CN105861526A - One group of DNA (Deoxyribose Nucleic Acid) molecules, recombinant vector, recombinant ketogulonigenium sp and method for producing 2-keto-L-gulonic acid - Google Patents
One group of DNA (Deoxyribose Nucleic Acid) molecules, recombinant vector, recombinant ketogulonigenium sp and method for producing 2-keto-L-gulonic acid Download PDFInfo
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- CN105861526A CN105861526A CN201610246687.6A CN201610246687A CN105861526A CN 105861526 A CN105861526 A CN 105861526A CN 201610246687 A CN201610246687 A CN 201610246687A CN 105861526 A CN105861526 A CN 105861526A
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Abstract
The invention relates to the technical field of genetic engineering, and discloses one group of DNA (Deoxyribose Nucleic Acid) molecules, a recombinant vector, a recombinant ketogulonigenium sp and a method for producing 2-keto-L-gulonic acid. The DNA molecules at least comprise one of nucleotide sequences shown by SEQ ID NO:1 and SEQ ID NO:2. The invention provides the DNA molecules capable of coding a key enzyme in a ketogulonigenium sp lipoic acid metabolic pathway, and the vector is transformed into the ketogulonigenium sp through an embedding way, so that the missing key enzyme can be expressed, endogenous lipoic acid is generated, the growth of bacterial strains is promoted, the acid yield and the acid production efficiency are improved, and the aim of carrying out monoxenie fermentation and acid production without adding exogenous lipoic acid nutritional factors with higher cost can be realized.
Description
Technical field
The present invention relates to gene engineering technology field, more particularly to one group DNA molecular and restructuring
Carrier, restructuring Ketogulonigenium sp and the method producing 2-keto-L-gulonic acid.
Background technology
Vitamin C is to maintain a kind of water soluble vitamins necessary to body growth and metabolism, the most extensively should
For medicine, food, feedstuff and cosmetic field, market stability.Current ascorbic main flow working system
It is two-step fermenting, refers mainly to, from the beginning of D-glucitol, be converted into vitamin C precursor through two-step fermentation--2-
The process of ketone-L-2-KLG.The first step, utilizes Gluconobacter oxvdans that D-glucitol is converted into L-mountain
Pears sugar, second step utilizes raw Ketogulonigenium sp and the mixed thalline of concomitance bacterium (predominantly bacillus cereus) thereof
L-sorbose is converted into 2-keto-L-gulonic acid by system.But, the mixed fungus fermentation of this size bacterium is to technique control
System adds difficulty, causes the waste of fermentation medium nutritional labeling and the energy, adds environmental pollution,
Improve production cost.
Realize saccharic acid conversion (by L-sorbose to 2-keto-L-gulonic acid) by single bacterium fermentation and always tie up life
The target that element C fermentation technology is pursued.Chinese patent CN102321698A discloses a kind of promotion ketone 2-KLG
Bacillus growth and the method producing acid, it adds trophic factors thioctic acid in the medium, improves ketone GULONG
The speed of growth of acidfast bacilli and acid producing ability, and then reached to utilize the method for single bacterium fermentation to realize saccharic acid and turn
The purpose changed.But the market quotes of current DL thioctic acid is at 550 yuan/kilogram on market, and R-
Thioctic acid reach 2000-3000 unit/kilogram, price difference huge.Thioctic acid produces and is currently in R-
Thioctic acid (dextrorotation) replaces among the process of DL thioctic acid, and the thioctic acid that antibacterial produces is LA,
Thus promote that its growth and product acid have more meaning biology in Ketogulonigenium sp construct in vitro thioctic acid module
Justice.
Although enzyme is imperfect in the thioctic acid metabolic pathway of synthesizing of Ketogulonigenium sp to have research to point out, but
Specifically the imperfect of which kind of enzyme does not draws definite conclusion, simultaneously Ketogulonigenium sp in the art
Being non-model organism, there is the biggest difficulty in molecule manipulation, there is great challenge, therefore about ancient at ketone
Dragon acidfast bacilli construct in vitro these aspects of thioctic acid module rarely have report.
