CN112250740A

CN112250740A - Glucose transport protein and application thereof in improving production of organic acid

Info

Publication number: CN112250740A
Application number: CN202011131531.6A
Authority: CN
Inventors: 郑平; 郑小梅; 孙际宾; 周文娟; 张立辉; 马延和
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2020-10-21
Filing date: 2020-10-21
Publication date: 2021-01-22
Anticipated expiration: 2040-10-21
Also published as: CN112250740B

Abstract

The invention discloses a protein with a glucose transport function in filamentous fungi and application thereof in improving the production of organic acid, belonging to the fields of bioengineering and genetic engineering. Meanwhile, the invention also discloses a construction method of the engineering strain and a method for producing organic acid, especially citric acid by using the strain.

Description

Glucose transport protein and application thereof in improving production of organic acid

Technical Field

The invention relates to the technical field of biological engineering and genetic engineering, in particular to a construction method and application of a glucose transport function protein and an organic acid production engineering strain.

Background

Aspergillus niger is an extremely important industrial strain for the organic acid and enzyme preparation industry. Aspergillus niger produces citric acid in a global annual production of 200 million tons, with a value in excess of 20 billion dollars, and at an increasing rate of 5% per year. Aspergillus niger has a strong hydrolase system which can rapidly decompose polyglucose into glucose, and the glucose is efficiently transported to cells by a glucose transport system and supplied to cell growth and fermentation production, so that the glucose transport and absorption directly influence the performance of producing organic acid by Aspergillus niger fermentation.

In the known genomic databases, the data annotated as glucose transporters of fungal origin are rare, most of them are annotated as sugar transporters. This is because the types of transport substrates for sugar transporters are complicated, and one sugar transporter is often capable of transporting only a specific sugar substance but not other sugar substances, involving hexoses such as glucose, fructose, mannose, and galactose, pentoses such as xylose, arabinose, and ribose, disaccharides such as sucrose and lactose, trisaccharides such as maltose, and oligosaccharides. It cannot be determined which sugar transporter has a transport function for which sugar only by sequence homology alignment and phylogenetic tree analysis, and a large number of molecular biological experiments are required. For example, Jiang et al predicted three potential sugar transporters in the genome of Trichoderma reesei by using gene comparison analysis, but molecular biological tests found that Garr 1p is a membrane protein but cannot transport sugars such as glucose, mannose, fructose and Xylose, Gltr1p only has weak glucose transport ability, and Xltr1p has strong ability to transport sugars such as glucose, mannose, fructose and Xylose (Jiang et al, Identification and Characterization of an effective d-Xylose Transporter in Saccharomyces cerevisiae 2020 Mar4; 68(9):2702 + 2710.). As another example, although 173 sugar Transporters were annotated in the genome of Clostridium phytofermentans, Cerisy et al confirmed by knockout experiments that only 4 Transporters are critical for carbohydrate transport, but there were significant differences in transport substrates, such as cphy2241 for glucose and galactose, cphy2465 for cellobiose, and cphy3589 for galactan (Tristan Cerisy et al, ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans.J.Bacteriol.2019Jul 10; 201(15): e 00241-19.).

At present, fewer glucose transporters derived from Aspergillus niger have been demonstrated. In 2004, vanKuyk et al identified the first high affinity glucose transporter MstA from a gene library. However, Aspergillus niger can still normally transport glucose after the MstA coding gene is knocked out, which indicates that the Aspergillus niger glucose transport system does have the phenomenon of multi-gene synergy (vanKuyk et al, Aspergillus niger mstA codes a high-affinity sugar/H + simple white regulated in response to extracellular pH. biochem J.2004Apr 15; 379(Pt 2): 375-83.). In 2007, Jorgensen et al further identified a low affinity Glucose transporter MstC in the MstA knockout strain and found that there were different expression patterns for both (Jorgensen et al, Glucose uptake and growth of Glucose-limited chemostat cultures of Aspergillus niger and a discrete enzyme MstA, a high-affinity Glucose transporter. microbiology (Reading).2007 Jun; 153(Pt 6): 1963-. Sloothaak et al combined the hidden Markov model with the membrane proteome and found two other high affinity glucose transporters MstG and MstH (Sloothaak et al, Aspergillus niger membrane-associated protein analysis for the identification of glucose transporters. Biotechnol biofuels.2015Sep 17; 8: 150.).

Meanwhile, in the prior art, the research on glucose transporters mostly focuses on the analysis of mechanisms, and the glucose transporter-integrated aspergillus niger engineering strain and the reports on the production of organic acids thereof are few. For example, CN106635847A discloses that overexpression of the endogenous glucose transporter LGT1 increases the yield of citric acid from 134g/L to 180g/L in A.niger.

Therefore, the problem to be solved in the field is to excavate a new glucose transporter, construct a high-efficiency aspergillus niger engineering strain and improve the yield of organic acid and citric acid.

Disclosure of Invention

The invention discovers a novel protein with a glucose transport function in a large amount of preliminary experimental researches, and the expression of the novel protein can be enhanced to promote the production of organic acid and improve the acid production level of a strain, thereby completing the invention.

In a first aspect, the present invention provides novel proteins having glucose transport function, including any one of the following proteins:

a) has the sequence shown in SEQ ID NO:1 to SEQ ID NO: 5 in the sequence of any one of the amino acids; or

b) Consisting of the sequence SEQ ID NO:1 to SEQ ID NO: 5 through substitution, deletion or addition of one or a plurality of amino acid residues, and has the functions of the protein a).

c) And the sequence SEQ ID NO:1 to SEQ ID NO: 5 is higher than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the protein which is derived from Aspergillus niger and has glucose transport function.

As used herein, "glucose transporter" refers to a transporter capable of transporting extracellular glucose into the cell.

In addition, the present invention provides a fusion protein, wherein the fusion protein comprises the glucose transporter described above, and a foreign polypeptide fused to the glucose transporter; optionally, the exogenous polypeptide comprises a tag polypeptide; preferably, the exogenous polypeptide comprises a tag polypeptide and a spacer polypeptide linking the tag polypeptide to the polypeptide having glucose transporter activity.

In a second aspect, the invention provides expression cassettes, recombinant vectors, and recombinant host cells comprising the glucose transporter-encoding gene. Among them, the vector to be used is not particularly limited, and may be any vector known in the art as long as it can replicate in a host, including plasmids, viruses, phages and transposons. Possible vectors for use in 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 combinations 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, a plasmid, a phage. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, integrate into the genome itself.

In a third aspect, the invention provides a method for constructing an engineering strain for producing organic acid, which comprises enhancing the activity of a glucose transporter in the strain, wherein the amino acid sequence of the glucose transporter is shown as SEQ ID NO:1 to SEQ ID NO: 5 or as shown in sequence SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 12 is shown in any one of the above.

Among them, the above-mentioned "enhancing the activity of a glucose transporter in a strain" means that the intracellular glucose transporter activity of a protein in a microorganism is improved by modification as compared with the activity of the protein in the natural state. Not only includes higher effects than the original functions due to the increase in the activity of the protein itself, but also it can be performed by at least one method selected from the following: increasing the copy number of a polynucleotide encoding a protein, modifying a regulatory sequence of a gene encoding a protein, replacing a 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 a protein, introducing a modification in a gene encoding a protein on a chromosome to enhance the activity of a protein, and may also include, without limitation, any method of inhibition as long as the activity of a protein can be enhanced or the activity of an introduced protein can be enhanced as compared with the endogenous activity.

In one embodimentWherein the activity of the glucose transporter in the strain is enhanced by introducing an expression vector containing the glucose transporter into the strain to achieve overexpression of the glucose transporter. "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 (CaPO)₄) Precipitation method, calcium chloride (CaCl)₂) Precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposome, and lithium acetate-DMSO.

Further, the recombinant host cell has enhanced activity of the citrate transporter CexA, wherein the sequence of CexA is as shown in SEQ ID NO: shown at 13. Overexpression is preferably achieved by introducing an expression vector containing the gene encoding the CexA. Wherein the CexA is capable of transporting intracellular citrate to an extracellular efflux protein.

