CN111411119A - Construction and application of recombinant escherichia coli for coupling production of pentanediamine and succinic acid - Google Patents
Construction and application of recombinant escherichia coli for coupling production of pentanediamine and succinic acid Download PDFInfo
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Abstract
The invention discloses construction and application of recombinant escherichia coli for coupling production of pentanediamine and succinic acid. The invention relates to lysine decarboxylase with improved pH tolerance after mutation modificationEcCadA‑M3Connected to an expression vector pCDFDuet-1 and introduced into a high-yield succinic acid production strainE.coliAFP111, a recombinant strain capable of coupling production of pentanediamine and succinic acid is obtainedE.coliSC 01. The cell coupling production of the pentanediamine and the succinic acid effectively solves the problem of pH neutralizer existing in the independent synthesis of the pentanediamine and the succinic acid, and is capable of fixing carbon dioxideAnd green synthesis of pentamethylene diamine and succinic acid with less greenhouse gas exhaust and carbon loss.
Description
Technical Field
The invention belongs to the field of preparation of pentanediamine and succinic acid, and particularly relates to construction and application of recombinant escherichia coli for coupling production of pentanediamine and succinic acid.
Background
1,5-pentanediamine (1, 5-pentanediamine), commonly known as 1, 5-diaminopentane, has a malodorous odor similar to ammonia gas, referred to as cadaverine. The substance is inflammable, highly toxic and highly irritant. Inhalation can be fatal by spasm, inflammation and edema of the larynx and bronchi, chemical pneumonia or pulmonary edema. The toxicities are manifested by cough, wheeze, laryngitis, shortness of breath, headache, nausea and vomiting, and will die in severe cases.
Intracellular pentanediamine is an extension of the lysine synthetic pathway, is produced by decarboxylation of lysine under the action of lysine decarboxylase, is present in putrefactive products as a carnitine amine, and is a component of odor generated by putrefaction of biological corpses like the decarboxylated product of ornithine, putrescine. This diamine is not only responsible for putrefactive action, but also produces a small amount of pentamethylenediamine in the living body's metabolism, and is a nitrogen-containing base having biological activity widely existing in the living body. Pentanediamine has wide application in agriculture, medicine and industry. In agriculture, the exogenously applied pentanediamine can improve fruit setting, promote fruit development and increase the fruit yield; medically, it can be used as a drug effective in the treatment of dysentery; industrially, the pentanediamine and the dibasic acid are polymerized to synthesize high-quality polymer materials. At present, 1,5-pentanediamine is synthesized by a chemical method mainly by taking glutaronitrile as a raw material, firstly converted into 5-aminopentanenitrile under the action of a catalyst, and further converted into the pentanediamine, wherein the reaction is usually carried out under high pressure. Through the selection and modification of the catalyst, the conversion rate of the glutaronitrile can reach 100 percent, and the selectivity of the 1,5-pentanediamine can reach 66.8 percent. The chemical method has high yield, but the reaction condition is severe, the requirement on equipment is high, the environmental pollution is large, and a certain amount of byproduct piperidine is contained in the final reaction product, so that the method is not suitable for large-scale popularization.
Succinic acid, also known as hyaluronic acid, is a common natural organic acid. From 1546
After being separated from the vitreous mesh for the first time in the year, the succinic acid is widely applied to the fields of agriculture, food, medicine and the like.
Succinic acid is an important chemical, can be used in the fields of surfactants, detergents, solvents and the like, can be used as a pH modifier, a flavor substance or an antibacterial agent in the food industry, and can also be used as an animal feed additive, a plant growth substance and the like. Succinic acid is also an important four-carbon compound in industry, and can be converted into a plurality of important chemicals, such as fatty acid, 1, 4-butanediol, tetrahydrofuran, y-butyrolactone and the like; the monomer can also be used for synthesizing biodegradable materials PBS (poly butyric acid hyaluronic acid) and polyurethane, and at present, more than 250 chemical products which take benzene as raw materials can be obtained by converting succinic acid, so the market demand of the succinic acid is continuously increased. The succinic acid sold in the market at present is produced by a chemical method by taking liquefied petroleum gas or petroleum as a raw material. The chemical method for producing the succinic acid has high cost, low succinic acid sales volume and yield and greatly limited application field.
