A kind of production method of citric acid and application thereof

Abstract

The invention discloses a method for increasing the citric acid yield of Aspergillus niger and its application, belonging to the fields of bioengineering and genetic engineering. At the same time, the invention also discloses a method for constructing an engineering strain and a method for producing citric acid using the strain.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering and genetic engineering, and in particular to a construction method and application of an engineering strain for producing citric acid, and a method for producing citric acid. Background Art

[0002] Citric acid is a very important organic acid, a natural preservative and food additive, widely used in industry, food and cosmetics. It is mainly produced by microbial fermentation, and its main production strains include yeast and Aspergillus niger. Aspergillus niger is the most competitive industrial strain in the citric acid industry. The annual global production of citric acid produced by Aspergillus niger reaches 2 million tons, with an output value of more than 2 billion US dollars, and it is increasing at an annual rate of 5%. 【1】 .

[0003] Although my country is a major producer and exporter of citric acid, due to the fierce competition in the citric acid industry, the current citric acid production strains are still mainly screened by mutagenesis, and the improvement of strain fermentation performance has entered a bottleneck period. With the development of genome editing technology, genetic manipulation of Aspergillus niger has been realized. The targets of modification include increasing the supply of its substrate acetyl-CoA, increasing the efflux of citric acid, increasing respiratory chain regulation, etc. 【1,2】 These genetic modifications have increased the production of citric acid in Aspergillus niger to varying degrees. However, with the growing demand for citric acid in the global market, how to discover new modification targets, construct efficient Aspergillus niger engineered strains to increase the production of citric acid, and further reduce production costs are issues that need to be addressed in this field.

[0004] References:

[0005] 【1】Zhenyu Tong, Xiaomei Zheng, Yi Tong, Yong-Cheng Shi, Jibin Sun. Systemsmetabolic engineering for citric acid production by Aspergillus niger in the post-genomic era. Microbial Cell Factories. 2019 Feb 4; 18(1):28.

[0006] 【2】Bikash Chandra Behera.Citric acid from Aspergillus niger:acomprehensive overview.Critical Reviews in Microbiology.2020;46(6):727 Summary of the invention

[0007] The present invention found in transcriptome analysis that the polypeptide with the amino acid sequence shown in SEQ ID NO: 1 is a potential protein affecting citric acid production. Further, by enhancing the activity of the polypeptide with the amino acid sequence shown in SEQ ID NO: 1, the production of citric acid was significantly promoted, and the present invention was completed on this basis.

[0008] In a first aspect, the present invention provides a use of a polypeptide in producing citric acid, characterized in that the polypeptide is selected from at least one group of the following a) to c), or a polypeptide having enhanced activity of these polypeptides:

[0009] a) having the amino acid sequence shown in SEQ ID NO: 1;

[0010] b) a derivative polypeptide formed by deleting or adding one or more amino acids at both ends of the polypeptide of the amino acid sequence shown in SEQ ID NO: 1 and having the function of the polypeptide described in a); or

[0011] c) A polypeptide having a homology higher than 90% with the polypeptide of the amino acid sequence shown in SEQ ID NO: 1 and derived from Aspergillus niger and having the same function as the polypeptide described in a).

[0012] According to the technical solution of the present invention, in c), the homology between the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 is between 90-99%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and is derived from Aspergillus niger and has the same function as the polypeptide in a).

[0013] According to the preferred technical solution of the present invention, the "enhanced polypeptide activity" refers to the modification that makes the intracellular activity of the polypeptide in the microorganism improve compared with the activity of the polypeptide protein in the natural state. It not only includes the effect higher than the original function due to the increase of the activity of the polypeptide protein itself, but also can be achieved by one or a combination of the following methods: increasing the copy number of the polynucleotide encoding the polypeptide protein, modifying the regulatory sequence of the gene encoding the polypeptide protein, replacing the regulatory sequence of the gene encoding the polypeptide protein on the chromosome with a sequence with strong activity, replacing the gene encoding the polypeptide protein with a mutant gene to increase the activity of the polypeptide protein, introducing modifications in the gene encoding the polypeptide protein on the chromosome to enhance the activity of the polypeptide protein, and can also include any inhibition method without limitation, as long as it can enhance the activity of the polypeptide protein or enhance the activity of the introduced polypeptide protein compared with the endogenous activity.

