CN1066291A - New microorganism and the method for utilizing these microorganisms producing vitamin H vitamers - Google Patents
New microorganism and the method for utilizing these microorganisms producing vitamin H vitamers Download PDFInfo
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Abstract
The invention provides a kind of mutant strain, the glucose consumption speed that has of this bacterial strain is at most 1/4th of wild type strain, this mutant strain belongs to Escherichia, bacillus, pseudomonas, or Serratia, the glucose consumption speed that has is at most 1/4th of corresponding wild type strain, and vitamin H feedback inhibition wherein is by way of removing.The present invention also provides the method for utilizing this mutant strain to produce vitamin H.
Description
The present invention relates to new mutant strain, the glucose consumption speed that these bacterial strains have is at most 1/4th of wild type strain, especially relates to the mutant strain that belongs to the Escherichia that is used to produce vitamin H.In addition, the present invention relates to utilize these mutant strains to produce the method for vitamin H vitamer.
In order to improve a kind of productivity of concrete useful matter, various mutant strains have been prepared.For example, (as this concrete this vitamin H of useful matter is a kind of important VITAMIN of animal, plant and microorganism with regard to the bacterium that is used to produce vitamin H.) microorganism that obtains through artificial mutation, bacillus for example, chromobacterium, pseudomonas and genus arthrobacter are known.More particularly, in recent years, belong to Escherichia to a bacterial strain of α-dehydrobiotin tool resistance (for example, patent disclosure (kokai) No.61-149091 referring to Japanese unexamined), belong to the bacterial strain (as patent disclosure (kokai) No.61-202686) that the vitamin H feedback inhibition approach wherein of Escherichia has been removed referring to Japanese unexamined, belong to the bacterial strain that the acetic acid production rates of Escherichia reduces (referring to, as the open No.0316229 of European patent) be known as the host micro-organism that belongs to the host who is used for the gene recombination process one carrier system of Escherichia.As the microorganism except that Escherichia, the known use of host that has as a kind of concrete recombinant plasmid belongs to bacillus, the microorganism of those genus of pseudomonas and Saccharomycodes (referring to patent disclosure (kokai) No.64-44889 of for example Japanese unexamined).It should be noted that patent application above-mentioned also discloses utilization and contained the method for producing vitamin H with a transformant of the corresponding recombinant plasmid of host.
On the other hand, everybody has generally acknowledged that the mutant of a kind of enzyme of undertaking the carbohydrate phosphorylation can influence the assimilation of several carbohydrates, and its model also is suggested [referring to for example: intestinal bacteria and Salmonella typhimurium, F.C.Neidhardt, Vol.1, American Society For Microbiology:pp.127-150, (1987)].But, do not have the mutual relationship between the productivity of document description they and concrete material.
With regard to vitamin H (vitamin H is an example by the useful matter of microorganisms producing), above all mutant strains of Miao Shuing can reach predetermined purpose, but still leave some room for improvement.For example, with regard to the open No.0316229 of European patent (one of above-described open source literature), disclosing a kind of with the former method of utilizing glucose to carry out de novo synthesis production vitamin H as matrix compares more cheap and effective means, this method is utilized gene engineering, improved the E.coli recombinant chou from technical ability, this recombinant chou the has comprised removal as indicated above feedback inhibition approach of vitamin H and mutant strain having increased the phenotype that reduces the acetic acid production ability is as the host, and containing a recombinant plasmid, the vitamin H operon DNA that will obtain from the intestinal bacteria with vitamin H throughput in this plasmid is inserted into the carrier DNA.
Yet, ubiquitous problem is need provide a kind of can improve useful matter, the mutant strain of the productivity of vitamin H particularly, therefore, an object of the present invention is to provide a kind of a kind of concrete useful matter of microorganisms producing that utilizes, especially utilize the more efficient methods of the microorganisms producing vitamin H that contains the recombinant plasmid that has inserted the vitamin H operon.
