CN109207373A - The method that one plant height produces the microbial strains and its fermentation starch saccharic production citric acid of citric acid - Google Patents

The method that one plant height produces the microbial strains and its fermentation starch saccharic production citric acid of citric acid Download PDF

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CN109207373A
CN109207373A CN201811104656.2A CN201811104656A CN109207373A CN 109207373 A CN109207373 A CN 109207373A CN 201811104656 A CN201811104656 A CN 201811104656A CN 109207373 A CN109207373 A CN 109207373A
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fermentation
citric acid
aspergillus niger
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acid
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CN109207373B (en
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王德培
张鸿飞
秦郦
张建华
侯莉
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Tianjin University of Science and Technology
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Abstract

The method that the microbial strains and its fermentation starch saccharic production citric acid of citric acid are produced the invention discloses a plant height, classify Aspergillus niger strain Aspergillus niger 101-HAC11, deposit number CGMCC No.12480.The culture medium prescription and fermentation processes technique that citric acid is produced using starch saccharic as carbon source through fermentation are also disclosed simultaneously.Using the yield of bacterial strain production citric acid up to 166.8g/L.Compared with prior art, which is 220.34 g/L, output increased 32.10%.This method has quickly, and the technological process of production is simple, product non-toxic residual, highly-safe feature.

Description

One plant height produces the microbial strains of citric acid and its fermentation starch saccharic produces citric acid Method
Technical field
The invention belongs to fermentation engineering fields, are related to the Aspergillus niger strain application that genetic engineering constructs high ferment strength The method that the fermentation of starch saccharic generates citric acid.In particular be: a strain gene engineering constructs the aspergillus niger of high ferment strength Bacterial strain adds other organic and inorganic nitrogen-sourced and necessary metal salt component fermenting and producing lemon by fermenting carbon source of starch saccharic The method of lemon acid.
Background technique
Citric acid (Citric acid) is that one kind common are machine acid, in food and drink, medication chemistry, cosmetic industry It is widely used, is a kind of maximum organic acid of current world demand amount.
C.W. house is strangled and citric acid is made using the calcium citrate precipitation method within 1784.At the end of the 19th century at the beginning of biological fermentation process blank At.1916, most of Aspergillus such as aspergillus oryzae, aspergillus awamori, Trichoderma viride and Wen Shi aspergillus and aspergillus niger by Tom and Curry experiment confirms all produce citric acid, and aspergillus niger then occupy the umber one.Before in 20th century, shallow tray fermentation method is mainstream, Nineteen twenty-three in the world first hand with aspergillus niger shallow tray fermentation method production citric acid factory in Fei Ze company of the U.S..After nineteen fifty, Tank fermentation method is gradually built up.Compared to shallow tray fermentation method, the period is shorter for submerged fermentation, and yield is higher, and occupied area is small, labor Power more saves, highly beneficial for industrialized production, has become the main method of Citric Acid Production.
China, which most rises earlier than nineteen forty-two soup, to be taught fermentation method and produces citric acid in the report of Chinese et al..Nineteen fifty-two starts to use Shallow tray fermentation legal system takes citric acid.The 200L scale submerged fermentation lemon of nineteen fifty-nine Ministry of Light Industry's fermentation industry Science Institute Sour Preliminary Experiment success, after 1966, Tianjin Research Institute of Industrial Microbiology, Shanghai City Industry Wei Biological Research Institute are successively used Using potato dry powder as raw material, citric acid is produced in submerged fermentation, and is succeeded in succession, so that it is determined that the one of domestic product citric acid Dominating process route.Since 2000 so far, China's Citric Acid Production is liquefied liquid glucose with corn flour through high-temperature injection, and application is black Aspergillus strain liquid submerged fermentation produces citric acid, using calcium salt method and sulfuric acid solution separation and Extraction citric acid.As China is sent out The technological progress of ferment industry, and the environmental protection requirement of production process is increasingly improved, technique production citric acid can not Reach high-intensitive citric acid fermented requirement.
Main cause has following problem:
(1) using corn flour liquefier and with part maize pulp co-fermentation, cause fermentation liquid sticky, at present fermentation liquid Total reducing sugar can be such that feed liquid viscosity further increases in 17%-18%, if increasing the total reducing sugar of mash, and fermentation process is to reach dissolved oxygen to want It asks ventilation and stirring energy consumption excessive, cannot achieve industrialization, tank single in this way produces acid and is limited to 18% or so, can not be promoted.
