CN107723317A - A kind of method that itaconic acid is produced in Escherichia coli - Google Patents

A kind of method that itaconic acid is produced in Escherichia coli Download PDF

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CN107723317A
CN107723317A CN201711230812.5A CN201711230812A CN107723317A CN 107723317 A CN107723317 A CN 107723317A CN 201711230812 A CN201711230812 A CN 201711230812A CN 107723317 A CN107723317 A CN 107723317A
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itaconic acid
escherichia coli
acid
gene
acna
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任宇红
魏东芝
杨中伟
王红玲
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East China University of Science and Technology
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Abstract

The method that a kind of method that itaconic acid is produced in Escherichia coli of present invention offer is a kind of while realizes the efficient production itaconic acid in Escherichia coli with metabolic pathway renovation technique using multienzyme package technique, comprises the following steps：1) three key enzymes for synthesizing itaconic acid are carried out being self-assembly of multienzyme complex in the cell, three key enzymes include：Citrate synthase gltA, cis-aconitic enzyme acnA and aconitate decarboxylase cadA；2) metabolic pathway transformation is carried out to Escherichia coli using Crispr/cas9 technologies；And 3) multienzyme complex is transferred to a kind of colibacillus engineering of Escherichia coli structure of metabolic pathway transformation, pass through the production for the progress itaconic acid that ferments.The present invention utilizes Crispr/cas9 technological transformation metabolic pathways using three key enzymes of synthesis itaconic acid as assembling object, realizes the efficient production of the itaconic acid in Escherichia coli, new way is provided for the industrialized production of itaconic acid.

Description

A kind of method that itaconic acid is produced in Escherichia coli
Technical field
The present invention relates to genetic engineering field, relates more specifically to a kind of method that itaconic acid is produced in Escherichia coli.
Background technology
Itaconic acid (methylene succinic acid, methene succinic acid) is a kind of unsaturated dicarboxylic acid, is the important of chemical field The raw material of industry and additive, it is widely used in producing plastics, chemical fibre, hypersorption agent, latex, scale preventative etc. as constructing module Aspect.Itaconic acid has obtained increasing concern as a kind of recyclable materials.2004, itaconic acid was by american energy It is chosen as one of most potential 12 kinds of bio-based platform chemicals in portion.At present, the production capacity of whole world itaconic acid is 50,000 Ton/year, at least 30,000 tons of breach is also faced with, and itaconic acid can substitute acrylic acid and methacrylic acid is used for degradation plastic Production, its tomorrow requirement amount continues to rapid growth, is that a kind of important biomolecule matter source with broad prospect of application has Machine acid.
At present, the predominant methods of domestic and international industrialized production itaconic acid are Aspergillus terreus fermentation methods.Biosynthesis itaconic acid Main flow theory is considered the route by glycolysis and TCA circulations, i.e., using citric acid and aconitic acid as metabolic intermediate, the latter Itaconic acid is formed by aconitate decarboxylase cadA decarboxylations.However, compared with other organic acid fermentations produce, it is sharp in the world at present Still relatively low with the fermentation unit of Aspergillus terreus fermentation production of itaconic acid, its reason is：1) Aspergillus terreus fermentation time is grown, space-time production Rate is not high；2) acnA is positioned in mitochondria in fungal cell, and cadA is positioned in kytoplasm, and two electrodes methods are not Diffusion and reaction with the substrate and intermediate product for making two enzymes become increasingly difficult.Therefore, researcher selects Escherichia coli As the Host Strains of production itaconic acid, the advantage is that：1) growth cycle is short, and space-time yield is high；2) genetic background understands, gene Transformation is relatively easy to, and in the absence of different electrodes methods the different organelles the problem of；3) Escherichia coli have good organic acid Tolerance, possess the potentiality of production higher concentration itaconic acid.But also simply concern uses traditional biological skill for these researchs at present Art means, such as improve expression and metabolic pathway transformation of key enzyme etc..Although gram level can be reached in Escherichia coli Fermentation level, but also have a certain distance apart from production requirement.
Multienzyme package technique is a kind of method that can significantly improve multienzyme cascade catalytic efficiency to grow up in recent years. More enzyme cascade systems can solve problem of transmission of the substrate between multiple enzymes, reduce mass transfer distance, increase subrange The concentration of interior substrate, shorten transhipment and the reaction time of substrate, improve the catalytic efficiency of each single enzyme, apply more and more In the biochemical reaction for needing the enzymatic of multistep list to complete.Wherein using to the more peptide or proteins of interaction be widely present in nature Boundary, and its type is rich and varied, and for molecular weight from several KD to hundreds of KD, the mode of interaction also respectively has feature, is adapted to As the PDZ interaction proteins pair from metazoa cell used in the assembling element of interaction, such as the present invention With the SHM interaction proteins pair from mouse signal albumen.Artificial constructed efficient heterologous more enzyme cascade systems are also One of main research and study hotspot of synthetic biology.
