Strains with improved organic acid production and construction method and application thereof

Abstract

The present invention discloses an organic acid producing strain with weakened orotidine-5-phosphate decarboxylase (PyrG). The organic acid yield of the strain is significantly improved. Furthermore, histone methyltransferase (LaeA) and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) in the strain can also be enhanced. The present invention also discloses a method for constructing the strain, and a method for producing organic acids, especially citric acid, using the strain.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering and genetic engineering; in particular, the invention relates to a strain with improved organic acid production, a construction method and an application thereof. Background Art

[0002] Aspergillus niger is the main production strain of organic acids such as citric acid. The selection and breeding of production strains is the core of the fermentation industry. The fermentation performance of the strain directly determines the success or failure of the fermentation. Taking citric acid as an example, citric acid is the organic acid with the largest production and sales volume in the world, and has been widely used in different fields such as food, medicine, and cosmetics. At present, most industrial production strains of organic acids are obtained through mutagenesis screening, and there are often coexistence of harmful mutations and beneficial mutations, resulting in the increase of yield and conversion rate of the strains, but often accompanied by problems such as slowed growth and longer fermentation cycle.

[0003] With the publication of Aspergillus niger genome data and the development of efficient genome editing tools, genome editing is used to rationally transform Aspergillus niger and effectively improve the comprehensive fermentation performance of the strain. In the rational design and transformation of strains, the selection of transformation targets is the key. In the existing technology, the transformation targets are mostly concentrated on genes related to the organic acid metabolic pathway, such as citrate synthase. Although the production of organic acids can be improved, the target selection is relatively limited.

[0004] Therefore, there is an urgent need in the art to develop a method for rationally modifying organic acid-producing strains, thereby significantly increasing the yield of organic acids. Summary of the invention

[0005] The object of the present invention is to provide a strain with improved organic acid production and application thereof in organic acid production.

[0006] Another object of the present invention is to provide a method for transforming organic acid producing strains.

[0007] In a first aspect, the present invention provides a method for constructing an organic acid-producing strain, the method comprising the step of attenuating orotidine-5-phosphate decarboxylase (PyrG) in the strain.

[0008] In a preferred embodiment, the organic acid is citric acid, succinic acid, malic acid or fumaric acid; preferably citric acid.

[0009] In a preferred embodiment, the orotidine-5-phosphate decarboxylase (PyrG) in the attenuated strain means that the orotidine-5-phosphate decarboxylase cannot function normally in the cells of the strain, or its activity in the cells is reduced or even completely inactivated compared with the orotidine-5-phosphate decarboxylase activity in the natural state.

[0010] In a preferred embodiment, attenuation of orotidine-5-phosphate decarboxylase (PyrG) in the attenuated strain can be achieved by partially or completely knocking out the gene encoding the enzyme, inactivating or partially inactivating the gene by mutation, changing the gene promoter or translation regulatory region to weaken its transcription or translation, changing the gene sequence to weaken its mRNA stability or destabilize the enzyme structure, regulating the gene by sRNA, and the like or a combination thereof.

[0011] In a preferred embodiment, a gene expression control system is inserted upstream of the coding sequence of the orotidine 5-phosphate decarboxylase gene, and the amount of effector added is controlled to weaken the orotidine 5-phosphate decarboxylase.

[0012] In a preferred embodiment, the gene expression regulation system includes but is not limited to Tet-on gene expression regulation system, RNA interference gene expression system, and small RNA regulation system.

[0013] In a further preferred embodiment, the attenuation of the orotidine-5-phosphate decarboxylase (PyrG) is compared with the activity of the wild-type orotidine-5-phosphate decarboxylase (PyrG), and the transcription and expression of the encoding gene of the orotidine-5-phosphate decarboxylase (PyrG) in the strain is reduced by at least 20%, preferably at least 30%, more preferably at least 40%, for example at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 530%, at least 540%, at least 550%, The activity of the orotidine-5-phosphate decarboxylase (PyrG) is reduced by at least 96%, at least 97%, at least 98% or at least 99%, or the activity of the orotidine-5-phosphate decarboxylase (PyrG) is reduced by at least 20%, preferably at least 30%, more preferably at least 40%, for example at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%; or the gene encoding the orotidine-5-phosphate decarboxylase (PyrG) is removed.

[0014] In a specific embodiment, the strain is a fungus; preferably, the strain is selected from Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Penicillium chrysogenum, Trichoderma reesei, Ustilago maydis or Myceliophthora thermophila; preferably Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei; more preferably Aspergillus niger.

[0015] In a specific embodiment, the method further comprises enhancing histone methyltransferase (LaeA) and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) in the strain.

[0016] In a preferred embodiment, the enhancement of histone methyltransferase (LaeA) and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) in the strain means that the intracellular activity is increased compared with the activity of the protein in the natural state.

[0017] In a preferred embodiment, methods for enhancing histone methyltransferase (LaeA) and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) in a strain include but are not limited to: increasing the copy number of a polynucleotide encoding a protein, modifying the regulatory sequence of a gene encoding a protein, replacing the regulatory sequence of a gene encoding a protein on a chromosome with a sequence having strong activity, replacing a gene encoding a protein with a mutant gene to increase the activity of the protein, and introducing modifications into a gene encoding a protein on a chromosome to enhance the activity of the protein.

[0018] In a preferred embodiment, the histone methyltransferase (LaeA) and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) gene is enhanced by inserting a gene expression regulation system upstream of the coding sequence of the histone methyltransferase (LaeA) and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) gene, and controlling the added amount of the corresponding effector of the gene expression regulation system.

[0019] In a preferred embodiment, different gene expression regulatory systems are respectively inserted upstream of the coding sequences of genes for orotidine-5-phosphate decarboxylase, histone methyltransferase (LaeA) and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA), thereby weakening orotidine-5-phosphate decarboxylase and enhancing histone methyltransferase (LaeA) and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) by controlling the added amounts of different effectors.

[0020] In a preferred embodiment, the strain has an increase in organic acid production by at least 20%, preferably at least 30%, more preferably at least 40%, for example at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 150%, at least 200%, or at least 210% compared to the wild-type strain.

[0021] In a preferred embodiment, the method further comprises overexpressing one or more genes selected from the group consisting of:

[0022] 1) Malate transport gene c4t318;

[0023] 2) malate dehydrogenase gene mdh;

[0024] 3) phosphoenolpyruvate carboxykinase;

[0025] 4) Succinyl-CoA synthetase;

[0026] 5) cytoplasmic fumarase gene fum; and

[0027] 6) succinate / fumarate mitochondrial transporter gene sfc;

[0028] and / or

[0029] Knock out one or more genes selected from the following:

[0030] 1) Succinate dehydrogenase gene sdh;

[0031] 2) Fumarate reductase gene fr;

[0032] 3) mitochondrial malate transporter gene moc; and

[0033] 4) Mitochondrial fumarase gene mtfum.

[0034] In a second aspect, the present invention provides an organic acid producing strain, in which orotidine-5-phosphate decarboxylase (PyrG) is attenuated.

[0035] In a specific embodiment, the strain is a fungus; preferably, the strain is selected from Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Penicillium chrysogenum, Trichoderma reesei, Ustilago maydis or Myceliophthora thermophila; preferably Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei; more preferably Aspergillus niger.

[0036] In a preferred embodiment, the organic acid is citric acid, succinic acid, malic acid or fumaric acid; preferably citric acid.

[0037] In a specific embodiment, histone methyltransferase (LaeA) and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) in the strain are enhanced.

[0038] In a specific embodiment, the strain is constructed by the method described in the first aspect.

[0039] In a preferred embodiment, the strain has an increase in organic acid production by at least 20%, preferably at least 30%, more preferably at least 40%, for example at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 150%, at least 200%, or at least 210% compared to the wild-type strain.

[0040] In a third aspect, the present invention provides a use of the strain described in the second aspect or the strain constructed by the method described in the first aspect for producing organic acids.

[0041] In a specific embodiment, the strain is a fungus; preferably, the strain is selected from Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Penicillium chrysogenum, Trichoderma reesei, Ustilago maydis or Myceliophthora thermophila; preferably Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei; more preferably Aspergillus niger.

[0042] In a preferred embodiment, the organic acid is citric acid, succinic acid, malic acid or fumaric acid; preferably citric acid.

[0043] In a fourth aspect, the present invention provides a method for producing an organic acid, the method comprising:

[0044] a. Cultivating the strain described in the second aspect or the strain constructed by the method described in the first aspect; and

[0045] b. optionally separating the produced organic acid from the culture system obtained in step a.

[0046] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The genomic PCR validation results of the pyrG gene insertion-inactivation strain are shown;

[0048] Figure 2 The citric acid fermentation results of the Aspergillus niger pyrG gene insertion-inactivated strain are shown; WT is the Aspergillus niger wild-type strain D, and AnP1-AnP5 are pyrG gene insertion-inactivated strains, respectively;

[0049] Figure 3 The results of genomic PCR validation of the strain with regulated expression of the pyrG gene are shown;

[0050] Figure 4 The citric acid fermentation results of the Aspergillus niger pyrG gene expression regulated strain are shown; wherein WT is the Aspergillus niger wild-type strain D, and AnTP1 is the pyrG gene expression regulated strain;

[0051] Figure 5 The figure shows the comparison of citric acid fermentation between the Aspergillus niger pyrG single gene editing strain and the pyrG and pkaC double gene editing strain; WT is the Aspergillus niger wild-type strain D, AnP1 is the pyrG gene inactivated strain, and AnTPkP1 is the pyrG and pkaC double gene editing strain;

[0052] Figure 6 The figure shows the comparison of citric acid fermentation between single-gene edited and double-gene edited strains of Aspergillus niger; WT is the wild-type strain D of Aspergillus niger, AnP1 is the pyrG gene inactivated strain, and AnTLP1 is the double-gene edited strain of pyrG and leaA;

[0053] Figure 7 A comparison of citric acid fermentation between single-gene edited and double-gene edited strains of Aspergillus niger is shown; WT is the wild-type strain D of Aspergillus niger, AnP1 is the pyrG gene inactivated strain, and AnTCP1 is the double-gene edited strain of pyrG and cexA. DETAILED DESCRIPTION

[0054] After extensive and in-depth research, the inventor unexpectedly found that weakening the pyrG gene in the organic acid production strain can significantly promote the production of organic acids and increase the acid production level of the strain, thereby providing a new technical means for the transformation of organic acid production strains. On this basis, the present invention was completed.

[0055] definition

[0056] The terms used herein have the meanings commonly understood by those skilled in the art. For the sake of clarity, some of the terms are defined as follows.

[0057] The term "exogenous" as used herein refers to the inclusion of substances that were not originally present in a system. For example, if a coding gene that was not originally present in a strain is introduced into the strain through transformation, the gene is "exogenous" to the strain.

[0058] The term "wild type / endogenous" as used herein refers to the activity of a polypeptide in an unmodified state in a microorganism, ie, the activity in the natural state.

[0059] The "modification" mentioned in the present invention refers to any genetic manipulation performed on a wild-type strain or a parent strain, including but not limited to various molecular biological methods.

[0060] The term "enhancing the activity of a protein" as used herein refers to the modification of a protein so that the intracellular activity of the protein in a microorganism is improved compared to the activity of the protein in the natural state. It not only includes the effect higher than the original function due to the increase in the activity of the protein itself, but also can be carried out by at least one method selected from the following: increasing the copy number of the polynucleotide encoding the protein, modifying the regulatory sequence of the gene encoding the protein, replacing the regulatory sequence of the gene encoding the protein on the chromosome with a sequence having strong activity, replacing the gene encoding the protein with a mutant gene to increase the activity of the protein, introducing a modification in the gene encoding the protein on the chromosome to enhance the activity of the protein, and can also include any known method without limitation, as long as the activity of the protein can be enhanced or the activity of the introduced protein can be enhanced compared to the endogenous activity.

