A strain for producing itaconic acid, its construction method and application

Abstract

This invention provides an itaconic acid-producing strain, its construction method, and its application. E.coli W3110, as the chassis strain, upregulated the gltA, acnA, ACS, and gatA genes, knocked out the aceA and Pta genes, downregulated the transcription level of the icd gene using the BBa_J23115 promoter, and carried the high-copy plasmid PET-28a-WAL. The plasmid was used with the T7 promoter to overexpress a gene derived from... Aspergillus terreus The strain constructed using the CAD1 gene exhibits excellent itaconic acid synthesis capabilities and stable performance. It can efficiently and stably synthesize itaconic acid de novo using glucose as a carbon source through fermentation, with low production costs and high acid production efficiency, thus achieving efficient itaconic acid production and showing excellent prospects for industrial applications.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and fermentation engineering technology, and in particular to an itaconic acid producing strain, its construction method, and its application. Background Technology

[0002] Itaconic acid (IA), also known as methylene succinic acid or methylene succinic acid, is an important unsaturated dicarboxylic acid and a crucial raw material in chemical production. Currently, itaconic acid is produced using both chemical and biological methods. Compared to chemical methods, biological methods offer advantages such as lower production costs, higher efficiency, mature technology, and environmental friendliness and energy conservation. Therefore, biological methods are the most commonly used method for industrial production of itaconic acid.

[0003] Currently, itaconic acid is mainly produced industrially through microbial fermentation, with Aspergillus terreus being the most widely used method. However, while traditional industrial Aspergillus terreus strains produce high yields of itaconic acid, they suffer from drawbacks such as high viscosity of the fermentation broth due to filamentous growth, extreme sensitivity to manganese ions, and potential pathogenicity. Therefore, developing a genetically engineered bacterium that uses glucose as a substrate for inexpensive and efficient itaconic acid production has significant practical value in production. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an itaconic acid production strain.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned itaconic acid producing strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned itaconic acid producing strain.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A plasmid named plasmid PET-28a-WAL has a nucleotide sequence as shown in SEQ ID NO.14 of the sequence listing.

[0008] Preferably, the aforementioned plasmid is derived from plasmid pET28a and carries plasmid elements such as a replication origin site, a kanamycin resistance gene, a T7 promoter, and a T7 terminator, while also carrying plasmid elements derived from... Aspergillus terreus The CAD1 gene was transcribed using the T7 promoter for enhanced transcription.

[0009] The above plasmids were constructed using the ClonExpress® rapid cloning technology from Nanjing Novizan Biotechnology Co., Ltd.: cloning sites were selected, and the pET28a linear vector was amplified using reverse PCR. The pET28a linear vector was then ligated with the cloning fragment T7-CAD1 using ClonExpress® recombinase to obtain the new plasmid PET-28a-WAL.

[0010] Preferably, the nucleotide sequence of the plasmid pET28a is shown in SEQ ID NO.11, the nucleotide sequence of the CAD1 gene is shown in SEQ ID NO.8, the nucleotide sequence of the T7 promoter is shown in SEQ ID NO.12, the nucleotide sequence of the T7 terminator is shown in SEQ ID NO.13, and the nucleotide sequence of PET-28a-WAL is shown in SEQ ID NO.14.

[0011] The application of the above plasmids in constructing itaconic acid-producing strains and / or producing itaconic acid.

[0012] A strain that produces itaconic acid, named engineered strain WAL8, is... E. coli W3110, a chassis strain, was obtained through metabolic engineering: the gltA, acnA, ACS, and gatA genes were upregulated. ， The aceA and Pta genes were knocked out, the icd gene was downregulated, and the plasmid PET-28a-WAL, which is a high copy number, was carried.