(2) containing various saccharides such as dextrin, oligosaccharides, oligosaccharide, maltose, isomaltoses in liquefied corn, and contain Different types of protein causes tunning complicated, generates a variety of heteroacid.Fermentation liquid ingredient multiplicity, separates for citric acid Extraction brings difficulty, and citric acid, which extracts, at present uses calcium salt method, and most beneficial for the removal of heteroacid, but it is useless to generate calcium sulfate simultaneously Slag can not be handled, and form industrial production waste residue.Since fermentation liquid is difficult to isolate and purify citric acid, so continuing to use calcium salt method always Citric acid is extracted, and calcium sulfate has become the solid waste hidden danger of each Citric Acid Production company.
(3) citric acid fermentation terminates at present, and black-koji mould pompon is blended in unavailable maize pulp in solid waste of fermenting Together, it can only dry into feed market, and black-koji mould pompon can be used to extract aminoglucose saccharic and sterol etc. completely High value added product.
(4) since citric acid fermentation uses corn flour liquefier for raw material, by the extreme influence of corn quality, such as corn Seed drying temperature, the corn place of production, corn variety, corn period of storage etc. can all influence the carbon-nitrogen ratio in liquefied corn, Cause fermentation process unstable, influences Citric Acid Production.
(5) citric acid fermentation uses corn flour liquefier for raw material at present, leads to dissolved oxygen and pH measurement and control in fermentation process Difficulty, fermentation process automation control and continuous production cannot achieve.
In summary reason, it is imperative as the citric acid fermentation of raw material using starch saccharic to develop.
There is following advantage based on the citric acid fermented technique of starch saccharic:
(1) pulverized sugar matter is object solid content in fermenting raw materials liquid, and fermentation liquid viscosity is low, and can increase initial sugar concentration and reach 25%, It realizes that single tank produces acid 25% or more, improves labor productivity.
(2) starch saccharic can be completely soluble, and fermentation liquid viscosity is low, is conducive to dissolved oxygen, and fermentation process is ventilated and stirred It mixes energy consumption to decline to a great extent, realizes the energy conservation object of Citric Acid Fermentation.
(3) heteroacid of the citric acid fermented generation of starch saccharic lacks citric acid purity is high, and other readily carbonizable substances concentration are low, have Conducive to citric acid separation and Extraction, calcium salt method can be thoroughly substituted using directly condensing crystallizing extracts citric acid after fermentation liquor treatment, Eliminate the calcium sulfate waste residues that calcium salt method generates.
(4) the black-koji mould pompon of the citric acid fermented generation of starch saccharic can be separated directly from fermentation liquid, be used for ammonia Sugar and sterol etc. extract, and realize high value added product.
(5) the citric acid fermented fermentation liquid of starch saccharic is in addition to black-koji mould pompon, without other solid contents, is conducive to ferment Online observation of the journey to mycelium pellet can carry out online composition detection to fermentation liquid, to realize automation control and ferment The accurate optimization of journey, final to realize that starch sugar mass flow adds, the realization of the continuous ferments technique such as mycelium pellet cutting duplication.
Realize the citric acid fermented key technical problems of starch saccharic:
(1) the organic nitrogen source type and concentration added during starch saccharic is citric acid fermented are to realize the conversion of citric acid fermentation height The key of rate.
(2) preferable mycelium pellet can be formed during starch saccharic is citric acid fermented.
(3) the high conversion bacterial strain citric acid fermented for starch saccharic.
Summary of the invention
The purpose of the present invention is to provide an Aspergillus niger strainsAspergillus niger101-HAC11(CGMCC No.12480 the culture medium prescription and fermentation processes technique of citric acid) are produced using starch saccharic as carbon source through fermentation.
It is a further object to provide the bacterial strain aspergillus nigers of one plant of microbial fermentation starch saccharic high yield citric acid Bacterial strainAspergillus niger101-HAC11, classification category black-koji mould (Aspergillus niger), deposit number is CGMCC No. 12480。
The present invention a further object is that is disclosed uses Aspergillus niger strainAspergillus niger 101-HAC11 (CGMCC No.12480) strain, the method for fermentation starch saccharic production citric acid.