[0061] The term "weakened protein" in the present invention refers to a protein that is modified so that it cannot function normally in the cell, or its intracellular activity is reduced compared to the protein activity in the natural state, or even completely loses the activity. In a specific embodiment, inactivation can be achieved by partially or completely knocking out the coding gene of the enzyme, inactivating or partially inactivating the gene by mutation, changing the gene promoter or translation regulatory region to weaken its transcription or translation, changing the gene sequence to weaken its mRNA stability or enzyme structure, regulating the gene by sRNA, etc. or a combination thereof, including but not limited to the above methods.

[0062] Orotidine-5'-phosphate decarboxylase (pyrG)

[0063] In fungi, the orotidine-5'-phosphate decarboxylase gene is a key enzyme in the de novo synthesis of uracil nucleotides (UMP), which catalyzes the removal of the 5-position carboxyl group of orotidine monophosphate to generate uracil nucleoside. The pyrG gene knockout or inactivated strains cannot grow on the basic medium because they cannot synthesize uracil nucleotides from scratch; however, when uridine is added to the basic medium, the strains can synthesize uracil nucleotides through the salvage synthesis pathway and grow. Therefore, this gene is usually used as a nutritional deficiency screening marker for genetic manipulation of fungi. However, the effect of the expression of this gene on the accumulation of organic acids has not been reported.

[0064] As used herein, "orotidine-5-phosphate decarboxylase" or "PyrG" refers to an enzyme that can catalyze the removal of the 5-position carboxyl group of orotidine monophosphate to generate uridine. Specifically, the orotidine-5-phosphate decarboxylase of the present invention has an amino acid sequence of SEQ ID NO: 38, or a derivative protein or polypeptide formed by substitution, deletion or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 38 and still having the function of orotidine-5-phosphate decarboxylase.

[0065] Histone methyltransferase (Methyltransferase; laeA)

[0066] "Histone methyltransferase" or "LaeA" as used herein refers to a methyltransferase that can catalyze the methylation of lysine sites such as histone H3K4 and H3K9. Specifically, the histone methyltransferase of the present invention has the amino acid sequence of SEQ ID NO: 39, or a derivative protein or polypeptide having the function of histone methyltransferase formed by substitution, deletion or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 39.

[0067] Catalytic subunit of cAMP-dependent protein kinase (pkaC)

[0068] As used herein, "cAMP-dependent protein kinase subunit" or "PkaC" refers to a protein kinase that can catalyze the phosphorylation modification of a target protein in the cAMP / PKA signal transduction pathway. Specifically, the cAMP-dependent protein kinase subunit of the present invention has an amino acid sequence of SEQ ID NO: 40, or a derivative protein or polypeptide formed by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 40 and still having the function of a cAMP-dependent protein kinase subunit.

[0069] Citrate exporter (CexA)

[0070] As used herein, "citrate transporter" or "CexA" refers to an efflux protein that can transport intracellular citrate to the extracellular space. Specifically, the citrate efflux protein of the present invention has an amino acid sequence of SEQ ID NO: 41, or a derivative protein or polypeptide formed by substitution, deletion or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 41 and still having the function of a citrate efflux protein.

[0071] The strain of the present invention and its construction method

[0072] The present invention provides a strain with improved organic acid production capacity, which performs gene editing on gene pyrG by a gene editing method to obtain a recombinant bacterium with a pyrG gene mutation, or further performs expression regulation on Aspergillus niger gene laeA to obtain a recombinant bacterium with a pyrG gene mutation and enhanced laeA expression, or further performs expression regulation on Aspergillus niger gene pkaC to obtain a recombinant bacterium with a pyrG gene mutation and enhanced pkaC expression, or further performs expression regulation on Aspergillus niger gene cexA to obtain a recombinant bacterium with a pyrG gene mutation and enhanced cexA expression. The single / double gene-edited engineered strain obtained by gene editing has a significantly improved ability to synthesize organic acids, thereby improving the production capacity of Aspergillus niger organic acids.

[0073] The term "organic acid production strain" used herein is a meaning generally understood by those of ordinary skill in the art, i.e., a strain wherein orotidine-5-phosphate decarboxylase (PyrG) is weakened. In view of the teachings of the present invention, it will be appreciated by those skilled in the art that organic acid production strains as described herein may include any strain, as long as the orotidine-5-phosphate decarboxylase (PyrG) in the strain is weakened. The production strain may be from any of the strains such as Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Penicillium chrysogenum, Trichoderma reesei, Ustilago maydis, Myceliophthora thermophila, preferably Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei, most preferably Aspergillus niger.

[0074] Specifically, the production strain of the present invention refers to a strain capable of producing organic acids, that is, when the bacteria are cultured in culture, they can produce organic acids and accumulate organic acids, or can secrete organic acids into the culture medium, that is, they can obtain extracellular free organic acids, especially the ability to accumulate more organic acids compared with the wild-type strain or the parent strain. In order to give the strain the ability to produce organic acids, traditional breeding methods can be used, such as cultivating auxotrophic mutants, analog-resistant strains, or metabolically controlled mutants capable of producing organic acids, as well as methods for cultivating recombinant strains with improved amino acid biosynthesis-related enzyme activities, or a combination of the above methods.

[0075] In a specific embodiment, the present invention weakens orotidine-5-phosphate decarboxylase (PyrG) in order to significantly improve the ability of Aspergillus niger to produce citric acid, and can also further enhance histone methyltransferase (LaeA) and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) in the strain. In addition, those skilled in the art can also carry out relevant transformations to increase the yield of other organic acids after obtaining the enlightenment of the present invention. For example, in order to more significantly improve the malic acid synthesis ability of Aspergillus niger, the malic acid transport gene c4t318 and the Aspergillus niger malate dehydrogenase gene mdh of Aspergillus oryzae can also be overexpressed by a genetic recombination method. In order to more significantly improve the succinic acid synthesis ability of Aspergillus niger, it is also possible to overexpress Aspergillus niger phosphoenolpyruvate carboxylation kinase and succinyl-CoA synthetase by a genetic recombination method and knock out the Aspergillus niger succinate dehydrogenase gene sdh by a gene editing method. In order to more significantly improve the fumarate synthesis ability of Aspergillus niger, the cytoplasmic fumarase gene fum and the succinate / fumarate mitochondrial transporter gene sfc can be overexpressed by gene recombination methods, and the Aspergillus niger fumarate reductase gene fr, the mitochondrial malate transporter gene moc and the mitochondrial fumarase gene mtfum can be knocked out by gene editing methods.

[0076] In view of the teaching of the present invention and the conventional technical means in this area, those skilled in the art can transform organic acid production strains using various methods known in the art. For example, the copy number of the polynucleotide encoding the protein can be increased, the regulatory sequence of the gene encoding the protein can be modified, the regulatory sequence of the gene encoding the protein on the chromosome can be replaced with a sequence with strong activity, the gene encoding the protein can be replaced with a mutant gene to increase the activity of the protein, and the modification can be introduced into the gene encoding the protein on the chromosome to enhance the activity of the protein. It is also possible to weaken the protein by partially or completely knocking out the coding gene of the enzyme, inactivating or partially inactivating the gene mutation, changing the gene promoter or translation control region to weaken its transcription or translation, changing the gene sequence to weaken its mRNA stability or the enzyme structure is unstable, and regulating the gene by sRNA.

[0077] In a specific embodiment, a gene expression control system is inserted upstream of the coding sequence of orotidine-5-phosphate decarboxylase, cAMP-dependent protein kinase subunit (PkaC), histone methyltransferase (LaeA), and citrate transporter (CexA) by using the CRISPR / Cas9 method, so that orotidine-5-phosphate decarboxylase can be weakened by controlling the amount of effector added, or the cAMP-dependent protein kinase subunit (PkaC), histone methyltransferase (LaeA), and citrate transporter (CexA) can be enhanced. The gene expression control system includes but is not limited to a Tet-on gene expression control system, an RNA interference gene expression system, and a small RNA control system.

[0078] Those skilled in the art know that the Tet-on gene expression regulation system achieves regulation of the target gene expression level by changing the conformation of the regulatory protein after the effector (such as doxycycline) binds to the regulatory protein, wherein the effector includes but is not limited to doxycycline, tetracycline, etc.; the RNA interference gene expression system uses double-stranded RNA (dsRNA) to induce specific degradation of the mRNA of the target gene, thereby achieving regulation of the target gene expression level; the small RNA regulation system refers to the use of smaller RNA such as antisense RNA or ribosomal switch to regulate the mRNA stability or translation rate of the target gene, thereby achieving regulation of the target gene expression level.

[0079] In a preferred embodiment, different gene expression control systems can be inserted upstream of the coding sequences of orotidine-5-phosphate decarboxylase, histone methyltransferase (LaeA) and / or cAMP-dependent protein kinase subunit (PkaC) genes, respectively, thereby weakening orotidine-5-phosphate decarboxylase by controlling the added amount of different effectors, while enhancing histone methyltransferase (LaeA) and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA).

[0080] Compared with the wild-type strain, the organic acid production of the strain of the present invention is significantly improved; for example, it is increased by at least 20%, preferably at least 30%, more preferably at least 40%, for example at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 150%, at least 200%, at least 210%.

[0081] The strain of the present invention can be used to produce organic acids, including but not limited to citric acid, succinic acid, malic acid or fumaric acid; preferably citric acid.

[0082] Based on the strain of the present invention, the present invention also provides a method for producing an organic acid, comprising:

[0083] a. Cultivating the strain of the present invention or the strain constructed by the method of the present invention; and

[0084] b. optionally separating the produced organic acid from the culture system obtained in step a.

[0085] Advantages of the present invention:

[0086] 1. The present invention provides a new idea for transforming organic acid-producing strains;

[0087] 2. The organic acid production strain of the present invention significantly increases the organic acid yield;

[0088] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0089] The reagents and raw materials used in the present invention are commercially available.

[0090] Example

[0091] Example 1. Construction of pyrG gene inactivated strain of Aspergillus niger and citric acid fermentation test

[0092] 1) Target sequence selection

[0093] According to the principle of genome localization of CRISPR / Cas9 system, it is necessary to find the target site containing PAM (NGG) in the genome. The present invention selects the following site of the PyrG encoding gene as the target site for the construction of the inactivated strain of Aspergillus niger pyrG gene (ID: 56726). The specific sequence is as follows:

[0094] sgRNA-pyrG1:GAAGACCAATGTGACTGTCT(SEQ ID NO:1)

[0095] 2) Construction of sgRNA expression cassette

[0096] The present invention uses Aspergillus niger's own 5S rRNA (recognizable by RNA polymerase III) as a promoter, and adds HDV as a ribozyme to complete the self-processing of sgRNA transcription, thereby constructing an sgRNA expression cassette. The construction of the target sequence sgRNA-pyrG1 uses the target sequence double-stranded synthesis, annealing and phosphorylation, and directly connects to the sgRNA expression cassette cloning vector p5S-sgRNA treated with enzyme cutting and dephosphorylation to obtain the sgRNA expression plasmid p5S-sgRNA-pyrG1.

[0097] sgRNA-pyrG1-F: caccGAAGACCAATGTGACTGTCT (SEQ ID NO: 2)

[0098] sgRNA-pyrG1-R: aaacAGACAGTCACATTGGTCTTC (SEQ ID NO: 3)

[0099] 3) CRISPR / Cas9 system for protoplast transformation of Aspergillus niger

[0100] The present invention adopts the method of co-transforming the Cas9 protein expression vector pCas9, the sgRNA expression cassette fragment sgRNA-pyrG1 and the donor DNA fragment MHi-pyrG1-hyh into the protoplasmic cells of Aspergillus niger D (purchased from the strain resource library of Shanghai Industrial Microbiology Technology Co., Ltd. (formerly Shanghai Institute of Industrial Microbiology), with public deposit number M202) to construct an Aspergillus niger pyrG insertion-inactivated strain.

