAGGGCSEQ NO 2 Glycerol kinase glpK nucleotide sequence

[0127] ATGCAGAAATCACCCACCATCACTTTTGACTCTATCAGTCTTCCCGATCCTCTCAACCCC

[0128] CCCACCAAGAAGACCAGAACGCCACCAATCACTACCTCCAAAATGAGGAACCCTTTCG

[0129] ATATGGCCGACCTGGACTCCGCCGTCGAACAGGAAGAACCTCAGCCTTTCATGAACATT

[0130] CACGCCTACCAGTCGCATGATGGCAATCGCTCCCTCTTACAACCCAACGAAAACCGCCA

[0131] CAGTATGAAGGATCGCTTTATCGGTGCCATTGACACTGGTACCACCAGCTCCCGTTTCAT

[0132] CATCTTCGACTGCACTGGTGTTCCTGTGGCCAAGTACCAGATGGAATTCCGCCAAATCCA

[0133] CGAGAAATCAGGATGGCACGAACAAGACCCCTTCGAACTTGTCGATTCCGTATACACCT

[0134] GCATTGAAGAGGCTATGAAGACCTTCCTCGCACTCGGCCACTCCCGGGACGATATCGAG

[0135] GCTATTGGTATTACCAGTCAACGTGAGACAACCGTGTGCTGGGATTGGGAGACTGGCGA

[0136] GCCTCTACACCACGCAATCGCGTGGCCAGATACCAGAACCACTGGGCTGGTTCGAGAAC

[0137] TCAAGACAAAGGAGGGTGCGGATGAGCTGTCAAACATTTGCGGCCTTCCCCTATCGACC

[0138] TACCCTTCCTCTGTCACCCTCATGTGGATGCTCCGCAACCTCCCCGAGGTTAAGAGGGCA

[0139] TATGATGAGGGCCGCCTTGCTTTTGGTACCATCGACACCTGGCTGATATATAACCTGAAC

[0140] GGTGGCCCGGAGGGCGGCCGGCTGGTGACTGACGTCACCAACGCCTCACGAACCATGT

[0141] TCATGAACCTCGAAACGCTTGATTACGATGATAAGTTGTTGAAATTCTTCGAAATTGATC

[0142] GGAACAAGATTAGACTGCCCAAGATTCTCCCTTCGTCTGACCCGGAGGGCTACGGATGG

[0143] GTTAGAGACGGCCCGCTTGAGGGCATTCCTATCACAAGCTGTCTCGGAGATCAGTCTGC

[0144] AGCCTTGGTCGGTCACTGTGCCTTCACGCCTGGATCTGCGAAGAACACCTATGGCACGG

[0145] GCTGTTTCCTTCTCTACAACGTTGGTGAGAAGCCAGTTATCTCCAAGCATGGCTTGCTTG

[0146] GCACCGTGGGCTTCCAGCTGGGCAAGCACCGCAAGCCAGTGTATGCCTTGGAGGGCAG

[0147] TGTCGCTGTTGCTGGAAGTGGTATCTCTTTCCTGATGAACAACATGGGTTTCTTCCGTGA

[0148] TTCCCGCAAAGTCAGTGAGGTCGCTGCGACCGTCCCCGACAGTGGCGGCTGTGTCTTCG

[0149] TGACGGCCTTCAGTGGTCTGTTCGCACCCTACTGGATTGATGATGCCAAGGGAACCATCT

[0150] TTGGTATCACCCAGCACACCCAGCGTGGACACATTGCACGAGCCACCATGGAAGCGGCC

[0151] TGCTTCCAGACCAAGGCCATCTTGGATGCCATGGAGATGGACAGTGGTCACAAATTGTC

[0152] GGAATTGGCCGTCGATGGCGGTATGAGCAATTCCGATATTTGCATGCAGACTCAAGCCGA

[0153] TATCATTCAGATTCCCGTTGAACGCCCGGCCATGCACGAGACGACGGCGCTGGGAGCCG

