CN109207373B - Microbial strain for high yield of citric acid and method for producing citric acid by fermenting starch sugar through microbial strain - Google Patents

Abstract

The invention discloses a microbial strain for high yield of citric acid and a method for producing citric acid by fermenting starch sugar by using the microbial strain, which is classified as Aspergillus niger 101-HAC11 with the collection number of CGMCC No. 12480. Also discloses a culture medium formula for producing citric acid by fermentation by using starch sugar as a carbon source and a fermentation process control process. The yield of citric acid produced by the strain can reach 166.8 g/L. Compared with the prior art, the yield of citric acid of the recombinant aspergillus niger strain is 220.34 g/L, and the yield is improved by 32.10%. The method has the advantages of high speed, simple production process, no toxic residue, and high safety.

Description

Microbial strain for high yield of citric acid and method for producing citric acid by fermenting starch sugar through microbial strain

Technical Field

The invention belongs to the technical field of fermentation engineering, and relates to a method for producing citric acid by constructing a high-fermentation-strength aspergillus niger strain through starch sugar fermentation by using genetic engineering. More specifically, the method comprises the following steps: a method for producing citric acid by fermentation of Aspergillus niger strain with high fermentation strength by using starch saccharine as fermentation carbon source and adding other organic and inorganic nitrogen sources and necessary metal salt components.

Background

Citric acid (Citric acid) is a common organic acid, is widely applied to food, beverage, pharmaceutical and chemical industries and cosmetic industries, and is an organic acid with the largest worldwide demand at present.

In 1784, citric acid was prepared by calcium citrate precipitation method in c.w. seler. The biological fermentation method is formed in the beginning at the end of the 19 th century. In 1916, most of Aspergillus such as Aspergillus oryzae, Aspergillus awamori, Trichoderma viride and Aspergillus wense, and Aspergillus niger were confirmed to produce citric acid by Thomus and Cokri experiments, while Aspergillus niger was in leaderboard. Tray fermentation was the mainstream in the past 20 th century, and the first factory in the world in 1923, which produced citric acid by aspergillus niger tray fermentation, was philippines in the united states. After 1950, submerged fermentation was gradually established. Compared with a tray fermentation method, the deep fermentation method has the advantages of short period, higher yield, small occupied area, less labor force and great benefit for industrial production, and is a main method for producing citric acid.

The report of Tangtenhan et al, which was originally 1942, taught the production of citric acid by fermentation. In 1952, the citric acid was prepared by tray fermentation. 1959 the experiment of preparing citric acid by 200L scale submerged fermentation of scientific research institute of fermentation industry of the young industrial department was initially successful, and after 1966, the institute of Industrial microorganisms in Tianjin and Shanghai respectively adopts dried potato powder as raw material to prepare citric acid by submerged fermentation, and the experiments are successful in succession, thereby determining a main process route for producing citric acid in China. Until now in 2000 years, in the citric acid production in China, corn flour is liquefied by high-temperature spraying, aspergillus niger strain liquid submerged fermentation is used for producing citric acid, and a calcium salt method and sulfuric acid acidolysis are adopted for separating and extracting the citric acid. With the technical progress of the fermentation industry in China and the increasing requirement on the environmental protection in the production process, the citric acid produced by the process cannot meet the requirement on high-strength fermentation of the citric acid.

The main reasons are the following problems:

(1) the corn flour liquefied liquid with partial corn residues is used for fermentation together, so that the fermented mash is viscous, the total sugar of the fermented mash is 17% -18% at present, if the total sugar of the mash is increased, the viscosity of the feed liquid is further increased, ventilation and stirring energy consumption are too large in the fermentation process for achieving dissolved oxygen, industrialization cannot be achieved, and the acid production in a single tank is limited to be about 18% and cannot be improved.

(2) The corn liquefied liquid contains various saccharides such as dextrin, oligosaccharide, maltose, isomaltose and the like, and contains different kinds of proteins, so that the fermentation product is complex, and various kinds of mixed acids are generated. The fermented mash has various components and brings difficulty to the separation and extraction of citric acid, and the existing citric acid extraction adopts a calcium salt method, which is most beneficial to the removal of mixed acid, but can generate calcium sulfate waste residue which can not be treated, thereby forming industrial production waste residue. Because the fermented mash is difficult to separate and purify the citric acid, the citric acid is extracted by a calcium salt method all the time, and calcium sulfate becomes a solid waste hidden trouble of various citric acid production companies.

(3) At present, after citric acid fermentation is finished, aspergillus niger mycelium pellets in fermentation solid waste are mixed with unusable corn residues and only can be dried to enter a feed market, and the aspergillus niger mycelium pellets can be completely used for extracting high value-added products such as glucosamine, sterol and the like.

(4) The corn flour liquefied liquid is used as a raw material for citric acid fermentation, so that the citric acid fermentation is greatly influenced by the quality of corn, and the carbon-nitrogen ratio in the corn liquefied liquid is influenced by the drying temperature of corn grains, the production area of the corn, the variety of the corn, the storage time of the corn and the like, so that the fermentation process is unstable, and the citric acid production is influenced.

(5) At present, the citric acid fermentation adopts corn flour liquefied liquid as a raw material, so that the measurement and control of dissolved oxygen and pH are difficult in the fermentation process, and the automatic control and continuous production of the fermentation process cannot be realized.

For these reasons, it is imperative to develop citric acid fermentation from starch sugars.

The citric acid fermentation process based on starch sugar has the following advantages:

(1) the powdery sugar is solid in the fermentation liquor of the raw material, the viscosity of the fermentation liquor is low, the initial sugar concentration can be increased by 25 percent, the single-tank acid production is realized by more than 25 percent, and the labor production efficiency is improved.

(2) The starch sugar can be completely dissolved in water, the viscosity of the fermented mash is low, the dissolved oxygen is facilitated, the aeration and stirring energy consumption in the fermentation process is greatly reduced, and the energy-saving purpose of the citric acid fermentation process is realized.

(3) The citric acid produced by fermenting the citric acid by the starch sugar has less mixed acid and high purity, and other easily-carbonized compounds have low concentration, so that the citric acid is separated and extracted, the citric acid can be directly concentrated and crystallized after being treated by fermentation liquor, a calcium salt method is thoroughly replaced, and calcium sulfate waste residues produced by the calcium salt method are eliminated.

(4) Aspergillus niger mycelium pellets generated by fermenting citric acid by using starch sugar can be directly separated from fermented mash, and are used for extracting glucosamine, sterol and the like, so that a high value-added product is realized.