According to the opinion that is different from conventional viewpoint, promptly according to following viewpoint: when containing glucose and cultivate as the fermention medium commonly used of carbohydrate, though the preferential consumption of glucose of wild type strain, and the lowered saltant of glucose assimilation ability is preferentially consumed other carbon sources (as amino acid).After removing the restraining effect of glucose, so the expectation latter plays advantageous effect to the production of concrete useful matter, the contriver has investigated the relation between this mutant and the vitamin H productivity, the result is, some has accumulated a large amount of vitamin Hs in the mutant of the plasmid of finding to contain above and being mentioned.
Therefore, can reach above described purpose with microbial mutation bacterial strain of the present invention, this mutant strain belongs to Escherichia, any of bacillus and Serratia belongs to, and it is characterized in that its maximum glucose consumption speed is at most 1/4th of corresponding wild type bacterial strain.
Especially, the problem that relates to vitamin H productivity, can solve by a kind of method of producing vitamin H, this method is included in cultivation mutant strain mentioned above in a kind of nutritional medium, this mutant strain contains a recombinant plasmid that carries a vitamin H operon that is inserted into the microorganism that derives from Escherichia in the carrier DNA, yet, collect the vitamin H and/or the desthiobiotin that form and accumulate in the substratum.
Mutant strain of the present invention can derive from but not limit as for belonging to Escherichia, and various types of microorganisms of bacillus and Serratia all can as long as can reach the bacterial strain of purpose of the present invention.As host's one carrier system, then preferential use belongs to escherich's bacillus, particularly in colibacillary mutant strain.In these bacterial strains, more preferably removed the bacterial strain that is used to produce vitamin H of vitamin H feedback inhibition approach, (after this also being referred to as " DR bacterial strain " herein sometimes).The concrete bacterial strain example that is used as the parental strain of mutant strain comprises, but be not limited to: intestinal bacteria (E.coli) bacterial strain DR-85(FERM P-8096), DRK-332(FERM BP-2113) and DRK-3323(FERM BP-2116) (referring to patent disclosure (kokai) No.61-202686 of Japanese unexamined, No.62-155081, and the open No.0316299 of European patent, as indicated above).
Here mentioned " FERM " numbering is meant in Japanese industry Science and Technology mechanism, the deposit numbers of fermentation research institute.
From these parental strains, can obtain the mutant strain that maximum value is at most 1/4th the maximum glucose consumption speed that has of corresponding wild type bacterial strain with known mutagenic treatment method, with following glucose consumption speed as index.
" glucose consumption speed " used herein is meant the amount [(S) grams per liter] of the glucose that per unit bacterial cell [(X) grams per liter] consumes in the time per unit [(T) hour], and the definition of the equation below available.Term used herein " corresponding to wild type strain " is meant a notion, and it has comprised the mutant strain of the sudden change that is different from the object of the invention that obvious variation has wherein taken place not show on the glucose consumption ability.
γ (gram glucose/gram cell X hour)=1/X * dS/dT
In order to obtain aforesaid mutant strain, for example, will be for example with ordinary method, differentiate on the Agar Plating cultivate that according to a kind of glucose assimilation that is described in of " product manual of Eiken chemistry company limited; the 5th edition " this mutant strain forms white colony and separate easily with the microorganism of a kind of mutagenic compound such as N-methyl-N-nitro-N-nitrosoguanidine mutagenic treatment on this substratum.This principle is based on the following fact, when growing on bacterium colony is containing the nutrient agar recited above of assimilable glucose, has reduced the pH value of substratum owing to materials such as producing organic acid.That is to say that because the dyestuff " tetrabromophenol sulfonphthalein " that adds in substratum, bacterium colony becomes orange, on the contrary, the bacterium colony display white that the mutant strain of the present invention that obtains by mutagenic treatment forms is not because pH value almost changes.
