Method for improving citric acid production of aspergillus niger, mcs2 gene and application

Abstract

The invention belongs to the technical field of biology, and discloses a method for improving citric acid production of aspergillus niger, an mcs2 gene and application, the nucleotide sequence of the mcs2 gene is shown as SEQ ID No.1, and the method comprises the step of introducing the mcs2 gene, an expression cassette containing the mcs2 gene or a carrier containing the mcs2 gene into the aspergillus niger. According to the invention, a new base mcs2 related to citric acid biosynthesis is successfully identified, and a new view angle and scientific basis are provided for understanding a biosynthesis pathway of citric acid. The invention not only provides a new gene target and a modification strategy for biosynthesis of citric acid, but also opens up a new way for biotechnology improvement of citric acid production, and is hopeful to significantly improve production efficiency and economic benefit.

Description

Technical Field

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

[0002] Citric acid, a multifunctional platform compound of natural origin, has built a comprehensive and three-dimensional application network across food, daily chemicals, medicine and advanced manufacturing with its unique chemical properties and excellent biocompatibility. In the food industry, citric acid, as a GRAS (generally recognized as safe) certified acidity regulator and preservative, occupies a core position and plays an indispensable role. In the field of daily chemicals, it drives the continuous innovation and development of the high-end skin care market with its dual effects of keratin remodeling and anti-oxidation. In medical scenarios, citric acid is constantly expanding the new boundaries of treatment with its unique functions of metabolic regulation and material modification.

[0003] In view of the huge economic potential and broad application prospects of citric acid in many fields, genetic improvement and process optimization of production strains have become the core focus of current scientific research. Aspergillus niger, as the main force of industrial citric acid production for a century, the study of its citric acid biosynthesis mechanism is particularly important and critical. 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 this series of reactions are completed in the mitochondria. Acetyl-CoA and oxaloacetate are derived from the metabolic conversion of pyruvate and malate in the cytoplasm, respectively. In addition, the mitochondrial citrate / malate antiporter plays a bridging role, responsible for the precise transport of citrate and malate between the cytoplasm and mitochondria. Although two citrate synthases, CitA and CitB, have been found 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 citrate synthases that have not yet been discovered. Related studies have reported that some methylcitrate synthases (Mcs) may have partial or similar functions of citrate synthase.

[0004] Based on this, we hypothesize that the mcs1 and mcs2 genes encoding Mcs in Aspergillus niger may play a certain role and influence in its citric acid biosynthesis process. The present invention systematically identified the functions of the mcs1 and mcs2 genes in Aspergillus niger through gene knockout and complementation technology, aiming to clarify the specific role of Mcs in citric acid biosynthesis and provide a solid scientific basis and theoretical support for the development of more efficient and economical citric acid production strategies. This research result is not only expected to promote the innovation of citric acid production technology, but also to lay a solid foundation for the sustainable development of the citric acid industry. Summary of the invention

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

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

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

[0008] The use 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 and used for improving the biosynthesis efficiency of citric acid, the amino acid sequence of the polypeptide is shown in SEQ ID No.2.

[0010] An expression cassette comprising the mcs2 gene described above and used for improving the biosynthesis efficiency of citric acid, wherein the expression cassette comprises a promoter, the mcs2 gene and a terminator.

[0011] A vector comprising the mcs2 gene as described above for improving the biosynthesis efficiency of citric acid, wherein the vector is selected from a plasmid, a viral vector or an artificial chromosome.

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

[0013] A recombinant Aspergillus niger strain comprising the mcs2 gene described above and capable of improving citric acid production in Aspergillus niger.

[0014] The method for constructing the recombinant strain as described above comprises the following steps:

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

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

[0017] The method for producing citric acid by fermentation using the recombinant strain as described above comprises the following steps:

[0018] The recombinant strain stored at ultra-low temperature was inoculated into PDA solid medium containing 200 μg / mL hygromycin B and incubated at 28 °C for 4-5 days. After the spores matured, they were scraped and suspended in sterile saline containing 0.05% Tween-80 by volume. The spore concentration was adjusted to 1×10 ^8 CFU / mL; then, the fermentation medium was inoculated with 1×10 ^8 Spores were cultured at 28°C and 200 r / min shaking for 5 days;

[0019] 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.

[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 biosynthetic pathway of citric acid. The present invention not only provides new gene targets and transformation strategies for the biosynthesis of citric acid, but also opens up a new path for the biotechnological improvement of citric acid production, which is expected to significantly improve production efficiency and economic benefits.

