CN106047913A - Construction method of dual-enzyme co-expression strains for producing [alpha]-ketoglutarate - Google Patents
Construction method of dual-enzyme co-expression strains for producing [alpha]-ketoglutarate Download PDFInfo
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Abstract
The invention discloses a construction method of dual-enzyme co-expression strains for producing [alpha]-ketoglutarate, and belongs to the technical fields of fermentation engineering and enzyme engineering. According to the construction method, on the basis of analyzing the production of the [alpha]-ketoglutarate from L-glutamic acid, a KatG dosage required by a transformation system is analyzed; and then, the co-enzyme co-expression strains, which are different in expression level, are constructed at a transcription level and a translation level, and the performances of the co-expression strains are evaluated by the production of the [alpha]-ketoglutarate through whole-cell transformation of L-glutamic acid at a shaker level, wherein transformation conditions are as follows: 110g/L of the L-glutamic acid, 2-2.5g/L of bacteria, a phosphate buffer system with pH value at 6.5, 30 DEG C, and 18-24h at 200rpm. The yield of the [alpha]-ketoglutarate from the optimum strain F006 can reach 107.2g/L or above, with a transformation rate of 98.4%, completely replacing exogenous catalase; bacterium concentration is increased, the yield of the [alpha]-ketoglutarate under the condition of 132g/L of a substrate can reach 127.1g/L and a transformation rate is 96.9%.
Description
Technical field
The present invention relates to the construction method of a kind of double enzyme coexpression bacterial strains producing α-ketoglutaric acid, belong to fermentation engineering and
Technical field of enzyme engineering.
Background technology
α-ketoglutaric acid (α-ketoglutarate is called for short α-KG) is as a kind of important high level fine chemicals (20-
250000 yuan/ton), it is widely used in the industrial circles such as food, medicine, chemical industry and cosmetics.In field of medicaments, α-KG can subtract
Fast quick-recovery after the kidney burden of light nephrotic, minimizing complication and promotion corrective surgery；Compound with the aminoacid such as arginine,
Can quickly help athlete to supplement energy, be widely used in functional nutrient hardening agent；In addition, due to change that α-KG is special
Learn character, be widely used in chemosynthesis industry.Along with the continuous expansion of α-KG application, cause domestic and international market pair
The demand of α-KG is continuously increased.The data recognized from customs, current international market reaches 50,000 to food stage α-KG demand breach
More than Dun, and the food stage α-KG market price is 20-25 ten thousand yuan/ton at present, and faces the awkward situation having price but no buyers.The production of α-KG
Method includes: chemical synthesis, enzyme catalysis method and microbe fermentation method.At present, industrially producing alpha-KG mainly uses organic conjunction
Cheng Fa, relates to the chemical reaction process of series of complex, thus causes the aspect series of problems such as raw material sources, environmental pollution.
Simultaneously because there is serious safety problem during chemical method synthesis α-KG, α-KG is caused to be difficult to be directly used in food, medicine
With fields such as cosmetics.Therefore, how to use biotechnology method to prepare the α-KG that safety is high on a large scale, be domestic and international academia
With industrial circle institute focus of attention problem.
This research department's early stage constructs L-GLOD efficient expression strain E.coli FMME089, and by highly dense
Degree fermentation means achieve the extensive preparation of L-GLOD, make enzymatic conversion method Pidolidone produce the industrialization of α-KG
Production is possibly realized.But need to add catalase during enzymatic conversion method, in industrial catalase, impurity is relatively
The isolated and purified of multipair α-KG product later stage causes puzzlement and external source interpolation catalase to cause relatively costly.
Summary of the invention
In order to solve the problems referred to above, reduce cost, it is contemplated that on the basis of original Pidolidone produces α-KG system
Realize converting without the high efficiency under the conditions of catalase, build qualified L-GLOD and hydrogen peroxide
Enzyme coexpression bacterial strain.
First purpose of the present invention is to provide the construction method of a kind of double enzyme coexpression bacterial strains producing α-ketoglutaric acid,
Described method prediction on the basis of resting cell Pidolidone produces α-ketoglutaric acid transformation system required Pidolidone oxidation
Enzyme and catalatic consumption, and then built from the double expression of enzymes of transcription and translation horizontal adjustment by molecular biology method
L-GLOD and catalatic coexpression bacterial strain, it is achieved the requirement of two kinds of enzyme variable expressions, to reach this turn
Under change system, α-ketoglutaric acid efficiently produces, during without catalatic extra interpolation.