Three enzymes of key in itaconic acid route of synthesis are assembled, structure is efficient more with certain compartmentation effect There is presently no research in terms of enzyme body to report.If can in Escherichia coli by three enzymes of key in itaconic acid synthesis path all Fit together, form a close structure, aligned orderly and the artificial multienzyme with certain compartmentation microenvironment, make clothing health Sour route of synthesis is closely connected with the part path that TCA is circulated, then intermediate-citric acid and cis-aconitic can be greatly decreased Mass transfer distance and haulage time, increase the concentration of substrate in multienzyme microenvironment, enable caused by intermediate immediately by under One enzyme is catalyzed, and is improved cascade catalytic reaction efficiency, is strengthened itaconic acid synthesis path metabolic fluxes, balance is synthesized towards itaconic acid Direction is carried out, and the space-time yield for being expected to make itaconic acid is largely increased, and is with a wide range of applications and industrial value.
The content of the invention
It is an object of the invention to provide a kind of method that itaconic acid is produced in Escherichia coli, so as to solve in the prior art The problem of method of production itaconic acid can not also meet high yield industrialized production.
In order to solve the above-mentioned technical problem, the present invention uses following technical scheme：
A kind of method that itaconic acid is produced in Escherichia coli is provided, methods described is a kind of while assembles skill using multienzyme Art realizes the method that itaconic acid is efficiently produced in Escherichia coli with metabolic pathway renovation technique, and specifically, methods described includes Following steps：1) three key enzymes for synthesizing itaconic acid are carried out being self-assembly of in the cell multienzyme complex, described three Key enzyme includes：Citrate synthase gltA, cis-aconitic enzyme acnA and aconitate decarboxylase cadA；2) Crispr/ is utilized Cas9 technologies carry out metabolic pathway transformation to Escherichia coli；And 3) multienzyme complex is transferred to and transformed by metabolic pathway Escherichia coli build a kind of colibacillus engineering, pass through to ferment and carry out the production of itaconic acid.
The step 1) is closed using two Thermodynamic parameters Protein S H3/SH3lig and PDZ/PDZlig fusion citric acids Enzyme gltA, cis-aconitic enzyme acnA and aconitate decarboxylase cadA, form gltA-PDZ-SH3, acnA-PDZlig respectively, cadA-SH3lig。
The step 2) is to knock out part glycolytic cycle to Escherichia coli using Crispr/cas9 technologies, and tricarboxylic acids follows Gene on ring and accessory substance branch road.
Include suppressing lactic acid accessory substance path, first using metabolic pathway transformation of the Crispr/cas9 technologies to Escherichia coli Sour accessory substance path, acetic acid by-product path, and enhancing pepC approach and PEP approach.
The gene knocked out in metabolic pathway transformation includes：Lactate dehydrogenase gene ldhA, pyruvate formate cracking In enzyme pflB, acetonic acid oxidase gene poxB, isocitrate lyase gene icL and isocitric acid dehydrogenase gene icd It is at least one.
In the metabolic pathway transformation gene that knocks out and meanwhile including：Lactate dehydrogenase gene ldhA, pyruvate formate Lyases pflB, acetonic acid oxidase gene poxB, isocitrate lyase gene icL and isocitric acid dehydrogenase gene icd.
The fermentation condition of the colibacillus engineering is：Using minimal medium, fermented and cultured 40 at 7.0,30 DEG C of pH ~100h.
The composition of the minimal medium includes：6.8g/LNa2HPO4, 3g/LKH2PO4, 0.5g/LNaCl, 1g/LNH4Cl, 0.49g/LMgCl, 0.1mmol/L CaCl2, 1mg/L thiamines, 30g/L glucose, 4g/L α-ketoglutaric acids, 1000 × micro- Secondary element (2.86g H3BO3, 1.81g MnCl2·4H2O, 0.222g ZnSO4·7H2O, 0.39g Na2MoO4·2H2O, 0.79g CuSO4·5H2O, 0.049gCo (NO3)2·6H2O)。
The fermentation of the colibacillus engineering comprises the following steps：1) trained first at 37 DEG C under the conditions of 300~500rpm Supporting 15~20h increases Fungal biodiversity；2) after 15~20h, temperature is changed to 30 DEG C, 550rpm fermentations, stream plus 4M NaOH are real When control zymotic fluid pH=6.7, be constantly passed through filtrated air make dissolved oxygen control more than 30%；3) sampled every 6h, measure Portugal Grape sugar concentration, when concentration of glucose is less than 10g/L, 300g/L glucose, 52.5g/L (NH are added manually4)2SO4, 4g/L α- Ketoglutaric acid so that concentration of glucose is not less than 20g/L；And after 4) fermenting 100 hours, measure glucose consumption and finish, send out Ferment terminates.
