CN115786342A

CN115786342A - Constitutive promoter, screening method of constitutive promoter element in aspergillus niger and application of constitutive promoter element

Info

Publication number: CN115786342A
Application number: CN202211444406.XA
Authority: CN
Inventors: 刘浩; 申洁; 朱洁; 曹威
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2022-11-18
Filing date: 2022-11-18
Publication date: 2023-03-14

Abstract

The invention discloses a constitutive promoter stably expressed in Aspergillus niger, which comprises an ANI _1_974074 promoter, namely a histone H4 promoter, an ANI _1_58174 promoter, namely an S- (hydroxymethyl) glutathione dehydrogenase promoter, an ANI _1_780184 promoter, namely a hypothetical protein promoter and an ANI _1_1402034 promoter, namely an L-arabitol dehydrogenase promoter. The identification and application of the novel constitutive promoter lay a foundation for the development and improvement of an Aspergillus niger expression system and the production of other metabolites, enrich a gene expression element library, and screen out constitutive promoter genes with different strengths stably expressed in Aspergillus niger, thereby providing more support for expressing internal/external genes by utilizing Aspergillus niger.

Description

Constitutive promoter, screening method of constitutive promoter element in aspergillus niger and application of constitutive promoter element

Technical Field

The invention belongs to the technical field of genetic engineering research, and particularly relates to a screening method and application of constitutive promoter elements in a constitutive promoter and aspergillus niger.

Background

Aspergillus niger is used as an important industrial fermentation strain, and has good industrial application prospects in aspects of protein expression, organic acid synthesis and the like. At present, a common strategy for improving the performance of industrial strains is to strengthen a synthetic approach of a target product through metabolic engineering. The promoter is an essential element of various expression systems, and is also an important component of protein expression vectors, and can directly determine the start time of transcription (expression) of a target gene and the concentration of mRNA in a cell at the transcription stage so as to influence the strength of gene expression. Since metabolism of substances and energy in the cells is very complicated, overexpression of a target gene sometimes results in not only an increase in the yield of a target product but also an increase in the load of cells due to excessive accumulation of metabolites, substrate deficiency, and metabolic imbalance. Therefore, the proper expression of the target gene can obtain better effect, and ideally, a series of promoters with proper strength can indirectly and effectively regulate metabolic flow and metabolic network to achieve specific targets.

At present, the reported constitutive promoter elements in the metabolic modification of aspergillus niger are limited, so that the invention excavates and identifies the aspergillus niger self promoter, and expects to screen more promoter elements, thereby enriching a gene expression original element library and providing element support for the construction of engineering strains.

Through searching, no patent publication related to the present patent application has been found.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provides a constitutive promoter, a screening method of constitutive promoter elements in Aspergillus niger and application thereof.

The technical scheme adopted by the invention for solving the technical problem is as follows:

a constitutive promoter stably expressed in a. Niger, comprising ANI _1_974074, a histone H4 promoter, ANI _1_58174, an S- (hydroxymethyl) glutathione dehydrogenase promoter, ANI _1_780184, a hypothetical protein promoter, and ANI _1_1402034, an L-arabitol dehydrogenase promoter.

Further, the nucleotide sequence of the ANI _1_974074 promoter, i.e., the histone H4 promoter, is SEQ ID No.6, the nucleotide sequence of the ANI _1_58174 promoter, i.e., the S- (hydroxymethyl) glutathione dehydrogenase promoter, is SEQ ID No.7, the nucleotide sequence of the ANI _1 _780184promoter, i.e., the hypothetical protein promoter, is SEQ ID No.8, and the nucleotide sequence of the ANI _1 _1402034promoter, i.e., the L-arabitol dehydrogenase promoter, is SEQ ID No.9.

Further, the constitutive promoter is a constitutive promoter of different strengths, and with reference to known constitutive promoters PgpdA, i.e., glyceraldehyde-3-phosphate dehydrogenase, ppptA, i.e., npgA protein, the promoter strengths are ordered from strong to weak as PgpdA > PpptA > ANI _1_974074 (histone H4) promoter > ANI _1_58174 (S- (hydroxymethyl) glutathione dehydrogenase) promoter > ANI _1_780184 (hypothetical protein) promoter > ANI _1_1402034 (L-arabitol dehydrogenase) promoter.

Further, the glyceraldehyde-3-phosphate dehydrogenase gene promoter PgpdA and the npgA protein gene promoter PpptA are promoters that control gene transcription.

Further, the constitutive promoter is derived from the genomes of aspergillus niger, aspergillus flavus, aspergillus oryzae, aspergillus sojae and aspergillus fumigatus, but is not limited to the promoter sequences of the same genes of other aspergillus species, for example. The similarity of the promoter sequences from aspergillus flavus, aspergillus oryzae, aspergillus sojae and aspergillus fumigatus and the promoter sequences from SEQ ID NO.6 to SEQ ID NO.9 is higher than 70%.

A method for assessing the expression strength of a constitutive promoter according to any one of claims 1 to 5 in A.niger, said method comprising the steps of:

(1) Extracting RNA from an Aspergillus niger strain S469, performing transcriptome sequencing, screening genes with stable gene expression quantity at different time points according to transcriptome data, grouping promoter genes screened according to the transcriptome data according to an FPKM value, selecting a promoter gene with the transcription intensity larger than PgpdA, a promoter gene with the transcription intensity between PgpdA and PpptA and a promoter gene with the transcription intensity smaller than PpptA, performing q-PCR verification on the screened promoter, and selecting genes with the transcription level difference smaller than 1 to obtain different promoter genes;

(2) Constructing a fixed-point expression cexA gene frame vector plasmid, and driving the expression of the citrate transporter cexA by using different screened promoter genes;

(3) Co-culturing agrobacterium containing promoter expression vectors with different strengths and an aspergillus niger host strain S834 to obtain aspergillus niger strains containing promoters with different strengths;

(4) The recombinant Aspergillus niger containing the expression vector is preliminarily applied: culturing the recombinant strain on PDA solid culture medium for 5 days, washing off spores with sterile water, collecting spores, counting, and culturing at 2 × 10 ⁶ Inoculating the cells/mL into 50mL of citric acid fermentation medium, culturing at 28 ℃ and 220rpm for 3 days, and measuring the yield of citric acid in fermentation liquor by using an HPLC method after sample preparation; meanwhile, the thalli is collected and crushed by a mortar, liquid nitrogen can be added for auxiliary grinding during the process, RNA is extracted by a reference RNA extraction kit FungalrNAKit (brand: OMEGA, product number: R6840-02), supernatant is obtained by centrifuging fermentation liquor of a reference RNA reverse transcription kit RevertaIdFirstStrandcDNAsSynthe, sisKit reverse transcription cDNA is used as a template, and the expression amount of the citrate transporter cexA under constitutive promoters with different strengths is verified.

Further, the sequence of the citrate transporter cexA in the step (2) is ANI _1 _478154at NCBI-locus _ tag.

A method for screening constitutive promoter genes with different strengths, which are stably expressed in Aspergillus niger, comprises the following steps:

(1) Screening of promoters at different transcription levels:

spores of the strain S469 (. DELTA.oahA) were collected and 1X 10 spores were collected ⁸ Inoculating each spore in 50mL Aspergillus niger liquid culture medium, culturing at 28 deg.C and 200rpm, respectively sampling at different time points, filtering the thallus with gauze, extracting RNA, performing transcriptome sequencing, and screening 168 genes stably transcribed at different time points in the fermentation process, wherein the two promoters with different strengths are PgpdA promoter, i.e. 3-glyceraldehyde phosphate dehydrogenase promoter, and PppTA promoter, i.e. npgA protein promoter, known in the art, and the PgpdA promoter strength is higher than the PppTA promoter strengthThe promoter strength is taken as a reference, and the promoter to be selected is divided into three parts, namely a promoter with an FPKM value higher than PgpdA, a promoter with an FPKM value between PgpdA and PppPTA, and a promoter with a partial FPKM value below PppPTA; performing q-PCR detection on the selected promoter gene, and selecting a promoter with the gene transcription difference multiple smaller than 1 according to the result to obtain promoters with different transcription levels;

(2) Evaluating the transcription intensity of the promoter, and constructing an expression vector of the expression cassette at a fixed point by the following steps:

on the basis of pLH924 (delta amyA), the cexA gene is amplified by taking an ATCC1015 genome as a template, and the cexA gene is inserted into an amyA site in a fixed point manner to form a frame vector pLH1099, namely cexA is expressed in the amyA site of the acid amylase gene in a fixed point manner; then, on the basis of pLH1099, promoter genes with different transcription levels obtained by screening in the step (1) are inserted into the site to start the expression of cexA;

(3) The promoter strength is characterized by the following construction method:

taking an aspergillus niger strain S834 (delta oahA, delta cexA) as a starting strain, taking a citrate transporter cexA as a reporter gene, and quantitatively analyzing the citric acid yield of each recombinant strain by using HPLC (high performance liquid chromatography), thereby verifying the strength of different screened constitutive promoters; (the recombinant strain contains promoters with different strengths, the promoters with different strengths drive the transcription of the cexA gene, cexA is a citrate transporter, and finally the yield of the citric acid is in direct proportion to the strength of the promoters, and the higher the yield is, the stronger the promoters are.)

