CN1886516A - Method to produce succinic acid from raw hydrolysates - Google Patents
Method to produce succinic acid from raw hydrolysates Download PDFInfo
- Publication number
- CN1886516A CN1886516A CNA028301420A CN02830142A CN1886516A CN 1886516 A CN1886516 A CN 1886516A CN A028301420 A CNA028301420 A CN A028301420A CN 02830142 A CN02830142 A CN 02830142A CN 1886516 A CN1886516 A CN 1886516A
- Authority
- CN
- China
- Prior art keywords
- hydrolysate
- succsinic acid
- mutant strain
- described method
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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- 239000001384 succinic acid Substances 0.000 title abstract description 18
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- 239000000413 hydrolysate Substances 0.000 claims abstract description 27
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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Abstract
A method for producing succinic acid from industrial-grade hydrolysates is provided, comprising supplying an organism that contains mutations for the genes ptsG, pflB, and ldhA, allowing said organism to accumulate biomass, and allowing said organism to metabolize the hydrolysate. Also provided is a bacteria mutant characterized in that it produces succinic acid from substrate contained in industrial-grade hydro-lysate in a ratio of between 0.6:1 and 1.3:1 succinic acid to substrate.
Description
Contract of the present invention source
According to USDOE with represent contract number W-31-109-ENG-38 between the Chicago University of Argonne National Laboratory, United States Government enjoys rights and interests to the present invention.
Background of invention
1. invention field
The present invention relates to a kind of fermentation process of producing succsinic acid, more specifically, the present invention relates to a kind ofly produce that to utilize multiple sugar to produce with succsinic acid be the method for the bacterial isolates of main tunning.
2. background of invention
Carboxylic acid is hopeful the potential precursor as many chemical.For example, succsinic acid can be used as plastics precursor as 1,4-butyleneglycol (BDO), the raw material of tetrahydrofuran (THF) and γ-Ding Suan acetone.Developed by succsinic acid deutero-product innovation, wherein foremost is that succsinic acid is connected the polyester made from BDO.Usually, the ester of succsinic acid is potential to become new, " green " solvent, and it can replace more deleterious solvent.On the whole, succsinic acid can be used as the precursor of annual millions of pounds chemical, and the market total value surpasses 1,000,000,000.As succsinic acid, other 4-carbon dicarboxylic acid also have potentiality as raw material as oxysuccinic acid and fumaric acid.
Producing these carboxylic acids by reproducible raw material (passing through fermentation process in this situation) is to substitute a kind of approach that is obtained the more energy intensive methods of these carboxylic acids by non-renewable source.Succinate is the intermediate product of pressing aerobe fermentation by the propionate production bacterium, but those methods cause low-yield and concentration.
Anaerobic rumen bacteria, bacteroides ruminicola (Bateroides ruminicola) for example, bacteroides amylophilus (Bacteroides amylophilus) also produces succinate.Yet, but the feature of cud biology is unstable during the fermentation.
Can produce mixing acid with regard to known Escherichia coli fermentation for a long time, as at Stokes, J.Bacteriol.57:147-158 described in detail J.L.1949 " intestinal bacteria suspension is to the fermentation (Fermentation of glucose by suspensionsof Escherichia coli) of glucose ".But every fermentation 1mol glucose only produces 1.2mol formic acid, the succsinic acid of 0.1-0.2mol lactic acid and 0.3-0.4mol.Similarly, the effort through fermentation generation carboxylic acid causes a large amount of relatively growth substrates such as glucose can not change into the purpose product.
Some bacteriums as A.succiniciproducens, as in U.S. Patent No. 5,143,834 general introductions of Glassner etc., utilize during the fermentation, only reach every liter of about 35-40 at the medium succsinic acid that rises natural generation in the number and restrain (g/L).Host strain A.succiniciproducens is impatient at high salt concentration and is further suppressed by the intermediate concentration product, shows that it is not highly anti-infiltration.At last, there is operational issue in A.succiniciproducens, because as obligatory anaerobic bacteria, uses this biological method to carry out under anoxic.In addition, the medium preparation of inoculum requires to add tryptophane.
The bacterium that before making great efforts cause separate and utilize sudden change of the inventor on the production succsinic acid.Can be used as the mutant strain that ATCC registration number 202021 obtains, is that United States Patent (USP) is issued application No.09/429,693 theme again.Again the application No.09/429 that issues, 693, it has instructed a strain to produce the bacterial isolates (AFP111) of succsinic acid by with reference to being incorporated into this, and it can be from its precursor spontaneous mutation.Mutant strain can be in glucose top fermentation growth, and high productivity is produced succsinic acid, and its precursor can not.But, utilize this method of producing succsinic acid, its open defect is to be limited to single mutant strain.