The term "host cell" as used herein means any cell type that is susceptible to transformation, transfection, transduction, and the like with a polynucleotide or recombinant expression vector comprising a glucose transporter, an encoded protein of the present invention. The host cell is selected from the group consisting of Aspergillus niger (Aspergillus niger), Aspergillus nidulans (Aspergillus nidulans), Aspergillus oryzae (Aspergillus oryzae), Penicillium chrysogenum (Penicillium chrysogenum), Trichoderma reesei (Trichoderma reesei), Ustilago zeae (Ustilago maydis), Myceliophthora thermophila (Myceliophthora thermophila), preferably Aspergillus niger (Aspergillus niger), Aspergillus nidulans (Aspergillus nidulans).

Wherein the organic acid is selected from citric acid, malic acid, succinic acid, itaconic acid and fumaric acid, and is preferably citric acid.

In a fourth aspect, the present invention also provides the use of a glucose transporter as described above, or a nucleic acid encoding a glucose transporter as described above, or an expression cassette or recombinant vector comprising a nucleic acid encoding as described above, or a recombinant host cell comprising said nucleic acid encoding as described above, for the production of an organic acid, preferably citric acid. Accordingly, the present invention provides a method for producing an organic acid, which comprises culturing the host cell constructed by the above method, and collecting the produced target product. Such organic acids include, but are not limited to, citric acid, malic acid, succinic acid, itaconic acid, fumaric acid, and the like.

The present inventors have made extensive and intensive studies and have unexpectedly found that a series of glucose transporters are present in filamentous fungi. The expression of the glucose transport protein in the organic acid production strain is enhanced, the production of the organic acid can be promoted, and the acid production level of the strain is improved, so that a brand new modification target point is provided for the modification of the organic acid production strain. Meanwhile, the transport protein can enable microorganisms such as yeast and the like to transport hexose such as glucose, fructose and the like. Through research experiments, the citric acid yield of the engineering strain constructed by culturing can be improved by 1-3 times compared with that of the original strain, and the engineering strain has better industrial application potential.

Drawings

FIG. 1 Green fluorescent labeling localization of glucose transporters in Saccharomyces cerevisiae

FIG. 2 growth of Saccharomyces cerevisiae expressing strains of glucose transporters on media with different carbon sources

Detailed Description

Definition of terms:

the terms "a" and "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but may also mean "one or more," at least one, "and" one or more than one.

As used herein, "comprising," "having," "including," or "containing" means inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

The term "about" as used herein means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.

The term "optional/preferred" numerical range "as used in the present invention includes both the numerical endpoints at the ends of the range, and all natural numbers subsumed within the middle of the numerical endpoints with respect to the aforementioned numerical endpoints.

The terms "polypeptide", "peptide" and "protein" as used herein are used interchangeably and are polymers of amino acids of any length. The polymer may be linear or branched, it may comprise 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).

The term "recombinant polypeptide" as used herein means that two or more polypeptides are expressed by fusion using genetic engineering techniques. In some embodiments, the recombinant polypeptide is a polypeptide that encodes SEQ ID NO: 1-5 and exogenous polypeptide. In some embodiments, the exogenous polypeptide comprises a tag polypeptide, and further, the exogenous polypeptide comprises a tag polypeptide, and a spacer polypeptide linking the mutant and the tag polypeptide, in particular, the spacer polypeptide can have less than 10 spacer amino acid residues.

The term "expression vector" as used herein refers to a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide operably linked to control sequences for its expression.

The term "expression cassette" as used herein comprises a polynucleotide encoding a polypeptide operably linked to suitable control sequences necessary for expression of the polynucleotide in a selected cell or strain. In the present invention, the transcription regulatory element includes a promoter, and may further include an enhancer, a silencer, an insulator, and the like. Modifications to regulatory sequences include, but are not limited to, such as: modifications introduced by deletions, insertions, conservative or non-conservative mutations, or combinations thereof in a polynucleotide sequence may also be made by replacing the original polynucleotide sequence with a polynucleotide sequence having enhanced activity.

The term "operably linked" as used herein refers to the following configuration: the control sequences are positioned at an appropriate location relative to the coding sequence of the polynucleotide such that the control sequences direct expression of the coding sequence. Illustratively, the regulatory sequence may be selected from sequences encoded by promoters and/or enhancers.

The term "sequence identity" or "percent identity" in the context of the present invention refers to nucleotide or amino acid residue sequences that are identical or have a specified percentage of the same sequence when compared and aligned for maximum correspondence as measured using nucleotide or amino acid sequence comparison algorithms or by visual inspection. That is, the identity of nucleotide or amino acid sequences can be defined by the ratio of the number of nucleotides or amino acids that are identical when two or more nucleotide or amino acid sequences are aligned in such a manner that the number of nucleotides or amino acids that are identical is maximized, and gaps are added as necessary, to the total number of nucleotides or amino acids in the aligned portion. The methods of determining "sequence identity" or "percent identity" to which the present invention relates include, but are not limited to: computer Molecular Biology (computerized Molecular Biology), Lesk, a.m. ed, oxford university press, new york, 1988; biological calculation: informatics and genomic Projects (Biocomputing: information and Genome Projects), Smith, d.w. eds, academic press, new york, 1993; computer Analysis of Sequence Data (Computer Analysis of Sequence Data), first part, Griffin, a.m. and Griffin, h.g. eds, Humana Press, new jersey, 1994; sequence Analysis in Molecular Biology (Sequence Analysis in Molecular Biology), von Heinje, g., academic Press, 1987 and Sequence Analysis primers (Sequence Analysis Primer), Gribskov, m. and Devereux, j. eds M Stockton Press, New York, 1991 and Carllo, h. and Lipman, d.s., SIAM j.applied Math., 48:1073 (1988). The preferred method of determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG program package (Devereux, J. et al, 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al, 1990). BLASTX programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al, NCBI NLM NIH Bethesda, Md.20894; Altschul, S. et al, 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

Furthermore, it will be appreciated by those of ordinary skill in The art that The alteration of a small number of amino acid residues in certain regions, e.g., non-critical regions, of a polypeptide does not substantially alter The biological activity, e.g., The sequence resulting from The appropriate substitution of certain amino acids does not affect The activity (see Watson et al, Molecular Biology of The Gene, fourth edition, 1987, The Benjamin/Cummings pub. Co. P224). Representative examples of such conservative mutations are conservative substitutions.

The term "conservative substitution" as used herein relates to the replacement of 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 those 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), beta-branches (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). "conservative substitutions" typically exchange one amino acid for one or more sites in a protein. Such substitutions may be conservative. Examples of the substitution regarded as conservative substitution include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His or Lys, substitution of Asn with Glu, Gln, Lys, His or Asp, substitution of Asp with Asn, Glu or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp or Arg, substitution of Glu with Gly, Asn, Gln, Lys or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg or Tyr, substitution of Ile with Leu, Met, Val or Phe, substitution of Leu with Ile, Met, Val or Phe, substitution of Lys with Asn, Glu, Gln, His or Arg, substitution of Met with Ile, Leu, Val or Phe, substitution of Phe with Trp, Tyr, Met, Ile or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe, Tyr, His or Phe, and substitution of Met with Met or Phe. Furthermore, conservative mutations include naturally occurring mutations due to individual differences in the origin of the gene, differences in strain, species, and the like.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental techniques and experimental procedures used in this example are, unless otherwise specified, conventional techniques, e.g., those in the following examples, in which specific conditions are not specified, and generally according to conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The materials, reagents and the like used in the examples are commercially available from normal sources unless otherwise specified.

Example 1

1. Construction of glucose Transporter expression plasmid

Based on genome data mining and transcriptome data analysis, 7 potential glucose transporters GT1, GT2, GT3, GT4, GT5, GT6 and GT7 are predicted, and the amino acid sequences are shown in SEQ ID NO. 1-7. PCR is performed to amplify cDNA fragments of each glucose transporter by using an upstream primer and a downstream primer of each glucose transporter and cDNA of a citric acid producing strain D (purchased from a strain resource library of Shanghai institute of Industrial microbiology, Japan, published as No. M202) as a template. Specific primer sequences are shown in table 1.

TABLE 1 primers for construction of glucose transporter expression plasmids in Saccharomyces cerevisiae

The PCR reaction system was 5 XFastPFu buffer 10. mu.L, 10mM dNTPs 1. mu.L, each of the upstream/downstream primers 2.5. mu.L, DNA template 0.5. mu.L, FastPFu (TransGene) 1.5. mu.L, and ultrapure water 32. mu.L.