[0014] For example, the present invention provides use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 in producing citric acid.

[0015] In a preferred embodiment, in the above use, the polypeptide has enhanced activity of the polypeptide with the amino acid sequence shown in SEQ ID NO: 1.

[0016] In a second aspect, the present invention provides a method for constructing a citric acid producing strain, characterized in that the activity of a polypeptide in the strain is enhanced, wherein the polypeptide is selected from at least one of the following a) to c): a) having an amino acid sequence shown in SEQ ID NO: 1;

[0017] b) a derivative polypeptide formed by deleting or adding one or more amino acids at both ends of the polypeptide of the amino acid sequence shown in SEQ ID NO: 1 and having the function of the polypeptide described in a); or

[0018] c) A polypeptide having a homology higher than 90% with the polypeptide of the amino acid sequence shown in SEQ ID NO: 1 and derived from Aspergillus niger and having the same function as the polypeptide described in a).

[0019] According to the technical solution of the present invention, in c), the homology of the polypeptide with the amino acid sequence shown in SEQ ID NO: 1 is between 90-99%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the polypeptide derived from Aspergillus niger has the same function as the polypeptide in a). For example, the present invention provides a method for constructing a citric acid production strain, characterized in that the activity of the polypeptide shown in SEQ ID NO: 1 in the strain is enhanced.

[0020] According to the technical solution of the present invention, the activity of the polypeptide in the strain is enhanced by modifying the intracellular activity of the polypeptide in the strain in the microorganism to be improved compared with the activity of the polypeptide protein in the natural state. It not only includes the effect higher than the original function due to the increase of the activity of the polypeptide protein itself, but also can be carried out by at least one method selected from the following: preferably, by introducing an expression vector containing the polypeptide into the strain, or increasing the copy number of the polypeptide in the host strain, or by modifying its regulatory sequence to achieve the enhancement of the polypeptide activity.

[0021] Furthermore, it also includes enhancing the activity of citrate transporter CexA in the strain.

[0022] Preferably, the amino acid sequence of the citrate transporter CexA is as shown in SEQ ID NO:2.

[0023] Furthermore, the strain includes but is not limited to any one of Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Penicillium chrysogenum, Trichoderma reesei, Ustilago maydis, Myceliophthora thermophila and the like, preferably Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei, and most preferably Aspergilus niger.

[0024] In a third aspect, the present invention provides a strain for producing citric acid, characterized in that the activity of a polypeptide in the strain is enhanced, wherein the polypeptide is selected from at least one group of the following a) to c):

[0025] a) having the amino acid sequence shown in SEQ ID NO: 1;

[0026] b) a derivative polypeptide formed by deleting or adding one or more amino acids at both ends of the polypeptide of the amino acid sequence shown in SEQ ID NO: 1 and having the function of the polypeptide described in a); or

[0027] c) A polypeptide having a homology higher than 90% with the polypeptide of the amino acid sequence shown in SEQ ID NO: 1 and derived from Aspergillus niger and having the same function as the polypeptide described in a).

[0028] According to the technical solution of the present invention, in c), the homology between the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 is between 90-99%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and is derived from Aspergillus niger and has the same function as the polypeptide in a).

[0029] According to a preferred technical solution of the present invention, the activity of the polypeptide in the strain is enhanced by introducing an expression vector containing the polypeptide into the strain, or increasing the copy number of the polypeptide in the host strain, or by modifying its regulatory sequence to achieve the enhancement of the polypeptide activity.

[0030] For example, the present invention provides a citric acid producing strain, characterized in that the activity of the polypeptide shown in SEQ ID NO: 1 in the strain is enhanced;

[0031] Furthermore, it also includes enhancing the activity of citrate transporter CexA in the strain.

[0032] According to a preferred technical solution of the present invention, the amino acid sequence of the citrate transporter CexA is shown in SEQ ID NO: 2.

[0033] Furthermore, the strain includes but is not limited to any one of Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Penicillium chrysogenum, Trichoderma reesei, Ustilago maydis, Myceliophthora thermophila and the like, preferably Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei, and most preferably Aspergilus niger.