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

[0023] 3. The present invention successfully identified the mcs2 gene, which is expected to be widely used in the genetic improvement of industrial Aspergillus niger strains, thereby significantly improving the production efficiency of citric acid. According to statistics, China's annual citric acid production is as high as about 3 million tons, with a market size of about 50 billion yuan. However, current industry data show that traditional strain improvement technology encounters a bottleneck in metabolic flux regulation. The application of the mcs2 gene provides a scalable solution for building a new generation of citric acid microbial cell factories, which is expected to bring breakthrough progress to citric acid production, not only to increase production, but also to effectively reduce production costs, thereby creating significant economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 This is the 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 The enzyme digestion verification maps of plasmids pLH2175 and pLH2177 in the present invention (the enzymes are KpnⅠ and Hind Ⅲ);

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

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

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

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

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

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

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

[0036] Fig.13The electrophoresis patterns of PCR verification after electrotransformation of plasmids pLH2176 and pLH2178 into Agrobacterium tumefaciens AGL-1 in the present invention; wherein, the electrophoresis of groups a and b correspond to the amplification verification of homologous arms of mcs1 and mcs2, respectively, and each group contains: M: DNA Marker; N: negative control (sterile ddH2O template); P: positive control (amplification product with recombinant plasmid as 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 equal size; mcs2-left arm: the positive control shows a characteristic band of 963 bp, and the bands of transformants 1-4 are in line with expectations; mcs1-right arm: the positive control has a band of 1034 bp, and transformants 1-4 are successfully verified; mcs2-right arm: the positive control has a band of 923 bp, and transformants 1-4 are successfully verified;

[0037] Fig.14 This is a phenotype analysis diagram of the multi-resistance screening of the transformants with the mcs1 gene knocked out in the present invention; wherein, this experiment used three different solid culture media to evaluate the resistance phenotype of the recombinant transformants of Aspergillus niger; wherein, the upper row shows transformants 1 to 52, and the lower row shows transformants 53 to 98, and the specific positions are shown in the schematic diagram on the far left of the figure; the functions of each culture medium are specifically described as follows: PDA is a non-selective culture medium, which serves as a positive growth control; PDA+HYG is a PDA culture medium supplemented with 250 μg / mL hygromycin B; MM+PPT is a basic culture 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; wherein N is a hygromycin-resistant sensitive strain as a negative control, and P is a hygromycin-resistant strain as a positive control.

[0038] Fig.15 This is a phenotype analysis diagram of the multi-resistance screening of the transformants with the mcs2 gene knocked out in the present invention; wherein, this experiment used three different solid culture media to evaluate the resistance phenotype of the recombinant transformants of Aspergillus niger; wherein, the upper row shows transformants 1 to 52, and the lower row shows transformants 53 to 98, and the specific positions are shown in the schematic diagram on the far left of the figure; the functions of each culture medium are specifically described as follows: PDA is a non-selective culture medium, which serves as a positive growth control; PDA+HYG is a PDA culture medium supplemented with 250 μg / mL hygromycin B; MM+PPT is a basic culture 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; wherein N is a hygromycin-resistant sensitive strain as a negative control, and P is a hygromycin-resistant strain as a positive control.

[0039] Fig.16This is a flow chart of the principle of knocking out the mcs1 (a) and mcs2 (b) genes in the present invention;

[0040] Fig.17 Figure 1 is a PCR verification diagram of the genome of the mcs1 gene knockout transformant in the present invention; wherein, this experiment verifies the homologous recombination event through five groups of PCR systems, wherein (a) left arm PCR amplification is used to detect the original sequence on the left side of the knockout site; (b) left arm-hyg PCR amplification is used to confirm the connection between the left arm and the inserted hyg screening marker gene; (c) right arm PCR amplification is used to detect the original sequence on the right side of the knockout site; (d) right arm-hyg PCR amplification is used to verify the connection between the right arm and the hyg screening marker gene; (e) full-length PCR amplification is used to evaluate the sequence changes in the knockout region as a whole; wherein, M: DNAMarker, all markers in the figure are of the same type, and the values ​​of each band are uniformly labeled; N: negative control, PCR amplification using the S469 genome as a template; P: positive control, pLH2176; the verification principle can be seen in detail. Fig.10 According to the verification results, if the left arm PCR and right arm PCR amplification bands are both negative, and the left arm-hyg PCR and right arm-hyg PCR amplification bands are both positive, and the full-length PCR amplification band shows a difference from the expected knockout sequence, then the transformant is judged as 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] Fig.18 This is the PCR verification diagram of the genome of the mcs2 gene knockout transformant in the present invention; wherein, this experiment verifies the homologous recombination event through five groups of PCR systems, wherein (a) left arm PCR amplification is used to detect the original sequence on the left side of the knockout site; (b) left arm-hyg PCR amplification is used to confirm the connection between the left arm and the inserted hyg screening marker gene; (c) right arm PCR amplification is used to detect the original sequence on the right side of the knockout site; (d) right arm-hyg PCR amplification is used to verify the connection between the right arm and the hyg screening marker gene; (e) full-length PCR amplification is used to evaluate the sequence changes in the knockout region as a whole; wherein, M: DNAMarker, all markers in the figure are of the same type, and the values ​​of each band are uniformly labeled; N: negative control, PCR amplification using the S469 genome as a template; P: positive control, pLH2178. The verification principle can be seen in detail. Fig.10According to the verification results, if the left arm PCR and right arm PCR amplification bands are both negative, and the left arm-hyg PCR and right arm-hyg PCR amplification bands are both positive, and the full-length PCR amplification band shows a difference from the expected knockout sequence, then the transformant is judged as a positive transformant, that is, the knockout of the mcs2 gene is successfully achieved; 70, 76, 77, 78, 85, 86, 89, and 98 are all mcs2 knockout recombinant bacteria;