(5) The citric acid fermentation liquor for starch sugar fermentation has no other solid matters except Aspergillus niger mycelium pellets, is favorable for the on-line observation of the mycelium pellets in the fermentation process, and can carry out on-line component detection on the fermentation liquor, thereby realizing the automatic control and the accurate optimization of the fermentation process, and finally realizing the realization of continuous fermentation processes such as starch sugar feeding, mycelium pellet cutting and copying and the like.

The key technical difficulty for realizing the fermentation of the starch saccharine citric acid is as follows:

(1) the type and concentration of the organic nitrogen source added in the process of fermenting citric acid by using starch sugar are the key points for realizing high conversion rate of citric acid fermentation.

(2) Better mycelium pellets can be formed in the process of fermenting citric acid by starch sugar.

(3) High conversion rate bacterial strain for starch saccharine fermentation citric acid.

Disclosure of Invention

The invention aims to provide an aspergillus niger strainAspergillus niger101-HAC11 (CGMCC No. 12480) is a culture medium formula for producing citric acid by fermentation by using starch sugar as a carbon source and a fermentation process control process.

Another purpose of the invention is to provide a strain Aspergillus niger strain for microbial fermentation of starch sugar to produce citric acid with high yieldAspergillus niger101-HAC11, which belongs to the genus Aspergillus niger (A. niger)Aspergillus niger) The preservation number is CGMCC number 12480.

It is also an object of the present invention to disclose the use of Aspergillus niger strainsAspergillus niger101-HAC11 (CGMCC No. 12480) and a method for producing citric acid by fermenting starch sugar.

In order to achieve the above object, the present invention discloses the following technical contents:

construction of Aspergillus niger by genetic engineeringAspergillus niger101-HAC11 microbial fermentation strain belonging to the genus Aspergillus niger (A. niger)Aspergillus niger) The preservation number is CGMCC number 12480.

The CGMCC number 12480 strain provided by the invention is a new Aspergillus niger strain which can utilize starch sugar to ferment citric acid and is constructed by genetic engineering, and the Aspergillus niger strain is used for fermenting sugar raw materials to produce citric acid and belongs to the genus Aspergillus niger (Aspergillus niger)Aspergillus niger) The preservation number is CGMCC number 12480, and the preservation date is 2016, 6 and 12. The storage place: china microbial strain preservationGeneral microbiological center of the regulatory Committee (address: Hospital No.3, West Lu No.1, Beijing, Chaoyang, Chao). The strain is Aspergillus niger which is a citric acid production strainAspergillus nigerWLG CGMCC No.10142, preservation date 2014, 12 months and 11 days, preservation place: china general microbiological culture Collection center (address: West Lu No.1 Hospital No.3, North Chen, Chaoyang, Beijing).Aspergillus nigerWLG CGMCC No.10142 is used as an original strain, and a high fermentation strength citric acid strain obtained by over-expressing aox1 gene, pyruvate carboxylase gene and pyruvate dehydrogenase gene is constructed through genetic engineering.

Aspergillus nigerAspergillus niger101-HAC11 microbial fermentation strain, wherein the preservation number is CGMCC number 12480, and the physicochemical properties are as follows: can efficiently express the key enzymes of pyruvate carboxylase, pyruvate dehydrogenase and alternative oxidase in the process of synthesizing the citric acid.

The invention uses inducible promoter Pgla to start PC protein to express in Aspergillus niger, further uses constitutive promoter PgpdA to start pyruvate dehydrogenase PD gene and alternative oxidase Aox1 gene, and improves the pyruvate content by enhancing the expression quantity and activity of pyruvate carboxylase PC gene. Further enhance the oxidation metabolism flow of the acetyl CoA and the NADH, thereby improving the yield of the citric acid and the conversion rate of the saccharic acid.

The method of the invention uses citric acid to produce the bacterial strain Aspergillus nigerAspergillus nigerWLG CGMCC No.10142 is used as an original strain to carry out genetic engineering transformation, the citric acid yield of the original strain is 166.8g/L, compared with the prior art, the citric acid yield of the recombined aspergillus niger strain is 220.34 g/L, and the yield is improved by 32.10%.

The invention discloses a method for producing citric acid by fermenting starch sugar with CGMCC number 12480 strain, which is characterized by comprising the following steps:

(1) the adopted fermentation strain is aspergillus niger strainAspergillus niger101-HAC11 preservation number is CGMCC No.12480, and the fermentation medium comprises (g/L): 200-260 g of starch sugar raw material, 60-80 g/L of corn steep liquor, (NH)₄)₂ SO ₄9~11 g/L, MgSO₄·7H₂O 0.05~ 0.15g/L，K₂HPO_, 4~6g/L，CuSO₄ 0.8 mg/L ZnSO₄ 0.02 g/L，CaCl₂ 2.0 g/L，KCl 0.1 g/L，FeSO₄ 0.34 mg/L pH 5.0～5.5；

(2) Fermentation culture control conditions: controlling the inoculation amount to be 10-15% (v/v), controlling the seed age to be 27 hours, inoculating the cultured seeds into a fermentation culture medium, wherein the culture temperature is 30-40 ℃, and the fermentation ventilation volume is 0.1-0.15 m³/ m³And min, the tank pressure is 0.07-0.12 MPa, the stirring speed is 80-100 r/min, the fermentation time is 50-60 hours, sterile air is introduced in the fermentation process, the yield of citric acid is 220-260 g/L, and the sugar-acid conversion rate is more than 102%. The method has the characteristics of high speed, simple production process flow, high saccharic acid conversion rate, high production efficiency, easy product separation and the like, and is suitable for automatic large-scale industrial production.

The preparation method of the invention comprises the following steps of: liquefying and saccharifying starch (including corn starch, potato starch and tapioca starch), and filtering to obtain sacchariferous solution, wherein the sacchariferous solution comprises: glucose, sucrose, fructose, maltose and non-chromogenic dextrin.

The invention is described in more detail below:

fermentation strain of microorganism for high yield of citric acid, and Aspergillus niger strain thereofAspergillus niger101-HAC11 with the preservation number of CGMCC No.12480, the construction method of the strain is as follows: integrating multiple copies of a pyruvate carboxylase PC gene, a pyruvate dehydrogenase PD gene and an alternative oxidase Aox1 gene in the Aspergillus niger on an Aspergillus niger genome, and fermenting and producing citric acid by using the recombinant Aspergillus niger strain; the expression of the pyruvate carboxylase PC gene is regulated by a Pgla promoter, and the pyruvate dehydrogenase PD gene and the alternative oxidase Aox1 gene are regulated by a PgpdA promoter.

The nucleotide sequence of the pyruvate carboxylase PC gene is shown as SEQ ID NO.1, the Pgla promoter is an inducible promoter, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 2.