(4) The constitutive promoters with different strengths and stable expression are obtained by screening, the promoter strength is ANI _1 _974074which is histone H4 from strong to weak, the promoter nucleotide sequence is SEQ ID NO.6, ANI _1 _58174which is S- (hydroxymethyl) glutathione dehydrogenase, the promoter nucleotide sequence is SEQ ID NO.7, ANI _1 _780184which is hypothetical protein, the promoter nucleotide sequence is SEQ ID NO.8, ANI _1 _1402034which is L-arabitol dehydrogenase, and the promoter nucleotide sequence is SEQ ID NO.9.

Further, the glyceraldehyde-3-phosphate dehydrogenase gene promoter PgpdA and the npgA protein gene promoter PpptA are promoters controlling gene transcription;

alternatively, the citrate transporter cexA sequence is ANI _1 _478154at NCBI-locus _ tag.

The constitutive promoter is applied to the construction of engineering strains and/or the utilization of Aspergillus niger expression internal/external genes.

The beneficial effects obtained by the invention are as follows:

1. the invention screens constitutive promoters with different strengths based on transcriptome data, establishes an evaluation method of the expression strength of the constitutive promoter in Aspergillus niger by taking a citrate transporter gene cexA as a reporter gene, and constructs a vector of the promoter site-specific expression gene. The identification and application of the novel constitutive promoter lay a foundation for the development and improvement of an Aspergillus niger expression system and the production of other metabolites, enrich a gene expression element library, and screen out constitutive promoter genes with different strengths stably expressed in Aspergillus niger, thereby providing more support for expressing internal/external genes by utilizing Aspergillus niger.

2. The constitutive promoter with different strengths and stable expression obtained by screening comprises ANI _1_974074 (histone H4), the nucleotide sequence is SEQ ID NO.6, the nucleotide sequence of an ANI _1_58174 (S- (hydroxymethyl) glutathione dehydrogenase) promoter is SEQ ID NO.7, the nucleotide sequence of an ANI _1_780184 (hypothetical protein) promoter is SEQ ID NO.8, and the nucleotide sequence of an ANI _1_1402034 (L-arabitol dehydrogenase) promoter is SEQ ID NO.9, so that more abundant promoter elements are provided for Aspergillus niger.

3. The invention screens constitutive promoters with different strengths based on transcriptomics technology, and establishes an evaluation method of the expression strength of the constitutive promoters in Aspergillus niger. The constitutive promoters obtained with different strengths of stable expression were screened and, with reference to the known constitutive promoters PgpdA (glyceraldehyde-3-phosphate dehydrogenase), ppptA (npgA protein), the promoter strengths were ranked from strong to weak for PgpdA > PpptA > ANI _1_974074 (histone H4) promoter > ANI _1_58174 (S- (hydroxymethyl) glutathione dehydrogenase) promoter > ANI _1_780184 (hypothetical protein) promoter > ANI _1 u 1402034 (L-arabitol dehydrogenase) promoter. The identification and application of the novel constitutive promoter lay a foundation for the development and improvement of an Aspergillus niger expression system and the iterative updating and reconstruction of metabolic engineering, and enrich a gene expression element library.

4. The invention provides a site-directed expression vector containing an Aspergillus niger constitutive expression element, which is characterized by taking an Aspergillus niger strain S834 (delta oahA, delta cexA) as an initial strain and a citric acid transporter cexA (NCBI-arcus _ tag is ANI _1_478154) as a reporter gene and quantitatively analyzing the citric acid yield of each strain by using HPLC (high performance liquid chromatography).

Drawings

FIG. 1 is a map of a plasmid pLH1099 of a fixed-point expression cexA gene frame vector constructed in the invention;

FIG. 2 is a double restriction enzyme digestion verification diagram of plasmid pLH1099 (Bgl II/SalI,12461bp/1621 bp) of the site-specific expression cexA gene frame vector constructed in the invention, wherein M is DNAmarker, and 1-2 is a Bgl II/SalI double restriction enzyme digestion verification plasmid;

FIG. 3 is a map of site-directed expression PcexA + cexA plasmid pLH1142 constructed in the present invention;

FIG. 4 is a diagram showing the single restriction enzyme digestion verification of the site-directed expression PcexA + cexA plasmid pLH1142 constructed in the present invention (SacI, 13626bp/1462 bp); wherein M is DNAmarker,1 is SacI which is single enzyme digestion verification plasmid;

FIG. 5 is a map of a site-directed expression PpptA + cexA plasmid pLH1143 constructed in the present invention;

FIG. 6 is a single restriction enzyme digestion verification map of pLH1143 plasmid pLH + cexA constructed in the present invention (Bgl II,14082bp/1006 bp), wherein M is DNArker, and 1-2 is a Bgl II single restriction enzyme digestion verification plasmid;

FIG. 7 is a map of the site-directed PgpdA + cexA plasmid pLH1145 constructed in the present invention;

FIG. 8 is the restriction enzyme digestion verification map of site-directed expression PgpdA + cexA plasmid pLH1145 constructed in the invention (Bgl II,14082bp/942 bp), wherein M is DNAmarker,1 is Bgl II single restriction enzyme digestion verification plasmid;

FIG. 9 is a map of site-directed expression ANI _1_974074 + cexA plasmid pLH1490 constructed in the present invention;

FIG. 10 is the restriction enzyme digestion verification map of site-directed expression ANI _1_974074 + cexA plasmid pLH1490 constructed in the present invention (BglII/KpnI, 122471bp/1835bp/1017 bp), wherein M is DNAmarker,1-2 is BglII/KpnI dual restriction enzyme digestion verification plasmid;

FIG. 11 is a map of site-directed expression ANI _1_58174 + cexA plasmid pLH1491 constructed in the present invention;

FIG. 12 shows the restriction enzyme digestion verification map of plasmid pLH1491 of site-directed expression ANI _1 \/58174 + cexA constructed in the present invention (Kpn I/SalI,10626bp/4483 bp), where M is DNArker and 1-2 is Kpn I/SalI restriction enzyme digestion verification plasmid;

FIG. 13 is a map of plasmid pLH1492 of site-directed expression ANI _1_780184+ cexA constructed in the present invention;

FIG. 14 is a restriction enzyme digestion verification map of site-directed expression ANI _1_780184 + cexA plasmid pLH1492 constructed in the present invention (SpeI, 9327bp/5761 bp), wherein M is DNAmarker, and 1-2 is SpeI single restriction enzyme digestion verification plasmid;

FIG. 15 is a map of plasmid pLH1493 of site-directed expression ANI _1_1402034+ cexA constructed in the present invention;

FIG. 16 is the restriction enzyme digestion verification map of site-directed expression ANI _1_1402034 + cexA plasmid pLH1493 (SalI/KpnI, 10626bp/4488 bp), wherein M is DNAmarker, and 1 is SalI/KpnI double restriction enzyme digestion verification plasmid;

FIG. 17 is a graph showing the results of relative expression of cexA under promoters of different transcription strengths in the present invention;

FIG. 18 is a graph showing citric acid production analysis of the Aspergillus niger starting strain S549 (S549 is a starting strain and has been disclosed, and the published articles (. DELTA.oahA) and S834 (. DELTA.cexA,. DELTA.oahA) and strains S2006, S2009, S2313, S2011, S2014, S2505, S2541, S2510, S2511 containing promoters of different strengths in the present invention.

Detailed Description

The present invention will be described in further detail with reference to examples for better understanding, but the scope of the present invention is not limited to the examples.

The raw materials used in the invention are all conventional commercial products unless otherwise specified, the methods used in the invention are all conventional in the field, and the quality of each substance used in the invention is conventional quality.

Preferably, the nucleotide sequence of the ANI _1 \/974074 promoter, i.e., histone H4 promoter, is SEQ ID No.6, the nucleotide sequence of the ANI _1 \/58174 promoter, i.e., S- (hydroxymethyl) glutathione dehydrogenase promoter, is SEQ ID No.7, the nucleotide sequence of the ANI _1 \/780184 promoter, i.e., the nucleotide sequence of the hypothetical protein promoter, is SEQ ID No.8, and the nucleotide sequence of the ANI _1 \/1402034 promoter, i.e., L-arabitol dehydrogenase promoter, is SEQ ID No.9.