Research before this laboratory shows, by three key enzymes in itaconic acid route of synthesis in Escherichia coli Two enzyme-acnA and cadA are assembled, and the efficiency and yield of its catalytic citric acid generation itaconic acid are all significantly increased.But It is the industrial level that is still not reaching to, therefore the invention provides a kind of new thinking, i.e., is assembled simultaneously using multienzyme Two kinds of strategies are transformed with metabolic pathway, by itaconic acid path structure in Escherichia coli, key enzyme is assembled and adjusts metabolic flux Flow direction, so as to reach the purpose efficiently produced.The main thought of the present invention is specific as follows：
(1) on the basis of gltA, acnA and cadA 3D structural models are established in computer simulation, according to homologous modeling point Do not found and PDZ or SHM domain or the appropriate site (or region) of ligand fused, Yi Jixuan in the body structure surface of three enzymes Select appropriate linkage element so that they are after with PDZ or SHM albumen to merging (terminal fusion or insertion are merged), knot Big change does not occur for structure and vigor, and keeps the structural integrity of interaction element and be sufficiently exposed to fusion protein surface, It is easily achieved and is mutually distinguishable and assembles, and shows a certain distance and direction between the activated centre of three enzymes after assembling. According to cascade reaction route and oligomerization enzyme autohemagglutination characteristic, scientific design assembles scheme, by the modeling and analysis of fusion protein, and Filter out optimal amalgamation mode.
(2) according to the design and rational scheme of acquisition, build plasmid respectively, realize gltA, acnA and cadA respectively with PDZ or The fusion of SHM domain or part and the tandem compound fusion for the element that interacts, express, purify and investigate fusion respectively The influence of vigor, stability to each enzyme etc., ideal amalgamation mode and combination are further screened, and obtain high work Property fusion protein, and in vitro carry out assembling checking and characterize.Utilize non denatured electrophoresis, molecular-exclusion chromatography, laser particle The technology such as measure, AFM (AFM), ESEM (SEM) and dynamic light scattering, is detected with reference to catalytic activity, is investigated The putting in order of each enzyme, ratio, amalgamation mode etc. to the structure of compartmentation, assembling process and the influence for cascading catalytic activity, It is determined that optimal packaging technology.
(3) different type (flexible, rigidity), the connection small peptide of different length (1nm~30nm) and different companies are studied Connect the active influence of formation of the site (or region) to compartmentation, space structure (geometric configuration) and multienzyme.Investigate simultaneously Before and after assembling, the change of substrate and the intermediate product transmission efficiency between three enzymes, and the distance between three enzymes, ratio and Activity center establishes reaction Kinetics Model, investigates and verify multienzyme towards the influence to substrate and intermediate product transmission efficiency The compartmentation effect of body, disclose its efficient cascade mechanism of catalytic reaction.
(4) the optimal assembling mode established according to extracellular assembling research institute, passes through fusion protein being total in Escherichia coli Expression, realize three enzymes in intracellular without support compartmentation self assembly, and investigate different expression vectors and assembling primitive table Influence up to amount, ratio, tandem compound mode etc. to the structure and catalytic activity of the multienzyme of formation, it is determined that the intracellular optimized Assembly model and condition.Determined using molecular fluorescence complementary technology and laser co-focusing, investigate expression, the interaction element of enzyme Influence of the combination to multienzyme self assembly, monitor self assembling process and form the dynamic of compartmentation multienzyme in the process State change, assembling process, which neutralize, is completed the distribution situation of rear multienzyme in the cell etc., discloses the formation of intracellular multienzyme Process and mechanism.
(5) Host Strains-Escherichia coli are carried out with gene editing using CRISPR/cas9 technologies, transformation is possible to influence clothing The metabolic pathway of health acid generation.The related gene being related in glycolytic cycle and TCA circulations, including suppress lactic acid accessory substance and lead to Road, formic acid by-product path, acetic acid by-product path etc., metabolic pathway is carried out according to optimal direction, reduce the damage of C atoms Consumption, so as to strengthen the concentration of intermediate product citric acid and cis-aconitic, reaches the purpose of high-yield itaconic acid.
