A method for improving citric acid production by Aspergillus niger, the mcs2 gene and its application

Abstract

The present invention belongs to the field of biotechnology, and discloses a method for improving citric acid production by Aspergillus niger, the mcs2 gene and its application. The nucleotide sequence of the mcs2 gene is shown as SEQ ID No.1. The method includes introducing the mcs2 gene, an expression cassette containing the mcs2 gene or a vector containing the mcs2 gene into Aspergillus niger. The present invention has successfully identified a new gene mcs2 related to citric acid biosynthesis, providing a new perspective and scientific basis for understanding the citric acid biosynthesis pathway. The present invention not only provides new gene targets and modification strategies for citric acid biosynthesis, but also opens up a new path for the biotechnological improvement of citric acid production, and is expected to significantly improve production efficiency and economic benefits.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular, to a method for improving citric acid production by Aspergillus niger, the mcs2 gene and its application. Background Art

[0002] Citric acid, this naturally sourced multifunctional platform compound, has built a comprehensive and three-dimensional application network spanning food, daily chemical, pharmaceutical, and advanced manufacturing industries, thanks to its unique chemical properties and excellent biocompatibility. In the food industry, citric acid, as an acidity regulator and preservative with GRAS (Generally Recognized as Safe) certification, occupies a core position and plays an indispensable role. In the daily chemical field, it drives continuous innovation and development in the high-end skincare market with its dual functions of keratin remodeling and antioxidant properties. In the medical scenario, citric acid continuously expands the new boundaries of treatment with its unique functions of metabolic regulation and material modification.

[0003] Given the huge economic potential and broad application prospects demonstrated by citric acid in many fields, the genetic improvement of production strains and process optimization have become the core focus of current scientific research. Aspergillus niger, as the main force in industrial citric acid production for over a century, the study of its citric acid biosynthesis mechanism is particularly important and crucial. The general scientific view is that citric acid in Aspergillus niger is mainly synthesized through the mitochondrial pathway. In this process, citrate synthase plays a catalytic role in converting acetyl-CoA and oxaloacetate into citric acid, and all these reactions are completed within the mitochondria. Acetyl-CoA and oxaloacetate are respectively derived from the metabolic conversion of pyruvate and malate in the cytoplasm. In addition, the mitochondrial citrate / malate antiporter acts as a bridge, responsible for the precise transport of citric acid and malate between the cytoplasm and mitochondria. Although two types of citrate synthases, CitA and CitB, have been discovered in Aspergillus niger, the existing research results are still insufficient to fully reveal the metabolic characteristics and mechanisms of citric acid in industrial strains. Therefore, it is reasonable to speculate that there may be other undiscovered citrate synthases. There are relevant research reports stating that some methyl citrate synthases (Mcs) may possess partial or similar functions to citrate synthase.

[0004] Based on this, we propose the hypothesis that the mcs1 and mcs2 genes encoding Mcs in Aspergillus niger may play a certain role and have an impact during the process of citric acid biosynthesis. In this invention, through gene knockout and complementation techniques, systematic functional identification of the mcs1 and mcs2 genes in Aspergillus niger was carried out, aiming to clarify the specific role of Mcs in citric acid biosynthesis and provide a solid scientific basis and theoretical support for developing more efficient and economical citric acid production strategies. This research result not only is expected to promote the innovation of citric acid production technology but also will lay a solid foundation for the sustainable development of the citric acid industry. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies in the prior art and provide a method for improving citric acid production by Aspergillus niger, the mcs2 gene and its application.

[0006] The technical solution adopted by the present invention to solve its technical problems is:

[0007] An mcs2 gene related to citric acid biosynthesis, the nucleotide sequence of the mcs2 gene is as shown in SEQ ID No.1.

[0008] The application of the mcs2 gene as described above in improving the biosynthesis efficiency of citric acid.

[0009] The Mcs2 active polypeptide encoded by the mcs2 gene as described above for improving the biosynthesis efficiency of citric acid, the amino acid sequence of the polypeptide is as shown in SEQ ID No.2.

[0010] An expression cassette for improving the biosynthesis efficiency of citric acid containing the mcs2 gene as described above, the expression cassette includes a promoter, the mcs2 gene and a terminator.

[0011] A vector for improving the biosynthesis efficiency of citric acid containing the mcs2 gene as described above, the vector is selected from plasmids, viral vectors or artificial chromosomes.

[0012] A method for improving citric acid production by Aspergillus niger using the mcs2 gene as described above, the method includes introducing the mcs2 gene, the expression cassette containing the mcs2 gene or the vector containing the mcs2 gene into Aspergillus niger.

[0013] An Aspergillus niger recombinant strain for improving citric acid production by Aspergillus niger containing the mcs2 gene as described above.

[0014] The construction method of the recombinant strain as described above includes the following steps:

[0015] First, the overexpression plasmid pLH2209 containing the mcs2 gene was introduced into Agrobacterium tumefaciens AGL1 competent cells by electroporation. The electroporation conditions were: 2.5 kV, 25 μF, 200 Ω. Positive clones were obtained by screening on LB medium containing 100 μg / mL kanamycin, and colony PCR verification was performed using primers P5420 / P5421. A specific band of 1398 bp was amplified to confirm that the plasmid had been transferred into Agrobacterium tumefaciens. Subsequently, the verified Agrobacterium tumefaciens was co-cultured with Aspergillus niger S469 spores at 1×10 ^6 CFU / mL in a volume ratio of 1:10 on IM solid medium containing 100 μg / mL kanamycin and 0.2 μM acetosyringone AS at 25 °C for 60 h. The grown transformants were transferred to CM selective medium containing 200 μM cefotaxime, 100 μg / mL ampicillin, 100 μg / mL streptomycin, and 250 μg / mL hygromycin B for screening transformants with hygromycin B resistance. The positive transformants were further verified for hygromycin resistance on PDA medium containing 250 μg / mL hygromycin, and genomic PCR detection was performed: a specific band of 1550 bp was amplified using primers P5422 / P5423 to confirm that the mcs2 expression cassette gene had been successfully integrated into the genome, obtaining the recombinant Aspergillus niger strain.

[0016] Application of the recombinant strain as described above in the fermentation production of citric acid.

[0017] A method for fermenting and producing citric acid using the recombinant strain as described above, comprising the following steps:

[0018] The cryopreserved recombinant strain was inoculated onto PDA solid medium containing 200 μg / mL hygromycin B and incubated at 28 °C in an inverted position for 4 - 5 days. After the spores matured, they were scraped and suspended in sterile physiological saline containing 0.05% (v / v) Tween-80, and the spore concentration was adjusted to 1×10 ^8 CFU / mL; subsequently, 1×10 ^8 spores were inoculated into the fermentation medium and incubated at 28 °C with shaking at 200 r / min for 5 days;

[0019] Among them, the formula of the fermentation medium was: sucrose 20 g / L, yeast extract 10 g / L, KH2PO4 1 g / L, MgSO4·7H2O 1 g / L, (NH4)2SO4 4.13 g / L, the solvent was water, and the pH was adjusted to 2.5.

[0020] The advantages and positive effects achieved by the present invention are:

[0021] 1. The present invention successfully identified a new gene mcs2 related to citric acid biosynthesis, providing a new perspective and scientific basis for understanding the citric acid biosynthesis pathway. The present invention not only provides new gene targets and modification strategies for citric acid biosynthesis, but also opens up a new path for the biotechnological improvement of citric acid production, and is expected to significantly improve production efficiency and economic benefits.