A pyruvate carboxylase PC gene expression cassette, the expression cassette comprises a Pgla promoter, a pyruvate carboxylase PC gene, a HYG resistance marker and a trp terminator, and the Pgla-PC-HYG-trp gene expression cassette is arranged in sequence. The nucleotide sequence of the trp terminator is shown in SEQ ID NO. 3. The nucleotide sequence of the pyruvate dehydrogenase PD gene is shown as SEQ ID NO. 4; the nucleotide sequence of the alternative oxidase Aox1 gene is shown as SEQ ID NO.5, the PgpdA promoter is a constitutive promoter, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 6. The nucleotide sequence of the HYG gene is SEQ ID NO. 7.

The preparation method of the recombinant aspergillus niger comprises the following steps:

constructing a pyruvate carboxylase PC gene expression frame Pgla-PC-HYG-trp;

constructing a pyruvate dehydrogenase PD gene and an alternative oxidase Aox1 gene expression frame PgpdA-PD-Aox 1-HYG-trp;

and thirdly, the gene expression frame prepared in the first step and the second step is transformed into aspergillus niger, and the recombinant aspergillus niger is obtained through resistance screening and PCR identification.

The nucleotide sequence of the HYG gene in the resistance expression frame in the step (i) is shown as SEQ ID NO. 7.

(2) Liquid state submerged fermentation aspergillus nigerAspergillus niger101-HAC11 citric acid production medium and fermentation process control:

a. aspergillus niger with different organic nitrogen source typesAspergillus niger101-HAC11 influence of fermentation of starchy sugar citric acid; aspergillus niger with different organic nitrogen sourceAspergillus niger101-HAC11 citric acid fermentation, sugar acid conversion rate and the like have significant effects, and the results are shown in FIG. 1.

As can be seen from FIG. 1, the sequence of the 4 nitrogen sources for acid production is that corn steep liquor is greater than soybean peptone is greater than protein feed is greater than yeast powder, the acid production can reach 9.27% at most when the corn steep liquor is added, the sugar acid conversion rate is higher than that of other nitrogen sources, residual reducing sugar is relatively less, the soybean peptone is inferior, and the fermentation performance of the added corn steep liquor and soybean peptone is superior to that of the added other organic nitrogen sources. And finally selecting corn steep liquor as the most suitable organic nitrogen source for citric acid corn clear liquid fermentation in the four organic nitrogen sources by combining the acid production, the conversion rate, the residual reducing sugar and the bacterial pellet shape consideration.

b. Determination of the amount of corn steep liquor added

Determining corn steep liquor as an optimal organic nitrogen source, wherein the concentration of the corn steep liquor is one of important factors related to whether the citric acid can be efficiently fermented, and determining the optimal concentration of the corn steep liquor by measuring the final acid production and the calculation of the conversion rate of saccharic acid. The results are shown in FIG. 2. As can be seen from fig. 2, when the concentration of the corn steep liquor is 0.7%, the aspergillus niger acidogenic and saccharic acid conversion rate is the highest, and the increase of the acidogenic rate of the corn steep liquor is not increased any more. Comprehensively comparing the indexes of acid production and conversion rate, and selecting 0.7 percent of corn steep liquor as the optimal organic nitrogen source of the fermentation medium.

c. Different concentrations (NH)₄)₂SO₄Influence on fermentation of citric acid clear solution

Adding 0.7% corn steep liquor to fermentation medium, and adding (NH) with different final concentrations₄)₂SO₄Determination of (NH) in the supernatant fermentation medium by determination of the last acid production₄)₂SO₄The results are shown in FIG. 3. As can be seen from FIG. 3, (NH) in the fermentation broth₄)₂SO₄When the total concentration of the compound is 0.1 percent, the acid yield and the saccharic acid conversion rate are both highest. After addition of other concentrations of (NH)₄)₂SO₄In this case, the acid yield and the conversion rate of sugar acid were not satisfactory, and 0.1% was selected as (NH)₄)₂SO₄The optimum total concentration of (c).

d. Effect of different concentrations of the trophic factor inositol on fermentation of citric acid serum

Inositol with different concentrations is added into the fermentation medium, the final acid production is finally measured, and the mycelium morphology is observed so as to determine the optimal addition amount of the inositol in the clear liquid fermentation medium, and the result is shown in figure 4. Fig. 4 shows that, from the aspect of acid production and sugar acid conversion rate, the addition of a certain amount of inositol has an obvious effect on improving acid production, and the fermentation acid production with the addition of a certain amount of inositol is higher than the fermentation acid production without the addition of inositol, wherein the acid production and sugar acid conversion rate is highest when the addition of inositol with a total concentration of 0.007%, and when the concentration of inositol is continuously increased, the acid production is not increased any more, the consistency of mycelium pellets is reduced, and the mycelium is diffused more. The factors are combined to select 0.007% of inositol as the optimal concentration.

e. Effect of Metal ion concentration on fermentation

Selecting MgSO₄、CuSO₄、ZnSO₄、CaCl₂、KCl、FeSO₄Orthogonal experiments were performed at various concentrations and the results are shown in table 1.

TABLE 1 results of orthogonal experiments

TABLE 2 analysis of variance in orthogonal tests

As can be seen from Table 2, MgSO₄、CuSO₄、ZnSO₄、CaCl₂、KCl、FeSO₄In different levels of the six factors, the primary and secondary sequences of the influencing factors are shown by range analysis: CaCl₂＞MgSO₄＞KCl＞FeSO₄＞ZnSO₄＞CuSO₄The optimal combination is A1B1C1D1E1F3, and the optimized optimal concentration is as follows: MgSO (MgSO)₄ 0.1 g/L，CuSO₄ 0.8 mg/L ZnSO₄ 0.02 g/L，CaCl₂ 2.0 g/L，KCl 0.1 g/L，FeSO₄ 0.34 mg/L。

And (3) carrying out a verification test on an optimization result under the culture condition with the optimal concentration, and carrying out citric acid fermentation for 72 hours at 36.5 ℃ and 330 r/min with the liquid loading capacity of a 500 mL triangular flask of 50 mL. The acid production of the verification result is 9.96%, which is higher than that of any group in the orthogonal experiment, and the advantage effect of the optimal combination is verified.

f. Optimal fermentation condition of starch saccharine clear liquid

(1) Effect of different initial sugar concentrations on citric acid fermentation

After different starch sugar clear liquids are selected for fermentation, the citric acid yield is measured, and the residual sugar results are shown in table 3.