Described method, determines double enzyme demand, by double enzymes building on the basis of single expression of enzymes bacterial strain measures zymologic property
The methods such as different building modes, interval between SD sequence and start codon ATG and RBS sequence strength optimization are done step-by-step
Two kinds of various required expression requirements of enzyme, are verified coexpression bacterial strain performance by conversion reaction simultaneously.
It is with L-GLOD recombinant bacterium that described resting cell Pidolidone produces α-ketoglutaric acid transformation system
Strain E.coli FMME089 carries out resting cell.
In one embodiment of the invention, described transformation system refers to Pidolidone or sodium glutamate as substrate
The method producing α-KG, at pH 6.0-8.0, temperature 30-42 DEG C, converts 18-24h, catalytic production α-ketoglutaric acid.
In one embodiment of the invention, described resting cell Pidolidone produces α-ketoglutaric acid transformation system
And conversion condition is: 110g/L Pidolidone, 2～2.5g/L thalline, pH 6.5 phosphoric acid buffer liquid system, 30～42 DEG C,
24h is converted under the conditions of 200rpm.
In one embodiment of the invention, described concentration of substrate is 110-135g/L.
In one embodiment of the invention, described recombination bacillus coli or coexpression bacterial strain are with E.coli BL21
(DE3) be Host Strains, with pET28a expression vector；L-GLOD derives from
StreptomycesghanaensisATCC14672, catalase derives from E.coli K12.
In one embodiment of the invention, described L-GLOD and catalatic character are by dividing
Do not build efficient expression strain FXC001 and FXC007 of enzyme, abduction delivering, broken, centrifugal, filtration, hanging column, eluting and desalination
Record etc. after process.
In one embodiment of the invention, described catalatic consumption is by directly adding in transformation system
Catalase after purification directly records.
In one embodiment of the invention, the described minimum 1000U/mL of catalatic demand.
Described molecular biology method refers to by gene clone technology, round pcr, amalgamation and expression technology, RBS intensity pre-
Survey technology, plasmid construction techniques etc., relate to some molecule manipulations of biology field.
The double expression of enzymes of described transcriptional level control refers to by double enzyme difference building modes enzyme duplex table as double in single promoter
Reach, the double enzyme expressing in series of double-promoter, single double enzyme amalgamation and expressions of promoter etc. build co-expression plasmid, wherein Pidolidone oxygen
Before change enzyme is respectively positioned on catalase, the three strain coexpression bacterial strains built are respectively designated as F001, F002 and F003.
The double expression of enzymes of described translation skill regulation and control refer to by change the interval between SD sequence and start codon or
RBS sequence intensity adjustments ribosome and the bond strength of mRNA, and then affect translation initiation speed, translation skill regulates egg
White matter expression.Interval optimization between described SD sequence and start codon ATG refers to by optimizing catalase gene
Interval between SD sequence and the start codon of front end regulates and controls catalatic variable expression, interval be set in 3bp～
12bp, constructs four strain coexpression bacterial strain named FXC003～FXC006 respectively.Described RBS sequence strength optimization refers to lead to
Cross RBS Calculator v1.1 (https://www.denovodna.com/software/) prediction reach certain expression
Required catalase gene front end RBS intensity and translation initiation speed TIF ＞ 24,000au, thus design 4 groups and conform to
The RBS sequence asked, builds double enzyme expressing in series plasmid, before L-GLOD is positioned at catalase, successfully constructs
Four strain coexpression bacterial strains are respectively designated as F004～F007.
The performance evaluation of the most all recombinant bacteriums all induction mensuration product enzyme effects under identical conditions, followed by
Thalline or enzyme system convert Pidolidone and produce α-KG.
In one embodiment of the invention, the derivant of described induction be added to OD600 between 0.6～1.2 time,
Add 0.3～0.5mmol/L IPTG or 3～7g/L lactose-induced.
In one embodiment of the invention, the inducing temperature of described induction is 25-30 DEG C.
In one embodiment of the invention, the induction time of described induction is 4-8h.
In described method, for fermentation medium, those skilled in the art can train according to existing escherichia coli completely
Support base, select the culture medium being suitable for producing enzyme or further culture medium be optimized.