Other effort of the inventor (U.S. Patent No. 6,159,738) cause making up the method for the bacterial isolates of the succinic acid production with increase.This method has been instructed the change of intestinal bacteria phosphoric acid transferase genes, causes that thalline produces more succsinic acids.This method defective is to be limited to single change.
Have the demand to the method for fermentative production succsinic acid in this area, this method does not belong to single mutant strain or gene.This method should be able to be used for any genotypic biology specific and that determine easily that has.This method should be able to use strong biology (promptly have high feedback inhibition threshold value those) to implement under the condition of relative inertness, and avoids needs to the complex environment control survey with this.This method should be able to utilize lignocellulosic materials hydrolytic action deutero-sugar mixture to produce superior result, because these substrates provide cheap sugar source, so their use can reduce the production cost of succsinic acid.
Summary of the invention
An object of the present invention is to provide a kind of method of producing succsinic acid, overcome many shortcomings of prior art with this.
Another object of the present invention provides the zymotechnique of production high yields of succinic acid.Feature of the present invention is to utilize to contain the bacterial genomes of a plurality of mutator genes to implement this method.An advantage of the present invention is that bacterium easily is operated to produce a plurality of mutant strains.Alternatively, can utilize the bacterium that has contained a plurality of sudden changes and needn't further operate.
The present invention also has another purpose to provide the operation bacterium to produce the method for a large amount of succsinic acids.A feature of the present invention is to destroy glycometabolic normal regulating in the bacterium.An advantage of the present invention is to operate various bacteriums, realizes that in producing the succsinic acid zymotechnique high relatively product is to the ratio of growth substrate (for example 1: 1 or be higher than this).Another advantage of the present invention ground is to utilize the bacterium that becomes glucose metabolism person and non-glucose metabolism person.
It is the fermentative production succsinic acid that the present invention also has another purpose.A feature of the present invention is to utilize to contain phosphotransferase (pts) system that changes, the bacterium of pyruvate formate-lyase (pfl) system and serum lactic dehydrogenase (ldh) system.An advantage of the present invention is that bacterium can be derived from many kinds of using these enzyme system sugar fermentation.
Concisely, provide the method for producing succsinic acid from industrial hydrolysate, this method comprises: provide and contain gene ptsG, the biology of pflB and ldhA sudden change; Allow described biological accumulation biomass; And allow described biological metabolism hydrolysate.
The mutant bacteria strain also is provided, it is characterized in that its substrate production succsinic acid from be contained in industrial hydrolysate, the ratio of succsinic acid and substrate is (for example, every gram consumes the succsinic acid of total reducing sugar generation between 0.6 gram restrains to 1.3) between 0.6: 1 to 1.3: 1.
The accompanying drawing summary
By the detailed description of following diagram embodiment of the present invention, can understand the present invention best together with above-mentioned and other purpose and advantage.Wherein:
Fig. 1 is the chart of representing with enhanced production of succinic acid behind the mutator gene transform bacteria, and it is consistent with feature of the present invention;
Fig. 2 is the chart of expression by the fermentation of the service water hydrolysis products of triple mutant organisms, and it is consistent with feature of the present invention; With
Fig. 3 is the chart of expression by the synthetic sugar-fermenting of triple mutant organisms, and it is consistent with feature of the present invention.
Detailed Description Of The Invention
The inventor has developed the method for fermenting and producing high yields of succinic acid. The method utilizes the selected biological catabolite product that changes to check mechanism, produces butanedioic acid with the mixture that allows this biological utilisation glucose and non-glucose feed.
The mutant strain that obtains and scheme make the ratio of butanedioic acid and raw material up to 1.3: 1, typically 0.9: 1. The accumulation of succinate reaches 60g/L between the 75g/L. The duration of typical scenario surpasses 70 hours, usually between 120 to 170 hours. For example obtaining productive rate after 160 hours is 70g/L. This technique is feasible between from about 25 ℃ to 45 ℃, and preferable range is between about 30 ℃ to 39 ℃. PH is to be suitable between 5 to 9, and more preferably scope is 6.1 to 7.2 approximately.
Mutant strain of the present invention is great-hearted especially component in the fermentation scheme, because they have the tolerance to the enhancing of tunning. For example, can realize that concentration is the butanedioic acid of 72g/L, the acetate of 22g/L, the ethanol of 14g/L, the lactate of 8g/L and do not induce feedback inhibition.