Although pentanediamine and succinic acid can be efficiently synthesized by a biological method, a large amount of acid-base solution is used for neutralization of pH in respective production and purification processes, thereby generating a large amount of waste salt, so that the subsequent process is complicated, the problems caused by a pH neutralizer can be effectively solved by coupling the synthesis processes of the pentanediamine and the succinic acid, because the pH of a CO-production system can be adjusted by an alkaline product of the pentanediamine and an acidic product of the succinic acid, and furthermore, L i and the like in earlier researches are crystallized by an alcohol precipitation method by using ethanol as a solvent to obtain the hexanediamine dodecanedioic acid salt and are used for synthesizing PA6.12, so that the pentanediamine and the succinic acid in a fermentation broth can be directly salified and separated and crystallized from the fermentation broth by the alcohol precipitation method to obtain the pentanediamine succinic acid salt by using a coupling production pH adjustment strategy, the polymerization process of PA5.4 can be greatly simplified, and CO is needed for producing the succinic acid by anaerobic fermentation2Is used as a substrate to participate in the metabolic synthesis pathway of succinic acid. In contrast, CO2Will be released as a by-product in the course of catalyzing L-lys decarboxylation by lysine decarboxylase to produce pentanediamine, thereby coupling pentanediamine and succinic acidAnother advantage of the production process is the development of a green synthetic route that can fix carbon dioxide, reduce greenhouse gas emissions and carbon losses.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the construction and the application of the recombinant escherichia coli for coupling production of the pentanediamine and the succinic acid.
Constructing recombinant Escherichia coli for coupling production of pentanediamine and succinic acid by adding lysine decarboxylaseEcCadA-M3Constructing a high-yield succinic acid production strainE. coliIn AFP111, the lysine decarboxylaseEcCadA-M3The recombinant Escherichia coli SC01 is obtained from the enzyme with improved pH tolerance after mutation modification.
As an improvement, the lysine decarboxylase gene with improved pH tolerance after mutation modificationEcCadA-M3And the DNA fragment is connected with a vector pCDFDuet-1, transferred into a cloning vector Trans1-T1, subjected to preliminary screening by a L B plate, picked up and subjected to colony PCR verification and then sequenced.
As an improvement, a succinic acid-producing strain with high yield is preparedE. coliConversion of AFP111 to competence.
As an improvement, the constructed pCDFDuet-CadA-M3Conversion toE.coliConversion of AFP111 into competence to obtainE.coliSC01。
The recombinant Escherichia coli SC01 obtained by the construction is applied to the synthesis of pentanediamine and succinic acid.
As an improvement, the application comprises the following steps:
Has the advantages that:
compared with the prior art, the construction and application of the recombinant escherichia coli for coupling production of the pentamethylene diamine and the succinic acid have the advantages that:
1. because the pH of the co-production system can be adjusted by the alkaline product of the pentanediamine and the acidic product of the succinic acid, the problem caused by a pH neutralizer can be effectively solved by coupling the synthetic process of the pentanediamine and the succinic acid;
2. by utilizing a coupling production pH adjustment strategy, the product of the pentamethylene diamine and the succinic acid in the fermentation liquor can be directly salified and directly separated and crystallized from the fermentation liquor by an alcohol analysis method to obtain the pentamethylene diamine succinate, so that the polymerization process of PA5.4 is greatly simplified;
3. the coupling of the production process of the pentanediamine and the succinic acid can fix carbon dioxide, reduce greenhouse gas emission and carbon loss, greatly simplify the process and save the cost.
Drawings
FIG. 1 shows recombinant strainsE.coliSDS-PAGE patterns of L-lysine decarboxylase expression in AFP111, M: marker, 1:E.coliSC01 uninduced group cell disruption supernatant; 2:E.coliSC01 did not induce precipitation of cell debris; 3:E.coliSC01 induced group cell disruption supernatant; and 4:E.coliSC01 induced precipitation of cell debris;
FIG. 2 shows recombinant strainsE.coliL-lysine decarboxylase enzyme activity in AFP111(ii) a condition;
FIG. 3 is a drawing showingEcCadA-M3Expression of (2) against the host strainE.coliEffect of biomass and succinic acid production during the metabolism of AFP111, wherein (a): over-expressionEcCadA-M3Biomass to the host strain; (B) the method comprises the following steps Over-expressionEcCadA-M3Influence on succinic acid yield.
Detailed Description
Technical means which are not mentioned in the following examples are all conventional technical means in the field. The materials and instruments used, are also commercial materials.
In the experiment, Escherichia coli AFP111 was donated by university of NanoIlno, Escherichia coli Trans1-T1 and plasmid pETDuet-1 were all commercial products and can be purchased conventionally.