The object lesson of the mutant strain that so obtains comprises intestinal bacteria (E.coli) DRG024 bacterial strain, this bacterial strain is protected the root of Dahurian angelica in Japanese industry science and technology mechanism mentioned above March 22 nineteen ninety, the patent microorganism of fermentation research institute protects root of Dahurian angelica center, protecting the root of Dahurian angelica number is 11366, to protect the root of Dahurian angelica is transferred to the budapest treaty regulation in this bacterial strain of domestic official mission world guarantor's root of Dahurian angelica mechanism on March 14th, 1991, provide and protect the root of Dahurian angelica and number be FEPM BP-3309, and DRG005 bacterial strain, the DRG014 bacterial strain, the DRG026 bacterial strain carries out identical guarantor's root of Dahurian angelica with the DRG101 bacterial strain.Utilize following method to be easy to judge, it is 1/4th the feature that is at most corresponding wild type strain that such mutant strain has maximum glucose consumption speed, this method is: contain in as initial concentration on the substratum of glucose of appropriate amount and cultivate this bacterial strain, behind certain incubation time, the instrument of glucose concn is measured in utilization by an enzymatic reaction, for example, measure remaining glucose amount by the 27 type quantitative determination instruments that YSI produces, then it is compared with the undiminished wild type strain of glucose assimilative capacity.Determine the more desirable method of glucose consumption speed to be described in embodiment 1(2 hereinafter by the glucose assimilative capacity of measuring microorganism) in.
By the mutant strain that obtains mentioned above (being referred to as " DR mutant strain " herein sometimes) industrial application value is arranged, for example, help using in other aspects of the present invention.More particularly, the invention provides mutant strain with vitamin H height throughput, (this recombinant plasmid carries the vitamin H operon of the microorganism that comes from Escherichia wherein to use recombinant plasmid transformed mutant strain mentioned above, this operon is inserted in the carrier DNA), the present invention has also mentioned the method for using this mutant strain to produce vitamin H.
Used herein term " vitamin H vitamer " is meant vitamin H itself, and its precursor desthiobiotin.For the recombinant plasmid that is transformed in the mutant strain of the present invention, can use any carrier, as long as expressing this vitamin H in host's carrier system, these carriers produce ability.Intestinal bacteria (E.coli) the bacterial strain DRK-3323(pXBA321 that utilizes conventional separation quality grain method to provide from us preferably will be provided) (FERM BP-2117) and DRK-332(pKHN 31) pXBA312 and the pKHN31 that obtain (FERM BP-2114).
Mutant strain of the present invention with such recombinant plasmid of above describing can obtain with following method, promptly according to a conventional method as Mandel M, the calcium chloride method that et.al describes in " J.Mol.Biol.53.109(1970) " above the described recombinant plasmid transformed that obtains in the DG mutant strain, on nutrient agar, cultivate the transformant that obtains subsequently, on this substratum owing to introduced the phenotype of carrier, the clone who has the bacterial cell of recombinant plasmid, the bacterium colony that occurs is gathered in optionally growth.
The DG mutant strain that has recombinant plasmid that obtains thus comprises, for example, intestinal bacteria (E.coli) DRG 024[pXBA312] bacterial strain, as indicated above it be deposited at Japanese industry Science and Technology mechanism on March 22nd, 1991, the patent microorganism of fermentation research institute protects root of Dahurian angelica center, and provide deposit numbers 11365, then, to protect the root of Dahurian angelica is transferred to the regulation of budapest treaty in the bacterial strain of domestic official mission the internal authority guarantor root of Dahurian angelica guarantor of the mechanism root of Dahurian angelica on August 14th, 1991, protecting the root of Dahurian angelica number is FERM BP-3308, to DRG005[pXBA312] bacterial strain, DRG014[pXBA312] bacterial strain, DRG026[pXBA312] bacterial strain and DRG101[pXBA312] bacterium also handled same guarantor's root of Dahurian angelica formality.
On the felicity condition of the microorganism that is generally used for cultivating Escherichia and nutritional medium, cultivate the above-mentioned microorganism that obtains, can in substratum, accumulate a large amount of vitamin Hs.For example can use the known carbon source that contains, the synthetic or natural medium of nitrogenous source and inorganic substance.Used carbon source comprises carbohydrate such as glycerine, fructose, sucrose, maltose, seminose, starch, hydrolyzed starch liquid, molasses etc.The preferable range of institute's consumption is about 0.5-5.0%.For the lowered microorganism of glucose assimilative capacity of the present invention, if utilize glucose not have a kind of substratum of strict restriction as single carbon source, for example, during the hereinafter described a kind of natural medium that contains yeast extractive substance or white hydrolyzate of junket dawn, glucose still also can be used as available carbon source.For this situation, satisfactory amount ranges is about 0.5-5.0%.