Feedstock detail
A notable feature of method of the present invention and mutant strain is directly to utilize the raw material of industry. Can utilize plurality of raw materials, include but not limited to light steep water, with the lignin fibre cellulose hydrolysate using that various method for hydrolysis are produced, derive from (corn-derived) sugar juice (for example corn steep liquor) of corn, be derived from the lactose of whey and the sugar of other technical grades. For example, the lignin fibre cellulose hydrolysate using of producing through Concentrated acid hydrolysis effect or dilute acid hydrolysis, enzyme hydrolysis, or the hydrolysate of the combinations produce by these methods all is fit to. The sugar juice that derives from corn is also applicable.
Usually, the raw material of industry is the mixture of glucose and other sugar, and the sugar of the most conventional non-glucose is wood sugar. Fig. 2 has described one of mutant strain of the present invention and has utilized glucose and xylose.
In sum, any glycogen material that contains glucose and/or non-glucose all is suitable for.Equally, comprise glucose, Sorbitol Powder, wood sugar, pectinose, seminose, lactose, glucuronic acid, semi-lactosi, fructose and combination thereof are suitable.
Organism detail
Method utilization of the present invention contains the biology that biological decomposition meta-bolites repressible system changes.Particularly, the contriver finds phosphotransferase (pts) system when bacterium, and pyruvate formate-lyase (pfl) system and serum lactic dehydrogenase (ldh) system exist when changing, and these bacteriums are suitable for production of succinic acid technology of the present invention.PflAB and ldhA are respectively the coding pyruvic acid: the gene of the serum lactic dehydrogenase of formic acid lyase and fermentation.
Therefore, the unique restriction for the biotype that is used for zymotechnique of the present invention is that this biology must have these systems at first.Naturally contain biology (for example spontaneous mutation strain) that these systems change or the biology of specific transformation all can use.
In the situation that bacterium is transformed, not or have the bacterium of very low production of succinic acid productive rate (for example, supply with every 1mol growth substrate productive rate and be lower than 0.5mol) fermentation to be converted to bacterium (promptly supply with every 1mol growth substrate productive rate and be equal to or higher than the 1mol succsinic acid) with high production of succinic acid productive rate.
Any bacterium that can carry out any succsinic acid fermentation particularly is suitable as the transduction candidates bacterium, includes but not limited to fermentable Gram-negative and gram positive bacterium.Preferably, the bacterial strain that is fit to includes but not limited to Colibacter (Escherichia coli), klebsiella (Klebsiella), and erwinia belongs to (Erwinia) and lactobacillus (Lactobacillus).
To be to change through transforming to comprise the biology of three rejectings (knockout) by a series of transductions of using phage P1.Can utilize the P1 transduction scheme of standard, a kind of typical scheme is in molecular genetics experiment (Experiments in Molecular Genetics) (cold spring harbor laboratory that J.H.Miller in 1972 edits, the cold spring port, open in N.Y.), it is by with reference to being incorporated into this.Utilize this method, wild-type or nearly wild-type bacterium bacterial strain (intestinal bacteria C600 bacterial strain for example; ATCC registration number 23724) can be used for producing lacking and be selected from pfl, ldh, among the ptsG one, the sudden change substrain of two or three functional genes.
Can be used for one of coli strain example that contains three sudden changes of the present invention and be named as AFP184 (feedstock program that AFP=is alternative, Alternative Feedstock Program).AFP 184 has pfl disappearance, and ldh rejects, and has a mind to insert the different mutant forms of the ptsG of nearly wild-type e. coli bacterial strain.Another bacterial strain that is called as AFP 415 also can use.The unique difference of AFP415 and AFP184 is to have ptsG and rejects.Its performance is similar to AFP184.
The contriver finds the metabolic rate of AFP184 and AFP415 unexpectedly and tires and be better than United States Patent(USP) Nos. 5,770 amazedly, 435 (issuing application number 09/429,693 now again) and 6,159, the 738 disclosed W1485 strain of deriving.
The comparison that table 1 provides AFP184 and W1485 to derive strain (AFP111) succinic acid production.Utilize very purified raw material though it should be noted that the W1485 strain of deriving, AFP184 still can provide higher value with industrial hydrolysate.
Use the bacterium that has contained one or two genetic abnormality, induce remaining rejecting then, also can produce the mutant strain that comprises all three rejectings.In this case, a feasible eozoan is W1485, ATCC registration number 12435.AFP400 is the triple knockout of having a mind to make.It comprises the pfl disappearance by AugustBock, by the David Clark insertion W1485 generation FMJ123 of illinois university.According to seeing (1997) Microbiology 143 such as P.K.Bunch, the scheme of 187-195 produces FMJ123, and it is by with reference to being incorporated into this.AFP400 comprises that also ldhA rejects, and is also finished and is inserted FMJ123 by Clark and produce DC1327.The generation of DC1327 is according to seeing Chatterjee etc., Appl.Environ.Microbiol.67, and the scheme of pp148-154, it is by with reference to being incorporated into this.As described in the Chatterjee reference, AFP400 contains ptsG and rejects.