(6) by determining intracellular substrate (glucose), intermediate product (citric acid, cis-aconitic), product (clothing health Acid) and accessory substance (formic acid, acetic acid, lactic acid, butanedioic acid, isocitric acid etc.) content change, research assembling and metabolic pathway The influence to itaconic acid synthesis path and metabolic fluxes is transformed, the condition and factor for influenceing itaconic acid synthesis is investigated, furthers elucidate In Escherichia coli itaconic acid synthesize path and molecular mechanism, and by regulate and control each enzyme expression quantity and formed multienzyme amount come Regulate and control the metabolic pathway of intermediate product, realize the optimum balance that TCA circulations synthesize with itaconic acid, reach itaconic acid synthesis path Optimize.
(7) to the colibacillus engineering obtained through multienzyme self assembly and metabolic pathway transformation and optimization, carry out shaking flask and Upper tank fermentation, determines optimal fermentation condition, foundation is provided for itaconic acid industrialized production.
In a word, according to the present invention, a kind of self assembly by producing itaconic acid relevant enzyme and metabolic pathway transformation skill are disclosed Art is combined, the method that itaconic acid is efficiently produced in Escherichia coli.Three enzymes of key of itaconic acid will be synthesized：Citrate synthase GltA, cis-aconitic enzyme acnA and aconitate decarboxylase cadA pass through design and rational selection and profit as main assembling object Three enzymes are subjected to group of connecting with two Thermodynamic parameters Protein S H3/SH3lig and PDZ/PDZlig respectively with polypeptide structure element Fusion is closed, using the interaction of albumen pair, three enzymes is self-assembled into close structure, aligned orderly in the cell, have one Determine the multienzyme complex of compartmentation microenvironment, and utilize Crispr/cas9 technological transformation metabolic pathways, regulate and control the metabolism of citric acid Approach, the optimum balance that TCA circulations synthesize with itaconic acid is realized, reaches the optimization of itaconic acid synthesis path, and finally realize The efficiently production itaconic acid, therefore the present invention provides new way for the industrialized production of itaconic acid in Escherichia coli.
Brief description of the drawings
Fig. 1 is according to gltA-PDZ-SH3 provided by the invention, acnA-PDZlig, and the enzyme intracellulars of cadA-SH3lig tri- are from group Fill simulation drawing；
Fig. 2 is e. coli k12 MG1655 metabolic pathways transformation schematic diagram.
Fig. 3 is the itaconic acid fermentation comparative result figure of different experiments bacterial strain.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described.It should be understood that following examples are merely to illustrate this Invention is not for limitation the scope of the present invention.
The construction and expression of the enzyme self assembly plasmid of embodiment 1 three
Glt A (being predicted as dimer enzyme) gene comes from corynebacterium glutamicum (GenBank ID:1018824), acnA (being predicted as monomeric enzyme) derives from corynebacterium glutamicum (GenBank ID:1019512), cadA (being predicted as dimer enzyme) comes Come from Aspergillus terreus (GenBank ID:BAG49047.1).Gene gltA, acnA, cadA are obtained by biotech firm's full genome synthesis , codon optimization.In addition, gene PDZ, SH3 have been implemented in this laboratory, -20 DEG C are preserved in.
The method that plasmid construction uses one-step cloning, all plasmid backbones insert strong promoter BBa_ in this experiment J23100 is transformed, and target gene and carrier are carried out into Gene Fusion.GltA and cadA is gene constructed in plasmid pQE-30 In, obtain plasmid pQE30-cadA-gltA, using the method for one-step cloning insert respectively SHM albumen pair aglucon SH3lig and Two Thermodynamic parameters albumen form pQE30-cadA-SH3lig-TAA-gltA-PDZ-SH3 to part PDZ-SH3；It is existing AcnA is gene constructed on pACYCDuet-1 plasmids, and the aglucon of PDZ interaction proteins pair is inserted using the method for one-step cloning PDZlig forms pACYCDuet-acnA-PDZlig.Three enzyme assembling forms are as shown in Figure 1.