To achieve the above object, the technology contents that the present invention is disclosed directly below:
Genetic engineering constructs aspergillus nigerAspergillus niger101-HAC11 microbial fermentation bacterial strain, classification belong to black song Mould (Aspergillus niger), deposit number is CGMCC No. 12480.
12480 bacterial strain of CGMCC No. provided by the invention is that one plant of energy is citric acid fermented using starch saccharic, passes through base Because of the aspergillus niger new strains of engineering building, citric acid is produced with the strain fermentation saccharine material, classification belongs to aspergillus niger (Aspergillus niger), deposit number is CGMCC No. 12480, preservation date on June 12nd, 2016.Preservation place: in State's Microbiological Culture Collection administration committee common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3). The bacterial strain is with Citric Acid Production bacterial strain aspergillus nigerAspergillus nigerWLG CGMCC No.10142, preservation date On December 11st, 2014, preservation place: China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: Beijing The institute 3 of city Chaoyang District North Star West Road 1).Aspergillus nigerWLG CGMCC No.10142 is starting strain, is passed through Genetic engineering building is overexpressed the high ferment strength that aox1 gene, pyruvate carboxylase gene, pyruvate dehydrogenase gene obtain Citric acid bacterial strain.
Aspergillus nigerAspergillus niger101-HAC11 microbial fermentation bacterial strain, deposit number are CGMCC No. 12480 physicochemical property is as follows: enzyme pyruvate carboxylase that can be crucial in high efficient expression citric acid synthesis process, pyruvic acid are de- Hydrogen enzyme, alternative oxidase.
The present invention is expressed in aspergillus niger using inducible promoter Pgla starting PC albumen, and then is started using composing type Sub- PgpdA starts pyruvic dehydrogenase PD gene, alternative oxidase Aox1 gene, by enhancing pyruvate carboxylase PC gene table Up to amount and activity, acetone acid content is improved.Acetyl-CoA, NADH oxidative metabolism stream are further enhanced, to improve citric acid Yield, saccharic acid conversion ratio.
The method of the present invention is with Citric Acid Production bacterial strain aspergillus nigerAspergillus niger WLG CGMCC No.10142 is that starting strain carries out genetic engineering transformation, and the lemon acid yield of the starting strain is 166.8g/L, with existing skill Art is compared, which is 220.34 g/L, output increased 32.10%.
Method disclosed by the invention using 12480 strain fermentation starch saccharic of CGMCC No. production citric acid, it is special Sign is:
(1) fermentation strain used is Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480, fermentation medium group become (g/L): starch 200 ~ 260g of saccharine material, corn pulp 60 ~ 80 g/L, (NH4)2SO4 9~11 g/L, MgSO4·7H2O 0.05 ~ 0.15g/L, K2HPO,4 ~ 6g/L, CuSO4 0.8 mg/L ZnSO4 0.02 g/ L, CaCl22.0 g/L, KCl 0.1 g/L, FeSO40.34 mg/L pH 5.0~5.5；
(2) fermented and cultured control condition: existing by the inoculum concentration control of 10% ~ 15%(v/v), and kind age control will be cultivated at 27 hours Good seed accesses fermentation medium, and 30 ~ 40 DEG C of cultivation temperature, fermentation ventilatory capacity is 0.1 ~ 0.15m3/ m3.min, tank pressure 0.07 ~ 0.12 MPa, fermentation time 50 ~ 60 hours, is passed through filtrated air by 80 ~ 100 revs/min of mixing speed in fermentation process, Fermentation results lemon acid yield is 220 ~ 260g/L, and saccharic acid conversion ratio is 102% or more.With quick, technological process of production letter List, the technological process of production is simple, saccharic acid conversion ratio is high, adaptation automation large-scale industrial production, high production efficiency, and product is easy The features such as separation.
Preparation method of the present invention, it (includes: cornstarch, potato shallow lake that wherein starch saccharine material, which includes: starch, Powder, tapioca) filtered after liquefying, being saccharified containing Saccharoid solution, wherein saccharic includes: glucose, sucrose, fructose, wheat Bud sugar, achrodextrin.