[0034] In a fourth aspect, the present invention provides a method for producing citric acid, the method comprising culturing the strain of the third aspect or the strain constructed according to the method of the second aspect, so that it produces citric acid;

[0035] Preferably, the method further comprises the step of separating citric acid from the fermentation broth.

[0036] In a fifth aspect, the present invention provides use of the strain constructed by the method described in the second aspect, the strain of the third aspect, and the production method of the fourth aspect in producing citric acid.

[0037] Beneficial effects of the present invention

[0038] The invention discloses the use of active polypeptides in the production of citric acid. The yield of citric acid is increased by enhancing the activity of the active polypeptides in the strain, so that the strain can efficiently utilize substrates to convert into products, thereby reducing the production cost of citric acid and having good industrial application potential.

[0039] The present invention also constructs an engineered strain for producing citric acid by enhancing the activity of an active polypeptide with unknown function in the strain, thus providing a new idea and transformation target for the transformation of the citric acid engineering strain. DETAILED DESCRIPTION

[0040] After extensive and in-depth research, the inventors unexpectedly found that enhancing the activity of the polypeptide of the amino acid sequence shown in SEQ ID NO: 1 in the strain can promote the production of citric acid, thus providing a new target for the transformation of citric acid production strains. On this basis, the present invention was completed.

[0041] The active polypeptides of the present invention respectively have the amino acid sequence of SEQ ID NO: 1, or the derivative polypeptides or polypeptides formed by deleting or adding one or more amino acid residues at both ends of the amino acid sequence shown in SEQ ID NO: 1, and the polypeptides derived from Aspergillus niger having the function of the polypeptides having a sequence higher than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the amino acid sequence shown in SEQ ID NO: 1. "Citrate transport polypeptide" or "CexA" used herein refers to an efflux polypeptide capable of transporting intracellular citric acid to the extracellular space. Specifically, the citric acid efflux polypeptide of the present invention has the amino acid sequence of SEQ ID NO: 2, or the derivative polypeptides or polypeptides formed by replacing, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 and still having the function of citric acid efflux polypeptides.

[0042] Definition of terms

[0043] When used in conjunction with the term "comprising" in the claims and / or the specification, the word "a" may mean "one", but may also mean "one or more", "at least one" and "one or more than one".

[0044] As used herein, "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0045] As used herein, the term "about" means that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0046] Although the disclosure supports a definition of the term "or" as only alternatives as well as "and / or," the term "or" in the claims means "and / or" unless explicitly stated as only alternatives or the alternatives are mutually exclusive.

[0047] The term optional / preferred "numerical range" used in the present invention includes both the numerical endpoints at both ends of the range and all natural numbers covered between the numerical endpoints relative to the aforementioned numerical endpoints.

[0048] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and are amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0049] The term "exogenous" used in the present invention refers to the inclusion of substances that did not originally exist in a system. For example, if a coding gene that did not originally exist in a strain is introduced into the strain through transformation, the gene is "exogenous" to the strain.

[0050] The term "wild type / endogenous" as used in the present invention refers to the activity of a polypeptide in an unmodified state in a microorganism, ie, the activity in a natural state.

[0051] The "modification / transformation" 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.

[0052] The "vector" of the present invention refers to a DNA construct containing a DNA sequence operably linked to a suitable control sequence to express the target gene in a suitable host. The vector used in the present invention is not particularly limited and may be any vector known in the art as long as it can be replicated in the host, including plasmids, viruses, phages and transposons. Possible vectors for the present invention include, but are not limited to, chromosomal, non-chromosomal and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from a combination of plasmids and phage DNA, DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40 and pseudorabies. That is, the vector includes, but is not limited to, plasmids and phages. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases be integrated into the genome itself.

[0053] The term "expression cassette" used in the present invention comprises a polynucleotide encoding a polypeptide operably linked to a suitable regulatory sequence, which is necessary for the expression of the polynucleotide in a selected cell or strain. In the invention, the transcriptional regulatory element comprises a promoter, and on this basis, may also comprise enhancers, silencers, insulators and other elements. Modifications to the regulatory sequence include, but are not limited to, deletions, insertions, conservative mutations or non-conservative mutations, or combinations thereof, in the polynucleotide sequence to introduce modifications, and may also be accomplished by replacing the original polynucleotide sequence with a polynucleotide sequence having enhanced activity.