[0013] Preferably, the above-mentioned itaconic acid producing strain is *Escherichia coli*. E. coli W3110 was used as the chassis strain. The aceA and Pta genes were knocked out of the strain's genome. The icd gene was downregulated using the BBa_J23115 promoter and integrated into the yeeL gene locus in the genome. The gltA gene was enhanced using the trc promoter and integrated into the ycgH gene locus in the genome. The acnA gene was enhanced using the trc promoter and integrated into the ylbE gene locus in the genome. The ACS gene was enhanced using the trc promoter and integrated into the ilvG gene locus in the genome. The gatA gene was enhanced using the trc promoter and integrated into the ygaY gene locus in the genome.

[0014] Preferably, the above-mentioned itaconic acid producing strain, specifically the chassis strain *Escherichia coli*. E. coli W3110 is Escherichia coli. E. coli W3110 ATCC 27325 (available for purchase through the ATCC cell bank).

[0015] Preferably, in the above-mentioned itaconic acid producing strain, the nucleotide sequence of the aceA gene is shown in SEQ ID NO.1, the nucleotide sequence of the Pta gene is shown in SEQ ID NO.2, the nucleotide sequence of the icd gene is shown in SEQ ID NO.3, the nucleotide sequence of the gltA gene is shown in SEQ ID NO.4, the nucleotide sequence of the acnA gene is shown in SEQ ID NO.5, the nucleotide sequence of the ACS gene is shown in SEQ ID NO.6, and the nucleotide sequence of the gatA gene is shown in SEQ ID NO.7.

[0016] Preferably, in the above-mentioned itaconic acid producing strain, the nucleotide sequence of the BBa_J23115 promoter is shown in SEQ ID NO.9, and the nucleotide sequence of the trc promoter is shown in SEQ ID NO.10.

[0017] The above method for constructing itaconic acid-producing strains involves starting with the strain... E. coli W3110 Based on this foundation, targeted modifications will be carried out, with the specific steps as follows: (1) Modification of the chassis bacteria to enhance the itaconic acid carbon synthesis pathway: gltA and acnA genes were expressed by using the trc promoter to enhance the mobility of the carbon source pathway; (2) Increase carbon flux: The Pta gene was knocked out and the ACS gene was expressed using the trc promoter, which enhanced the reflux of acetyl-CoA and increased the accumulation of acetyl-CoA. (3) Reduce the catabolism of isocitrate: Use the BBa_J23115 promoter to weakly express the icd gene to weaken the conversion of isocitrate to oxaloylsuccinate; knock out the aceA gene to ensure sufficient precursors without affecting normal metabolism. (4) Enhanced acid resistance mechanism: Overexpression of the gatA gene using the trc promoter maintained intracellular pH homeostasis; (5) Transformation of PET-28a-WAL plasmid system: Enhance itaconic acid synthesis pathway.

[0018] Application of the above-mentioned itaconic acid producing strains in the fermentation production of itaconic acid.

[0019] Preferably, in the above application, the strain is fermented in a culture medium, which includes, but is not limited to, carbon sources, nitrogen sources, inorganic salts, vitamins, etc.; the fermentation conditions include fermentation temperature, fermentation pH, fermentation dissolved oxygen conditions, fermentation pressure, fermentation time, etc.

[0020] Preferably, the above application follows these steps: ① Slant culture: Inoculate the itaconic acid producing strain onto a slant culture medium and incubate at 32-35℃ for 12-16 hours; ② Shake flask seed culture: Take the slant culture and inoculate it into the shake flask culture medium for fermentation. The culture temperature is 32-35℃, the culture time is 12-20h, the shaking speed is 200-240r / min, and the pH is 6.4-6.7. ③ Shake flask fermentation culture: The inoculum size is 15-20%, the culture temperature is 32-35℃, the pH is 6.4-6.7, the culture time is 24-36h, and the shaker speed is 200-240r / min.