[0022] 2. In order to deeply explore the specific role of the mcs2 gene in the process of citric acid biosynthesis, the present invention carefully designed control experiments of gene knockout and overexpression. The experimental results showed that compared with the original Aspergillus niger strain, the citric acid yield of the recombinant strain with the mcs2 gene knocked out decreased sharply to 7.2 g / L, a decrease of up to 82.65%. This data strongly proves the indispensability of the mcs2 gene for citric acid synthesis. On the other hand, the recombinant strain with the mcs2 gene overexpressed showed an amazing yield increase, which was 41% higher than the original strain, fully demonstrating the great potential and broad prospects of this gene in optimizing the production performance of citric acid.

[0023] 3. The present invention successfully identified the mcs2 gene, which is expected to be widely applied to the genetic improvement of industrial Aspergillus niger strains, thereby significantly improving the production efficiency of citric acid. According to statistics, the annual citric acid output in China is as high as about 3 million tons, and the market scale is about 50 billion yuan. However, current industry data shows that traditional strain improvement technologies encounter metabolic flux regulation bottlenecks. The application of the mcs2 gene provides an expandable solution for constructing a new generation of citric acid microbial cell factories, and is expected to bring breakthrough progress to citric acid production, not only improving the yield, but also effectively reducing the production cost, thus creating significant economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a phylogenetic tree construction and evolutionary relationship analysis diagram of mcs2 and citA proteins in the present invention; Note: This figure constructs a phylogenetic tree using the neighbor-joining method to show the evolutionary correlation of citrate synthase; the scale length of the phylogenetic tree is marked as . The specific numerical values of the branch lengths in the figure have also been marked. The branch length between two nodes multiplied by the scale number is equal to the actual evolutionary distance. The value 0.5 is the scale length. The scale is a scale-like diagram at the bottom of the phylogenetic tree, used to represent the actual unit length of the evolutionary distance. It is similar to the scale in a map, converting the branch lengths in the phylogenetic tree diagram into quantifiable biological differences. The scale marked "0.5" means that on average 0.5 substitutions occur at each position. For example, if the total branch length between two nodes is 1.5 and the scale is 0.5, the actual evolutionary distance is 1.5 multiplied by 0.5, which is equal to 0.75.

[0025] Figure 2 It is a map of the starting plasmid pLH594 in the present invention;

[0026] Figure 3 This is the map of the intermediate plasmid pLH2175 in the present invention;

[0027] Figure 4 This is the map of the intermediate plasmid pLH2177 in the present invention;

[0028] Figure 5 This is the restriction enzyme digestion verification map of plasmids pLH2175 and pLH2177 in the present invention (the enzymes are both KpnⅠ and Hind Ⅲ);

[0029] Figure 6 This is the map of the plasmid pLH2176 with the mcs1 gene knocked out in the present invention;

[0030] Figure 7 This is the map of the plasmid pLH2178 with the mcs2 gene knocked out in the present invention;

[0031] Figure 8 This is the restriction enzyme digestion verification map of pLH2176 and pLH2178 in the present invention (the left enzymes are PstⅠ and EcoRⅠ, and the right enzyme is PstⅠ);

[0032] Figure 9 This is the map of the starting plasmid pLH454 in the present invention;

[0033] Figure 10 This is the map of the plasmid pLH2208 overexpressing the mcs1 gene in the present invention;

[0034] Figure 11 This is the map of the plasmid pLH2209 overexpressing the mcs2 gene in the present invention;

[0035] Figure 12 This is the restriction enzyme digestion verification map of plasmids pLH2208 and pLH2209 in the present invention (enzyme a is EcoRⅠ and EcoRⅤ, and enzyme b is Hind Ⅲ);

[0036] Figure 13PCR verification electrophoresis patterns after electrotransforming Agrobacterium tumefaciens AGL-1 with plasmids pLH2176 and pLH2178 in the present invention; among them, the electrophoreses of groups a and b correspond to the amplification verification of the mcs1 and mcs2 homologous arms respectively. Each group includes: M: DNA Marker; N: negative control (sterile ddH2O template); P: positive control (amplification product using the recombinant plasmid as the template); 1-4: PCR products of Agrobacterium transformant colonies; mcs1-left arm: the positive control shows a characteristic band of 956 bp, and transformants 1-4 all show bands of the same size; mcs2-left arm: the positive control shows a characteristic band of 963 bp, and the bands of transformants 1-4 are as expected; mcs1-right arm: the positive control has a 1034 bp band, and the verification of transformants 1-4 is successful; mcs2-right arm: the positive control has a 923 bp band, and the verification of transformants 1-4 is successful;

[0037] Figure 14 Phenotypic analysis diagram of multi-resistance screening of transformants with the mcs1 gene knocked out in the present invention; among them, three different solid media were used in this experiment to evaluate the resistance phenotypes of Aspergillus niger recombinant transformants; among them, the upper row shows transformants 1 to 52, and the lower row shows transformants 53 to 98. The specific positions are shown in the schematic diagram on the far left; regarding the functions of each medium, the specific descriptions are as follows: PDA is a non-selective medium and serves as a positive growth control; PDA+HYG is a PDA medium supplemented with 250 μg / mL hygromycin B; MM+PPT is a minimal medium containing 500 μg / mL phosphinothricin; in this screening system, transformants that can grow on PDA+HYG but not on MM+PPT are identified as positive clone transformants; among them, N is a hygromycin-resistant sensitive strain as a negative control, and P is a hygromycin-resistant strain as a positive control.

[0038] Figure 15 Phenotypic analysis diagram of multi-resistance screening of transformants with the mcs2 gene knocked out in the present invention; among them, three different solid media were used in this experiment to evaluate the resistance phenotypes of Aspergillus niger recombinant transformants; among them, the upper row shows transformants 1 to 52, and the lower row shows transformants 53 to 98. The specific positions are shown in the schematic diagram on the far left; regarding the functions of each medium, the specific descriptions are as follows: PDA is a non-selective medium and serves as a positive growth control; PDA+HYG is a PDA medium supplemented with 250 μg / mL hygromycin B; MM+PPT is a minimal medium containing 500 μg / mL phosphinothricin; in this screening system, transformants that can grow on PDA+HYG but not on MM+PPT are identified as positive clone transformants; among them, N is a hygromycin-resistant sensitive strain as a negative control, and P is a hygromycin-resistant strain as a positive control.