[0054] As used in the present invention, the term "operably linked" refers to the following configuration: a regulatory sequence is placed in an appropriate position relative to a coding sequence of a polynucleotide so that the regulatory sequence directs the expression of the coding sequence. Exemplarily, the regulatory sequence can be selected from sequences encoded by a promoter and / or an enhancer.

[0055] "Sequence homology" and "identity percentage" of the present invention refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. The polynucleotides can be different at one position by, for example, containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). The polypeptides can be different at one position by, for example, containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, the identity percentage can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0056] In addition, it is not difficult for a person skilled in the art to know that changing a few amino acid residues in certain regions of a polypeptide, such as non-important regions, will not substantially change the biological activity. For example, a sequence obtained by appropriately replacing certain amino acids will not affect its activity (see Watson et al., Molecular Biology of The Gene, 4th edition, 1987, The Benjamin / Cummings Pub. Co. P224). A representative example of this type of conservative mutation is conservative substitution.

[0057] The term "conservative substitution" used in the present invention relates to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, and include having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). "Conservative substitution" typically exchanges an amino acid at one or more sites of a protein. This substitution can be conservative. Examples of substitutions considered to be conservative substitutions include substitutions of Ala for Ser or Thr, substitutions of Arg for Gln, His or Lys, substitutions of Asn for Glu, Gln, Lys, His or Asp, substitutions of Asp for Asn, Glu or Gln, substitutions of Cys for Ser or Ala, substitutions of Gln for Asn, Glu, Lys, His, Asp or Arg, substitutions of Glu for Gly, Asn, Gln, Lys or Asp, substitutions of Gly for Pro, substitutions of His for Asn, Lys, Gln, Arg or Tyr. Conservative mutations include substitutions of Ile to Leu, Met, Val or Phe, substitutions of Leu to Ile, Met, Val or Phe, substitutions of Lys to Asn, Glu, Gln, His or Arg, substitutions of Met to Ile, Leu, Val or Phe, substitutions of Phe to Trp, Tyr, Met, Ile or Leu, substitutions of Ser to Thr or Ala, substitutions of Thr to Ser or Ala, substitutions of Trp to Phe or Tyr, substitutions of Tyr to His, Phe or Trp, and substitutions of Val to Met, Ile or Leu. In addition, conservative mutations also include naturally occurring mutations caused by individual differences, strains, species differences, etc. from which genes are derived.

[0058] The term "polypeptide activity enhancement" used in the present invention refers to the modification of the intracellular activity of the polypeptide in the microorganism to be improved compared with the activity of the polypeptide protein in the natural state. It not only includes the effect higher than the original function due to the increase of the activity of the polypeptide 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 polypeptide protein, modifying the regulatory sequence of the gene encoding the polypeptide protein, replacing the regulatory sequence of the gene encoding the polypeptide protein on the chromosome with a sequence with strong activity, replacing the gene encoding the polypeptide protein with a mutant gene to increase the activity of the polypeptide protein, introducing modifications in the gene encoding the polypeptide protein on the chromosome to enhance the activity of the polypeptide protein, and can also include any inhibitory method without limitation, as long as it can enhance the activity of the polypeptide protein or enhance the activity of the introduced polypeptide protein compared with the endogenous activity.

[0059] In a specific embodiment, the activity of the active polypeptide in the enhanced strain is achieved by introducing an expression vector containing the polypeptide into the strain to achieve overexpression of the active polypeptide. "Transformation" herein has the meaning generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The method of transformation includes any method of introducing nucleic acid into a cell, including but not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method. "Citrate production strain" of the present invention has the meaning generally understood by those of ordinary skill in the art, i.e., a strain that can produce citric acid under specific culture conditions. In other words, the citric acid production strain of the present invention can be any strain, including wild-type strains and engineered strains obtained through genetic modification. For example, the citric acid producing strain of the present invention can be a eukaryotic cell, including but not limited to any one of Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Penicillium chrysogenum, Trichoderma reesei, Ustilago maydis, Myceliophthora thermophila and the like, preferably Aspergilus niger, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei, most preferably Aspergillus niger, and mutants or strains producing citric acid prepared from the above strains.