On the basis of the above-mentioned pQE30-cadA-SH3lig-TAA-gltA-PDZ-SH3 for having been built up completing, a step is utilized Cloning process builds two plasmids pQE30-cadA-SH3lig and pQE30-gltA-PDZ-SH3 in addition, by this successfully constructed Two plasmids and pACYCDuet-acnA-PDZlig convert into e. coli bl21 (DE3) respectively, from inducing temperature, initially Many-sided research gltA, acnA and cadA such as concentration of glucose, medium pH and fermentation time merges different interaction proteins The expression of element, condition optimizing is carried out, and isolates and purifies and obtain each fusion protein, studied for extracellular multienzyme self assembly, Experiment finds that optimal inductive condition is：Inducing temperature is 30 DEG C, and MM medium pHs are 7.0, induction time 20h.Isolate and purify Each fusion protein acnA-PDZlig, cadA-SH3lig, gltA-PDZ-SH3 are obtained, is studied for extracellular multienzyme self assembly.
Embodiment 2gltA, acnA and cadA zymologic property characterize
The plasmid of single expression gltA, acnA and cadA albumen is built, as control after isolating and purifying.Three are determined respectively The catalytic activity of enzyme and its protoenzyme is merged, studies its zymologic property.Enzyme activity assay method is as follows：
1) aconitate decarboxylase cadA enzyme activity is detected by itaconic acid concentration, using cis-aconitic and enzyme reaction, Using the growing amount of HPLC methods detection itaconic acid.Testing conditions are：Inertsil ODS -3column, 0.1M ammonium dihydrogen phosphates (pH 2.6) is used as mobile phase, and column temperature is 40 DEG C.One unit enzyme activity, 1 micromole of catalysis generation per minute under the conditions of being defined as 37 DEG C The enzyme amount of itaconic acid.
2) aconitase acnA detection methods are that enzyme reacts with sodium citrate under pH7.0 buffer solutions, utilize isocitric acid Kit is detected.One unit enzyme activity is defined as the enzyme amount of 1 micromole's isocitric acid of catalysis generation per minute under the conditions of 37 DEG C.
3) citrate synthase gltA Enzyme activity assay can utilize the citrate synthase testing cassete of commercialization to be measured.
Testing result finds to add interaction protein to rear, and the enzyme activity influence on each single enzyme is smaller, keeps original enzyme activity More than 75%.
The multienzyme assembled in vitro of embodiment 3 is tested
In extracellular progress multienzyme assembling and verify and characterize, i.e., using non denatured electrophoresis, molecular-exclusion chromatography, laser particle size The technologies such as analysis, dynamic light scattering and Activity determination, concentration, ratio and the built-up time of each enzyme assembling are investigated to assembling The influence of journey and cascade catalytic activity, and determined by means such as AFM (AFM), SEM (SEM) The form and surface topography of multienzyme after assembling, characterize the structure and compartmentation geometric properties of multienzyme.Intracellular assembling activity Verified by the amount (HPLC detections) of the product itaconic acid for synthesis of finally fermenting.
The checking and optimization of the intracellular self assembly of embodiment 4
First, separation assembling checking is carried out to albumen using the method for Ni-NTA ni-sepharose purifications.Obtained according to above structure It is cadA-SH3lig-gltA- (linker-PDZ-linker- that plasmid, which carries out cotransformation Host Strains and obtains one group of assembling bacterial strain, SH3)+acnA-PDZlig and corresponding control strain gltA-cadA+acnA, wherein, linker is that common 5nm is rigidly linked Arm ER/K, alternatively, protein sequence KAKLKEEEERKQREEEERIKRLEELAKRKEEERK.Although we have design excessive Kind assembly model, but above-mentioned this assembly model is that selected effect is optimal at present.Three kinds of albumen are carried out in Escherichia coli Coexpression, by the albumen of solvable coexpression in the method purifying supernatant of histidine-tagged purifying after smudge cells, eluting During destination protein, assembling albumen elutes under a certain concentration imidazole elution, and reference protein gltA-cadA+acnA Due to and it is unassembled, in the absence of the connection in structure, therefore three albumen can be eluted under various concentrations imidazole elution, then It can verify that albumen completes self assembly in the cell, form the multienzyme reactor of relative close.
Three fusion proteins are co-expressed in e. coli k12 MG1655, investigate the enzyme self assembly situation of intracellular three, And utilize three molecular fluorescence complementary technologies detection assembling process.By fluorescin, mIrisFP points are mIN150, mIN (151-165) With mIC166 three parts, merged respectively with assembling albumen, build plasmid：pQE-cadA-SH3lig-mIN(151-165)- TAA-gltA-PDZ-SH3-mIC166-TAA, pACYCDuet-acnA-mIN150-PDZlig-TAA.The double fluorescence that will be built Plasmid converts to be co-expressed into Host Strains, after protein expression, the effect of each self-interaction element and gltA, Self assembly under cadA dimerization, intracellular change in fluorescence situation is determined using laser co-focusing, observe three enzyme compartmentations from group The dynamic changing process and its assembling position of dress, thalline are red under 561nm exciting lights, are green under 488nm exciting lights, sharp Monitor intracellular compartmentation assembling process in real time with laser confocal fluorescence microscope and form the dynamic of multienzyme in the process State change, assembling process, which neutralize, is completed the distribution situation of rear multienzyme in the cell, discloses the formation of multienzyme complex Journey and mechanism.