[0042] Fig.19 The electrophoresis diagram of PCR verification of plasmid pLH2208 / pLH2209 transforming Agrobacterium AGL-1 in the present invention; wherein, the electrophoresis of groups a and b respectively verifies the integration of mcs1 and mcs2, and each group contains: M: DNA Marker; N: negative control (sterile ddH2O template); P: positive control (amplification product with recombinant plasmid as template); 1-2: PCR product of Agrobacterium transformant colony; mcs1 verification: positive control P shows a 1429 bp characteristic band, and transformants 1 and 2 both show a target band as large as the positive control; mcs2 verification: positive control P shows a 1398 bp characteristic band, and transformants 1 and 2 successfully amplify a target band as large as the control;

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

[0044] Fig.21It is a verification diagram of the double resistance phenotype of the mcs2 gene overexpression transformant in the present invention; wherein, the leftmost schematic diagram 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; this experiment uses PDA medium and PDA medium supplemented with 250 μg / mL hygromycin B (i.e., PDA+HYG medium) to evaluate whether the transformant is positive; the left side of the figure shows the growth of the transformant on the PDA medium, while the right side shows the growth of the transformant on the PDA medium containing 250 μg / mL hygromycin B; wherein, the transformant that can grow normally on the PDA+HYG medium is the positive transformant;

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

[0046] Fig.23 The figure is a PCR verification diagram of the genome of the mcs1 gene overexpression transformant in the present invention; wherein, M: DNA Marker; N: negative control, PCR amplification is performed using the S469 genome as a template; P: positive control, pLH2208 is used as a template, and 12, 15, 21, 44, 47 respectively represent the PCR amplification results performed using the genome of the mcs1 overexpression transformant as a template; wherein 15, 21, 44, 47 are correct transformants;

[0047] Fig.24 The figure is a PCR verification diagram of the genome of the mcs2 gene overexpression transformant in the present invention; wherein, M: DNA Marker; N: negative control, PCR amplification is performed using the S469 genome as a template; P: positive control, pLH2209 is used as a template, and 8, 20, 24, 27, and 32 represent the PCR amplification results using the genome of the mcs2 overexpression transformant as a template, respectively; wherein 8, 20, 24, 27, and 32 are correct transformants;

[0048] Fig.25 The graph is a graph of the production of citric acid during the fermentation process of the recombinant strains S4036 and S4040 obtained by knocking out the mcs1 and mcs2 genes respectively in the present invention;

[0049] Fig.26 A graph showing the production of citric acid during fermentation of the recombinant strains S4127 and S4130 obtained by overexpressing the mcs1 and mcs2 genes, respectively, in the present invention. DETAILED DESCRIPTION

[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 descriptive examples and are not intended to limit the scope of protection of the present invention. Therefore, the boundaries of the present invention cannot be defined solely by these embodiments.

[0051] All experimental operations involved in the examples are conventional techniques in the art. For parts not specifically noted herein, those skilled in the art may refer to relevant commonly used reference books, scientific literature, instructions or manuals before the filing date of the present invention for specific implementation.

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

[0053] The 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 and used for improving the biosynthesis efficiency of citric acid, the amino acid sequence of the polypeptide is shown in SEQ ID No.2.

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

[0056] A vector comprising the mcs2 gene as described above for improving the biosynthesis efficiency of citric acid, wherein the vector is selected from a plasmid, a viral vector or an artificial chromosome.

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

[0058] A recombinant Aspergillus niger strain comprising the mcs2 gene described above and capable of improving citric acid production in Aspergillus niger.