[0021] Preferably, the above application follows these steps: ① Slant culture: The itaconic acid producing strain was inoculated onto a slant culture medium and cultured at 32℃ for 12 hours; ② Shake flask seed culture: Take the slant culture and inoculate it into the shake flask culture medium for fermentation. The culture temperature is 32℃, the culture time is 14h, the shaking speed is 220r / min, and the pH is controlled at 6.7. ③ Shake flask fermentation culture: The inoculum size is 15-20%, the culture temperature is 32℃, the pH is controlled at 6.5, the culture time is 36h, and the shaker speed is 220r / min.

[0022] Preferably, in the above application, the slant culture medium used in step ① is general LB solid culture medium.

[0023] Preferably, in the above application, the seed culture medium used in step ② is: glucose 30g / L, yeast 5g / L, peptone 2g / L, (NH4)2SO4 1g / L, KH2PO4·3H2O 2g / L, MgSO4·7H2O 1g / L, citric acid 5g / L, glutamic acid 3g / L, and the remainder is water.

[0024] Preferably, in the above application, the fermentation medium used in step ③ is: glucose 15g / L, yeast powder 4g / L, peptone 2g / L, (NH4)2SO4 1.5g / L, KH2PO4·3H2O 3.5g / L, MgSO4·7H2O 2g / L, glutamic acid 2.5g / L, citric acid 10g / L, FeSO4·7H2O 20mg / L, MnSO4·H2O 10mg / L, with the remainder being water.

[0025] All of the above-mentioned culture media can be prepared using standard methods.

[0026] Beneficial effects: The aforementioned itaconic acid-producing strain upregulated the gltA, acnA, ACS, and gatA genes, knocked out the aceA and Pta genes, downregulated the transcription level of the icd gene using the BBa_J23115 promoter, and carried the high-copy plasmid PET-28a-WAL. The plasmid was used to overexpress a gene derived from [a specific source - likely a specific strain]. Aspergillus terreus The strain constructed using the CAD1 gene exhibits excellent itaconic acid synthesis capability and stable performance. It achieves efficient and stable de novo itaconic acid synthesis via fermentation using glucose as a carbon source, with low production cost and high acid production efficiency, realizing high-efficiency itaconic acid production and demonstrating excellent industrial application prospects. Specifically: (1) By upregulating the ACS gene and knocking out the aceA and Pta genes, the supply of aconitine, the precursor of itaconic acid, and acetyl-CoA, a key substrate of the TCA cycle, is increased, resulting in the accumulation of carbon sources flowing to itaconic acid and effectively reducing the catabolism of carbon sources in the synthetic pathway.

[0027] (2) Upregulation of gltA and acnA genes enhances the mobility of carbon sources in the pathway.

[0028] (3) Upregulation of the gatA gene increased intracellular ATP concentration and promoted H + -ATPase pumps out accumulated protons from the cell, maintains pH homeostasis, enhances the strain's acid resistance mechanism, and improves its tolerance to itaconic acid.

[0029] (4) By constructing the PET-28a-WAL plasmid, the T7 promoter on the plasmid is overexpressed from Aspergillus terreus The CAD1 gene was used to express the key enzyme of itaconic acid using a highly stable plasmid system, which enhanced the synthetic pathway from cis-aconitic acid to itaconic acid, enabling itaconic acid to accumulate efficiently. This effectively improved the itaconic acid synthesis efficiency and production level, achieving high-efficiency production of itaconic acid. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the structure of plasmid pET28a.

[0031] Figure 2 This is a schematic diagram of the structure of plasmid PET-28a-WAL.

[0032] Figure 3 This diagram illustrates the process of modifying the de novo synthesis pathway of itaconic acid-producing genetically engineered bacteria. Detailed Implementation

[0033] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0034] Unless otherwise specified, the percentage sign "%" used in the examples refers to the mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0035] The starting strain used in the examples was wild-type. E. coliW3110 ATCC 27325 (commercially available), the corresponding promoter and gene are shown in the sequence listing. Primers used in the construction of the involved strains are shown in Table 1.