[0101] (1) Preparation of sgRNA expression cassette fragment sgRNA-pyrG1

[0102] The sgRNA-pyrG1 fragment was obtained by PCR using 5S-Fm and sgRNA-Rm as primers and the sgRNA expression plasmid p5S-sgRNA-pyrG1 as a template.

[0103] 5S-F:GGTTGGAGATTCCAGACTCAG(SEQ ID NO:4)

[0104] sgRNA-R:AAAAAAGCACCGACTCGGTGCCAC(SEQ ID NO:5)

[0105] The PCR reaction system was 10 μL of 5×FastPfu buffer, 1 μL of 10 mM dNTPs, 2.5 μL of upstream / downstream primers, 0.5 μL of DNA template, 1.5 μL of FastPfu (TransGene), and 32 μL of ultrapure water.

[0106] The PCR reaction conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 sec, 55°C annealing for 30 sec, 72°C extension for 45 sec, 35 cycles; and finally 72°C extension for 10 min. The PCR product was purified and then subjected to PEG-mediated protoplast transformation.

[0107] (2) Preparation of donor DNA fragment MHi-pyrG1-hyh

[0108] In the donor DNA fragment MHi-pyrG1-hyh sequence containing the ultra-short homology arm pyrG gene, the upstream and downstream homology arms are located immediately upstream and downstream of the target sequence sgRNA-pyrG1, respectively. In order to avoid being recognized and cut again by Cas9 after homologous recombination of the targeting fragment, the PAM sequence is not included in the design of the downstream homology arm. The construction of the donor DNA fragment MHi-pyrG1-hyh was completed by a one-step PCR method, directly using MHi-pyrG1-Fm and MHi-pyrG1-Rm as primers and pSilent-1 as a template for PCR amplification to obtain the donor DNA fragment MHi-pyrG1-hyh.

[0109] MHi-pyrG1-Fm:ttcgagattgccgaggccaagaagaccaatgtgactgtctGACGTTAACTGATATTGAAGGAGC (SEQ ID NO: 6)

[0110] MHi-pyrG1-Rm:tcagcaagatctagtagctccttagtggtggtaacgtcagAACCCAGGGGCTGGTGACGG(SEQ ID NO:7)

[0111] The PCR reaction system was 10 μL of 5×FastPfu buffer, 1 μL of 10 mM dNTPs, 2.5 μL of upstream / downstream primers, 0.5 μL of DNA template, 1.5 μL of FastPfu (TransGene), and 32 μL of ultrapure water.

[0112] The PCR reaction conditions were as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 65°C annealing for 30 s, 72°C extension for 1 min, 10 cycles, with the annealing temperature decreasing by 1°C in each cycle; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 25 cycles; and finally 72°C extension for 10 min. The PCR product was purified and then subjected to PEG-mediated protoplast transformation.

[0113] (3) PEG-mediated protoplast transformation

[0114] Take wild-type D spores of Aspergillus niger and suspend them in 200 mL CMA liquid medium (Dextrose 20 g / L, DifcoBrand Malt Extract 20 g / L, Bacto Peptone 1 g / L). The final concentration of the spore suspension is 10 5 / mL, culture at 30℃, 200r / min for 12-16h. Collect the cells with sterile Micro-cloth filter cloth under sterile conditions, rinse once with solution A (K2HPO4 5mM, KH2PO4 5mM, MgSO4 96.312g / L, pH 5.8, filter sterilize), transfer it to 20mL lysis solution (0.4g lysis enzyme in 20mL solution A) with a sterile cotton swab, and lyse for about two hours at 37℃, 75r / min. Filter the above lysis solution with sterile Micro-cloth filter cloth, collect the protoplasts with two 50mL sterile centrifuge tubes, and rinse with solution B (Tris-HCl 10mM, CaCl2 5.54g / L, D-Sorbitol 218.64g / L, pH 7.5, filter sterilize) to make each tube about 25mL. Centrifuge at 2000r / min for 5min, discard the supernatant, and resuspend the precipitate twice with 20mL solution B. Resuspend the precipitate in 10mL solution B, combine the two tubes into one tube, and count the protoplasts with a hemocytometer. Centrifuge and add an appropriate amount of solution B to resuspend once according to the counting results. Add 100μL of protoplast suspension, 2μg pCas9, 2μg sgRNA-pyrG1 and 2μgMHi-pyrG1-hyh to a pre-cooled 15mL centrifuge tube on ice, add 1mL solution C (Tris-HCl 10mM, CaCl2 5.54g / L, PEG600050% (w / v), pH 7.5, filtered and sterilized) to it, ice bath for 10min, 2mL solution B and mix. Mix with the preheated upper culture medium MMSH containing hygromycin, and then spread it on the lower culture medium MMSH plate. The plates were incubated at 30°C for 3-5 days until transformants emerged.

[0115] 4) Verification of the pyrG gene inactivated strain of Aspergillus niger

[0116] The genomic DNA of the transformants after secondary subculture and purification was extracted using Tiangen's new plant genome extraction kit DP350. Then, the transformed cells were verified by PCR using primers pyrG1-gF and pyrG1-gR using the extracted genomic DNA as a template.

[0117] The primer sequences are as follows:

[0118] pyrG1-gF:CATGTGCAGCAGGGAATACGAG (SEQ ID NO:8)

[0119] pyrG1-gR:GTTTCCGCTTCCGTATCCGTTG (SEQ ID NO:9)

[0120] The PCR reaction system was: 10 μL of 2×Taq Buffer, 1 μL of upstream / downstream primers, 1 μL of DNA template, and 7 μL of deionized water.

[0121] The PCR reaction conditions were: 94°C for 5 min, 94°C for 30 sec, 55°C for 30 sec, 72°C for 2 min, 30 cycles, and 72°C for 10 min. The PCR amplification products were subjected to 1% agarose gel electrophoresis (150 V, 20 min).

[0122] Cas9 cleaved the pyrG gene at a specific site under the mediation of sgRNA-pyrG1 to produce a DNA double-strand break, and then used the donor DNA fragment MHi-pyrG-hyh as a repair template to accurately repair the site-specific DNA double-strand break through homologous recombination, resulting in an insertion-inactivation mutant of the pyrG gene. Figure 1 As shown, using pyrG1-gF and pyrG1-gR as primers, the DNA fragment amplified in the wild-type strain was 1345 bp, while the pyrG gene inactivated strain after CRISPR / Cas9 editing amplified a target fragment containing donor DNA with a size of 3240 bp, indicating that the detected transformants were all positive pyrG gene insertion inactivation strains, and the obtained positive mutant strains were named AnP1-AnP5 strains.

[0123] 5) Organic acid fermentation of Aspergillus niger pyrG gene inactivated strain

[0124] The pyrG gene-inactivated strain and wild-type strain D were inoculated on PDA medium at 30°C for 5 days, and then spores were collected with 0.9% saline and counted using a hemocytometer. 6 / mL inoculation amount was inoculated into citric acid fermentation medium (corn starch medium, total sugar content 12%), 34 ℃, 250r / min culture for 96h. The fermentation supernatant was collected by rapid filtration, diluted 10 times, heated and boiled for 10min, filtered with a filter membrane and then tested for citric acid content by HPLC. The specific detection conditions were: using a chromatographic column Aminex HPX-87H (300mmX7.8mmX9um, BioRad), Shimadzu UFLC high performance liquid chromatograph (equipped with Shimadzu LC-20AD infusion pump, SPD-20A UV detector, CTO-20A / AC column oven, SIL-20ACHT UFLC specification automatic sampler, Shimadzu LCsution workstation), mobile phase A was ultrapure water, mobile phase B was 2.75mM H2SO4, flow rate was 0.6mL / min, injection volume was 10uL, column temperature was 50℃, and ultraviolet detection wavelength was 210nm. The results showed that the present invention can significantly promote the accumulation of citric acid after using the CRISPR / Cas9 system to perform insertion inactivation editing on Aspergillus niger pyrG. Figure 2 As shown, when fermented to 96h, the citric acid production of the pyrG gene insertion-inactivated strains AnP1 to AnP5 reached 32.8, 33.6, 36.9, 34.5 and 32.7 g / L, respectively, which were 93.0%, 97.9%, 117.5%, 103.1% and 92.4% higher than the citric acid production (17.0 g / L) of the wild strain D. This example shows that the insertion-inactivation editing of pyrG in Aspergillus niger can significantly enhance the production of citric acid.

[0125] Example 2. Construction of Aspergillus niger pyrG gene expression regulation strain and citric acid fermentation test

[0126] 1) Target sequence selection

[0127] According to the principle of genome localization of the CRISPR / Cas9 system, it is necessary to find a target site containing PAM (NGG) in the genome. The inventors selected the following site of the pyrG gene as the target site for the construction of the Aspergillus niger pyrG gene expression regulation strain. The specific sequence is as follows:

[0128] sgRNA-pyrG2:GAGTAGTTCGAAGTTTCGAC (SEQ ID NO:10)

[0129] 2) Construction of sgRNA expression cassette

[0130] The present invention uses Aspergillus niger's own 5S rRNA (recognizable by RNA polymerase III) as a promoter, and adds HDV as a ribozyme to complete the self-processing of sgRNA transcription, thereby constructing an sgRNA expression cassette. The construction of the target sequence sgRNA-pyrG2 uses the target sequence double-stranded synthesis, annealing and phosphorylation, and directly connects to the sgRNA expression cassette cloning vector p5S-sgRNA treated with enzyme cutting and dephosphorylation to obtain the sgRNA expression plasmid p5S-sgRNA-pyrG2.

[0131] sgRNA-pyrG2-F: caccGAGTAGTTCGAAGTTTCGAC (SEQ ID NO: 11)

[0132] sgRNA-pyrG2-R:aaacGTCGAAACTTCGAACTACTC (SEQ ID NO:12)

[0133] 3) CRISPR / Cas9 system for protoplast transformation of Aspergillus niger

[0134] The present invention adopts the method of co-transforming a Cas9 protein expression vector pCas9, an sgRNA expression box fragment sgRNA-pyrG2 and a donor DNA fragment MHi-pyrG2-Tet-on:hyh into the protoplasmic cells of Aspergillus niger D to construct an Aspergillus niger pyrG gene expression regulation strain.

[0135] (1) Preparation of sgRNA expression cassette fragment sgRNA-pyrG2

[0136] The sgRNA-pyrG2 fragment was obtained by PCR using 5S-Fm and sgRNA-Rm as primers and the sgRNA expression plasmid p5S-sgRNA-pyrG2 as a template.

[0137] 5S-F:GGTTGGAGATTCCAGACTCAG(SEQ ID NO:4)

[0138] sgRNA-R:AAAAAAGCACCGACTCGGTGCCAC(SEQ ID NO:5)

[0139] The reaction system and reaction conditions of PCR are as described in Example 1. PCR products were purified and then subjected to PEG-mediated protoplast transformation.

[0140] (2) Preparation of donor DNA fragment MHi-pyrG2-Tet-on:hyh

[0141] In the donor DNA fragment MHi-pyrG2-Tet-on:hyh sequence containing the ultra-short homology arm pyrG gene, the upstream homology arm is located immediately upstream of the target sequence sgRNA-pyrG2, and the downstream homology arm is a 40bp coding sequence including ATG. The construction of the donor DNA fragment MHi-pyrG2-Tet-on:hyh was completed by a one-step PCR method, and PCR amplification was performed directly using MHi-pyrG2-Fm and MHi-pyrG2-Rm as primers and pTC1.13 as a template to obtain the donor DNA fragment MHi-pyrG2-Tet-on:hyh.