Available nitrogenous source comprises ammonia, various inorganic or organic ammonium salt such as ammonium chlorides, ammonium phosphate, ammonium sulfate, and natural organic nitrogen source such as amino acid, meat medicinal extract, yeast extract, corn impregnation liquid, white hydrolyzate of junket dawn, and degreasing soya bean or its digest.In many cases, natural organic nitrogen source not only can be used as nitrogenous source, also can be used as carbon source.
As no mechanism matter, can utilize dipotassium hydrogen phosphate, potassium primary phosphate, sal epsom, sodium-chlor, ferrous sulfate, calcium chloride, zinc chloride, copper sulfate, magnesium chloride, cobalt chloride, ammonium molybdate, boric acid etc.
When cultivating DG bacterial strain of the present invention, also can be as adding L-Ala as described in the European Patent Publication No .0316229.The L-Ala that uses can be a D type compound, L type compound or DL-type compound, and wherein each can produce same effect.
Consider that cost issues is more suitable with DL type mixture.The concentration that joins the L-Ala in the substratum with the 1-10 grams per liter for well, 3-7 grams per liter more preferably.Can once add L-Ala immediately when cultivating beginning, also can merotomize adds in the training period.
As for the introducing that obtains to the microorganism of the phenotype of microbiotic tool resistance, can prevent other microbiological contamination by corresponding microbiotic is joined in the cultivation.Cultivation is preferably under the aerobic condition to be carried out, and for example sways and cultivates or aerated culture.Preferred culture temperature is 25-37 ℃, and the pH value of substratum preferably remains on and is essentially neutral in culturing process.The incubation time that produces vitamin H is about 24-48 hour usually, continues if do not add new nutrition to cultivate 48 hours again, and desthiobiotin is accumulated effectively.After cultivate finishing, extracting from crude substance and the method for purifying routinely utilizes the character of vitamin H or desthiobiotin, can be from nutrient medium collection of biological element or desthiobiotin.For example, by from the material of having cultivated, removing bacterial cell, with the remaining nutritive medium of activated carbon treatment, elution gac then, utilize the ion-exchange resin purification eluant, or directly spent ion exchange resin processing culture filtered liquid makes it purifying, then recrystallization from water or ethanol.
According to the present invention, can obtain to reduce the novel microorganism of glucose assimilative capacity, utilize this microorganism can produce specific useful matter easily.Utilize this microorganism to do the host, and a recombinant plasmid that will be for example insert the vitamin H operon is incorporated among the host and the microorganism that obtains can be advantageously used in the generation of vitamin H.
Embodiment
(1) preparation DG mutant strain from the intestinal bacteria (E.coli) of having removed vitamin H feedback inhibition approach.
The intestinal bacteria DRK3323(FERM BP-2116 that the feedback inhibition approach has been removed) L-substratum (10 grams per liter dawn white peptone, 5 grams per liter yeast extracts, 1 grams per liter glucose, 5 grams per liter sodium-chlor, transferring PH is 7.2) at 37 ℃, oscillating condition was cultivated 3 hours down.Collection is in the bacterial cell of logarithmic phase, and washed cell is suspended in TM damping fluid (0.61%Tris, the bundle acid of 0.6% horse of the N-methyl-N '-nitro-N-nitrosoguanidine that contains 100ug/ml then, transferring pH value is 6.0) in, keep 30 fen second month in a season so that mutagenesis takes place down at 37 ℃.After bacterial cell is collected and is washed, again the solution that suspends is added in the culture dish of a stock size, this suspension covered (2 grams per liter dawn are white on the Agar Plating, 10 grams per liter glucose, 5 grams per liter sodium-chlor, 0.3 grams per liter dipotassium hydrogen phosphate, 0.08 grams per liter tetrabromophenol sulfonphthalein, PH7.0) this substratum is with Eiken chemistry company limited product manual, the concentration that the agar concentration of the substratum that is used for decomposition glucose of the 5th edition description is varied on the Agar Plating commonly used prepares, and has obtained about 200-800 bacterium colony thus.37 ℃ cultivate 48 hours after, white or be bordering on white and do not transfer orange bacterium colony to, separate by acquisition method, with the feedback inhibition that obtains vitamin H DG mutant strain by way of the intestinal bacteria (E.coli) of removing, be DRG024 bacterial strain (FERM BP-3309), DRG 005 bacterial strain, DRG 014 bacterial strain, DRG 026 bacterial strain, and DRG 101 bacterial strains.