The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 2min, 35 cycles; finally, extension is carried out for 10min at 72 ℃. After PCR purification of the PCR products, the plasmids pRS426-PGK1 were digested with the restriction enzymes SpeI and HindIII, respectively. The PCR product of gene GT5 was cleaved with plasmid pRS426-PGK1(Galazka et al.2010) using the restriction enzymes SpeI and EcoRI. Then, each gene was ligated with the corresponding enzyme cleaved product of plasmid pRS426-PGK1, and transformed into E.coli. Then, the corresponding recombinant plasmids were extracted, and the expression plasmids pRS-GT1, pRS-GT2, pRS-GT3, pRS-GT4, pRS-GT5, pRS-GT6, pRS-GT7, pRS-MstA, pRS-MstC, pRS-MstE, pRS-MstG, pRS-MstH of each glucose transporter were obtained by digestion and sequencing verification. The correct recombinant plasmid and the empty plasmid pRS426-PGK1 were transformed into Saccharomyces cerevisiae EBY.VW4000(Wieczorke et al, 1999) to obtain Saccharomyces cerevisiae expression strains of glucose transporters, which were named EBY.GT1, EBY.GT2, EBY.GT3, EBY.GT4, EBY.GT5, EBY.GT6, EBY.GT7, EBY.MstA, EBY.MstC, EBY.MstE, EBY.MstG, and EBY.MstH, respectively. After expression of each glucose transporter, it is localized to the plasma membrane of s.cerevisiae, as shown in FIG. 1.

2. Experiment for endowing microorganism with glucose utilization capability by glucose transporter

Single colonies of yeast expression strains of each glucose transporter and a control strain containing an empty plasmid were picked, inoculated into SC-URA liquid medium (6.7 g/L nitrogen source without amino yeast, 1.4g/L yeast synthetic deletion culture supplement, 20g/L maltose, 20mg/L leucine, 20mg/L histidine, 20mg/L tryptophan) containing 2% maltose, and cultured at 30 ℃ for about 12 to 14 hours. After collecting the cells by centrifugation, the cells were washed with sterilized ultrapure water, and the cell concentration was adjusted to OD₆₀₀Is 0.3. The thalli is respectively diluted by 10 times and 100 times,after 1000 times, the cells were spotted on SC-URA solid medium containing maltose, SC-URA solid medium containing glucose as a sole carbon source, and SC-URA containing fructose as a sole carbon source, respectively, and cultured at 30 ℃ to observe the growth of the cells. In saccharomyces cerevisiae eby.vw4000, 17 hexose transporters and 3 maltose/glucose transporters were knocked out, and the ability to transport glucose was lost. Thus, a control strain containing an empty plasmid can grow on medium containing maltose, but not on medium with glucose or fructose as the sole carbon source. As shown in FIG. 2, yeast expressing strains of each transporter were able to grow on glucose compared to the control strains, indicating that overexpression of each transporter allows the yeast strain to gain glucose utilization. It can be seen that the 7 novel transporters GT1, GT2, GT3, GT4, GT5, GT6 and GT7 of the present invention all have glucose transport functions. In addition, yeast expressing strains of GT1, GT2, GT3, GT4, GT6 can also grow on media with fructose as the sole carbon source, indicating that GT1, GT2, GT3, GT4 and GT6 also have the function of transporting fructose, enabling the yeast to reuse fructose.

Example 2

1. Construction of glucose transporter Aspergillus niger expression plasmid

And performing PCR amplification on each glucose transporter gene fragment by adopting an upstream primer and a downstream primer of each glucose transporter and taking the genome of the citric acid producing strain D as a template. Specific primer sequences are shown in table 2. Although several glucose transporters have been reported in A.niger, the effect of overexpression on citric acid fermentation is not clear. Therefore, in this example, 5 reported glucose transporters (MstA, MstC, MstE, MstG, MstH) were simultaneously sequenced to have the sequences of SEQ ID NOs: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12 and detecting the overexpression and citric acid fermentation.

TABLE 2 primers for construction of glucose transporter Aspergillus niger expression plasmids

The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 2min, 35 cycles; finally, extension is carried out for 10min at 72 ℃.

After PCR product purification, the PCR product was subjected to recombination reaction with plasmid pGm-hyh (Zheng et al, 2019) digested with restriction enzyme XholI using Novozam non-ligase-dependent single-fragment rapid cloning kit (cat # C112-01), and then transformed into E.coli. After extracting corresponding recombinant plasmids, enzyme digestion and sequencing verification are adopted to obtain expression plasmids pGm-GT1, pGm-GT2, pGm-GT3, pGm-GT4, pGm-GT5, pGm-GT6, pGm-GT7, pGm-MstA, pGm-MstC, pGm-MstE, pGm-MstG and pGm-MstH of each glucose transporter.

2. Transformation of glucose transporter expression plasmids to Aspergillus niger by PEG-mediated protoplast transformation

Taking Aspergillus niger wild type M202 spores to suspend in 200mL CMA liquid medium (glucose 20g/L, malt extract 20g/L, peptone 1g/L), the final concentration of the spore suspension is 10⁵/mL, culturing at 30 deg.C and 200r/min for 12-16 h. The cells were collected under sterile conditions on a sterile Micro-cloth and the solution A (K)₂HPO₄ 5mM,KH₂PO₄5mM,MgSO₄96.312g/L, pH 5.8, filter sterilized) was washed once, transferred to 20mL of lysis buffer (0.4g of lyase in 20mL of solution A) using a sterile cotton swab, and lysed at 37 ℃ at 75r/min for about two hours. The lysate was filtered through sterile Micro-cloth and protoplasts were collected in two 50mL sterile centrifuge tubes and applied to solution B (Tris-HCl10mM, CaCl)₂5.54g/L, D-Sorbitol 218.64g/L, pH 7.5, filter sterilized) to a volume of approximately 25mL per tube. Centrifuging at 2000r/min for 5min, discarding supernatant, and adding 20mL of solutionAnd B, resuspending the pellet twice more. The pellet was resuspended in 10mL of solution B, the tubes were combined into one tube and the protoplasts were counted using a hemocytometer. Centrifuging and adding an appropriate amount of solution B to resuspend once according to counting results. Add 100. mu.L of protoplast suspension to a pre-cooled 15mL centrifuge tube on ice, then add 5. mu.g of glucose transporter over-expression plasmid pGm-GT1, pGm-GT2, pGm-GT3, pGm-GT4, pGm-GT5, pGm-GT6, pGm-GT7, pGm-MstA, pGm-MstC, pGm-MstE, pGm-MstG, pGm-MstH and empty plasmid pGm, add 1mL of solution C (Tris-HCl10mM, CaCl 10. sup. g, and₂5.54g/L, PEG 600050% (w/v), pH 7.5, filter sterilize) ice bath for 10min, 2mL solution B and mix well. Uniformly mixing the culture medium with preheated upper layer culture medium MMSH containing hygromycin, and then spreading the mixture on a lower layer culture medium MMSH plate. The plates were incubated in an incubator at 30 ℃ for 3-5 days until transformants grew.

3. Genotype verification of aspergillus niger glucose transporter expression-enhanced strain

Extracting the genome DNA of the transformant after secondary passage and purification by adopting a novel Tiangen plant genome extraction kit DP350, and then carrying out gene PCR verification on the transformant by using primers pGm-F and pGm-R by taking the extracted genome DNA as a template. Specific sequences of the primers are shown in Table 3.

TABLE 3 validation primers for glucose transporter expression-enhanced transformants

The PCR reaction system is as follows: 10 mu L of 2 xTaq Buffer, 1 mu L of each upstream/downstream primer, 1 mu L of DNA template and 7 mu L of deionized water.

The PCR reaction conditions are as follows: 5min at 94 ℃; 30 cycles of 94 ℃ for 30sec, 55 ℃ for 30sec, 72 ℃ for 4 min; 10min at 72 ℃. The PCR amplification product was subjected to 1% agarose gel electrophoresis (150V voltage, 20 min).

The PCR amplification products were subjected to 1% agarose gel electrophoresis (150V voltage, 20 min), and gene amplification bands were observed under a gel imaging system, and the results showed that expression plasmids of each glucose transporter had been integrated into the Aspergillus niger genome, to obtain Aspergillus niger expression-enhanced strains of each glucose transporter, which were designated as AnGT1, AnGT2, AnGT3, AnGT4, AnGT5, AnGT6, AnGT7, AnMstA, AnMstC, AnMstE, AnMstG, and AnMstH, respectively.