[0154] CGATCGCAGCTGGCTTTGCAGTCAACATCTGGAAGGATTTCAGCGAGCTCAAGGACATG

[0155] AACCGGGCCAACCGCACTACCTTCCTTCCCCATGCCTCTCCAGTGCAGAGCAAGAAGAT

[0156] GTACCGTCAATGGAGCAAGGCCGTCGAGATGTCGCGTGGCTGGCTCGACACGAACGAG

[0157] TCGGAAGAAGAAGAAGAAGAAGGACAGAACGGTGCATAG

[0158] SEQ NO 3 Nucleotide sequence of glycerol-3-phosphate dehydrogenase gpdA

[0159] ATGGCCTCCCGCCAGTCTCGTAGGGTTCTGCGACCTCTGCTTTATACATCGCTGGCGGCT

[0160] GCCACAGGAGCCGGAGTTCTCTACATCTCTTATCGTCCCCGCAATATACCTGGTTCAGAA

[0161] GCCCCCGCAGTTCCGCCGCCGGGCTACCATGAAGGCAAGCTCGTGCCCCCAAGCTTTCC

[0162] CAATATCAAATCCCGCCTCGAGCAGATCCAAGACTTAAAGCGATCTTCTGCTGGAAGCA

[0163] ACTCTGAAGAGTATGATCTGCTCGTCATTGGCGGTGGTGCCACTGGATCGGGTATTGCCC

[0164] TTGATGCTGCCACACGGGGGTTGAAGGTTGCCATTGTTGAGAGGGACGACTTCAGCGCC

[0165] GGAACGAGCAGCAAGAGTACCAAACTCGTTCACGGTGGTGTTCGATATTTGGAAAAGG

[0166] CTGTTTGGGAGTTGGATTACAACCAGTACAAGCTAGTCAAAGAGGCTCTGCGGGAGCG

[0167] AAAGTATTTCTTGAACACAGCACCTCATCTGTCTAGCTGGCTTCCTATTATGGTTCCGCTA

[0168] CAGAAATGGTGGCAGGCGCCTTATTTCTGGGCGGGAACAAAGTTCTACGATTACTTGGC

[0169] TGGGTCTGAAGGTATCGAGAGCTCCTACTTCCTCACCAAGAGCAAGGCCATCGATGCCT

[0170] TCCCTATGTTAAGAAAGGACAACCTGTTTGGAGCCATGGTATACTATGATGGTGCCCACA

[0171] ACGACTCTCGAATGAATGTTTCTCTGGCCATGACAGCTGCGTTATACGGTGCCACTGTTG

[0172] TCAATCACATGCAAGTCACTGGCCTGGAAAAGGACTCCTCCGGGAAGTTGAACGGCGC

[0173] TCGTGTCAAGGATCTTATCCCCGGAAGAGATGGCCAAGAGACGGAGGAGTTCACGATCA

[0174] AAGCAAAGGGTATCATTAACGCAACTGGCCCCTTCACCGACTCGATCAGAAAGATGGAC

[0175] GAACCCGGCATCAAGGAGATCGTGGCTCCTAGCTCAGGTGTTCATGTGATCTTACCTGG

[0176] CTATTACAGCCCTTCGGATATGGGCCTCATTGACCCATCTACGTCGGACGGCCGTGTGAT

[0177] CTTCTTCCTTCCCTGGCAAGGGAACACCATTGCCGGTACAACGGACCAACCGACCGAGA

[0178] TCACCCCTCAACCCCAGCCTTCTGAGAAGGATATCAATTGGATCATTTCCGAAGTCCGTA

[0179] ATTACTTGGCTCCAGACATCAATATTGAGCGGAGTGACGTCCTTGCTGCCTGGGCTGGAA

[0180] TTCGCCCGCTGGTGCGTGATCCTAAGGTAAAGAGCTCCGAGGCTCTGGTCCGCAACCAT

[0181] CTCATTACTGTCTCACCGTCCGGACTCTTGACCTGTGCTGGTGGCAAGTGGACTACCTAC