[0036] Table 1 Primers used in strain construction

[0037]

[0038]

[0039]

[0040]

[0041] The following examples demonstrate the construction of itaconic acid-producing strains using the following five modules: (1) Chassis bacteria modification: increase the transcription level of gltA gene and increase the transcription level of acnA gene; (2) Increase carbon flux: The Pta gene was knocked out and the ACS gene was expressed using the trc promoter, which enhanced the reflux of acetyl-CoA and increased the accumulation of acetyl-CoA. (3) Reduce the catabolism of isocitrate: The icd gene is weakly expressed using the BBa_J23115 promoter, and the aceA gene is knocked out to ensure sufficient precursors without affecting normal metabolism. (4) Enhanced acid resistance mechanism: Overexpression of the gatA gene using the trc promoter maintained intracellular pH homeostasis; (5) Construction of the PET-28a-WAL plasmid system: to enhance the itaconic acid synthesis pathway (e.g. Figure 2 As shown, it is specifically noted that, except for the plasmid elements specifically mentioned in this invention, other elements shown in the diagram can be replaced by any known equivalent effect element. The PET-28a-WAL plasmid is composed of plasmid pET28a (e.g., ...). Figure 1 (As shown) It was modified and carries plasmid elements such as the replication start site, kanamycin resistance gene, T7 promoter, and terminator, and also carries genes derived from... Aspergillus terreus The CAD1 gene was transcribed using the T7 promoter.

[0042] The gene editing methods used in the gene manipulation are referenced in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genomeediting. Metabolic Engineering, 2015, 31: 13-21.). Unless otherwise specified, all technical terms used in this invention are explained in this article. "Introduction" refers to the insertion of a foreign gene into the genome of an engineered bacterium after ligating it with a promoter and terminator.

[0043] Example 1 like Figure 3 As shown, this embodiment aims to illustrate the specific construction steps of strain WAL8. In particular, if there are similar gene manipulation methods in the embodiment, they will only be provided once and annotated, without further elaboration.

[0044] (1) Transformation of plasmid PET-28a-WAL to obtain engineered bacteria: Transform the complete plasmid PET-28a-WAL into wild-type E. coli W3110 competent cells by electrotransformation (or chemical transformation, etc.) to obtain engineered bacteria WAL1.

[0045] (2) Knockout of the aceA gene: Using the E. coli W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using aceA-US, aceA-UA and aceA-DS, aceA-DA respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were obtained by overlapping PCR amplification using aceA-US and aceA-DA as primers. The gRNA fragment was obtained by annealing using pGRB-aceA-S and pGRB-aceA-A as primers and ligated with the pGRB vector to obtain aceA-pGRB. E. coli W3110 electroporation competent cells were prepared, and the overlapping fragment and aceA-pGRB were electroporated into competent cells together. Positive transformants were screened to obtain strain WAL2.

[0046] (3) Knockout of Pta gene: Using the E. coli W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using Pta-US, Pta-UA and Pta-DS, Pta-DA respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were obtained by overlapping PCR amplification using Pta-US and Pta-DA as primers. Using pGRB-Pta-S and pGRB-Pta-A as primers, gRNA fragments were annealed and ligated with the pGRB vector to obtain Pta-pGRB. E. coli W3110 electroporation competent cells were prepared, and the overlapping fragments and Pta-pGRB were electroporated into competent cells together. Positive transformants were screened to obtain strain WAL3.

[0047] (4) Downregulation of the icd gene at the yeeL pseudogene locus using the BBa_J23115 promoter: E. coli W3110 Using the genome as a template, upstream and downstream homologous arms were amplified by PCR using yeeL-US, yeeL-UA, yeeL-DS, and yeeL-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were amplified by PCR using icd-S and icd-A primers. Annealing was then performed using pGRB-yeeL-S and pGRB-yeeL-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain yeeL-pGRB. Preparation... E. coli W3110 The overlapping fragment was electrotransformed into competent cells along with yeeL-pGRB, and positive transformants were obtained by screening to obtain strain WAL4.