As preceding cultivation, to above described DRG024 bacterial strain with a platinum loop from keeping the agar plate culture, DRG005, DRG 026, DRG101 and DRK3323 are inoculated into L substratum (10 grams per liter dawn white peptones, 5g/e yeast extract, 1 grams per liter glucose medium, 5 grams per liter sodium-chlor, PH transfers to 7.0) cultivated 8-12 hour at 37 ℃ then.
(2) measure glucose consumption speed
After this pre-culture of 0.2ml is inoculated into the Sakaguchi flask that volume is 500ml (production of Iwaki Glass Co., Ltd.), carry out shaking culture at 37 ℃, every sampling from this bottle in 2 hours, measure the turbidity of bacterial cell, calculate the weight (X) of dried bacterial cell and represent with grams per liter.Simultaneously, the aliquots containig of the culture that obtains is centrifugal, and get the instrument (YsI preparation, 27 types) that 200 μ l supernatant liquors are injected into measure glucose concentration.The amount of the glucose that measurement stays is with the amount (S) of the glucose of decision consumption.
Utilize these numerical value,, utilize the general formula of Gregory-Newton can draw the speed (ν) of each time durations internal consumption glucose according to the number of degrees differential method
ν (gram/glucose X hour)=1/XXds/dT(is referring to engineering mathematics, Vol.1 Wily Ed.Brain Book Publisher, PP.96).
DG mutant strain that obtains and parent strain DRK3323(FERM BP-2116) the maximum value of glucose consumption speed be shown in the table 1.
Culture medium A (grams per liter)
Sodium phosphate dibasic (12H
2O) 17.6
Potassiumphosphate 2.4
Ammonium sulfate 4.0
Yeast extractive substance 10.0
Dawn white peptone 10.0
Ferrous sulfate (7H
2O) 0.1
Calcium chloride (2H
2O) 0.05
Magnesium chloride (4H
2O) 0.05
Sal epsom (7H
2O) 0.1
Glucose 5.0
DL-L-Ala 5.0
Table 1
Bacterial strain glucose consumption speed maximum value (ν)
DRG024 0.2
DRG005 0.05
DRG014 0.1
DRG026 0.5
DRG101 0.1
DRK3323 2.0
(3) determine carbohydrate assimilation ability
With DRG005, DRG101 and DRK3323 bacterial strain were cultivated 12 hours in the L-substratum, washed secondary with the PBS damping fluid then.With each cell transfer of equal quantities to the basic synthetic medium of Mg, this substratum contain concentration be respectively 0.2% suitable carbohydrate or be listed in the table below in a kind of metabolic intermediate as single carbon source.After cultivating 24 hours, measure cell and whether grow.The results are shown in the table 2.For DRG005 bacterial strain and DRG101 bacterial strain, can see that clearly the ability of assimilation glucose or other several carbohydrates reduces.
Table 2
Bacterial strain DRK3323 DRG005 DRG101
(carbohydrate of adding)
Glucose+--
Glycerine+-+
Maltose+--
Fructose+--
Pectinose+++
Sodium.alpha.-ketopropionate+++
Embodiment 2 preparations contain the DG mutant strain of recombinant plasmid
Adopt ordinary method, calcium chloride method [Mol.Gen.Genet.Vol.124 for example, PP1-10(1973)] with recombinant plasmid pXBA312(wherein the vitamin H operon be inserted in the carrier DNA) transformed into escherichia coli (E.coli) DRG024 bacterial strain, the DRG005 bacterial strain, the DRG014 bacterial strain, the DRG026 bacterial strain, or DRG101 bacterial strain, containing the formed bacterium colony of separation on the LB solid plate substratum of 10ug/ml tsiklomitsin then, obtain DRG024[pXBA312 respectively] bacterial strain (FERM BP-3308), DRG005[pXBA312] bacterial strain, DRG014[pXBA312] bacterial strain, DRG026[pXBA312] bacterial strain, or DRG101[pXBA312] bacterial strain.
