CN115466758A - Method for producing D-psicose by fermenting escherichia coli with methanol and D-xylose - Google Patents

Method for producing D-psicose by fermenting escherichia coli with methanol and D-xylose Download PDF

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CN115466758A
CN115466758A CN202211129170.0A CN202211129170A CN115466758A CN 115466758 A CN115466758 A CN 115466758A CN 202211129170 A CN202211129170 A CN 202211129170A CN 115466758 A CN115466758 A CN 115466758A
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psicose
gene
coli
methanol
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范立海
刘美茗
郭强
郑辉东
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Qingyuan Innovation Laboratory
Fuzhou University
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Fuzhou University
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Abstract

The invention provides a method for producing D-psicose by fermentation of escherichia coli by using methanol and D-xyloseThe enterobacter is used as a host cell, and a path from methanol and D-xylose to synthesis of D-psicose is established by expressing exogenous genes of methanol dehydrogenase Mdh, 3-hexose-6-phosphate synthase Hps, 6-phospho-3-hexose isomerase Phi, D-psicose-6-phosphate epimerase AlsE and D-psicose-6-phosphate phosphatase A6PP in wild escherichia coli. The protein tag SUMO is introduced to improve the expression quantity of the AlsE protein. By knocking out byproduct pathway genesfrmABRKey genes of the pentose phosphate pathwayrpiAAnd key genes of glycolytic pathwaypfkA，pfkBThe recombinant strain is obtained to realize rational control of a carbon source, so that the Escherichia coli can efficiently synthesize D-psicose by using methanol and D-xylose.

Description

Method for producing D-psicose by fermenting escherichia coli by using methanol and D-xylose
Technical Field
The invention belongs to the field of metabolic engineering, and particularly relates to a method for producing D-psicose by fermenting escherichia coli by using methanol and D-xylose.
Background
D-psicose (D-Allulose), which is the C-3 epimer of D-fructose, is a rare zero-energy sugar, and has a sweet taste of 70% of that of sucrose. A large number of researches prove that the D-psicose has unique physicochemical properties and physiological functions, such as the effects of reducing blood fat and blood sugar, resisting inflammation, protecting nerves, eliminating the activity of Reactive Oxygen Species (ROS), treating atherosclerosis and the like, so the D-psicose has good market potential as an ideal sweetening agent and sucrose substitute, especially in the health care product and food industry.
D-psicose is a naturally occurring rare monosaccharide that is present in very small amounts in nature. The preparation method comprises a chemical synthesis method and a biological synthesis method. The chemical synthesis method comprises a selective aldol condensation synthesis method, a catalytic hydrogenation method, an addition reaction method, a Ferrier rearrangement method and the like. The chemical synthesis method has the problems of poor economy, serious environmental pollution, insufficient stereoselectivity and the like, and thus cannot be a mainstream method for preparing D-psicose. Compared with a chemical method, the biological enzyme catalysis method has the advantages of mild reaction conditions, high activity, low dosage, no toxicity, environmental friendliness and the like. However, the enzyme catalysis still has the problems of high production cost, long construction period, poor thermal stability and the like. In contrast, the microbial fermentation method is not only favorable for reducing the industrial production cost, but also conforms to the current green and environment-friendly production principle, and indicates the direction for synthesizing the D-psicose. Escherichia coli is transformed by a genetic engineering technology, so that the Escherichia coli can efficiently utilize methanol and D-xylose to synthesize D-psicose, and the yield of the D-psicose is greatly improved.
Disclosure of Invention
The invention aims to solve the problems and provides a method for producing D-psicose by fermenting Escherichia coli by using methanol and D-xylose. By expressing foreign genesmdh，hps，phi，alsE，a6PPObtaining the recombinant escherichia coli capable of preparing the D-psicose by using the methanol and the D-xylose as carbon sources, and knocking out a byproduct pathway genefrmABR， rpiA， pfkA，pfkBFinally, the recombinant strain for producing the D-psicose by using the methanol and the D-xylose is obtained, so that the high-efficiency utilization of the carbon source is realized, and the yield of the D-psicose is improved.