Summary of the invention
In view of this, it is an object of the invention to provide one group of DNA molecular so that described DNA molecular
The enzyme that the albumen of coding makes up in Ketogulonigenium sp body thioctic acid metabolic pathway of synthesizing is imperfect, it is achieved sulfur
Octanoic acid anabolism.
A kind of recombinant vector comprising described DNA molecular of offer is provided so that
Described recombinant vector can be transformed in Ketogulonigenium sp, it is achieved Ketogulonigenium sp thioctic acid synthesis generation
Thank, improve its acid producing ability, shorten its fermentation period.
Further object is that offer one restructuring Ketogulonigenium sp so that described restructuring ketone
2-KLG bacillus can carry out thioctic acid anabolism, has higher acid producing ability and efficiency.
Further object is that a kind of method producing 2-keto-L-gulonic acid of offer so that described
Method can improve yield and the production efficiency of 2-keto-L-gulonic acid.
For achieving the above object, the present invention provides following technical scheme:
One group of DNA molecular, including at least following a kind of nucleotide sequence:
Nucleotide sequence shown in SEQ ID NO:1；
Nucleotide sequence shown in SEQ ID NO:2.
Which kind of enzyme is lacked by and prior art imperfect for enzyme in Ketogulonigenium sp metabolism of lipoic acid approach
Lose the defect without definite conclusion, the invention provides one group of DNA molecular of the key enzyme of coding disappearance, i.e.
Nucleotide sequence shown in SEQ ID NO:1-2, the key enzyme present invention of coding be respectively designated as lipA and
lipB.The key enzyme of described coding can make Ketogulonigenium sp metabolism of lipoic acid approach improve and synthesize endogenous
Thioctic acid, it is achieved Ketogulonigenium sp list bacteria growing and product acid, it is not necessary to add the thioctic acid of external source.
In some technical schemes of the present invention, described DNA molecular comprises core shown in SEQ ID NO:1-2
Nucleotide sequence.And in other technical scheme, described DNA molecular is by shown in SEQ ID NO:1-2
Nucleotide sequence forms.
Effect based on described DNA molecular, the invention provides it and is producing 2-keto-L-gulonic acid, with
And in preparation application in the recombinant vector producing 2-keto-L-gulonic acid and recombinant bacterial strain.Should for this
With, the present invention provides a kind of recombinant vector accordingly, comprises described in any one technical scheme of the present invention
DNA molecular, the most described DNA molecular can be following any case:
(1) nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2；
(2) nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2, comprises simultaneously and does not affect this
Invent other DNA moleculars of described DNA molecular coding expressing protein；
(3) nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:2；
(4) nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:2, comprises simultaneously and does not affect
Other DNA moleculars of DNA molecular of the present invention coding expressing protein.
DNA molecular of the present invention can be embedded in carrier in any appropriate site, SEQ ID NO:1
Both can adjacent can also be separately present on carrier with nucleotide sequence shown in SEQ ID NO:2, this can
Accommodation is carried out with the selection according to carrier, simultaneously in order to encode expression smoothly, described restructuring
In carrier, on nucleotide sequence shown in nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2
Trip also should have promoter, and downstream has terminator.In some technical schemes of the present invention, described startup
Son selects 16S promoter, and described terminator selects 5S terminator.
The present invention gives the most concrete selection for the selection of carrier, and described carrier may be selected to be
PBBR1MCS2 plasmid vector, according to restriction enzyme site on this plasmid vector, can be to DNA of the present invention
The corresponding restriction enzyme site of two ends design of molecule embeds, to form recombinant vector.As restriction enzyme site KpnI,
HindIII, BamHI etc., equally, changing other plasmid vectors can be corresponding according to its restriction enzyme site having
Adjust.
Effect based on DNA molecular of the present invention, described recombinant vector could be used for producing 2-
Ketone-L-2-KLG, and for preparing the recombinant bacterial strain producing 2-keto-L-gulonic acid.
Additionally, present invention also offers a kind of restructuring Ketogulonigenium sp, its conversion has above-mentioned of the present invention
The recombinant vector mentioned in meaning technical scheme, described conversion can use the mode of electricity conversion to carry out.The present invention
The restructuring Ketogulonigenium sp obtained i.e. can reach by adding external source thioctic acid without adding external source thioctic acid
Original strain acid yield, be better than the original Ketogulonigenium sp being not added with external source thioctic acid simultaneously.?