More detailed description of the present invention is as follows:
One plant height produces the fermentation strain of citric acid microorganism, Aspergillus niger strain of classifyingAspergillus niger 101- HAC11, deposit number CGMCC No.12480, the construction method of the bacterial strain is: by aspergillus niger pyruvate carboxylase PC gene, It is black using the recombination on pyruvic dehydrogenase PD gene and alternative oxidase Aox1 gene multi-copy integration to aspergillus niger genome Aspergillus strain fermentation production of citric acid；Regulation of the expression of the pyruvate carboxylase PC gene by Pgla promoter, pyruvic acid Dehydrogenase PD gene and alternative oxidase Aox1 gene are by PgpdA promoter regulation.
The nucleotide sequence of the pyruvate carboxylase PC gene such as SEQ ID NO.1, the Pgla promoter are induction Type promoter, the nucleotide sequence of the promoter is as shown in SEQ ID NO.2.
A kind of pyruvate carboxylase PC gene expression frame, the expression cassette include Pgla promoter, pyruvate carboxylase PC Gene, HYG resistance marker and trp terminator are arranged according to the sequence of Pgla-PC-HYG-trp.The nucleosides of the trp terminator Acid sequence is as shown in SEQ ID NO.3.The nucleotide sequence of the pyruvic dehydrogenase PD gene such as SEQ ID NO.4；It is described The nucleotide sequence of alternative oxidase Aox1 gene such as SEQ ID NO.5, the PgpdA promoter are constitutive promoter, should The nucleotide sequence of promoter is as shown in SEQ ID NO.6.The nucleotides sequence of HYG gene is classified as SEQ ID NO:7.
The preparation method of recombinant aspergillus niger bacterium of the present invention includes the following steps:
2. constructing pyruvate carboxylase PC gene expression frame Pgla-PC-HYG-trp；
2. constructing pyruvic dehydrogenase PD gene and alternative oxidase Aox1 gene expression frame PgpdA-PD-Aox1- HYG-trp；
3. by step, 1. 2. gene expression frame obtained converts aspergillus niger with step, through described in resistance screening and PCR identification acquisition Recombinant aspergillus niger bacterium.
The step 1. described in resistance expression's frame HYG gene nucleotide sequence as shown in SEQ ID NO.7.
(2) liquid submerged fermentation aspergillus nigerAspergillus niger101-HAC11 produce citric acid culture medium and Fermentation processes:
A. different organic nitrogen source types are to aspergillus nigerAspergillus niger101-HAC11 starch saccharic citric acid fermentation It influences；Different organic nitrogen sources are to aspergillus nigerAspergillus niger101-HAC11 citric acid fermentation, the tool such as saccharic acid conversion ratio There is significant impact, as a result as shown in Figure 1.
As shown in Figure 1,4 kinds of nitrogen sources are corn pulp > soy peptone > protein feed for the influence sequencing for producing acid > yeast powder produces acid and reaches as high as 9.27% when adding corn pulp, saccharic acid conversion ratio is above other nitrogen sources, and residual reduced sugar Relatively fewer, soy peptone takes second place, and adds corn pulp and soy peptone fermenting property better than other organic nitrogen sources of addition.It is comprehensive It closes above acid, conversion ratio, residual reduced sugar and the pellet form of producing to consider, in four kinds of organic nitrogen sources, final corn pulp of choosing is lemon The most suitable organic nitrogen source of lemon acid corn liquor fermentation.
B. the determination of corn pulp additional amount
Determine corn pulp be best organic nitrogen source, the concentration of corn pulp be relationship citric acid whether be capable of high-efficiency fermenting it is important because One of element, by measuring the calculating of last production acid and saccharic acid conversion ratio, so that it is determined that corn pulp optimum concentration.As a result as schemed Shown in 2.As shown in Figure 2, Aspergillus Niger acid and the equal highest of saccharic acid conversion ratio when corn pulp concentration is 0.7%, continue growing corn Slurry produces acid and is not further added by.It is comprehensively compared and produces acid and conversion ratio index, choose 0.7% corn pulp most preferably having as fermentation medium Machine nitrogen source.
C. various concentration (NH4)2SO4Influence to citric acid liquor fermentation
It is added after 0.7% corn pulp in the fermentation medium plus (the NH of different final concentrations4)2SO4, by measuring last production acid, So that it is determined that (NH in liquor fermentation culture medium4)2SO4Optimum concentration, as a result such as Fig. 3.From the figure 3, it may be seen that (NH in fermentation liquid4)2SO4Total concentration 0.1% when, produce acid amount and saccharic acid conversion ratio be highest.In (the NH for adding other concentration4)2SO4When, produce acid Amount and saccharic acid conversion ratio are undesirable, so selecting 0.1% as (NH4)2SO4Most suitable total concentration.