[0059] The method for constructing the recombinant strain as described above comprises the following steps:

[0060] First, the overexpression plasmid pLH2209 containing the mcs2 gene was introduced into Agrobacterium AGL1 competent cells by electroporation. The electroporation conditions were: 2.5 kV, 25 μF, 200 Ω. Positive clones were screened in LB medium containing 100 μg / mL kanamycin and verified by colony PCR using primers P5420 / P5421. A specific band of 1398 bp was amplified, confirming that the plasmid was transferred into Agrobacterium. Subsequently, the verified Agrobacterium was mixed with 1×10^6 CFU / mL Aspergillus niger S469 spores were co-cultured in IM solid medium containing 100 μg / mL kanamycin and 0.2 μM acetosyringone AS at a volume ratio of 1:10 at 25°C for 60 h, and 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 to screen transformants with resistance to hygromycin B. Positive transformants were further verified for hygromycin resistance on PDA medium containing 250 μg / mL hygromycin, and genomic PCR was used to detect: a 1550 bp specific band was amplified using primers P5422 / P5423, confirming that the mcs2 expression cassette gene was successfully integrated into the genome, and the recombinant Aspergillus niger strain was obtained.

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

[0062] The method for producing citric acid by fermentation using the recombinant strain as described above comprises the following steps:

[0063] The recombinant strain stored at ultra-low temperature was inoculated into PDA solid medium containing 200 μg / mL hygromycin B and incubated at 28 °C for 4-5 days. After the spores matured, they were scraped and suspended in sterile saline containing 0.05% Tween-80 by volume. The spore concentration was adjusted to 1×10 ^8 CFU / mL; then, the fermentation medium was inoculated with 1×10 ^8 Spores were cultured at 28°C and 200 r / min shaking for 5 days;

[0064] 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 MCS protein sequence analysis

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

[0067] In order to further explore the potential associations and functional differences between these enzymes, the present invention conducted a detailed amino acid sequence comparison analysis. Based on the neighbor-joining method, a phylogenetic tree was constructed, such as Figure 1 The analysis results show that Mcs2 and CitA have a close evolutionary relationship, while Mcs1 and CitB also have a close evolutionary origin. This discovery provides valuable clues for analyzing the specific role of Mcs1 and Mcs2 in the citric acid biosynthesis 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 Aspergillus niger ATCC1015 genomic DNA as template, high-quality DNA (OD260 / 280=1.7-1.9) was extracted using the Bio-Sky Genomic DNA Mini-Extraction Kit (D0063). High-fidelity PCR amplification was performed using the specific primers listed in Table 1 and Novozyme PhantaMax Master Mix (P515): for the mcs1 gene, primers P5402 / P5403 amplified a 956 bp left arm fragment (SEQ ID NO.6), and primers P5406 / P5407 amplified a 923 bp right arm fragment (SEQ ID NO.8); for the mcs2 gene, primers P5404 / P5405 amplified a 963 bp left arm fragment (SEQ ID NO.7), and primers P5408 / P5409 amplified a 1034 bp right arm fragment (SEQ ID NO.9). PCR conditions were as follows: 95°C pre-denaturation 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. The amplified product was verified by 1% agarose gel electrophoresis and purified using the Tiangen Gel Recovery Kit (DP203) for later use.

[0071] Plasmid pLH594 (structure see Figure 2, supported by the literature: 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) was linearized by double digestion with Thermo Scientific FastDigest XbaⅠ / PstⅠ (37 ℃, 15 min), and the digestion product was purified by Tiangen DP203 kit, mixed with the mcs1 or mcs2 right arm fragment in a 3:1 molar ratio, and seamlessly cloned by Vazyme ClonExpress II Homologous Recombination Kit (C112) (50 ℃, 30 min). The ligation product was transformed into E. coli JM109 competent cells (TIANGEN CB101), and after heat shock at 42 °C for 45 seconds, ice bath for 2 minutes, and recovery culture at 37 °C for 1 hour, it was spread on LB plates containing 100 μg / mL kanamycin (cultured at 37 °C for 16 hours). Five single colonies were randomly selected for colony PCR verification (primers P5404 / P5405, P5408 / P5409), and the plasmid was extracted using the Tiangen Plasmid Extraction Kit (DP103) to obtain plasmid pLH2175 (structure see Figure 3 ) and pLH2177 (structure see Figure 4 ). Further, KpnⅠ / HindⅢ double restriction enzyme digestion confirmed that pLH2175 produced 8960 bp / 2418 bp fragments, and pLH2177 produced 7682 bp / 3807 bp fragments ( Figure 5 ), indicating that the recombinant plasmid was successfully constructed.

[0072] pLH2175 and pLH2177 were further linearized by EcoRⅠ / BamHⅠ double restriction enzyme digestion and ligated with the left arm fragments of mcs1 and mcs2 by homologous recombination, respectively, using the same method as above. The final plasmid pLH2176 (structure see Figure 6 ) was digested with PstⅠ / EcoRⅠ to produce 8925 bp / 3398 bp fragments, pLH2178 (structure see Figure 7 ) was digested with PstⅠ to produce 9552 bp / 2889 bp fragments ( Figure 8 ), agarose gel electrophoresis showed that the sizes of all bands were consistent with the theoretical values, indicating that the knockout plasmid of methylcitrate synthase had been successfully constructed.