In described method, for transformation system, those skilled in the art can select suitably according to existing conversion completely
It is similar to buffer solution system and conversion condition or adds cofactor further.
Second object of the present invention is to provide a kind of coexpression L-GLOD and catalatic DNA sheet
Section, described DNA fragmentation is by the gene sequence encoding the gene order of L-GLOD, catenation sequence, encoding catalase
Row are connected in sequence.
The aminoacid sequence of described L-GLOD as shown in SEQ ID NO.1, nucleotide sequence such as SEQ ID
Shown in NO.2.
Described catalatic aminoacid sequence as shown in SEQ ID NO.3, nucleotide sequence such as SEQ ID NO.4 institute
Show.
The nucleotide sequence of described catenation sequence is as shown in SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7.
Third object of the present invention is to provide recombinant vector or the weight coexpression bacterial strain expressing described DNA fragmentation.
Described recombinant vector, is connected to described DNA fragmentation on pET28a expression vector obtain.
Described coexpression bacterial strain, is that described recombinant vector is transformed into the coexpression bacterial strain obtained in host e. coli.
Fourth object of the present invention is to provide the enzyme system that described DNA fragmentation obtains after expressing.
5th purpose of the present invention is to provide described enzyme system or coexpression bacterial strain and is converting production α-ketoglutaric acid side
The application in face.
In one embodiment of the invention, described application, is with Pidolidone or sodium glutamate as substrate, at pH
6.0-8.0, at temperature 30-42 DEG C, converts 18-24h, utilizes described enzyme system or coexpression bacterial strain catalytic production α-one penta 2
Acid.
In one embodiment of the invention, described application needs to add the MnCl of 1～5mmol/L2As two kinds of enzymes
Activator.
In one embodiment of the invention, the concentration of described substrate is 110-135g/L.
Beneficial effects of the present invention:
1, application the inventive method successively obtains 11 strain L-GLODs and catalase coexpression bacterial strain, it is achieved
The high efficient expression of the different expressions of two kinds of enzymes；
2, the application present invention rationally achieves target enzyme specific expression scope；
3, part coexpression bacterial strain is additionally adding Mn2+Under the conditions of, it is also possible to realize under non-catalase adding conditional
The efficient production of α-ketoglutaric acid；
4, the somatic cells that coexpression bacterial strain F006 fermentation obtains is used directly for converting and produces α-KG, substrate conversion efficiency
Up to 98.7%, both non-catalase added without adding Mn2+；
5, at 110g/L Pidolidone, 2～2.5g/L thalline, pH 6.5 phosphoric acid buffer liquid system, 30 DEG C, 200rpm bar
Converting 18～24h under part, the α-ketoglutaric acid yield of optimum bacterial strain F006 can reach 107.2g/L, and conversion ratio is 98.4%,
Substitute external source completely and add catalase and without adding Mn2+；Increase cell concentration, α-one penta 2 under the conditions of 132g/L substrate
The yield of acid can reach 127.1g/L, conversion ratio 96.9%.
Accompanying drawing explanation
Fig. 1: restructuring LGOX purification and SDS-PAGE analyze (1 is fermentation liquid, and 2 is recombinant bacterium, and 3 is LGOX after Ni column purification,
4 is LGOX after desalination)；
Fig. 2: restructuring KatG purification and SDS-PAGE analyze that (1 is BL21 empty plasmid bacterium, and 2 is recombinant bacterium, and 3 is egg after purification
In vain)；
The temperature stability of LGOX at Fig. 3: 30 DEG C；
Fig. 4: Lineweaver-Burk double reciprocal curve；
The impact on converting of Fig. 5: the KatG addition；
Fig. 6: the structure of different interval co-expression plasmid；
Fig. 7: different RBS intensity coexpression strain protein electrophoretograms；
The impact on converting of Fig. 8: the whole-cell catalyst.