Embodiment 5 is transformed based on CRISPR/cas9 technology Metabolism of E. coli approach
CRISPR/cas9 technologies are a kind of new gene editing means, easy to operate, and editorial efficiency is high, and cost is relatively low, Miss rate is low, and multiple genes can be practiced shooting simultaneously, therefore the present invention uses side of the technology as gene editing Method, the gene being related to are as shown in Figure 2.
Knock out plasmid pCas and pTargetF and be purchased from addgene, be preserved in the refrigerator-freezer of -40 DEG C of this laboratory.Specific side Method is as follows：
1) pTargetF-N20 plasmids are built：According to gene order to be knocked out, its N20 is designed using crispr.mit.edu Sequence, choose the N20 that score is high and miss rate is low and be building up on pTargetF plasmids, obtain plasmid pTargetF-N20；
2) one-step cloning method structure pTargetT- Δ gene plasmids：Using Escherichia coli MG1655 genomes as template, PCR Upstream and downstream sequence fragment up, down of gene to be knocked out are expanded, the connection of multiple clips one-step cloning method is double through EcoR I/Hind III PTargetF-N20 linear fragments that digestion obtains, pTargetF-N20+up and pTargetF-N20+down fragments, conversion The α competence of E.coli DH 5, the LB solid mediums containing Spec are coated on, 37 DEG C are inverted culture, picking monoclonal, bacterium solution PCR Verify and be sequenced, upgrading grain obtains pTargetT- Δ gene plasmids after being sequenced successfully.
3) gene editing：Plasmid pTargetT- Δ gene electricity is transferred into Escherichia coli MG1655-pCas electricity, and to turn competence thin Born of the same parents, the LB solid mediums containing kanamycins and spectinomycin are coated on, 30 DEG C are inverted culture, and picking monoclonal, bacterium solution PCR is tested Card；
4) Plasmid elimination：1. eliminate pTargetT：Monoclonal of the picking containing pCas and pTargetT, be inoculated in containing Kan and IPTG LB fluid nutrient mediums, 30 DEG C of shaken cultivations, after appropriate dilution, it is coated on the LB solid mediums containing Kan, 30 DEG C of inversions Culture, picking monoclonal, pTargetT eradicating efficacies are verified by detecting its sensitiveness to Spec；2. eliminate pCas：Will be upper State bacterium solution to be inoculated in non-resistant LB fluid nutrient mediums, shaken cultivation is stayed overnight to eliminate temperature sensitive type plasmid pCas.
5) bacterium checking is knocked out：Select monoclonal and enter performing PCR checking, carried out from two groups of primers, one group special to knock out gene Specific primer, another group is knockout upstream region of gene and the primer of design, and first group of PCR is verified without band if successful knockout, Second group of PCR stripe size is up+down；If knock out it is unsuccessful if first group of PCR stripe size be gene original size, second Group PCR stripe sizes are that gene original size adds up+down.Select the correct monoclonal of checking and knock out bacterium progress in next step in fact Test.
6) prepared by gene knockout bacterium competence cell：TSS methods prepare knock-out bacterial strain competence, take a pipe competence during conversion Bacterium, DNA is added, ice bath after gently mixing.LB nutrient solutions are added, 37 degree of gentle shaken cultivations, coating, are put in after room temperature placement 37 DEG C of constant incubators are incubated overnight, and select the transformed bacteria progress subsequent experimental that work(is had been converted to through PCR checking double-mass models.
The Escherichia coli fermentation condition optimizing of embodiment 6 and activity checking
Each assembling plasmid built is co-expressed in through the improved Escherichia coli of metabolic pathway, with unassembled Engineering bacteria is control group, carries out fermenting experiment.Carbon source is used glucose as, is fermented some time at 30 DEG C, monitoring pH changes, Zymotic fluid is taken to be analyzed in Each point in time.Organic acid detection method is as previously described.