[0073] Special note: In this experiment, the Bio-Sky Genomic DNA Mini-Extraction Kit (D0063) was used to efficiently extract the genomic DNA of Aspergillus niger. PCR amplification was performed using Phanta Max Master Mix (P515) from Novozymes. Plasmid extraction was performed using the Tiangen Plasmid Mini-Extraction Kit (DP103). DNA purification and recovery was performed using the Tiangen Plasmid Mini-Extraction Kit (DP103). Enzyme digestion of the vector and the insert fragment was performed using the commercial enzyme digestion system Thermo Scientific FastDigest fast endonuclease. The connection of the DNA fragments was achieved using the commercial homologous recombination kit Vazyme ClonExpress II One Step Cloning Kit (C112). For specific operation details, please refer to the latest version of the instructions for each reagent and kit.

[0074] Table 1 Primer sequences used in Example 1

[0075]

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

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

[0078] Next, plasmid pLH454 (structure see Fig. 9) was treated with Thermo Scientific FastDigest EcoRⅠ / BamHⅠ double enzyme digestion (37℃, 15 minutes) to linearize it. The digestion products were also purified using the Tiangen Gum Recovery 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 seamlessly cloned using the Vazyme ClonExpress II Homologous Recombination Kit (C112) (50℃, 30 minutes).

[0079] The cloned products were 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 plated on LB plates 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 Extraction Kit (DP103) to obtain pLH2208 (structure see Fig.10 ) and pLH2209 (structure see Fig.11 ). To further verify the correctness of the plasmid, pLH2208 (mcs1 overexpression plasmid) and pLH2209 (mcs2 overexpression plasmid) were double-enzyme digested. Among them, pLH2208 produced 9053 bp and 2342 bp fragments after double digestion with FastDigest EcoRⅠ / EcoRⅤ, while pLH2209 produced 10219 bp and 1155 bp fragments after single digestion with HindⅢ. Agarose gel electrophoresis results (see Fig.12 ) showed that the methylcitrate synthase overexpression plasmid was successfully constructed.

[0080] Special note: For relevant information about the plasmid pLH454 used, please refer to the literature: Development of a Cre-loxP-based genetic system in Aspergillus niger ATCC1015 and its applicationto construction of efficient organic acid-producing cell factories. ApplMicrobiol 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 was constructed according to the literature Appl Microbiol Biotechnol. 2019, 103(19):8105-8114.

[0086] 1) Electroporation of Agrobacterium and plasmid verification: Based on the literature method, the knockout plasmids pLH2176 and pLH2178 were transformed into Agrobacterium AGL1 competent cells by electroporation. The electroporation parameters were set as follows: voltage 2.5 kV, capacitance 25 μF, resistance 200 Ω, pulse time 4-5 ms (Gene Pulser Xcell system, Bio-Rad). The transformed bacterial solution was spread on LB solid medium containing 100 μg / mL kanamycin (cultured at 28 ℃ for 48 h) to obtain a single clone. In the colony PCR verification, the plasmid was used as a positive control, the original constructed primers were used, and Phanta Max Master Mix (P515) of Novozymes was used. pLH2176: Primers P5402 / P5403 amplified a 956 bp left arm fragment, and P5406 / P5407 amplified a 923 bp right arm fragment; pLH2178: Primers P5404 / P5405 amplified a 963 bp left arm fragment, and P5408 / P5409 amplified a 1034 bp right arm fragment. Agarose gel electrophoresis ( Fig.13 ) showed that the positive clone bands were exactly the same as expected, indicating that the plasmid was successfully transformed into Agrobacterium.

[0087] 2) Agrobacterium induction and co-cultivation with Aspergillus niger: The verified Agrobacterium single clone was inoculated into LB liquid medium (3 mL) containing 100 μg / mL kanamycin, and cultured at 28°C and 200 r / min for 20 h until OD 600 =0.8. Then the cells were transferred to IM induction medium (containing 100 μg / mL kanamycin and 0.2 μM acetosyringone AS) and cultured at 28 °C and 100 r / min for 5 h. At the same time, a fresh spore suspension of Aspergillus niger S469 was prepared (concentration 1×10 ^6CFU / mL), mixed with induced Agrobacterium at a volume ratio of 1:10, evenly coated on IM solid medium (containing 0.45 μm filter membrane), and co-cultured at 25 ℃ for 60 h. After the bacteria on the filter membrane surface turned yellow-green, they were transferred to 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 ℃ for 5-7 days until single colonies were formed.