[0142] MHi-pyrG2-Fm:gtatccgcgcacgtctctggatttacgaatcagggtccaGACGTTAACTGATATTGAAG(SEQ ID NO:13)

[0143] MHi-pyrG2-Rm:ggcacgggcagtgtaggtcaatcgcgacttggaggacatGGTGTTTAAAACGGTGATGTC (SEQ ID NO: 14)

[0144] The reaction system and reaction conditions of PCR were as described in Example 1. The PCR product was purified and then subjected to PEG-mediated protoplast transformation.

[0145] (3) PEG-mediated protoplast transformation

[0146] As in the operation method of Example 1, the Cas9 protein expression plasmid pCas9, the sgRNA expression cassette fragment sgRNA-pyrG2 and the donor DNA fragment MHi-pyrG2-Tet-on:hyh were mixed in equal proportions and co-transformed into the protoplast cells of the wild-type strain D of Aspergillus niger. Cas9 performed a site-specific cut downstream of the promoter of the pyrG gene under the mediation of sgRNA-pyrG2 to produce a DNA double-strand break, and then the donor DNA fragment MHi-pyrG2-Tet-on:hyh was used as a repair template to accurately repair the site-specific DNA double-strand break through homologous recombination, thereby inserting the Tet-on gene expression regulation system into the upstream of the coding sequence of the pyrG gene, thereby obtaining a mutant strain that can use the effector doxycycline to strictly regulate the expression level of pyrG.

[0147] 4) Verification of the Aspergillus niger pyrG gene expression regulation strain

[0148] The genomic DNA of the transformants after secondary subculture and purification was extracted using Tiangen's new plant genome extraction kit DP350. Then, the transformed cells were verified by PCR using primers pyrG1-gF and pyrG1-gR using the extracted genomic DNA as a template.

[0149] The primer sequences are as follows:

[0150] PyrG2-gF:GTCGGAGCCGGAGCAATCCAC (SEQ ID NO: 15)

[0151] pyrG2-gR:AGCTTATCTCCCTTGGAAGA (SEQ ID NO:16)

[0152] The reaction system and reaction conditions of PCR were as described in Example 1. The PCR amplification products were subjected to 1% agarose gel electrophoresis (150 V voltage, 20 minutes).

[0153] like Figure 3 As shown, using pyrG2-gF and pyrG2-gR as primers, the DNA fragment amplified in the wild-type strain was 1004 bp, while the pyrG gene expression regulation strain edited by CRISPR / Cas9 amplified the target fragment containing the donor DNA, with a size of 5424 bp, indicating that the strains tested were all positive pyrG gene expression regulation strains, and the obtained positive mutant strains were named AnTP1-AnTP6 strains.

[0154] 5) Organic acid fermentation of Aspergillus niger pyrG gene expression regulated strain

[0155] The pyrG gene expression regulated strain AnTP1 and the wild-type strain D were inoculated on PDA medium and cultured at 30°C for 5 days, and then the spores were collected with 0.9% saline and counted using a hemocytometer. 6 / mL inoculated into citric acid fermentation medium containing different effectors of doxycycline, and cultured at 34°C, 250r / min for 96h. The fermentation supernatant was collected by rapid filtration, diluted 10 times, heated and boiled for 10min, filtered through a filter membrane, and then the citric acid content was detected by HPLC. The specific detection conditions are as shown in Example 1.

[0156] It was found that the present invention utilizes the CRISPR / Cas9 system to replace the promoter insertion of the pyrG gene of Aspergillus niger with the Tet-on gene expression regulation promoter system. Due to the rigor of this system, the amount of effector doxycycline added is positively correlated with gene expression. Through the correlation analysis between the addition of effectors and the expression level of the pyrG gene, it was found that the less effectors were added, the higher the weakening level of the pyrG gene. In the doxycycline concentration ranges of 10μg / ml to 5μg / ml, 5μg / ml to 2μg / ml, 2μg / ml to 1μg / ml and 1μg / ml to 0μg / ml, the weakening level of the pyrG gene of the pyrG gene expression regulation strain AnTP1 reached 10-50%, 50-70%, 70-80%, 80-90% and 90-100%, respectively. Figure 4 As shown, when the fermentation reached 96h, the citric acid production gradually increased with the decrease of the effector doxycycline. At doxycycline concentrations of 5, 2, 1 and 0 μg / ml, the citric acid production reached 21.2, 23.5, 27.5 and 32.8 g / L, respectively, which was 20.7%, 26.9%, 55.8% and 88.3% higher than the citric acid production of wild strain D (about 17.0 g / L). This example shows that the downregulation of the expression level of the Aspergillus niger pyrG gene helps to significantly enhance the production of citric acid.

[0157] Example 3. Construction of a double gene editing strain of Aspergillus niger pyrG gene and pkaC gene and citric acid fermentation test

[0158] 1) Target sequence selection

[0159] According to the principle of genome localization of the CRISPR / Cas9 system, it is necessary to find a target site containing PAM (NGG) in the genome. The inventors selected the following site of the gene encoding the PkaC protein shown in sequence 3 (ID: 36874) as the target site for the construction of a double gene editing strain of Aspergillus niger pkaC gene expression regulation and pyrG inactivation. The specific sequence is as follows:

[0160] sgRNA-pkaC: GCATGGAGAACACGCTGCTG (SEQ ID NO: 17)

[0161] 2) Construction of sgRNA expression cassette

[0162] The present invention uses the 5S rRNA of Aspergillus niger itself (which can be recognized by RNA polymerase III) as a promoter, and adds HDV as a ribozyme to complete the self-processing of sgRNA transcription in the middle, so as to construct an expression cassette of sgRNA. The construction of the target sequence sgRNA-pkaC adopts the double-stranded synthesis of the target sequence, annealing and phosphorylation, and directly connects to the sgRNA expression cassette cloning vector p5S-sgRNA treated with enzyme cutting and dephosphorylation to obtain the sgRNA expression plasmid p5S-sgRNA-pkaC.

[0163] sgRNA-pkaC-F: caccGCATGGAGAACACGCTGCTG (SEQ ID NO: 18)

[0164] sgRNA-pkaC-R: aaacCAGCAGCGTGTTCTCCATGC (SEQ ID NO: 19)

[0165] 3) CRISPR / Cas9 system for protoplast transformation of Aspergillus niger

[0166] The present invention adopts the method of co-transforming the Cas9 protein expression vector pCas9-pyrG, the sgRNA expression box fragment sgRNA-pkaC and the donor DNA fragment MHi-pkaC-Tet-on:DR-pyrG into the protoplasmic cells of Aspergillus niger AnP1 to construct the Aspergillus niger pkaC gene expression regulation strain.

[0167] (1) Preparation of sgRNA expression cassette fragment sgRNA-pkaC

[0168] The sgRNA-pkaC fragment was obtained by PCR using 5S-Fm and sgRNA-Rm as primers and the sgRNA expression plasmid p5S-sgRNA-pkaC as a template.

[0169] 5S-F:GGTTGGAGATTCCAGACTCAG(SEQ ID NO:4)

[0170] sgRNA-R:AAAAAAGCACCGACTCGGTGCCAC(SEQ ID NO:5)

[0171] The reaction system and reaction conditions of PCR are as described in Example 1. PCR products were purified and then subjected to PEG-mediated protoplast transformation.

[0172] (2) Preparation of donor DNA fragment MHi-pkaC-Tet-on:DR-pyrG

[0173] In the donor DNA fragment MHi-pkaC-Tet-on:DR-pyrG sequence containing the ultra-short homology arm pkaC gene, the upstream homology arm is located immediately upstream of the target sequence sgRNA-pkaC, and the downstream homology arm is a 40bp coding sequence including ATG. The construction of the donor DNA fragment MHi-pkaC-Tet-on:DR-pyrG was completed by a one-step PCR method, and PCR amplification was performed directly using MHi-pkaC-Fm and MHi-pkaC-Rm as primers and pTet-on:DR-pyrG as a template to obtain the donor DNA fragment MHi-pkaC-Tet-on:DR-pyrG.

[0174] MHi-pyrG2-Fm:atacccatcgtgggctgctctatcattttaattttactgcGACGTTAACTGATATTGAAG(SEQ ID NO:20)

[0175] MHi-pyrG2-Rm:cgttcgccgtttcttcagcaaacctcctaaactaggcatGGTGTTTAAAACGGTGATGTC (SEQ ID NO: 21)

[0176] The reaction system and reaction conditions of PCR were as described in Example 1. The PCR product was purified and then subjected to PEG-mediated protoplast transformation.

[0177] (3) PEG-mediated protoplast transformation

[0178] As in the operation method of Example 1, the Cas9 protein expression plasmid pCas9, the sgRNA expression cassette fragment sgRNA-pkaC and the donor DNA fragment MHi-pkaC-Tet-on:DR-pyrG were mixed in equal proportions and co-transformed into the protoplasmic cells of Aspergillus niger AnP1. Cas9 performed a site-specific cut downstream of the promoter of the pkaC gene under the mediation of sgRNA-pkaC, resulting in a DNA double-strand break, and then the donor DNA fragment MHi-pkaC-Tet-on:DR-pyrG was used as a repair template to accurately repair the site-specific DNA double-strand break through homologous recombination, thereby inserting the Tet-on gene expression regulation system into the upstream of the coding sequence of the pkaC gene, thereby obtaining a mutant strain that can use the effector doxycycline to strictly regulate the expression level of pkaC.

[0179] 4) Verification of the pkaC gene expression regulation strain of Aspergillus niger

[0180] The genomic DNA of the transformants after secondary subculture and purification was extracted using Tiangen's new plant genome extraction kit DP350. Then, the transformed cells were verified by PCR using primers pkaC-gF and pkaC-gR, using the extracted genomic DNA as a template.

[0181] The primer sequences are as follows:

[0182] pkaC-gF:TTTCCGTTGACCGCCGTTC (SEQ ID NO:22)

[0183] pkaC-gR:GCTCTACGTCAAAGGTAGCCA (SEQ ID NO:23)

[0184] The reaction system and reaction conditions of PCR are as described in Example 1. The PCR amplification products were subjected to 1% agarose gel electrophoresis (150V voltage, 20 minutes). The results showed that with pkaC-gF and pkaC-gR as primers, the DNA fragment amplified in the wild-type strain was 1004bp, while the pkaC gene expression regulation strain edited by CRISPR / Cas9 amplified the target fragment containing the donor DNA, with a size of 5424bp, which indicated that the detected transformants were all positive pkaC gene expression regulation strains, and the obtained positive mutant strains were named AnTPk1 strains. In order to further reverse screen the spores of AnTPk1 strain on a basic medium containing 5-fluoroorotic acid and uracil on the basis of the AnTPk1 strain, the 200bp directional repeat sequence carried by the upstream and downstream of the pyrG gene was used to self-recombine and further obtain the pyrG inactivated strain, and the double gene mutant strain of pkaC gene expression regulation and pyrG inactivation was named AnTPkP1.

[0185] 5) Organic acid fermentation of Aspergillus niger strain with double mutations of pyrG gene and pkaC gene

[0186] The pkaC gene and pyrG gene double mutant strain AnTPkP1 and the pyrG inactivated strain AnP1 were inoculated on PDA medium at 30°C for 5 days, and then the spores were collected with 0.9% saline and counted using a hemocytometer. 6 / mL inoculated into citric acid fermentation medium containing 20μg / mL effector doxycycline, and cultured at 34°C, 250r / min for 96h. The fermentation supernatant was collected by rapid filtration, diluted 10 times, heated and boiled for 10min, filtered through a filter membrane, and then the citric acid content was detected by HPLC. The specific detection conditions are as shown in Example 1.

[0187] The results showed that after the inactivation of the pyrG gene of Aspergillus niger and the regulation of pkaC gene expression were edited simultaneously in the present invention, when the pyrG gene was inactivated and pkaC was overexpressed, the production of citric acid could be significantly promoted. When the fermentation lasted for 96 hours, the citric acid production of the double gene mutant strain AnTPkP1 was 38.9 g / L, which was 123.2% and 31.1% higher than that of the wild-type strain D and the pyrG gene inactivated strain AnP1, respectively (Fig. Figure 5 ). This example illustrates that the simultaneous editing of the inactivation of the pyrG gene of Aspergillus niger and the regulation of the expression of the pkaC gene helps to significantly enhance the production of citric acid.