4. Citric acid fermentation of aspergillus niger glucose transporter expression-enhanced strain

Aspergillus niger glucose transporter expression enhancing strains AnGT1, AnGT2, AnGT3, AnGT4, AnGT5, AnGT6, AnGT7, AnMstA, AnMstC, AnMstE, AnMstG, AnMstH and a control strain AnControl integrated with pGm empty plasmid were inoculated on PDA medium at 30 ℃ for 5 days, respectively, and then spores were collected with 0.9% physiological saline and counted using a blood count plate. At 10⁶The inoculation amount of the strain/mL is inoculated in a citric acid fermentation culture medium (corn starch culture medium, the total sugar content is 12 percent), and the culture is carried out for 96h at 34 ℃ and 250 r/min.

Collecting fermentation supernatant by rapid suction filtration, diluting by 10 times, heating and boiling for 10min, filtering with filter membrane, and detecting citric acid content with HPLC. The specific detection conditions include chromatographic column Aminex HPX-87H (300mM X7.8 mM X9 μm, BioRad), Shimadzu UFLC high performance liquid chromatograph (equipped with Shimadzu LC-20AD infusion pump, SPD-20A UV detector, CTO-20A/AC column incubator, SIL-20ACHT UFLC specification autosampler, Shimadzu LCproposal workstation), mobile phase A as ultrapure water, and mobile phase B as 2.75mM H₂SO₄The flow rate is 0.6mL/min, the sample amount is 10uL, the column temperature is 50 ℃, and the ultraviolet detection wavelength is 210 nm. As shown in Table 4, it was found that the expression of the Aspergillus niger glucose transporter GT1-GT5 of the present invention was enhanced compared with the expression of the reported 5 glucose transporters MstA, MstC, MstE, MstG, and MstH, and the production of citric acid was significantly promoted compared with the starting strain. However, the enhancement of the expression of the glucose transporters GT6 and GT7 did not have any effect on the production of citric acid.

TABLE 4 Aspergillus niger glucose transporter expression enhancement for citric acid fermentation

Example 3

1. Construction of glucose transporter/citric acid efflux protein Aspergillus niger expression plasmid

Firstly, an upstream primer and a downstream primer of the citrate efflux protein CexA (the amino acid sequence of which is SEQ ID NO: 13) are adopted, and the genome of the citrate-producing strain D is taken as a template to carry out PCR amplification on the citrate efflux protein gene fragment. Specific primer sequences are shown in table 5.

TABLE 5 primers for construction of glucose transporter Aspergillus niger expression plasmids

After PCR product purification, the PCR product was subjected to recombination reaction with plasmid pGm-hyh (Zheng et al, 2019) digested with restriction enzyme XholI using Novozam non-ligase-dependent single-fragment rapid cloning kit (cat # C112-01), and then transformed into E.coli. After extracting corresponding recombinant plasmids, carrying out enzyme digestion and sequencing verification to obtain the Aspergillus niger expression plasmid pGm-CexA of the citrate efflux protein.

The expression cassettes of the respective glucose transporters were PCR-amplified using pGm-F2 and pGm-R2 as primers and the expression plasmids pGm-GT1, pGm-GT2, pGm-GT3, pGm-GT4, pGm-GT5, pGm-MstA, pGm-MstC, pGm-MstE, pGm-MstG, pGm-MstH of the respective glucose transporters obtained in example 2 as templates, respectively. pGm-F2 and pGm-R2 have the specific sequences shown in Table 5.

The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 4min, 35 cycles; finally, extension is carried out for 10min at 72 ℃.

After PCR product purification, the PCR product was subjected to recombination reaction with plasmid pGm-CexA digested with restriction enzyme SphI using Novozam non-ligase-dependent single-fragment rapid cloning kit (cat # C112-01), and then transformed into E.coli. After extracting corresponding recombinant plasmids, adopting enzyme digestion and sequencing verification to obtain Aspergillus niger co-expression plasmids of each glucose transporter and citrate efflux protein, which are respectively named as pGm-CexA/GT1, pGm-CexA/GT2, pGm-CexA/GT3, pGm-CexA/GT4, pGm-CexA/GT5, pGm-CexA/MstA, pGm-CexA/MstC, pGm-CexA/MstE, pGm-CexA/MstG and pGm-CexA/MstH.

Taking Aspergillus niger wild type M202 spores to suspend in 200mL CMA liquid medium (glucose 20g/L, malt extract 20g/L, peptone 1g/L), the final concentration of the spore suspension is 10⁵/mL, culturing at 30 deg.C and 200r/min for 12-16 h. The cells were collected under sterile conditions on a sterile Micro-cloth and the solution A (K)₂HPO₄ 5mM,KH₂PO₄5mM,MgSO₄96.312g/L, pH 5.8, filter sterilized) was washed once, transferred to 20mL of lysis buffer (0.4g of lyase in 20mL of solution A) using a sterile cotton swab, and lysed at 37 ℃ at 75r/min for about two hours. The lysate was filtered through sterile Micro-cloth and protoplasts were collected in two 50mL sterile centrifuge tubes and applied to solution B (Tris-HCl10mM, CaCl)₂5.54g/L, D-Sorbitol 218.64g/L, pH 7.5, filter sterilized) to a volume of approximately 25mL per tube. The supernatant was discarded by centrifugation at 2000r/min for 5min, and the pellet was resuspended twice more with 20mL of solution B. The pellet was resuspended in 10mL of solution B, the tubes were combined into one tube and the protoplasts were counted using a hemocytometer. Centrifuging and adding an appropriate amount of solution B to resuspend once according to counting results. Add 100. mu.L of protoplast suspension to a pre-cooled 15mL centrifuge tube on ice, then add 5. mu.g of glucose transporter and citrate efflux protein, respectively, of the Aspergillus niger co-expression plasmids pGm-CexA/GT1, pGm-CexA/GT2, pGm-CexA/GT3, pGm-CexA/GT4, pGm-CexA/GT5, pGm-CexA/MstA, pGm-CexA/MstE, pGm-CexA/MstG, pGm-CexA/MstH, to which 1mL of solution C (Tris-HCl10mM, CaCl) was added₂5.54g/L, PEG 600050% (w/v), pH 7.5, filter sterilize) ice bath for 10min, 2mL solution B and mix well. Uniformly mixing the culture medium with preheated upper layer culture medium MMSH containing hygromycin, and then spreading the mixture on a lower layer culture medium MMSH plate. The plates were incubated in an incubator at 30 ℃ for 3-5 days until transformants grew.

Extracting the genome DNA of the transformant after secondary subculture and purification by using a Tiangen novel plant genome extraction kit DP350, carrying out PCR verification on the transformant by using the extracted genome DNA as a template and using universal primers pGm-F and CexA-R to verify the genome integration condition of the citrate efflux protein CexA, and carrying out PCR verification on the transformant by using universal primers pGm-F2 and various glucose transporter specific downstream primers such as GT-R2 to verify the genome integration condition of various glucose transporter expression cassettes. Specific sequences of the primers are shown in Table 2, Table 3 and Table 5.

And (3) carrying out 1% agarose gel electrophoresis (150V voltage, 20 minutes) on the PCR amplification product, observing a gene amplification band under a gel imaging system, wherein the result shows that the expression cassette of each glucose transporter and the expression cassette of the citrate efflux protein are integrated into the Aspergillus niger genome, and obtaining the co-expression enhancing strains of each glucose transporter and the citrate efflux protein, which are respectively named as AnCGT1, AnCGT2, AnCGT3, AnCGT4, AnCGT5, AnCMstA, AnCMstC, AnCMstE, AnCMstG and AnCMstH.

Aspergillus niger glucose transporter expression-enhanced strains AnCGT1, AnCGT2, AnCGT3, AnCGT4, AnCGT5, AnCMstA and AnCMstC, AnCMstE, AnCMstG, AnCMstH and a control strain AnControl integrated with pGm empty plasmid were inoculated on PDA medium for 5 days at 30 ℃ respectively, and then spores were collected with 0.9% physiological saline and counted using a hemocytometer. At 10⁶The inoculation amount of the strain/mL is inoculated in a citric acid fermentation culture medium (corn starch culture medium, the total sugar content is 12 percent), and the culture is carried out for 96h at 34 ℃ and 250 r/min.