[0048] (5) Controlling gltA gene overexpression at the ycgH pseudogene site using the trc promoter: E. coli W3110 Using the genome as a template, upstream and downstream homologous arms were amplified by PCR using ycgH-US, ycgH-UA, ycgH-DS, and ycgH-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were amplified by PCR using gltA-S and gltA-A primers. Annealing was then performed using pGRB-ycgH-S and pGRB-ycgH-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain ycgH-pGRB. Preparation... E. coli W3110 The overlapping fragment was electroporated into competent cells along with ycgH-pGRB, and positive transformants were obtained by screening to obtain strain WAL5.

[0049] (6) Controlling acnA gene overexpression at the ylbE pseudogene site using the trc promoter: E. coliUsing the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using ylbE-US, ylbE-UA, ylbE-DS, and ylbE-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were obtained by PCR amplification using acnA-S and acnA-A primers. Annealing was then performed using pGRB-ylbE-S and pGRB-ylbE-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain ylbE-pGRB. Preparation... E. coli W3110 The overlapping fragments were electrotransformed into competent cells along with ylbE-pGRB, and positive transformants were screened to obtain strain WAL6.

[0050] (7) Controlling ACS gene overexpression using the trc promoter at the ilvG pseudogene site: E. coli Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using ilvG-US, ilvG-UA, ilvG-DS, and ilvG-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were obtained by PCR amplification using ACS-S and ACS-A primers. Annealing was then performed using pGRB-ilvG-S and pGRB-ilvG-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain ilvG-pGRB. Preparation... E. coli W3110 Competent cells were electrotransformed, and the overlapping fragment was electrotransformed together with ilvG-pGRB into competent cells. Positive transformants were then screened to obtain strain WAL7.

[0051] (8) Controlling gatA gene overexpression at the ygaY pseudogene site using the trc promoter: E. coli W3110 Using the genome as a template, upstream and downstream homologous arms were amplified by PCR using ygaY-US, ygaY-UA, ygaY-DS, and ygaY-DA, respectively. Then, using these homologous arms as templates, overlapping fragments were amplified by PCR using gGRB-S and gGRB-A primers. Annealing was then performed using pGRB-ygaY-S and pGRB-ygaY-A primers to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain ygaY-pGRB. Preparation... E. coli W3110 The overlapping fragment was electrotransformed into competent cells along with ygaY-pGRB, and positive transformants were obtained by screening to obtain strain WAL8.

[0052] Example 2 This embodiment aims to illustrate the construction method of plasmid PET-28a-WAL in Example 1. The specific steps are as follows: Using the Aspergillus terreus genome as a template and CAD1-pet-S and CAD1-pet-A as primers, the CAD1 gene was amplified. The pET28a plasmid linearized vector was obtained using reverse PCR. The gene fragment was ligated to the pET28a plasmid linearized vector using recombinase. The pET28a linearized vector was then ligated to the T7 promoter-CAD1-T7 terminator of the cloned fragment using ClonExpress® recombinase to obtain the new plasmid PET-28a-WAL, the nucleotide sequence of which is shown in SEQ ID NO.14 of the sequence listing.

[0053] Example 3 This embodiment aims to illustrate the application of the engineered strain WAL8 in shake-flask fermentation. The specific steps are as follows: ① Slant culture: Take the bacterial strain preserved in the -80℃ refrigerator and inoculate it on the slant culture medium. Incubate at 32℃ for 12h. The slant culture medium is the general LB solid medium (2g glucose, 10g tryptone, 5g yeast extract, 2.5g sodium chloride, 15g agar powder, add double distilled water to 1000mL, adjust the pH to 7.2 with 5mol / L NaOH (about 0.2ml), and sterilize at 121℃ for 20min).