TABLE 3 citric acid production at the end of fermentation

As can be seen from table 3, the sugar-acid conversion rate reached 100% or more at the end of the fermentation when the initial sugar concentration was 20% to 22%, and the residual sugar concentration was at a lower level.

(2) Effect of age of the seed on fermentation of citric acid serum

Seed solutions of different ages were inoculated for fermentation experiments, the final citric acid content, residual reducing sugars and the corresponding fermentation period were determined and the results are shown in table 4.

TABLE 4 Effect of age on citric acid fermentation

As can be seen from Table 4, the yield of citric acid gradually increases with the age of the species, wherein the acid production is highest at the age of 27 h, so that the optimal time for the aspergillus niger to move is selected as the age of 27 h.

(3) Effect of different Process controls on fermentation of citric acid serum

Different process controls make the concentration difference of dissolved oxygen in fermentation liquor larger in the fermentation process, and the concentration of dissolved oxygen has great influence on citric acid fermentation. Sampling every 8h from the beginning of fermentation, and measuring the change conditions of the citric acid concentration and the pH value in the fermentation liquid. The results are shown in FIG. 5. As can be seen from FIG. 5, the large ventilation rate dissolved oxygen is superior to the sectional ventilation at 0-16 h; and the dissolved oxygen in the scheme A is higher than that in the scheme B at 16-64 h. Referring to fig. 5, in the scheme B, too high dissolved oxygen in the pre-fermentation period easily causes excessive proliferation of the thallus, so that aspergillus niger enters the citric acid accumulation period too early, and the pH value drops too fast. The low pH value can affect the activity of the saccharifying enzyme, further affect the utilization rate of raw materials and cause the residual sugar content of the fermentation liquor to be too high, so that the acid production of the scheme B in the later period of fermentation is inferior to that of the scheme A. And finally selecting the three-section ventilation volume of the scheme A.

As can be seen from FIG. 5, the ventilation rate was controlled to be 0.15m in the early stage of fermentation (0 to 16 hours)³/ m³Min, the thalli are massively proliferated and normally metabolized, the pH value of the fermentation liquor is 3.5, and the pH value is favorable for maintaining the sugar of the fermentation liquorActivating enzyme activity to increase the content of reducing sugar, fermenting for 16 hours, and then allowing aspergillus niger to enter a rapid acid production period, wherein the ventilation volume of 17-48 hours is adjusted as follows: 0.225 m³/ m³At the speed of 90-100 r/min, the aspergillus niger mycelium pellets are more vigorously metabolized, more reducing sugar is converted into citric acid, the content of the reducing sugar begins to be greatly reduced, and the acid yield begins to be greatly increased; after 48 hours, the fermentation is finished, and the ventilation volume of 49-58 hours is adjusted as follows: ventilation 0.10 m³/ m³Min, the rotating speed is 85-95 r/min, at the moment, the reducing sugar is in a lower level, the dissolved oxygen is higher, the pH value is relatively stable, and the ventilation and the rotating speed can be reduced. At the end of the 58 h fermentation, the yield of citric acid was 220.3g/L, the sugar-acid conversion rate was 102.8%, and the amount of residual reducing sugar was 0.1%.

The citric acid-producing microbial fermentation strain and the preparation method thereof disclosed by the invention have the positive effects that:

(1) the viscosity of the fermentation mash which is prepared from the powdery sugar is low, the initial sugar concentration can be increased by 25 percent, the acid production in a single tank is realized by more than 25 percent, and the labor production efficiency is improved.

(2) The starch sugar can be completely dissolved in water, so that oxygen dissolution is facilitated, the aeration and stirring energy consumption in the fermentation process is greatly reduced, and the energy-saving aim of the citric acid fermentation process is fulfilled.

(3) The citric acid produced by fermenting the citric acid by the starch sugar has high purity, and the citric acid can be directly concentrated and crystallized to extract the citric acid after being treated by the fermentation liquid, so that a calcium salt method is thoroughly replaced, and the calcium sulfate waste residue produced by the calcium salt method is eliminated.

(4) Aspergillus niger wire balls generated by fermenting citric acid with starch sugar are used for extracting glucosamine, sterol and the like, and high value-added products are realized.

(5) The citric acid fermentation of the starch saccharine fermentation can be used for on-line component detection, so that the automatic control and the accurate optimization of the fermentation process are realized, and the realization of continuous fermentation processes such as starch saccharine feeding, mycelium pellet cutting and copying and the like is finally realized.

The SEQUENCE triggering of the present invention is as follows:

nucleotide sequence of PC gene SEQ ID NO 1

Nucleotide sequence of Pgla promoter SEQ ID NO 2

Nucleotide sequence of trp terminator SEQ ID NO 3

Nucleotide sequence SEQ ID NO4 of PD gene

Nucleotide sequence SEQ ID NO 5 of Aox1 gene

Nucleotide sequence of PgpdA promoter SEQ ID NO 6

Nucleotide sequence SEQ ID NO 7 of HYG gene

Drawings

FIG. 1 influence of different organic nitrogen sources on citric acid fermentation;

FIG. 2 the effect of different corn steep liquor contents on citric acid fermentation;

FIG. 3 shows a difference (NH)₄)₂SO₄Influence of content on citric acid fermentation;

FIG. 4 effect of different inositol contents on citric acid fermentation;

FIG. 5 effect of different processes on 8 hours citric acid accumulation; a: scheme A three-stage ventilation B: scheme B has a large ventilation rate acid production curve.

Detailed Description

The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.

Example 1

The construction method of the fermentation strain of the microorganism for producing the citric acid with high yield comprises the steps of integrating multiple copies of a pyruvate carboxylase PC gene, a pyruvate dehydrogenase PD gene and an alternative oxidase Aox1 gene in the Aspergillus niger on an Aspergillus niger genome, and fermenting and producing the citric acid by utilizing the recombinant Aspergillus niger strain; the expression of the pyruvate carboxylase PC gene is regulated by a Pgla promoter, and the pyruvate dehydrogenase PD gene and the alternative oxidase Aox1 gene are regulated by a PgpdA promoter.

Example 2

Using Aspergillus niger strainsAspergillus nigerThe method for preparing the citric acid by the strain with the preservation number of 101-HAC11 being CGMCC No.12480 comprises the following steps:

(1) the adopted fermentation strain is aspergillus niger strainAspergillus niger101-HAC11 preservation number is CGMCC No.12480, and the fermentation medium comprises (g/L): glucose 200g, corn steep liquor 60g/L, (NH)₄)₂SO₄ 9g/L, MgSO₄·7H₂O 0.05g/L，K₂HPO_, 4g/L，CuSO₄ 0.8 mg/L ZnSO₄ 0.02 g/L，CaCl₂ 2.0 g/L，KCl 0.1 g/L，FeSO₄ 0.34 mg/L pH 5.0～5.5；

(2) Fermentation culture control conditions: controlling the inoculation amount according to 10% (v/v), controlling the seed age at 27 hr, inoculating the cultured seeds into fermentation medium at 30 deg.C, and fermenting with ventilation of 0.1m³/ m³Min, the tank pressure is 0.07 MPa, the stirring speed is 80 r/min, the fermentation time is 50 hours, sterile air is introduced in the fermentation process, the yield of citric acid is 220g/L as a result of fermentation, and the conversion rate of saccharic acid is 102%.