Collecting fermentation supernatant by rapid suction filtration, diluting by 10 times, heating and boiling for 10min, filtering with filter membrane, and detecting citric acid content with HPLC. The specific detection conditions include chromatographic column Aminex HPX-87H (300mM X7.8 mM X9 μm, BioRad), Shimadzu UFLC high performance liquid chromatograph (equipped with Shimadzu LC-20AD infusion pump, SPD-20A UV detector, CTO-20A/AC column incubator, SIL-20ACHT UFLC specification autosampler, Shimadzu LCproposal workstation), mobile phase A as ultrapure water, and mobile phase B as 2.75mM H₂SO₄The flow rate is 0.6mL/min, the sample amount is 10uL, the column temperature is 50 ℃, and the ultraviolet detection wavelength is 210 nm. The results are shown in Table 6.

TABLE 6 enhanced citric acid fermentation by co-expression of Aspergillus niger glucose transporter and citric acid transporter

The results show that when the expression of the Aspergillus niger glucose transporter is enhanced, compared with the original strain, the Aspergillus niger glucose transporter can obviously promote the production of citric acid, and particularly, the promoting effect reaches 2-3 times of the original promoting effect.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> glucose transporter and application thereof in improving production of organic acid

<130> 202001015

<160> 67

<170> PatentIn version 3.5

<210> 1

<211> 563

<212> PRT

<213> Aspergillus niger

<400> 1

MGDNYEEKPD AIGKEFSDEL TPEEREHEIE ARDLQSNQYL DNEPLYDRIA FAQIPVHMRS 60

PRMLVITLGV FAAFSGMLSG LDQSVISGAL PGIRKHFIAS GEWASMDDPK LATDISLISS 120

LMPLGAMAGA LIMTPLNFYF GRRNSIIISC LWYTLGSGLC AGSRSVGMLF AGRFILGIGI 180

GIEGGCVGIY ISECVPPEVR GNLVSVYQLM IAFGEIIGYA AGGVFFDVPS GSWRWMLGSS 240

LLFSTILLVG MAFLPESPRW LASKGKYGRA WRVWKSLRDM ADPKSLDEYL AMEVTVKHDV 300

DQSANVKWHQ RYLEVCRTPR NRRALVYATA MIFFGQMTGI NAVMYNMSNL MAKMNFDDRE 360

SVLMSMVGGG ALFLGTIPAV FTMDKFGRRV WAQNILMFIV GLALVGVGYL YTNNGVDYFN 420

EHKATALGLF FSGLVLYMAF FGAYSCLTWV VPAESFSFNT RSQGMAICSV FLYLWSFIVT 480

YNFEGMQKAM TYTGLTIGFF GGLAALGFFY QLFFMPETKD KTLEEIDELF LMPTSKLVAL 540

NTSNLLKPLR RRHRPEPVDN YNA 563

<210> 2

<211> 554

<212> PRT

<213> Aspergillus niger

<400> 2

MGVVHDLDTS HDDHKEGTAL EMDDVLKDPK AIEEFNEAAM DAELLQLEEQ LQERKPGWFV 60

GFSNPSVYTY VLVAFASMGG LLSGLDQSLI SGANIYLPDA LNLTDNQSSL VDAGMPLGAV 120

AGALILSPAN EYLGRRMAII VSCILYTIGA ALEAGAISFG MIFAGRFVLG MGVGLEGGTV 180

PVYVAESVPR KMRGNLVSLY QLNIALGEVL GYAVAAIFLD VKGNWRYILG SSLVFSTILL 240

VGMLFLPESP RYLMHKGRAV EAYGVWKRIR GFNDYDAKDE FLGMRQAVDA EREEQAATKK 300

YAWMDFITVP RARRAMVYAN IMVFLGQFTG VNAIMYYMST LMDAIGFDER DSVFMSLVGG 360

GSLLIGTIPA VLYMERFGRR YWANAMLPGF FIGLVLVGIG YQINYQTHPK AAQGVYLTGI 420

ILYQAFFGSY ACLTWVVPAE VFPTYLRSYG MTTADANLFL CSFIVTYNFT RMMKAMTRIG 480

LTLGFYGGIA FIGWFYQIIF MPETKNKSLE EIDELFSRPT SAIVKENLKS TGEVIDDLFH 540

LRFKKVFSPP PKED 554

<210> 3

<211> 484

<212> PRT

<213> Aspergillus niger

<400> 3

MAKLSTLLSA VFLAIGGFLF GYDSGIITST IGQTEFIRYF NNPNDTVTGG VVSAFQGGAI 60

LGTIINIFTG DRLGRKMSVF VGACISIVGC ALQGGAINMT MLIIGRFIAG VAVGMLTATV 120

PMYAGEMAEA ASRGMMSGLL QWMLSWGSLV AQWLGYGCSF NSTDFQWRFP LAFQCVPGLV 180

LMTGVWFLQE SPRWLMEQDR HEEALATLHH LHGDGTPEKS QYIELEYQEI RDTIEAERAH 240

NTITWTSILT KPSWRRRLIL GCGVQAFGPL SGINVINYYG TRIYSSLGID TRTTLMIIGI 300

SGGLSIIYCT IGLWALERVG RIRPLIVSAA GMAAALVCNA AMSQHYNESN TNQLRAMVAM 360

NFVFSLFYTS PGIISWVYPA EIFPVDIRNQ GNSITTFTNW TVNLVFAQFS PTALSNIGFR 420

YFYVFFVFNL IAMICYIFFF PETKGKTLEQ MDELFGDVMT PPGDLKGTDE PQVTMIEDAM 480

NVSS 484

<210> 4

<211> 484

<212> PRT

<213> Aspergillus niger

<400> 4

MFGFDIEFRR HRWALIYCSI SAIGALVFGY DNTYYSGILG MQEFKNDYGD HYEDGAKALA 60

TSFTSLTTSS IYIGDMLGAL IAGPLNDRFG RKTVLWIASA FVLAGGVTQV ADTNIEGVIV 120

LGRILIGLGV GNFTVTSLLY IGEIAPMEIR SPALYMYQFL QSCSQLVASG LTQGTNSIHS 180

SLSYKLPMGG LVILPLFMLF LLPFIPESPT WYVFHNRLED ARRSLLKINR NNRTYDPRAD 240

LAFLTQARRY EEAQGEESSW LSLLLDPIER KKLIWASGGM YAQQICGIIF FYNYGVVFAE 300

ALGVSQPFTI TLITMILQIF AVAVSILTGN RLRRRTNLLA STSLILVAFI IIGGLGTQST 360

LTTASKYIIV VFSYVVICAY NFGLGPLTYA VSREISVGVN QNKIMSVSIV ALYFFLWVIS 420

FTAPYLYYDA GLGPMVGFVY AGTTLTSLIW VWFCVGETQG RTRLEVAMFF TERVPARRWR 480

THVFAGSGGD EKDDVRAEHV ENKA 504

<210> 5

<211> 510

<212> PRT

<213> Aspergillus niger

<400> 5

MTAPMSATAA ARADWRAIAI CLFITIAAFQ FGFDSSYYSG ILVMEPFIKA YGHYDNATGG 60

YVLSSSIQSL TTSIINVGEL VGAVSSYLVD DRIGRRGGLF VSSTFVVLGV IFQVSADKLA 120

LLIVGRLLLG YAVGLISCLV PLYVADCAPA RFRGALVSLY QFDVGLGLLL GVIVDNATKN 180

RNDSGAYRIP MAVQLVFPVI LVPGLILFAP ESPRWLLKKG KTEQARAALR RLHGNRPDVI 240

ESEVLYISGT IEEERRAEGS WRELLHWHQD GRKAYLGMAL QAWQQASGIN FITSYGIVFF 300

SAIGMTNSFL IQMGLYLVPM PAVWLNQYCV ERFGRRPMLL LSCVLVAMVL LIVGGAGTAS 360

HKTTTLDRLI VAMVYIYMIV YNLSLGPAVW VVTSEISAGP NRSKLMATST GVNWFCSWMV 420

TFTFPYLFDS DAANLSARVG FIYGSLMVAA AVWIYFLLPE TSGRTLEEIQ ILFKNGVSAW 480

NFKSYEIPEM PGGSVIDEKK EAHATEVEEV 510

<210> 6

<211> 510

<212> PRT

<213> Aspergillus niger

<400> 6

MYRISNIYVL AGFGTIGGAL FGFDVSSMSA WIGTDQYLEY FNHPDSDLQG GITASMSAGS 60

FAGALAAGFI SDRIGRRYSL MLACCIWVIG AAIQCSAQNV AHLVAGRVIS GLSVGITSSQ 120

VCVYLAELAP ARIRGRIVGI QQWAIEWGML IMYLISYGCG QGLAGAASFR VSWGVQGIPA 180

LILLAALPFF PESPRWLASK ERWEEALDTL ALLHAKGDRN DPVVQVEYEE VQEAARIAQE 240

AKDISFFSLF GPKIWKRTLC GVSAQVWQQL LGGNVAMYYV VYIFNMAGMS GNTTLYSSAI 300

QYVIFLVTTG TILPFVDRIG RRLLLLTGSV LCMACHFAIA GLMASRGHHV DSVDGNANLK 360

WSITGPPGKG VIACSYIFVA VYGFTWAPVA WIYASEVFPL KYRAKGVGLS AAGNWIFNFA 420

LAYFVAPAFT NIQWKTYIIF GVFCTVMTFH VFFFYPETAR RSLEDIDLMF ETDMKPWKTH 480

