CN107916246A - The method that DHDPR improves lysine production in genetic modification Corynebacterium glutamicum - Google Patents
The method that DHDPR improves lysine production in genetic modification Corynebacterium glutamicum Download PDFInfo
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Abstract
The present invention is that the method for DHDPR (dihydrodipicolinate reductase) raisings L lysine productions in genetic modification Corynebacterium glutamicum belongs to genetic engineering and enzyme engineering field.The present invention applies gene engineering method, DHDPR encoding genes dapB in rite-directed mutagenesis Corynebacterium glutamicum JL 6, change DHDPR protein structures, so as to adjust its affinity to different redox cofactors, release in L lysine building-up processes to NADP (H/+) demand deficiency the shortcomings that, improve strain for accumulating L lysine abilities.Recombinant bacterium reaches 17.6g/L through shake flask fermentation, L lysine accumulations amount.The invention successful change of the redox cofactors affinity of DHDPR in Corynebacterium glutamicum, alleviate NADP (H/ in L lysine building-up processes+) demand deficiency, provide brand-new thinking for selection and breeding L lysine high-yielding strains.
Description
Technical field
The invention belongs to genetic engineering and enzyme engineering field, and in particular to DHDPR is carried in genetic modification Corynebacterium glutamicum
The method of high-lysine yield.
Technical background
L-lysine be human and animal necessary to, the amino acid that itself cannot synthesize, belong to eight big essential amino acids
One of.Since L-lysine has different physiological roles, such as amino acid composition, adjust internal metabolic balance, improve body
Absorption and utilization rate and promotion body growth development to grain protein etc., thus it is widely used in feed industry, medicine
In industry and food industry.The method of industrial production L-lysine mainly has three kinds：Albumen hydrolysis, chemical synthesis and micro-
Biological fermentation process, wherein microbe fermentation method have production cost is low, production intensity is high, high specific and environmental pollution are small etc.
Advantage and become the current most widely used method of industrial production L-lysine.Therefore, selection and breeding have the height of independent intellectual property right
L-lysine synthesis, the production bacterial strain of low byproducts build-up, the L- of high-purity low color level of the exploitation with independent intellectual property right rely
Propylhomoserin production new technology, new process, to widening products application scope and profit margin, it is important to realize that Sustainable Development of Enterprises has
Meaning.
Microorganism for producing L-lysine includes more kinds, wherein both at home and abroad for industrialized production L-lysine
Bacterial strain is mostly Corynebacterium glutamicum (Corynebacterium glutamicum) and its subspecies and Escherichia coli
The reworked bacterial strain of (Escherichia coli).L-lysine biosynthesis is synthesized different from Pidolidone,
1mol L-lysines are synthesized in C.glutamicum biosynthesis pathways to be needed to consume 4mol NADPH, and forms 1mol L- paddy
Propylhomoserin only needs 1mol NADPH.Therefore, in order to improve the accumulation of L-lysine in C.glutamicum biosynthesis pathways, carry
In high C.glutamicum metabolic pathways NADPH amounts or reduce L-lysine route of synthesis in NADPH demands be extremely important
Strategy.
Dihydrodipicolinate reductase (Dihydrodipicolinate reductase, DHDPR) is bacterium and high
Second key enzyme during plant biological synthesizing diamino pimelic acid and L-lysine, is catalyzed the NAD of dihydrodipicolinic acid
(P) the reproducibility reaction that H- is relied on, generates hexahydropyridine dicarboxylic acids.The enzyme plays the role of key in cell membrane is formed.Have
The DHDPR of activity exists generally in the form of tetrad, and single molecular weight subunit size is about 30kDa, and each subunit is by two
Region forms, i.e. C- ends substrate (i.e. dihydrodipicolinic acid) or repressor binding domain and N- ends dinucleotides binding domain is (i.e. auxiliary
Factor binding domain), two functional domains are connected by a variable ring.N- ends dinucleotides binding domain is by flat positioned at 7 of center
Row β-pleated sheet and 4 spiral compositions of α positioned at periphery, co-factor binding site is located at the C- end margins of β-pleated sheet, and C- ends substrate
Or repressor binding domain is by 4 β-pleated sheets and 2 α are spiral forms.DHDPR is not only with NADH but also using NADPH as confactor, no
It is also different to the affinity of different co-factors with the DHDPR of bacterium, as E.coli more favors NADH, and in C.glutamicum
DHDPR mainly participates in the synthesis of L-lysine using NADPH as co-factor.DHDPR mono- in the organism being had found with other
Sample, DHDPR is encoded by gene dapB in C.glutamicum, its enzyme activity from end-product in route of synthesis adjustment effect, but
Suppressed by 2,6- pyridinedicarboxylic acids (2,6-PDC).Previous karyotype studies show that changing NADPH- dependent form enzymes by fixed point can
To change its affinity to different co-factors.Therefore, by DHDPR encoding genes in rite-directed mutagenesis C.glutamicum, change
Become affinity of the DHDPR to NADPH, so as to increase L-lysine yield.
This laboratory is by classic mutagenesis method, the one plant of production L-lysine mutant strain Corynebacterium glutamicum screened
JL-6 (C.glutamicum JL-6), the bacterium is through shake flask fermentation production L-lysine 14.5g/L.However, to the Strains L-lysine
Found when intracellular redox cofactors measure in fermentation process, intracellular NADPH contents are still insufficient, so that limiting L- relies ammonia
The further increase of acid.In addition, DHDPR encoding genes dapB is also generation missense mutation in the bacterial strain.