Example 3

Using Aspergillus niger strainsAspergillus nigerThe method for preparing the citric acid by the strain with the preservation number of 101-HAC11 being CGMCC No.12480 comprises the following steps:

(1) the adopted fermentation strain is aspergillus niger strainAspergillus niger101-HAC11 preservation number is CGMCC No.12480, and the fermentation medium comprises (g/L): 260g of glucose, 80 g/L of corn steep liquor, (NH)₄)₂SO₄ 11 g/L, MgSO₄·7H₂O 0.15g/L，K₂HPO_, 6g/L，CuSO₄ 0.8 mg/L ZnSO₄ 0.02 g/L，CaCl₂ 2.0 g/L，KCl 0.1 g/L，FeSO₄ 0.34 mg/L pH 5.0～5.5；

(2) Fermentation culture control conditions: controlling the inoculation amount according to 15% (v/v), controlling the seed age at 27 hr, inoculating the cultured seeds into fermentation medium at 40 deg.C, and fermenting with ventilation of 0.15m³/ m³Min, the tank pressure is 0.12 MPa, the stirring speed is 100 r/min, the fermentation time is 60 hours, sterile air is introduced in the fermentation process, the yield of citric acid is 260g/L as a result of fermentation, and the conversion rate of saccharic acid is 100%.

Example 4

Using Aspergillus niger strainsAspergillus nigerThe method for preparing the citric acid by the strain with the preservation number of 101-HAC11 being CGMCC No.12480 comprises the following steps:

(1) the adopted fermentation strain is aspergillus niger strainAspergillus niger101-HAC11 accession numberCGMCC No.12480, the fermentation medium comprises (g/L): 60g/L (NH) of corn steep liquor calculated by 200g of glucose contained in corn or potato starch saccharification liquid₄)₂SO₄ 9g/L, MgSO₄·7H₂O 0.05g/L，K₂HPO_, 4g/L，CuSO₄ 0.8 mg/L ZnSO₄ 0.02 g/L，CaCl₂ 2.0 g/L，KCl 0.1 g/L，FeSO₄ 0.34 mg/L pH 5.0～5.5；

(2) Fermentation culture control conditions: controlling the inoculation amount according to 10% (v/v), controlling the seed age at 27 hr, inoculating the cultured seeds into fermentation medium at 30 deg.C, and fermenting with ventilation of 0.1m³/ m³Min, the tank pressure is 0.07 MPa, the stirring speed is 80 r/min, the fermentation time is 50 hours, sterile air is introduced in the fermentation process, the yield of citric acid is 220g/L as a result of fermentation, and the conversion rate of saccharic acid is 102%.

Example 5

Using Aspergillus niger strainsAspergillus nigerThe method for preparing the citric acid by the strain with the preservation number of 101-HAC11 being CGMCC No.12480 comprises the following steps:

(1) the adopted fermentation strain is aspergillus niger strainAspergillus niger101-HAC11 preservation number is CGMCC No.12480, and the fermentation medium comprises (g/L): corn or potato starch saccharification liquid contains 80 g/L (NH) of corn steep liquor calculated by 260g of glucose₄)₂SO₄ 11 g/L, MgSO₄·7H₂O 0.15g/L，K₂HPO_, 6g/L，CuSO₄ 0.8 mg/L ZnSO₄ 0.02 g/L，CaCl₂ 2.0 g/L，KCl 0.1 g/L，FeSO₄ 0.34 mg/L pH 5.0～5.5；

(2) Fermentation culture control conditions: controlling the inoculation amount according to 15% (v/v), controlling the seed age at 27 hr, inoculating the cultured seeds into fermentation medium at 40 deg.C, and fermenting with ventilation of 0.15m³/ m³Min, the tank pressure is 0.12 MPa, the stirring speed is 100 r/min, the fermentation time is 60 hours, sterile air is introduced in the fermentation process, the yield of citric acid is 260g/L as a result of fermentation, and the conversion rate of saccharic acid is 100%.

Example 6

Using Aspergillus niger strainsAspergillus nigerThe method for preparing the citric acid by the strain with the preservation number of 101-HAC11 being CGMCC No.12480 comprises the following steps:

(1) the adopted fermentation strain is aspergillus niger strainAspergillus niger101-HAC11 preservation number is CGMCC No.12480, and the fermentation medium comprises (g/L): 200g of sucrose or maltose, 60g/L of corn steep liquor, (NH)₄)₂SO₄ 9g/L, MgSO₄·7H₂O 0.05g/L，K₂HPO_, 4g/L，CuSO₄ 0.8 mg/L ZnSO₄ 0.02 g/L，CaCl₂ 2.0 g/L，KCl 0.1 g/L，FeSO₄ 0.34 mg/L pH 5.0～5.5；

(2) Fermentation culture control conditions: controlling the inoculation amount according to 10% (v/v), controlling the seed age at 27 hr, inoculating the cultured seeds into fermentation medium at 30 deg.C, and fermenting with ventilation of 0.1m³/ m³Min, the tank pressure is 0.07 MPa, the stirring speed is 80 r/min, the fermentation time is 50 hours, sterile air is introduced in the fermentation process, the yield of citric acid is 220g/L as a result of fermentation, and the conversion rate of saccharic acid is 102%.

Example 7

Using Aspergillus niger strainsAspergillus nigerThe method for preparing the citric acid by the strain with the preservation number of 101-HAC11 being CGMCC No.12480 comprises the following steps:

(1) the adopted fermentation strain is aspergillus niger strainAspergillus niger101-HAC11 preservation number is CGMCC No.12480, and the fermentation medium comprises (g/L): 260g of sucrose or maltose, 80 g/L of corn steep liquor, (NH)₄)₂SO₄ 11 g/L, MgSO₄·7H₂O 0.15g/L，K₂HPO_, 6g/L，CuSO₄ 0.8 mg/L ZnSO₄ 0.02 g/L，CaCl₂ 2.0 g/L，KCl 0.1 g/L，FeSO₄ 0.34 mg/L pH 5.0～5.5；

(2) Fermentation culture control conditions: controlling the inoculation amount according to 15% (v/v), controlling the seed age at 27 hr, inoculating the cultured seeds into fermentation medium at 40 deg.C, and fermenting with ventilation of 0.15m³/ m³Min, the tank pressure is 0.12 MPa, the stirring speed is 100 r/min, the fermentation time is 60 hours, and no air is introduced in the fermentation processThe yield of citric acid is 260g/L and the conversion rate of saccharic acid is 100% as a result of fermentation.