QIHDRFGEEV ERHKHKDMAD QEKGVVSTHD EMA 513

<210> 7

<211> 498

<212> PRT

<213> Aspergillus niger

<400> 7

MPYLLVTLCC LFACLGSFLF GYDSGVISSV IDQDSFRYRF HNPSAAATGG IVASYNGGAI 60

LGSVFVSYLS DPWGRRPVLF TGGLLASLGA ALQAGAVNVA MLIAGRLIAG LAIGLMSAII 120

PVYCSELSPP RIRGFLGSLQ QWMIGLGVVV AQWVGYGCSL RTGDFSWSFP LAFQAVPAVI 180

LSCGVWFLPE SPRWLIEKGN PDAGWAVLNR LHLPRGQLNA LPVESEFERI SAGIAEARHS 240

ANHSWRQLLF AQPNWRKRVL LACGMQVFTQ CSGTNVLQNY NPGLYRSLGL SQSTSLILQG 300

IWGALAQFWN TVFILFIDRV DRRKLLIPSL LGMGATMCVE AILGQVYNNF ESVASPNHSA 360

VRAAIAVFFV FSFFYTSLGL ISWIYQSEIF PTAIRARGSS VATATNWSLN LVFAQCSPIA 420

QSRIQFKYFY CFAAFNWVAA GLVWTFYPET AGKSLEEIDR LFTAETSMPA YDADKPHVSL 480

PNSKAKDSLV VFSTQDTT 498

<210> 8

<211> 530

<212> PRT

<213> Aspergillus niger

<400> 8

MAEGFVDASR VEAPVTLKTY LMCAFAAFGG IFFGYDSGYI SGVMGMRYFI EEFEGLDYNT 60

TPTDSFVLPS WKKSLITSIL SAGTFFGALI AGDLADWFGR RTTIVSGCVV FIVGVILQTA 120

STSLGLLVAG RLVAGFGVGF VSAIIILYMS EIAPRKVRGA IVSGYQFCIT IGLMLASCVD 180

YGTENRLDSG SYRIPIGLQL AWALILGGGL LCLPESPRYF VKKGDLAKAA EVLARVRGQP 240

QDSDYIKDEL AEIVANHEYE MQVIPEGGYF VSWMNCFRGS IFSPNSNLRR TVLGTSLQMM 300

QQWTGVNFVF YFGTTFFQSL GTIDDPFLIS MITTIVNVCS TPVSFYTIEK FGRRSLLLWG 360

ALGMVICQFI VAIVGTVDGS NKHAVSAEIS FICIYIFFFA STWGPGAWVV IGEIFPLPIR 420

SRGVALSTAS NWLWNCIIAV ITPYMVDKDK GDLKAKVFFI WGSLCACAFV YTYFLIPETK 480

GLTLEQVDKM MEETTPRTSA KWTPHGTFTA EMGLTANAVA EKATAVHQEV 530

<210> 9

<211> 560

<212> PRT

<213> Aspergillus niger

<400> 9

MGVSNMMSRF KPQADHSESS TEAPTPARSN SAVEKDNVLL DDSPVKYLTW RSFILGIVVS 60

MGGFIFGYST GQISGFETMD DFLQRFGQEQ ADGSYAFSNV RSGLIVGLLC IGTMIGALVA 120

APIADRMGRK LSICLWSVIH IVGIIIQIAT DSNWVQVAMG RWVAGLGVGA LSSIVPMYQS 180

ESAPRQVRGA MVSAFQLFVA FGIFISYIIN FGTERIQSTA SWRITMGIGF AWPLILAVGS 240

LFLPESPRFA YRQGRIDEAR EVMCKLYGVS PNHRVIAQEM KDMKDKLDEE KAAGQAAWHE 300

LFTGPRMLYR TLLGIALQSL QQLTGANFIF YYGNSIFTST GLSNSYVTQI ILGAVNFGMT 360

LPGLYVVEHF GRRNSLMVGA AWMFICFMIW ASVGHFALDL ADPQATPAAG KAMIIFTCFF 420

IVGFATTWGP IVWAICGEMY PARYRALCIG IATAANWTWN FLISFFTPFI SSSIDFAYGY 480

VFAGCCFAAI FVVFFFVNET QGRTLEEVDT MYVLHVKPWQ SASWVPPEGI VQDMHRPPSS 540

SKQEGQAEMA EHTEPTELRE 560

<210> 10

<211> 558

<212> PRT

<213> Aspergillus niger

<400> 10

MPRKRPQDEL NGDVLQQEAQ ATGPNDQKSE EGIVDTPIPL LTWRSFLMGI FVSMGGFLFG 60

YDTGQISGFL EMPNFLQRYG QQQADGTYYF SNARSGLIVA LLSIGTLIGA LIAAPIADRV 120

GRKWSISGWS AMVCVGITIQ ISSPFGKWYQ VAMGRWVAGL GVGALSLLVP MYQAETGPRH 180

IRGSLVSTYQ LFITLGIFVA NCINFGTEAR NDTGSWRIPM GITYIWAMIL GFGIALFPES 240

ARYDYRHGRE AKAARTLSRM YGIPENHRML KLEIGEIRQK FVEEQERGEI TWSHLLHAPR 300

MKYRVAVGVA LQALQQLTGA NYFFYYGTTI FRGAGISNSY VTQMILGGVN FGTTFLGLYL 360

IENYGRRRSL ITGALWMFVC FMVFASVGHF SLDHENPERT HTAGVVMVVF ACLFILGFAS 420

TWGPMVWTII AELYPSEFRA RAMSLATASN WLWNFLLAFF TPFITSAIDF RLGYVFAGCL 480

FLAAGLVYVA VIEGRGRTLE EIDTMYVMKV PPWKSSKYVF PDIDPYDRRG SILSKAGASH 540

IPQVSGGNET LSPHTTEV 558

<210> 11

<211> 542

<212> PRT

<213> Aspergillus niger

<400> 11

MGVETSADHG ENISPAVAPQ QHSSQESFQK PLGGVDTPVP RVTLRAFIMA VFVSMGGLLF 60

GYDTGQISGF EQESDYLRRY GMQNAKGEWY LSDVRSGLLT SLLSIGTLVG ALVAAPIANK 120

VGRKWSITIW CVILMVGLIV QISAPSGNWV QMVMGRWTTG LGVGACSLLV PMYQGESAPR 180

HVRGAMVSCY QLFVTFGIFL AYLINLGTNT LEGTAQWRIT LGLTFLFAIV LGGGMAFFPE 240

SPRFDFRHGR VDRARATMAK LYGVPENHQV ILQELDEIQN QLEAETGSEK WYEFLTAPRM 300

FYRICLGMGL QTLQQLTGSN YFFYYGTTIF KGAGLSNSFV TQCILGAVNF ACTFGGLYTV 360

ENFGRRKSLI FGALWMFVCF MIFASIGHFM LDVAEPENTP GVGKGMIVLA CFFIAGYAMT 420

WAPMVWTITA ELYPSKYRAQ GMALAVAANW AWNFLIGFFT PFITSAIDFA YGYVFAGCMF 480

VGAFVVYFFV MEGKGRTLEE LYWLYVNKIK PWKSSNFEIP ALHSFQYEEE RKQSRSYHAE 540

NA 542

<210> 12

<211> 527

<212> PRT

<213> Aspergillus niger

<400> 12

MGFLLKRPDD AVGSAAPAIM IGLFVSFGGI LFGYDTGTIS GILAMKFWRK MFSTGYINPA 60

DDYPDVTSSQ SSMIVSLLSA GTFFGALASA PVADYFGRRI AMIIESFVFC FGVILQTAAT 120

SIPLFVAGRF FAGFGVGLLS ATIPLYQSET APKWIRGTIV GAYQLAITIG LLLASVVNNA 180

TKDRMDTGCY RIPVAVQFAW AIILVVGMSV LPETPRFLIK KDRHEAAAKA LARLRRMNVD 240

DQAVVDELVE IRASHEYEMS VGKASFREIV TGSLGKRLAT GCAVQALQQL AGVNFIFYYG 300

TTFFQRSGIQ NSFTITLITN IVNVVSTFPG LYMVEKWGRR PLLLFGAVGM CVCQLIVAIV 360

GMVASSDVAN KVLIAFVCIY IFFFASSWGP VAWVVTGELY PLKARAKCLS ITTATNWLLN 420

WAIAYATPYM VDSGPGNANL QSKVFFIWGG FCFIAGVFVY TCIYETKGLS LEQVDELYSK 480

VSSAWRSPGF IPSAHFAGAD TEKAGPSVYE VEGELPQKRE SQHIETA 527

<210> 13

<211> 524

<212> PRT

<213> Aspergillus niger

<400> 13

MSSTTSSSRS DLEKVPVPQV IPRDSDSDKG SLSPEPSTLE AQSSEKPPHH IFTRSRKLQM 60

VCIVSLAAIF SPLSSNIYFP ALDDVSKSLN ISMSLATLTI TVYMIVQGLA PSFWGSMSDA 120

TGRRPVFIGT FIVYLVANIA LAESKNYGEL MAFRALQAAG SAATISIGAG VIGDITNSEE 180

RGSLVGIFGG VRMLGQGIGP VFGGIFTQYL GYRSIFWFLT IAGGVSLLSI LVLLPETLRP 240

IAGNGTVKLN GIHKPFIYTI TGQTGVVEGA QPEAKKTKTS WKSVFAPLTF LVEKDVFITL 300

FFGSIVYTVW SMVTSSTTDL FSEVYGLSSL DIGLTFLGNG FGCMSGSYLV GYLMDYNHRL 360

TEREYCEKHG YPAGTRVNLK SHPDFPIEVA RMRNTWWVIA IFIVTVALYG VSLRTHLAVP 420

IILQYFIAFC STGLFTINSA LVIDLYPGAS ASATAVNNLM RCLLGAGGVA IVQPILDALK 480

PDYTFLLLAG ITLVMTPLLY VEDRWGPGWR HARERRLKAK ANGN 524

<210> 14

<211> 34

<212> DNA

<213> Artificial sequence

<400> 14

gcatactagt atgggcgata attatgagga aaag 34

<210> 15

<211> 36

<212> DNA

<213> Artificial sequence

<400> 15

gcctaagctt ggcgttgtag ttatcgaccg gctctg 36

<210> 16

<211> 33

<212> DNA

<213> Artificial sequence

<400> 16

gcatactagt atgggtgtcg tacacgacct gga 33

<210> 17

<211> 35

<212> DNA

<213> Artificial sequence

<400> 17

gcctaagctt gtcctccttc ggcggcggcg aaaac 35

<210> 18

<211> 32

<212> DNA

<213> Artificial sequence

<400> 18

gcatactagt atggccaaac tatcaacatt ac 32