Based on research department's early-stage study basis, present invention analysis first compares DHDPR protein structures in different microorganisms,
It has found the potential amino acid residue for adjusting DHDPR and combining different co-factors：11st Lys residue and the 13rd Arg residue.
Using gene engineering method, rite-directed mutagenesis DHDPR encoding gene dapB, make DHDPR amino acid sequences the 11st Lys residue and
13rd Arg residue mutations obtain recombinant bacterial strain JL-6dapB into Ala residuesA31G,A32C(i.e. Lys11Ala mutant strains, K11A)
And JL-6dapBC37G,G38C(i.e. Arg13Ala mutant strains, R13A).These recombinant bacterial strains improve affinity of the DHDPR to NADH
And the affinity to NADPH is reduced, NADPH is supplied during relieving C.glutamicum JL-6 fermentation production of L-lysine
Should be insufficient the shortcomings that, improve the ability of strain for accumulating L-lysine.
The content of the invention
It is an object of the invention to：Rite-directed mutagenesis C.glutamicum JL-6L- lysine synthetic pathway key enzymes DHDPR
Encoding gene dapB, changes the structure of co-factor binding domain in DHDPR protein structures, obtains with different co-factor affinity
Recombinant bacterial strain JL-6dapBA31G,A32CAnd JL-6dapBC37G,G38C.These recombinant bacterial strains improve affinity of the DHDPR to NADH
And the affinity to NADPH is reduced, NADPH is supplied during relieving C.glutamicum JL-6 fermentation production of L-lysine
Should be insufficient the shortcomings that, improve the ability of strain for accumulating L-lysine.The invention is in successful modification L-lysine route of synthesis
The protein structure of key enzyme, changes its co-factor affinity, release NADPH insufficient supply in building-up process provide one it is fine
New thinking.
Technical scheme：Using genetic engineering as means, by varying the oxidation of dihydrodipicolinate reductase
Reduced cofactor affinity, alleviates NADP (H/ in L-lysine building-up process+) insufficient, obtain the weight of high-yield L-lysine
Group bacterial strain.
(1) change the redox cofactors affinity of dihydrodipicolinate reductase, can efficient accumulation L- rely ammonia
The restructuring Corynebacterium glutamicum of acid, its Classification And Nomenclature is respectively C.glutamicum JL-6dapBA31G,A32CWith
C.glutamicum JL-6dapBC37G,G38C。
(2) construction method of the recombinant bacterium, first with online software comparative analysis different microorganisms source
DHDPR, determines the potential amino acid residue for adjusting DHDPR and combining different co-factors：11st Lys residue and the 13rd Arg
Residue；DHDPR encoding gene dapB are mutated respectively using site-directed mutagenesis kit, followed by carrying antibiotic resistance and condition
The new integrating vector pK18mobsacB of lethal phenotypic marker sacB double labellings, is replaced by homologous recombination twice
Autogene dapB in C.glutamicum JL-6, obtains recombinant bacterial strain C.glutamicumJL-6dapB respectivelyA31G,A32CWith
C.glutamicum JL-6dapBC37G,G38C。
1. potentially adjust DHDPR to determine with reference to the amino acid residue of different co-factors
DHDPR from E.coli and tubercle bacillus (Mycobacterium tuberculosis) more favors utilization
NADH as co-factor, and from C.glutamicum and Thermotoga maritima (Thermotoga maritima) DHDPR more
Favor is used as co-factor by the use of NADPH.For this by using 3.0 comparative analysis of online software Clustalw and ESPript they
Common ground and difference in DHDPR structures, so that it is residual to find out the amino acid that potential adjusting DHDPR combines different co-factors
Base：11st Lys residue and the 13rd Arg residue.Finally, using the DHDPR albumen knots of PyMOL software analysis different mutants
Structure finds, respectively by the 11st Lys residue and the 13rd Arg residue mutations into Ala, can change in DHDPR with the 2 ' of NADPH-
The structure of phosphate moiety, so as to influence the compatibility of DHDPR and NADPH.Therefore, 11st is selected in DHDPR amino acid sequences
Lys residues and the 13rd Arg residue transform site as later experiments.
2. the external rite-directed mutagenesis of DHDPR encoding genes dapB in C.glutamicum
According in GenBank C.glutamicum ATCC13032dapB gene orders design dapB genes upstream and downstream and
Rite-directed mutagenesis primer, primer sequence such as table 1：
Primer sequence needed for table 1.PCR amplifications (underscore is restriction enzyme site)
Using C.glutamicum JL-6 genomes as template, using dapB-F/dapB-R as primer PCR, dapB pieces are obtained
Section, its total length 747bp.Above-mentioned dapB fragments are connected with carrier T (Pucm-T), convert Escherichia coli, extract recombinant plasmid
Pucm-T/dapB.Using plasmid Pucm-T/dapB as template, respectively with mutant primer MCB1-F/MCB1-R and MCB2-F/MCB2-
R carries out PCR reactions for primer, and with after DNA fragmentation Purification Kit, with restriction enzyme DpnI digestions, (DpnI is only identified
The DNA to methylate, and the DNA newly synthesized is not methylated), digestion products are then transformed into E.coli JM106 senses after purification
In by state cell, and it is coated in ammonia benzyl resistant panel and screens mutant strain, finally provokes in ammonia benzyl resistant panel well-grown
Strain culturing, extraction plasmid and sequencing identification, obtains purpose recombinant plasmid Pucm-T/dapBA31G,A32CAnd Pucm-T/
dapBC37G,G38C。
3. DHDPR mutant DHDPRK11AAnd DHDPRR13AKinetic determination
Using corresponding restriction enzyme difference digestion recombinant plasmid Pucm-T/dapBA31G,A32CAnd Pucm-T/
dapBC37G,G38C.Then using plastic recovery kit recycling dapBA31G,A32CAnd dapBC37G,G38CFragment.By dapBA31G,A32CWith
dapBC37G,G38CFragment is connected with the expression plasmid pET28a with His labels after identical digestion with restriction enzyme respectively
Construction recombination plasmid pET28a/dapBA31G,A32CAnd pET28a/dapBC37G,G38C.Correct plasmid pET28a/ will be verified respectively
dapBA31G,A32CAnd pET28a/dapBC37G,G38CIt is transformed into E.coli BL21 competent cells, through LB+Km solid mediums
Screening, obtains recombinant conversion.Purpose transformant is forwarded to IPTG induced expressions in LB fluid nutrient mediums, and utilizes protein
Purify instrument purifying destination protein.Finally examine or check mutant DHDPRK11A、DHDPRR13AWith wild type DHDPR to NADH's and NADPH
Kinetic parameter.