[0054] ② Shake flask seed culture: Solid slant culture was inoculated into shake flask culture medium for fermentation. The culture temperature was 32℃, the culture time was 14h, the shaking speed was 220r / min, and the pH was 6.7. The seed culture medium consisted of 30g / L glucose, 5g / L yeast, 3g / L peptone, 1g / L (NH4)2SO4, 2g / L KH2PO4·3H2O, 1g / L MgSO4·7H2O, 5g / L citric acid, 3g / L glutamic acid, and the remainder was water.

[0055] ③ Shake-flask fermentation culture: The inoculum size was 15-20%, the culture temperature was 32℃, the pH was 6.5, the culture time was 36h, and the shaker speed was 220r / min. The fermentation medium used was: glucose 15g / L, yeast powder 4g / L, peptone 2g / L, (NH4)2SO4 1.5g / L, KH2PO4·3H2O 3.5g / L, MgSO4·7H2O 2g / L, glutamic acid 2.5g / L, citric acid 10g / L, FeSO4·7H2O 20mg / L, MnSO4·H2O 10mg / L, and the remainder was water.

[0056] wild type E. coli W3110 As a control group, after 36 hours of fermentation verification, the wild type... E. coli W3110 Itaconic acid failed to accumulate. The engineered strain WAL8 described in Example 1 accumulated 4.2 g / L of itaconic acid, which proves the effectiveness of the strain.

[0057] Example 4 Itaconic acid tolerance test of strain after 36 hours of fermentation The cell density, intracellular pH, and itaconic acid yield of E. coli engineered strains introduced with the acid-resistant gene gatA were compared with those of control strains without the acid-resistant gene during itaconic acid production, thereby verifying the effect of the acid-resistant gene on improving the strain's tolerance and fermentation performance.

[0058] Two strains were designed for the experiment, with three parallel experiments for each strain. The experimental strain WAL8 was designated as WAL8-1, WAL8-2, and WAL8-3, and the control strain WAL7 was designated as WAL7-1, WAL7-2, and WAL7-3. The culture conditions and culture medium were as described in Example 3, and the detection method is as follows: Cell density: Take an appropriate amount of fermentation broth, dilute it, and measure the absorbance at 600 nm using a UV-Vis spectrophotometer.

[0059] Intracellular pH: The BCECF-AM fluorescent probe method was used for measurement. The specific procedure was as follows: the bacterial cells were collected by centrifugation, washed twice with PBS, loaded with fluorescent probes, and the fluorescence ratio was measured by a fluorescent microplate reader and converted into intracellular pH value (for details, please refer to the instructions of Beyotime's Intracellular pH Detection Kit (BCECF AM)).

[0060] Itaconic acid yield: The supernatant of the fermentation broth was taken, appropriately diluted, and determined by high-performance liquid chromatography (HPLC). HPLC conditions were: Aminex HPX-87H column, 5 mM H2SO4 as mobile phase, flow rate 0.6 mL / min, column temperature 50℃, and detection wavelength 210 nm. The results of itaconic acid tolerance test are shown in Table 2.

[0061] Table 2. Itaconic acid tolerance test of strains after 36 hours of fermentation.

[0062] Example 5 This embodiment aims to illustrate the impact of some key modification steps in Embodiment 1 on yield. The shake flask fermentation method is the same as in Embodiment 3. The data results of four groups of shake flask fermentation for 36 hours are shown in Table 3.

[0063] Table 3 Impact of Key Modification Steps on Final Output

[0064] As shown in Table 3, the key modification steps synergistically and significantly increased the yield of itaconic acid. When the strain WAL8 was modified, the yield of the target product reached its highest level, with a total accumulation of 4.2 g / L of itaconic acid.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A plasmid, characterized in that: Its nucleotide sequence is shown in the sequence listing SEQ ID NO.

14.