D. influence of the various concentration trophic factors inositol to citric acid liquor fermentation
The inositol for adding various concentration in the fermentation medium finally measures last production acid and observes mycelium morphology to really The most suitable additive amount of liquor fermentation culture medium mysoinositol is determined, as a result such as Fig. 4.Fig. 4 is it is found that in terms of producing acid and saccharic acid conversion ratio It sees, adding a certain amount of inositol has obvious effect to high acid is mentioned, and the fermentation and acid for adding a certain amount of inositol, which is above, to be not added with The fermentation and acid of inositol continues growing flesh wherein producing acid and saccharic acid conversion ratio highest when the inositol of addition total concentration 0.007% When determining alcohol, production acid is not further added by and mycelium pellet consistency declines, and mycelia diffusion is more.In summary factor considers to choose 0.007% inositol is optimal concentration.
E. influence of the concentration of metal ion to fermentation
Choose MgSO4、CuSO4、ZnSO4、CaCl2、KCl、FeSO4A variety of concentration carry out orthogonal test, and orthogonal experiments are shown in Table 1。
1 orthogonal experiments of table
2 orthogonal test variance analysis of table
As shown in Table 2, MgSO4、CuSO4、ZnSO4、CaCl2、KCl、FeSO4In the different level of this six factors, by very poor Analyze the primary and secondary sequence it is found that impact factor are as follows: CaCl2> MgSO4> KCl > FeSO4> ZnSO4> CuSO4, optimum combination is A1B1C1D1E1F3, the optimal concentration after optimization: MgSO40.1 g/L, CuSO4 0.8 mg/L ZnSO40.02 g/L, CaCl22.0 g/L, KCl 0.1 g/L, FeSO4 0.34 mg/L。
Under optimal concentration condition of culture, to optimum results carry out verification test, 500 mL triangular flask liquid amount, 50 mL, 36.5 DEG C, 330 r/min carry out 72 h of citric acid fermentation.It is 9.96% that verification result, which produces acid, is higher than any group of in orthogonal experiment Acid is produced, the advantageous effect of optimal combination is demonstrated.
F. starch saccharic clear liquid optimal conditions of fermentation
(1) influence of the different initial sugar concentrations to citric acid fermentation
It chooses after different starch saccharic clear liquids are fermented, measures lemon acid yield, residual sugar the results are shown in Table 3.
3 fermentation ends lemon acid yield of table
As shown in Table 3, when initial sugar concentration is 20%-22%, saccharic acid conversion ratio reaches 100% or more when fermentation ends, and residual sugar is dense Degree is in reduced levels.
(2) influence of the kind age to citric acid liquor fermentation
The seed liquor for being inoculated with age not of the same race carries out fermenting experiment, measures final citric acid content, and residual reduced sugar simultaneously records corresponding hair In the ferment period, it the results are shown in Table 4.
Influence of the 4 kinds of ages of table to citric acid fermentation
As shown in Table 4, with the increase in kind of age, citric acid yield is gradually increased, and sour highest is wherein produced when 27 h of cell age, therefore Selecting kind of 27 h of age is the aspergillus niger strain best culture transferring time.
(3) different process controls the influence to citric acid liquor fermentation
Different process control, makes oxyty in fermentation process fermentation liquid differ greatly, and oxyty has citric acid fermentation Strong influence.It is sampled since fermentation every 8 h, measures the situation of change of citric acid concentration in fermentation liquid, pH value.As a result such as Shown in Fig. 5.As shown in Figure 5, when 0-16h, big ventilation quantity dissolved oxygen is better than sectionalized ventilation；When 16-64h, option A dissolved oxygen is higher than scheme B.From the point of view of Fig. 5, in option b, earlier fermentation dissolved oxygen is excessively high, easily causes growing microorganism superfluous, aspergillus niger is caused to enter too early Citric acid accumulated phase, pH value decline are too fast.Low pH will affect diastatic activity, and then influence the utilization rate of raw material, make fermentation liquid Residual sugar too high levels, therefore later period option b of fermenting produces acid not as good as option A.Final Choice A three-stage ventilation quantity.