[0188] Example 4. Construction of a double gene editing strain of Aspergillus niger pyrG gene and laeA gene and citric acid fermentation test

[0189] 1) Target sequence selection

[0190] According to the principle of genome localization of the CRISPR / Cas9 system, it is necessary to find a target site containing PAM (NGG) in the genome. The inventors selected the following site of the gene encoding the LaeA protein (ID: 170198) as the target site for the construction of a double gene editing strain for Aspergillus niger laeA gene expression regulation and pyrG inactivation.

[0191] The specific sequence is as follows:

[0192] sgRNA-laeA:GAGTAGTTCGAAGTTTCGAC (SEQ ID NO: 24)

[0193] 2) Construction of sgRNA expression cassette

[0194] The present invention uses Aspergillus niger's own 5S rRNA (recognizable by RNA polymerase III) as a promoter, and adds HDV as a ribozyme to complete the self-processing of sgRNA transcription, thereby constructing an sgRNA expression cassette. The construction of the target sequence sgRNA-laeA uses the target sequence double-stranded synthesis, annealing and phosphorylation, and directly connects to the sgRNA expression cassette cloning vector p5S-sgRNA treated with enzyme cutting and dephosphorylation to obtain the sgRNA expression plasmid p5S-sgRNA-laeA.

[0195] sgRNA-laeA-F: caccAAGTCGGCTGATTTCAAACA (SEQ ID NO: 25)

[0196] sgRNA-laeA-R:aaacTGTTTGAAATCAGCCGACTT(SEQ ID NO:26)

[0197] 3) CRISPR / Cas9 system for protoplast transformation of Aspergillus niger

[0198] The present invention adopts the method of co-transforming a Cas9 protein expression vector pCas9-pyrG, an sgRNA expression cassette fragment sgRNA-laeA and a donor DNA fragment MHi-laeA-Tet-on:DR-pyrG into the protoplasmic cells of Aspergillus niger AnP1 to construct an Aspergillus niger laeA gene expression regulation strain.

[0199] (1) Preparation of sgRNA expression cassette fragment sgRNA-laeA

[0200] The sgRNA-laeA fragment was obtained by PCR using 5S-Fm and sgRNA-Rm as primers and the sgRNA expression plasmid p5S-sgRNA-laeA as a template.

[0201] 5S-F:GGTTGGAGATTCCAGACTCAG(SEQ ID NO:4)

[0202] sgRNA-R:AAAAAAGCACCGACTCGGTGCCAC(SEQ ID NO:5)

[0203] The reaction system and reaction conditions of PCR are as described in Example 1. PCR products were purified and then subjected to PEG-mediated protoplast transformation.

[0204] (2) Preparation of donor DNA fragment MHi-laeA-Tet-on:DR-pyrG

[0205] In the donor DNA fragment MHi-laeA-Tet-on:DR-pyrG sequence containing the ultra-short homology arm laeA gene, the upstream homology arm is located immediately upstream of the target sequence sgRNA-laeA, and the downstream homology arm is a 40bp coding sequence including ATG. The construction of the donor DNA fragment MHi-laeA-Tet-on:DR-pyrG was completed by a one-step PCR method, and PCR amplification was performed directly using MHi-laeA-Fm and MHi-laeA-Rm as primers and pTet-on:DR-pyrG as a template to obtain the donor DNA fragment MHi-laeA-Tet-on:DR-pyrG.

[0206] MHi-laeA-Fm:aatctcttcccagcgaaccgatccttggacttcagggttGACGTTAACTGATATTGAAG(SEQ ID NO:27)

[0207] MHi-laeA-Rm:agtaattggctgatgcaaaagtcggctgatttcaaacatGGTGTTTAAAACGGTGATGTC (SEQ ID NO: 28)

[0208] The reaction system and reaction conditions of PCR were as described in Example 1. The PCR product was purified and then subjected to PEG-mediated protoplast transformation.

[0209] (3) PEG-mediated protoplast transformation

[0210] As in the operation method of Example 1, the Cas9 protein expression plasmid pCas9, the sgRNA expression cassette fragment sgRNA-laeA and the donor DNA fragment MHi-laeA-Tet-on:DR-pyrG were mixed in equal proportions and co-transformed into the protoplasmic cells of Aspergillus niger AnP1. Cas9 performed a site-specific cut downstream of the promoter of the laeA gene under the mediation of sgRNA-laeA, resulting in a DNA double-strand break, and then the donor DNA fragment MHi-laeA-Tet-on:DR-pyrG was used as a repair template to accurately repair the site-specific DNA double-strand break through homologous recombination, thereby inserting the Tet-on gene expression regulation system into the upstream of the coding sequence of the laeA gene, thereby obtaining a mutant strain that can use the effector doxycycline to strictly regulate the expression level of laeA.

[0211] 4) Verification of the Aspergillus niger laeA gene expression regulation strain

[0212] The genomic DNA of the transformants after secondary subculture and purification was extracted using Tiangen's new plant genome extraction kit DP350. Then, the transformed cells were verified by PCR using primers laeA-gF and laeA-gR, using the extracted genomic DNA as a template.

[0213] The primer sequences are as follows:

[0214] laeA-gF:GTTGTTGCCGTCTTTTCGTC(SEQ ID NO:29)

[0215] laeA-gR:GCCTAGATTAAACGAACAACGGA(SEQ ID NO:30)

[0216] The reaction system and reaction conditions of PCR are as described in Example 1. The PCR amplification products were subjected to 1% agarose gel electrophoresis (150V voltage, 20 minutes). The results showed that with laeA-gF and laeA-gR as primers, the DNA fragment amplified in the wild-type strain was 1000bp, while the laeA gene expression regulation strain edited by CRISPR / Cas9 amplified the target fragment containing donor DNA, with a size of 5420bp, which indicated that the detected transformants were all positive laeA gene expression regulation strains, and the obtained positive mutant strains were named AnTL1 strains. In order to further reverse screen the spores of AnTL1 strain on a basic medium containing 5-fluoroorotic acid and uracil on the basis of AnTL1 strain, the 200bp directional repeat sequence carried by the upstream and downstream of the pyrG gene was used to self-recombine and further obtain the pyrG inactivated strain, and the double gene mutant strain of pyrG inactivation and laeA gene expression regulation was named AnTLP1.

[0217] 5) Organic acid fermentation of Aspergillus niger strain with double mutations of pyrG gene and laeA gene

[0218] The pyrG gene and laeA gene double mutant strain AnTLP1 and the pyrG inactivated strain AnP1 were inoculated on PDA medium at 30°C for 5 days, and then the spores were collected with 0.9% saline and counted using a hemocytometer. 6 / mL inoculated into citric acid fermentation medium containing 20μg / mL effector doxycycline, and cultured at 34°C, 250r / min for 96h. The fermentation supernatant was collected by rapid filtration, diluted 10 times, heated and boiled for 10min, filtered through a filter membrane, and then the citric acid content was detected by HPLC. The specific detection conditions are as shown in Example 1.

[0219] In the present invention, after the inactivation of the pyrG gene of Aspergillus niger and the regulation of laeA gene expression are edited simultaneously, when the pyrG gene is inactivated and laeA is overexpressed, the production of citric acid can be significantly promoted. When the fermentation is continued for 96 hours, the citric acid production of the double gene mutant strain AnTLP1 is 50.9 g / L, which is 206.1% and 62.9% higher than that of the wild-type strain D and the pyrG gene inactivated strain AnP1, respectively (e.g. Figure 6 ). This example illustrates that the simultaneous editing of the inactivation of the pyrG gene of Aspergillus niger and the regulation of the expression of the laeA gene helps to significantly enhance the production of citric acid.

[0220] Example 4. Construction of a double gene editing strain of Aspergillus niger pyrG gene and cexA gene and citric acid fermentation test

[0221] 1) Target sequence selection

[0222] According to the principle of genome localization of CRISPR / Cas9 system, it is necessary to find the target site containing PAM (NGG) in the genome. The inventors selected the following site of the gene encoding CexA protein (ID: 121799) as the target site for the construction of a double gene editing strain of Aspergillus niger with cexA gene expression regulation and pyrG inactivation. The specific sequence is as follows:

[0223] sgRNA-cexA: CATGATTGTGGATATGACTCGGG (SEQ ID NO:31)

[0224] 2) Construction of sgRNA expression cassette

[0225] The present invention uses the 5S rRNA of Aspergillus niger itself (which can be recognized by RNA polymerase III) as a promoter, and adds HDV as a ribozyme to complete the self-processing of sgRNA transcription, thereby constructing an expression cassette of sgRNA. The construction of the target sequence sgRNA-cexA uses the target sequence double-stranded synthesis, annealing and phosphorylation, and directly connects to the sgRNA expression cassette cloning vector p5S-sgRNA treated with enzyme cutting and dephosphorylation to obtain the sgRNA expression plasmid p5S-sgRNA-cexA.

[0226] sgRNA-cexA-F: caccCATGATTGTGGATATGACTC (SEQ ID NO:32)

[0227] sgRNA-cexA-R:aaacGAGTCATATCCACAATCATG (SEQ ID NO:33)

[0228] 3) CRISPR / Cas9 system for protoplast transformation of Aspergillus niger

[0229] The present invention adopts the method of co-transforming a Cas9 protein expression vector pCas9-pyrG, an sgRNA expression box fragment sgRNA-cexA and a donor DNA fragment MHi-cexA-Tet-on:DR-pyrG into the protoplasmic cells of Aspergillus niger AnP1 to construct an Aspergillus niger cexA gene expression regulation strain.

[0230] (1) Preparation of sgRNA expression cassette fragment sgRNA-cexA

[0231] The sgRNA-cexA fragment was obtained by PCR using 5S-Fm and sgRNA-Rm as primers and the sgRNA expression plasmid p5S-sgRNA-cexA as a template.

[0232] 5S-F:GGTTGGAGATTCCAGACTCAG(SEQ ID NO:4)

[0233] sgRNA-R:AAAAAAGCACCGACTCGGTGCCAC(SEQ ID NO:5)

[0234] The reaction system and reaction conditions of PCR are as described in Example 1. PCR products were purified and then subjected to PEG-mediated protoplast transformation.

[0235] (2) Preparation of donor DNA fragment MHi-cexA-Tet-on:DR-pyrG

[0236] In the donor DNA fragment MHi-cexA-Tet-on:DR-pyrG sequence containing the ultra-short homology arm cexA gene, the upstream homology arm is located immediately upstream of the target sequence sgRNA-cexA, and the downstream homology arm is a 40bp coding sequence including ATG. The construction of the donor DNA fragment MHi-cexA-Tet-on:DR-pyrG was completed by a one-step PCR method, and PCR amplification was performed directly using MHi-cexA-Fm and MHi-cexA-Rm as primers and pTet-on:DR-pyrG as a template to obtain the donor DNA fragment MHi-cexA-Tet-on:DR-pyrG.

[0237] MHi-cexA-Fm:aggtggttcacagattgccaacgtgcttcgaggctggccGACGTTAACTGATATTGAAG(SEQ ID NO:34)

[0238] MHi-cexA-Rm:ttcaaggtctgatcttgatgaagacgtggttgaagacatGGTGTTTAAAACGGTGATGTC (SEQ ID NO: 35)

[0239] The reaction system and reaction conditions of PCR were as described in Example 1. The PCR product was purified and then subjected to PEG-mediated protoplast transformation.