[0182] CGGCAGATGGCTGAAGAGGCGGTGGACGAAGCCATTGGCGTGTTCGGCTTGAAGCCTC

[0183] GCGGAGTCACCGGCGCTCCGGATATCAGTGGTGTCGGCGGCAGTGGCTTGGTCTCCGAC

[0184] GGTGCTGTTCTCGACGGATCTTGCCAAACCCACCAGGTTCGCTTGATTGGTGCCCATGG

[0185] CTTCTCCAAAACCCTTTTCATCAACCTCATCCAGCACTTCGGACTCGAGGTTGATGTGGC

[0186] TAAGCATCTAACGGAGTCGTACGGTGACCGAGCCTGGCAGGTTGCGGCTTTGTCTTCCC

[0187] CCACAAATGCTCGCTTCCCTGTCCGCGGCCAACGCATCTCCAGACTATACCCCTTCGTTG

[0188] ATGGTGAAATCAGGTATGCTGTGCGCCACGAGTATGCACAGACTGCCACCGATGTCATT

[0189] GCTCGCAGAACACGGTTGGCATTTTTGAACGCCGAAGCAGCACTCGAAGCCCTTCCTAA

[0190] CATTATCGATCTCATGGGTGAGGAGCTGAAGTGGGATAAGAACCGGAAAGATTCTGAAT

[0191] GGAAGGAGAGCGTCCAGTTTCTTTCTTCAATGGGTCTACCAAAGGCTTTCCTGAGCATG

[0192] ACGCGGGATGATGTCGAGGCGGGTAGAGTAAAGGATCTGGATATCGCACAAAGAAAGG

[0193] CCTTTGCTCGGTCCGATCCCCCAGCGGATGTGCTCAATAGTGACATCCCCTCGTCCGATT

[0194] CTAACAAGCTGATACATCCCGAGTCCCCGGCAAACAAATAG

[0195] SEQ NO 4 Nucleotide Sequence of Glycerol Dehydrogenase gdh

[0196] ATGGAATTCGCTCCTCCCACTCACTTCAAGCTCAATACGGGCGCCCTCATTCCCGCTGTC

[0197] GGCCTTGGAACCTGGAAATCCGAGCCAGGTGAGGTGGCAAGGGCAGTGGCCTTCGCCC

[0198] TCAAGAATGGATACCGGCACATTGATGCGGCATTGATCTACGGTAACGAAAACGAGGTC

[0199] GGCCAGGGAATTAGGGATAGCGGTGTGCCTCGCAAGGACATATTCATCACCAGTAAGCT

[0200] TTGGAACACACATCACCCAAATGTCAAGGAGGGTCTACAGAAGACTCTAGATAGCCTGG

[0201] GAACCGACTATCTTGATCTTTACCTTATTCACTGGCCCGTTCGGCTGGTTCCTAATGAGAC

[0202] GAGCGAGCTTCTGCCGGTAAACCCGGATGGCACCCGCTCAGTGGACCGATCGTGGGATC

[0203] AGAATGAGACCTGGCGCCAGATGGAGGAAGTTTACAAAGCTGGTATGGTCAAGGCGGT

[0204] CGGGCTCGCCAACTGGTCCATTCCCTATCTGGAGGAGCTTCGCAAGACATGGACAGTCG

[0205] TGCCGGCTGTAAACCAAGTTGAGCTGCATCCTTTCCTCCCTCAACATGAACTCCGAGAG

[0206] TATTGCGACAAGCTTGGTATACTCCTCGAAGCATACTCCCCTCTTGGTTCTACCGGGGCT

[0207] CCTATCATGTCAGATCCTGAGATCCAACAGATTGCGGACAAGAATGGGGTCTCCGCCGC

[0208] CACGATACTCATCAGTTACCACGTCAACAAAGGCGTGGTTGTGCTTCCGAAATCTGTGA

[0209] GTGAGAAGCGCATCGCTAGTAACAGAGAGGTTATTTCACTATCCAAGGAGGATCTGTATG