[0039] Figure 16This is the principle flowchart for knocking out mcs1 (a) and mcs2 (b) genes in the present invention;

[0040] Figure 17 This is the genomic PCR verification diagram of the mcs1 gene knockout transformant in the present invention. In this experiment, five groups of PCR systems were used to verify homologous recombination events. Among them, (a) left-arm PCR amplification was used to detect the original sequence on the left side of the knockout site; (b) left-arm-hyg PCR amplification was used to confirm the connection between the left arm and the inserted hyg selection marker gene; (c) right-arm PCR amplification was used to detect the original sequence on the right side of the knockout site; (d) right-arm-hyg PCR amplification was used to verify the connection between the right arm and the hyg selection marker gene; (e) full-length PCR amplification was used to comprehensively evaluate the sequence changes in the knockout region. Among them, M: DNA Marker, and all the Markers in the figure are of the same kind, and the numerical values of each band are uniformly marked; N: negative control, PCR amplification was carried out using the S469 genome as a template; P: positive control, pLH2176. The verification principle can be detailed in Figure 10 the description; According to the verification results, if the amplification bands of both left-arm PCR and right-arm PCR are negative, while the amplification bands of both left-arm-hyg PCR and right-arm-hyg PCR are positive, and the full-length PCR amplification band shows a difference from the expected sequence after knockout, then this transformant is determined to be a positive transformant, that is, the knockout of the mcs1 gene is successfully achieved; Strains 43, 45, and 46 are correct transformants, and 50 is a false positive transformant;

[0041] Figure 18 This is the genomic PCR verification diagram of the mcs2 gene knockout transformant in the present invention. In this experiment, five groups of PCR systems were used to verify homologous recombination events. Among them, (a) left-arm PCR amplification was used to detect the original sequence on the left side of the knockout site; (b) left-arm-hyg PCR amplification was used to confirm the connection between the left arm and the inserted hyg selection marker gene; (c) right-arm PCR amplification was used to detect the original sequence on the right side of the knockout site; (d) right-arm-hyg PCR amplification was used to verify the connection between the right arm and the hyg selection marker gene; (e) full-length PCR amplification was used to comprehensively evaluate the sequence changes in the knockout region. Among them, M: DNA Marker, and all the Markers in the figure are of the same kind, and the numerical values of each band are uniformly marked; N: negative control, PCR amplification was carried out using the S469 genome as a template; P: positive control, pLH2178. The verification principle can be detailed in Figure 10Description; According to the verification results, if the PCR amplification bands of both the left arm and the right arm are negative, while the PCR amplification bands of both the left arm - hyg and the right arm - hyg are positive, and the full - length PCR amplification band shows a difference from the expected sequence after knockout, then the transformant is determined to be a positive transformant, that is, the knockout of the mcs2 gene is successfully achieved; 70, 76, 77, 78, 85, 86, 89, 98 are all mcs2 - knockout recombinant bacteria;

[0042] Figure 19 This is the PCR verification electrophoresis pattern diagram of the transformation of Agrobacterium tumefaciens AGL - 1 with plasmid pLH2208 / pLH2209 in the present invention; among them, the electrophoresis of groups a and b verifies the integration of mcs1 and mcs2 respectively. Each group contains: M: DNA Marker; N: negative control (sterile ddH2O template); P: positive control (amplification product using the recombinant plasmid as the template); 1 - 2: PCR products of Agrobacterium tumefaciens transformant colonies; mcs1 verification: the positive control P shows a characteristic band of 1429 bp, and the transformants 1 and 2 both show target bands of the same size as the positive control; mcs2 verification: the positive control P shows a characteristic band of 1398 bp, and the transformants 1 and 2 successfully amplify target bands of the same size as the control.

[0043] Figure 20 This is the double - resistance phenotype verification diagram of the mcs1 gene over - expression transformant in the present invention; among them, the schematic diagram on the far left shows the specific positions of transformants 1 to 49. N is a hygromycin - resistant sensitive strain as the negative control, and P is a hygromycin - resistant strain as the positive control; in this experiment, PDA medium and PDA medium supplemented with 250 μg / mL hygromycin B (i.e., PDA + HYG medium) are used to evaluate whether the transformant is positive; the left side of the figure shows the growth of the transformant on PDA medium, while the right side shows the growth of the transformant on PDA medium containing 250 μg / mL hygromycin B; among them, the transformant that can grow normally on PDA + HYG medium is a positive transformant.

[0044] Figure 21This is the verification diagram of the dual resistance phenotype of the mcs2 gene overexpression transformant in the present invention. Among them, the schematic diagram on the far left shows the specific positions of transformants 1 to 53. N is a hygromycin-resistant sensitive strain as a negative control, and P is a hygromycin-resistant strain as a positive control. In this experiment, PDA medium and PDA medium supplemented with 250 μg / mL hygromycin B (i.e., PDA+HYG medium) were used to evaluate whether the transformant is positive. The growth of the transformant on PDA medium is shown on the left side of the figure, while the growth of the transformant on PDA medium containing 250 μg / mL hygromycin B is shown on the right side. Among them, the transformant that can grow normally on PDA+HYG medium is a positive transformant.

[0045] Figure 22 This is the principle flow chart of overexpressing mcs1 (a) and mcs2 (b) genes in the present invention.

[0046] Figure 23 This is the genomic PCR verification diagram of the mcs1 gene overexpression transformant in the present invention. Among them, M: DNA Marker; N: negative control, PCR amplification was carried out using the S469 genome as a template; P: positive control, using pLH2208 as a template, and 12, 15, 21, 44, 47 respectively represent the PCR amplification results using the genomes of mcs1 overexpression transformants as templates. Among them, 15, 21, 44, 47 are correct transformants.

[0047] Figure 24 This is the genomic PCR verification diagram of the mcs2 gene overexpression transformant in the present invention. Among them, M: DNA Marker; N: negative control, PCR amplification was carried out using the S469 genome as a template; P: positive control, using pLH2209 as a template, and 8, 20, 24, 27, 32 respectively represent the PCR amplification results using the genomes of mcs2 overexpression transformants as templates. Among them, 8, 20, 24, 27, 32 are correct transformants.

[0048] Figure 25 This is the yield diagram of citric acid produced by the recombinant strains S4036 and S4040 obtained by knocking out mcs1 and mcs2 genes respectively during fermentation in the present invention.

[0049] Figure 26 This is the yield diagram of citric acid during fermentation of the recombinant strains S4127 and S4130 obtained by overexpressing mcs1 and mcs2 genes respectively in the present invention. Detailed implementation mode

[0050] To further illustrate the present invention, the following will be described in conjunction with specific embodiments. It should be emphasized that these embodiments are only narrative examples and are not used to limit the protection scope of the present invention. Therefore, the boundary of the present invention cannot be defined solely based on these embodiments.

[0051] All experimental operations involved in the embodiments belong to the conventional technical means in the technical field. For parts not specifically noted in this article, those of ordinary skill in the art can refer to relevant common reference books, scientific and technical literature, specifications or manuals before the application date of the present invention for specific implementation.

[0052] An mcs2 gene related to citric acid biosynthesis, wherein the nucleotide sequence of the mcs2 gene is as shown in SEQ ID No.1.

[0053] Use of the mcs2 gene as described above in improving the biosynthesis efficiency of citric acid.

[0054] The Mcs2 active polypeptide encoded by the mcs2 gene as described above for improving the biosynthesis efficiency of citric acid, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID No.2.

[0055] An expression cassette for improving the biosynthesis efficiency of citric acid, comprising the mcs2 gene as described above, the expression cassette comprising a promoter, the mcs2 gene and a terminator.

[0056] A vector for improving the biosynthesis efficiency of citric acid, comprising the mcs2 gene as described above, the vector selected from plasmids, viral vectors or artificial chromosomes.

[0057] A method for improving citric acid production by Aspergillus niger using the mcs2 gene as described above, the method comprising introducing the mcs2 gene, an expression cassette comprising the mcs2 gene or a vector comprising the mcs2 gene into Aspergillus niger.