Table 1: the comparison of the colibacillary production of succinic acid by different pedigrees
| Bacterial strain | Peak concentration | Peak performance | Productive rate (g/g glucose) |
| AFP111 AFP184 | 51g/L 72g/L | 0.87g/Lh 1.00g/Lh | 0.70 1.00 |
Also can make up triple knockout AFP404 by rejecting introducing bacterial strain C600 with three.AFP404 and AFP184 are similar, have just rejected ptsG rather than this gene mutations.It also can about 1mol/mol glucose productive rate produce succsinic acid.
Also see R.Chatterjee etc. from the scheme of wild type strain development triple mutant.Typical each antibiotic marker of rejecting existence that shows includes but not limited to paraxin, tetracycline, and kantlex.New the coli strain AFP400 and the AFP404 that contain rejecting and antibiotic marker have so been produced.This scheme is as follows:
Make up and import the ptsG gene that inserts inactivation
Use the N-of targeting proteins matter and the primer of C-end, by cloning colibacillary natural ptsG gene from the genomic dna of W1485 preparation by PCR, other genome sequence does not increase.This gene clone is obtained pJFptsG to carrier pFJ118EH.By obtaining plasmid pTSGK with the MfeI site that the kanamycin resistance cassette of the pUC-4K (Pharmacia) of EcoRI excision is inserted the ptsG gene in the pJFptsG.Because NZN111 comprised the kalamycin resistance mark, make up equivalent strains by importing FMJ123 from the ldhA gene of the Tn10-inactivation of bacterial strain SE1752.The bacterial strain that obtains, DC1327 can not distinguish on physiology with NZN111.By using the pTSGK transformant, change destructive ptsG gene over to DC1327, kantlex is being arranged, in culturing cell about 30 generations under the no penbritin, then coating culture and anaerobism incubation on the LB flat board of glucose are being arranged.The bacterium colony that purifying can ferment and grow screens them to two kinds of antibiotic susceptibility.
As stable anti-kantlex, penbritin responsive type bacterial strain, isolated strains AFP400, its glucose fermentation generates succinate, acetate and ethanol.Verify the suitable integration of the ptsG gene of destroyed by PCR.With the primer that is complementary with the overseas about 110 base pair flanking sequences of gene coding region, from the DNA cloning destructive gene of AFP400.In integrative vector, there are not these sequences.The product size that obtains is 3.0kb, predicts as inserting segment from known ptsG sequence, its side areas and kantlex.With ClaI (kantlex box in site) and AgeI (site in the ptsG) digestion product, produced the MfeI site of box being inserted ptsG of expecting segment (for ClaI is 1.95 and 1.05kb, for Agel be 2.3 and 0.7kb).
With above-mentioned same scheme, another strains A FP404 from the nearly wild type strain C600 of e. coli k12 derives also comprises three rejectings.
From the inventor's discussed above early-stage Study (U.S. Patent No. 6,159,738 and Chatteriee etc.) can known rejecting the position.By copy of rejecting gene of putting into antibiotic marker, will reject transfered cell.Allow it that homologous recombination takes place, as promoting by host enzyme.Select to comprise the karyomit(e) of mark then.PtsG rejects promptly and imports with this approach.Verify that by PCR it inserts, before seeing for details by with reference to the Chatterjee that is incorporated into this etc.
Growth detail
The triple mutant organisms of inventor's preparation is not an obligate anaerobic.Therefore, early stage biomass accumulation can take place at aerobic condition, sets up fermentation condition afterwards.The advantage of two-stage process (being aerobic anaerobic then) is compared with the growth curve of Fig. 1 one stage anaerobism scheme as shown in Figure 2, and the productivity of succsinic acid wherein is much bigger.
Usually, reach every milliliter about 10 of trim point when biomass
8To 10
11During individual cell (or every liter of about 2-5 gram dry cell weight), make fermentor tank become anaerobic.Approximately be to reach this concentration point after six hours in the laboratory.