[0210] TGCTCGACGGTATGGCGGCCGCGGGGAAGGCGAAACGCTTAAATACGCCACTGTGGGG

[0211] ATTCGATCTGGGCTTCGCGGACTGGTACGGTCCGGTGAAGTCGCAGTAAGGATCC

[0212] SEQ NO 5 Nucleotide sequence of glycerol-3-phosphate dehydrogenase dhaK

[0213] ATGCAGACCAAACACTTCTTCTCGGACCCTACCCATCTGGTCCAGACCGCCCTGAACTC

[0214] CCTGACTCTCACAAACCCTTCTCTTGCCTATGACCCCGAAAACAAAATTATTTTCCGTCG

[0215] TCCGGATACGACTTCAAAGTCCAAGGTCGCGATTGTGTCTGGAGGAGGATCCGGCCATG

[0216] AGCCTGCCTTTGCCGGCTATGTTGGCAAGGGTTTCCTAGATGCCTCGGCCGCCGGGACC

[0217] ATTTTCGCCTCTCCTTCCGCAGAGCAGATCCGTAAGGCTGTCATGGACTGCGTCGACAA

[0218] CGAGAAGGGTGTCCTCATCATCCCCATGAACTACACTGGTGATGTCCTCAACTTCGGTAT

[0219] GGCTACGGAGAAGGCCCGCGCCGCCGGTATCAAGACTGAGTTCTTCGCCATCAACGAC

[0220] GACGTTGGTGTCGGCAAGAAGAAGGGCGGCAAGGTCGGCCGTCGCGGTATTAGTGGTG

[0221] GTATCCTAATCTTGAAGATCGTCAGCGCGCTGGCCGAAGCTGGTGGATCTCTCGAGGAT

[0222] GTTTACCGCACCGCTCAGTTGGCAAACAAGAACCTCGTCTCAGTTGGCTCTTCTCTGGA

[0223] GCACGTCCACGTCCCTGGCAGAGCTCCGCCAACGGACACCATCCCCGATGGTGAGGTC

[0224] GAGGTCGGCATGGGCATCCACAATGAACCGGGATCTCACCGGGAAAAGTTCGAGCTGA

[0225] CCGAACTGGTCGCTACCATGCTCCTTCAGCTCTTGGACCACAACGATCCCGACCGCGCC

[0226] TGGATCACTCGCAAGCCCGATGATGACTTTGTGCTGCTGATCAACAACTTGGGTGGTGT

[0227] GAGCGCTCTGGAACTTTCGGGCATCACCGATGAGGTCTACCGCCAGCTGAACAGAGACT

[0228] ACAACGTTCGGCCCATCCGGGTGATCCAGGGAACCTTCCTCACCAGTCTGAACGGCCTA

[0229] GGCTTCAGCATTTCCTTGATGAAGCTCGTGGACTCTGGATTGGGCGAGGGCAAGTCCTT

[0230] CCTTGAACTCCTTGATGCCCCCGCTGAGGCGGTTGGCTGGTCTGCTCCCATCCGTCCTGA

[0231] GACCTGGGAGCACCGCTCTGATGCTCCTGTTGAGCTGAAGAGGACCAAGATGCCCGAG

[0232] GATCAGCCCAGCAACGTCAAGTTGGATGTTGATTCCCTCAAGAAGGTCCTGGGCAGTGC

[0233] ACTCAAGCGTGTCATTGCCGCCGAGCCCGAAGTCACCCGCTTCGACACCATCGTCGGAG

[0234] ACGGAGACTGCGGCGTAGGCTTGAAGCGTGGCGCCGAGGCCGTCCTTAAGCTCGTCGA

[0235] AGACCCCTCGTCTCCGCTCACCGACGATGTCGTGAACGCAGTGAACCGCATCGTCAGCA

[0236] TTGTCGAGAACGTCATGGATGGAACCTCCGGAGCCATCTACGCTATCTTCCTGAACGCCC

[0237] TGGCCCACGGTCTTCGCGAGCAAGACCGAGGCACCACCACCCCCGCCACGGTGGATGT