[0058] An Aspergillus niger recombinant strain for improving citric acid production by Aspergillus niger, comprising the mcs2 gene as described above.

[0059] A method for constructing the recombinant strain as described above, comprising the following steps:

[0060] First, the overexpression plasmid pLH2209 containing the mcs2 gene was introduced into the competent cells of Agrobacterium tumefaciens AGL1 by electroporation. The electroporation conditions were: 2.5 kV, 25 μF, 200 Ω. Positive clones were obtained by screening with LB medium containing 100 μg / mL kanamycin, and colony PCR verification was performed with primers P5420 / P5421. A specific band of 1398 bp was amplified to confirm the transfer of the plasmid into Agrobacterium tumefaciens. Subsequently, the verified Agrobacterium tumefaciens was combined with 1×10^6 The spores of Aspergillus niger S469 at a concentration of CFU / mL were co-cultured with a volume ratio of 1:10 in an IM solid medium containing 100 μg / mL kanamycin and 0.2 μM acetosyringone (AS) at 25 °C for 60 h. The grown transformants were transferred to a CM selective medium containing 200 μM cefotaxime, 100 μg / mL ampicillin, 100 μg / mL streptomycin, and 250 μg / mL hygromycin B for screening transformants with hygromycin B resistance. The positive transformants were further verified for hygromycin resistance on a PDA medium containing 250 μg / mL hygromycin, and detected by genomic PCR: specific bands of 1550 bp were amplified using primers P5422 / P5423 to confirm the successful integration of the mcs2 expression cassette gene into the genome, obtaining a recombinant strain of Aspergillus niger.

[0061] Application of the recombinant strain as described above in the fermentation production of citric acid.

[0062] A method for fermenting and producing citric acid using the recombinant strain as described above, comprising the following steps:

[0063] The cryopreserved recombinant strain was inoculated onto a PDA solid medium containing 200 μg / mL hygromycin B and cultured in an inverted position at 28 °C for 4 - 5 days. After the spores matured, they were scraped and suspended in sterile physiological saline containing 0.05% (v / v) Tween-80, and the spore concentration was adjusted to 1×10 ^8 CFU / mL; subsequently, 1×10 ^8 spores were inoculated into the fermentation medium and cultured with shaking at 28 °C and 200 r / min for 5 days;

[0064] Among them, the formula of the fermentation medium is: sucrose 20 g / L, yeast extract 10 g / L, KH2PO4 1 g / L, MgSO4·7H2O 1 g / L, (NH4)2SO4 4.13 g / L, the solvent is water, and the pH is adjusted to 2.5.

[0065] Example 1 Analysis of MCS protein sequence

[0066] In Aspergillus niger, two citrate synthases, CitA (SEQ ID No.3) and CitB (SEQ ID No.4), which are crucial for citric acid synthesis, have been reported and confirmed, and they play a core role in the citric acid synthesis pathway. In this invention, through in-depth genomic mining work, two novel methyl citrate synthases, Mcs1 (SEQ ID No.5) and Mcs2 (SEQ ID No.2), were first identified and reported. The functions of these two enzymes have not been disclosed in the existing literature, so their specific mechanisms of action need to be explored urgently.

[0067] To deeply explore the potential associations and functional differences among these enzymes, the present invention conducted a detailed amino acid sequence alignment analysis. And based on the neighbor-joining method, a phylogenetic tree was constructed, as Figure 1 shown. The analysis results show that there is a relatively close evolutionary relationship between Mcs2 and CitA, and there is also a relatively close evolutionary origin between Mcs1 and CitB. This finding provides valuable clues for analyzing the specific roles of Mcs1 and Mcs2 in the citric acid synthesis pathway, and also lays a solid foundation for subsequent functional research and application development.

[0068] Example 2 Construction of mcs1 and mcs2 gene knockout plasmids and overexpression plasmids

[0069] (1)Construction of mcs1 and mcs2 gene knockout plasmids

[0070] Using the genomic DNA of Aspergillus niger ATCC1015 as a template, high-quality DNA (OD260 / 280 = 1.7 - 1.9) was extracted using the Beyotime Genomic DNA Mini Extraction Kit (D0063). High-fidelity PCR amplification was performed using the specific primers listed in Table 1 and Novoprotein PhantaMax Master Mix (P515): For the mcs1 gene, the left arm fragment of 956 bp (SEQ ID NO.6) was amplified with primers P5402 / P5403, and the right arm fragment of 923 bp (SEQ ID NO.8) was amplified with primers P5406 / P5407; for the mcs2 gene, the left arm fragment of 963 bp (SEQ ID NO.7) was amplified with primers P5404 / P5405, and the right arm fragment of 1034 bp (SEQ ID NO.9) was amplified with primers P5408 / P5409. The PCR conditions were: pre-denaturation at 95 °C for 5 min, 30 cycles (98 °C for 10 s, 60 °C for 15 s, 72 °C for 1 min / kb), and final extension at 72 °C for 5 min. After the amplified products were verified by 1% agarose gel electrophoresis, they were purified using the Tiangen Gel Extraction Kit (DP203) and reserved for use.

[0071] Plasmid pLH594 (the structure is shown in Figure 2, Literature support: Development of a Cre-loxP-based geneticsystem in Aspergillus niger ATCC1015 and its application to construction ofefficient organic acid-producing cell factories. Appl Microbiol Biotechnol.2019, 103(19):8105-8114). It was linearized by double digestion with Thermo Scientific FastDigest XbaⅠ / PstⅠ (37 °C, 15 min). After the digestion products were purified by Tiangen DP203 kit, they were mixed with the right arm fragments of mcs1 or mcs2 at a molar ratio of 3:1, and seamless cloning was carried out using Vazyme ClonExpress II homologous recombination kit (C112) (50 °C, 30 min). The ligation products were transformed into E. coli JM109 competent cells (TIANGEN CB101). After heat shock at 42 °C for 45 seconds, ice bath for 2 min and recovery culture at 37 °C for 1 h, they were spread on LB plates containing 100 μg / mL kanamycin (cultured at 37 °C for 16 h). Five single colonies were randomly selected for colony PCR verification (primers P5404 / P5405, P5408 / P5409), and plasmids were extracted using Tiangen plasmid mini kit (DP103) to obtain plasmid pLH2175 (structure shown in Figure 3 ) and pLH2177 (structure shown in Figure 4 ). Further, double digestion verification with KpnⅠ / HindⅢ showed that pLH2175 produced 8960 bp / 2418 bp fragments and pLH2177 produced 7682 bp / 3807 bp fragments ( Figure 5 ), indicating successful construction of the recombinant plasmids.

[0072] Furthermore, pLH2175 and pLH2177 were linearized by double digestion with EcoRⅠ / BamHⅠ and ligated with the left arm fragments of mcs1 and mcs2 by homologous recombination respectively. The specific method was the same as above. The final plasmid pLH2176 (structure shown in Figure 6 ) produced 8925 bp / 3398 bp fragments by PstⅠ / EcoRⅠ digestion, and pLH2178 (structure shown in Figure 7 ) produced 9552 bp / 2889 bp fragments by PstⅠ single digestion ( Figure 8 ). Agarose gel electrophoresis showed that the sizes of all bands were completely consistent with the theoretical values, indicating successful construction of the knockout plasmids of methylcitrate synthase.