In industrial protocols, light steep water is added the lignocellulose hydrolyzate fermentor tank of packing into.Comprise essential microbiotic, its concentration is as follows: 100 μ g/ml Pyocianils, 30 μ g/ml kantlex, 10 μ g/ml tetracyclines and 30 μ g/ml paraxin.Add rich meat soup and contain (every liter) 10 gram Tryptoness, 5 gram sodium-chlor and 1 gram yeast powder.The flat-plate solid substratum comprises 1.5% (wt/vol) Difco Bacto-agar.According to Vogel, H.J.1956 Acetylornithinase in E.coli, the description of Biol.Chem.218:97-103 prepares minimum medium E, and it is incorporated into this by reference.
The laboratory condition of fermentation is as follows:
In the airtight serum tube that contains 10ml LB substratum, replenish 0.5g MgCO
3(adding to keep the pH of substratum between yeast phase), microbiotic and about 10g/L glucose are implemented the fermentation growth.Can use various growth substrates, include but not limited to sugar, sugar alcohol, saccharic acid and combination thereof.In anaerobic growth, to test with following sugared place of glucose, its concentration is 5g/L: trehalose, seminose, fructose, Sorbitol Powder and glucuronic acid.
The inoculum for preparing the anaerobism liquid culture by the aerobic cultivation bacterial strain that in being supplemented with antibiotic LB substratum, spends the night.The sample of overnight culture is diluted 100 times in fresh culture, and make aerobic growth to A600 be about 1; With the 1ml inoculum anaerobic culture medium is inoculated.
In due course anaerobic ground from sealed tube sampling with the level of the tunning of analyzing remaining glucose (or alternative sugared substrate) and formation.For anaerobic growth on solid medium, with agar plate under 37 ℃, by using incubation in the anaerobism bottle under the H2-CO2 atmosphere that Gas-Pak produces.
Measure the existence of checking normal catabolite repression in the bacterial strain with the flat board of betagalactosidase activity.LB or E-nutrient agar are two kinds in utilizable several substratum.The E-nutrient agar is the basic nutritional medium of using always, at Vogel, and H.J., 1956 Acetylornithase in E.coli come into question among the J.Bio/Chem 218:97-103, list reference at this.In typical scheme, replenish 4g/L glucose, 4g/L lactose, 5-bromo-4-chloro-3-indoles-β-D-galactoside (X-gal) and microbiotic of 3mg/L in LB or the E-nutrient agar.Below these substratum can be called the X-Gal/ agar glucose.Forming blue colonies represents to express beta-galactosidase enzymes owing to there is not normal catabolite repression when glucose exists.On the contrary, form white colony and represent to exist normal catabolite repression, therefore do not have enzyme to exist with cracking disaccharides lactose.
The inventor has also designed the method for utilizing mutant strain in continuous processing.Carry out the multiple experiment, wherein after culture has been produced about 50g/L succsinic acid, the adding of 1 ml mixture is contained in the fresh inclusion (enclosure) of LB substratum, glucose and MgCO3.This new inoculum production succsinic acid that remains valid.This process repeats 3-4 time, all causes effective production of succsinic acid in each situation.
Embodiment 1 utilizes industrial hydrolysate to produce succsinic acid
AFP184 is put into fermentor tank from the true hydrolysate of rice straw.Typical hydrolysate is a commercial production, and can be available from Arkenol Inc., of Mission Viejo, and CA., it obtains by the concentrated acid method for hydrolysis.Rice straw medium comprises about 600g/L glucose and 169g/L wood sugar as two kinds of main sugar components, adds a small amount of other sugar.Experimental data sees Table 2 and Fig. 2.
Be based on the zymotechnique scheme of AFP184 below: fermention medium comprises following component: Difco yeast extract 5g/L, Tryptones 10g/L, (NH
4)
2SO
42g/L, MgSO
47H
2O0.2g/L, NaCl 10g/L, K
2HPO
47g/L, KH
2PO
43g/L, Arkenol ' s hydrolysate 16.5mg/L and kantlex 30mg/L.Industrial hydrolysate comprises 607g/L glucose and 169g/L wood sugar as two kinds of main sugar components, adds a small amount of other sugar.Other all components of substratum were 121 ℃ of autoclavings 20 minutes except that microbiotic.After cooling off, add kantlex.Inoculation is shaken in bottle and 1 liter of fermentor tank and is all used this fermention medium.During inoculation, the 50mg substratum is inserted 250mg shake in the bottle, inoculation 0.2mg is-70 ℃ of AFP 184 stock cultures of preserving down in 30% glycerine.Shake bottle on shaking table 37 ℃, overnight incubation under the 250rpm (about 16 hours).All shake bottle content and be used for the inoculation fermentation jar then, its temperature maintenance is at 37 ℃.Ventilation is gone into the interior substratum of fermentor tank to allow rapid microbial growth.When reaching required cell concentration, adopt following measures: 1. close ventilation to reach anaerobic condition, it will cause the generation of succsinic acid after six hours; 2. inject CO with the speed of per minute 0.03mg
2Gas; With 3. will contain and be diluted to total glucose with deionized water to add xylose concentration be that the material solution of Arkenol ' the s hydrolysate of 500g/L adds fermentor tank, make that total sugar concentration reaches 50g/L in the fermention medium.In experimentation, when sugared concentration in the fermentor tank was low, adding more raw materials provided enough substrates for production of succinic acid.Because producing succsinic acid pH, cell can descend.By the effect of automatic pH controller, add 1.5M Na
2CO
3Solution is kept pH 6.5.Interval sampling analysis optical density(OD), glucose, wood sugar, succsinic acid, acetate, lactic acid and ethanol.