It as shown in Figure 5, is 0.15m in earlier fermentation (0 ~ 16 h) control ventilation quantity3/ m3.min, thallus is largely proliferated, Metabolism of Normal, fermentation liquid pH are 3.5, which is conducive to keep fermentation liquid saccharifying enzymic activity, increase content of reducing sugar, are fermented Aspergillus niger enters the sour phase that quickly produces, 17 ~ 48 h ventilation quantities adjustment are as follows: 0.225 m after 16 h3/ m3.min, revolving speed 90 ~ 100 R/min, black-koji mould pompon metabolism at this time is more vigorous, and more reduced sugars are converted into citric acid, and content of reducing sugar is opened at this time Beginning sharp fall produces acid amount and starts to increase significantly；Close to fermentation ends, 49 ~ 58 h ventilation quantities adjustment are as follows: ventilation after 48h 0.10 m3/ m3.min, 85 ~ 95 r/min of revolving speed, reduced sugar is in reduced levels at this time, and dissolved oxygen is higher, and pH value is relatively stable, Ventilation and revolving speed can be reduced.In 58 h fermentation ends, lemon acid yield is 220.3g/L, and saccharic acid conversion ratio 102.8% is residual Restoring sugar amount is 0.1%.
It is had the active effect that possessed by microbial fermentation bacterial strain disclosed by the invention for producing citric acid and preparation method thereof
(1) pulverized sugar matter is that fermenting raw materials mash viscosity is low, and can increase initial sugar concentration and reach 25%, realizes that single tank produces acid 25% More than, improve labor productivity.
(2) starch saccharic can be completely soluble, is conducive to dissolved oxygen, and fermentation process ventilation and stirring energy consumption decline to a great extent, Realize the energy conservation object of Citric Acid Fermentation.
(3) the citric acid purity is high of the citric acid fermented generation of starch saccharic, is directly concentrated after can using fermentation liquor treatment Citric acid is extracted in crystallization, thoroughly substitutes calcium salt method, eliminates the calcium sulfate waste residues that calcium salt method generates.
(4) the black-koji mould pompon of the citric acid fermented generation of starch saccharic is extracted for ammonia sugar and sterol etc., is realized high attached Value-added product.
(5) the citric acid fermented fermentation of starch saccharic can carry out online composition detection, to realize automation control and hair The accurate optimization of ferment process, final to realize that starch sugar mass flow adds, the realization of the continuous ferments technique such as mycelium pellet cutting duplication.
SEQUENCE LISTING of the invention is as follows:
The nucleotide sequence SEQ ID NO:1 of PC gene
The nucleotide sequence SEQ ID NO:2 of Pgla promoter
The nucleotide sequence SEQ ID NO:3 of trp terminator
The nucleotide sequence SEQ ID NO4 of PD gene
The nucleotide sequence SEQ ID NO:5 of Aox1 gene
The nucleotide sequence SEQ ID NO:6 of PgpdA promoter
The nucleotide sequence SEQ ID NO:7 of HYG gene
Detailed description of the invention
The influence of Fig. 1 difference Organic Nitrogen Sources on Citric Acid Fermentation；
Influence of Fig. 2 difference corn pulp content to citric acid fermentation；
Fig. 3 difference (NH4)2SO4Influence of the content to citric acid fermentation；
Influence of Fig. 4 difference inositol content to citric acid fermentation；
Influence of Fig. 5 different process to 8 hours lemon acid accumulations；A: option A three-stage ventilation quantity B: the big ventilation volume production of option b Love song line.
Specific embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this Under the premise of invention spirit and scope, to the various changes or change of material component and dosage progress in these embodiments It belongs to the scope of protection of the present invention.The raw materials used in the present invention and reagent are commercially available.
Embodiment 1
The construction method of the fermentation strain of high yield citric acid microorganism, the bacterial strain are by the pyruvate carboxylase PC base in aspergillus niger It is heavy using this on cause, pyruvic dehydrogenase PD gene and alternative oxidase Aox1 gene multi-copy integration to aspergillus niger genome Group Aspergillus niger strain fermentation production of citric acid；The expression of the pyruvate carboxylase PC gene by Pgla promoter regulation, third Ketoacid dehydrogenase PD gene and alternative oxidase Aox1 gene are by PgpdA promoter regulation.