Detailed description of the invention
The research of embodiment 1L-glucose oxidation enzymatic property and catalase demand determine
L-GLOD (LGOX) gene of StreptomycesghanaensisATCC14672 will be derived from respectively
(, as shown in SEQ ID NO.1, nucleotide sequence is as shown in SEQ ID NO.2 for aminoacid sequence), derives from E.coli K12's
Catalase gene KatG (, as shown in SEQ ID NO.3, nucleotide sequence is as shown in SEQ ID NO.4 for aminoacid sequence),
By primer 1 (forward primer: 5 '-CATGCCATGGCAATGCTGCCCGCACCGGCCGCCT-3 ', sequence such as SEQ ID
NO.8；Downstream primer: 5 '-CCCAAGCTTGTCACGCTGTGTGGATCTCCAAG-3 ', sequence such as SEQ ID NO.9) and draw
Thing 2 (forward primer: 5 '-CATGCCATGGCAATGAGCACGTCAGACGAT-3 ', sequence such as SEQ ID NO.10；Downstream is drawn
Thing: 5 '-CCCAAGCTTGCAGCAGGTCGAAACGGTC-3 ', sequence such as SEQ ID NO.11) clonal expression is in plasmid pET28a
In, recombiant plasmid proceeds to, in E.coli BL21 (DE3), filter out positive strain and be respectively designated as FXC001 and FXC007.Restructuring
Bacterial strain is abduction delivering in TB culture medium, works as OD60030 DEG C of inductions are carried out for adding the IPTG of 0.4mmol/L time between 0.6-1.2
5～6h, obtain two kinds of enzyme (Fig. 1 after purification respectively after then passing through broken, centrifugal, filtration, hanging column, eluting and desalting processing
And Fig. 2), albumen size is respectively 65kDa and 80kDa.Finally, two kinds of enzymes are carried out character research.
1) under the conditions of measuring 30 DEG C, the activity of LGOX is worth over time, and matching inactivation equation curve (Fig. 3) calculates
The half-life of this enzyme is 4.62h；Subsequently its kinetic parameter is determined, obtains Lineweaver-Burk shown in Fig. 4 double
Reciprocal curve, calculates the binding constant K of LGOXmFor 6.32mmol L-1, VmaxIt is 40 μm ol min-1·mg-1, corresponding
kcatFor 1.23min-1。
2) at transformation system, (pH 6.5 phosphoric acid delays for 110g/L Pidolidone, 2～2.5g/LE.coli FMME089 thalline
Rush liquid) on the basis of, by the yield of α-KG in interpolation 0～2000U/mL KatG mensuration 24h after purification, result is shown in Fig. 5, roughly
Estimate that this transformation system needs such catalatic consumption to be 1250U/mL or above (now conversion ratio is more than 95%) altogether,
Determine the expression of this gene required in coexpression bacterial strain.
2 pairs of enzyme coexpression bacterial strain transcriptional level controls of embodiment
Setting three kinds of Different Strategies with pET28a for expression vector and build coexpression bacterial strain, strategy 1 uses single startup mould
Formula, by LGOX and KatG expressing in series, is connected KatG by the RBS sequence identical with before LGOX, the coexpression bacterial strain successfully constructed
Named F001；Strategy 2 uses double-promoter pattern promoter as adding before LGOX and KatG and correlated series, structure
Building coexpression Strain Designation is F002；Strategy 3 uses single promoter pattern directly to be connected by Hind III by LGOX and KatG
Together, the coexpression Strain Designation successfully constructed is F003.Wherein, strategy 1 can transcribe one mRNA of production, containing two cores
Thuja acid binding site, translates two albumen；Strategy 2 can transcribe out small one and large one two mRNA, all contains nucleotide bound site
Point, translates two albumen；Strategy 3 can transcribe out a mRNA, contains only a nucleotide binding site, translates one and melt
Hop protein.Wherein, the KatG expressing quantity of strategy 2 is due to strategy 1, it would be desirable to obtain the bacterial strain of different KatG expression；Plan
The peptide chain number that summary 3 can realize LGOX with KatG is consistent, it is desirable to one merges enzyme and can complete target response, the work of two kinds of enzymes
Property relevant with foldable structure be difficult to judge.
Recombinant bacterium is inoculated on LB seed culture medium after 10-12h, is transferred in TB culture medium with the inoculum concentration of 4%, when
OD60030 DEG C of inductions 5～6h are carried out for adding the IPTG of 0.4mmol/L time between 0.6-1.2；Centrifugal acquisition coexpression bacterium subsequently
Body, reacts 24h under the conversion conditions such as 110g/L Pidolidone, 2～2.5g/L thalline；The product enzyme effect of 3 strain coexpression bacterial strains
It is organized into form 1 with changing effect, the F002 > F001 > F003 it appeared that KatG enzyme is lived, LGOX activity F003 > F001 > F002,
α-KG yield F003 > F001 > F002, and α-KG yield F001 is more or less the same with F003.Therefore, it can be seen that identical promoters string
Connection expression is unfavorable for double expression of enzymes, and large fragment gene fusion expression operability is poor and cannot determine double enzymatic activity, single startup
The double enzyme expressing in series of son can be workable by the expression of RBS controlling gene, and can preferably preserve double enzymatic activity, is next
The basis that step optimizes.