<210> 19

<211> 32

<212> DNA

<213> Artificial sequence

<400> 19

gcctaagctt ggatgaaaca ttcatagcgt ct 32

<210> 20

<211> 28

<212> DNA

<213> Artificial sequence

<400> 20

gcatactagt atgtttggct tcgacatc 28

<210> 21

<211> 31

<212> DNA

<213> Artificial sequence

<400> 21

gcctaagctt agccttattt tcaacatgct c 31

<210> 22

<211> 28

<212> DNA

<213> Artificial sequence

<400> 22

gcatactagt atgacggctc caatgtcg 28

<210> 23

<211> 32

<212> DNA

<213> Artificial sequence

<400> 23

gcctgaattc tacctcctct acctcagtag ca 32

<210> 24

<211> 37

<212> DNA

<213> Artificial sequence

<400> 24

gcatactagt atgtatcgca tttcgaatat ctacgtc 37

<210> 25

<211> 30

<212> DNA

<213> Artificial sequence

<400> 25

gcctaagctt cgccatttcg tcatgggtcg 30

<210> 26

<211> 32

<212> DNA

<213> Artificial sequence

<400> 26

attcggatcc atgccctatc ttctcgttac gc 32

<210> 27

<211> 35

<212> DNA

<213> Artificial sequence

<400> 27

gccaatcgat tgtagtatcc tgtgtgctaa atacc 35

<210> 28

<211> 36

<212> DNA

<213> Artificial sequence

<400> 28

gcatactagt atggctgaag gcttcgttga cgcctc 36

<210> 29

<211> 32

<212> DNA

<213> Artificial sequence

<400> 29

gcctaagctt cacctcctgg tgaaccgcag ta 32

<210> 30

<211> 28

<212> DNA

<213> Artificial sequence

<400> 30

gcctaagctt ctcgcggagc tcagtggg 28

<210> 31

<211> 28

<212> DNA

<213> Artificial sequence

<400> 31

gcatactagt atgggtgtcg aaacctcc 28

<210> 32

<211> 32

<212> DNA

<213> Artificial sequence

<400> 32

gcatactagt atgcctcgta agaggccgca ag 32

<210> 33

<211> 27

<212> DNA

<213> Artificial sequence

<400> 33

gcctaagctt cacctcagtt gtgtggg 27

<210> 34

<211> 28

<212> DNA

<213> Artificial sequence

<400> 34

gcatactagt atgggtgtcg aaacctcc 28

<210> 35

<211> 30

<212> DNA

<213> Artificial sequence

<400> 35

gcctaagctt cgcattctca gcgtggtaag 30

<210> 36

<211> 30

<212> DNA

<213> Artificial sequence

<400> 36

gcatactagt atgggtttct tgttgaagag 30

<210> 37

<211> 28

<212> DNA

<213> Artificial sequence

<400> 37

gcctaagctt agcggtctca atgtgctg 28

<210> 38

<211> 50

<212> DNA

<213> Artificial sequence

<400> 38

catccccagc atcattacac ctcgagatgg gcgataatta tgaggaaaag 50

<210> 39

<211> 58

<212> DNA

<213> Artificial sequence

<400> 39

tattaattaa ggccggcctt aagctcgagt caggcgttgt agttatcgac cggctctg 58

<210> 40

<211> 49

<212> DNA

<213> Artificial sequence

<400> 40

catccccagc atcattacac ctcgagatgg gtgtcgtaca cgacctgga 49

<210> 41

<211> 57

<212> DNA

<213> Artificial sequence

<400> 41

tattaattaa ggccggcctt aagctcgagt cagtcctcct tcggcggcgg cgaaaac 57

<210> 42

<211> 48

<212> DNA

<213> Artificial sequence

<400> 42

catccccagc atcattacac ctcgagatgg ccaaactatc aacattac 48

<210> 43

<211> 54

<212> DNA

<213> Artificial sequence

<400> 43

tattaattaa ggccggcctt aagctcgagt caggatgaaa cattcatagc gtct 54

<210> 44

<211> 44

<212> DNA

<213> Artificial sequence

<400> 44

catccccagc atcattacac ctcgagatgt ttggcttcga catc 44

<210> 45

<211> 53

<212> DNA

<213> Artificial sequence

<400> 45

tattaattaa ggccggcctt aagctcgagt caagccttat tttcaacatg ctc 53

<210> 46

<211> 44

<212> DNA

<213> Artificial sequence

<400> 46

catccccagc atcattacac ctcgagatga cggctccaat gtcg 44

<210> 47

<211> 54

<212> DNA

<213> Artificial sequence

<400> 47

tattaattaa ggccggcctt aagctcgagt catacctcct ctacctcagt agca 54

<210> 48

<211> 53

<212> DNA

<213> Artificial sequence

<400> 48

catccccagc atcattacac ctcgagatgt atcgcatttc gaatatctac gtc 53

<210> 49

<211> 52

<212> DNA

<213> Artificial sequence

<400> 49

tattaattaa ggccggcctt aagctcgagt cacgccattt cgtcatgggt cg 52

<210> 50

<211> 48

<212> DNA

<213> Artificial sequence

<400> 50

catccccagc atcattacac ctcgagatgc cctatcttct cgttacgc 48

<210> 51

<211> 57

<212> DNA

<213> Artificial sequence

<400> 51

tattaattaa ggccggcctt aagctcgagt catgtagtat cctgtgtgct aaatacc 57

<210> 52

<211> 52

<212> DNA

<213> Artificial sequence

<400> 52

catccccagc atcattacac ctcgagatgg ctgaaggctt cgttgacgcc tc 52

<210> 53

<211> 54

<212> DNA

<213> Artificial sequence

<400> 53

tattaattaa ggccggcctt aagctcgagt cacacctcct ggtgaaccgc agta 54

<210> 54

<211> 49

<212> DNA

<213> Artificial sequence

<400> 54

catccccagc atcattacac ctcgagatgg gtgtctctaa tatgatgtc 49

<210> 55

<211> 50

<212> DNA

<213> Artificial sequence

<400> 55

tattaattaa ggccggcctt aagctcgagt cactcgcgga gctcagtggg 50

<210> 56

<211> 48

<212> DNA

<213> Artificial sequence

<400> 56

catccccagc atcattacac ctcgagatgc ctcgtaagag gccgcaag 48

<210> 57

<211> 49

<212> DNA

<213> Artificial sequence

<400> 57

tattaattaa ggccggcctt aagctcgagt cacacctcag ttgtgtggg 49

<210> 58

<211> 44

<212> DNA

<213> Artificial sequence

<400> 58

catccccagc atcattacac ctcgagatgg gtgtcgaaac ctcc 44

<210> 59

<211> 52

<212> DNA

<213> Artificial sequence

<400> 59

tattaattaa ggccggcctt aagctcgagt cacgcattct cagcgtggta ag 52

<210> 60

<211> 46

<212> DNA

<213> Artificial sequence

<400> 60

catccccagc atcattacac ctcgagatgg gtttcttgtt gaagag 46

<210> 61

<211> 50

<212> DNA

<213> Artificial sequence

<400> 61

tattaattaa ggccggcctt aagctcgagt caagcggtct caatgtgctg 50

<210> 62

<211> 22

<212> DNA

<213> Artificial sequence

<400> 62

cggagattcg tcgcctaatg tc 22

<210> 63

<211> 22

<212> DNA

<213> Artificial sequence

ccgtcggtcg caatacaatc ac 22

<400> 63

<210> 64

<211> 47

<212> DNA

<213> Artificial sequence

<400> 64

catccccagc atcattacac ctcgagatgt cttcaaccac gtcttca 47

<210> 65

<211> 48

<212> DNA

<213> Artificial sequence

<400> 65