Embodiment 2
Using Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480 bacterial strain prepares lemon The method of acid:
(1) fermentation strain used is Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480, fermentation medium group become (g/L): glucose 200g, corn pulp 60 g/L, (NH4)2SO4 9g/L, MgSO4· 7H2O 0.05g/L, K2HPO,4g/L, CuSO4 0.8 mg/L ZnSO40.02 g/L, CaCl20.1 g/ of 2.0 g/L, KCl L, FeSO40.34 mg/L pH 5.0~5.5；
(2) fermented and cultured control condition: by the inoculum concentration control of 10%(v/v), kind age control was at 27 hours, by cultured kind Son access fermentation medium, 30 DEG C of cultivation temperature, fermentation ventilatory capacity is 0.1m3/ m3.min, tank presses 0.07 MPa, mixing speed 80 revs/min, fermentation time 50 hours, filtrated air is passed through in fermentation process, fermentation results lemon acid yield is 220g/L, sugar Sour conversion ratio is 102%.
Embodiment 3
Using Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480 bacterial strain prepares lemon The method of acid:
(1) fermentation strain used is Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480, fermentation medium group become (g/L): glucose 260g, corn pulp 80 g/L, (NH4)2SO4 11 g/L, MgSO4·7H2O 0.15g/L, K2HPO,6g/L, CuSO4 0.8 mg/L ZnSO40.02 g/L, CaCl22.0 g/L, KCl 0.1 g/L, FeSO40.34 mg/L pH 5.0~5.5；
(2) fermented and cultured control condition: by the inoculum concentration control of 15%(v/v), kind age control was at 27 hours, by cultured kind Son access fermentation medium, 40 DEG C of cultivation temperature, fermentation ventilatory capacity is 0.15m3/ m3.min, tank presses 0.12 MPa, stirring speed 100 revs/min of degree fermentation time 60 hours, is passed through filtrated air in fermentation process, fermentation results lemon acid yield is 260g/ L, saccharic acid conversion ratio is 100%.
Embodiment 4
Using Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480 bacterial strain prepares lemon The method of acid:
(1) fermentation strain used is Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480, fermentation medium group become (g/L): corn or potato starch saccharified liquid are calculated with wherein 200g containing glucose, Corn pulp 60 g/L, (NH4)2SO4 9g/L, MgSO4·7H2O 0.05g/L, K2HPO,4g/L, CuSO4 0.8 mg/L ZnSO40.02 g/L, CaCl22.0 g/L, KCl 0.1 g/L, FeSO40.34 mg/L pH 5.0~5.5；
(2) fermented and cultured control condition: by the inoculum concentration control of 10%(v/v), kind age control was at 27 hours, by cultured kind Son access fermentation medium, 30 DEG C of cultivation temperature, fermentation ventilatory capacity is 0.1m3/ m3.min, tank presses 0.07 MPa, mixing speed 80 revs/min, fermentation time 50 hours, filtrated air is passed through in fermentation process, fermentation results lemon acid yield is 220g/L, sugar Sour conversion ratio is 102%.
Embodiment 5
Using Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480 bacterial strain prepares lemon The method of acid:
(1) fermentation strain used is Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480, fermentation medium group become (g/L): corn or potato starch saccharified liquid are calculated with wherein 260g containing glucose, Corn pulp 80 g/L, (NH4)2SO4 11 g/L, MgSO4·7H2O 0.15g/L, K2HPO,6g/L, CuSO4 0.8 mg/L ZnSO40.02 g/L, CaCl22.0 g/L, KCl 0.1 g/L, FeSO40.34 mg/L pH 5.0~5.5；
(2) fermented and cultured control condition: by the inoculum concentration control of 15%(v/v), kind age control was at 27 hours, by cultured kind Son access fermentation medium, 40 DEG C of cultivation temperature, fermentation ventilatory capacity is 0.15m3/ m3.min, tank presses 0.12 MPa, stirring speed 100 revs/min of degree fermentation time 60 hours, is passed through filtrated air in fermentation process, fermentation results lemon acid yield is 260g/ L, saccharic acid conversion ratio is 100%.