Preferably, the constitutive promoter is a constitutive promoter of different strengths, with reference to the known constitutive promoters PgpdA, i.e. glyceraldehyde-3-phosphate dehydrogenase, ppptA, i.e. npgA protein, the promoter strengths being ordered from strong to weak as PgpdA > PpptA > ANI _1_974074 (histone H4) promoter > ANI _1_58174 (S- (hydroxymethyl) glutathione dehydrogenase) promoter > ANI _1_780184 (hypothetical protein) promoter > ANI _1_1402034 (L-arabitol dehydrogenase) promoter.

Preferably, the glyceraldehyde-3-phosphate dehydrogenase gene promoter PgpdA and the npgA protein gene promoter PpptA are promoters controlling gene transcription.

Preferably, the constitutive promoter is derived from the genomes of aspergillus niger, aspergillus flavus, aspergillus oryzae, aspergillus sojae, and aspergillus fumigatus, but is not limited to promoter sequences of the same genes of other aspergillus species, for example. The similarity of the promoter sequences from aspergillus flavus, aspergillus oryzae, aspergillus sojae and aspergillus fumigatus and the promoter sequences from SEQ ID NO.6 to SEQ ID NO.9 is higher than 70%.

A method for evaluating the expression strength of a constitutive promoter as described above in A.niger, said method comprising the steps of:

(1) Extracting RNA from an Aspergillus niger strain S469, performing transcriptome sequencing, screening genes with stable gene expression quantity at different time points according to transcriptome data, grouping promoter genes screened according to the transcriptome data according to an FPKM value, selecting a promoter gene with the transcription intensity greater than PgpdA, a promoter gene with the transcription intensity between PgpdA and PpptA and a promoter gene with the transcription intensity less than PpptA, performing q-PCR verification on the screened promoter, and selecting genes with the transcription level difference less than 1 to obtain different promoter genes;

(4) The recombinant Aspergillus niger containing the expression vector is applied preliminarily: culturing the recombinant strain on PDA solid culture medium for 5 days, washing off spores with sterile water, collecting spores, counting, and culturing at 2 × 10 ⁶ Inoculating the cells/mL into 50mL of citric acid fermentation medium, culturing at 28 ℃ and 220rpm for 3 days, and measuring the yield of citric acid in fermentation liquor by using an HPLC method after sample preparation; meanwhile, collecting thalli, crushing the thalli by using a mortar, adding liquid nitrogen for auxiliary grinding during the process, extracting RNA by using a reference RNA extraction kit FungalRNAKit (brand: OMEGA, product number: R6840-02), centrifuging fermentation liquor by using a reference RNA reverse transcription kit RevertAidFirstStrandcDNAsSynthe, taking supernatant, carrying out reverse transcription of cDNA by using a sisKit, and verifying the expression amount of the citrate transporter cexA under constitutive promoters with different strengths by using recombinant strain cDNA as a template.

Preferably, the sequence of the citrate transporter cexA in the step (2) is ANI _1 _478154at NCBI-locus _ tag.

(1) Screening of promoters at different transcription levels:

spores of the strain S469 (. DELTA.oahA) were collected and 1X 10 spores were collected ⁸ Inoculating the spores into 50mL Aspergillus niger liquid culture medium, and culturing at 28 deg.C for 200rpm culture, respectively sampling at different time points, filtering thalli by gauze, extracting RNA, performing transcriptome sequencing, screening 168 genes which are stably transcribed at different time points in the fermentation process, dividing the promoters to be selected into three parts, namely a promoter with an FPKM value higher than PgpdA, a promoter with an FPKM value between PgpdA and PppPTA and a promoter with a partial FPKM value below PppPTA according to two promoters with different strengths, namely a 3-glyceraldehyde phosphate dehydrogenase promoter and a PppPTA promoter which are known in the field, namely an npgA protein promoter; performing q-PCR detection on the selected promoter gene, and selecting a promoter with the gene transcription difference multiple smaller than 1 according to the result to obtain promoters with different transcription levels;

on the basis of pLH924 (delta amyA), the cexA gene is amplified by taking an ATCC1015 genome as a template, and the cexA gene is inserted into an amyA site in a fixed point manner to form a frame vector pLH1099, namely cexA is expressed in the amyA site of the acid amylase gene in a fixed point manner; then, on the basis of pLH1099, promoter genes with different transcription levels obtained by screening in the step (1) are inserted in a fixed point manner to start the expression of cexA;

taking an aspergillus niger strain S834 (delta oahA, delta cexA) as a starting strain, taking a citrate transporter cexA as a reporter gene, and quantitatively analyzing the citric acid yield of each recombinant strain by using HPLC (high performance liquid chromatography), thereby verifying the strength of different screened constitutive promoters; (the recombinant strain contains promoters with different strengths, the promoters with different strengths drive the transcription of the cexA gene, cexA is a citrate transporter, and finally the yield of the citric acid is in direct proportion to the strength of the promoters, and the higher the yield, the stronger the promoters are)

Preferably, the glyceraldehyde-3-phosphate dehydrogenase gene promoter PgpdA and the npgA protein gene promoter Ppppta are promoters for controlling gene transcription;

Specifically, the preparation and detection are as follows:

example 1-a method for screening constitutive promoter elements that can stably express genes, the specific construction process is as follows:

screening and providing a constitutive promoter element capable of stably expressing gene, culturing strain S469 on PDA solid culture medium for 5 days, washing S469 spores with sterile water, collecting spores, counting, collecting 1 × 10 ⁸ Inoculating spores into an Aspergillus niger liquid culture medium, culturing at 28 ℃, 200rpm, sampling at different time points of 10, 14, 18, 22, 26h and the like, filtering thallus warps, washing with sterile water, crushing the thallus by using a mortar, adding liquid nitrogen for auxiliary grinding during the period, extracting RNA by using a reference RNA extraction kit Fungalrnakt (brand: OMEGA, product number: R6840-02), sequencing transcriptome, screening genes with stable gene expression at different time points according to transcriptome data, grouping constitutive promoters screened according to the transcriptome data, and selecting a certain amount of promoters for q-PCR verification.

The reference RNA reverse transcription kit RevertAidFirstStrandcDNAsynthesis kit reverses cDNA. The expression amounts of the selected constitutive promoters at different time points are verified by using S469 strain cDNA as a template, and the promoter strengths are arranged in sequence from strong to weak, namely ANI _1_974074 (histone H4), ANI _1_58174 (S- (hydroxymethyl) glutathione dehydrogenase), ANI _1_780184 (hypothetical protein) and ANI _1_1402034 (L-arabitol dehydrogenase).

Example 2 construction of site-directed expression of cexA Gene framework vector plasmid: plasmid pLH1099 (FIG. 1) was engineered from the pLH924 vector.

The specific construction process is as follows:

taking ATCC1015 genome as a template, cexA-R and cexA-F as primers to carry out PCR amplification on a cexA gene sequence fragment, wherein the nucleotide sequence is SEQ ID NO.1, the length is 1575bp, the amino acid sequence is SEQ ID NO.2, and the length is 525aa; the cexA gene sequence fragment was ligated to the starting vector PLH924 linearized by BglII and BamHI using Novozan C113-Clon express-MultiSONeStepCloning kit, the ligation product was transformed into E.coli JM109 competent cells, which were then spread evenly on LB plates containing 100. Mu.g/mL kanamycin, cultured overnight at 37 ℃, single clones were selected, verified by double digestion (FIG. 2) to obtain plasmid pLH1099, and primers designed for amplifying the cexA gene sequence fragment were cexA-R and cexA-F (Table 1).

Example 3 construction of site-directed expression of the cexA Gene-framework A.niger strains: agrobacterium containing plasmid pLH1099 (published, see article (DOI: 10.27359/d.cnki.gtqgu.2020.000424)) was co-cultured with Aspergillus niger host strain S834 (published, see article (DOI: 10.27359/d.cnki.gtqgu.2020.000424)) in an IM plate, and the co-culture was then transferred to CM plates containing 200. Mu.M cefotaxime, 100. Mu.g/mL ampicillin, 100. Mu.g/mL streptomycin, 250. Mu.g/mL hygromycin B and cultured at 28 ℃ until monoclones were formed. Selecting a single clone to transfer to a PDA plate B containing hygromycin B and an MM plate containing glufosinate-ammonium, screening transformants with hygromycin B resistance and glufosinate-ammonium sensitivity, extracting a genome, and verifying to obtain a cexA gene site-directed expression strain S2006.

Example 4 construction of a plasmid expressing PcexA + cexA Gene at a site: plasmid pLH1142 (FIG. 3) was engineered from the PLH1099 vector.