Example 1E.coliConstruction of SC01 expression Strain
1、pCDFDuet-CadA-M3Construction of plasmids
Selecting the existing lysine decarboxylase with improved pH tolerance after mutation modification in a laboratoryEcCadA-M3(see patent application No. 201810787121.3), connecting with vector pCDFDuet-1, transferring into cloning vector Trans1-T1, primarily screening by L B plate, picking up points, performing colony PCR verification, and sequencing;
inoculating the positive strain to 5ml of L B/Sm liquid culture medium, wherein the L B/Sm liquid culture medium comprises 10 g/L of peptone, 5 g/L of yeast powder and 5 g/L of sodium chloride, performing shaking culture at 37 ℃ and 200rpm overnight, extracting plasmid pCDFDuet-CadA-M3。
2、E.coliPreparation of AFP111 chemocompetence
Selecting a production strain with high succinic acid yieldE.coliAFP111, inoculated into 5ml L B/Cm/KanR liquid medium, L B/Cm/KanR liquid medium composed of peptone 10 g/L, yeast powder 5 g/L, sodium chloride 5 g/L, at 37 ℃, under 200rpm conditions oscillation culture overnight to obtain seed liquid.
Transferring the seed liquid to 50ml L B/Cm/KanR liquid culture medium, wherein the L B/Cm/KanR liquid culture medium comprises peptone 10 g/L, and yeastPerforming shake culture of mother powder 5 g/L and sodium chloride 5 g/L at 37 deg.C and 200rpm until OD600 is about 0.6, ice-cooling for 15min, centrifuging at 4 deg.C for 10min at 6000r, re-suspending with 20ml sterilized 0.1mM calcium chloride, ice-cooling for 10min, centrifuging at 4 deg.C for 10min, collecting the thallus (repeating twice), re-suspending with 1ml sterilized 0.1mM calcium chloride +15% glycerol, and ice-cooling for 5min to obtain the final productE.coliAFP111 transformation competent Each 100. mu. L was sub-packaged in sterile centrifuge tubes of 1.5m L and stored at-80 ℃.
3. Construction ofE.coliSC01
The constructed pCDFDuet-CadA-M3Conversion toE.coliConversion of AFP111 into competence to obtainE. coliSC01。
Example 2 verification of coupled production of pentanediamine and succinic acid
1. L-lysine decarboxylase expression verification
Succinic acid production strainE.coliAFP111 is a strain with gene knockoutpfl、ldhAAndptsGthe genetically engineered strain of (1). To strengthenE. coliA catalytic synthesis route from L-lysine to pentanediamine in AFP111, which has improved pH tolerance after mutation modificationEcCadA-M3Connected to an expression vector pCDFDuet-1 and introducedE. coliIn AFP111, a recombinant strain for co-production of pentanediamine and succinic acid is constructedE. coliSC 01. The strain SC01 is induced and then subjected to cell disruption and SDS-PAGE protein electrophoresis.
As a result, as shown in FIG. 1, a clear soluble protein overexpression band was observed around the molecular weight of 78 kDa, which is consistent with the size of the target protein. However, becauseEcCadA-M3Is derived fromE.coliSo that the decarboxylase is even inE.coliLittle bulk expression of CadA was also observed, but not induced during metabolism.
2. L-lysine decarboxylase in host strainE. coliEnzyme activity verification after expression in AFP111
Will be provided withE. coliSC01 was used to catalyze the decarboxylation of 150 g/L of L-lysine to pentanediamine the results are shown in FIG. 2, with exogenous addition of 0.1mM P L P, half of the reactionAfter hours, the resulting pentamethylenediamine concentration was 101.2 g/L, the yield was 1.775 mol/g (DCW)/h.Compare, as reported in the literature, Tonomri et al, for recombinant L DC overexpressionE. coliThe cells are used for whole-cell transformation to generate the pentanediamine after heat treatment, low-temperature freeze thawing and other treatments, the final yield of the pentanediamine is only 0.187 mol/g (DCW)/h, and the strain containing L DC mutant is utilized by the plum ketone et alE. coliL N3014, to produce pentamethylenediamine with a final pentamethylenediamine yield of 0.887 mol/g (DCW)/h,EcCadA- M3in thatE. coliThe expression and the enzyme activity of the AFP111 are not influenced at all, and the AFP111 can be used as a biocatalyst to efficiently catalyze and generate an appreciable amount of pentamethylene diamine.
3. L-lysine decarboxylase expression versus host strainE. coliVerification of influence of biomass and succinic acid yield in AFP111 metabolic process
The results in fig. 3A show that there was no significant difference in cell growth tendency and biomass between the two strains. Likewise, as shown in FIG. 3B, after anaerobic fermentation for 62 h,E. colithe succinic acid yield of SC01 metabolic synthesis is 11.6 g/L, and the yield to glucose is 0.85 g/g, wherein the succinic acid yield is equivalent to the succinic acid yield (10.81 g/L) synthesized by a control strain AFP 111.