Embodiment 6
Using Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480 bacterial strain prepares lemon The method of acid:
(1) fermentation strain used is Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480, fermentation medium group become (g/L): sucrose or maltose 200g, corn pulp 60 g/L, (NH4)2SO4 9g/L, MgSO4·7H2O 0.05g/L, K2HPO,4g/L, CuSO4 0.8 mg/L ZnSO40.02 g/L, CaCl22.0 g/L, KCl 0.1 g/L, FeSO40.34 mg/L pH 5.0~5.5；
(2) fermented and cultured control condition: by the inoculum concentration control of 10%(v/v), kind age control was at 27 hours, by cultured kind Son access fermentation medium, 30 DEG C of cultivation temperature, fermentation ventilatory capacity is 0.1m3/ m3.min, tank presses 0.07 MPa, mixing speed 80 revs/min, fermentation time 50 hours, filtrated air is passed through in fermentation process, fermentation results lemon acid yield is 220g/L, sugar Sour conversion ratio is 102%.
Embodiment 7
Using Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480 bacterial strain prepares lemon The method of acid:
(1) fermentation strain used is Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480, fermentation medium group become (g/L): sucrose or maltose 260g, corn pulp 80 g/L, (NH4)2SO4 11 g/L, MgSO4·7H2O 0.15g/L, K2HPO,6g/L, CuSO4 0.8 mg/L ZnSO40.02 g/L, CaCl22.0 g/L, KCl 0.1 g/L, FeSO40.34 mg/L pH 5.0~5.5；
(2) fermented and cultured control condition: by the inoculum concentration control of 15%(v/v), kind age control was at 27 hours, by cultured kind Son access fermentation medium, 40 DEG C of cultivation temperature, fermentation ventilatory capacity is 0.15m3/ m3.min, tank presses 0.12 MPa, stirring speed 100 revs/min of degree fermentation time 60 hours, is passed through filtrated air in fermentation process, fermentation results lemon acid yield is 260g/ L, saccharic acid conversion ratio is 100%.
Sequence table
<110>University Of Science and Technology Of Tianjin
The method that<120>one plant heights produce the microbial strains and its fermentation starch saccharic production citric acid of citric acid
<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 (4)

1. a kind of fermentation strain of high yield citric acid microorganism, Aspergillus niger strain of classifyingAspergillus niger 101- HAC11, deposit number CGMCC No.12480.
2. the construction method of the fermentation strain of high yield citric acid microorganism described in claim 1, it is characterised in that the bacterial strain be by Pyruvate carboxylase PC gene, pyruvic dehydrogenase PD gene and alternative oxidase Aox1 gene integration in aspergillus niger are to black song On mould genome, citric acid is produced using the recombinant aspergillus niger strain fermentation；The expression of the pyruvate carboxylase PC gene by The regulation of Pgla promoter, pyruvic dehydrogenase PD gene and alternative oxidase Aox1 gene are by PgpdA promoter regulation.
3. a kind of using Aspergillus niger strain described in claim 1Aspergillus niger101-HAC11 deposit number CGMCC The method that No.12480 bacterial strain prepares citric acid:
(1) fermentation strain used is Aspergillus niger strainAspergillus niger101-HAC11 deposit number CGMCC No.12480, fermentation medium group become (g/L): starch 200 ~ 260g of saccharine material, corn pulp 60 ~ 80 g/L, (NH4)2SO4 9~11 g/L, MgSO4·7H2O 0.05 ~ 0.15g/L, K2HPO,4 ~ 6g/L, CuSO4 0.8 mg/L ZnSO4 0.02 g/ L, CaCl22.0 g/L, KCl 0.1 g/L, FeSO40.34 mg/L pH 5.0~5.5；
(2) fermented and cultured control condition: inoculum concentration controls 10% ~ 15%(v/v), kind age control, will be cultured at 24 ~ 27 hours Seed accesses fermentation medium, and 30 ~ 40 DEG C of cultivation temperature, fermentation ventilatory capacity is 0.1 ~ 0.15m3/ m3.min, tank pressure 0.07 ~ 0.12 MPa, fermentation time 50 ~ 60 hours, is passed through filtrated air by 80 ~ 100 revs/min of mixing speed in fermentation process, fermentation As a result lemon acid yield is 220 ~ 260g/L, and saccharic acid conversion ratio is 100% ~ 102%.
4. preparation method as claimed in claim 3, wherein starch saccharine material includes: starch, wherein having cornstarch, potato What starch, tapioca filtered after liquefying, being saccharified contains Saccharoid solution, and saccharic includes: glucose, sucrose, fructose, malt Sugar, achrodextrin.
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