4. recombinant bacterium C.glutamicum JL-6dapBA31G,A32CWith C.glutamicum JL-6dapBC37G,G38CObtain
Using corresponding restriction enzyme difference digestion recombinant plasmid Pucm-T/dapBA31G,A32CAnd Pucm-T/
dapBC37G,G38C.Then using plastic recovery kit recycling dapBA31G,A32CAnd dapBC37G,G38CFragment.By dapBA31G,A32CWith
dapBC37G,G38CFragment is connected with the linearisation pK18mobsacB integrating vectors after identical digestion with restriction enzyme respectively
Construction recombination plasmid pK18mobsacB/dapBA31G,A32CAnd pK18mobsacB/dapBC37G,G38C.Correct matter will be verified respectively
Grain pK18mobsacB/dapBA31G,A32CAnd pK18mobsacB/dapBC37G,G38CElectroporated C.glutamicum JL-6, warp
LBHIS+Km solid mediums screen, and obtain first time homologous recombination transformant.Again respectively by targeted transformation containing sucrose
Carry out coercing secondary restructuring screening in culture medium, line separation and the multiple transformants of picking are finally carried out on LBG tablets.To hair
The bacterial strain of raw second of homologous recombination carries out the identification of reply wild type/genic mutation type.Extraction conversion daughter chromosome, with target
The upstream and downstream primer of gene dapB carries out PCR and carries out sequencing identification to PCR product.Identify that correct transformant is named as
C.glutamicumJL-6dapBA31G,A32CWith C.glutamicum JL-6dapBC37G,G38C。
(3) recombinant bacterium C.glutamicum JL-6dapBA31G,A32CWith C.glutamicum JL-6dapBC37G,G38CFermentation
Produce L-lysine
Picking single bacterium colony is seeded to liquid seed culture medium, 30 DEG C, 100r min-1Reciprocal shaker culture 12h.With 10%
Seed culture fluid is forwarded to fermentation medium by inoculum concentration, 30 DEG C, 100r min-1Reciprocal shaker culture 40h.Detection hair in real time
The parameter such as L-lysine, glucose residual quantity and biomass during ferment.
(4) monitoring of the quantification and qualification and thalli growth situation of substrate and product
The real-time detection of glucose and L-lysine passes through SBA-40B bio-sensing analysis-e/or determinings in zymotic fluid.Bacterium solution
Concentration mensuration：Pipette samples bacterium solution, dilutes certain multiple, using distilled water as blank control, using spectrophotometric with distilled water
Count and measure OD in 1cm light paths600nm。
(5) quantification and qualification of accessory substance
Accessory substance mainly examines or check organic acid and amino acid in zymotic fluid.For the measure of organic acid, using high-efficient liquid phase color
Spectrometer (HLPC) measures；For the measure of amino acid, measured using amino acid determining instrument.Using high performance liquid chromatograph or amino
Acidity test instrument distinguishes bioassay standard sample and the respective appearance time of conversion fluid and peak area, you can with to determinand in conversion fluid
Matter carries out qualitative and quantitative detection.
Beneficial effects of the present invention：Pass through rite-directed mutagenesis C.glutamicum JL-6L- lysine synthetic pathway key enzymes
DHDPR encoding gene dapB, change the structure of co-factor binding domain in DHDPR protein structures, obtain more favor and utilize NADH
Recombinant bacterial strain JL-6dapB as co-factorA31G,A32CAnd JL-6dapBC37G,G38C, so as to relieve L-lysine fermenting and producing
During NADPH it is insufficient the shortcomings that, improve the ability of strain for accumulating L-lysine.
Brief description of the drawings
The amino acid alignment of the DHDPR in Fig. 1 different microorganisms source；
Abbreviation explanation：CgDHDPR- derives from the DHDPR of C.glutamicum；MtDHDPR- is derived from
The DHDPR of M.tuberculosis；EcDHDPR-from the DHDPR of E.coli；TmDHDPR-from T.maritima's
DHDPR。
Expression of Fig. 2 DHDPR in E.coli BL21；
Swimming lane explanation：M swimming lanes are protein molecular weight standard Marker；No. 1 swimming lane is E.coli BL21 (pET28a/
DapB) extraction liquid of cell adds IPTG to induce；No. 2 swimming lanes purify egg for E.coli BL21 (pET28a/dapB) extraction liquid of cell
In vain；No. 3 swimming lanes do not induce for E.coli BL21 (pET28a/dapB) extraction liquid of cell；No. 4 swimming lanes are E.coli BL21
(pET28a) extraction liquid of cell；No. 5 swimming lanes are E.coli BL21 extraction liquid of cell.