[0238] CTGGGCTGCTGCACTGTCCTACTCCCTCACCGCGCTGTCCCGCTATACCCCTGCCCAACC

[0239] CGGTGACCGTACCCTCCTGGACGCCCTTGTACCCTTCTGCACGCAGCTCCAAAAGGCCA

[0240] AGGACGTGCACATCGCCGCCAAAGCAGCACAGGAAGGCACCGACTCCACCAAGAACAT

[0241] GAAGGCCAGCCTAGGACGGTCCGTCTACGTCGGCGGCGAAGAAGAATGGGTGGGCAAG

[0242] ATCCCCGACCCCGGCGCTTACGGACTCAGCGTGTTCCTTAATGGTCTGTCTGAGGCGTTG

[0243] TAA

[0244] Amino acid sequence encoded by SEQ NO 6 stl1

[0245] MKDLKLSNFKGKFISRTSHWGLTGKKLRYFITIASMTGFSLFGYDQGLMASLITGKQFNYEF

[0246] PATKENGDHDRHATVVQGATTSCYELGCFAGSLFVMFCGERIGRKPLILMGSVITIIGAVIST

[0247] CAFRGYWALGQFIIGRVVTGVGTGLNTSTIPVWQSEMSKAENRGLLVNLEGSTIAFGTMIAY

[0248] WIDFGLSYTNSSVQWRFPVSMQIVFALFLLAFMIKLPESPRWLISQSRTEEARYLVGTLDDA

[0249] DPNDEEVITEVAMLHDAVNRTKHEKHSLSSLFSRGRSQNLQRALIAASTQFFQQFTGCNAAI

[0250] YYSTVLFNKTIKLDYRLSMIIGGVFATIYALSTIGSFFLIEKLGRRKLFLLGATGQAVSFTITFA

[0251] CLVKENKENARGAAVGLFLFITFFGLSLLSLPWIYPPEIASMKVRASTNAFSTCTNWLCNFA

[0252] VVMFTPIFIGQSGWGCYLFFAVMNYLYIPVIFFFYPETAGRSLEEIDIIFAKAYEDGTQPWRVA

[0253] NHLPKLSLQEVEDHANALGSYDDEMEKEDFGEDRVEDTYNQINGDNSSSSSNIKNEDTVNDKANFEG*

[0254] Amino acid sequence of glycerol kinase glpK, SEQ NO 7

[0255] MQKSPTITFDSISLPDPLNPPTKKTRTPPITTSKMRNPFDMADLDSAVEQEEPQPFMNIHAYQ

[0256] SHDGNRSLLQPNENRHSMKDRFIGAIDTGTTSSRFIIFDCTGVPVAKYQMEFRQIHEKSGWH

[0257] EQDPFELVDSVYTCIEEAMKTFLALGHSRDDIEAIGITSQRETTVCWDWETGEPLHHAIAWP

[0258] DTRTTGLVRELKTKEGADELSNICGLPLSTYPSSVTLMWMLRNLPEVKRAYDEGRLAFGTI

[0259] DTWLIYNLNGGPEGGRLVTDVTNASRTMFMNLETLDYDDKLLKFFEIDRNKIRLPKILPSSD

[0260] PEGYGWVRDGPLEGIPITSCLGDQSAALVGHCAFTPGSAKNTYGTGCFLLYNVGEKPVISK

[0261] HGLLGTVGFQLGKHRKPVYALEGSVAVAGSGISFLMNNMGFFRDSRKVSEVAATVPDSGGC

[0262] VFVTAFSGLFAPYWIDDAKGTIFGITQHTQRGHIARATMEAACFQTKAILDAMEMDSGHKL

[0263] SELAVDGGMSNSDICMQTQADIIQIPVERPAMHETTALGAAIAAGFAVNIWKDFSELKDMNRANRTTFLPHASPVQSKKMYRQWSKAVEMSRGWLDTNESEEEEEEGQNGA*

[0264] Amino acid sequence of glycerol-3-phosphate dehydrogenase gpdA, SEQ ID NO 8