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

[0089] Extract an appropriate number of positive transformants, use the kit to extract the genome, and follow the gene knockout principle flow chart ( Fig.16 ) Design verification primers, see Table 3 for details. For mcs1 knockout verification: Primers P5410 / P5411 (left arm) and P5412 / P5413 (right arm) amplify bands in the positive control, and the correct transformant should have no bands; Primers P5410 / P641 (left arm-Hyg) and P642 / P5413 (Hyg-right arm) should amplify the expected bands in the transformant; the product amplified by primers P5410 / P5413 should be 100 bp shorter than the positive control, indicating that homologous recombination is successful. For specific results, see Fig.17 , strains No. 43, 45 and 46 are transformants with correct knockout of mcs1. For mcs2 knockout verification: primers P5414 / P5415 (left arm) and P5416 / P5417 (right arm) did not amplify in the transformants; primers P5414 / P641 (left arm-Hyg) and P642 / P5417 (Hyg-right arm) amplified the target band; the length of the amplified product of primers P5414 / P5417 was consistent with the positive control, indicating the specificity of mcs2 knockout. For specific results, see Fig.18 The strains numbered 70, 76, 77, 78, 85, 86, 89, and 98 are all mcs2 knockout recombinant bacteria.

[0090] The transformants numbered 45 and 76 were named S4036 and S4040, which were mcs1 and mcs2 knockout recombinant bacteria, respectively.

[0091] Special Notes:

[0092] LB medium: trypsin: 10.0 g / L, yeast extract: 5.0 g / L, NaCl: 10.0 g / L, dissolved in deionized water, fixed to 1.0 L, pH adjusted to 7.0-7.2, solid medium plus 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 into small pieces, add distilled water and boil with continuous stirring for 30 min, collect the filtrate through double-layer gauze, add 50 g of glucose and stir until completely dissolved, make up to 2.5 L with distilled water and dispense into wide-mouth bottles, add 1.5% agar, and sterilize under high pressure at 121°C for 20 min.

[0094] The preparation method of IM solid culture medium is as follows: add water to 15 g agar to make up to 905.7 mL, sterilize at 121℃ for 20 min, add 0.8 mL of 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, and 5 mL of 20% glucose, and when the temperature cools to about 50℃, add kanamycin to a final concentration of 100 µg / mL and acetosyringone to a final concentration of 200 µM.

[0095] The preparation method of IM induction medium is as follows: add water to make up to 900.7 mL, sterilize at 121℃ for 20 min, add the sterile K buffer 0.8 mL, MN buffer 20 mL, 1% CaCl2·2H2O 1 mL, 0.01% FeSO410mL, IM Trace elements 5 mL, 20% NH4NO3 2.5 mL, 50% glycerol 10 mL, 1M MES 40 mL, 20% glucose 10 mL, add kanamycin to a final concentration of 100 µg / mL and acetosyringone to a final concentration of 200 µM when the temperature cools to about 50℃.

[0096] The preparation method of CM medium is as follows: add water to 20 g agar to make up to 897 mL, sterilize at 121℃ for 20 min, add 20 mL of sterile ASPN solution prepared in advance (the ingredients are 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 ingredients are ZnSO4·7H2O 2.1g, H3BO3 1.1g, MnCl2·4H2O 0.5 g, FeSO4·7H2O 0.5g, CoCl2·6H2O 0.17g, CuSO4·5H2O 0.16g, Na2MoO4·2H2O 0.15g, EDTA 5.1 g per 100 mL), 10 mL 10% casein hydrolysate, 50 mL 10% yeast extract, when the temperature cools to about 50℃, add hygromycin to make the final concentration 250 µg / mL, add streptomycin to make the final concentration 100 µg / mL, add ceftriaxone to make the final concentration 100 µg / mL, and add ampicillin to make the final concentration 100 µg / mL.

[0097] Among them, more specific information of the above-mentioned 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 document "Establishment of a genetic transformation system of Aspergillus niger using amdS as a screening marker". China Agricultural Science and Technology Herald, 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 electroporated into Agrobacterium competent AGL-1, and single clones were grown and PCR colony verification was performed. The verification primers P5418 / P5419 were used to amplify the mcs1 gene fragment (length 1429 bp), and the primers P5420 / P5421 were used to amplify the mcs2 gene fragment (length 1398 bp). The specific results are shown in Fig.19 The positive clone bands were completely consistent with the expected ones, indicating that the plasmid was successfully transferred into Agrobacterium. According to the steps and methods in 2) of the construction of mcs1 and mcs2 gene knockout strains, the co-culture of Agrobacterium and the starting strain S469 was completed until a single colony was formed.