In order to achieve the purpose, the technical scheme of the invention is as follows:
d-psicose was produced using methanol and D-xylose in the metabolically engineered strain Escherichia coli JM109 (DE 3). Firstly, firstlyE.coliMethanol dehydrogenase gene required for expression in JM109 (DE 3)mdh3-hexose-6-phosphate synthase genehps6-phospho-3-hexose isomerase genephiD-psicose-6-phosphate epimerase genealsED-psicose-6-phosphate phosphatase genea6PP. The protein tag SUMO is introduced to improve the expression quantity of the AlsE protein. Then, by-product pathway gene was knocked outfrmABRKey genes of the pentose phosphate pathwayrpiAAnd key genes of glycolytic pathwaypfkA，pfkBAnd rational control of a carbon source is realized, so that the D-psicose is efficiently produced.
Further, the method for producing D-psicose by fermenting Escherichia coli with methanol and D-xylose specifically comprises the following steps:
(1) In thatE. coliRequired for expressing a path in JM109 (DE 3)Methanol dehydrogenase genemdh3-hexose-6-phosphate synthase genehps6-phospho-3-hexose isomerase genephiD-psicose-6-phosphate epimerase genealsED-psicose-6-phosphate phosphatase genea6PP. Getting through a path for synthesizing D-psicose by escherichia coli by using methanol and D-xylose to obtain a recombinant strainE. coli (Mdh, Hps, Phi, AlsE, A6PP)；
(2) Introducing protein tag SUMO, fusion expressing AlsE-SUMO protein, enhancing expression amount of AlsE protein in Escherichia coli, respectively expressing exogenous proteins AlsE and A6PP and exogenous proteins AlsE-SUMO and A6PP in wild type Escherichia coli to obtain recombinant strainE. coli(AlsE, A6 PP) and recombinant strainsE. coli(SUMO-AlsE, A6 PP). By using extracellular enzyme catalysis experiment, determining the effective action of adding SUMO label on the high-efficiency expression of AlsE protein in Escherichia coli, and expressing proteins Mdh, hps, phi, SUMO-AlsE and A6PP in Escherichia coli to obtain recombinant strainE. coli (Mdh, Hps, Phi, SUMO-AlsE, A6PP)；
(3) Knocking out a byproduct pathway gene of methanol utilization by using a lambda Red homologous recombination method on the basis of the step (2)frmABR(Gene ID：944988、944991、944986)Obtaining a recombinant strainE. coli(Mdh, hps, phi, SUMO-AlsE, A6PP, delta FrmABR) and product fermentation verification, and further constructing recombinant strainsE. coli(Mdh,. DELTA.FrmABR) and recombinant strainsE. coli(Mdh) validation of the knockout Gene by fermentationfrmABRThe effect on methanol consumption;
(4) In a bacterial strainE. coli(Mdh, hps, phi, SUMO-AlsE, A6PP,. DELTA.FrmABR) generpiA(Gene ID：947407)Obtaining a recombinant strainE. coli(Mdh, hps, phi, SUMO-AlsE, A6PP,. DELTA.FrmABR,. DELTA.RpiA) and performing product fermentation validation;
(5) In a bacterial strainE. coli(Mdh, hps, phi, SUMO-AlsE, A6PP,. DELTA.FrmABR,. DELTA.RpiA) knock-out of glycolytic pathway genespfkA(Gene ID:948412 Obtaining a recombinant strainE. coli(Mdh, hps, phi, SUMO-AlsE, A6PP,. DELTA.FrmABR,. DELTA.RpiA,. DELTA.PfkA) and performing product fermentation validation;
(6) In bacterial strainsE. coliKnocking out glycolytic pathway gene based on (Mdh, hps, phi, SUMO-AlsE, A6PP,. DELTA.FrmABR,. DELTA.RpiA,. DELTA.PfkA)pfkB(Gene ID：946230)，Obtaining the target strainE. coli(Mdh, hps, phi, SUMO-AlsE, A6PP,. DELTA.FrmABR,. DELTA.RpiA,. DELTA.PfkA,. DELTA.PfkB) and verified by fermentation.
Further, a key gene in the synthesis pathway of the product expressed in step (1). Wherein the genemdhFrom hookworm bacteria, geneshpsFrom Bacillus methanolicus, genephiFrom Methylococcus capsulatus, genesalsEFrom Escherichia coli, genea6PPFrom bacteroides fragilis. Sequence reference NCBI database, genesmdh、hps、phi、alsE、a6PPAre WP _013953014.1, WP _014707664, WP _064496767, S101520_02794 and BF9343_0892, respectively.
Furthermore, the protein tag SUMO in the step (2) is artificially synthesized, and forms a fusion protein SUMO-AlsE with the protein AlsE, so as to enhance the expression level of the protein AlsE.