[0265] MASRQSRRVLRPLLYTSLAAATGAGVLYISYRPRNIPGSEAPAVPPPGYHEGKLVPPSFPNIKS

[0266] RLEQIQDLKRSSAGSNSEEYDLLVIGGGATGSGIALDAATRGLKVAIVERDDFSAGTSSKSTK

[0267] LVHGGVRYLEKAVWELDYNQYKLVKEALRERKYFLNTAPHLSSWLPIMVPLQKWWQAPY

[0268] FWAGTKFYDYLAGSEGIESSYFLTKSKAIDAFPMLRKDNLFGAMVYYDGAHNDSRMNVSL

[0269] AMTAALYGATVVNHMQVTGLEKDSSGKLNGARVKDLIPGRDGQETEEFTIKAKGIINATGP

[0270] FTDSIRKMDEPGIKEIVAPSSGVHVILPGYYSPSDMGLIDPSTSDGRVIFFLPWQGNTIAGTTD

[0271] QPTEITPQPQPSEKDINWIISEVRNYLAPDINIERSDVLAAWAGIRPLVRDPKVKSSEALVRNH

[0272] LITVSPSGLLTCAGGKWTTYRQMAEEAVDEAIGVFGLKPRGVTGAPDISGVGGSGLVSDGA

[0273] VLDGSCQTHQVRLIGAHGFSKTLFINLIQHFGLEVDVAKHLTESYGDRAWQVAALSSPTNA

[0274] RFPVRGQRISRLYPFVDGEIRYAVRHEYAQTATDVIARRTRLAFLNAEAALEALPNIIDLMGE

[0275] ELKWDKNRKDSEWKESVQFLSSMGLPKAFLSMTRDDVEAGRVKDLDIAQRKAFARSDPPADVLNSDIPSSDSNKLIHPESPANK*

[0276] SEQ NO 9 Amino acid sequence of glycerol dehydrogenase gdh

[0277] MEFAPPTHFKLNTGALIPAVGLGTWKSEPGEVARAVAFALKNGYRHIDAALIYGNENEVGQ

[0278] GIRDSGVPRKDIFITSKLWNTHHPNVKEGLQKTLDSLGTDYLDLYLIHWPVRLVPNETSELL

[0279] PVNPDGTRSVDRSWDQNETWRQMEEVYKAGMVKAVGLANWSIPYLEELRKTWTVVPAV

[0280] NQVELHPFLPQHELREYCDKLGILLEAYSPLGSTGAPIMSDPEIQQIADKNGVSAATILISYH

[0281] VNKGVVVLPKSVSEKRIASNREVISLSKEDLYVLDGMAAAGKAKRLNTPLWGFDLGFADWYGPVKSQ*

[0282] SEQ NO 10 Amino acid sequence of glycerol-3-phosphate dehydrogenase dhaK

[0283] MQTKHFFSDPTHLVQTALNSLTLTNPSLAYDPENKIIFRRPDTTSKSKVAIVSGGGSGHEPAFA

[0284] GYVGKGFLDASAAGTIFASPSAEQIRKAVMDCVDNEKGVLIIPMNYTGDVLNFGMATEKA

[0285] RAAGIKTEFFAINDDVGVGKKKGGKVGRRGISGGILILKIVSALAEAGGSLEDVYRTAQLAN

[0286] KNLVSVGSSLEHVHVPGRAPPTDTIPDGEVEVGMGIHNEPGSHREKFELTELVATMLLQLLD

[0287] HNDPDRAWITRKPDDDFVLLINNLGGVSALELSGITDEVYRQLNRDYNVRPIRVIQGTFLTS

[0288] LNGLGFSISLMKLVDSGLGEGKSFLELLDAPAEAVGWSAPIRPETWEHRSDAPVELKRTKM

[0289] PEDQPSNVKLDVDSLKKVLGSALKRVIAAEPEVDTRFDTIVGDGDCGVGLKRGAEAVLKLVE

[0290] DPSSPLTDDVVNAVNRIVSIVENVMDGTSGAIYAIFLNALAHGLREQDRGTTTPATVDVWAA

[0291] ALSYSLTALSRYTPAQPGDRTLLDALVPFCTQLQKAKDVHIAAKAAQEGTDSTKNMKASLGRSVYVGGEEEWVGKIPDPGAYGLSVFLNGLSEAL*