Fig. 3 difference DHDPR mutant strain DHDPR enzyme activity determinations；
Fig. 4 difference DHDPR mutant strain fermentation production of L-lysine conditional curves；
Number explanation：A-L- lysine change curves；B- glucose change curves；C- thalli growth curves.
Fig. 5 difference DHDPR mutant strain zymotic fluid accessory substance measurement results；
Number explanation：A- Accumulation of Organic Acids amounts；B- amino acid accumulations
Specific implementation method
Embodiment 1：The external rite-directed mutagenesis of DHDPR encoding genes dapB in C.glutamicum
Using C.glutamicum JL-6 genomes as template, using dapB-F/dapB-R as primer, reacted and expanded by PCR
The dapB fragments of 747bp, EcoRI and HindIII restriction enzyme sites are introduced at the 5 ' of PCR product and 3 ' ends respectively.
Above-mentioned dapB fragments are connected with carrier T (Pucm-T), convert Escherichia coli, extraction recombinant plasmid Pucm-T/
dapB.Using plasmid Pucm-T/dapB as template, respectively using mutant primer MCB1-F/MCB1-R and MCB2-F/MCB2-R as primer
PCR reactions are carried out, (DpnI is only identified and methylated with restriction enzyme DpnI digestions with after DNA fragmentation Purification Kit
DNA, and the DNA newly synthesized is not methylated), it is thin that digestion products are then transformed into E.coli JM106 competence after purification
In born of the same parents, and it is coated in ammonia benzyl resistant panel and screens mutant strain, finally provokes in the well-grown bacterial strain of ammonia benzyl resistant panel
Culture, extraction plasmid and sequencing identification, obtain purpose recombinant plasmid Pucm-T/dapBA31G,A32CAnd Pucm-T/dapBC37G,G38C。
PCR reaction systems (50 μ L)：25 μ L of Ex Taq enzymes, 100 μ g of DNA profiling, forward primer (20 μm of olL-1) 1 μ L,
Reverse primer (20 μm of olL-1) 1 μ L, add ddH2O to 50 μ L；PCR reaction conditions：94 DEG C of pre-degeneration 5min first, then 94 DEG C
30s is denatured, anneal 30s, 72 DEG C of extensions, counts 35 circulations, and last 72 DEG C re-extend 10min and 4 DEG C of preservations.Annealing in reaction
Temperature and extension of time are as shown in table 1.
Embodiment 2：Expression and purifying of the DHDPR mutant in E.coli BL21
Pointed out according to table 1, using restriction enzyme EcoRI and HindIII difference digestion recombinant plasmid Pucm-T/
dapBA31G,A32CAnd Pucm-T/dapBC37G,G38C.Then using plastic recovery kit recycling dapBA31G,A32CAnd dapBC37G,G38C
Fragment.By dapBA31G,A32CAnd dapBC37G,G38CFragment is connected structure with the pET28a after identical digestion with restriction enzyme respectively
Build recombinant plasmid pET28a/dapBA31G,A32CAnd pET28a/dapBC37G,G38C；By recombinant plasmid pET28a/dapBA31G,A32CWith
pET28a/dapBC37G,G38CE.coli BL21 are transferred to, picking positive transformant is to liquid on kalamycin resistance LB tablets
Thalline is collected after LB, IPTG induction after ultrasonic disruption, supernatant SDS-PAGE, detects molecular weight about 30kDa's
Specific band (Fig. 2), it is in the same size with the target protein of report, and control strain E.coli BL21 (pET28a) are without this
Band.Illustrate recombinant plasmid pET28a/dapBA31G,A32CAnd pET28a/dapBC37G,G38CCorrectly expressed in Escherichia coli.With
Afterwards, using protein purification instrument, (the figure of DHDPR albumen in liquid is crushed according to the His label purifying cells on expression plasmid pET28a
2)。
Embodiment 3：The measure of DHDPR mutant kinetic parameters
The mutant DHDPR of above-mentioned purifying is taken respectivelyK11A、DHDPRR13ADHDPR enzyme activity determinations are added with wild type DHDPR
In reaction system, absorbance change at 340nm is monitored in real time, and according to K at absorbance change calculatingmAnd Kcat(table 2).
Reaction system：100mmol L-1MOPS buffer solutions (pH 7.2), 50 μm of ol L-1Dihydrodipicolinic acid, 0.01~
0.04mmol L-1NADPH or 0.004~0.032mmol L-1NADH；Reaction temperature：30℃；Reaction time：≥300s.
2. wild type DHDPR of table and its mutant kinetic parameter
Embodiment 4：Recombinant bacterium C.glutamicum JL-6dapBA31G,A32CWith C.glutamicum JL-6dapBC37G ,G38CAcquisition
Pointed out according to table 1, using restriction enzyme EcoRI and HindIII difference digestion recombinant plasmid Pucm-T/
dapBA31G,A32CAnd Pucm-T/dapBC37G,G38C.Then using plastic recovery kit recycling dapBA31G,A32CAnd dapBC37G,G38C
Fragment.By dapBA31G,A32CAnd dapBC37G,G38CFragment respectively with the linearisation after identical digestion with restriction enzyme
PK18mobsacB integrating vectors are connected construction recombination plasmid pK18mobsacB/dapBA31G,A32CAnd pK18mobsacB/
dapBC37G,G38C.Correct recombinant plasmid pK18mobsacB/dapB will be verified respectivelyA31G,A32CAnd pK18mobsacB/
dapBC37G,G38CConversion fluid, is then coated on containing 25 μ g mL by electroporated C.glutamicum JL-6-1Kanamycins
LBHIS solid mediums screen, and select the good bacterial strain of LBHIS+Km plated growths, obtain first time homologous recombination transformant.