[0073] Special note: In this experiment, the high-efficiency extraction of Aspergillus niger genomic DNA was completed using the Genomic DNA Mini Kit (D0063) from Beyotime. The PCR amplification was carried out using Phanta Max Master Mix (P515) from Novoprotein. The plasmid extraction was performed using the Plasmid Mini Kit (DP103) from Tiangen. The DNA purification and recovery were also carried out using the Plasmid Mini Kit (DP103) from Tiangen. The enzymatic digestion of the vector and the inserted fragment both adopted the commercial enzymatic digestion system of Thermo Scientific FastDigest restriction enzymes. The ligation of DNA fragments was achieved through the commercial homologous recombination kit Vazyme ClonExpress II One Step Cloning Kit (C112). The specific operation details need to be referred to the latest version of the instructions of each reagent and kit.

[0074] Table 1 Primer sequences used in Example 1

[0075]

[0076] (2) Construction of overexpression plasmids for mcs1 and mcs2 genes:

[0077] First, using Aspergillus niger ATCC1015 strain as the material, high-quality genomic DNA was extracted using the Genomic DNA Mini Kit (D0063) from Beyotime. Subsequently, using this DNA as a template, the primers listed in Table 2 were used, and high-fidelity PCR amplification was carried out using Novoprotein Phanta Max Master Mix (P515). Specifically, primers P5418 / P5419 were used to amplify the mcs1 gene fragment (with a length of 1429 bp, SEQ ID NO.10), while primers P5420 / P5421 were used to amplify the mcs2 gene fragment (with a length of 1398 bp, SEQ ID NO.1). The amplification conditions were set as pre-denaturation at 95°C for 5 minutes, followed by 30 cycles (98°C for 10 seconds, 60°C for 15 seconds, 72°C for 1.5 minutes). After the amplification products were verified by 1% agarose gel electrophoresis, they were purified using the Gel Extraction Kit (DP203) from Tiangen and reserved for use.

[0078] Next, for plasmid pLH454 (the structure is shown in Figure 9)(1) It was digested with Thermo Scientific FastDigest EcoRⅠ / BamHⅠ double enzymes (at 37 °C for 15 minutes) to linearize it. The digestion product was also purified using the Tiangen Gel Extraction Kit (DP203). Subsequently, the purified mcs1 and mcs2 gene fragments were mixed with the linearized pLH454 plasmid at a molar ratio of 3:1, and seamless cloning was performed using the Vazyme ClonExpress II One Step Cloning Kit (C112) (at 50 °C for 30 minutes).

[0079] The cloning product was then transformed into E. coli JM109 competent cells (TIANGEN CB101). After heat shock at 42 °C for 45 seconds, ice bath for 2 minutes, and recovery culture at 37 °C for 1 hour, the cells were spread on an LB plate containing 100 μg / mL kanamycin and cultured at 37 °C for 16 hours. Subsequently, 5 single colonies were randomly selected for colony PCR verification, and plasmids were extracted using the Tiangen Plasmid Mini Kit (DP103) to obtain pLH2208 (structure shown in Figure 10 ) and pLH2209 (structure shown in Figure 11 ). To further verify the correctness of the plasmids, double enzyme digestion verification was performed on pLH2208 (mcs1 overexpression plasmid) and pLH2209 (mcs2 overexpression plasmid) respectively. Among them, pLH2208 produced fragments of 9053 bp and 2342 bp after double digestion with FastDigest EcoRⅠ / EcoRⅤ, while pLH2209 produced fragments of 10219 bp and 1155 bp after single digestion with HindⅢ. The results of agarose gel electrophoresis (shown in Figure 12 ) indicated that the overexpression plasmids of methylcitrate synthase were successfully constructed.

[0080] Special note: The relevant information of the plasmid pLH454 used can be found in the reference: 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.

[0081] Table 2 Primer sequences used in the examples

[0082]

[0083] Example 3 Construction of mcs1 and mcs2 Gene Knockout Strains and Overexpression Plasmid Strains

[0084] (1)Construction of mcs1 and mcs2 Gene Knockout Strains

[0085] The strain construction was carried out according to Appl Microbiol Biotechnol. 2019, 103(19):8105-8114 in the literature.

[0086] 1)Agrobacterium electrotransformation and plasmid verification: Based on the literature method, the knockout plasmids pLH2176 and pLH2178 were transformed into competent cells of Agrobacterium tumefaciens AGL1 by electroporation. The electroporation parameters were set as: voltage 2.5 kV, capacitance 25 μF, resistance 200 Ω, pulse time 4 - 5 ms (Gene Pulser Xcell system, Bio-Rad). After transformation, the bacterial solution was spread on LB solid medium containing 100 μg / mL kanamycin (cultured at 28 °C for 48 h), and monoclonal colonies were obtained. In colony PCR verification, the plasmid was used as a positive control, and the original construction primers were used. Phanta Max Master Mix (P515) from Novoprotein was used. For pLH2176: primers P5402 / P5403 amplified a 956 bp left arm fragment, and P5406 / P5407 amplified a 923 bp right arm fragment; for pLH2178: primers P5404 / P5405 amplified a 963 bp left arm fragment, and P5408 / P5409 amplified a 1034 bp right arm fragment. Agarose gel electrophoresis ( Figure 13 )showed that the positive clone bands were completely consistent with the expected ones, indicating that the plasmid was successfully transferred into Agrobacterium.

[0087] 2)Agrobacterium induction and co-culture with Aspergillus niger: The verified Agrobacterium monoclonal colonies were inoculated into LB liquid medium (3 mL) containing 100 μg / mL kanamycin and cultured with shaking at 28 °C and 200 r / min for 20 h until OD 600 = 0.8. Subsequently, it was transferred to IM induction medium (containing 100 μg / mL kanamycin and 0.2 μM acetosyringone AS) and induced at 28 °C and 100 r / min for 5 h. At the same time, a fresh spore suspension of the starting strain Aspergillus niger S469 (concentration 1×10 ^6(CFU / mL), mixed with the induced Agrobacterium at a volume ratio of 1:10, and evenly spread on the IM solid medium (containing a 0.45 μm filter membrane), and co-cultured at 25 °C for 60 h. After the bacteria on the filter membrane surface showed yellowish green, they were transferred to the CM selective medium (containing 200 μM cefotaxime, 100 μg / mL ampicillin, 100 μg / mL streptomycin, and 250 μg / mL hygromycin B), and cultured at 28 °C for 5 - 7 days until single colonies formed.

[0088] 3) Screening and molecular verification of transformants: Single colonies were picked and inoculated into the following media for phenotypic screening: PDA + Hyg (PDA medium containing hygromycin B (250 μg / mL)) to verify hygromycin resistance; PDA medium to exclude contamination by miscellaneous bacteria; MM + PPT (MM medium containing glufosinate (100 μg / mL)) to verify double exchange events. In this screening system, transformants that could grow on PDA + HYG but not on MM + PPT were identified as positive clone transformants. The specific results are shown in Figure 14 and Figure 15 (growth diagrams of knockout transformants of mcs1 and mcs2 respectively), and there were many positive transformants.