On fermentation period, restructuring Ketogulonigenium sp of the present invention is compared original Ketogulonigenium sp and is shortened 5
Hour, improve production efficiency.Thus, present invention also offers described restructuring Ketogulonigenium sp giving birth to
Produce the application in 2-keto-L-gulonic acid.
The related application provided corresponding to the present invention, the present invention each provides a kind of shake flask fermentation and produces 2-
A kind of method that the method for ketone-L-2-KLG and ferment tank produce 2-keto-L-gulonic acid.Wherein, shaking flask
Fermentation process is as follows:
Restructuring Ketogulonigenium sp of the present invention is joined in solid seed culture medium and activate, then turn
Receive and seed culture medium carries out shake flask fermentation.
Wherein, described solid seed culture medium is the seed culture medium after adding agar, described seed culture
Basis set become:
Carnis Bovis seu Bubali cream 3.0g/L, Semen Maydis pulp 6.0g/L, yeast leaching powder 3.0g/L, peptone 10.0g/L, nothing
Water magnesium sulfate 0.2g/L, carbamide 1.0g/L, dipotassium hydrogen phosphate 1.0g/L, calcium carbonate 1.0g/L, separately take mountain
Pears sugar 20g/L, pH=6.8.
Described activation is cultivation 24h at 30 DEG C；Described shake flask fermentation for 30 DEG C, shake under 250r/min
Bottle fermentation.
Ferment tank method is as follows:
Restructuring Ketogulonigenium sp of the present invention is joined fermentation medium carries out ferment tank,
Sorbose is added during fermentation.
Wherein, described fermentation medium is:
Semen Maydis pulp 10g/L, carbamide 12g/L, potassium dihydrogen phosphate 1g/L, magnesium sulfate 0.2g/L, calcium carbonate 1g/L,
Sorbose 80g/L, defoamer 1ml/3L, pH value is 6.7-7.0.
Adding sorbose during described fermentation is the sorbose that fermentation 24h-30h mends 80g/L.
Ferment tank condition is 30 DEG C, and 500r/min, pH control 7.0, and ventilate 1.5vvm.PH controls
7.0, regulate pH with 2.5mol sulphuric acid and 5mol sodium hydroxide.
From above technical scheme, the present invention provides and can encode Ketogulonigenium sp metabolism of lipoic acid way
The DNA molecular of the key enzyme in footpath, proceeds to Ketogulonigenium sp by the form embedding carrier so that it is energy
Enough express the key enzyme lacked, generate endogenous thioctic acid, promote the growth of bacterial strain, improve acid yield
With product acid efficiency, it is achieved that without adding relatively costly external source thioctic acid trophic factors and still can enter
The purpose of the single bacterium fermentation and acid of row.
Accompanying drawing explanation
Fig. 1 show the recombinant vector collection of illustrative plates based on PBBR1MCS2 plasmid vector；
Fig. 2 show the different time points shake flask fermentation bacteria concentration broken line graph of kv group and lip-kv group；Kv group
For original Ketogulonigenium sp, lip-kv group is restructuring Ketogulonigenium sp of the present invention；
Fig. 3 show the different time points shake flask fermentation of kv group and lip-kv group and produces acid amount broken line graph；Kv group
For original Ketogulonigenium sp, lip-kv group is restructuring Ketogulonigenium sp of the present invention；
Fig. 4 show different disposal group 72h shake flask fermentation and produces acid bar diagram；Kv: original ketone 2-KLG bar
Bacterium；Kv-lxs: add the original Ketogulonigenium sp of external source thioctic acid；Lip-kv: restructuring of the present invention
Ketogulonigenium sp；Lip-kv-lxs: add the restructuring Ketogulonigenium sp of the present invention of external source thioctic acid；
Fig. 5 show the different time points ferment tank bacteria concentration broken line graph of kv group and lip-kv group；kv
Group is original Ketogulonigenium sp, and lip-kv group is restructuring Ketogulonigenium sp of the present invention；
Fig. 6 show the different time points ferment tank of kv group and lip-kv group and produces acid amount broken line graph；kv
Group is original Ketogulonigenium sp, and lip-kv group is restructuring Ketogulonigenium sp of the present invention；
Fig. 7 show the different time points ferment tank substrate sorbose consumption folding of kv group and lip-kv group
Line chart；Kv group is original Ketogulonigenium sp, and lip-kv group is restructuring Ketogulonigenium sp of the present invention.