[0292] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A strain of Aspergillus niger LH-S8 that efficiently utilizes glycerol to produce malic acid, characterized in that: The Aspergillus niger LH-S8 is constructed by enhancing the glycerol transport and glycerol metabolism and decomposition capabilities through genetic engineering.

2. The Aspergillus niger LH-S8 according to claim 1, characterized in that: The Aspergillus niger LH-S8 is constructed by sequentially expressing a glycerol transporter gene stl1, a glycerol kinase gene glpK, a glycerol-3-phosphate dehydrogenase gene gpdA, a glycerol dehydrogenase gene gdh and a dihydroxyacetone kinase gene dhaK in Aspergillus niger LH-11 by means of genetic engineering.

3. Aspergillus niger LH-S8 according to claim 2, characterized in that: The nucleotide sequences of the glycerol transporter gene stl1, glycerol kinase gene glpK, glycerol-3-phosphate dehydrogenase gene gpdA, glycerol dehydrogenase gene gdh and dihydroxyacetone kinase gene dhaK are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQID NO.4 and SEQ ID NO.5, respectively, and the corresponding amino acid sequences are SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

4. The Aspergillus niger LH-S8 according to any one of claims 1 to 3, characterized in that: The Aspergillus niger LH-S8 is suitable for producing malic acid using glycerol or a mixture containing more than 30% glycerol as a raw material.

5. The method for constructing Aspergillus niger LH-S8 according to any one of claims 1 to 4, characterized in that: The specific steps for sequentially expressing the above genes in Aspergillus niger LH-11 are as follows: The glycerol transporter gene stl1, glycerol kinase gene glpK, glycerol-3-phosphate dehydrogenase gene gpdA, glycerol dehydrogenase gene gdh and dihydroxyacetone kinase gene dhaK were cloned into the multiple cloning site of vector pLH454, respectively, to construct the corresponding expression plasmids; the expression plasmids were successively transferred into Aspergillus niger LH-11 by Agrobacterium-mediated method, and after each transfer, the hygromycin resistance marker hph was lost before being transferred into the next plasmid, and finally Aspergillus niger LH-S8 was obtained.