The specific construction process is as follows: PCR amplification is carried out on Pcexa gene sequence fragments by taking ATCC1015 genome as a template and Pcexa-R and Pcexa-F as primers, the nucleotide sequence is SEQ ID NO.3, the length is 1000bp, the Pcexa gene sequence fragments are connected with a starting vector PLH1099 linearized by BglII single enzyme digestion by using a Novozac 113-Clon express-multisoneStepCringing kit, a connecting product is transformed into escherichia coli JM109 competent cells, the competent cells are uniformly coated in an LB culture dish containing 100 mu g/mL kanamycin and cultured overnight at 37 ℃, single clones are selected, and the plasmid PLH1142 is obtained by single enzyme digestion verification (figure 4). Primers were designed to amplify the PcexA gene sequence fragment as PcexA-R, pcexA-F (Table 1).

Example 5 construction of site-directed expression of PcexA + cexA plasmid Aspergillus niger strains: agrobacterium containing plasmid pLH1142 (published, see article (DOI: 10.27359/d.cnki.gtqgu.2020.000424) was co-cultured with Aspergillus niger host strain S834 in IM plates, and the co-culture was then transferred to CM plates containing 200. Mu.M cefotaxime, 100. Mu.g/mL ampicillin, 100. Mu.g/mL streptomycin, 250. Mu.g/mL hygromycin B and cultured at 28 ℃ until monoclonals were formed. And (3) selecting a single clone to be transferred to a PDA (personal digital assistant) plate containing hygromycin B and an MM (methyl methacrylate) plate containing glufosinate-ammonium, screening transformants resistant to hygromycin B and sensitive to glufosinate-ammonium, and extracting a genome for verification to obtain PcexA + cexA gene site-specific expression strains S2009 and S2313.

Example 6 construction of a plasmid for site-directed expression of PpptA + cexA Gene, plasmid pLH1143 (FIG. 5) was engineered from PLH1099 vector.

The specific construction process is as follows: PCR amplification is carried out by taking ATCC1015 genome as template and PpptA-R and PpptA-F as primers to obtain PpptA promoter sequence fragment, the nucleotide sequence of which is SEQ ID NO.4 and the length of which is 1000bp, the PpptA gene sequence fragment is connected with an original vector PLH1099 linearized by BglII single enzyme digestion by using a Novonophen C113-Clonexpress-multisoneStepCringing kit, the connection product is transformed into Escherichia coli JM109 competent cells, the competent cells are evenly coated in an LB culture dish containing 100 mu g/mL kanamycin and cultured overnight at 37 ℃, single clone is picked, and the plasmid PLH1143 is obtained by single enzyme digestion verification (figure 6). Primers were designed to amplify PcaxA gene sequence fragments as PpptA-R and PpptA-F (Table 1).

Example 7 construction of site-directed expression of PpptA + cexA plasmid aspergillus niger strains: agrobacterium containing plasmid pLH1143 (published, see article (DOI: 10.27359/d.cnki.gtqgu.2020.000424) was co-cultured with Aspergillus niger host strain S834 in IM plates, and the co-culture was then transferred to CM plates containing 200. Mu.M cefotaxime, 100. Mu.g/mL ampicillin, 100. Mu.g/mL streptomycin, 250. Mu.g/mL hygromycin B and cultured at 28 ℃ until monoclonals were formed. And selecting a single clone to transfer to a PDA plate containing hygromycin B and a MM plate containing glufosinate-ammonium, screening transformants with hygromycin B resistance and glufosinate-ammonium sensitivity, extracting a genome, and verifying to obtain the PpptA + cexA gene site-directed expression strain S2011.

Example 8 construction of a plasmid for site-directed expression of the PgpdA + cexA Gene, plasmid pLH1145 (FIG. 7) was engineered from the PLH1099 vector.

The specific construction process is as follows: PCR amplification is carried out by taking ATCC1015 genome as template and PgpdA-R and PgpdA-F as primers to obtain PgpdA promoter sequence fragment, the nucleotide sequence is SEQ ID NO.5, the length is 936bp, a Novozan C113-Clon express-multisoneStepClingkit kit is used to connect the PgpdA gene sequence fragment with a starting vector PLH1099 after BglII single enzyme digestion linearization, a connecting product is transformed into JM Escherichia coli 109 competent cells and is evenly coated in an LB culture dish containing 100 mu g/mL kanamycin for overnight culture at 37 ℃, a single clone is picked up, and the plasmid PLH1145 is obtained through single enzyme digestion verification (figure 8). Primers were designed for PgpdA-R and PgpdA-F (Table 1).

Example 9 construction of site-directed PgpdA + cexA plasmid Aspergillus niger strains: agrobacterium containing plasmid pLH1145 (published, see article (DOI: 10.27359/d.cnki.gtqgu.2020.000424) was co-cultured with Aspergillus niger host strain S834 in IM plates, and the co-culture was then transferred to CM plates containing 200. Mu.M cefotaxime, 100. Mu.g/mL ampicillin, 100. Mu.g/mL streptomycin, 250. Mu.g/mL hygromycin B and cultured at 28 ℃ until monoclonals were formed. Selecting a single clone to transfer to a PDA plate containing hygromycin B and an MM plate containing glufosinate-ammonium, screening transformants with hygromycin B resistance and glufosinate-ammonium sensitivity, extracting a genome for verification, and obtaining a PgpdA + cexA gene site-directed expression strain S2014.

Example 10 construction of plasmid for site-directed expression of ANI _1 \/974074 + cexA Gene, plasmid pLH1490 (FIG. 9) was modified from PLH1099 vector.

The specific construction process is as follows: PCR amplification of gene ANI _1 _974074promoter sequence fragment with nucleotide sequence SEQ ID NO.6 and length 1011bp using Primer4505/4506 as template, ANI _1 _974074gene sequence fragment was ligated with BglII single enzyme linearized starting vector PLH1099 using Novozam C113-Clon express-multisoneStepcloning kit, the ligation product was transformed into E.coli JM109 competent cells, spread evenly on kanamycin LB culture dish containing 100. Mu.g/mL, cultured overnight at 37 ℃, single clone was picked, and plasmid PLH1490 was obtained by single enzyme digestion verification (FIG. 10). Primers 4505 and 4506 (table 1) were designed for amplifying the ANI _1_974074 gene sequence fragment.

Example 11 construction of site-directed expression of ANI _1_974074+ cexA plasmid Aspergillus niger strains: agrobacterium containing plasmid pLH1490 (published, see article (Chinese Notification, DOI:10.27359/d.cnki. Gtqgu.2020.000424)) was co-cultured with Aspergillus niger host strain S834 on IM plates, and the co-culture was then transferred to CM plates containing 200. Mu.M cefotaxime, 100. Mu.g/mL ampicillin, 100. Mu.g/mL streptomycin, 250. Mu.g/mL hygromycin B and cultured at 28 ℃ until monoclonals were formed. Selecting a single clone to transfer to a PDA plate containing hygromycin B and an MM plate containing glufosinate-ammonium, screening transformants with hygromycin B resistance and glufosinate-ammonium sensitivity, extracting a genome and verifying, and obtaining the ANI _1_974074 + cexA gene site-directed expression strain S2505.

Example 12 construction of plasmid for site-directed expression of ANI _1 \/58174 + cexA Gene, plasmid pLH1491 (FIG. 11) was modified from PLH1099 vector.

The specific construction process is as follows: PCR amplification of the gene ANI _1 _58174promoter sequence fragment with the nucleotide sequence of SEQ ID NO.7 and the length of 1687bp was carried out using ATCC1015 genome as template and Primer4507/4508 as Primer, the ANI _1 _58174gene sequence fragment was ligated with BglII single-enzyme linearized starting vector PLH1099 using Novozam C113-Clon express-multisoneStepcloning kit, the ligation product was transformed into competent cells of Escherichia coli JM109, and spread evenly on LB culture dish containing 100. Mu.g/mL kanamycin, cultured overnight at 37 ℃, single clone was picked, and plasmid PLH1491 was obtained by single-enzyme digestion verification (FIG. 12). Primers 4507 and 4508 (table 1) were designed for amplifying the ANI _1 _58174gene sequence fragment.

Example 13 construction of site-directed expression of ANI _1_58174+ cexA plasmid Aspergillus niger strains: agrobacterium containing plasmid pLH1491 (published, see article (Chinese Notification, DOI:10.27359/d.cnki. Gtqgu.2020.000424)) was co-cultured with Aspergillus niger host strain S834 on IM plates, and the co-culture was then transferred to CM plates containing 200. Mu.M cefotaxime, 100. Mu.g/mL ampicillin, 100. Mu.g/mL streptomycin, 250. Mu.g/mL hygromycin B and cultured at 28 ℃ until monoclonals were formed. Selecting a single clone to transfer to a PDA plate containing hygromycin B and an MM plate containing glufosinate-ammonium, screening transformants with hygromycin B resistance and glufosinate-ammonium sensitivity, extracting a genome and verifying, and obtaining an ANI _1_58174 + cexA gene site-directed expression strain S2541.