2. The use of the plasmid of claim 1 in constructing itaconic acid producing strains and / or producing itaconic acid.

3. An itaconic acid-producing strain, characterized in that: Therefore E. coli W3110, a chassis strain, was obtained through metabolic engineering: the gltA, acnA, ACS, and gatA genes were upregulated. ， The aceA and Pta genes were knocked out, the icd gene was downregulated, and the plasmid described in claim 1 was carried.

4. The itaconic acid-producing strain according to claim 3, characterized in that: With Escherichia coli E. coli W3110 was used as the chassis strain. The aceA and Pta genes were knocked out in the strain genome. The icd gene was downregulated using the BBa_J23115 promoter and integrated into the yeeL gene locus in the genome. The gltA gene was enhanced using the trc promoter and integrated into the ycgH gene locus in the genome. The acnA gene was enhanced using the trc promoter and integrated into the ylbE gene locus in the genome. The ACS gene was enhanced using the trc promoter and integrated into the ilvG gene locus in the genome. The gatA gene was enhanced using the trc promoter and integrated into the ygaY gene locus in the genome.

5. The itaconic acid-producing strain according to claim 3, characterized in that: The nucleotide sequences of the aceA gene are shown in SEQ ID NO.1, the Pta gene in SEQ ID NO.2, the icd gene in SEQ ID NO.3, the gltA gene in SEQ ID NO.4, the acnA gene in SEQ ID NO.5, the ACS gene in SEQ ID NO.6, and the gatA gene in SEQ ID NO.

7.

6. The itaconic acid-producing strain according to claim 4, characterized in that: The nucleotide sequence of the BBa_J23115 promoter is shown in SEQ ID NO.9 of the sequence listing, and the nucleotide sequence of the trc promoter is shown in SEQ ID NO.10 of the sequence listing.

7. The method for constructing the itaconic acid-producing strain according to any one of claims 3-6, characterized in that: In the originating strain E. coli W3110 Based on this foundation, targeted modifications will be carried out, with the specific steps as follows: (1) The gltA and acnA genes were expressed using the trc promoter, respectively; (2) The Pta gene was knocked out, and the ACS gene was expressed using the trc promoter; (3) Use the BBa_J23115 promoter to weakly express the icd gene; knock out the aceA gene; (4) Overexpressing the gatA gene using the trc promoter; (5) Transform the plasmid described in claim 1.

8. The application of the itaconic acid producing strain according to any one of claims 3-6 in the fermentation production of itaconic acid.

9. The application according to claim 8, characterized in that: The specific steps are as follows: ① Slant culture: Inoculate the itaconic acid producing strain onto a slant culture medium and incubate at 32-35℃ for 12-16 hours; ② Shake flask seed culture: Take the slant culture and inoculate it into the shake flask culture medium for fermentation. The culture temperature is 32-35℃, the culture time is 12-20h, the shaking speed is 200-240r / min, and the pH is 6.4-6.

7. ③ Shake flask fermentation culture: The inoculum size is 15-20%, the culture temperature is 32-35℃, the pH is 6.4-6.7, the culture time is 24-36h, and the shaker speed is 200-240r / min.

10. The application according to claim 9, characterized in that: The slant culture medium used in step ① is general-purpose LB solid medium; the seed culture medium used in step ② is: glucose 30g / L, yeast 5g / L, peptone 2g / L, (NH4)2SO4 1g / L, KH2PO4·3H2O 2g / L, MgSO4·7H2O 1g / L, citric acid 5g / L, glutamic acid 3g / L, with the remainder being water; the fermentation culture medium used in step ③ is: glucose 15g / L, yeast extract 4g / L, peptone 2g / L, (NH4)2SO4 1.5g / L, KH2PO4·3H2O 3.5g / L, MgSO4·7H2O 2g / L, glutamic acid 2.5g / L, citric acid 10g / L, FeSO4·7H2O 20mg / L, MnSO4·H2O 10mg / L, with the remainder being water.

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