Detailed description of the invention
The invention discloses one group of DNA molecular and recombinant vector, restructuring Ketogulonigenium sp and produce 2-
The method of ketone-L-2-KLG, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter
Realize.Special needs to be pointed out is, all similar replacements and change are for a person skilled in the art
It will be apparent that they are considered as being included in the present invention.DNA molecular of the present invention, recombinant vector,
Recombinant bacterial strain and production method are described by preferred embodiment, and related personnel substantially can be
Without departing from present invention, spirit and scope, method described herein and application being modified or suitably
Change and combination, realize and apply the technology of the present invention.
Involved in the present invention some plasmid vectors, bacterial strain are all commercially available, such as PBBR1MCS2
Plasmid can be by China plasmid vector strain cell pnca gene preservation center (Biovector Science
Lab, Inc) to buy, original Ketogulonigenium sp can obtain from North China Wei Erkang pharmaceutical Co. Ltd of pharmacy group
?.
According to the summary of the invention of the present invention, those skilled in the art are after knowing DNA molecular of the present invention
Can be embedded it in suitable vector plasmid by this area disclosed technical operation.The present invention is real
Execute in example involved recombinant vector based on commercially available PBBR1MCS2 plasmid, at its KpnI and
It is respectively embedded in DNA molecular of the present invention between HindIII, HindIII and BamHI restriction enzyme site
SEQ ID NO:1 and SEQ ID NO:2 shown in nucleotide sequence, the most also include opening of two sequences
The necessary Genetic elements such as mover and terminator sequence, the overall nucleotide sequence embedded is followed successively by KpnI enzyme
Cut sequence-5S terminator-HindIII restriction enzyme site-6S shown in the-16S promoter-SEQ ID NO:2 of site to open
Sequence-5S terminator-BamHI restriction enzyme site shown in mover-SEQ ID NO:1, can be public by biotechnology
(as shown in SEQ ID NO:3,1-6bp is KpnI restriction enzyme site, and 7-383bp is 16S to have taken charge of synthesis
Promoter, 384-1067bp is the sequence of coding lipB shown in SEQ ID NO:2, and 1068-1395bp is
5S terminator, 1396-1401bp is HindIII restriction enzyme site, and 1402-1778bp is 16S promoter,
1779-2747bp is the sequence of coding lipA shown in SEQ ID NO:1, and 2746-3075bp is that 5S terminates
Son, 3076-3081bp is BamHI restriction enzyme site).
By enzyme action, being introduced in preparation process of restriction enzyme site verifies that genes of interest becomes the most
Merit proceeds to and recombiant plasmid whether successful conversion, and the PBBR1MCS2 plasmid map after restructuring is shown in Fig. 1.
The present invention proceeds to after the preparation completing recombinant vector under the conditions of 2000V by the way of electricity converts
Ketogulonigenium sp, concrete method is as follows:
The preparation of 1.K.vulgare (Ketogulonigenium sp) competent cell
K.vulgare glycerol stock activates through solid plate, transfers in 50mL HJ culture medium, cultivates
13h arrives late exponential phase to thalline；Take 40mL thalline in the centrifuge tube of 50mL pre-cooling, ice bath
15min；In 4 DEG C of refrigerated centrifugers, 4500rpm is centrifuged 15min, outwells supernatant；With pre-cooling
The glycerol of 10% washs, re-suspended cell, and 4500rpm is centrifuged 15min, removes supernatant equally；
Repeat previous step；Adding the ultra-pure water re-suspended cell of the sterilizing pre-cooling of 30mL, 4500rpm is centrifuged 15
Min, outwells supernatant；Adding 900 μ L and shift to an earlier date 10% glycerol of pre-cooling, re-suspended cell, with the rifle of pre-cooling
Head subpackage competent cell, in 1.5mL EP manages, often has competent cell 90 μ L in pipe.Prepare
Competent cell preserves in being positioned over-80 DEG C of refrigerators, takes out and use when needing.