Fermentation medium is optimized, including：Composition, C/N ratios, pH, temperature, incubation time, the substrate of culture medium are dense Degree, substrate addition manner etc., timing sampling, HPLC detect the generation of product itaconic acid, continue to optimize condition of culture to determine Optimal conditions of fermentation and production technology.Fermentation medium is improvement MM culture mediums, and composition includes：6.8g/LNa2HPO4, 3g/ LKH2PO4, 0.5g/LNaCl, 1g/LNH4Cl, 0.49g/LMgCl, 0.1mmol/L CaCl2, 1mg/L thiamines, 30g/L Portugal Grape sugar, 4g/L α-ketoglutaric acids, 1000 × trace element (2.86g H3BO3, 1.81g MnCl2·4H2O, 0.222g ZnSO4· 7H2O, 0.39g Na2MoO4·2H2O, 0.79g CuSO4·5H2O, 0.049gCo (NO3)2·6H2) and corresponding antibiotic O.Hair During ferment, cultivating 20h first at 37 DEG C, under the conditions of 400rpm increases Fungal biodiversity.After 20h, temperature is changed to 30 DEG C, 550rpm ferments, and stream plus 4M NaOH control zymotic fluid pH=6.7 in real time, and being constantly passed through filtrated air makes dissolved oxygen control 30% More than.Sampled every 6h, measure glucose concentration, when concentration of glucose is less than 10g/L, add 300g/L glucose manually, 52.5g/L(NH4)2SO4, 4g/L α-ketoglutaric acids so that concentration of glucose is not less than 20g/L.After fermentation 100 hours, Portugal is measured Grape sugar consumption finishes, fermentation ends.
Self assembly bacterium and experiment contrast bacterial strain α-ketoglutaric acid, citric acid, different during the fermentation are determined using HPLC methods The change of citric acid, cis-aconitic and itaconic acid content, cascade catalysis path and itaconic acid synthesis of the research assembling to three enzymes The influence of metabolic fluxes, the strategy in previous work using PDZ interaction proteins to progress multienzyme assembling, enters in original strain Go and compared, as shown in figure 3, assembling bacterium is compared with the unassembled bacterium, output increased 63%；Further in original wild-type host bacterium Middle knockout icL, icd, poxB, pflB and ldhA genes, and multienzyme assembling is carried out in bacterium is knocked out, shake flask fermentation is carried out, such as Shown in Fig. 3, Escherichia coli knock out bacterium in, using albumen to assembling after, its synthesize itaconic acid ability obtained significantly Improve, be original assemble bacterium 4.3 times after finally knocking out 5 genes, 100h shake flask fermentations have finally reached 3g/L, this knot Fruit also demonstrates that the transformation of metabolic pathway is equally very significant considering that the transformation of optimal metabolic pathway is same to itaconic acid production When knock out icd, icL, poxB, pflB and ldhA, so as to obtain gene knock-out bacterial strain.
Above-described, only presently preferred embodiments of the present invention is not limited to the scope of the present invention, of the invention is upper Stating embodiment can also make a variety of changes.What i.e. every claims and description according to the present patent application were made Simply, equivalent changes and modifications, the claims of patent of the present invention are fallen within.The not detailed description of the present invention is Routine techniques content.

Claims (10)

  1. A kind of 1. method that itaconic acid is produced in Escherichia coli, it is characterised in that methods described is one kind while utilizes multienzyme Package technique and metabolic pathway renovation technique realize in Escherichia coli the efficiently method of production itaconic acid, methods described include with Lower step：
    1) three key enzymes for synthesizing itaconic acid are carried out being self-assembly of multienzyme complex, three key enzymes in the cell Including：Citrate synthase gltA, cis-aconitic enzyme acnA and aconitate decarboxylase cadA；
    2) metabolic pathway transformation is carried out to Escherichia coli using Crispr/cas9 technologies；And
    3) Escherichia coli that the multienzyme complex is transferred to by metabolic pathway transformation are built into a kind of colibacillus engineering, led to Everfermentation carries out the production of itaconic acid.
  2. 2. according to the method for claim 1, it is characterised in that the step 1) is using two Thermodynamic parameters albumen SH3/SH3lig and PDZ/PDZlig fusion citrate synthases gltA, cis-aconitic enzyme acnA and aconitate decarboxylase cadA, GltA-PDZ-SH3, acnA-PDZlig, cadA-SH3lig are formed respectively.
  3. 3. according to the method for claim 2, it is characterised in that by citrate synthase gltA, cis-aconitic enzyme The multienzyme complex that acnA and aconitate decarboxylase cadA are self-assembly of is cadA-SH3lig-gltA- (linker- PDZ-linker-SH3)+acnA-PDZlig。
  4. 4. according to the method for claim 1, it is characterised in that the step 2) is to big using Crispr/cas9 technologies Enterobacteria knocks out part glycolytic cycle, the gene in tricarboxylic acid cycle and accessory substance branch road.