Embodiment 3 makes vitamin H
As a kind of pre-culture, the DRG mutant strain that contains this recombinant plasmid that utilizes a platinum loop that the maintenance agar plate is cultivated is inoculated into L substratum (10 grams per liter dawn white peptones, 5 gram/long yeast extractive substances, 1 grams per liter glucose, 5 grams per liter sodium-chlor; Transfer PH to 7.0; When cultivating, add the 29ug/ml tsiklomitsin again for the bacterial cell that contains recombinant plasmid), cultivated 8-12 hour at 37 ℃.This pre-culture of 0.2ml is inoculated into the Sakaguchi flask (being made by Iwaki Glass Co., Ltd.) of the 500ml volume that contains the A substratum that 20ml above describes, cultivates then.After 96 hours, take a sample, measure turbidity and the vitamin H of accumulation and the amount of desthiobiotin of bacterial cell then.The results are shown in the table 3.
For vitamin H is carried out quantitative analysis, be diluted to suitable degree with removing the supernatant liquor that bacterial cell obtains after centrifugal, (ATCC8014) carry out biological assay with Lactobacillus plantarum (Lactobacillus plan-tarum).Quantitative analysis for desthiobiotin, utilize total concentration (the Metbod in Enzymology of white avidin of antibiotin dawn by colorimetric method for determining vitamin H and desthiobiotin, the Vol.XV III PP.49) deducts the amount of above describing the vitamin H that bioassay method measures and the amount that calculates desthiobiotin from total concn.
Table 3
The life of the desulfurization accumulation of strain cell concentration accumulation
The amount of the amount thing element of (grams per liter) vitamin H
(mg/ liter) (mg/ liter)
DRG024 5 82 10
[pXBA312]
DRG005 4 73 9
[pxBA312]
DRG014 4 69 9
[pXBA312]
DRG026 5 80 10
[pxBA312]
DRG101 5 80 10
[pxBA312]
DRK3323 4 13 9
[pxBA312]
(contrast)
Claims (7)
1, a kind of escherich's bacillus that belongs to, the microbial mutation bacterial strain of arbitrary genus of genus bacillus and Sai Shi bacillus is characterized in that its maximum glucose consumption speed is at most 1/4th of corresponding wild type strain.
2, according to the said mutant strain of claim 1, wherein said microorganism belongs to Escherichia.
3, according to the said mutant strain of claim 2, wherein said microorganism is used to produce the vitamin H vitamer.
4, according to the said mutant strain of claim 3, the vitamin H feedback inhibition approach of this bacterial strain is removed.
5, according to the said mutant strain of claim 4, this bacterial strain contains a recombinant plasmid that carries a vitamin H operon that is inserted into the microorganism that derives from Escherichia in the carrier DNA.
6, according to the said mutant strain of claim 5, this bacterial strain is selected from following one group of bacterial strain: DRG024 [pXBA312] bacterial strain, DRG005 [pXBA312] bacterial strain, DRG014 [pXBA312] bacterial strain, DRG026 [pXBA312] bacterial strain, and DRG101 [pXBA312] bacterial strain.
7, a kind of method for preparing vitamin H is characterized in that cultivating the mutant strain of claim 5 in a kind of nutritional medium, collect the vitamin H and/or the desthiobiotin that form and accumulate in the nutrient solution then.
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| JP2075683A JP3006847B2 (en) | 1990-03-27 | 1990-03-27 | Novel microorganism and method for producing biotin using the same |
| CN91103221A CN1053217C (en) | 1990-03-27 | 1991-04-27 | Microorganisms and process for producing biotin vitamers using same |
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| CN1050153C (en) * | 1992-09-10 | 2000-03-08 | 住友化学工业株式会社 | Process for production of biotin |
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