2. electricity turns
From-80 DEG C of refrigerators, take out the K.vulgare competent cell prepared, be placed in thawed on ice；
Add 10 μ L plasmids (concentration is preferably at more than 100ng) with freezing rifle head, notice that plasmid need to slowly add
Enter, and can not pressure-vaccum competent cell repeatedly, carefully flick EP pipe outer wall mixing cell and plasmid, then
Stand 5min；Freezing for mixture in EP pipe rifle head is transferred in the electric revolving cup of prior pre-cooling, electricity
Revolving cup carries out ice bath all the time；Shock voltage 2.0kV is set, controls the electric shock time 4～6ms, fast after electric shock
Speed adds the nonreactive HJ culture medium of pre-cooling；At 30 DEG C, 150rpm shaking table carries out recovery 3h, centrifugal
Thalline is coated in the resistant panel corresponding with plasmid resistance gene.
Correct transformant is obtained by proposing plasmid enzyme restriction checking.
HJ culture medium: weigh yeast leaching powder 5.0g, peptone 15.0g, carbamide 5.0g, anhydrous magnesium sulfate
2.5g, is settled to 0.8L, separately weighs 15.0g sorbose and is settled to 0.2L, equal 115 DEG C of sterilizing 15min.
Connecing 200ml before bacterium, 7.5% sugar juice adds in HJ culture medium and mixes.
Below in conjunction with embodiment, the present invention is expanded on further.
Embodiment 1: the preparation of restructuring Ketogulonigenium sp of the present invention
1, the amplification in vitro of described recombinant vector
The DNA molecular provided according to the present invention and the preparation scheme of recombinant vector, pass through biotech company
On behalf of synthesis, Fig. 1 is shown in by the vector plasmid collection of illustrative plates after restructuring, the sequence wherein embedded such as SEQ ID NO:3
Shown sequence, i.e. sequence-5S terminator shown in KpnI restriction enzyme site-16S promoter-SEQ ID NO:1-
Sequence-5S terminator-BamHI enzyme action position shown in HindIII restriction enzyme site-6S promoter-SEQ ID NO:2
Point.
By large intestine, described recombinant vector is converted conventional classical heat shock method, and the competent cell of use is purchased from
The limited public BDH5 α of Beijing Bo Maide development in science and technology.After conversion, according to the resistance (institute proceeded to entrained by plasmid
Be kana resistance by resistance) select suitable LB resistant panel coated plate after carry out incubated overnight.Incubated overnight
After growing single bacterium colony, then by the way of bacterium colony PCR, carry out the screening (mould of bacterium colony PCR of positive transformant
Plate is single bacterium colony, and as other, bacterium colony PCR programming is similar with regular-PCR program).PCR completes
After, carry out electrophoresis, the positive (correctly) transformant can be picked out by stripe size.Further turn
Beggar's verification mode proposes plasmid enzyme restriction checking and order-checking, by further checking (digestion verification means)
Obtain final correct transformant.
Shock step is as follows:
1) take 100 μ L competent cells to be placed in ice bath, as needed subpackage can be dispensed into just melting cell suspension
In the centrifuge tube of aseptic pre-cooling, it is placed in ice bath.
2) taking 10 μ L connection products and add in 100 μ L competent cells, rotating centrifugal pipe is with in mixing gently
Tolerant, ice bath stands 30min.
3) centrifuge tube is placed in 42 DEG C of water-baths placement 90s, then quickly pipe is transferred in ice bath, make thin
Born of the same parents cool down 2min, and this process does not shake centrifuge tube.
4) in each centrifuge tube, 500 LB culture medium (without antibiotic) aseptic for μ L are added, after mixing
It is placed in 37 DEG C, 150rpm shaking table shaken cultivation 45min, makes resistant maker gene relevant on plasmid express,
Thalline is made to recover.