Table 2: with containing ptsG, the mutant strain that ldh and pfl are unusual is produced succsinic acid by Arkenol, acetate and ethanol
| Time | Glucose | Wood sugar | Succsinic | Acetate | Ethanol | |
| 0 2 4.2 6 6.05 24 24.05 28.1 29.5 | 7.04 6.85 4.41 0 29.27 9.56 27.25 23.7 22.8 | 1.94 1.53 0 0 7.1 7 1.69 5.60 14.69 14.17 | 0 0 0 0 0 26.12 26.39 28.42 27.20 | 0 0.41 0.85 0.55 0 2.24 2.82 2.98 3.04 | 1.60 1.35 1.13 1.04 0.68 0.49 0.72 0.71 0.67 |
| 29.55 48 54 54.05 72 80 104.25 120 192 | 34.77 20.98 19.13 46.73 35.14 33.60 23.02 19.73 13.04 | 7.95 4.72 4.30 10.85 8.85 8.45 7.20 6.77 5.87 | 26.41 37.98 43.82 43.51 48.52 51.44 50.99 54.12 63.21 | 2.63 3.71 4.10 3.69 4.01 4.10 4.64 4.83 4.88 | 0.52 0.62 0.71 0.59 0.63 0.50 0 0 0 |
Embodiment 2 produces succsinic acid from synthetic sugar mixture
Utilize AFP 184 to develop the fermentation scheme in conjunction with the glycogen biosynthesis material.As shown in Figure 3, up to 80 hours, succinate was produced fast, and kept stable until finally reached 60g/L after about 140 hours a little.
Fermention medium comprises following component: Difco yeast extract 5g/L, Tryptones 10g/L, (NH
4)
2SO
42g/L, MgSO
47H
2O 0.2g/L, NaCl 10g/L, K
2HPO
47g/L, KH
2PO
43g/L, glucose 7.6g/L, wood sugar 1.85g/L and kantlex 30mg/L.Other all components of substratum were 121 ℃ of autoclavings 20 minutes except that microbiotic.After cooling, add kantlex again.Bottle is shaken in inoculation and 1 liter of fermentor tank all uses this fermention medium.During inoculation, the 50mg substratum is inserted 250mg shake in the bottle, be kept at-70 ℃ of following AFP184 stock culture inoculations in 30% glycerine with 0.2mg.Shake bottle and cultivating on the shaking table 37 ℃, overnight incubation under the 250rpm (about 16 hours).To all shake bottle content then and be used for the fermentor tank that inoculation temp maintains 37 ℃.
Ventilation is gone into the interior substratum of fermentor tank to allow biological quick growth.When reaching required cell concentration, adopt following measures after six hours:
1. close ventilation to apply anaerobic condition, it can cause the production of succsinic acid;
With the speed of per minute 0.03mg with CO
2Gas feeds substratum;
3. will contain the glucose of 400g/L and the material solution of 84g/L wood sugar and add fermentor tank, total sugar concentration reaches 50g/L in the fermention medium.
In experimentation, when sugared concentration in the fermentor tank was low, adding more raw materials provided enough substrates for production of succinic acid.Descend because cell produces succsinic acid pH,, add 1.5M Na by the effect of automatic pH controller
2CO
3Solution is kept pH6.5.Interval sampling analysis optical density(OD), glucose, wood sugar, succsinic acid, acetate, lactic acid and ethanol.
Table 3 is hereinafter for example understood the succinic acid production that utilization obtains when synthesizing sugar mixture with Fig. 3.
As embodiment 1 and embodiment 2 as seen, when with industrial hydrolysate when comparing with the synthetic raw material, the production of succinic acid of mutant strain is the suitable moment 120 and 122 of table 2 and 3 (respectively referring to).Any poisonous raw material of inherent can not reduce productive rate in this presentation of results the present invention program's the firm feature, industrial hydrolysate.