The different building mode cell concentration of table 1, product enzyme and changing effect compare
Embodiment 3SD sequence (Shine-Dalgarno sequence) and the impact at ATG interval
According to Fig. 6 plasmid construction mode construction recombination plasmid (wherein RBS* represents the sequence at different SD and ATG interval), if
Fixed interval is respectively 3bp, 6bp, 9bp and 12bp, forward primer be respectively KatG-rbs1 (sequence such as SEQ ID NO.12),
KatG-rbs2 (sequence such as SEQ ID NO.13), (sequence is such as KatG-rbs3 (sequence such as SEQ ID NO.14) and KatG-rbs4
SEQ ID NO.15), downstream primer is KatG-A (sequence such as SEQ ID NO.16) (table 2), by traditional building mode structure
Build and verify recombinant bacterial strain, the bacterial strain built and be proved to be successful is respectively designated as FXC003, FXC004, FXC005 (i.e. L-paddy
Amino acid oxidase and catalase are attached by the catenation sequence of SEQ ID NO.5), FXC006.
Table 2 primer used in the present invention
Four strain recombinant bacterial strains are cultivated in TB culture medium OD600To 0.6～1.2, add 0.4mmol L-1IPTG induces
5～6h, measure now cell concentration, LGOX activity and KatG activity, and collect cell and carry out protein electrophoresis and resting cell
Produce α-KG (110g L-1Pidolidone, full cell, pH 6.5 phosphate-buffered liquid system, 200r min-1, 30 DEG C
Convert 24h).The cell concentration OD of different recombinant bacterial strains600, LGOX enzyme live, KatG activity and resting cell α-KG yield be listed in
Table 3, it can be seen that LGOX enzyme FXC003 > FXC005 > FXC006 > FXC004 alive, and FXC004, FXC005, FXC006 difference is not
Greatly, and KatG activity FXC005 > FXC004 > FXC006 > FXC003, wherein FXC003 is only 56U mL far below other three strain-1.Wherein the KatG activity of FXC005 bacterial strain is closest to testing predictive value 1250U mL-1, α-KG yield is up to 86.7g L-1, conversion ratio is 79.6%.Although be enhanced still without meet substitute catalatic purpose completely, need into
One-step optimization.
Table 3 different interval coexpression strain cell concentration, product enzyme and changing effect compare
The impact on producing enzyme of embodiment 4 ribosome binding site (RBS sequence) intensity
In FXC003 (RBS sequence, i.e. two enzyme catenation sequences, for rbs1), LGOX is with Nco I and HindIII as enzyme action
Site is connected to pET28a, corresponding Δ GtotFor 4.21kcal mol-1, TIR is 375.4au；KatG is with HindIII and Xhol
I is inserted into pET28a-LGOX, corresponding Δ Gtot=1.85kcal mol-1, TIR=1087.31au；KatG activity is again
56U·mL-1, about (required KatG activity is 1250U mL to the 4.5% of demand-1), therefore the TIF of required RBS exists
24433.9au be more than advisable.
Separately design 4 groups of rbs sequences, be respectively designated as rbs5 (sequence such as SEQ ID NO.17), rbs6 (sequence such as SEQ
ID NO.18), rbs7 (sequence such as SEQ ID NO.19) and rbs8 (sequence such as SEQ ID NO.20), its sequence is shown in Table 4, its
TIR is all at more than 24,000au.And separately design forward primer KatG-rbs5 (sequence such as SEQ ID NO.21), KatG-rbs6
(sequence such as SEQ ID NO.22), KatG-rbs7 (sequence such as SEQ ID NO.23) and KatG-rbs8 (sequence such as SEQ ID
NO.24), downstream primer is KatG-A (table 2), is built by traditional building mode and verifies recombinant bacterial strain, just will construct
True positive strain is also named F004, F005 respectively (i.e. L-GLOD and catalase is by SEQ ID NO.6's
Catenation sequence is attached), F006 (i.e. L-GLOD and the catalase catenation sequence by SEQ ID NO.7
Be attached), F007.