These results show that it is possible to determine,EcCadA-M3does not affect the growth of the host strain and the metabolic synthesis of succinic acid. Thus, use of recombinant strainsE. coliSC01 is feasible to couple succinic acid biofermentation and pentanediamine bioconversion processes.
Example 3 coupled production of pentanediamine and succinic acid
100 μ L was cultured overnightE.coliThe SC01 seed solution is inoculated in L B culture medium containing 34 mg/L Cm resistance, 50 mg/L Kana resistance, 80 mg/L Sm resistance and 15 g/L glucose in 100 m L, and is cultured under the conditions of 37 ℃ and 200rpm, when OD600 reaches 0.6, IPTG with the final concentration of 0.5 mM is added to induce the strain, and the strain is continuously induced under the conditions of 37 ℃ and 200rpm for 20 h, the fermentation liquor supplemented with 20 g/L glucose is transferred to a sterilized and sealed blue-covered flask, CO is introduced into the blue-covered flask, and the fermentation liquor is dried and sterilized in water, and then the fermentation liquor is dried and is added with water to obtain the fermented liquor, wherein the fermentation liquor is prepared by adding the IPTG with the final concentration of 02Maintaining for 2min to ensure physical integrityThe method is characterized in that the pentamethylene diamine and the succinic acid are produced in an anaerobic environment by anaerobic culture, and the molar yields of the pentamethylene diamine and the succinic acid reach 25 percent and 37 percent respectively.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Claims (6)
1. The construction of recombinant Escherichia coli for coupling production of pentanediamine and succinic acid is characterized in that lysine decarboxylase is usedEcCadA-M3Constructing a high-yield succinic acid production strainE.coliIn AFP111, the lysine decarboxylaseEcCadA-M3The recombinant Escherichia coli SC01 is obtained from the enzyme with improved pH tolerance after mutation modification.
2. The construction of the recombinant Escherichia coli for coupling production of pentanediamine and succinic acid as claimed in claim 1, wherein the lysine decarboxylase gene with improved pH tolerance after mutation modification is usedEcCadA-M3And the DNA fragment is connected with a vector pCDFDuet-1, transferred into a cloning vector Trans1-T1, subjected to preliminary screening by a L B plate, picked up and subjected to colony PCR verification and then sequenced.
3. The construction of the recombinant Escherichia coli coupled to produce pentanediamine and succinic acid according to claim 1, wherein: preparation of high-yield succinic acid production strainE.coliConversion of AFP111 to competence.
4. The construction of the recombinant Escherichia coli coupled to produce pentanediamine and succinic acid according to claim 1, wherein: the constructed pCDFDuet-CadA-M3Conversion toE.coliConversion of AFP111 into competence to obtainE.coliSC01。
5. The use of the recombinant E.coli SC01 constructed on the basis of claim 1 for synthesizing pentanediamine and succinic acid.
6. Use according to claim 5, characterized in that it comprises the following steps:
step 1, L-lysine decarboxylase inE.coliValidation of expression in AFP 111;
step 2, L-lysine decarboxylase in host strainE.coliVerifying the enzyme activity after expression in AFP 111;
step 3, verifyingEcCadA-M3Expression of (2) against the host strainE.coliThe effect of biomass and succinic acid production during the metabolism of AFP 111;
step 4, 100. mu. L was cultured overnightE.coliThe SC01 seed liquid is inoculated in L B culture medium containing 34 mg/L Cm resistance, 50 mg/L Kana resistance, 80 mg/L Sm resistance and 15 g/L glucose in 100 m L, cultured under the conditions of 37 ℃ and 200rpm, when OD600 reaches 0.6, IPTG with the final concentration of 0.5 mM is added to induce the strain, the culture is continued under the conditions of 37 ℃ and 200rpm to synthesize pentamethylene diamine, after 20 h of induction, the fermentation liquid supplemented with 20 g/L glucose is transferred to a sterilized and sealed blue-covered flask, CO is introduced into the blue-covered flask, the fermentation liquid is added with the glucose with the final concentration of 0.5 mM, and the fermentation liquid is cultured and synthesized under the conditions of 37 ℃ and 200rpm, and after 20 h of induction, the fermentation liquid2And maintaining for 2min to ensure the anaerobic environment of the system, and performing anaerobic culture to produce the succinic acid.
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| CN113005155A (en) * | 2021-03-17 | 2021-06-22 | 南京工业大学 | Method for in-situ regulation of pH in decarboxylation process by using carbonic acid glycosidase |
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| CN113005155A (en) * | 2021-03-17 | 2021-06-22 | 南京工业大学 | Method for in-situ regulation of pH in decarboxylation process by using carbonic acid glycosidase |
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