Targeted transformation is inoculated in respectively again after cultivating 24h in LBG fluid nutrient mediums, L containing 100g is coated on after doubling dilution-1Sugarcane
Carry out coercing secondary restructuring screening in the LBG solid mediums of sugar, line separation is finally carried out on LBG tablets and picking is multiple
Transformant.Extraction conversion subgenom, carries out PCR with the upstream and downstream primer of target gene dapB and PCR product is sequenced
Identification, determines that rite-directed mutagenesis occurs for C.glutamicum JL-6dapB genes.Identify that correct transformant is named as
C.glutamicum JL-6dapBA31G,A32CWith C.glutamicum JL-6dapBC37G,G38C。
Embodiment 5：Recombinant bacterium C.glutamicum JL-6dapBA31G,A32CWith C.glutamicum JL-6dapBC37G ,G38CDHDPR enzyme activity determinations
Take the strain of frozen pipe preservation to be inoculated with and contain 0.25g L-1L-Methionine and 40g L-1The CgXII cultures of glucose
In base (i.e. CgXIIMG culture mediums), 30 DEG C of shaken cultivations are stayed overnight, and in 10000r min-1Thalline is collected by centrifugation.Then, by bacterium
Body is suspended in MOPS buffer solutions and prepares crude enzyme liquid in ultrasonic fragmentation.Crude enzyme liquid carries out enzyme activity determination using colorimetric method
(A320nm).Enzyme reaction system：100mmol L-1MOPS buffer solutions (pH 7.2), 50 μm of ol L-1Dihydrodipicolinic acid,
0.162mmol L-1NADPH or 0.162mmol L-1NADH；Reaction temperature：30℃；Reaction time：≥300s.One enzyme activity
Unit (U) is defined as the enzyme amount needed for oxidation per minute 1 μm of ol NADPH or NADH under determination condition.After measured, recombinant bacterium
DHDPR enzyme activities will be higher than control strain, and the results are shown in Figure 3.
Embodiment 6：Recombinant bacterium C.glutamicum JL-6dapBA31G,A32CWith C.glutamicum JL-6dapBC37G ,G38CThe measure of intracellular co-factor
Corynebacterium glutamicum single bacterium colony is taken to be inoculated in CgXIIMG fluid nutrient mediums, 30 DEG C, 100rmin-1Shaking table culture
About 10h, 4 DEG C, 6000rmin-1Thalline is collected by centrifugation, and washing thalline is three times, removes remaining extracellular metabolin.Then,
With acid extract (0.5molL-1HCl oxidized form pyridine nucleotide (NAD) is extracted+And NADP+), with alkaline extract
(0.5mol·L-1NaOH reduced form pyridine nucleotide (NADH and NADPH)) is extracted.Then, purchased by from BioVision companies
The quantification assay kit put, utilizes enzyme parameters measure NAD (P)+With the concentration of NAD (P) H and calculate NADH/NAD+With
NADPH/NADP+, wherein with NAD/NADH Quantification Colorimeteric Kit specific detections NAD+With
NADH, with NADP/NADPH Quantification Colorimeteric Kit specific detections NADP+And NADPH, specifically
Step is carried out with reference to the method for kit specification, and the results are shown in Table 3.
Embodiment 7：C.glutamicum JL-6dapBA31G,A32CWith C.glutamicum JL-6dapBC37G,G38CFermentation
Produce L-lysine
Culture medium：1. seed culture medium (gL-1)：Glucose 5, peptone 10, yeast extract 5, NaCl 10, pH 7.0,
121 DEG C of sterilizing 20min；2. fermentation medium (i.e. CgXIIMG；g·L-1)：Glucose 40, (NH4)2SO420, urea 5,
KH2PO41, K2HPO4·3H2O 1, MgSO4·7H2O 0.25,3- (N- morpholines)-propane sulfonic acid 42, L-Methionine 0.25,
CaCl20.010, FeSO4·7H2O 0.01, MnSO4·H2O 0.01, ZnSO4·7H2O 0.01, CuSO4·5H2O
0.0002, NiCl2·6H2O 0.00002, biotin 0.0002,0.00003,115 DEG C of sterilizing 10min of protocatechuic acid.Fermented
By adding 10gL in journey-1CaCO3Adjust pH.
By the recombinant C .glutamicum JL-6dapB of above-mentioned empirical testsA31G,A32CWith C.glutamicum JL-
6dapBC37G,G38CShake flask fermentation experiment is carried out respectively, and picking one expires ring from the slant medium of fresh activation
C.glutamicum (control bacterium and recombinant bacterium) is in seed culture medium (25mL/250mL), 30 DEG C, 1000r min-1It is reciprocating
Shaking table culture 12h；With 10% inoculum concentration, it is inoculated in fermentation medium, 30 DEG C, 1000r min-1Reciprocal shaker culture 40h,
Divide time section measure L-lysine, glucose and biomass, as a result compared with control strain C.glutamicum JL-6, knot
Fruit is as shown in Figure 4.
In addition, high performance liquid chromatograph and amino-acid analyzer measure recombinant bacterium C.glutamicum JL- is respectively adopted
6dapBA31G,A32CWith C.glutamicum JL-6dapBC37G,G38CAnd accessory substance accumulates in control bacterium C.glutamicum JL-6
Tired situation (including organic acid and amino acid), the results are shown in Figure 5.
Approach described above is applicable not only to utilize Corynebacterium glutamicum fermentation production of L-lysine, to other any utilizations
Microbial fermentation production NADPH- dependent form products are all suitable for, therefore these contents belong to protection scope of the present invention.