Sequence listing

<110> Tianjin science and technology university

<120> microbial strain for high yield of citric acid and method for producing citric acid by fermenting starch sugar through microbial strain

<141> 2018-09-21

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3639

<212> DNA

<213> Artificial sequence ()

<400> 1

atggctgctc cccgccagcc cgaggaggcg gtcgatgaca ccgagttcat cgatgaccac 60

catgaccagc accgggactc tgtccacacc cgtctgcgtg ccaattcggc catcatgcag 120

ttccaaaaga tccttgttgc caaccgtggt gaaatcccca ttcgtatctt ccggacggct 180

cacgagctgt ccctgcagac tgtcgccgtc tactcccatg aggaccgtct ctccatgcac 240

cgtcagaagg ccgacgaggc ctacatgatt ggcaagcgcg gtcaatatac accggtcggg 300

gcctacttgg ccattgacga gatcgtcaag attgctctgg agcatggtgt acacctgatc 360

cacccgggtt acggtttcct gtccgagaac gccgagtttg cccgcaaggt ggagcagtcc 420

ggcatggttt tcgtcggccc taccccccag accatcgaga gcctcggtga caaggtctcc 480

gcccgtcagc tggctatccg ctgcgatgtg cccgtcgtgc ccggtacccc gggccctgtc 540

gagcgctacg aggaggtcaa ggccttcacc gacacctacg gcttccccat catcatcaag 600

gccgcctttg gtggtggtgg tcgtggtatg cgtgtcgttc gcgaccaggc cgaactgcgt 660

gactccttcg agcgtgccac ctccgaggcc cgctctgcct ttggcaacgg caccgtcttc 720

gtcgagcgct tcctcgaccg ccccaagcac atcgaagtcc agctgctggg tgacaaccac 780

ggcaacgtcg tccacctgtt cgagcgtgac tgctccgtcc aacgtcgcca ccagaaggtc 840

gttgaaattg ccccggccaa ggacctgcct gccgatgtcc gtgaccgcat cctggctgat 900

gctgtcaagc tggccaagtc ggtcaactac cgcaacgccg gtactgcaga gttcctggtt 960

gaccagcaga accgttacta cttcattgag atcaaccccc gtatccaggt cgagcacact 1020

atcaccgaag agatcacggg tatcgatatt gttgctgctc agatccagat tgcggccggt 1080

gctaccctgg aacagctggg tctgacccag gaccgcatct ccactcgtgg attcgccatt 1140

cagtgccgta tcaccaccga ggacccctcc aagggcttct cccccgacac cggtaagatc 1200

gaggtctacc gctccgccgg tggtaacggt gtccgtctgg atggtggaaa cggtttcgcc 1260

ggtgccatca tcacccctca ctacgactcc atgttggtca agtgtacctg ccgtggttcc 1320

acctatgaga tcgctcgccg caaggtcgtt cgtgctttgg tcgagttccg tatccgtggt 1380

gtcaagacca acattccttt cctcacctct cttctgagtc accctgtgtt cgtggatggt 1440

acctgctgga ccacgttcat tgatgacact cccgagctgt tcgcccttgt cggcagtcag 1500

aaccgtgccc agaagctgct ggcttacctg ggtgatgtgg ctgtcaacgg cagcagcatc 1560

aagggccaga tcggcgagcc caagctcaag ggcgatatca tcaagcccgt tctgcatgac 1620

gctgccggca agcccctcga cgtctctgtc cccgccacca agggatggaa gcagatcctg 1680

gacagtgagg gtcccgaggc ttttgcccgc gctgtgcgtg ccaacaaggg ctgcttgatc 1740

atggatacta cctggcgtga tgcccaccag tcgctgctgg ccactcgtgt gcgtaccatt 1800

gacctcctga acatcgccca cgagacgagc cacgccctcg ccaacgccta cagtttggaa 1860

tgctggggtg gtgccacctt cgatgtcgcc atgcgcttcc tgtacgagga cccctgggac 1920

cgtcttcgca agctgcgcaa ggccgttccc aacatcccct tccagatgtt gctccgtggt 1980

gccaacggtg ttgcttactc ctccctccct gacaacgcca tctaccactt ctgtaagcag 2040

gccaagaagt gcggtgtcga catcttccgt gtcttcgatg ctctcaacga cgttgaccag 2100

ctcgaggtcg gtatcaaggc tgtccacgct gccgagggtg ttgttgaggc tactatttgc 2160

tacagtggtg atatgctcaa ccccagcaag aagtacaacc tgccttacta cctcgacctt 2220

gttgataagg tggtccagtt caagccccac gtcttgggta tcaaggacat ggctggtgtg 2280

ctgaagcccc aggctgctcg tctgctgatc ggttccatcc gcgagcgcta ccccgacctc 2340

cccatccacg tgcacaccca cgactctgct ggtaccggtg tggcttccat gattgcttgc 2400

gctcaggccg gtgctgatgc cgttgatgct gccacggaca gcctttccgg tatgacctcc 2460

cagcccagca ttggtgctat cctcgcttcc ctcgagggaa ctgagcacga ccccggcctc 2520

aactcggccc aggttcgcgc ccttgacacc tactgggcgc agctgcgtct cctctactcg 2580

cccttcgagg caggtctgac tggtcccgac cccgaggttt acgagcatga gatccctggt 2640

ggtcaattga ccaacctgat cttccaggcc agccagcttg gtctgggcca gcagtgggcg 2700

gagacgaaga aggcttacga gtctgccaac gatcttttgg gcgatgtcgt caaggtcacc 2760

cccacttcca aggtggtcgg tgatttggct cagttcatgg tctccaacaa gttgactgct 2820

gaagatgtga ttgctcgcgc cggcgagctt gacttccccg gttccgttct ggagttcctc 2880

gagggtctca tgggccagcc ctacggtggg ttccccgagc ctctgcgctc tcgcgctctg 2940

cgtgaccgtc gcaagctcga caagcgccct ggtctgtacc tggagcccct tgacctggcc 3000

aagatcaaga gccagatccg ggagaactat ggcgcagcta ccgagtacga tgtggccagc 3060

tatgccatgt accccaaggt cttcgaggat tacaagaagt ttgtcgccaa gttcggtgat 3120

ttgtccgtcc tgcccacccg ttacttcttg gccaagcccg agatcggcga ggaattccac 3180

gtcgagctgg agaagggtaa ggtgctgatc ctgaagttgt tggccattgg tcccctctcc 3240