5) under aseptic condition, take appropriate bacterium solution and be added on the LB solid culture flat board containing corresponding antibiotic, use
Aseptic antibacterial spreader or bead are the most spreadable by cell.After fully absorbing Deng the liquid in flat board,
It is inverted flat board, cultivates 12-16h for 37 DEG C.
6) retain remaining bacterium solution in 4 DEG C of refrigerators, determine going or staying depending on colony growth situation on flat board.
LB culture medium: 10.0g/L sodium chloride, 10.0g/L tryptone, 5.0g/L yeast extract.
Corresponding solid medium adds 2% agar powder.Sterilising conditions: 121 DEG C, 20min.
2, the conversion of Ketogulonigenium sp
Utilize LB culture medium culturing to contain the escherichia coli of correct transformant, carry again after obtaining finite concentration containing
The plasmid of genes of interest, then thioctic acid gene electricity under the conditions of 2000V of acquisition is proceeded to ketone 2-KLG bar
Bacterium, it is thus achieved that the Ketogulonigenium sp of coding thioctic acid genophore.
The preparation of 1.K.vulgare (Ketogulonigenium sp) competent cell
K.vulgare glycerol stock activates through solid plate, transfers in 50mL HJ culture medium, cultivates
13h arrives late exponential phase to thalline；Take 40mL thalline in the centrifuge tube of 50mL pre-cooling, ice bath
15min；In 4 DEG C of refrigerated centrifugers, 4500rpm is centrifuged 15min, outwells supernatant；With pre-cooling
The glycerol of 10% washs, re-suspended cell, and 4500rpm is centrifuged 15min, removes supernatant equally；
Repeat previous step；Adding the ultra-pure water re-suspended cell of the sterilizing pre-cooling of 30mL, 4500rpm is centrifuged 15
Min, outwells supernatant；Adding 900 μ L and shift to an earlier date 10% glycerol of pre-cooling, re-suspended cell, with the rifle of pre-cooling
Head subpackage competent cell, in 1.5mL EP manages, often has competent cell 90 μ L in pipe.Prepare
Competent cell preserves in being positioned over-80 DEG C of refrigerators, takes out and use when needing.
2. electricity turns
From-80 DEG C of refrigerators, take out the K.vulgare competent cell prepared, be placed in thawed on ice；
Add 10 μ L plasmids (concentration is preferably at more than 100ng) with freezing rifle head, notice that plasmid need to slowly add
Enter, and can not pressure-vaccum competent cell repeatedly, carefully flick EP pipe outer wall mixing cell and plasmid, then
Stand 5min；Freezing for mixture in EP pipe rifle head is transferred in the electric revolving cup of prior pre-cooling, electricity
Revolving cup carries out ice bath all the time；Shock voltage 2.0kV is set, controls the electric shock time 4～6ms, fast after electric shock
Speed adds the nonreactive HJ culture medium of pre-cooling；At 30 DEG C, 150rpm shaking table carries out recovery 3h, centrifugal
Thalline is coated in the resistant panel corresponding with plasmid resistance gene.
Correct transformant is obtained by proposing plasmid enzyme restriction checking.
Embodiment 2: the shake flask fermentation test of restructuring Ketogulonigenium sp of the present invention
1, test packet
Kv: original Ketogulonigenium sp；
Kv-lxs: add the original Ketogulonigenium sp of external source thioctic acid；
Lip-kv: restructuring Ketogulonigenium sp of the present invention；
Lip-kv-lxs: add the restructuring Ketogulonigenium sp of the present invention of external source thioctic acid；
2, the different time points shake flask fermentation contrast test of kv group and lip-kv group
Each group of bacterial strain is charged first in solid seed culture medium cultivate 24h at 30 DEG C, activates,
Then it is transferred in seed culture medium at 30 DEG C, shake flask fermentation under 250r/min with identical initial OD,
The different time points sample analysis such as 0h, 3h, 6h, 9h, 12h, 19h, 24h, 30h.