tattaattaa ggccggcctt aagctcgagc tagttgccgt tggctttg 48

<210> 66

<211> 47

<212> DNA

<213> Artificial sequence

<400> 66

acacgaacat cgacctgcag gcatgctgcc attggcggag gggtccg 47

<210> 67

<211> 48

<212> DNA

<213> Artificial sequence

<400> 67

cgacggccag tgccaagctt gcatgcggcg ggacgaacaa tccaattc 48

Claims

1. A glucose transporter protein, wherein the protein comprises any one of the following proteins:

b) Consisting of the sequence SEQ ID NO:1 to SEQ ID NO: 5 through substitution, deletion or addition of one or a plurality of amino acid residues, and has a) the function of the protein;

c) and a) the sequence of SEQ ID NO:1 to SEQ ID NO: 5 is higher than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the protein which is derived from aspergillus niger and has glucose transport function.

2. A fusion protein, wherein the fusion protein comprises the glucose transporter of claim 1, and a foreign polypeptide fused to the glucose transporter; optionally, the exogenous polypeptide comprises a tag polypeptide; preferably, the exogenous polypeptide comprises a tag polypeptide and a spacer polypeptide linking the tag polypeptide to the polypeptide having glucose transporter activity.

3. An expression cassette comprising a nucleotide sequence encoding the glucose transporter of claims 1-2.

4. An expression vector comprising a nucleic acid encoding the glucose transporter of claims 1-2, or the expression cassette of claim 3.

5. A recombinant host cell comprising the protein of claims 1-2, or the expression cassette of claim 3, or the expression vector of claim 4; further, the host cell is a host cell selected from the group consisting of Aspergillus niger (Aspergillus niger), Aspergillus nidulans (Aspergillus nidulans), Aspergillus oryzae (Aspergillus oryzae), Penicillium chrysogenum (Penicillium chrysogenum), Trichoderma reesei (Trichoderma reesei), Ustilago zeae (Ustilago maydis), Myceliophthora thermophila (Myceliophthora thermophila), preferably Aspergillus niger (Aspergillus niger), Aspergillus nidulans (Aspergillus nidulans).

6. A method of constructing an engineered strain for the production of organic acids, said method comprising enhancing the activity of a glucose transporter in the strain, said glucose transporter being a glucose transporter according to claims 1-2 or a polypeptide as set forth in sequence SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 12 is a glucose transporter represented by the amino acid sequence of any one of the above.

7. The method of claim 6, wherein the activity of the citrate transporter CexA in the strain is further enhanced, and the sequence of the CexA is shown in SEQ ID NO: 13 is shown in the figure; further, the activity of the citrate transporter CexA is enhanced by introducing an expression vector containing the CexA protein into the strain, or increasing the copy number in a host strain, or modifying a regulatory sequence thereof so as to achieve overexpression.

8. The method of claim 6 or 7, wherein the glucose transporter activity in the strain is enhanced by introducing an expression vector containing the glucose transporter into the strain, or by increasing the copy number in the host strain, or by modifying the regulatory sequences thereof, to achieve overexpression of the glucose transporter.

9. Construction process according to claim 6 or 7, wherein the host cell is selected from Aspergillus niger (Aspergillus niger), Aspergillus nidulans (Aspergillus nidulans), Aspergillus oryzae (Aspergillus oryzae), Penicillium chrysogenum (Penicillium chrysogenum), Trichoderma reesei (Trichoderma reesei), Ustilago zeae (Ustilago maydis), Myceliophthora thermophila (Myceliophthora thermophila), preferably Aspergillus nidulans (Aspergillus niger).

10. Construction process according to claim 6 or 7, wherein the organic acid is selected from citric acid, malic acid, succinic acid, itaconic acid, fumaric acid, preferably citric acid.

11. An organic acid producing strain in which the activity of a glucose transporter protein is enhanced and the activity of a citrate transporter protein is enhanced. Wherein the host cell is selected from the group consisting of Aspergillus niger (Aspergillus niger), Aspergillus nidulans (Aspergillus nidulans), Aspergillus oryzae (Aspergillus oryzae), Penicillium chrysogenum (Penicillium chrysogenum), Trichoderma reesei (Trichoderma reesei), Ustilago zeae (Ustilago maydis), Myceliophthora thermophila (Myceliophthora thermophila), preferably Aspergillus niger (Aspergillus niger), Aspergillus nidulans (Aspergillus nidulans).

12. The glucose transporter according to claims 1-2, the expression cassette according to claim 3, the vector according to claim 4, the host cell according to claim 5, the method of construction according to claims 6-10, the use of the producer strain according to claim 11 for the production of an organic acid selected from the group consisting of citric acid, malic acid, succinic acid, itaconic acid, fumaric acid and the like, preferably citric acid.

13. A method for producing an organic acid, comprising culturing the host cell according to claim 5, or culturing the strain constructed by the construction method according to claims 6 to 10, or the organic acid-producing strain according to claim 11 to produce an organic acid, and isolating the organic acid; the organic acid is selected from citric acid, malic acid, succinic acid, itaconic acid, fumaric acid and the like, and preferably citric acid.