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

[0101] Extract an appropriate number of positive transformants, extract the genome using a kit, and follow the gene overexpression principle flow chart ( Fig. 22 ), the upstream primer P5422 was designed to be located at the 3' end of the promoter PgpdA, and the downstream primer P5423 was designed to be located at the 5' end of the terminator TtrpC. If the mcs1 or mcs2 gene is successfully overexpressed, when the transformant genomic DNA is used as a template for PCR amplification, the target bands appear when P5422 / P5423 are used as primers, and the band lengths are 1571 bp and 1550 bp respectively. Otherwise, it means that mcs is not successfully overexpressed. The specific primers are shown in Table 3. The specific verification results are shown in Fig.23 and Fig.24 , the transformants with successful overexpression were determined, and transformants 15# and 20# were named S4127 and S4130, which were the correct transformants for 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 ultra-low temperature preserved Aspergillus niger strains (including recombinant strains and wild-type controls) were inoculated into PDA solid medium containing 200 μg / mL hygromycin B and incubated at 28 °C for 4-5 days. After the spores matured, they were scraped and suspended in sterile saline containing 0.05% Tween-80 by volume. The spore concentration was adjusted to 1×10 by counting with a hemocytometer. ^8 CFU / mL. Subsequently, 1×10 CFU / mL was inoculated in a 250 mL conical flask containing 50 mL of fermentation medium (sucrose 20 g / L, yeast extract 10 g / L, KH2PO4 1 g / L, MgSO4·7H2O 1 g / L, (NH4)2SO4 4.13 g / L, solvent was water, pH adjusted to 2.5). ^8Spores were cultured at 28°C and 200 r / min for 5 days. Three biological replicates were set for each strain, and 2 mL of fermentation liquid was sampled on the 3rd day (logarithmic growth phase) and the 5th day (stationary phase).

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

[0107] Comparison of fermentation performance of genetically engineered strains: e.g. Fig.25 and 26 As shown, the citric acid production of the wild-type strain S469 on the 5th day was 41.5 g / L; the production 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 production of the overexpression strain S4130 (OEmcs2) was significantly increased to 58.5±1.5 g / L (an increase of 41.0%), while that of S4127 (OEmcs1) decreased to 19.3 g / L (a decrease of 53.5%).

[0108] The above results show that knocking out methylcitrate synthase significantly reduces citric acid production, indicating that both genes encoding methylcitrate synthase have the properties of citrate synthase, among which the mcs2 gene has a significant effect on citric acid synthesis, which strongly proves the indispensability of the mcs2 gene for citric acid synthesis. It is worth noting that overexpression of mcs2 significantly increases citric acid accumulation, fully demonstrating the great potential and broad prospects of genetic engineering in optimizing citric acid production performance.

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

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

[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 mcs2 gene:

[0123] AATTCCAAAGAGGGGAGATTTGGAGGAGAGGAGAGGAGAGGAGAGGCGAGGGAAGTTGAAAGGCGAGCGCAAGAGGAAAAACAAGATGGAGGCGGGGCCGATAACGAGCCTGAGGATTTTTAGGCGGCGGCCCATTGGATCGGGCGGCGATGAAATCTCTGCCTCAGGCGAGGGGCTGATAGCCTATCATCCAGGCACGCGTGGCTGGGTTGTGGCGGTGCCAATCCCTTGCTAGCCTGATGTATCAGGTGTCATTTATACCATCATTGCCTAATATATCAGGTGTCAGTTTAGTCAGTTTCTGCCTGAAACGCGGTAATATAATGCCTGAGGCTGCCATAACGCCGCCGTTGCCCGATTGGGCTTACCCCGCACGGGGGCTCCACCTCGCCGGACCAAAAATGGAGTCAAGCATCCACTGCCAAATGGGAGGATCAGCTTCCCTTTCTTCCCTCTGCTGTTCCGAACATCCATCGTCCATGGACTTGTGAAGGGAATGCATGGTACCAAAGCACTTCTCTGCTCTTGCGGTTGGATCTTCCTCATCCCCGATGTCGCCTCACCGCATTGTACGATCGATCGACCCCTCACAGGAGTCCCCCGAGGCCCGGCGTGACGGGATCTGCAGCCAAATGGATCTAATTACACCATGATGATAACTAAAAGTGCAGCGCGCGCTCTACTGCACACTACCGATTTAGTCCGACGGCCGGCTCGGCCTCGCATCGGCGGTGAGGATGTGCACGACAGAGGGTGGATTGGACGCTGACTGCTTATGGCTGATTGTGTTGACCGTCCGCATGCAGCCGTGCCTTGCCTCGGCTCCTCAACACCGCGGCCAACCGAACGGCCACTCGCTCTCCTTCCGGGTGAATTTCTCCTGACTCTGCTTTGTACTCTGCTCCGTGTACATAATTCCTACTCTCTCTCGTCTTTGTTTGCACATTCAGCCTTCCCGAACACTCATCGGATC