Example 14 construction of plasmid for site-directed expression of ANI _1_780184+ cexA Gene, plasmid pLH1492 (FIG. 13) was engineered from PLH1099 vector.

The specific construction process is as follows: a PCR amplification gene ANI _1 _780184promoter sequence fragment is carried out by taking ATCC1015 genome as a template and Primer4509/45010 as a Primer, the nucleotide sequence is SEQ ID NO.8, the length is 836bp, the ANI _1 _780184gene sequence fragment is connected with a starting vector PLH1099 linearized by BglII single enzyme digestion by using a Novozam C113-Clon express-multisoneStepcloning kit, a connection product is transformed into an Escherichia coli JM109 competent cell and is evenly coated on an LB culture dish containing 100 mug/mL kanamycin for overnight culture at 37 ℃, a single clone is picked up, and the plasmid pLH1492 is obtained by single enzyme digestion verification (figure 14). Primers for amplifying ANI _1 \/780184 gene sequence fragment were designed as Primer4509 and Primer4510 (Table 1).

Example 15 construction of site-directed expression of ANI _1_780184+ cexA plasmid Aspergillus niger strains: agrobacterium containing plasmid pLH1492 (published, see article (Chinese Notification, DOI:10.27359/d.cnki. Gtqgu.2020.000424)) was co-cultured with Aspergillus niger host strain S834 on IM plates, and the co-culture was then transferred to CM plates containing 200. Mu.M cefotaxime, 100. Mu.g/mL ampicillin, 100. Mu.g/mL streptomycin, 250. Mu.g/mL hygromycin B and cultured at 28 ℃ until monoclonals were formed. A single clone is selected to be transferred to a PDA plate containing hygromycin B and an MM plate containing glufosinate-ammonium, transformants resistant to hygromycin B and sensitive to glufosinate-ammonium are screened, and a genome is extracted for verification, so that an ANI _1_780184 + cexA gene site-directed expression strain S2510 is obtained.

Example 16 construction of plasmid for site-directed expression of ANI _1_1402034+ cexA Gene, plasmid pLH1493 (FIG. 15) was modified from PLH1099 vector.

The specific construction process is as follows: a gene ANI _1_1402034 promoter sequence fragment is amplified by PCR by taking an ANI _1_1402034 genome as a template and a Primer Primer4511/4512 as a Primer, the nucleotide sequence of the gene ANI _1_1402034 promoter sequence fragment is SEQ ID NO.9, the length of the gene ANI _1_1402034 sequence fragment is 1356bp, the ANI _1_1402034 gene sequence fragment and a starting vector pLH1099 after BglII single enzyme digestion linearization kit are connected by applying a Novonauzin C113-Clonexpress-MultiSONe StepClingkit kit, a connection product is transformed into Escherichia coli JM109 competent cells, the competent cells are evenly spread in a culture dish containing 100 mu g/mL LB kanamycin and are cultured overnight at 37 ℃, a single clone is selected, and the plasmid PLH1493 is obtained through single enzyme digestion verification (figure 16). Primers for amplifying ANI _1 \/1402034 gene sequence fragment were designed as Primer4511 and Primer4512 (Table 1).

Example 17 construction of site-directed expression of ANI _1_1402034+ cexA plasmid A. Niger strains: agrobacterium containing plasmid pLH1493 (published, see article (Chinese Notification, DOI:10.27359/d.cnki. Gtqgu.2020.000424)) was co-cultured with Aspergillus niger host strain S834 on IM plates, and the co-culture was then transferred to CM plates containing 200. Mu.M cefotaxime, 100. Mu.g/mL ampicillin, 100. Mu.g/mL streptomycin, 250. Mu.g/mL hygromycin B and cultured at 28 ℃ until monoclonals were formed. Selecting a single clone to be transferred to a PDA plate containing hygromycin B and an MM plate containing glufosinate-ammonium, screening transformants with hygromycin B resistance and glufosinate-ammonium sensitivity, extracting a genome and verifying, and obtaining the ANI _1_1402034 + cexA gene site-directed expression strain S2511.

Example 18-The recombinant Aspergillus niger containing the expression vector is preliminarily applied: collecting spores of the Aspergillus niger strain at a ratio of 2 × 10 ⁶ Inoculating the strain/mL into 50mL of citric acid fermentation medium, culturing at 28 ℃ and 220rpm for 3 days, centrifuging fermentation liquor, taking supernatant, preparing a sample, measuring the yield of citric acid in the fermentation liquor by using an HPLC method, collecting thalli, crushing the thalli by using a mortar, extracting RNA, inverting cDNA, taking the recombinant strain cDNA as a template, and verifying the expression quantity of the citrate transporter cexA under constitutive promoters with different strengths.

Through analysis, the citric acid yield of the recombinant strain containing a stronger promoter is higher than that of the strain containing a weaker promoter, and the relative expression quantity of the citrate transporter cexA is known to be consistent with the strength of the promoter of the recombinant strain from a q-PCR result, namely, the stronger the promoter is, the higher the expression quantity of the cexA is, and under an ideal state, the specific target of indirectly and effectively regulating and controlling metabolic flow and metabolic network can be realized by using a series of promoters with proper strength.

Preferably, the citric acid fermentation medium comprises the following main components: sucrose 100g/L, (NH) ₄ ) ₂ SO ₄ 4.125g/L，MgSO ₄ ·7H ₂ O1g/L，KH ₂ PO ₄ 1g/L and yeast extract 0.5g/L.

Table 1 primer sequences used in the examples

Wherein (1) the above LB medium, PDA medium, IM medium, CM medium, MM medium are the same as those described in the publication of Susbania et al, in the establishment of the Aspergillus niger genetic transformation system using amdS as a screening marker (Susbania et al, chinese agricultural science and technology, 2020,22 (09): 179-187.).

The strain S469 is a strain used in an issued patent (Chinese patent, patent number: ZL 201810985901.9) obtained by the inventor in the prior art.

The starting strain S834 (. DELTA.oahA,. DELTA.cexA) was used in a prior published article (Chinese information network, DOI:10.27359/d.cnki. Gtqgu.2020.000424).

The Aspergillus niger liquid medium used in the above is the medium used in the published literature (DOI: 10.27359/d.cnki.gtqgu.2020.000424).

The plasmid pLH924 is a vector used in the published literature (Chinese Notification, DOI: 10.3389/fmicb.2022.1009491).

The related sequences of the invention are as follows:

SEQ ID NO.1: nucleotide sequence 1575bp of cexA

ATGTCTTCAACCACGTCTTCATCAAGATCAGACCTTGAAAAGGTCCCCGTACCACAG

GTCATCCCTAGAGACAGTGACTCCGATAAGGGATCCCTCTCTCCGGAGCCTTCGACCC

TAGAGGCTCAGTCATCCGAGAAGCCACCGCATCATATCTTCACACGGTCTCGCAAGCT

GCAAATGGTTTGCATCGTCTCCCTCGCTGCCATATTTTCTCCGCTTTCGTCGAACATTT

ACTTCCCTGCCCTGGATGATGTCTCGAAATCCCTCAACATCAGCATGTCGCTCGCAAC

ACTCACCATCACGGTGTACATGATCGTCCAAGGCCTCGCTCCCAGCTTCTGGGGTTCC

ATGTCAGACGCCACAGGTAGACGGCCTGTCTTTATTGGAACATTCATTGTTTACCTCGT

AGCCAATATTGCTCTGGCCGAATCCAAGAACTATGGTGAGCTCATGGCCTTCCGAGCC

TTGCAGGCTGCTGGTAGCGCGGCCACCATCTCAATCGGAGCTGGAGTGATTGGTGATA

TCACAAACTCGGAAGAAAGAGGTAGCTTGGTGGGTATCTTCGGTGGAGTTCGCATGC

TTGGACAGGGAATCGGGCCGGTTTTCGGCGGCATTTTCACCCAGTATCTCGGATATCG

ATCTATCTTTTGGTTCCTCACGATTGCTGGAGGCGTGAGTCTCCTGTCCATTCTGGTGC

TTCTTCCGGAGACATTGAGACCAATTGCTGGAAATGGAACTGTGAAGCTCAATGGCA

TTCATAAGCCCTTCATCTACACGATCACCGGCCAGACGGGGGTTGTCGAGGGAGCGC

AACCGGAAGCGAAAAAGACCAAAACCAGCTGGAAGTCTGTTTTTGCTCCTTTGACAT

TCCTCGTCGAAAAGGACGTTTTCATCACCCTGTTCTTTGGAAGTATCGTGTACACAGT

GTGGAGCATGGTGACATCCAGTACCACCGACCTCTTCAGCGAAGTGTACGGCCTGTC

ATCCCTGGACATTGGACTCACTTTCCTAGGCAATGGCTTTGGATGTATGTCTGGCTCTT

ATCTGGTCGGCTACCTTATGGATTACAACCACCGTCTTACCGAACGCGAATATTGCGA

GAAACACGGTTATCCGGCAGGCACACGTGTCAATCTGAAATCACACCCCGACTTCCC

CATTGAGGTCGCCCGGATGCGCAATACCTGGTGGGTGATTGCGATCTTCATCGTGACA

GTTGCTTTGTACGGCGTGTCTTTGCGGACACATCTGGCGGTGCCTATCATTCTGCAGT

ACTTCATTGCGTTCTGCTCAACAGGACTCTTCACCATCAACAGCGCCCTGGTCATCGA

TCTTTACCCAGGTGCTAGCGCCAGTGCGACAGCAGTGAACAATCTGATGCGGTGCCT

GCTTGGAGCTGGCGGTGTGGCTATCGTGCAACCTATCCTGGACGCCTTGAAGCCGGA

TTATACTTTCCTCTTGCTTGCCGGCATCACCCTCGTGATGACTCCGTTGCTGTACGTCG

AAGATCGATGGGGTCCTGGCTGGCGACATGCCCGCGAAAGGAGACTCAAGGCCAAA

GCCAACGGCAACTAG

SEQ ID No.2: amino acid sequence 525aa of cexA

MSSTTSSSRSDLEKVPVPQVIPRDSDSDKGSLSPEPSTLEAQSSEKPPHHIFTRSRKLQMV

CIVSLAAIFSPLSSNIYFPALDDVSKSLNISMSLATLTITVYMIVQGLAPSFWGSMSDATGR

RPVFIGTFIVYLVANIALAESKNYGELMAFRALQAAGSAATISIGAGVIGDITNSEERGSLV

GIFGGVRMLGQGIGPVFGGIFTQYLGYRSIFWFLTIAGGVSLLSILVLLPETLRPIAGNGTV

KLNGIHKPFIYTITGQTGVVEGAQPEAKKTKTSWKSVFAPLTFLVEKDVFITLFFGSIVYT

VWSMVTSSTTDLFSEVYGLSSLDIGLTFLGNGFGCMSGSYLVGYLMDYNHRLTEREYCE

KHGYPAGTRVNLKSHPDFPIEVARMRNTWWVIAIFIVTVALYGVSLRTHLAVPIILQYFIAF

CSTGLFTINSALVIDLYPGASASATAVNNLMRCLLGAGGVAIVQPILDALKPDYTFLLLAG

ITLVMTPLLYVEDRWGPGWRHARERRLKAKANGN*

SEQ ID NO.3: pcexa nucleotide sequence 1000bp

GTAACCCACTACACCTGAGCAGTTCGCCAATCAGGCTGATCTCAGATTGCTCATACTG

GTGCGCGTGCGTGCTCCCTGTTTCCTACAACTACTCCTCCTGAAGAGGGGGGATAGG

GACCCTGGCGCCCTGAGTCGTCAATTGATGACCTGCCTTGCCGTCTCGCGTTGCATCG

GGCCTCTCCGTGTTGATCACAGCTTAGCTTCTGCGTGGGAGACAGCCTTCTCCGTCAA

CACATGTGGGAGATGTTGGCTGAGAAGAGTCGACGGCCTATCTACTCCATACCATGTA

GCCCAACGCTCCGCCACGGCGCCACTGAATAGCCTGCTCAGCTCCCTTATCACGGGC

TCCGCTTCCATCTAGACCCTTGCGCATGACAGGCGTGCCCGGTCCTTCAAACACACCA

TTCGCTGGAAATCTGTATGCTAAGTTGACTAAATCCGTCAGCTCTTGAGGTGCAGGCG

CTAGTCGTAGTCCAGGATGGCCTGGAAAGCATGCTTGTTCTGGAATTTCATCACCACG

CCGGGCCCACGTCATGTATGCAGATCTTGGTAGCTCCGCCCTTTTGTCCTTTCAATTTT

ATTTTTTTTTCTCCCTCTTTCTTCGTCGGCTGCCCGACGGCTTGGACTCTCTCGGATGT

GACCTAGACTACTGGTCGCCAAGTAAGACCGGCCGAAGAGAAACTCCTAAACCCAC

GTCTCCGTTCATACCTTGGCGATAACACCGGCTCTTGCCACCCACATTTGCCCGCTTT

GGGAAGGTCATTGATGATGGATAGCCCCCCGTCTGTCCAAGTTGCTCCGCAGTGGAGT

AATCCTGCATCTGCTTTTTAATCGAGTTTTCGAAGATGTTTTGGCCCAGTGCCAGATCA

ATTATAAAACTCCGGAACATAAAGTGCATTGGCCTCGCACTTCTGGTCTTGCAATCTC

AGCTTTGATATTCCTGATTCACAGATTGCCAACGTGCTTCGAGGCTGGCCCCCGAGTC

ATATCCACAATCATG

SEQ ID NO.4: nucleotide sequence 1000bp of Ppppta

GTGAGGGAGGATTTTCTCCACGACGGGTGCGCGGATAAAGACGGCGGGGAAAGCGG

GTTGATTAGCGCCCAGGAAGGGCAAGTCGAGAGGAGCCTGGAAACTCTCCGTCTGA

CGGCCAAAATGATTGCGATTGACGCGCACGTCCAGCCCACCGATCAGATCCTGGCCA

CCCTTCTTTGTGCGGTTGGCTGACTCGGCGAGGAGGATCAGACCGGCGCAGGTACCC

CAAGTAGGCCTCCGGTGGACCCTGAGATGGTAATTAGCATACACATAGAGCAACTGT

GCAGAACACCGATAAGACATACTTGACAAAGTCTCGCAAAGGCTCCAAGAGGTTGG

ACCTGGCGGCAACAAGGGCCATGGTTGTGCTTTCACCTCCGGGCAGCACCAGCGCAT

CGCATCGCTCGAGTTCCTGCGGGGTGCGCACTTCGATGAAGTCCCATTGCGAGGCGG

AACTGCGGTCTTTGGCGGGCAAATCGGCGGCCGCACTCTTCAGCAATTGGATATGTTC

GTAGAAGGCGCCTTGCAAGGCGAGCACGCCGACGGTGATGCAGCCCATGGCGAAGT

ATAGGATCTGAGAGTGTGGCGGCAGCAACAGAACTATGACTCCAAACTCTCTATACTT

TATTTGATGGGAGCAACGCCGCCTTATGTCAGCGGATCAATGTCGGACCGCTTATCAC

CACGTGCTGCCCCGCGTTCAGCCTCGGACATTTGGGGGCCATCATTAACATCATGAGT

TCAATGTCTTTTTTGCTTTGCTGTGATAATACGCTGGTTGCGGTCTTTAACATAAGACT

CCGAGATTCGTCATTAGAACAGTTTAATCTCAGAATGTATCAATGACCCTCGTGGAGA

ACTAACCCAACCCCTCACTTCACCTCATCTCACCTCCGCATAGACGCCCGATCTCCTC

ACATCAGCTACACCACTACATCTCACTCAATTGAACACACCACCACCACAACAGCCT

CATACCCAACCCAACCAACCCACAATG

SEQ ID No.5: pgpdA nucleotide sequence 936bp

TGGACTAACATTATTCCAGCACCGGGATCACGGGCCGAAAGCGGCAAGGCCGCGCAC

TGCCCCTCTTTTTGGGTGAAAGAGCTGGCAGTAACTAAACTGTACTTTCTGGAGTGAA

TAATACTACTACTATGAAAGACCGCGATGGGCCGATAGTAGTAGTTACTTCCATTACAT

CATCTCATCCGCCCGGTTCCTCGCCTCCGCGGCAGTCTACGGGTAGGATCGTAGCAAA

AACCCGGGGGATAGACCCGTCGTCCCGAGCTGGAGTTCCGTATAACCTAGGTAGAAG

GTATCAATTGAACCCGAACAACTGGCAAAACATTCTCGAGATCGTAGGAGTGAGTAC

CCGGCGTGATGGAGGGGGAGCACGCTCATTGGTCCGTACGGCAGCTGCCGAGGGGG

AGCAGGAGATCCAAATATCGTGAGTCTCCTGCTTTGCCCGGTGTATGAAACCGGAAA

GGACTGCTGGGGAACTGGGGAGCGGCGCAAGCCGGGAATCCCAGCTGACAATTGAC

CCATCCTCATGCCGTGGCAGAGCTTGAGGTAGCTTTTGCCCCGTCTGTCTCCCCGGTG

TGCGCATTCGACTGGGCGCGGCATCTGTGCCTCCTCCAGGAGCGGAGGACCCAGTAG

TAAGTAGGCCTGACCTGGTCGTTGCGTCAGTCCAGAGGTTCCCTCCCCTACCCTTTTT

CTACTTCCCCTCCCCCGCCGCTCAACTTTTCTTTCCCTTTTACTTTCTCTCTCTCTTCCT

CTTCATCCATCCTCTCTTCATCACTTCCCTCTTCCCTTCATCCAATTCATCTTCCAAGTG

AGTCTTCCTCCCCATCTGTCCCTCCATCTTTCCCATCATCATCTCCCTTCCCAGCTCCTC

CCCTCCTCTCGTCTCCTCACGAAGCTTGACTAACCATTACCCCGCCACATAGACACAT

CTAAACAATG

SEQ ID NO.6: nucleotide sequence 1011bp of ANI _1_974074

TTGGAGGCGAGCTGCTTACGGGGAGCCTTGCCACCAGTGGACTTACCTATATTGAGAT

GATAGTTAGTGATGATGTTCCTCATGTGAGACACGGGTGTCGCGACCATGACGCGGTC

GAACGACACGAGGGGAAAGGACAGTGAGGTTGCACTTACGGGCAGTCTGCTTAGTG