[0060] In the present invention, the culture of the strain can be carried out according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and batch fed culture, and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

[0061] 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 techniques and experimental methods used in the present embodiment are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples that do not specify specific conditions are usually carried out according to conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the embodiments, unless otherwise specified, can all be obtained through regular commercial channels.

[0062] Example 1 Enhanced expression of active polypeptides to improve citric acid fermentation level in Aspergillus niger

[0063] 1. Construction of active peptide expression plasmid

[0064] Based on the analysis of transcriptome data, a potential active polypeptide affecting citric acid production was found, and its amino acid sequence is shown in SEQ ID NO: 1. To construct the expression plasmid of the above active polypeptide, the genome of the citric acid producing fungus Aspergillus niger M202 strain (purchased from the strain resource library of Shanghai Institute of Microorganisms Technology Co., Ltd., with public accession number M202) was used as a template, and the upstream primer and downstream primer of the polypeptide were used to perform PCR amplification of the gene fragment of the polypeptide. The specific primer sequences are shown in Table 1.

[0065] Table 1 Primers for constructing active polypeptide expression plasmids

[0066]

[0067] 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.

[0068] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 2 min, 35 cycles; and final extension at 72°C for 10 min.

[0069] After the PCR product was purified, it was recombined with the plasmid pGm-hyh (Zheng et al., 2019) digested with the restriction endonuclease XholI using the non-ligase-dependent single-fragment rapid cloning kit (Cat. No.: C112-01) of Nanjing Novogene Biotech Co., Ltd. (“Novogene”), and then transformed into Escherichia coli Trans-T1. After extracting the corresponding recombinant plasmid, it was verified by enzyme digestion and sequencing to obtain the expression plasmid pOE1 of the polypeptide.

[0070] 2. Construction of strains with enhanced expression of active peptides

[0071] The protoplast suspension of Aspergillus niger M202 was prepared with reference to patent CN112250740A. 100 μL of protoplast suspension was added to a pre-cooled 15 mL centrifuge tube on ice, and then 5 μg of the expression plasmid pGm-PglA and the empty plasmid pGm of the polypeptide were added respectively, 1 mL of solution C (Tris-HCl 10 mM, CaCl2 5.54 g / L, PEG6000 50% (w / v), pH 7.5, filtered and sterilized) was added thereto and ice-bathed for 10 min, and 2 mL of solution B was mixed. After mixing with the preheated upper culture medium MMSH containing hygromycin, it was spread on the lower culture medium MMSH plate. The plate was cultured in a 30°C incubator for 3-5 days until transformants grew. The genotype of the obtained transformants was verified with reference to the method of patent CN112250740A, and finally the Aspergillus niger expression enhanced strain of the active polypeptide was obtained, which was named AnOE1.

[0072] 3. Citric acid fermentation of active peptide expression-enhanced strains

[0073] The active polypeptide expression enhanced strain AnOE1 and the control strain AnControl integrated with the pGm empty plasmid 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 The inoculum size of 1 mL was inoculated into citric acid fermentation medium (corn starch medium, total sugar content 12%) and cultured at 34°C and 250 r / min for 96 h.

[0074] The fermentation supernatant was collected by rapid filtration, diluted 10 times, heated and boiled for 10 minutes, filtered through a filter membrane, and then the citric acid content was detected by HPLC. The specific detection conditions refer to patent CN112250740A. As shown in Table 2, it was found that when the expression of the active polypeptide in Aspergillus niger in the present invention was enhanced, compared with the empty plasmid control strain, the production of citric acid can be significantly promoted, and the citric acid fermentation level is increased to 1.15-1.40 times that of the control strain.

[0075] Example 2 Active polypeptide / citric acid efflux polypeptide co-expression enhances the level of citric acid fermentation by Aspergillus niger

[0076] 1. Construction of active polypeptide / citric acid efflux polypeptide co-expression plasmid

[0077] Using pGm-F2 and pGm-R2 as primers, and the expression plasmid pOE1 of the active polypeptide obtained in Example 1 as a template, PCR was performed to amplify the expression cassette of the active polypeptide. The specific sequences of pGm-F and pGm-R are shown in Table 2.