gagcagaccg gccagcgtga ggtcttctac gaagtcaacg gtgaggttcg ccaggttagt 3300

gtggacgaca agaaggcgtc cgtcgagaac accgcccgcc ccaaggccga gctgggtgac 3360

agcagccagg ttggtgctcc tatgagcggt gtggttgtcg agatccgcgt ccacgacggc 3420

ctggaagtca agaagggtga ccccattgcc gtcctgagcg ccatgaagat ggtatgtata 3480

accccaaaaa ctatcaacca aaactcctga ccggctgcta acatctgtta ggaaatggtt 3540

atctctgctc cccacagtgg caaggtctcc agcttgctgg tcaaggaagg tgactcggtg 3600

gatggccagg atcttgtctg caagatcgtc aaggcctag 3639

<210> 2

<211> 625

<212> DNA

<213> Artificial sequence ()

<400> 2

tcaggaactt agccttatga gatgaatgat ggacgtgtct ggcctcggaa aaggatatat 60

ggggatcatg atagtactag ccatattaat gaagggcata taccacgcgt tggacctgcg 120

ttatagcttc ccgttagtta tagtaccatc gttataccag ccaatcaagt caccacgcac 180

gaccggggac ggcgaatccc cgggaattga aagaaattgc atcccaggcc agtgaggcca 240

gcgattggcc acctctccaa ggcacagggc cattctgcag cgctggtgga ttcatcgcaa 300

tttcccccgg cccggcccga caccgctata ggctggttct cccacaccat cggagattcg 360

tcgcctaatg tctcgtccgt tcacaagctg aagagcttga agtggcgaga tgtctctgca 420

ggaattcaag ctagatgcta agcgatattg catggcaata tgtgttgatg catgtgcttc 480

ttccttcagc ttcccctcgt gcagatgagg tttggctata aattgaagtg gttggtcggg 540

gttccgtgag gggctgaagt gcttcctccc ttttagacgc aactgagagc ctgagcttca 600

tccccagcat cattacacct cagca 625

<210> 3

<211> 362

<212> DNA

<213> Artificial sequence ()

<400> 3

aagagtaaca ccgttataac ggcaaggttt gaaaagctaa tgtcgatgtt gatgtccttg 60

caaccggctt atttgtcaac aaggacacta tgccaatatt ctttttctag attagagcgt 120

ctgttatgtt tatgaatatt ttgggttggg tttgggttat ttccatattt atgccacagc 180

ttatttacga tcaatggttg tcacggacat acaatgtcaa ccatacgaat gacaagtgat 240

ttcaaaacat tctttcagat atattcgtag tatctaggaa acacagcata tatagtcatg 300

atggatatgt acaggatcaa gaccaacctc catttattca gaagtattga atctcttctc 360

cc 362

<210> 4

<211> 1856

<212> DNA

<213> Artificial sequence ()

<400> 4

ccttcctctc ctccagctcc tctataccct ccccggttgg accctccctc tcgtccatca 60

tgttgttccg cgctgcggtc cgtcagtccg cgcccttgag gcgtcaggcc ctcactcctc 120

ttgctcgtcg ctccgtcacc accgacgccg cctcgtcgca tgctgaaaat gtcccccagg 180

ttcgttgaga gctccctgga tatcccggat tgacctccgc aatggccctg gctaatgcga 240

tttccatcta tctacaggag gatgacaagc ccttcactgt ccgcctctcc gacgagagct 300

tcgagaccta cgagatcgac cctcccccgt acaccctgga gatcaccaag aaggagctca 360

agcagatgta ctacgacatg gtttcgatga ggtatggcca ccgtttggcc ctttgctttt 420

tctttccaca agcctcatgc agaatctaat ctatcgctca gacgcatgga gatggccgcc 480

gaccgtcttt acaaggagaa gaagatcaga ggtttctgcc acttgtctac cggtcaggaa 540

gccgtcgcca ccggtatcga gcacgctatc acccgcgacg acaaggtcat cactgcctac 600

cgttgccacg gtttcgccat gatgcgcggt ggtaccgtcc gctccatcat cggtgagctg 660

ctcggtcgcc gtgagggtat cgcctacgga aagggtggtt ccatgcacat gttcgccccc 720

aacttctacg gcggtaacgg tatcgtcggt gcccaggtcc ccgtcggtgc tggtctcgct 780

ttcgcccagc agtacaacga ggagcccacc accagtatcg tcctctacgg tgacggtgcc 840

tccaaccagg gtcaggtctt cgaggccttc aacatggcca agctgtggaa cctccccgtt 900

ctgttcggtt gtgagagtaa gtcatagtcc tcacgccact ccatccaaga cagccaaagc 960

taattccttt tctactagac aacaagtacg gtatgggtac ctccgccgct cgctcctccg 1020

ccttgaccga gtactacaag cgtggtcagt acatccctgg tatcaaggtc aacggcatgg 1080

acgtcctggc caccaaggcc gccgtcaagt acggtaagga ctgggccgtg gccggcaacg 1140

gccccctggt ctacgagtac gtcacctacc gttacggtgg tcactccatg tccgaccccg 1200

gtaccaccta ccgtagccgt gaggagatcc agcgcatgcg cagcaccaac gaccccatcg 1260

ccggtctcaa gcagaagatc ctcgactgga acgtctgctc cgaggacgag ctcaagtccc 1320

tcgacaaggc cgctcgtgcc cacgtcgacg aggaggtcgc catcgccgag aagatgcccg 1380

ctcccgagaa cacctcccgc attctcttcg aggacatcta cgtccgtggc tccgagccca 1440

agtggatgcg tggtcgcact gtcgacgaga ccttctacta ctagatatac ccataccact 1500

caaatccaaa tctagatatg tttctttaaa acacaaggct ccctattttc cagtgtgaag 1560

aatgaagctc agttggaaga actctggatg atgatgacaa ggcgagagaa gaaaaagaaa 1620

gaaataggag gagcgagtga gaggaaggtc atccttttgt ttttcttgtt aaacacaaaa 1680

gtttagaccg cgcacacaca tcacgcacca taccatttgg gattggcagg ctcctaccta 1740

cttactacgc aaaggcatcc ataccatgga tatgtttact atgtaattta gcccgggaaa 1800

tttttctgtt tttatttata tttttgcatt actccaaacc tcatcttgaa gtttca 1856

<210> 5

<211> 1056

<212> DNA

<213> Artificial sequence ()