The different RBS of table 4 and feature thereof
The coexpression bacterial strain being proved to be successful is cultivated D in TB culture medium600To 0.6～1.2, add 0.4mmol L- 1IPTG induction 5～6h, collects cell and carries out protein electrophoresis.Result as it is shown in fig. 7, with compare (E.coli BL21 and FXC001)
Comparing, coexpression bacterial strain production LGOX albumen size is about 65kDa, KatG albumen size and is about 80kDa, and two kinds of albumen have table
Reach.Wherein the KatG band of F004 and F005 is apparently higher than LGOX, and in F006, KatG and LGOX is more or less the same, LGOX in F007
Band is significantly more than KatG.The band brightness F004 > F005 > F006 > F007 of overall KatG, meets TIR prediction.
Carrying out producing enzyme Characterization result to F004, F005, F006 and F007 with reference to embodiment 3, it the results are shown in Table 5.Find
LGOX enzyme F007 > F006 > F004 > F005 alive, KatG activity F005 > F006 > F004 > F007, and the KatG of F005 and F006 is alive
Property is close to predictive value 1250U mL-1, find that KatG expressing quantity is disproportionate with KatG activity in conjunction with SDS-PAGE collection of illustrative plates,
And TIF prediction has certain deviation (F006 and F007).Four strain bacterium are carried out resting cell and produces α-KG (110g L-1L-paddy
Propylhomoserin, full cell, pH 6.5 phosphate-buffered liquid system, 200r min-1, 30 DEG C convert 24h), find α-KG yield
F006 > F005 > F004 > F007, KatG activity is more significant to changing effect, and wherein the changing effect of F006 is best, α-KG yield
Reach 103.1g L-1, conversion ratio reaches 94.6%, preferably achieves double enzymatic conversion effect.
Table 5 recombinant bacterium produces enzyme effect
Embodiment 5 coexpression bacterial strain F006 transformation system optimizes
With 132g L-1Pidolidone is substrate, at 1mmol L-1Mn2+Under the conditions of, the full cell adding different proportion is urged
Agent is tested, and as 1 times of whole-cell catalyst, (cell concentration exists the cell wherein obtained using same volume fermentation liquid
Between 2.5g/L～3.5g/L).Add respectively 1 times, 1.5 times and 2 times of whole-cell catalysts carry out transformation experiment, 30 DEG C,
200r·min-1Under the conditions of 24h changing effect see Fig. 8, when 1.5 times, α-KG yield is 121.8g L-1, conversion ratio is 92.9%,
When 2 times, α-KG yield is 127.1g L-1, now conversion ratio is 96.9%, α-KG output increased 23.2%.
The application of embodiment 6 enzyme
Obtain thalline by centrifugal for above-mentioned all coexpression bacterial strain fermentation liquors, be used for converting Pidolidone and produce α-KG.
At the Pidolidone that substrate is 110～135g/L, use the phosphate buffer of pH 7.0, add variable concentrations
Mn2+(1mM～5mM), 30 DEG C of yield converting 18～24h mensuration α-KG.Find that FXC005, F005 and F006 all can reach to turn
Rate is more than 95%, it is achieved efficiently produce without the α-KG under the conditions of catalase.
May determine that according to said method successfully construct satisfactory coexpression bacterial strain FXC005, F005 and
F006, most having bacterial strain is F006.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any is familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be with being as the criterion that claims are defined.
Claims (10)
1. the construction method of the double enzyme coexpression bacterial strains producing α-ketoglutaric acid, it is characterised in that described method is complete thin
Dysuria with lower abdominal colic Pidolidone produces the required L-GLOD of prediction and catalase on the basis of α-ketoglutaric acid transformation system
Consumption, and then build L-GLOD by molecular biology method from the double expression of enzymes of transcription and translation horizontal adjustment
With catalatic coexpression bacterial strain, it is achieved the requirement of two kinds of enzyme variable expressions, to reach α-one penta under this transformation system
Diacid efficiently produces, during without catalatic extra interpolation；Described method is building single expression of enzymes bacterial strain mensuration enzyme
Learn and determine double enzyme demand on the basis of character, by double enzyme difference building modes, between SD sequence and start codon ATG between
Every and/or the method for RBS sequence strength optimization two kind enzymes various required expression requirements are done step-by-step, pass through conversion reaction simultaneously
Coexpression bacterial strain performance is verified.