CGGGCCATCTTAGTGGATTAAGTTTGATGGATTTAGTTGTTTTAGAGATGGAAAGATG

ACGTTGAGGATGAAAGATTGATGGAGGTTGATAGATAAGGGTAGACGGGATGAGGAG

ATGGAAGAAGGATGGAGGAGGCGGGTGGGGAGAGGTATTTGTAGTAAGCGAAAGGG

TAGGTGGTGATCTGGGTTTGCCAGCAAAGGGAGGGGGCCTTAGCGTGCCGTTCCTTT

GATCGAAAAAGTGTTCGGAAAGCAGCACACCATAAGCCGTGATTTCGGGGCTCACTT

TTGGCTGGGAAAATCAAAAGTGCCCAAAGGCGGTCAGTCCCCTTTCTGCCTGCTCGG

AGCGCTAAGCGAGCGCCCTAATGCCCGCCAGCCAATAGGAAGCCAGGATCACGGATC

TAGAAGCTGCCCCTGCCTCACATTGATCCCAACCTCAGGCCCGGAATGTGATCCTATC

AACCTGTTGCAGATGCTCATGGCCATCGACAGGAATGTCCCTGACACCCTCTGATTGC

TGATCTGGGTGGTTGGGAGCATTCCCGTGGGAATCAGTCTCATCCCAGCGGAATCATC

CCTGCCCGGTCACGTTGCCGCCAGATCAGTCCGCCGGCCGGGATCCTGCGCTCTGATT

GGTCGATCCGCGCTCTAGCGTGTGATTCCAACGCCCTCCCCGCGGCGCGCCTTCTGCT

ATACAAATATCTCCCGTCCCATGGATGCTCTCTGCCCTCTTCCCTTTACATCCATCCTCA

CTCTAAAACGCTTAACTTATCTCCACCATCTTCACCCTCAACTTTAATTCAACTCTATTT

CCGTTTACTTTTTAGACAACCTTCACAATG

SEQ ID NO.7: 1687bp nucleotide sequence of ANI _1_58174

CATCGGCCAACTCCACCGTCTTTGCCGACCCGACCACTCTGCTGCCAATTTATTGCTG

GCTCAACCTCGAGCTTCATTCTTCCTCTTCTTTCATCCTTTTCTTCCCTTTTATCATCCC

TTTTCTTCCCTTGATTCCCTCTTCCGGTTCCTTCCGGTTCAGTGATTGCCACTATCTTTG

CCTGATTCATCATGGCCAGCACTGTCGGACAGGTGAGTTTGTTGGTTCCAGCTCGAA

GCAAAGACCAGCATTGACGCGTGCGTTGACTAACTCATCTAGACCATCACCTGCAAG

GTAAGAAAGCCACTCGAACAATGACCTCCACCATCGAGTATACCAATGAACTTTTGTA

GGCTGCGGTTGCCTGGGGCGCCGGAGAGCCTCTCTCCGTTGAGGACGTCGAAGTTGC

TCCTCCCAAGGCCCACGAGGTCCGCATTCAGGTCCTTCACACTGGTGTCTGCCACAC

AGGTCGGTGATTGTGAGACAGCTGGGATTGAAAGCCAGGCTGACCGAGACAGATGC

CTACACTCTCTCCGGAAAGGACCCCGAAGGTGCTTTCCCCGTTGTTCTGGGACACGA

GGGTGCCGGTATCGTCGAGTCCGTCGGCGAGGGCGTCACCTCCGTGAAGCCCGGTGA

CTATGTCGTTGCTCTCTAGTAAGCGACACCTGGTGCAATTGCGCACTCCGCTGCTAAC

GTTTCTAGCACCCCCGAATGCCGTGAGTGCAAGTTCTGCAAGTCCGGCAAGACCAAC

CTCTGCGGTAAGATTCGTGCCACTCAGGGCAAGGGTGTGATGCCCGACGGCACCTCC

CGTTTCAAGGCCCGCGGCAAGGACCTGCTGCACTTCATGGGTACCTCCACCTTCTCC

CAGTACACCGTCGTGGCCGACATCTCCGTTGTCGCTGTGACCCCCAAGATCCCCACG

GACCGGTCCTGCCTGCTTGGCTGCGGTATCACCACCGGTTACGGTGCGGCCGTCGTC

ACCGCCAAGGTGGAGGAGGGCTCCAACATTGCCGTCTTCGGTGCCGGCTGTGTCGGT

CTCTCAGTCATCCAGGGTGCTGTGAAGAACAAGGCTGGCCGCATCATCGTGGTCGAT

GTCAACGACGGCAAGGAGGCGTGGGCCCGTAAGTTCGGCGCCACGGACTTTGTCAA

CCCCACCAAGCTCGGTGGCAAGACCATCCAGGAGCAGCTGATTGAGATGACGGACG

GTGGTTGCGACTACACTTTCGACTGCACTGGTAACGTTGGTGTGATGCGGGCTGCCCT

GGAGGCGTGCCACAAGGGATGGGGTGAGAGTATTATCATCGGTGTTGCTGCTGCTGG

CCAGGAGATTTCCACTCGCCGTAAGTATTCTTTGAATGTGTAAAAAAGCGCGCAATGC

TAACTGGTTGCAGCATTCCAGCTGGTCACTGGCCGTGTGTGGAAGGGTTGCGCCTTT

GGCGGTATCAAGGGCCGTACGCAGCTGCCGGGATTGGTTGATGACTACCTGAACGGA

CAGCTGAAGGTTGATGAGTTCATTACGCACCGCGAGAAGCTGTCGGACATCAACAAG

GCGTTTGAGCAGATGAAGGCGGGCGACTGCGTTCGCTGCGTTGTTGACCTGTCATAA

ACCTGCCATAATGTACACTGATGAATAAATTGAATGAATAAAATTACGAGTTAGCTGTT

AGAAACGATGTCTATATATATGTG

SEQ ID No.8: nucleotide sequence 836bp of ANI _1_780184

CTTCAATCCCATATACCCCTACTTCAACGTTCATTGTCACATTTTCTGCGAAGCACTGA

TTCGCCTATCGAAAAATGCCTCGAACTGCTATCTTCGTCATCGACATCCAGAACCACC

TGGCCGTTGATTCAACCTCTCGCGTCCCTCAAGCGGATCGCGTCATCAAAGCCTCTGA

AGAGATTCTACAGACAGCTCGGTCCATCACCGACTCCAATGGAAACAGCAGCAGTCC

ACTCATTGTTTTTATTCAGCACGAAGAGTCTCCGAATGATGGAGCTCTGGTAAAGGGG

ACTGAGCCCTGGGAGCTCGTCTTTCATCCTCGCGCGGACGCAGAGGGTGAAATTTTG

GTAGCGAAGAAGACAGGTACAGTACTCAACATGCTCGATGAGAATCGATCTAGCTAA

TCATCTCGTTACATTTTACAGGTGACACATTCAAATCCAACCGCGACCTCGCGCAGAG

GCTGCGCAACGCCAACGTCACCGAAATCGTCGCGTTCGGCCTGCAGAGCGATGCCTG

CGTCGAAGCAACATGTACCGGCGCGCTGGCAGCAGGTTTCCACGTAACTGTACTGGC

AGGCGCACATTCTACCTACGACGCCGATGGAAAGACCGCCCAGGAAATCGAGCGTGA

AGTCGAACTTCGCCTCTCGACACGCGGCGCTCGTGTTGTCCGGTGGGAGAAGGCGAT

CGCGAAATGGGTGGAGAAGCAACAGGTTGTCTGATTCGCTTCCTTTGATTTCGATTTA

TTTCATCATTGTACATGTTAGAATTGCCAGGAACTTTGCCTGAAATGCGGGCATCATCG

AGATACACCACAAACGATATTATCCCCAC

SEQ ID No.9: nucleotide sequence 1356bp of ANI _1_1402034

AAGGTCCACCCTTCTGTCACGACACTCGCAGTGGGAGATCGCGTCGCGATCGAACCTCACATTGTATGCAAGGCATGCGAACCGTGTCTTACCGGACGCTACAATGGGTGCAAGAATCTGCAATTCCGCTCAAGTCCACCATCGCATGGTCTCCTGCGGACCTACGTGAACCATCCAGCCATCTGGTGCCACACAATCGGCGATCTATCATATCAGAAAGGCGCCTTGTTGGAGCCACTAAGTGTCGCACTCACCGCAGTTACTCGATCTGGGGTCAGAATTGGAGATCCGGTTTTGATTTGTGGCGCGGGACCGATTGGGTTGGTGGTGCTGCAATGCTGCCGCGCTGCTGGGGCGTATCCCACTGTGATCAGCGATGTTAACCCGGCGCGGTTGCAATTTGCGCGACAGTTCGTGCCTGCTGCTCGAACACTGCAGGTTCGGCCGGAGGAGACCGACAAGGAATTTGCAGCTCGGGTGGTGCAGTTGATGGGTGGGGAGGATTGCGAGCCCGTGGTTGCCCTGGAATGCACGGGAGTCGAAAGTTCGATCGCCGGTGCTATTCAGAGTGTCAAGTTTGGGGGCAAAGTGTTTGTGGTTGGCGTGGGCAAGGATAAGCTTCAGTTTCCATTCATGCGACTGAGCGAGCGCGAGATCGATCTGCAGTTCCAGCAGCGCTATGTCAACATGTGGCCCCGCGCCATCCGGGTGATGTCCAGCGGAGCCATCGATCTAGACCAGATGATCACTCATGTCTATGCCTTTGAAGACGCTCTCAAGGCGTTTCAGACGGCGTCAGATCCGAGTAGCGGCTCAATTAAGGTGCTGATTGAGGGGCCACAAGGATGA

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, alterations and modifications are possible without departing from the spirit and scope of this disclosure and appended claims, and accordingly, the scope of this disclosure is not limited to the embodiments disclosed.

Claims

1. A constitutive promoter that is stably expressed in aspergillus niger, characterized in that: the constitutive promoters include ANI _1_974074, the histone H4 promoter, ANI _1_58174, the S- (hydroxymethyl) glutathione dehydrogenase promoter, ANI _1_780184, the hypothetical protein promoter, and ANI _1_1402034, the L-arabitol dehydrogenase promoter.

2. The constitutive promoter stably expressed in A.niger according to claim 1, wherein: the nucleotide sequence of the ANI _1_974074 promoter, namely the histone H4 promoter, is SEQ ID No.6, the nucleotide sequence of the ANI _1 _58174promoter, namely the S- (hydroxymethyl) glutathione dehydrogenase promoter, is SEQ ID No.7, the nucleotide sequence of the ANI _1 _780184promoter, namely the hypothetical protein promoter, is SEQ ID No.8, and the nucleotide sequence of the ANI _1 _1402034promoter, namely the L-arabitol dehydrogenase promoter, is SEQ ID No.9.