Further, in step (3), the gene knockout step is carried out by the lambda-red homologous recombination methodfrmABRInhibiting methanol byproduct pathway, and knocking out gene for verificationfrmABRInfluence on methanol consumption in knocking out genesfrmABRExpression of foreign genes in Escherichia coli JM109 (DE 3) and wild Escherichia coli JM109 (DE 3)mdhObtaining a strainE. coli(Mdh,. DELTA.FrmABR) and strainsE. coli(Mdh), fermentation in LB medium supplemented with 240mM methanol to verify the effect of methanol consumption.
Further, the gene knockout sequence in the steps (4), (5), (6) and (7) is required to be in accordance withfrmABR、rpiA、pfkA、pfkBTo avoid metabolic pathway errors.
Compared with the prior art, the invention has the beneficial effects that:
in the invention, a recombinant strain is obtained by expressing an exogenous gene in wild escherichia coli, opening a way that the escherichia coli synthesizes D-psicose by using methanol and D-xylose, and knocking out a product way geneE. coli(Mdh, hps, phi, SUMO-AlsE, A6PP, delta FrmABR, delta RpiA, delta PfkA, delta PfkB), the strain can effectively utilize cheap methanol and D-xylose to produce D-psicose, the yield of the D-psicose is greatly improved, and finally the yield of the D-psicose reaches 23.92 mM.
Drawings
FIG. 1 is a diagram of a cell factory.
FIG. 2 shows the effect of AlsE protein expression optimization,
(A) The SDS-polyacrylamide gel electrophoresis method verifies that the fusion protein AlsE-SUMO increases the expression of the protein, the first lane is empty plasmid, the second lane is protein AlsE, the third lane is protein AlsE-A6PP, and the fourth lane is protein AlsE-SUMO-A6PP;
(B) The dephosphorylation effect of the protein AlsE and the fusion protein AlsE-SUMO is verified through extracellular enzyme catalysis;
(C) The extracellular enzyme catalytic reaction verifies the influence of the fusion protein AlsE-SUMO on the yield of D-psicose;
(D) And a mass spectrum of the product D-psicose.
FIG. 3 knock-out GenefrmABRThe effect on methanol consumption and product formation;
(A) Recombinant strainE. coli(Mdh, hps, phi, SUMO-AlsE, A6 PP) product fermentation profile;
(B) Recombinant strainE. coli(Mdh, hps, phi, SUMO-AlsE, A6PP,. DELTA.FrmABR) product fermentation profile;
(C) Recombinant strainE. coli(Mdh,. DELTA.FrmABR) and recombinant strainsE. coli(Mdh) verify the methanol consumption fermentation profile.
FIG. 4 is a fermentation diagram of the strain;
(A) Recombinant strainE. coli(Mdh, hps, phi, SUMO-AlsE, A6PP,. DELTA.FrmABR,. DELTA.RpiA) product fermentation profile;
(B) Recombinant strainE. coli(Mdh, hps, phi, SUMO-AlsE, A6PP,. DELTA.FrmABR,. DELTA.RpiA,. DELTA.PfkA) product fermentation profile;
(C) Recombinant strainE. coli(Mdh, hps, phi, SUMO-AlsE, A6PP,. DELTA.FrmABR,. DELTA.RpiA,. DELTA.PfkA,. DELTA.PfkB) product fermentation profile.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
Recombinant strainE. coli(AlsE, A6 PP), and recombinant strainsE. coli(SUMO-AlsE, A6 PP), in 37 ℃ containing 60 u g/mL Kanna resistant 30 u g/mL chloramphenicol resistant LB medium until OD600 reaches 0.8-1.0, adding 0.2 mM isopropyl-beta-D-thiogalactoside (IPTG) protein at 37 ℃ to over-express for 16 h,4 ℃ centrifugation method to collect cells, using 50 mM pH7.0 HEPES buffer with 0.1M sodium chloride added, washing cells twice. The cells were resuspended in the same buffer to a final cell concentration OD600 of 50. Taking a proper amount of cell suspension, and carrying out ultrasonic cell disruption in an ice water bath. Centrifuging, collecting supernatant for enzyme activity experiment, and storing the rest supernatant at-20 deg.C.
A molybdate reagent is prepared, the solvent is ultrapure water, the solutes are 15 mM ammonium molybdate and 100 mM anhydrous zinc acetate, and the pH is adjusted to 5.0 by concentrated hydrochloric acid. Formulation 10% ascorbic acid reagent was adjusted to pH 5.0 with 40% NaOH.