Method the most according to claim 1, it is characterised in that described method builds L-paddy ammonia respectively by pET28a plasmid
Acid oxidase and catalase efficient expression strain, Host Strains is E.coli BL21 (DE3), in research Pidolidone oxidation
On the basis of enzyme and catalatic zymologic property, determined altogether by the catalase added in transformation system after purification
Required catalatic expression in expression strain.
Method the most according to claim 1, it is characterised in that described resting cell Pidolidone produces α-ketoglutaric acid
Transformation system and conversion condition be: 110g/L Pidolidone, 2～2.5g/L thalline, pH 6.5 phosphoric acid buffer liquid system, 30～
42 DEG C, under the conditions of 200rpm, convert 24h.
Method the most according to claim 1, it is characterised in that the double expression of enzymes of the transcriptional level control of described method refers to lead to
Cross enzyme duplex as double in the single promoter expression of double enzyme difference building mode, the double enzyme expressing in series of double-promoter or the double enzyme of single promoter
Amalgamation and expressions etc. build co-expression plasmid, and expression plasmid is pET28a, and Host Strains is E.coli BL21 (DE3).
Method the most according to claim 1, it is characterised in that the interval between described SD sequence and start codon ATG
Optimize and refer to that the interval regulation and control by optimizing between SD sequence and catalase gene start codon are catalatic not
Same expression, interval is set in 3bp～12bp, and before L-GLOD is connected in series to catalase, expression plasmid is
PET28a, Host Strains is E.coli BL21 (DE3).
Method the most according to claim 1, it is characterised in that described RBS sequence strength optimization refers in claim 5
On the basis of the coexpression bacterial strain constructed by the optimization of interval between SD sequence and start codon ATG, calculate RBS intensity and turn over
Translate the RBS intensity needed for initial rate TIF and then prediction catalase gene front end, thus build satisfactory RBS sequence
Row, build double enzyme expressing in series plasmid, and before L-GLOD is connected in series to catalase, expression plasmid is pET28a,
Host Strains is E.coli BL21 (DE3).
7. a coexpression L-GLOD and catalatic DNA fragmentation, it is characterised in that described DNA fragmentation by
The coding gene order of L-GLOD, catenation sequence, the gene order of encoding catalase are connected in sequence；Institute
State the nucleotide sequence of catenation sequence as shown in SEQ ID NO.6.
8. contain recombinant vector or the coexpression bacterial strain of DNA fragmentation described in claim 1.
9. the enzyme system that the DNA fragmentation described in claim 7 obtains after expressing.
10. the enzyme system described in claim 9 or the coexpression bacterial strain described in claim 8 are converting production α-ketoglutaric acid side
The application in face.
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| CN106399216A (en) * | 2016-11-16 | 2017-02-15 | 江南大学 | Single-cell plant for efficiently synthesizing alpha-aminobutyric acid, as well as construction and application thereof |
| CN107365782A (en) * | 2017-08-29 | 2017-11-21 | 廊坊梅花生物技术开发有限公司 | A kind of genetic engineering bacterium and its application |
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| CN109679979A (en) * | 2019-01-11 | 2019-04-26 | 四川吉晟生物医药有限公司 | The production method of the recombinant vector of expression L-GLOD and catalase, engineering bacteria and application and α-ketoglutaric acid |
| CN109679979B (en) * | 2019-01-11 | 2023-03-17 | 四川吉晟生物医药有限公司 | Recombinant vector for expressing L-glutamate oxidase and catalase, engineering bacteria and application thereof, and production method of alpha-ketoglutaric acid |
| CN112941116A (en) * | 2021-01-04 | 2021-06-11 | 上海朴颐化学科技有限公司 | Method for preparing alpha-ketoglutarate calcium by enzyme method |
| CN112625993A (en) * | 2021-01-05 | 2021-04-09 | 雅本化学股份有限公司 | Preparation of alpha-ketoglutaric acid by microbial conversion method |
| CN112625993B (en) * | 2021-01-05 | 2023-01-20 | 雅本化学股份有限公司 | Preparation of alpha-ketoglutaric acid by microbial conversion method |
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