[0240] (3) PEG-mediated protoplast transformation

[0241] As in the operation method of Example 1, the Cas9 protein expression plasmid pCas9, the sgRNA expression cassette fragment sgRNA-cexA and the donor DNA fragment MHi-cexA-Tet-on:DR-pyrG were mixed in equal proportions and co-transformed into the protoplasmic cells of Aspergillus niger AnP1. Cas9 performed a site-specific cut downstream of the promoter of the cexA gene under the mediation of sgRNA-cexA, resulting in a DNA double-strand break, and then the donor DNA fragment MHi-cexA-Tet-on:DR-pyrG was used as a repair template to accurately repair the site-specific DNA double-strand break through homologous recombination, thereby inserting the Tet-on gene expression regulation system into the upstream of the coding sequence of the cexA gene, thereby obtaining a mutant strain that can use the effector doxycycline to strictly regulate the expression level of cexA.

[0242] 4) Verification of the Aspergillus niger cexA gene expression regulation strain

[0243] The genomic DNA of the transformants after secondary subculture and purification was extracted using Tiangen's new plant genome extraction kit DP350. Then, the transformed cells were verified by PCR using primers cexA-gF and cexA-gR using the extracted genomic DNA as a template.

[0244] The primer sequences are as follows:

[0245] cexA-gF:GCCGAAGAGAAACTCCTAAAC (SEQ ID NO:36)

[0246] cexA-gR:CGGTGGCTTCTCGGATGACT(SEQ ID NO:37)

[0247] The reaction system and reaction conditions of PCR are as described in Example 1. The PCR amplification products were subjected to 1% agarose gel electrophoresis (150V voltage, 20 minutes). The results showed that with cexA-gF and cexA-gR as primers, the DNA fragment amplified in the wild-type strain was 482bp, while the cexA gene expression regulation strain edited by CRISPR / Cas9 amplified the target fragment containing the donor DNA, with a size of 4902bp, which indicated that the detected transformants were all positive cexA gene expression regulation strains, and the obtained positive mutant strains were named AnTC1 strains. In order to further reverse screen the spores of AnTC1 strain on a basic medium containing 5-fluoroorotic acid and uracil on the basis of AnTC1 strain, the 200bp directional repeat sequence carried by the upstream and downstream of the pyrG gene was used to self-recombine and further obtain the pyrG inactivated strain, and the double gene mutant strain of pyrG inactivation and cexA gene expression regulation was named AnTCP1.

[0248] 5) Organic acid fermentation of Aspergillus niger strain with double mutations of pyrG gene and cexA gene

[0249] The pyrG gene and cexA gene double mutant strain AnTCP1 and the pyrG inactivated strain AnP1 were inoculated on PDA medium at 30°C for 5 days, and then the spores were collected with 0.9% saline and counted using a hemocytometer. 6 / mL inoculated into citric acid fermentation medium containing 20μg / mL effector doxycycline, and cultured at 34°C, 250r / min for 96h. The fermentation supernatant was collected by rapid filtration, diluted 10 times, heated and boiled for 10min, filtered through a filter membrane, and then the citric acid content was detected by HPLC. The specific detection conditions are as shown in Example 1.

[0250] In the present invention, after the inactivation of the pyrG gene of Aspergillus niger and the regulation of cexA gene expression are edited simultaneously, when the pyrG gene is inactivated and cexA is overexpressed, the production of citric acid can be significantly promoted. When the fermentation is continued for 96 hours, the citric acid production of the double gene mutant strain AnTCP1 is 48.2 g / L, which is 192.2% and 51.6% higher than that of the wild-type strain D and the pyrG gene inactivated strain AnP1, respectively (e.g. Figure 7 ). This example shows that the simultaneous editing of pyrG gene inactivation and cexA gene overexpression in Aspergillus niger helps to significantly enhance the production of citric acid.

[0251] The amino acid sequences of pyrG, LaeA, PkaC and CexA used in the present invention are as follows:

[0252] pyrG: MSSKSRLTYTARASKHPNALAKRLFEIAEAKKTNVTVSADVTTTKELLDLADRLGPYIAVIKTHIDILSDFSNETIEGLKALAQKHNFLIFEDRKFIDIGNTVQKQYHGGTLRISEWAHIINCSILPGEGIVEALAQTA SAPDFAYGPERGLLILAEMTSKGSLATGQYTTSSVDYARKYKNFVMGFVSTRALGEVQSEVSSPSDEEDFVVFTTGVNISSKGDKLGQQYQTPGSAIRGADFIIAGRGIYAAPDPVQAAQQYQKEGWEAYLARVGGKLIL(SEQ ID NO:38)

[0253] LaeA:MFEISRLLHQPITMASPNRNNYSYQGIESYDSGRSRQNSDAMDIHVITAQEPPREPPDNNDPYDGHGGPAGTSHYSKPPNRWLFYEENGRTYHGYRRGVYPLPCDEQEQDRLDIFHKLFTVARMSESLIYAPHPPNGRFLDLGCGTGIWAIDVAHKYPNAFVAGVDLAPIQPPNHPDNCEFYAPFDFEAPWTLGENSWDLIHLQMGCGSVLGWQNLYKRILRHLQPGAWFEQVEIDFEPRCDDRSLNGLALREWYQYLKQATQDTMRPIAHSSRDTIRHLEEAGFTQIDHQMVGLPLNPWHRDEHEQKVARWYNLAISESIETLSLAPFSRIFHWDLDRIRQITAEVKSQAFNKEIHAYNILHIYQARKPGGPSL(SEQ ID NO:39)

[0254] PkaC:MPSLGGLLKKRRTKDSQTLSKELEAGSAQTQTSPNAAEDHHNHNHHQHHHHLFHHHHQPQPATNSGSAANTPSQPQDSVPQQSNRSSGAEKSSDGQVASMQSAVTQASPSAHHTSGLPQPNANAASIQNIINPSQQGAMHSASSGQTQSHHAGRSDARTTKGKYSLDDFSLQRTLGTGSFGRVHLVQSKHNHRFYAVKVLKKAQVVKMKQIEHTNDERRMLNRVRHPFLITLWGTWQDSRNLYMVMDFVEGGELFSLLRKSQRFPNPVAKFYAAEVTLALEYLHTQNIIYRDLKPENLLLDRHGHLKITDFGFAKEVPDITWTLCGTPDYLAPEVVSSKGYNKSVDWWSLGILIFEMLCGFTPFWDSGSPVKIYENILRGRVKYPPYLHPDAVDLLSQLITADLTKRLGNLHGGSDDVKNHPWFAEVTWDRLARKDIDAPYVPPIRGGQGDASQYDRYPEETEQYGMAGEDPHGHLFPDF(SEQ ID NO:40)

[0255] CexA: (SEQ ID NO: 41)