The enzyme-activated reaction system is 600 mu L, and contains 100 mM HEPES buffer solution with pH7.0 and 5 mM MgCl 2 The method comprises the steps of taking 10 mM fructose-6-phosphate as a substrate, adding a proper amount of crude enzyme liquid, carrying out enzyme activity reaction in a thermostatic water bath at 37 ℃, starting the reaction from the addition of the crude enzyme liquid, carrying out reaction for 25 min, immediately placing a centrifugal tube into an ice-water bath after the reaction is finished, stopping the reaction, adding 2 mL of molybdic acid reagent and 500 μ L of ascorbic acid reagent into 200 μ L of a sample, mixing uniformly, allowing the system to react fully for 15 min at 30 ℃, testing an absorption peak at a wavelength of 850 nm by using an ultraviolet spectrophotometer after the reaction is finished, and displaying that the dephosphorylation effect of the fusion protein SUMO-AlsE is 2-3 times that of the protein AlsE by using an experimental result. The remaining sample after completion of the enzymatic reaction was subjected to a 0.2 μm membrane filtration treatment, and the sample analysis was carried out by HPLC, and D-psicose was determined by using a chromatographic column Waters Sugar-Pak TM I (RID) with water as a mobile phase at a flow rate of 0.5 mL/min and a sample retention time of 20 min. The determination result shows that the yield of D-psicose after the addition of the tag protein SUMO is the yield of the tag-free egg4 times white.
Example 2
Verification of knockout genesfrmABRInfluence on methanol consumption and products
Methanol consumption verification, gene knockoutfrmABRExpression of foreign genes in Escherichia coli JM109 (DE 3) and wild Escherichia coli JM109 (DE 3)mdhObtaining a strainE. coli(Mdh,. DELTA.FrmABR) and strainsE. coli(Mdh), adding 250 mM methanol to 50 mL LB medium of the system, 60. Mu.g/mL Carna resistance, growing at 37 ℃ to OD600 of 0.4-0.6, adding 0.2 mM IPTG, sampling every 12 h during 60 h of culture period for cell concentration determination and measuring the concentration of methanol by HPLC. The methanol measurement conditions were an Aminex HPX-87H column (75X 300 mM RID) as a column chromatography and 0.5 mM H as a mobile phase 2 SO 4 The flow rate of the ultrapure water solution is 0.6 mL/min, and the sample retention time is 25 min. The experimental result shows that the gene is knocked outfrmABRThe methanol consumption of the strain was reduced by 30 mM.
Product yield verification, recombinant strainsE. coli(Mdh, hps, phi, SUMO-AlsE, A6PP,. DELTA.FrmABR) and recombinant strainsE. coli(Mdh, hps, phi, SUMO-AlsE, A6 PP) in LB medium supplemented with 70 Mm D-xylose, 160 mM methanol, 60. Mu.g/mL kanamycin resistance, 30. Mu.g/mL chloramphenicol resistance, at 37 ℃ until OD600 reaches 0.4-0.6, 0.2 mM IPTG was added, and samples were taken every 12 h during the incubation period of 60 h to determine the cell concentration and the fermentation broth was subjected to HPLC analysis. The analysis method of D-xylose and D-psicose is the same, and the result shows that the gene is knocked outfrmABRAfter that, the yield of D-psicose increased from 1.039 mM to 2.21 mM.
Example 3
There are various combinations of more than one of the knocked-out strains. The final D-psicose yield is shown in the following table, consistent with the expectation, the culture medium is LB culture medium, the substrate is 70 mMD-xylose, 160 mM methanol, the resistance is 60 mug/mL kanamycin resistance and 30 mug/mL chloramphenicol resistance, 0.2 mM IPTG is added when the OD600 of the cells reaches 0.4-0.6 during the initial fermentation, the culture time is 60 h, the culture temperature is 37 ℃, and the experimental result shows that the recombinant DNA is compared with the recombinant DNAStrain of bacillusE. coli(Mdh, hps, phi, SUMO-AlsE, A6 PP), recombinant strainsE. coliThe yield of D-psicose (Mdh, hps, phi, SUMO-AlsE, A6PP,. DELTA.FrmABR,. DELTA.RpiA,. DELTA.PfkA,. DELTA.PfkB) increased from 1.039 mM to 23.92 mM.
Figure 118633DEST_PATH_IMAGE001
The above description is only a preferred embodiment of the present invention, and all the equivalent changes and modifications made according to the claims of the present invention should be covered by the present invention.