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

[0125] CTAGAGGATAGGGGATAGGGATGGGATTGTACAGAATGGGTCATCATAGGAGTCTATTGTTTTCGGGTCTGGGTCTTGGTTACTTCATAGTTTGCGCTATACTCATTAGTGGTTAATACTTTTTCAACAGACTGCTCCTTTTCTTCTTAAATACACTGGCAGTTGGTGCATTTTTATTTTGGAACAATACATGCAGTGATAATCATCATATTGACTGTCGATTAGTGGTACCTGAAAGAACAATACTATCGATGTAAGTAGTTTCTATGTCTGAGGTAGCTGCTTAATATCCATATTTGGCAATTATAAGATGATACATATGAATTATATTCAGAATCATAGTAGACTGGCAGTAGCCGATAATCAATGTTTGCAGCAGCAGACATCCCAAGAGCACTGGTAGTGATATAAAGTAAAATAAACTTTGCAAAGCCAAGTCCACAGGACTCTCTCTGTAGTCACTCTTGCGTTGCTTCGTAGGGGAAGAAGCTTCATAGTCTCAATGAGTAGTCTTCATGCCCTATAGGGCTATACAGGAAATCTCTCTTTAGATCATCTTCTGCTTCGTCAACTTTAATTGTTTAACCAAAAACGCCTAACCCACCTCTCCACGAACTACAACTTGCCACCAGCCTCTTACAACCGACCTTTTGACTTTAGTTTCAATATATATTTCCATAACTAAGCAACATATAGACATCAGAATAATGTAGACCATCGGTATATGCCTGCTGGGCAGAAACCACCGCCAGAATCCATCCCATTTCTTTTGTTTCCAACTCCACATAACCCAAAAATATGCCTGTAGTCTTAAAATTTAAGTCCTAAGCTCGTCGATAGTGGTTCTGCATAGGTAGCCTTATCTCGTATAATGGTAAACTTCACTAGTTCCAATCGGGCGGAAGTGATCGACACGCCGGAAACAAGCCTGCA

[0126] SEQ ID NO.9 mcs2 gene right arm nucleotide sequence:

[0127]

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

[0129]

[0130] 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 mcs2 gene related to citric acid biosynthesis, characterized in that: The nucleotide sequence of the mcs2 gene is shown in SEQ ID No.

1.

2. Use of the mcs2 gene as claimed in claim 1 in improving the biosynthesis efficiency of citric acid.

3. The Mcs2 active polypeptide encoded by the mcs2 gene for improving the biosynthesis efficiency of citric acid as claimed in claim 1, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID No.

2.

4. An expression cassette for improving the biosynthesis efficiency of citric acid comprising the mcs2 gene according to claim 1, characterized in that: The expression cassette comprises a promoter, the mcs2 gene and a terminator.

5. A vector for improving the biosynthesis efficiency of citric acid comprising the mcs2 gene as claimed in claim 1, characterized in that: The vector is selected from a plasmid, a viral vector or an artificial chromosome.

6. A method for improving citric acid production by Aspergillus niger using the mcs2 gene as claimed in claim 1, characterized in that: The method comprises introducing the mcs2 gene, an expression cassette comprising the mcs2 gene or a vector comprising the mcs2 gene into Aspergillus niger.

7. A recombinant strain of Aspergillus niger comprising the mcs2 gene as claimed in claim 1 for improving citric acid production in Aspergillus niger.

8. The method for constructing a recombinant strain according to claim 7, characterized in that: The steps include: First, the overexpression plasmid pLH2209 containing the mcs2 gene was introduced into Agrobacterium AGL1 competent cells by electroporation. The electroporation conditions were: 2.5 kV, 25 μF, 200 Ω. Positive clones were screened in LB medium containing 100 μg / mL kanamycin and verified by colony PCR using primers P5420 / P5421. A specific band of 1398 bp was amplified, confirming that the plasmid was transferred into Agrobacterium. The verified Agrobacterium was then mixed with 1×10 ^6 CFU / mL Aspergillus niger S469 spores were co-cultured in IM solid medium containing 100 μg / mL kanamycin and 0.2 μM acetosyringone AS at a volume ratio of 1:10 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 to screen resistant transformants; positive transformants were further verified for hygromycin resistance on PDA medium containing 250 μg / mL hygromycin, and genomic PCR was performed: a 1550 bp specific band was amplified using primers P5422 / P5423, confirming that the mcs2 expression cassette gene was successfully integrated into the genome, and the recombinant Aspergillus niger strain was obtained.

9. Use of the recombinant strain as claimed in claim 7 in citric acid fermentation production.

10. A method for producing citric acid by fermentation using the recombinant strain according to claim 7, characterized in that: The steps include: The recombinant strain stored at ultra-low temperature was inoculated into PDA solid medium containing 200 μg / mL hygromycin B and incubated at 28 °C for 4-5 days. After the spores matured, they were scraped and suspended in sterile saline containing 0.05% Tween-80 by volume. The spore concentration was adjusted to 1×10 ^8 CFU / mL; then, the fermentation medium was inoculated with 1×10 ^8 Spores were cultured at 28°C and 200 r / min shaking for 5 days; 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.

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