3. The constitutive promoter for stable expression in aspergillus niger according to claim 1 or 2, wherein: the constitutive promoter is constitutive promoters with different strengths, and the promoter strengths are ordered from strong to weak as PgpdA > PpptA > ANI _1_974074 (histone H4) promoter > ANI _1_58174 (S- (hydroxymethyl) glutathione dehydrogenase) promoter > ANI _1_780184 (hypothetical protein) promoter > ANI _1_1402034 (L-arabitol dehydrogenase) promoter by referring to PgpdA, namely 3-glyceraldehyde phosphate dehydrogenase, and PpptA, namely npgA protein.

4. The constitutive promoter stably expressed in A.niger according to claim 3, wherein: the glyceraldehyde-3-phosphate dehydrogenase gene promoter PgpdA and the npgA protein gene promoter PpptA are promoters for controlling gene transcription.

5. The constitutive promoter stably expressed in A.niger according to claim 1 or 2, wherein: the constitutive promoter is derived from genomes of Aspergillus niger, aspergillus flavus, aspergillus oryzae, aspergillus sojae and Aspergillus fumigatus.

6. Method for assessing the expression strength of a constitutive promoter according to any one of claims 1 to 5 in A.niger, characterized in that: the method comprises the following steps:

(1) Extracting RNA from an Aspergillus niger strain S469, sequencing transcriptome, screening genes with stable gene expression quantity at different time points according to the transcriptome data, grouping promoter genes screened according to the transcriptome data according to an FPKM value, selecting promoter genes with the transcription intensity larger than PgpdA, promoter genes with the transcription intensity between PgpdA and PpptA and promoter genes with the transcription intensity smaller than PpptA, carrying out q-PCR verification on the screened promoters, and selecting genes with the transcription level difference smaller than 1 to obtain different promoter genes;

(4) The recombinant Aspergillus niger containing the expression vector is preliminarily applied: culturing the recombinant strain on PDA solid culture medium for 5 days, washing off spores with sterile water, collecting spores, counting, and culturing at 2 × 10 ⁶ Inoculating the cells/mL into a citric acid fermentation culture medium, culturing at 28 ℃ and 220rpm for 3 days, and measuring the yield of citric acid in fermentation liquor by using an HPLC method after sample preparation; meanwhile, collecting thalli, crushing the thalli by using a mortar, extracting RNA, centrifuging fermentation liquor, taking supernatant, carrying out reverse transcription on cDNA by the sis Kit, and verifying the expression quantity of the citrate transporter cexA under constitutive promoters with different strengths by taking the recombinant strain cDNA as a template.

7. The evaluation method according to claim 6, characterized in that: the sequence of the citrate transporter cexA in the step (2) is ANI _1_478154 at NCBI-locus _ tag.

8. A method for screening constitutive promoter genes with different strengths stably expressed in Aspergillus niger, which is characterized in that: the screening method comprises the following steps:

(1) Screening of promoters at different transcription levels:

spores of the strain S469 (. DELTA.oahA) were collected and 1X 10 spores were collected ⁸ Inoculating spores into an Aspergillus niger liquid culture medium, culturing at 28 ℃ and 200rpm, sampling at different time points respectively, filtering thalli by gauze, extracting RNA, performing transcriptome sequencing, screening 168 genes which are stably transcribed at different time points in the fermentation process, dividing the selected promoters into three parts according to two promoters with different strengths known in the field, namely a PgpdA promoter, namely a 3-glyceraldehyde phosphate dehydrogenase promoter, and a PpptA promoter, namely an npgA protein promoter, wherein the PgpdA promoter strength is higher than the PpptA promoter, taking the two promoter strengths as references, namely the promoter with the FPKM value higher than the PgpdA, the promoter with the FPKM value between the PgpdA and the PpptA, and the promoter with the partial FPKM value below the PptA; performing q-PCR detection on the selected promoter gene, and selecting a promoter with the gene transcription difference multiple smaller than 1 according to the result to obtain promoters with different transcription levels;

on the basis of pLH924 (delta amyA), the cexA gene is amplified by taking the ATCC1015 genome as a template, and the cexA gene is inserted into an amyA site at a fixed point to form a frame vector pLH1099, namely, cexA is expressed at the amyA site of the acid amylase gene at a fixed point; then, on the basis of pLH1099, promoter genes with different transcription levels obtained by screening in the step (1) are inserted into the site to start the expression of cexA;

taking an aspergillus niger strain S834 (delta oahA, delta cexA) as a starting strain, taking a citrate transporter cexA as a reporter gene, and quantitatively analyzing the citric acid yield of each recombinant strain by using HPLC (high performance liquid chromatography), thereby verifying the strength of different screened constitutive promoters;

9. The constitutive promoter for stable expression in aspergillus niger according to claim 8, wherein: the promoter PgpdA of the glyceraldehyde-3-phosphate dehydrogenase gene and the promoter PpptA of the npgA protein gene are promoters for controlling gene transcription;

10. Use of a constitutive promoter according to any one of claims 1 to 5 in the construction of engineered strains and/or in the utilization of expression of endo/foreign genes in A.niger.