Claims (6)

1. A method for producing D-psicose by fermentation of Escherichia coli with methanol and D-xylose is characterized by introducing exogenous genemdh、hps、phi、alsE、a6PPEstablishing a synthetic path for preparing D-psicose by using methanol and D-xylose as carbon sources, and knocking out by-product pathway genesfrmABRKey genes of the pentose phosphate pathwayrpiAAnd key genes of glycolytic pathwaypfkA、pfkBFinally, the recombinant strain for efficiently synthesizing the D-psicose is obtained.
2. The method for producing D-psicose by fermentation of Escherichia coli using methanol and D-xylose according to claim 1, wherein: the method specifically comprises the following steps:
1) By constructing recombinant vector, expressing exogenous proteins Mdh, hps, phi, alsE, A6PP in wild type Escherichia coli JM109 (DE 3) to obtain recombinant strainE. coli(Mdh, hps, phi, alsE, A6 PP), establishing a cell factory for synthesizing D-psicose with methanol and D-xylose as carbon sources;
2) By utilizing the SUMO label, fusion expression AlsE-SUMO protein is performed, the expression quantity of the AlsE protein in escherichia coli is enhanced, exogenous proteins AlsE and A6PP and exogenous proteins AlsE-SUMO and A6PP are respectively expressed in wild escherichia coli, and the recombinant strain is obtainedE. coli(AlsE, A6 PP) and recombinant strainsE. coli(SUMO-AlsE, A6 PP); further with the aid of extracellular enzyme catalysis experimentsAdding an SUMO label to effectively express the AlsE protein in the escherichia coli;
3) Knock-out of byproduct pathway genesfrmABR(Gene ID：944988、944991、944986)Key genes of the pentose phosphate pathwayrpiA(Gene ID：947407)And key genes of glycolytic pathwaypfkA(Gene ID：948412)，pfkB (Gene ID：946230)Obtaining a recombinant strainE. coli(Mdh, hps, phi, alsE, A6PP,. DELTA.FrmABR,. DELTA.RpiA,. DELTA.PfkA,. DELTA.PfkB), thereby achieving efficient synthesis of D-psicose.
3. The method for producing D-psicose by fermentation of E.coli with methanol and D-xylose according to claim 2, wherein the foreign gene expressed in E.coli JM109 (DE 3) in step (1)mdh、hps、phiWhereinmdhThe gene is from the hookworm bacteria, and the gene is from the hookworm bacteria,hpsthe gene is from the bacillus methanolicus,phithe gene is from Methylococcus capsulatus, the gene sequence is referred to NCBI database,mdh、hps、phiNCBI sequence numbers of the genes are WP _013953014.1, WP _014707664 and WP _064496767 respectively,alsEthe gene is from Escherichia coli K-12 strain,a6PPthe genes are from Bacteroides fragilis, the gene sequences are referenced to NCBI database,alsEanda6PPthe NCBI sequence numbers of the genes are S101520_02794 and BF9343_0892 respectively.
4. The method for producing D-psicose by fermentation of E.coli using methanol and D-xylose according to claim 2, wherein the AlsE protein in step (2) is fusion-expressed by SUMO tag in order to enhance the intracellular expression effect of AlsE protein, E.coli JM109 (DE 3) is cultured at 37 ℃ and induces the expression of AlsE-SUMO protein, centrifuged at 4 ℃, cells are suspended twice using HEPES buffer solution at pH7.0, then disrupted by sonication, centrifuged and the supernatant is taken, and the protein expression effect is analyzed by SDS-polyacrylamide gel electrophoresis.
5. The method for producing D-psicose by fermentation of E.coli with methanol and D-xylose according to claim 2, wherein the reaction system of the extracellular enzyme catalysis experiment in step (2) is 600. Mu.L, in which fructose-6-phosphate is 10 mM, magnesium chloride is 5 mM, HEPES buffer solution is 100 mM at pH7.0, and crude enzyme solution is 20. Mu.L, and the reaction conditions of the enzyme catalysis experiment are 37 ℃ for 25 min, and 400. Mu.L of the reaction solution is taken and analyzed by HPLC.
6. The method for producing D-psicose by fermentation of E.coli with methanol and D-xylose according to claim 2, wherein the gene knockout in step (3) is performed according tofrmABR、rpiA、pfkA、pfkBThe sequence of (2) is carried out, and the specific knockout method refers to a lambda-red homologous recombination method.
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