Sequence table
<110>Southern Yangtze University
<120>The method that DHDPR improves lysine production in genetic modification Corynebacterium glutamicum
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 747
<212> DNA
<213> Corynebacterium glutamicum
<400> 1
atgggaatca aggttggcgt tctcggagcc aaaggccgtg ttggtcaaac tattgtggca 60
gcagtcaatg agtccgacga tctggagctt gttgcagaga tcggcgtcga cgatgatttg 120
agccttctgg tagacaacgg cgctgaagtt gtcgttgact tcaccactcc taacgctgtg 180
atgggcaacc tggagttctg catcaacaac ggcatttctg cggttgttgg aaccacgggc 240
ttcgatgatg ctcgtttgga gcaggttcgc gactggcttg aaggaaaaga caatgtcggt 300
gttctgatcg cacctaactt tgctatctct gcggtgttga ccatggtctt ttccaagcag 360
gctgcccgct tcttcgaatc agctgaagtt attgagctgc accaccccaa caagctggat 420
gcaccttcag gcaccgcgat ccacactgct cagggcattg ctgcggcacg caaagaagca 480
ggcatggacg cacagccaga tgcgaccgag caggcacttg agggttcccg tggcgcaagc 540
gtagatggaa tcccggttca tgcagtccgc atgtccggca tggttgctca cgagcaagtt 600
atctttggca cccagggtca gaccttgacc atcaagcagg actcctatga tcgcaactca 660
tttgcaccag gtgtcttggt gggtgtgcgc aacattgcac agcacccagg cctagtcgta 720
ggacttgagc attacctagg cctgtaa 747
<210> 2
<211> 747
<212> DNA
<213> Corynebacterium glutamicum
<400> 2
atgggaatca aggttggcgt tctcggagcc gcaggccgtg ttggtcaaac tattgtggca 60
gcagtcaatg agtccgacga tctggagctt gttgcagaga tcggcgtcga cgatgatttg 120
agccttctgg tagacaacgg cgctgaagtt gtcgttgact tcaccactcc taacgctgtg 180
atgggcaacc tggagttctg catcaacaac ggcatttctg cggttgttgg aaccacgggc 240
ttcgatgatg ctcgtttgga gcaggttcgc gactggcttg aaggaaaaga caatgtcggt 300
gttctgatcg cacctaactt tgctatctct gcggtgttga ccatggtctt ttccaagcag 360
gctgcccgct tcttcgaatc agctgaagtt attgagctgc accaccccaa caagctggat 420
gcaccttcag gcaccgcgat ccacactgct cagggcattg ctgcggcacg caaagaagca 480
ggcatggacg cacagccaga tgcgaccgag caggcacttg agggttcccg tggcgcaagc 540
gtagatggaa tcccggttca tgcagtccgc atgtccggca tggttgctca cgagcaagtt 600
atctttggca cccagggtca gaccttgacc atcaagcagg actcctatga tcgcaactca 660
tttgcaccag gtgtcttggt gggtgtgcgc aacattgcac agcacccagg cctagtcgta 720
ggacttgagc attacctagg cctgtaa 747
<210> 3
<211> 747
<212> DNA
<213> Corynebacterium glutamicum
<400> 3
atgggaatca aggttggcgt tctcggagcc aaaggcgctg ttggtcaaac tattgtggca 60
gcagtcaatg agtccgacga tctggagctt gttgcagaga tcggcgtcga cgatgatttg 120
agccttctgg tagacaacgg cgctgaagtt gtcgttgact tcaccactcc taacgctgtg 180
atgggcaacc tggagttctg catcaacaac ggcatttctg cggttgttgg aaccacgggc 240
ttcgatgatg ctcgtttgga gcaggttcgc gactggcttg aaggaaaaga caatgtcggt 300
gttctgatcg cacctaactt tgctatctct gcggtgttga ccatggtctt ttccaagcag 360
gctgcccgct tcttcgaatc agctgaagtt attgagctgc accaccccaa caagctggat 420
gcaccttcag gcaccgcgat ccacactgct cagggcattg ctgcggcacg caaagaagca 480
ggcatggacg cacagccaga tgcgaccgag caggcacttg agggttcccg tggcgcaagc 540
gtagatggaa tcccggttca tgcagtccgc atgtccggca tggttgctca cgagcaagtt 600
atctttggca cccagggtca gaccttgacc atcaagcagg actcctatga tcgcaactca 660
tttgcaccag gtgtcttggt gggtgtgcgc aacattgcac agcacccagg cctagtcgta 720
ggacttgagc attacctagg cctgtaa 747
Claims (9)
1. in genetic modification Corynebacterium glutamicum DHDPR improve lysine production method, it is characterised in that using genetic engineering as
Means, by varying the redox cofactors affinity of DHDPR (dihydrodipicolinate reductase, encoding gene dapB),
The restructuring Corynebacterium glutamicum C.glutamicum JL-6 dapB of efficient accumulation L-lysineA31G,A32CAnd C.glutamicum
JL-6 dapBC37G,G38C。
2. the method that DHDPR improves lysine production in genetic modification Corynebacterium glutamicum according to claim 1, it is special
Sign is, the genetic engineering bacterium C.glutamicum JL-6 dapBA31G,A32CWith C.glutamicum JL-6 dapBC37G ,G38CConstructed wetlands, be to utilize one plant of production L-lysine mutant strain C.glutamicum JL-6, rite-directed mutagenesis DHDPR coding
Gene dapB, obtains genetic engineering bacterium C.glutamicum JL-6 dapBA31G,A32CWith C.glutamicum JL-6
dapBC37G,G38C, this two plants of engineering bacterias, which improve DHDPR, reduces the affinity of NADH affinity to NADPH, releases
The shortcomings that NADPH is insufficient during C.glutamicum JL-6 fermentation production of L-lysine, improves strain for accumulating L-
The ability of lysine.