[0078] The PCR reaction system and reaction conditions are the same as above. After the PCR product is purified, the PCR product is recombined with the overexpression plasmid pGm-CexA (refer to patent CN112250740A) of the citric acid efflux polypeptide treated by restriction endonuclease SphI, using the non-ligase-dependent single fragment rapid cloning kit (Cat. No.: C112-01) of Novozymes, and then transformed into Escherichia coli Trans-T1. After extracting the corresponding recombinant plasmid, enzyme digestion and sequencing verification were used to obtain the Aspergillus niger co-expression plasmid of the active polypeptide and the citric acid efflux polypeptide, which was named pOE2.

[0079] Table 2 Primers used for construction of active polypeptide / citric acid efflux polypeptide co-expression plasmid

[0080]

[0081] 2. Construction of high-yield engineered strains of Aspergillus niger

[0082] The protoplasts of the M202 strain were prepared by the method in Example 1, and then the protoplasts were resuspended in solution B to obtain a protoplast suspension. 100 μL of the protoplast suspension was added to a pre-cooled 15 mL centrifuge tube on ice, and then 5 μg of the co-expression plasmid pOE2 of the active polypeptide and the citric acid efflux polypeptide was added, 1 mL of solution C was added thereto for an ice bath of 10 min, and 2 mL of solution B was mixed. After mixing with the preheated upper culture medium MMSH containing hygromycin, it was then spread on the lower culture medium MMSH flat plate. The plate was cultured in a 30°C incubator for 3-5 days until transformants were grown. The transformants obtained were genotyped with reference to the method of patent CN112250740A, and finally the co-expression enhanced strain of the active polypeptide and the citric acid efflux polypeptide was obtained, and the strain was named AnOE2.

[0083] 3. Citric acid fermentation of high-yield engineered strain of Aspergillus niger

[0084] The high-yield engineered strain AnOE2 of Aspergillus niger and the control strain AnControl with the pGm empty plasmid were inoculated on PDA medium and cultured at 30°C for 5 days. Then, spores were collected with 0.9% saline and counted using a hemocytometer. 6 / mL inoculated into citric acid fermentation medium (corn starch medium, total sugar content 12%), 34 ° C, 250r / min culture for 96h. Then the HPLC method of Example 1 was used to detect the organic acid content in the fermentation supernatant. The results showed that when the Aspergillus niger active polypeptide and the citric acid efflux polypeptide were co-expressed and strengthened, the production of citric acid could be further significantly promoted compared with the control strain of the empty plasmid, and the citric acid fermentation level was increased to 2.15-2.35 times that of the control strain. SEQUENCE LISTING <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> A kind of production method of citric acid and application thereof <130> 20211014 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 266 <212> PRT <213> Aspergillus niger <400> 1 Met Ala Gly Ser Ala Pro Asn Leu Tyr Ser Phe Leu Ser Asn Asp Ala 1 5 10 15 Leu Ala Gln Gln Leu Arg Pro Tyr Val Leu Arg Cys Gln Asn Ser Ala 20 25 30 Leu Ser Arg His Ser Thr Phe Arg Val Ala Val Ser Gly Gly Ser Leu 35 40 45 Pro Lys Val Leu Ala Gln Ala Leu Leu Ala Pro Gly Asn Gly Ser Pro 50 55 60 Glu Asp Thr Ala Gln Phe Ser Lys Trp Asp Ile Phe Phe Ala Asp Glu 65 70 75 80 Arg Ala Val Pro Leu Asp His Glu Asp Ser Asn Tyr Arg Leu Leu Lys 85 90 95 Glu Glu Leu Leu Asn Lys Ile Pro Ser Glu Leu Gly Ala Pro Thr Val 100 105 110 His Thr Ile Asp Ala Asp His Val Asn Asp Glu Asp Pro Gln Glu Leu 115 120 125 Ala Asp Leu Tyr Gln Glu Glu Leu Met Arg Ser Phe Ala Ala Lys Asp 130 135 140 Ser Val Lys Leu Pro Val Phe Asp Leu Ile Leu Leu Gly Cys Gly Pro 145 150 155 160 Asp Gly His Thr Cys Ser Leu Phe Pro Gly His Glu Leu Leu Arg Glu 165 170 175 Lys Asp Ala Trp Val Ala Ala Ile Ser Asp Ser Pro Lys Pro Pro Pro 180 185 190 Lys Arg Ile Thr Leu Thr Leu Pro Val Val Thr His Ala Leu Asn Ile 195 200 205 Ala Phe Val Ala Thr Gly Gly Gly Lys Lys Asp Ile Met Lys Gln Ile 210 215 220 Phe Asp Ala Glu Glu Gly Arg Asp Leu Pro Ser Thr Leu Val Asn Gln 225 230 235 240 Gly Gly Gly Glu Lys Val Ser Trp Phe Thr Asp Asn Ala Ala Val Glu 245 250 255 Gly Val Ala Phe Pro Arg Arg Gly Ser Leu 260 265 <210> 2 <211> 524 <212> PRT <213> Aspergillus niger <400> 2 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 Leu Gln Tyr Phe Ile 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 Path Ser Path Ser Path Thr Path 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 Path 500 505 510 Arg Glu Arg Arg Leu Lys Ala Lys Ala Asn Gly Asn 515 520 <210> 3 <211> 47 <212> DNA <213> Artificial synthesis <400> 3 catccccagc atcattacac ctcgagtcac ccacgtctta tctagcc 47 <210> 4 <211> 49 <212> DNA <213> Artificial synthesis <400> 4 tattaattaa ggccggcctt aagctcgaga cgttacaggc ttccacgtc 49 <210> 5 <211> 47 <212> DNA <213> Synthetic <400> 5 acacgaacat cgacctgcag gcatgctgcc attggcggag gggtccg 47 <210> 6 <211> 48 <212> DNA <213> Synthetic <400> 6 cgacggccag tgccaagctt gcatgcggcg ggacgaacaa tccaattc 48