<400> 5

atgaactcgt taacagccac ggcgcccatt cgggctgcta ttccaaagtc atatatgcat 60

atcgcgactc gaaattactc caatgtcatt gctatgagcg gtctgcgctg tagcgggtcg 120

ttggtggcaa acagacatca gacagctgga aagcgattca tatcaaccac acccaagtcg 180

cagatcaagg aattctttcc tcccccgaca gctcctcatg tgaaggaggt ggaaacagct 240

tgggtccatc ctgtctatac tgaagagcag atgaaacaag tcgcaatcgc tcaccgagac 300

gcaaagaatt gggccgactg ggtagcgttg ggaacggtgc ggatgctgag atggggcatg 360

gatcttgtga ctggatatcg gcaccctcca ccaggaaggg aacacgaggc taggttcaag 420

atgacagagc agaagtggct tacgcgcttt atcttcctgg aaagcgtggc tggcgtacca 480

ggcatggtcg gaggcatgct gagacatttg agaagtttgc ggcgcatgaa gagagacaat 540

ggatggattg agacactgct agaggaagca tacaatgaac gtatgcattt gctgaccttc 600

ctcaagctcg cagagcccgg atggttcatg cgcctgatgg tccttggagc acagggggta 660

ttcttcaacg gattcttcct gtcttacctc atgtcgccac gcatctgcca ccggttcgtc 720

ggttatctcg aagaggaagc ggtgatcaca tatactcggg cgatcaagga gattgaagct 780

ggaagtcttc ccgtgtggga gaagacagag gcccctgaga tcgccgtgca atattggaag 840

atgccagagg gtcagcgcag catgaaggat ctcttgctgt atgttcgggc ggatgaagcc 900

aaacatcggg aggtgaacca tacactggga aacctaaacc aggcgatcga ccccaaccca 960

tatgccgcaa agtacaagga tccgacaaag gcgcatccga acaaggggat tgcagacctg 1020

aaacccatgg gatgggagcg ggaggaggtg atctga 1056

<210> 6

<211> 1261

<212> DNA

<213> Artificial sequence ()

<400> 6

acagaggcca gagcatcacc aacatgggta ccctcagcaa taatatgcat gcattgtgcc 60

ccccctatgg agccgtagct ttcaagcaat tagacacgcg cccggccgaa tgagatgaac 120

cgttggagcc atcatcccac tcatcccgct ccagaaagga gagaaagaaa aaaaaaaaat 180

atgaccgagc gcgtgatgac cggtgaggac tccggtgaat tgatttgggt gacgggagag 240

acccaagagg ggccagaata ataagaatgg ggaaggcgaa ggtaccgcct ttggggtcca 300

gccacgcgac tccaacatgg aggggcactg gactaacatt attccagcac cgggatcacg 360

ggccgaaagc ggcaaggccg cgcactgccc ctctttttgg gtgaaagagc tggcagtaac 420

ttaactgtac tttctggagt gaataatact actactatga aagaccgcga tgggccgata 480

gtagtagtta cttccattac atcatctcat ccgcccggtt cctcgcctcc gcggcagtct 540

acgggtagga tcgtagcaaa aacccggggg atagacccgt cgtcccgagc tggagttccg 600

tataacctag gtagaaggta tcaattgaac ccgaacaact ggcaaaacat tctcgagatc 660

gtaggagtga gtacccggcg tgatggaggg ggagcacgct cattggtccg tacggcagct 720

gccgaggggg agcaggagat ccaaatatcg tgagtctcct gctttgcccg gtgtatgaaa 780

ccggaaagga ctgctgggga actggggagc ggcgcaagcc gggaatccca gctgacaatt 840

gacccatcct catgccgtgg cagagcttga ggtagctttt gccccgtctg tctccccggt 900

gtgcgcattc gactgggcgc ggcatctgtg cctcctccag gagcggagga cccagtagta 960

agtaggcctg acctggtcgt tgcgtcagtc cagaggttcc ctcccctacc ctttttctac 1020

ttcccctccc ccgccgctca acttttcttt cccttttact ttctctctct cttcctcttc 1080

atccatcctc tcttcatcac ttccctcttc ccttcatcca attcatcttc caagtgagtc 1140

ttcctcccca tctgtccctc catctttccc atcatcatct cccctcccag ctcctcccct 1200

cctctcgtct cctcacgaag cttgactaac cattaccccg ccacatagac acatctaaac 1260

a 1261

<210> 7

<211> 1395

<212> DNA

<213> Artificial sequence ()

<400> 7

tcgacgttaa ctgatattga aggagcattt tttgggcttg gctggagcta gtggaggtca 60

acaatgaatg cctattttgg tttagtcgtc caggcggtga gcacaaaatt tgtgtcgttt 120

gacaagatgg ttcatttagg caactggtca gatcagcccc acttgtagca gtagcggcgg 180

cgctcgaagt gtgactctta ttagcagaca ggaacgagga cattattatc atctgctgct 240

tggtgcacga taacttggtg cgtttgtcaa gcaaggtaag tggacgaccc ggtcatacct 300

tcttaagttc gcccttcctc cctttatttc agattcaatc tgacttacct attctaccca 360

agcatccaaa tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa 420

aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc 480

agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc 540

tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg 600

cttgacattg gggagttcag cgagagcctg acctattgca tctcccgccg tgcacagggt 660

gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctccagcc ggtcgcggag 720

gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga 780

ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc 840

catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct 900

ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcatgcg 960

gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg 1020

agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcctctt ctggaggccg 1080

tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca 1140

ggatcgccgc gcctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc 1200

ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc 1260

cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg 1320

accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg 1380

agggcaaagg aatag 1395

Claims (2)

1. Aspergillus niger (A) for producing citric acidAspergillus niger) 101-HAC11 strain with preservation number of CGMCC No. 12480.

2. A method for preparing citric acid using the citric acid-producing aspergillus niger 101-HAC11 of claim 1:

(1) the adopted fermentation strain is Aspergillus niger 101-HAC11 strain;

(2) the fermentation culture control conditions are as follows: controlling the inoculation amount to be 10-15% (v/v), controlling the seed age to be 24-27 hours, inoculating the cultured seeds into a fermentation tank, controlling the culture temperature to be 30-40 ℃, and controlling the fermentation ventilation amount to be 0.1-0.15 m³/ m³Min, the tank pressure is 0.07-0.12 MPa, the stirring speed is 80-100 r/min, the fermentation time is 50-60 hours, and sterile air is introduced in the fermentation process.

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