Table 3 utilization is synthesized the production of succinic acid in the sugared fermentation scheme
| Time | Glucose | Wood | Succinate | Acetate | |
| 0 2 4.2 4.45 6 6.25 24 30 30.25 48 54 54.25 72 78 78.25 96.5 122 144 | 7.65 7.19 3.15 6.03 1.04 40.2 7.76 9.18 39.3 18.6 14.8 27.4 19.7 17.6 35.5 30.2 24.1 22.8 | 1.85 1.03 0 0.84 0 7.57 3.92 2.63 8.2 5.5 4.95 8.1 6.04 5.42 9.49 8.25 6.48 5.67 | 0 0 0 0 0 0 24.55 29.34 29.34 39.8 42.33 40.77 48.33 50.27 48.87 53.62 55.1 59.35 | 0 0.32 1.1 1.1 2.02 2.02 3.43 4.11 4.11 4.6 5.26 4.9 5.76 6 5.75 5.87 5.87 5.43 |
Although described the present invention with reference to the details of illustrational embodiment, these details are not to be intended to restriction as the defined scope of the present invention of appended claim.
The PCT/RO/134 table
The Chinese translation of PCT/RO/134 table
PCT/US2002/035761
| Applicant or the file references 6321-240WO of agency | International application no PCT/US02/35761 |
Relate to the microorganism of preservation or the indication of other biomaterial
(PCT detailed rules and regulations 13bis)
The Chinese translation of PCT/RO/134 table
PCT/US2002/035761
| Applicant or the file references 6321-240WO of agency | International application no PCT/US02/35761 |
Relate to the microorganism of preservation or the indication of other biomaterial
(PCT detailed rules and regulations 13bis)
The Chinese translation of PCT/RO/134 table
PCT/US2002/035761
| Applicant or the file references 6321-240WO of agency | International application no PCT/US02/35761 |
Relate to the microorganism of preservation or the indication of other biomaterial
(PCT detailed rules and regulations 13bis)
Claims
(according to the modification of the 19th of treaty)
1. method of producing succsinic acid by industrial hydrolysate comprises:
A) provide and contain gene ptsG, the biology of pflB and ldhA sudden change;
B) allow described biological accumulation biomass; With
C) allow described biological metabolism hydrolysate.
2. the described method of claim 1, wherein said biogenetic derivation is in being selected from by Colibacter (Escherichia coli), klebsiella (Klebsiella), erwinia belong to the genus of the group of (Erwinia) and lactobacillus (Lactobacillus) composition.
3. the described method of claim 1, wherein biomass accumulation is to every milliliter about 10
8To 10
11Individual cell.
4. the described method of claim 1, wherein industrial hydrolysate is lignin fibre cellulose hydrolysate using or the sugar soln that derives from corn.
5. the described method of claim 1, wherein temperature is selected between about 25 ℃ to 45 ℃.
6. the described method of claim 1, wherein biomass accumulates under aerobic environment.
7. the described method of claim 1, wherein pH is selected between about 5 to 9.
8. the described method of claim 1, wherein hydrolysate is included in first in the material quantity, and wherein makes that by adding the secondary raw material amount this method is continuous.
9. the described method of claim 9, wherein the secondary raw material amount is to add during greatly about 50g/L when succsinic acid concentration.
10. mutant bacteria strain is characterized in that the substrate from be included in industrial hydrolysate is produced succsinic acid, and succsinic acid and substrate ratio are between 0.6: 1 and 1.3: 1.
11. the described mutant strain of claim 10, wherein substrate is to be selected from by glucose, lactose, Sorbitol Powder, wood sugar, pectinose, seminose, glucuronic acid, semi-lactosi, the sugar of the group of fructose or combinations thereof.
12. the described mutant strain of claim 10, wherein mutant strain comprises inoperative phosphotransferase system, inoperative pyruvate formate-lyase system and inoperative lactic dehydrogenase enzyme system.
13. the described mutant strain of claim 12, wherein inoperative phosphotransferase system are the results of point mutation.
14. the described mutant strain of claim 10, wherein mutant strain can utilize more than one substrates to produce succsinic acid simultaneously simultaneously.
15. the described mutant strain of claim 10, wherein said mutant strain comprises gene ptsG, the sudden change of pflB and ldhA.
16. the described mutant strain of claim 10, wherein said mutant strain derive from and are selected from by Colibacter, klebsiella, the genus of the group that erwinia genus and lactobacillus are formed.
17. the described mutant strain of claim 15, the ptsG gene of wherein said sudden change can not return back to its standard state.