3. the method that DHDPR improves lysine production in genetic modification Corynebacterium glutamicum according to claim 2, it is special
Sign is that the thinking of the rite-directed mutagenesis DHDPR encoding genes dapB, is that verification is correctly recombinated linear plasmid respectively
pK18mobsacB/dapBA31G,A32CAnd pK18mobsacB/dapBC37G,G38CElectroporated C.glutamicum JL-6, through containing
There are 25 μ g mL-1The LBHIS solid mediums screening of kanamycins, obtains first time homologous recombination transformant, then respectively by target
Transformant is in L containing 100g-1Carry out coercing secondary restructuring screening in the LBG solid mediums of sucrose, it is finally enterprising in LBG tablets
Row line separation and the multiple transformants of picking, are realized in C.glutamicum JL-6 the 31st and 32 in dapB gene orders
Base is mutated into G and C and sequence the 37th by A respectively and 38 bit bases are mutated into G and C by C and G respectively.
4. the method that DHDPR improves lysine production in genetic modification Corynebacterium glutamicum according to claim 3, it is special
Sign is, described to recombinate linear plasmid pK18mobsacB/dapBA31G,A32CAnd pK18mobsacB/dapBC37G,G38CStructure think
Road, it is characterized in that utilizing restriction enzyme EcoRI and HindIII difference digestion recombinant plasmid Pucm-T/dapBA31G,A32C、
Pucm-T/dapBC37G,G38CWith linearisation integrating vector pK18mobsacB, then by enzyme even in the form of by endonuclease bamhi
dapBA31G,A32CWith the pK18mobsacB and endonuclease bamhi dapB of digestionC37G,G38CWith the pK18mobsacB of digestion, structure restructuring
Plasmid pK18mobsacB/dapBA31G,A32CAnd pK18mobsacB/dapBC37G,G38C。
5. the method that DHDPR improves lysine production in genetic modification Corynebacterium glutamicum according to claim 4, it is special
Sign is, the recombinant plasmid Pucm-T/dapBA31G,A32CAnd Pucm-T/dapBC37G,G38CConstructed wetlands, with Pucm-T/
DapB is template, carries out PCR reactions by primer of mutant primer MCB1-F/MCB1-R and MCB2-F/MCB2-R respectively, uses DNA
Restriction enzyme DpnI digestions are used after fragment purification kits, digestion products are then transformed into E.coli after purification
In JM106 competent cells, and it is coated in ammonia benzyl resistant panel and screens mutant strain, finally provokes and given birth in ammonia benzyl resistant panel
Long good strain culturing, extraction plasmid and sequencing identification, obtain recombinant plasmid Pucm-T/dapBA31G,A32CWith
Pucm-T/dapBC37G,G38C；
MCB1-F：5’-CGTTCTCGGAGCCGCAGGCCGTGTTGGTCAAAC-3’
MCB1-R：5’-GTTTGACCAACACGGCCTGCGGCTCCGAGAACG-3’
MCB2-F：5’-CGGAGCCAAAGGCGCTGTTGGTCAAACTATTGTG-3’
MCB2-R：5’-CACAATAGTTTGACCAACAGCGCCTTTGGCTCCG-3’；
PCR reaction systems (50 μ L)：Ex Taq enzymes 25 μ L, Pucm-T/dapB 100 μ g, forward primer (20 μm of olL-1) 1 μ L,
Reverse primer (20 μm of olL-1) 1 μ L, add ddH2O to 50 μ L；PCR reaction conditions：94 DEG C of pre-degeneration 5min first, then 94 DEG C
30s is denatured, 58 DEG C of annealing 30s, 72 DEG C of extension 220s, count 35 circulations, last 72 DEG C re-extend 10min and 4 DEG C of preservations.
6. the method that DHDPR improves lysine production in genetic modification Corynebacterium glutamicum according to claim 5, it is special
Sign is, the Constructed wetlands of the middle recombinant plasmid Pucm-T/dapB, it is characterized in that by dapB genes and Pucm-T after purification
Connection, reaction system are as follows：0.5 μ L, dapB genes of plasmid Pucm-T, 4.5 μ L, Ligation Solution, 5 μ L, mixing connect
Liquid is connect, is placed on connection more than 4h, Transformed E .coli JM109 in 16 DEG C of incubators, extraction plasmid Pucm-T/dapB.
7. the method that DHDPR improves lysine production in genetic modification Corynebacterium glutamicum according to claim 5, it is special
Sign is that the acquisition of the dapB genes, shows using C.glutamicum JL-6 genomes as template, using dapB-F/dapB-R as
Primer, the dapB fragments of amplification 747bp are reacted by PCR, and EcoRI and HindIII is introduced respectively at the 5 ' of PCR product and 3 ' ends
Restriction enzyme site；
dapB-F(EcoRI)：5’-CGGAATTCGAAAGGAGATATACCATGGGAATCAAGGTTGGCGTTC-3’
dapB-R(HindIII)：5’-CCCAAGCTTTTACAGGCCTAGGTAATGC-3’；
PCR reaction systems (50 μ L)：25 μ L, C.glutamicum JL-6 genomes of Ex Taq enzymes 100 μ g, forward primer (20 μ
mol·L-1) 1 μ L, reverse primer (20 μm of olL-1) 1 μ L, add ddH2O to 50 μ L；PCR reaction conditions：94 DEG C of pre-degenerations first
5min, is then denatured 30s for 94 DEG C, 56 DEG C of annealing 30s, 72 DEG C of extension 45s, count 35 circulations, last 72 DEG C re-extend 10min simultaneously