[0089] Appropriate numbers of positive transformants were extracted, and genomic DNA was extracted using a kit. Verification primers were designed according to the flowchart of the gene knockout principle ( Figure 16 ), as shown in Table 3 specifically. For the verification of mcs1 knockout: Primers P5410 / P5411 (left arm) and P5412 / P5413 (right arm) amplified bands in the positive control, and no bands should be present in correctly transformed strains; Primers P5410 / P641 (left arm - Hyg) and P642 / P5413 (Hyg - right arm) should amplify the expected bands in transformants; The amplification product of primers P5410 / P5413 should be 100 bp shorter than the positive control, indicating successful homologous recombination. The specific results are shown in Figure 17 , and strains numbered 43, 45, and 46 were transformants with correct mcs1 knockout. For the verification of mcs2 knockout: Primers P5414 / P5415 (left arm) and P5416 / P5417 (right arm) had no amplification in transformants; Primers P5414 / P641 (left arm - Hyg) and P642 / P5417 (Hyg - right arm) amplified the target bands; The length of the amplification product of primers P5414 / P5417 was the same as that of the positive control, indicating the specificity of mcs2 knockout. The specific results are shown in Figure 18 , and strains numbered 70, 76, 77, 78, 85, 86, 89, and 98 were all mcs2 knockout recombinant strains.

[0090] The transformants numbered 45 and 76 were named S4036 and S4040 respectively, which are the recombinant strains with mcs1 and mcs2 knocked out.

[0091] Special note:

[0092] LB medium: Tryptone: 10.0 g / L, Yeast extract: 5.0 g / L, NaCl: 10.0 g / L. After dissolving with deionized water, make up the volume to 1.0 L, adjust the pH to 7.0 - 7.2. For solid medium, add 1.5% agar powder. Sterilize at 121℃ for 20 min.

[0093] Preparation method of Potato Dextrose Agar (PDA): Accurately weigh 500 g of peeled potatoes, cut them into small pieces, add distilled water and stir continuously until boiling for 30 min. Filter with double - layer gauze to collect the filtrate. Add 50 g of glucose and stir until completely dissolved. Make up the volume to 2.5 L with distilled water and dispense into wide - mouth bottles. Add 1.5% agar, and sterilize at 121℃ under high pressure for 20 min.

[0094] Preparation method of IM solid medium: Dissolve 15 g of agar in water and make up the volume to 905.7 mL, sterilize at 121℃ for 20 min. Add 0.8 mL of pre - prepared sterile K buffer, 20 mL of MN buffer, 1 mL of 1% CaCl₂·2H₂O, 10 mL of 0.01% FeSO₄, 5 mL of IM Trace elements, 2.5 mL of 20% NH₄NO₃, 10 mL of 50% glycerol, 40 mL of 1M MES, 5 mL of 20% glucose. When the temperature cools to about 50℃, add kanamycin to make its final concentration 100 µg / mL and add acetosyringone to make its final concentration 200 µM.

[0095] Preparation method of IM induction medium: Make up the volume to 900.7 mL with water, sterilize at 121℃ for 20 min. Add 0.8 mL of pre - prepared sterile K buffer, 20 mL of MN buffer, 1 mL of 1% CaCl₂·2H₂O, 10 mL of 0.01% FeSO₄, 5 mL of IM Trace elements, 2.5 mL of 20% NH₄NO₃, 10 mL of 50% glycerol, 40 mL of 1M MES, 10 mL of 20% glucose. When the temperature cools to about 50℃, add kanamycin to make its final concentration 100 µg / mL and add acetosyringone to make its final concentration 200 µM.

[0096] The preparation method of CM medium is as follows: 20 g of agar is added with water to a final volume of 897 mL, sterilized at 121 °C for 20 min, and then 20 mL of sterile ASPN solution (the composition is KCl 26.1 g / L, KH2PO4 74.8 g / L, NaNO3 297.5 g / L, adjusted to pH 5.5), 20 mL of 50% glucose, 2 mL of 1M MgSO4, 1 mL of CM Trace elements (the composition is per 100 mL containing ZnSO4·7H2O 2.1 g, H3BO3 1.1 g, MnCl2·4H2O 0.5 g, FeSO4·7H2O 0.5 g, CoCl2·6H2O 0.17 g, CuSO4·5H2O 0.16 g, Na2MoO4·2H2O 0.15 g, EDTA 5.1 g), 10 mL of 10% casein hydrolysate, and 50 mL of 10% yeast extract are added. When the temperature cools to about 50 °C, hygromycin is added to make its final concentration 250 µg / mL, streptomycin is added to make its final concentration 100 µg / mL, cefotaxime sodium is added to make its final concentration 100 µg / mL, and ampicillin is added to make its final concentration 100 µg / mL.

[0097] Among them, more specific information about the above LB medium, potato dextrose agar medium, IM solid and induction medium, CM medium, MM medium, etc. is the same as that recorded in the public literature "Establishment of Aspergillus niger Genetic Transformation System with amdS as a Screening Marker". China Agricultural Science & Technology Bulletin, 2020, 22(9):9. DOI:CNKI:SUN:NKDB.0.2020-09-020..

[0098] (2)Construction of mcs1 and mcs2 gene overexpression strains:

[0099] According to the steps and methods in 1) of the construction of mcs1 and mcs2 gene knockout strains, the constructed methylcitrate synthase overexpression plasmids pLH2208 and pLH2209 were electrotransformed into Agrobacterium tumefaciens competent AGL-1. After monoclonal colonies grew, PCR colony verification was carried out. The verification primers P5418 / P5419 were used to amplify the mcs1 gene fragment (length 1429 bp), while the primers P5420 / P5421 were used to amplify the mcs2 gene fragment (length 1398 bp). The specific results are shown in Figure 19 , showing that the positive clone bands are completely consistent with the expected ones, indicating that the plasmid was successfully transferred into Agrobacterium tumefaciens. According to the steps and methods in 2) of the construction of mcs1 and mcs2 gene knockout strains, the co-culture of Agrobacterium tumefaciens and the starting strain S469 was completed until single colonies formed.

[0100] Transformant screening and molecular verification: Single colonies were picked and inoculated into the following media for phenotypic screening: PDA + Hyg (PDA medium containing hygromycin B (250 μg / mL)) to verify hygromycin resistance; PDA medium to exclude contamination by miscellaneous bacteria. In this screening system, those that could grow on PDA + HYG were identified as positive clone transformants. The specific results are shown in Figure 20 and Figure 21 (growth diagrams of overexpression transformants of mcs1 and mcs2 respectively), and there were many positive transformants.

[0101] Genomes were extracted from an appropriate number of positive transformants using a kit. According to the principle flow chart of gene overexpression ( Figure 22 ), the upstream primer P5422 was designed at the 3'-end of the promoter PgpdA, and the downstream primer P5423 was designed at the 5'-end of the terminator TtrpC. If the mcs1 or mcs2 gene was successfully overexpressed, when PCR amplification was performed using the transformant genomic DNA as a template, target bands would appear when using P5422 / P5423 as primers, and the band lengths were 1571 bp and 1550 bp respectively. Otherwise, it indicated that mcs was not successfully overexpressed. The specific primers are shown in Table 3. The specific verification results are shown in Figure 23 and Figure 24 , and the transformants with successful overexpression were determined. The 15# and 20# transformants were named S4127 and S4130, which were the correct transformants overexpressing the mcs1 and mcs2 genes respectively.