6. The construction method according to claim 5, characterized in that: The specific steps are as follows: First, the glycerol transporter gene stl1, the glycerol kinase gene glpK, the glycerol-3-phosphate dehydrogenase gene gpdA, the glycerol dehydrogenase gdh, and the dihydroxyacetone kinase dhaK were cloned into the multiple cloning site of the pLH454 plasmid to construct the following expression plasmid: pLH454-stl1 is a glycerol transporter expression plasmid, pLH454-glpK is a glycerol kinase expression plasmid, pLH454-gpdA is a glycerol-3-phosphate dehydrogenase expression plasmid, pLH454-gdh is a glycerol dehydrogenase expression plasmid, and pLH454-dhaK is a dihydroxyacetone kinase expression plasmid; Next, these plasmids were sequentially transformed into Aspergillus niger LH-11 using the Agrobacterium-mediated method: pLH454-stl1 was transformed into Aspergillus niger LH-11, and the strain Aspergillus niger LH-11-stl1 overexpressing the glycerol transporter was obtained by screening with 250 μg / ml hygromycin resistance; pLH454-glpK was transferred into Aspergillus niger LH-11-stl1, and the strain Aspergillus niger LH-11-stl1-glpK overexpressing glycerol transporter and glycerol kinase was obtained by screening; pLH454-gpdA was transferred into Aspergillus niger LH-11-stl1-glpK, and Aspergillus niger LH-11-stl1-glpK-gpdA was obtained by screening; pLH454-gdh was transferred into Aspergillus niger LH-11-stl1-glpK-gpdA, and Aspergillus niger LH-11-stl1-glpK-gpdA-gdh was obtained by screening; Finally, pLH454-dhaK was transferred into Aspergillus niger LH-11-stl1-glpK-gpdA-gdh, and the final engineered strain Aspergillus niger LH-11-S8 was obtained by screening; After each transfer, the strain was screened for hygromycin resistance at 250 μg / ml to ensure that the target gene was successfully expressed, and the resistance marker of the previous plasmid was removed by losing the marker hph before the next plasmid was transferred.

7. Use of Aspergillus niger LH-S8 according to any one of claims 1 to 4 in producing malic acid by glycerol fermentation.

8. The method for producing malic acid by fermentation of Aspergillus niger LH-S8 according to any one of claims 1 to 4, characterized in that: In the method for producing malic acid, the culture medium contains 5 to 20 g / L of glucose and 60 to 200 g / L of glycerol.

9. The method according to claim 8, characterized in that: Here are the steps: Aspergillus niger LH-S8 was inoculated on a PDA medium solid plate and cultured for 3 to 5 days; then, sterile water was added and the spores were washed off with a cotton swab; the number of spores in the spore solution was counted using a hemocytometer and inoculated into a seed medium with a final spore concentration of 1.0×10 5 ~2.0×10 6 / mL, culture at 28-37°C and 200-300rpm shaking table for 20-40h; then take 50-140mL seed culture solution and inoculate it into a 2L fermenter with a malic acid fermentation medium filling volume of 1.26L, the fermentation temperature is set at 28-37°C, the ventilation volume is 0.5-2.0vvm, the rotation speed is 200-500rpm, and the fermentation cycle is 7 days; on the 7th day of fermentation, the malic acid concentration reaches 100-120g / L, the average glycerol consumption rate is 0.5-0.9g / L / h, and the carbon conversion rate is 0.9-1.2C-mol / C-mol, which is close to 70-90% of the theoretical maximum value.

10. The method according to claim 9, characterized in that: The seed culture medium has a formula of: per liter of seed culture medium components: 40 g of glucose, 6 g of bacterial peptone, 0.1 g of magnesium sulfate heptahydrate, 0.1 g of calcium chloride dihydrate, 0.75 g of potassium dihydrogen phosphate, 0.75 g of dipotassium hydrogen phosphate, 0.005 g of ferrous sulfate heptahydrate, 0.005 g of sodium chloride, 0.001 g of anhydrous citric acid, pH=6.5, and the solvent is water; The formula of the malic acid fermentation medium is as follows: the components of each liter of malic acid fermentation medium are: 20g of glucose, 81.7g of glycerol, Bacterial peptone 6g, magnesium sulfate heptahydrate 0.1g, calcium chloride dihydrate 0.1g, potassium dihydrogen phosphate 0.15g, dipotassium hydrogen phosphate 0.15g, 0.005 g of ferrous sulfate heptahydrate, 0.005 g of sodium chloride, 0.001 g of anhydrous citric acid, 80 g of calcium carbonate, pH = 6.5, the solvent is water.

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