  5. 5. according to the method for claim 4, it is characterised in that utilize metabolism of the Crispr/cas9 technologies to Escherichia coli Approach transformation includes suppressing lactic acid accessory substance path, formic acid by-product path, acetic acid by-product path.
  6. 6. according to the method for claim 5, it is characterised in that the gene knocked out in metabolic pathway transformation includes： Lactate dehydrogenase gene ldhA, pyruvate formate-lyase pflB, acetonic acid oxidase gene poxB, isocitrate lyase base Because of at least one of icL and isocitric acid dehydrogenase gene icd.
  7. 7. according to the method for claim 6, it is characterised in that the gene knocked out in metabolic pathway transformation includes： Lactate dehydrogenase gene ldhA, pyruvate formate-lyase pflB, acetonic acid oxidase gene poxB, isocitrate lyase base Because of icL and isocitric acid dehydrogenase gene icd.
  8. 8. according to the method for claim 1, it is characterised in that the fermentation condition of the colibacillus engineering is：Using Minimal medium, 40~100h of fermented and cultured at 7.0,30 DEG C of pH.
  9. 9. according to the method for claim 8, it is characterised in that the composition of the minimal medium includes：6.8g/ LNa2HPO4, 3g/LKH2PO4, 0.5g/LNaCl, 1g/LNH4Cl, 0.49g/LMgCl, 0.1mmol/L CaCl2, 1mg/L thiamines Element, 30g/L glucose, 4g/L α-ketoglutaric acids, 1000 × trace element (2.86g H3BO3, 1.81g MnCl2·4H2O, 0.222g ZnSO4·7H2O, 0.39g Na2MoO4·2H2O, 0.79g CuSO4·5H2O, 0.049g Co (NO3)2·6H2O)。
  10. 10. according to the method for claim 8, it is characterised in that the fermentation of the colibacillus engineering includes following step Suddenly：
    1) first at 37 DEG C, the 15~20h of colibacillus engineering is cultivated under the conditions of 300~500rpm increases Fungal biodiversity It is long；
    2) after 15~20h, temperature is changed to 30 DEG C, 550rpm fermentations, stream plus 4M NaOH control zymotic fluid pH=6.7 in real time, no It is open close enter filtrated air make dissolved oxygen control more than 30%；
    3) sampled every 6h, measure glucose concentration, when concentration of glucose is less than 10g/L, add 300g/L glucose manually, 52.5g/L(NH4)2SO4, 4g/L α-ketoglutaric acids so that concentration of glucose is not less than 20g/L；
    4) after fermenting 100 hours, measure glucose consumption and finish, fermentation ends.
CN201711230812.5A 2017-11-29 2017-11-29 A kind of method that itaconic acid is produced in Escherichia coli Pending CN107723317A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11208671B2 (en) 2019-07-12 2021-12-28 City University Of Hong Kong Recombinant cell and method of producing itaconic acid
CN114717170A (en) * 2021-01-05 2022-07-08 中国科学院分子植物科学卓越创新中心 Host cell for heterogeneously synthesizing flavonoid compound and application thereof

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CN101886045A (en) * 2009-05-11 2010-11-17 财团法人工业技术研究院 Genetically modified microorganisms for producing itaconic acid with high yields
CN104928272A (en) * 2014-03-19 2015-09-23 财团法人工业技术研究院 Fusion polypeptide, nucleic acid molecule encoding the same, and method for producing itaconic acid using the same

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Publication number Priority date Publication date Assignee Title
CN101886045A (en) * 2009-05-11 2010-11-17 财团法人工业技术研究院 Genetically modified microorganisms for producing itaconic acid with high yields
CN104928272A (en) * 2014-03-19 2015-09-23 财团法人工业技术研究院 Fusion polypeptide, nucleic acid molecule encoding the same, and method for producing itaconic acid using the same

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11208671B2 (en) 2019-07-12 2021-12-28 City University Of Hong Kong Recombinant cell and method of producing itaconic acid
CN114717170A (en) * 2021-01-05 2022-07-08 中国科学院分子植物科学卓越创新中心 Host cell for heterogeneously synthesizing flavonoid compound and application thereof
WO2022148377A1 (en) * 2021-01-05 2022-07-14 中国科学院分子植物科学卓越创新中心 Host cell of heterologous synthetic flavonoid compound, and use thereof
CN114717170B (en) * 2021-01-05 2024-06-04 中国科学院分子植物科学卓越创新中心 Host cell for heterologous synthesis of flavonoid compounds and application thereof

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