[0102] Table 3 Primer sequences used in the examples

[0103]

[0104] Example 4 Citric acid shake flask fermentation

[0105] The Aspergillus niger strains (including recombinant strains and wild-type controls) stored at ultra-low temperature were inoculated onto PDA solid medium containing 200 μg / mL hygromycin B and cultured inverted at 28 °C for 4 - 5 days. After the spores matured, they were scraped and suspended in sterile physiological saline containing 0.05% (v / v) Tween-80, and the spore concentration was adjusted to 1×10 ^8 CFU / mL using a hemocytometer. Subsequently, 1×10 ^8Spores were cultured by shaking at 28 °C and 200 r / min for 5 days. Three biological replicates were set for each strain, and 2 mL of fermentation broth was sampled on the 3rd day (logarithmic growth phase) and the 5th day (stationary phase), respectively.

[0106] The fermentation broth samples were centrifuged at 4 °C and 12,000×g for 20 min, and the supernatant was collected, filtered through a 0.22 μm aqueous filter membrane (Millipore SLGV033RS), diluted 20-fold with ultrapure water, and then injected into a special HPLC vial. High performance liquid chromatography (HPLC) system (Agilent 1260 Infinity II) was used for analysis. The chromatographic column was a Bio-Rad HPX-87H organic acid analysis column (300×7.8 mm), the mobile phase was 5 mM H2SO4 (flow rate 0.6 mL / min, column temperature 60 °C), the ultraviolet detection wavelength was 210 nm, and the injection volume was 20 μL.

[0107] The fermentation performance of the genetically engineered strains was compared: as Figure 25 and 26 shown, the citric acid yield of the wild-type strain S469 on the 5th day was 41.5 g / L; the yields of the knockout strains S4036 (Δmcs1) and S4040 (Δmcs2) decreased to 32.6 g / L (a decrease of 21.4%) and 7.2 g / L (a decrease of 82.7%), respectively; while the yield of the overexpression strain S4130 (OEmcs2) increased significantly to 58.5±1.5 g / L (an increase of 41.0%), and S4127 (OEmcs1) decreased to 19.3 g / L (a decrease of 53.5%).

[0108] The above results indicate that knocking out methylcitrate synthase significantly reduces the citric acid yield, indicating that both of the two coding genes of methylcitrate synthase have the properties of citrate synthase. Among them, the mcs2 gene has a significant impact on citric acid synthesis, strongly proving the indispensability of the mcs2 gene for citric acid synthesis. It is worth noting that overexpressing mcs2 significantly accumulates citric acid, fully demonstrating the great potential and broad prospects of genetic engineering means in optimizing the production performance of citric acid.

[0109] The sequences used in the present invention are as follows:

[0110] SEQ ID NO.1 Nucleotide sequence of mcs2 gene:

[0111]

[0112] Amino acid sequence of SEQ ID NO.2 Mcs2 active peptide:

[0113] MSFSMPIRPTTRHASRLAQAKIRSGRLYSTESDLKTALKSVIPEKRELFKQVKARSDDVVGEVKVGNIIGGMRGLKSMLWEGSVLDPEEGIRFHGKTIKDCQRELPKGTTGTEMLPEAMFWLLLTGQVPTTSQVRAFSRELAEKSHLPDHILGLIKSFPKDMHPMTQLSVAVAALNTESTFAKAYERGLNKADYWEPTFDDSISLLAKIPRVAALVFRSNEIDQVGTQALDATQDWSHNFAELLGKGGAEHADFHDLLRLYLALHGDHEGGNVSAHATHLVGSALSDPFLSYSAGLLGLAGPLHGLAAQEVLRWILAMQEKIGTQVTDDSVRTYLWDTLKSGRVVPGYGHGVLRKPDPRFEALMDFAATRPDVQANPVFQLVKKNSEIAPEVLTQHGKTKNPHPNVDAASGVLFYHYGFQQPLYYTVTFGVSRALGPLVQLIWDRALGLPIERPKSINLKGLIGN*

[0114] Amino acid sequence of SEQ ID NO.3 Citrate synthase CitA:

[0115] MASTLRLGTSALRSTSIAAKPVVQSAAFNGLRCYSTGKAKSLKETFAEKLPAEIEKVKKLRKEHGSKVIGEVTLDQAYGGARGVKCLVWEGSVLDSEEGIRFRGRTIPECQELLPKAPGGQEPLPEGLFWLLLTGEIPTEQQVRDLSAEWAARSDLPKFIEELIDRCPSTLHPMSQFSLAVTALEHESAFAKAYAKGINKKDYWNYTFEDSMDLIAKLPTIAAKIYRNVFKDGKVAPIQKDKDYSYNLANQLGYGDNNDFVELMRLYLTIHSDHEGGNVSAHTTHLVGSALSSPMLSLAAGLNGLAGPLHGLANQEVLNWLTKMKAAIGNDLSDEAIKNYLWSTLNAGQVVPGYGHAVLRKTDPRYVSQREFALRKLPDDPMFKLVSQVYKIAPGVLTEHGKTKNPYPNVDAHSGVLLQYYGLTEANYYTVLFGVSRALGVLPQLIIDRALGAPIERPKSYSTEAFAKLVGAKL*

[0116] SEQ ID NO.4 Citrate synthase CitB amino acid sequence:

[0117] MPDIASNGARNGASQNAETKPEPPVLHVVDSRTGKYFPIPIVRNAINASEFKKLKSPEDPAHPEDQNEQGIRVFDPGYSNTAVSESQVTYIDGLKGTIQYRGYNIEDIVGKKKFIDTAHLLIWGEWPTPEQAKSLQEKLSSVPVLDESVFKVIQAFPPNSSIIGMMIAALSAVQSTQMDRIPAHAAKNLYLGNPKAVDDEIVRLMGSLSMITAAVYCHHTGREFTPPRPELSYIENFLLMMGHVESSTGLPNPQYVDRIERLWVLIADHEMTCSTAAFLQTASSLPDVFSCMISALSALYGPLHGGAIEVAYKNFEEIGSVENVAAKIERVKAGKERLYGYGHRIYRVTDPRFIFIRQILDELKEEIARNPLLKVAFEVDRVASEDEYFVTRKLRPNADLFAALVYSAMGFPTEFILPLSLLSRTQGFLAHWKEAMSSTARIWRPGQIYTGHLNREMA*

[0118] SEQ ID NO.5 Amino acid sequence of methylcitrate synthase Mcs1:

[0119] MAYTLASWLGRLFDAGKSLLPLQGNYINALLEQELPGEREGTLTVRDNRTGSKYTIPIVRNSVPAMGFRQICVDRAGKSPRQQFEDGLRLIDPGYRNTAVKMSSITYINGNEGVILYRGHPLASLIGKSYEEITHLLIWGSLPTPEQRLRFQRRIAEAMMVVPENVKQLVATFPRNTPPMVILCAVLTGYLADQPELIPAHAGANLYNRRPEMVDEQIIRTLAVTAIAGSIAHCHMKGEELRMADPNLSYIENILWMGRYVDNNPAVTREKAAEILTKAWSLYADHEMTNSTSAFLHVSSSLADPLSAMAACCMSGYGLLHGGAIDAAYRGMREIGGPQNVPKLIEKVINKECRLSGYGHRIYKQVDPRAKYVREMLDELTRDRDIREMDPVLQVAMEIDRIASTHEYFVKRNLQANADLYGSFVYTALGIDSQFATVLAATARVSGVMAHWKEQTERAPDLWRPLQVYVPN*