[0256] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> Strains with improved organic acid production and construction method and application thereof <130> P2019-2325 <160> 41 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <400> 1 gaagaccaat gtgactgtct 20 <210> 2 <211> twenty four <212> DNA <213> Artificial sequence <400> 2 caccgaagac caatgtgact gtct 24 <210> 3 <211> twenty four <212> DNA <213> Artificial sequence <400> 3 aaacagacag tcacattggt cttc 24 <210> 4 <211> twenty one <212> DNA <213> Artificial sequence <400> 4 ggttggagat tccagactca g 21 <210> 5 <211> twenty four <212> DNA <213> Artificial sequence <400> 5 aaaaaagcac cgactcggtg ccac 24 <210> 6 <211> 64 <212> DNA <213> Artificial sequence <400> 6 ttcgagattg ccgaggccaa gaagaccaat gtgactgtct gacgttaact gatattgaag 60 gagc 64 <210> 7 <211> 60 <212> DNA <213> Artificial sequence <400> 7 tcagcaagat ctagtagctc cttagtggtg gtaacgtcag aacccagggg ctggtgacgg 60 <210> 8 <211> twenty two <212> DNA <213> Artificial sequence <400> 8 catgtgcagc agggaatacg ag 22 <210> 9 <211> twenty two <212> DNA <213> Artificial sequence <400> 9 gtttccgctt ccgtatccgt tg 22 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <400> 10 gagtagttcg aagtttcgac 20 <210> 11 <211> twenty four <212> DNA <213> Artificial sequence <400> 11 caccgagtag ttcgaagttt cgac 24 <210> 12 <211> twenty four <212> DNA <213> Artificial sequence <400> 12 aaacgtcgaa acttcgaact actc 24 <210> 13 <211> 59 <212> DNA <213> Artificial sequence <400> 13 gtatccgcgc acgtctctgg atttacgaat cagggtccag acgttaactg atattgaag 59 <210> 14 <211> 59 <212> DNA <213> Artificial sequence <400> 14 ggcacgggca gtgtaggtca atcgcgactt ggaggacatg gtgtttaaac ggtgatgtc 59 <210> 15 <211> twenty one <212> DNA <213> Artificial sequence <400> 15 gtcggaggcg gagcaatcca c 21 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <400> 16 agcttatctc ccttggaaga 20 <210> 17 <211> 20 <212> DNA <213> Artificial sequence <400> 17 gcatggagaa cacgctgctg 20 <210> 18 <211> twenty four <212> DNA <213> Artificial sequence <400> 18 caccgcatgg agaacacgct gctg 24 <210> 19 <211> twenty four <212> DNA <213> Artificial sequence <400> 19 aaaccagcag cgtgttctcc atgc 24 <210> 20 <211> 59 <212> DNA <213> Artificial sequence <400> 20 ataccatcgt gggctgctct atcattttaa ttttactgcg acgttaactg atattgaag 59 <210> twenty one <211> 59 <212> DNA <213> Artificial sequence <400> twenty one cgttcgccgtttcttcagca aacctcctaa actaggcatg gtgtttaaac ggtgatgtc 59 <210> twenty two <211> 19 <212> DNA <213> Artificial sequence <400> twenty two tttccgttga ccgccgttc 19 <210> twenty three <211> twenty one <212> DNA <213> Artificial sequence <400> twenty three gctctacgtc aaaggtagcc a 21 <210> twenty four <211> 20 <212> DNA <213> Artificial sequence <400> twenty four gagtagttcg aagtttcgac 20 <210> 25 <211> twenty four <212> DNA <213> Artificial sequence <400> 25 caccaagtcg gctgatttca aaca 24 <210> 26 <211> twenty four <212> DNA <213> Artificial sequence <400> 26 aaactgtttg aaatcagccg actt 24 <210> 27 <211> 59 <212> DNA <213> Artificial sequence <400> 27 aatctcttcc cagcgaaccg atccttggac ttcagggttg acgttaactg atattgaag 59 <210> 28 <211> 59 <212> DNA <213> Artificial sequence <400> 28 agtaattggc tgatgcaaaa gtcggctgat ttcaaacatg gtgtttaaac ggtgatgtc 59 <210> 29 <211> 20 <212> DNA <213> Artificial sequence <400> 29 gttgttgccg tcttttcgtc 20 <210> 30 <211> twenty three <212> DNA <213> Artificial sequence <400> 30 gcctagatta aacgaacaac gga 23 <210> 31 <211> twenty three <212> DNA <213> Artificial sequence <400> 31 catgattgtg gatatgactc ggg 23 <210> 32 <211> twenty four <212> DNA <213> Artificial sequence <400> 32 cacccatgat tgtggatatg actc 24 <210> 33 <211> twenty four <212> DNA <213> Artificial sequence <400> 33 aaacgagtca tatccacaat catg 24 <210> 34 <211> 59 <212> DNA <213> Artificial sequence <400> 34 aggtggttca cagattgcca acgtgcttcg aggctggccg acgttaactg atattgaag 59 <210> 35 <211> 59 <212> DNA <213> Artificial sequence <400> 35 ttcaaggtct gatcttgatg aagacgtggt tgaagacatg gtgtttaaac ggtgatgtc 59 <210> 36 <211> 21 <212> DNA <213> Artificial sequence <400> 36 gccgaagaga aactcctaaa c 21 <210> 37 <211> 20 <212> DNA <213> Artificial sequence <400> 37 cggtggcttc tcggatgact 20 <210> 38 <211> 280 <212> PRT <213> Aspergillus niger <400> 38 Met Ser Ser Lys Ser Arg Leu Thr Tyr Thr Ala Arg Ala Ser Lys His 1 5 10 15 Pro Asn Ala Leu Ala Lys Arg Leu Phe Glu Ile Ala Glu Ala Lys Lys 20 25 30 Thr Asn Val Thr Val Ser Ala Asp Val Thr Thr Thr Lys Glu Leu Leu 35 40 45 Asp Leu Ala Asp Arg Leu Gly Pro Tyr Ile Ala Val Ile Lys Thr His 50 55 60 Ile Asp Ile Leu Ser Asp Phe Ser Asn Glu Thr Ile Glu Gly Leu Lys 65 70 75 80 Ala Leu Ala Gln Lys His Asn Phe Leu Ile Phe Glu Asp Arg Lys Phe 85 90 95 Ile Asp Ile Gly Asn Thr Val Gln Lys Gln Tyr His Gly Gly Thr Leu 100 105 110 Arg Ile Ser Glu Trp Ala His Ile Ile Asn Cys Ser Ile Leu Pro Gly 115 120 125 Glu Gly Ile Val Glu Ala Leu Ala Gln Thr Ala Ser Ala Pro Asp Phe 130 135 140 Ala Tyr Gly Pro Glu Arg Gly Leu Leu Ile Leu Ala Glu Met Thr Ser 145 150 155 160 Lys Gly Ser Leu Ala Thr Gly Gln Tyr Thr Thr Ser Ser Val Asp Tyr 165 170 175 Ala Arg Lys Tyr Lys Asn Phe Val Met Gly Phe Val Ser Thr Arg Ala 180 185 190 Leu Gly Glu Val Gln Ser Glu Val Ser Ser Pro Ser Asp Glu Glu Asp 195 200 205 Phe Val Val Phe Thr Thr Gly Val Asn Ile Ser Ser Lys Gly Asp Lys 210 215 220 Leu Gly Gln Gln Tyr Gln Thr Pro Gly Ser Ala Ile Gly Arg Gly Ala 225 230 235 240 Asp Phe Ile Ile Ala Gly Arg Gly Ile Tyr Ala Ala Pro Asp Pro Val 245 250 255 Gln Ala Ala Gln Gln Tyr Gln Lys Glu Gly Trp Glu Ala Tyr Leu Ala 260 265 270 Arg Val Gly Gly Lys Leu Ile Leu 275 280 <210> 39 <211> 375 <212> PRT <213> Aspergillus niger <400> 39 Met Phe Glu Ile Ser Arg Leu Leu His Gln Pro Ile Thr Met Ala Ser 1 5 10 15 Pro Asn Arg Asn Asn Tyr Ser Tyr Gln Gly Ile Glu Ser Tyr Asp Ser 20 25 30 Gly Arg Ser Arg Gln Asn Ser Asp Ala Met Asp Ile His Val Ile Thr 35 40 45 Ala Gln Glu Pro Pro Arg Glu Pro Pro Asp Asn Asn Asp Pro Tyr Asp 50 55 60 Gly His Gly Gly Pro Ala Gly Thr Ser His Tyr Ser Lys Pro Pro Asn 65 70 75 80 Arg Trp Leu Phe Tyr Glu Glu Asn Gly Arg Thr Tyr His Gly Tyr Arg 85 90 95 Arg Gly Val Tyr Pro Leu Pro Cys Asp Glu Gln Glu Gln Asp Arg Leu 100 105 110 Asp Ile Phe His Lys Leu Phe Thr Val Ala Arg Met Ser Glu Ser Leu 115 120 125 Ile Tyr Ala Pro His Pro Pro Asn Gly Arg Phe Leu Asp Leu Gly Cys 130 135 140 Gly Thr Gly Ile Trp Ala Ile Asp Val Ala His Lys Tyr Pro Asn Ala 145 150 155 160 Phe Val Ala Gly Val Asp Leu Ala Pro Ile Gln Pro Pro Asn His Pro 165 170 175 Asp Asn Cys Glu Phe Tyr Ala Pro Phe Asp Phe Glu Ala Pro Trp Thr 180 185 190 Leu Gly Glu Asn Ser Trp Asp Leu Ile His Leu Gln Met Gly Cys Gly 195 200 205 Ser Val Leu Gly Trp Gln Asn Leu Tyr Lys Arg Ile Leu Arg His Leu 210 215 220 Gln Pro Gly Ala Trp Phe Glu Gln Val Glu Ile Asp Phe Glu Pro Arg 225 230 235 240 Cys Asp Asp Arg Ser Leu Asn Gly Leu Ala Leu Arg Glu Trp Tyr Gln 245 250 255 Tyr Leu Lys Gln Ala Thr Gln Asp Thr Met Arg Pro Ile Ala His Ser 260 265 270 Ser Arg Asp Thr Ile Arg His Leu Glu Glu Ala Gly Phe Thr Gln Ile 275 280 285 Asp His Gln Met Val Gly Leu Pro Leu Asn Pro Trp His Arg Asp Glu 290 295 300 His Glu Gln Lys Val Ala Arg Trp Tyr Asn Leu Ala Ile Ser Glu Ser 305 310 315 320 Ile Glu Thr Leu Ser Leu Ala Pro Phe Ser Arg Ile Phe His Trp Asp 325 330 335 Leu Asp Arg Ile Arg Gln Ile Thr Ala Glu Val Lys Ser Gln Ala Phe 340 345 350 Asn Lys Glu Ile His Ala Tyr Asn Ile Leu His Ile Tyr Gln Ala Arg 355 360 365 Lys Pro Gly Gly Pro Ser Leu 370 375 <210> 40 <211> 480 <212> PRT <213> Aspergillus niger <400> 40 Met Pro Ser Leu Gly Gly Leu Leu Lys Lys Arg Arg Thr Lys Asp Ser 1 5 10 15 Gln Thr Leu Ser Lys Glu Leu Glu Ala Gly Ser Ala Gln Thr Gln Thr 20 25 30 Ser Pro Asn Ala Ala Glu Asp His His Asn His Asn His His Gln His 35 40 45 His His His Leu Phe His His His His Gln Pro Gln Pro Ala Thr Asn 50 55 60 Ser Gly Ser Ala Ala Asn Thr Pro Ser Gln Pro Gln Asp Ser Val Pro 65 70 75 80 Gln Gln Ser Asn Arg Ser Ser Gly Ala Glu Lys Ser Ser Asp Gly Gln 85 90 95 Val Ala Ser Met Gln Ser Ala Val Thr Gln Ala Ser Pro Ser Ala His 100 105 110 His Thr Ser Gly Leu Pro Gln Pro Asn Ala Asn Ala Ala Ser Ile Gln 115 120 125 Asn Ile Ile Asn Pro Ser Gln Gln Gly Ala Met His Ser Ala Ser Ser 130 135 140 Gly Gln Thr Gln Ser His His Ala Gly Arg Ser Asp Ala Arg Thr Thr 145 150 155 160 Lys Gly Lys Tyr Ser Leu Asp Asp Phe Ser Leu Gln Arg Thr Leu Gly 165 170 175 Thr Gly Ser Phe Gly Arg Val His Leu Val Gln Ser Lys His Asn His 180 185 190 Arg Phe Tyr Ala Val Lys Val Leu Lys Lys Ala Gln Val Val Lys Met 195 200 205 Lys Gln Ile Glu His Thr Asn Asp Glu Arg Arg Met Leu Asn Arg Val 210 215 220 Arg His Pro Phe Leu Ile Thr Leu Trp Gly Thr Trp Gln Asp Ser Arg 225 230 235 240 Asn Leu Tyr Met Val Met Asp Phe Val Glu Gly Gly Glu Leu Phe Ser 245 250 255 Leu Leu Arg Lys Ser Gln Arg Phe Pro Asn Pro Val Ala Lys Phe Tyr 260 265 270 Ala Ala Glu Val Thr Leu Ala Leu Glu Tyr Leu His Thr Gln Asn Ile 275 280 285 Ile Tyr Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu Asp Arg His Gly 290 295 300 His Leu Lys Ile Thr Asp Phe Gly Phe Ala Lys Glu Val Pro Asp Ile 305 310 315 320 Thr Trp Thr Leu Cys Gly Thr Pro Asp Tyr Leu Ala Pro Glu Val Val 325 330 335 Ser Ser Lys Gly Tyr Asn Lys Ser Val Asp Trp Trp Ser Leu Gly Ile 340 345 350 Leu Ile Phe Glu Met Leu Cys Gly Phe Thr Pro Phe Trp Asp Ser Gly 355 360 365 Ser Pro Val Lys Ile Tyr Glu Asn Ile Leu Arg Gly Arg Val Lys Tyr 370 375 380 Pro Pro Tyr Leu His Pro Asp Ala Val Asp Leu Leu Ser Gln Leu Ile 385 390 395 400 Thr Ala Asp Leu Thr Lys Arg Leu Gly Asn Leu His Gly Gly Ser Asp 405 410 415 Asp Val Lys Asn His Pro Trp Phe Ala Glu Val Thr Trp Asp Arg Leu 420 425 430 Ala Arg Lys Asp Ile Asp Ala Pro Tyr Val Pro Pro Ile Arg Gly Gly 435 440 445 Gln Gly Asp Ala Ser Gln Tyr Asp Arg Tyr Pro Glu Glu Thr Glu Gln 450 455 460 Tyr Gly Met Ala Gly Glu Asp Pro His Gly His Leu Phe Pro Asp Phe 465 470 475 480 <210> 41 <211> 524 <212> PRT <213> Aspergillus niger <400> 41 Met Ser Ser Thr Thr Ser Ser Ser Arg Ser Asp Leu Glu Lys Val Pro 1 5 10 15 Val Pro Gln Val Ile Pro Arg Asp Ser Asp Ser Asp Lys Gly Ser Leu 20 25 30 Ser Pro Glu Pro Ser Thr Leu Glu Ala Gln Ser Ser Glu Lys Pro Pro 35 40 45 His His Ile Phe Thr Arg Ser Arg Lys Leu Gln Met Val Cys Ile Val 50 55 60 Ser Leu Ala Ala Ile Phe Ser Pro Leu Ser Ser Asn Ile Tyr Phe Pro 65 70 75 80 Ala Leu Asp Asp Val Ser Lys Ser Leu Asn Ile Ser Met Ser Leu Ala 85 90 95 Thr Leu Thr Ile Thr Val Tyr Met Ile Val Gln Gly Leu Ala Pro Ser 100 105 110 Phe Trp Gly Ser Met Ser Asp Ala Thr Gly Arg Arg Pro Val Phe Ile 115 120 125 Gly Thr Phe Ile Val Tyr Leu Val Ala Asn Ile Ala Leu Ala Glu Ser 130 135 140 Lys Asn Tyr Gly Glu Leu Met Ala Phe Arg Ala Leu Gln Ala Ala Gly 145 150 155 160 Ser Ala Ala Thr Ile Ser Ile Gly Ala Gly Val Ile Gly Asp Ile Thr 165 170 175 Asn Ser Glu Glu Arg Gly Ser Leu Val Gly Ile Phe Gly Gly Val Arg 180 185 190 Met Leu Gly Gln Gly Ile Gly Pro Val Phe Gly Gly Ile Phe Thr Gln 195 200 205 Tyr Leu Gly Tyr Arg Ser Ile Phe Trp Phe Leu Thr Ile Ala Gly Gly 210 215 220 Val Ser Leu Leu Ser Ile Leu Val Leu Leu Pro Glu Thr Leu Arg Pro 225 230 235 240 Ile Ala Gly Asn Gly Thr Val Lys Leu Asn Gly Ile His Lys Pro Phe 245 250 255 Ile Tyr Thr Ile Thr Gly Gln Thr Gly Val Val Glu Gly Ala Gln Pro 260 265 270 Glu Ala Lys Lys Thr Lys Thr Ser Trp Lys Ser Val Phe Ala Pro Leu 275 280 285 Thr Phe Leu Val Glu Lys Asp Val Phe Ile Thr Leu Phe Phe Gly Ser 290 295 300 Ile Val Tyr Thr Val Trp Ser Met Val Thr Ser Ser Thr Thr Asp Leu 305 310 315 320 Phe Ser Glu Val Tyr Gly Leu Ser Ser Leu Asp Ile Gly Leu Thr Phe 325 330 335 Leu Gly Asn Gly Phe Gly Cys Met Ser Gly Ser Tyr Leu Val Gly Tyr 340 345 350 Leu Met Asp Tyr Asn His Arg Leu Thr Glu Arg Glu Tyr Cys Glu Lys 355 360 365 His Gly Tyr Pro Ala Gly Thr Arg Val Asn Leu Lys Ser His Pro Asp 370 375 380 Phe Pro Ile Glu Val Ala Arg Met Arg Asn Thr Trp Trp Val Ile Ala 385 390 395 400 Ile Phe Ile Val Thr Val Ala Leu Tyr Gly Val Ser Leu Arg Thr His 405 410 415 Leu Ala Val Pro Ile Ile Leu Gln Tyr Phe Ile Ala Phe Cys Ser Thr 420 425 430 Gly Leu Phe Thr Ile Asn Ser Ala Leu Val Ile Asp Leu Tyr Pro Gly 435 440 445 Ala Ser Ala Ser Ala Thr Ala Val Asn Asn Leu Met Arg Cys Leu Leu 450 455 460 Gly Ala Gly Gly Val Ala Ile Val Gln Pro Ile Leu Asp Ala Leu Lys 465 470 475 480 Pro Asp Tyr Thr Phe Leu Leu Leu Ala Gly Ile Thr Leu Val Met Thr 485 490 495 Pro Leu Leu Tyr Val Glu Asp Arg Trp Gly Pro Gly Trp Arg His Ala 500 505 510 Arg Glu Arg Arg Leu Lys Ala Lys Ala Asn Gly Asn 515 520