Claims

1. Use of a polypeptide in producing citric acid, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1; The strain producing citric acid is Aspergillus niger; The activity of the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 in Aspergillus niger is enhanced; The polypeptide activity is enhanced by at least one of the following methods: increasing the copy number of the polynucleotide encoding the polypeptide protein, modifying the regulatory sequence of the gene encoding the polypeptide protein, and replacing the regulatory sequence of the gene encoding the polypeptide protein on the chromosome with a sequence with strong activity.

2. A method for constructing a citric acid producing strain, characterized in that: The method comprises enhancing the activity of a polypeptide in a strain, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1; The citric acid producing strain is Aspergillus niger; The polypeptide activity is enhanced by at least one of the following methods: increasing the copy number of the polynucleotide encoding the polypeptide protein, modifying the regulatory sequence of the gene encoding the polypeptide protein, and replacing the regulatory sequence of the gene encoding the polypeptide protein on the chromosome with a sequence with strong activity.

3. The construction method according to claim 2, further comprising enhancing the activity of the citrate transporter CexA in the strain.

4. The construction method according to claim 3, wherein the amino acid sequence of the citrate transporter CexA is shown in SEQ ID NO:

2.

5. A strain for producing citric acid, characterized in that The activity of the polypeptide in the strain is enhanced, wherein The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1; The strain producing citric acid is Aspergillus niger; The polypeptide activity is enhanced by at least one of the following methods: increasing the copy number of the polynucleotide encoding the polypeptide protein, modifying the regulatory sequence of the gene encoding the polypeptide protein, and replacing the regulatory sequence of the gene encoding the polypeptide protein on the chromosome with a sequence with strong activity.

6. The strain according to claim 5, characterized in that The method also includes enhancing the activity of citrate transporter CexA in the strain.

7. The strain according to claim 6, characterized in that The amino acid sequence of the citrate transporter CexA is shown in SEQ ID NO:

2.

8. A method for producing citric acid, comprising culturing the strain according to any one of claims 5 to 7 or the strain constructed according to the method according to any one of claims 2 to 4 to produce citric acid.

9. The method according to claim 8, characterized in that The method further comprises the step of separating citric acid from the fermentation broth.

10. Use of the strain constructed by the method according to any one of claims 2 to 4, the strain according to any one of claims 5 to 7, and the production method according to any one of claims 8 to 9 in producing citric acid.

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