[0120] SEQ ID NO.6 Nucleotide sequence of the left arm of the mcs1 gene:

[0121] AATTCGAAGGCATTGCGGATCTGGTAGCAGCCTTGAATCTCGTAGGCTTTGATCATCGCGATCAGGACCGTCCGGACGGTCAGTGGCTGGCTGGTGGCGCCTGTACGGCTGAGCCAATCTGTGACCGCCAAGATGGCGCCCAGGTTATCTGTTTTGTCGATCGGTTAGCCCTATATTCAACCAAGCTTCTGGGAGCGTAGATCCCGCAGACGGGCATACCAGACGGGTGGCCCCATTCAGCACCAGGGTATGCATCGTTGTGGTCCAGGAAGCGAATCAAGGTTGCCATATCAAAGGCACCCTTGACTGGATCAAGTTGGTGGCAGGTGCCCGGCAGCTTGAACCCATAAGGGGCGACCAGACCTGGTGTGTATGGGCCGATCAGAGCGCGACACTCGGGGCTTTGCTGAAGAGTCTCCAGCGCGCAACCAAGAGCATCCATGAGGGCATAGCGGGCCCGTAGGATAGCGAGGTCGCTGGTCACTTCGTAGTGATAAACATACTCAACTAGGCGAGTTATCGTTTCGTCATACTGTCGATCGGAAGTGGAAGACATGGTCCTCGGAGTGTGCACGGAAAGGCTTTTGAGTAGATAGGTTTCGGAAGAAAAGTTTGGATTGGCCCAGGAAGTGACGAGAGTGATGGCGAACGAAACAGACCGCAAAGGGAACCGCCCTTTTATGCTCCGTCGGGCAGACCGTGTCAGCCAGATGCCTTTCGATCGCCGGTGATAGTCTCTGTGGGCTGTCACTAGTCGATGGACCAAAGCTTGACAGTCTATCTCGGCGTATCCATTCGTCCTTTTGAACTATTCCGTCCGAGACTGCATGGTCTCGGACAGATGGCTGCTGCCATGCCAGACAAGGTACAAATATGTGCTTCAGAGCCCGACAAACCCCAACAGAATCAGGACAACAGCAGCGACAATCCTTCGCAGACTCATCCGATTCCCTTCTACAACGGATC

[0122] SEQ ID NO.7 Nucleotide sequence of the left arm of the mcs2 gene:

[0123] AATTCCAAAGAGGGGAGATTTGGAGGAGAGGAGAGGAGAGGAGAGGCGAGGGAAGTTGAAAGGCGAGCGCAAGAGGAAAAACAAGATGGAGGCGGGGCCGATAACGAGCCTGAGGATTTTTAGGCGGCGGCCCATTGGATCGGGCGGCGATGAAATCTCTGCCTCAGGCGAGGGGCTGATAGCCTATCATCCAGGCACGCGTGGCTGGGTTGTGGCGGTGCCAATCCCTTGCTAGCCTGATGTATCAGGTGTCATTTATACCATCATTGCCTAATATATCAGGTGTCAGTTTAGTCAGTTTCTGCCTGAAACGCGGTAATATAATGCCTGAGGCTGCCATAACGCCGCCGTTGCCCGATTGGGCTTACCCCGCACGGGGGCTCCACCTCGCCGGACCAAAAATGGAGTCAAGCATCCACTGCCAAATGGGAGGATCAGCTTCCCTTTCTTCCCTCTGCTGTTCCGAACATCCATCGTCCATGGACTTGTGAAGGGAATGCATGGTACCAAAGCACTTCTCTGCTCTTGCGGTTGGATCTTCCTCATCCCCGATGTCGCCTCACCGCATTGTACGATCGATCGACCCCTCACAGGAGTCCCCCGAGGCCCGGCGTGACGGGATCTGCAGCCAAATGGATCTAATTACACCATGATGATAACTAAAAGTGCAGCGCGCGCTCTACTGCACACTACCGATTTAGTCCGACGGCCGGCTCGGCCTCGCATCGGCGGTGAGGATGTGCACGACAGAGGGTGGATTGGACGCTGACTGCTTATGGCTGATTGTGTTGACCGTCCGCATGCAGCCGTGCCTTGCCTCGGCTCCTCAACACCGCGGCCAACCGAACGGCCACTCGCTCTCCTTCCGGGTGAATTTCTCCTGACTCTGCTTTGTACTCTGCTCCGTGTACATAATTCCTACTCTCTCTCGTCTTTGTTTGCACATTCAGCCTTCCCGAACACTCATCGGATC

[0124] SEQ ID NO.8 Nucleotide sequence of the right arm of the mcs1 gene:

[0125] CTAGAGGATAGGGGATAGGGATGGGATTGTACAGAATGGGTCATCATAGGAGTCTATTGTTTTCGGGTCTGGGTCTTGGTTACTTCATAGTTTGCGCTATACTCATTAGTGGTTAATACTTTTTCAACAGACTGCTCCTTTTCTTCTTAAATACACTGGCAGTTGGTGCATTTTTATTTTGGAACAATACATGCAGTGATAATCATCATATTGACTGTCGATTAGTGGTACCTGAAAGAACAATACTATCGATGTAAGTAGTTTCTATGTCTGAGGTAGCTGCTTAATATCCATATTTGGCAATTATAAGATGATACATATGAATTATATTCAGAATCATAGTAGACTGGCAGTAGCCGATAATCAATGTTTGCAGCAGCAGACATCCCAAGAGCACTGGTAGTGATATAAAGTAAAATAAACTTTGCAAAGCCAAGTCCACAGGACTCTCTCTGTAGTCACTCTTGCGTTGCTTCGTAGGGGAAGAAGCTTCATAGTCTCAATGAGTAGTCTTCATGCCCTATAGGGCTATACAGGAAATCTCTCTTTAGATCATCTTCTGCTTCGTCAACTTTAATTGTTTAACCAAAAACGCCTAACCCACCTCTCCACGAACTACAACTTGCCACCAGCCTCTTACAACCGACCTTTTGACTTTAGTTTCAATATATATTTCCATAACTAAGCAACATATAGACATCAGAATAATGTAGACCATCGGTATATGCCTGCTGGGCAGAAACCACCGCCAGAATCCATCCCATTTCTTTTGTTTCCAACTCCACATAACCCAAAAATATGCCTGTAGTCTTAAAATTTAAGTCCTAAGCTCGTCGATAGTGGTTCTGCATAGGTAGCCTTATCTCGTATAATGGTAAACTTCACTAGTTCCAATCGGGCGGAAGTGATCGACACGCCGGAAACAAGCCTGCA

[0126] Nucleotide sequence of the right arm of the mcs2 gene of SEQ ID NO.9:

[0127]

[0128] SEQ ID NO.10 Nucleotide sequence of mcs1 gene:

[0129]

[0130] Although embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand 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 content disclosed in the embodiments.

Claims

1. A method for improving citric acid production by Aspergillus niger, characterized in that: The method includes introducing mcs2 gene, an expression cassette containing mcs2 gene or a vector containing mcs2 gene into Aspergillus niger, and the nucleotide sequence of the mcs2 gene is as shown in SEQ ID No. 1.

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