Described solid seed culture medium is the seed culture medium after adding agar, and described seed culture medium forms
For:
Carnis Bovis seu Bubali cream 3.0g/L, Semen Maydis pulp 6.0g/L, yeast leaching powder 3.0g/L, peptone 10.0g/L, nothing
Water magnesium sulfate 0.2g/L, carbamide 1.0g/L, dipotassium hydrogen phosphate 1.0g/L, calcium carbonate 1.0g/L, separately take mountain
Pears sugar 20g/L, pH=6.8.
3, the 72h shake flask fermentation contrast test of 4 groups
Each group of bacterial strain is charged first in solid seed culture medium cultivate 24h at 30 DEG C, activates,
Then it is transferred in seed culture medium at 30 DEG C, shake flask fermentation under 250r/min with identical initial OD,
72h point in time sampling is analyzed.The group adding external source thioctic acid is needed to add in seed culture medium, final concentration
For 0.64mg/L.
4, detection is analyzed
Bacteria concentration measures: use 75-6 type ultraviolet spectrophotometer to measure the growth of bacterial strain in incubation bent
Line, wavelength uses 600nm, i.e. OD600.
Ketone 2-KLG determination of yield: high performance liquid chromatography detects, uses Aminex HPX-87H chromatographic column,
Composition distribution.0.5mM sulphuric acid makees flowing phase, column temperature 65 DEG C, detector temperature 50 DEG C.
5, result of the test
The different time points shake flask fermentation comparative test result of kv group and lip-kv group is shown in Fig. 2 and Fig. 3, by scheming
Understanding, under identical time point, restructuring Ketogulonigenium sp of the present invention is at bacteria concentration and produces sour water
Original Ketogulonigenium sp will be higher than on Ping.
The 72h shake flask fermentation comparative test result of 4 groups is shown in the table 1 of Fig. 4 and correspondence, by figure and the test of table
Result is it can be seen that the acid producing ability of restructuring Ketogulonigenium sp of the present invention is slightly better than adding external source sulfur
The original Ketogulonigenium sp of octanoic acid, compares original Ketogulonigenium sp product acid amount and improves 6.5%.
Table 1 72h shake flask fermentation comparative test result
| kv | kv-lxs | lip-kv | lip-kv-lxs | |
| 72h | 8.069971 | 8.565651 | 8.599779 | 8.653318214 |
| Standard deviation | 0.070387 | 0.076786 | 0.110913 | 0.247988552 |
Embodiment 3: the ferment tank test of restructuring Ketogulonigenium sp of the present invention
1, test packet
Kv: original Ketogulonigenium sp；
Lip-kv: restructuring Ketogulonigenium sp of the present invention；
2, the different time points shake flask fermentation contrast test of kv group and lip-kv group
By two groups of bacterial strains through overactivation, access ferment tank with identical initial density, during fermentation
24h-30h mends the sorbose of 80g/L.
Described fermentation medium is:
Semen Maydis pulp 10g/L, carbamide 12g/L, potassium dihydrogen phosphate 1g/L, magnesium sulfate 0.2g/L, calcium carbonate 1g/L,
Sorbose 80g/L, defoamer 1ml/3L, pH value is 6.7-7.0.
Ferment tank condition is 30 DEG C, and 500r/min, pH control 7.0, and ventilate 1.5vvm.PH controls
7.0, regulate pH with 2.5mol sulphuric acid and 5mol sodium hydroxide.
3, detection is analyzed
Bacteria concentration measures: use 75-6 type ultraviolet spectrophotometer to measure the growth of bacterial strain in incubation bent
Line, wavelength uses 600nm, i.e. OD600.
Base consumption and product acidity test: high performance liquid chromatography detects, and uses Aminex HPX-87H chromatograph
Post, Composition distribution.0.5mM sulphuric acid makees flowing phase, column temperature 65 DEG C, detector temperature 50 DEG C.
4, result of the test
Result is shown in that Fig. 5-7, result show, under identical time point, and restructuring ketone 2-KLG bar of the present invention
Bacterium will be higher than original Ketogulonigenium sp on bacteria concentration and acid yield.