Claims (14)
1. method of producing succsinic acid by industrial hydrolysate comprises:
A) provide and contain gene ptsG, the biology of pflB and ldhA sudden change;
B) allow described biological accumulation biomass; With
C) allow described biological metabolism hydrolysate.
2. the described method of claim 1, wherein said biogenetic derivation is in being selected from by Colibacter (Escherichia coli), klebsiella (Klebsiella), erwinia belong to the genus of the group of (Erwinia) and lactobacillus (Lactobacillus) composition.
3. the described method of claim 1, wherein biomass accumulation is to every milliliter about 10
8To 10
11Individual cell.
4. the described method of claim 1, wherein industrial hydrolysate is lignin fibre cellulose hydrolysate using or the sugar soln that derives from corn.
5. the described method of claim 1, wherein temperature is selected between about 25 ℃ to 45 ℃.
6. the described method of claim 1, wherein biomass accumulates under aerobic environment.
7. the described method of claim 1, wherein pH is selected between about 5 to 9.
8. the described method of claim 1, wherein hydrolysate is included in first in the material quantity, and wherein makes that by adding the secondary raw material amount this method is continuous.
9. the described method of claim 9, wherein the secondary raw material amount is to add during greatly about 50g/L when succsinic acid concentration.
10. mutant bacteria strain is characterized in that its substrate production succsinic acid from be included in industrial hydrolysate, and succsinic acid and substrate ratio are between 0.6: 1 and 1.3: 1.
11. the described mutant strain of claim 10, wherein substrate is to be selected from by glucose, lactose, Sorbitol Powder, wood sugar, pectinose, seminose, glucuronic acid, semi-lactosi, the sugar of the group of fructose or combinations thereof.
12. the described mutant strain of claim 10, wherein mutant strain comprises inoperative phosphotransferase system, inoperative pyruvate formate-lyase system and inoperative lactic dehydrogenase enzyme system.
13. the described mutant strain of claim 12, wherein inoperative phosphotransferase system are the results of point mutation.
14. the described mutant strain of claim 8, wherein mutant strain can utilize more than one substrates to produce succsinic acid simultaneously simultaneously.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/US2002/035761 WO2004043881A2 (en) | 2001-03-30 | 2002-11-07 | A method to produce succinic acid from raw hydrolysates |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1886516A true CN1886516A (en) | 2006-12-27 |
Family
ID=34311708
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA028301420A Pending CN1886516A (en) | 2002-11-07 | 2002-11-07 | Method to produce succinic acid from raw hydrolysates |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1558743A4 (en) |
| JP (1) | JP2006505276A (en) |
| CN (1) | CN1886516A (en) |
| AU (1) | AU2002340410A1 (en) |
| BR (1) | BR0215933A (en) |
| CA (1) | CA2505423A1 (en) |
| HU (1) | HUP0500960A2 (en) |
| MX (1) | MXPA05004865A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105051181A (en) * | 2013-03-18 | 2015-11-11 | Gs加德士 | Recombinant microorganism with increased productivity of 2,3-butanediol, and method for producing 2,3-butanediol using same |
-
2002
- 2002-11-07 BR BR0215933-3A patent/BR0215933A/en not_active Application Discontinuation
- 2002-11-07 CN CNA028301420A patent/CN1886516A/en active Pending
- 2002-11-07 JP JP2004551362A patent/JP2006505276A/en active Pending
- 2002-11-07 EP EP02778771A patent/EP1558743A4/en not_active Withdrawn
- 2002-11-07 CA CA002505423A patent/CA2505423A1/en not_active Abandoned
- 2002-11-07 MX MXPA05004865A patent/MXPA05004865A/en unknown
- 2002-11-07 AU AU2002340410A patent/AU2002340410A1/en not_active Abandoned
- 2002-11-07 HU HU0500960A patent/HUP0500960A2/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105051181A (en) * | 2013-03-18 | 2015-11-11 | Gs加德士 | Recombinant microorganism with increased productivity of 2,3-butanediol, and method for producing 2,3-butanediol using same |
Also Published As
| Publication number | Publication date |
|---|---|
| BR0215933A (en) | 2005-09-27 |
| EP1558743A4 (en) | 2007-11-14 |
| JP2006505276A (en) | 2006-02-16 |
| CA2505423A1 (en) | 2004-05-27 |
| HUP0500960A2 (en) | 2006-01-30 |
| AU2002340410A1 (en) | 2004-06-03 |
| EP1558743A2 (en) | 2005-08-03 |
| MXPA05004865A (en) | 2005-07-22 |
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