4 DEG C of preservations.
8. the restructuring Corynebacterium glutamicum C.glutamicum JL-6 dapB described in claim 1A31G,A32CWith
C.glutamicum JL-6 dapBC37G,G38CThe method of fermentation production L-lysine, it is characterized in that by recombinant bacterium with compareing bacterium difference
It is inoculated in containing 0.25g L-1L-Methionine and 40g L-1In the CgXII culture mediums (i.e. CgXIIMG culture mediums) of glucose,
With by adding 10gL-1CaCO3Adjust pH.
9. the method that DHDPR improves lysine production in the genetic modification Corynebacterium glutamicum according to claims 8, its
It is characterized in that, the recombinant bacterium C.glutamicum JL-6 dapBA31G,A32CWith C.glutamicum JL-6 dapBC37G,G38C
DHDPR enzyme activity determination methods：The strain of frozen pipe preservation is taken to be inoculated with CgXIIMG fluid nutrient mediums, 30 DEG C of shaken cultivation mistakes
Night, and in 10000r min-1Thalline is collected by centrifugation, then, thalline is suspended in MOPS buffer solutions and in ultrasonic fragmentation
Crude enzyme liquid is prepared, crude enzyme liquid carries out enzyme activity determination (A using colorimetric method320nm), enzyme reaction system：100mmol L-1MOPS is buffered
Liquid (pH 7.2), 50 μm of ol L-1Dihydrodipicolinic acid, 0.162mmol L-1NADPH or 0.162mmol L-1NADH；Reaction
Temperature：30℃；Reaction time：>=300s, an enzyme activity unit (U) are defined as 1 μm of ol of oxidation per minute under determination condition
Enzyme amount needed for NADPH or NADH；
The recombinant bacterium C.glutamicum JL-6 dapBA31G,A32CWith C.glutamicum JL-6 dapBC37G,G38CBorn of the same parents
Interior co-factor assay method：Corynebacterium glutamicum single bacterium colony is taken to be inoculated in CgXIIMG fluid nutrient mediums, 30 DEG C, 100r
min-1Shaking table culture about 10h, 4 DEG C, 6000rmin-1Thalline is collected by centrifugation, and washing thalline is three times, removes remaining cell
Outer metabolin；
Then, with acid extract (0.5molL-1HCl oxidized form pyridine nucleotide (NAD) is extracted+And NADP+), taken out with alkalescence
Extract (0.5molL-1NaOH reduced form pyridine nucleotide (NADH and NADPH)) is extracted；Then, by public from BioVision
The quantification assay kit purchased is taken charge of, utilizes enzyme parameters measure NAD (P)+With the concentration of NAD (P) H and calculate NADH/NAD+With
NADPH/NADP+, wherein with NAD/NADH Quantification Colorimeteric Kit specific detections NAD+With
NADH, with NADP/NADPH Quantification Colorimeteric Kit specific detections NADP+And NADPH, specifically
Step is carried out with reference to the method for kit specification；
The quantification and qualification of the substrate and product and the monitoring method of thalli growth situation, it is characterized in that in zymotic fluid
The real-time detection of glucose and L-lysine passes through SBA-40B bio-sensing analysis-e/or determinings；Bacterial concentration measures：Pipette samples
Bacterium solution, dilutes certain multiple with distilled water, using distilled water as blank control, is measured using spectrophotometer in 1cm light paths
OD600；
The monitoring method of the quantification and qualification of the accessory substance (including organic acid and amino acid)：Efficient liquid phase is respectively adopted
Chromatograph and amino-acid analyzer measure recombinant bacterium C.glutamicum JL-6 dapBA31G,A32CWith C.glutamicum JL-6
dapBC37G,G38CAnd byproducts build-up situation in control bacterium C.glutamicum JL-6 zymotic fluids.
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| CN114729338A (en) * | 2019-09-26 | 2022-07-08 | Cj第一制糖株式会社 | Mutant dihydrodipicolinate reductase polypeptide and method for producing L-threonine using the same |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114729338A (en) * | 2019-09-26 | 2022-07-08 | Cj第一制糖株式会社 | Mutant dihydrodipicolinate reductase polypeptide and method for producing L-threonine using the same |
| JP2022534403A (en) * | 2019-09-26 | 2022-07-29 | シージェイ チェイルジェダング コーポレイション | Dihydrodipicolinate reductase mutant polypeptide and method for producing L-threonine using the same |
| EP3960851A4 (en) * | 2019-09-26 | 2022-08-31 | CJ Cheiljedang Corporation | Modified polypeptide of dihydrodipicolinate reductase, and method for producing l-threonine by using same |
| JP7311634B2 (en) | 2019-09-26 | 2023-07-19 | シージェイ チェイルジェダング コーポレイション | Dihydrodipicolinate reductase mutant polypeptide and method for producing L-threonine using the same |
| CN114729338B (en) * | 2019-09-26 | 2024-01-26 | Cj第一制糖株式会社 | Variant dihydropyridine dicarboxylic acid reductase polypeptides and methods of producing L-threonine using the same |
| AU2020356063B2 (en) * | 2019-09-26 | 2024-02-29 | Cj Cheiljedang Corporation | Modified polypeptide of dihydrodipicolinate reductase, and method for producing L-threonine by using same |
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