By the situation of the sorbose base consumption of Fig. 7 it can be seen that restructuring ketone 2-KLG bar of the present invention
Bacterium shortens fermentation period 5h than original strain, decreases the cost added required for allogenic material, promotes
The growth of Ketogulonigenium sp and produce acid
The above is only the preferred embodiment of the present invention, it is noted that general for the art
For logical technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvement and profit
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (18)
1. one group of DNA molecular, it is characterised in that including at least following a kind of nucleotide sequence:
Nucleotide sequence shown in SEQ ID NO:1；
Nucleotide sequence shown in SEQ ID NO:2.
DNA molecular the most according to claim 1, it is characterised in that comprise SEQ ID NO:1-2
Shown nucleotide sequence.
DNA molecular the most according to claim 2, it is characterised in that by SEQ ID NO:1-2 institute
Show that nucleotide sequence forms.
4. DNA molecular described in claim 1-3 any one is producing 2-keto-L-gulonic acid, Yi Ji
Preparation application in the recombinant vector producing 2-keto-L-gulonic acid and recombinant bacterial strain.
5. a recombinant vector, it is characterised in that comprise DNA described in claim 1-3 any one
Molecule.
Recombinant vector the most according to claim 5, it is characterised in that core shown in SEQ ID NO:1
Nucleotide sequence upstream shown in nucleotide sequence or SEQ ID NO:2 is promoter, and downstream is terminator.
Recombinant vector the most according to claim 6, it is characterised in that described promoter is that 16S starts
Son.
Recombinant vector the most according to claim 6, it is characterised in that described terminator is that 5S terminates
Son.
Recombinant vector the most according to claim 5, it is characterised in that described carrier is PBBR1MCS2
Plasmid vector.
10. recombinant vector described in claim 5-9 any one is producing 2-keto-L-gulonic acid, Yi Ji
Preparation application in the recombinant bacterial strain producing 2-keto-L-gulonic acid.
11. 1 kinds of restructuring Ketogulonigenium sps, it is characterised in that convert requirement 5-9 any one of having the right
Described recombinant vector.
Restructuring Ketogulonigenium sp application in producing 2-keto-L-gulonic acid described in 12. claim 11.
The method that 13. 1 kinds of shake flask fermentations produce 2-keto-L-gulonic acid, it is characterised in that by claim
Ketogulonigenium sp of recombinating described in 11 joins in solid seed culture medium and activates, and is then transferred to seed training
Support in base and carry out shake flask fermentation.
14. according to method described in claim 13, it is characterised in that described solid seed culture medium is for adding
Entering the seed culture medium after agar, described seed culture medium consists of:
Carnis Bovis seu Bubali cream 3.0g/L, Semen Maydis pulp 6.0g/L, yeast leaching powder 3.0g/L, peptone 10.0g/L, nothing
Water magnesium sulfate 0.2g/L, carbamide 1.0g/L, dipotassium hydrogen phosphate 1.0g/L, calcium carbonate 1.0g/L, separately take mountain
Pears sugar 20g/L, pH=6.8.
The method that 15. 1 kinds of ferment tanks produce 2-keto-L-gulonic acid, it is characterised in that right is wanted
Ask Ketogulonigenium sp of recombinating described in 11 to join and fermentation medium carries out ferment tank, during fermentation
Add sorbose.
16. according to method described in claim 15, it is characterised in that described fermentation medium is:
Semen Maydis pulp 10g/L, carbamide 12g/L, potassium dihydrogen phosphate 1g/L, magnesium sulfate 0.2g/L, calcium carbonate 1g/L,
Sorbose 80g/L, defoamer 1ml/3L, pH value is 6.7-7.0.
17. according to method described in claim 15, it is characterised in that add sorbose during described fermentation
The sorbose of 80g/L is mended for the 24h-30h that ferments.
18. according to method described in claim 15, it is characterised in that the condition of described ferment tank is:
30 DEG C, 500r/min, pH control 7.0, and ventilate 1.5vvm.
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| CN109810975A (en) * | 2017-11-22 | 2019-05-28 | 沈阳药科大学 | A method of the production 2-keto-L-gulonic acid based on metabolic pathway transformation |
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| CN109810975A (en) * | 2017-11-22 | 2019-05-28 | 沈阳药科大学 | A method of the production 2-keto-L-gulonic acid based on metabolic pathway transformation |
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