Claims

1. A method for producing citric acid, characterized in that: The method comprises: a. Cultivating a strain constructed by attenuating orotidine-5-phosphate decarboxylase (PyrG) in a single strain; and b. optionally separating the citric acid produced from the culture system obtained in step a; The strain is Aspergillus niger.

2. The production method according to claim 1, characterized in that The orotidine 5-phosphate decarboxylase (PyrG) in the weakened strain means that the orotidine 5-phosphate decarboxylase cannot function normally in the cells of the strain, or its activity in the cells is reduced or even completely inactivated compared with the orotidine 5-phosphate decarboxylase activity in the natural state.

3. The production method according to claim 1, characterized in that The attenuation of orotidine-5-phosphate decarboxylase (PyrG) in the attenuated strain can be achieved by partially or completely knocking out the gene encoding the enzyme, inactivating or partially inactivating the gene through mutation, changing the gene promoter or translation regulatory region to weaken its transcription or translation, regulating the gene through sRNA, or a combination thereof.

4. The production method according to claim 1, characterized in that The gene expression regulatory system is inserted into the upstream of the coding sequence of the orotidine-5-phosphate decarboxylase gene, and the orotidine-5-phosphate decarboxylase is weakened by controlling the added amount of the effector.

5. The production method according to claim 4, characterized in that The gene expression regulation system includes a Tet-on gene expression regulation system or a small RNA regulation system.

6. The production method according to claim 5, characterized in that The small RNA regulatory system is an RNA interference gene expression system.

7. The production method according to claim 1, characterized in that The weakening of the orotidine 5-phosphate decarboxylase (PyrG) means that, compared with the activity of the wild-type orotidine 5-phosphate decarboxylase (PyrG), the transcription and expression of the gene encoding the orotidine 5-phosphate decarboxylase (PyrG) in the strain is reduced by at least 20%, or the activity of the orotidine 5-phosphate decarboxylase (PyrG) is reduced by at least 20%; or the gene encoding the orotidine 5-phosphate decarboxylase (PyrG) is removed.

8. The production method according to claim 7, characterized in that The weakening of the orotidine-5-phosphate decarboxylase (PyrG) means that the transcription and expression of the gene encoding the orotidine-5-phosphate decarboxylase (PyrG) in the strain is reduced by at least 30% compared with the activity of the wild-type orotidine-5-phosphate decarboxylase (PyrG), or the activity of the orotidine-5-phosphate decarboxylase (PyrG) is reduced by at least 30%.

9. The production method according to claim 8, characterized in that The weakening of the orotidine-5-phosphate decarboxylase (PyrG) means that the transcription and expression of the gene encoding the orotidine-5-phosphate decarboxylase (PyrG) in the strain is reduced by at least 40% compared with the activity of the wild-type orotidine-5-phosphate decarboxylase (PyrG), or the activity of the orotidine-5-phosphate decarboxylase (PyrG) is reduced by at least 40%.

10. The production method according to claim 9, characterized in that The weakening of the orotidine 5-phosphate decarboxylase (PyrG) means that the transcription and expression of the gene encoding the orotidine 5-phosphate decarboxylase (PyrG) in the strain is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% compared with the activity of the wild-type orotidine 5-phosphate decarboxylase (PyrG), or the activity of the orotidine 5-phosphate decarboxylase (PyrG) is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

11. The production method according to claim 1, characterized in that In the strain described in step a, orotidine-5-phosphate decarboxylase (PyrG) is weakened and cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) in the strain are enhanced.

12. The production method according to claim 11, characterized in that Enhancing the cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) in the strain means that the intracellular activity thereof is increased compared to the activity of the protein in the natural state.

13. The production method according to claim 11, characterized in that Methods for enhancing cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) in a strain include: increasing the copy number of a polynucleotide encoding the protein, modifying the regulatory sequence of a gene encoding the protein, and replacing the regulatory sequence of a gene encoding the protein on a chromosome with a sequence having strong activity.

14. The production method according to claim 11, characterized in that By inserting a gene expression regulatory system upstream of the coding sequence of the cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) gene, the cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) can be enhanced by controlling the added amount of the corresponding effector of the gene expression regulatory system.

15. The production method according to claim 11, characterized in that By inserting different gene expression regulatory systems upstream of the coding sequences of orotidine-5-phosphate decarboxylase, and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) genes, the orotidine-5-phosphate decarboxylase is weakened by controlling the added amounts of different effectors, while the cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) are enhanced.

16. The production method according to claim 1, characterized in that Compared with the wild-type strain, the strain has a citric acid production increased by at least 20%.

17. The production method according to claim 16, characterized in that Compared with the wild-type strain, the citric acid production of the strain is increased by at least 30%.

18. The production method according to claim 17, characterized in that Compared with the wild-type strain, the strain has a citric acid production increased by at least 40%.

19. The production method according to claim 18, characterized in that The strain has an increase in citric acid production by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 150%, at least 200%, or at least 210% compared to the wild-type strain.

20. Use of the strain constructed by the method described below for producing citric acid, the method comprising: Attenuating orotidine-5-phosphate decarboxylase (PyrG) in the strain alone; The strain is Aspergillus niger.

21. The use according to claim 20, characterized in that The orotidine 5-phosphate decarboxylase (PyrG) in the weakened strain means that the orotidine 5-phosphate decarboxylase cannot function normally in the cells of the strain, or its activity in the cells is reduced or even completely inactivated compared with the orotidine 5-phosphate decarboxylase activity in the natural state.

22. The use according to claim 20, characterized in that The attenuation of orotidine-5-phosphate decarboxylase (PyrG) in the attenuated strain can be achieved by partially or completely knocking out the gene encoding the enzyme, inactivating or partially inactivating the gene through mutation, changing the gene promoter or translation regulatory region to weaken its transcription or translation, regulating the gene through sRNA, or a combination thereof.

23. The use according to claim 20, characterized in that The gene expression regulatory system is inserted into the upstream of the coding sequence of the orotidine-5-phosphate decarboxylase gene, and the orotidine-5-phosphate decarboxylase is weakened by controlling the added amount of the effector.

24. The use according to claim 23, characterized in that The gene expression regulation system includes a Tet-on gene expression regulation system or a small RNA regulation system.

25. The use according to claim 24, characterized in that The small RNA regulatory system is an RNA interference gene expression system.

26. The use according to claim 20, characterized in that The weakening of the orotidine 5-phosphate decarboxylase (PyrG) means that, compared with the activity of the wild-type orotidine 5-phosphate decarboxylase (PyrG), the transcription and expression of the gene encoding the orotidine 5-phosphate decarboxylase (PyrG) in the strain is reduced by at least 20%, or the activity of the orotidine 5-phosphate decarboxylase (PyrG) is reduced by at least 20%; or the gene encoding the orotidine 5-phosphate decarboxylase (PyrG) is removed.

27. The use according to claim 26, characterized in that The weakening of the orotidine-5-phosphate decarboxylase (PyrG) means that the transcription and expression of the gene encoding the orotidine-5-phosphate decarboxylase (PyrG) in the strain is reduced by at least 30% compared with the activity of the wild-type orotidine-5-phosphate decarboxylase (PyrG), or the activity of the orotidine-5-phosphate decarboxylase (PyrG) is reduced by at least 30%.

28. The use according to claim 27, characterized in that The weakening of the orotidine-5-phosphate decarboxylase (PyrG) means that the transcription and expression of the gene encoding the orotidine-5-phosphate decarboxylase (PyrG) in the strain is reduced by at least 40% compared with the activity of the wild-type orotidine-5-phosphate decarboxylase (PyrG), or the activity of the orotidine-5-phosphate decarboxylase (PyrG) is reduced by at least 40%.

29. The use according to claim 28, characterized in that The weakening of the orotidine 5-phosphate decarboxylase (PyrG) means that the transcription and expression of the gene encoding the orotidine 5-phosphate decarboxylase (PyrG) in the strain is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% compared with the activity of the wild-type orotidine 5-phosphate decarboxylase (PyrG), or the activity of the orotidine 5-phosphate decarboxylase (PyrG) is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

30. The use according to claim 20, characterized in that In the strain described in step a, orotidine-5-phosphate decarboxylase (PyrG) is weakened and cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) in the strain are enhanced.

31. The use according to claim 30, characterized in that Enhancing the cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) in the strain means that the intracellular activity thereof is increased compared to the activity of the protein in the natural state.

32. The use according to claim 30, characterized in that Methods for enhancing cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) in a strain include: increasing the copy number of a polynucleotide encoding the protein, modifying the regulatory sequence of a gene encoding the protein, and replacing the regulatory sequence of a gene encoding the protein on a chromosome with a sequence having strong activity.

33. The use according to claim 30, characterized in that By inserting a gene expression regulatory system upstream of the coding sequence of the cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) gene, the cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) can be enhanced by controlling the added amount of the corresponding effector of the gene expression regulatory system.

34. The use according to claim 30, characterized in that By inserting different gene expression regulatory systems upstream of the coding sequences of orotidine-5-phosphate decarboxylase, and / or cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) genes, the orotidine-5-phosphate decarboxylase is weakened by controlling the added amounts of different effectors, while the cAMP-dependent protein kinase subunit (PkaC) and / or citrate transporter (CexA) are enhanced.

35. The use according to claim 20, characterized in that Compared with the wild-type strain, the strain has a citric acid production increased by at least 20%.

36. The use according to claim 35, characterized in that Compared with the wild-type strain, the citric acid production of the strain is increased by at least 30%.

37. The use according to claim 36, characterized in that Compared with the wild-type strain, the strain has a citric acid production increased by at least 40%.

38. The use according to claim 37, characterized in that The strain has an increase in citric acid production by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 150%, at least 200%, or at least 210% compared to the wild-type strain.

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