CN117448245A - Recombinant genetic engineering bacterium and preparation method of (6S) -5-methyltetrahydrofolate - Google Patents

Recombinant genetic engineering bacterium and preparation method of (6S) -5-methyltetrahydrofolate Download PDF

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CN117448245A
CN117448245A CN202311351532.5A CN202311351532A CN117448245A CN 117448245 A CN117448245 A CN 117448245A CN 202311351532 A CN202311351532 A CN 202311351532A CN 117448245 A CN117448245 A CN 117448245A
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recombinant
bacillus
genetically engineered
reductase
serine
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夏颖
张磊
赵华
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Shanghai Ruikang Biotechnology R & D Co ltd
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Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to a recombinant genetic engineering bacterium and a preparation method of (6S) -5-methyltetrahydrofolate. The invention combines key reaction enzyme and glucose dehydrogenase coenzyme circulation system in the natural (6S) -5-methyltetrahydrofolate synthesis path, and constructs a new process path for synthesizing (6S) -5-methyltetrahydrofolate by an enzymatic method in one pot. Furthermore, the invention also constructs recombinant genetic engineering bacteria capable of preparing the enzyme. The method for preparing (6S) -5-methyltetrahydrofolate has the advantages of mild condition, safety and high efficiency, and has good application prospect.

Description

Recombinant genetic engineering bacterium and preparation method of (6S) -5-methyltetrahydrofolate
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to a recombinant genetic engineering bacterium and a preparation method of (6S) -5-methyltetrahydrofolate.
Background
(6S) -5-methyltetrahydrofolate, (6S) -5-methyl tetrahydrofolate, (6S) -5-MTHF) is also known as levomethylfolic acid, active methylfolic acid, etc., which is the final active form of folic acid in the body, contains two chiral carbon atoms, and usually forms optical isomers at the 6-position carbon atom when the 5, 6-double bond is hydrogenated. (R, S) -5-MTHF is a chemically synthesized racemate of methyl folic acid, and (6S) -5-MTHF is a naturally occurring form of methyl folic acid. Only natural (6S) -5-MTHF can directly act on human body to exert corresponding curative effect, R type has no drug effect on human body, and excessive accumulation of R type in human body can also lead to increased risk of cardiovascular diseases.
The (6S) -5-MTHF is slightly soluble in water, sensitive to light and oxygen, and the (6S) -5-methyl calcium folate (Levomefolate calcium) is in the form of calcium salt, so that the (6S) -5-methyl calcium folate is better in water solubility and high in stability, and is the final preservation form of the current (6S) -5-MTHF bulk drug. The synthetic methods of (6S) -5-MTHF and its salts are mainly classified into three kinds, namely, chemical method, dihydrofolate reductase method and metabolic engineering method.
Chemical processes are also divided into chemical resolution processes and asymmetric hydrogenation. The chemical resolution method uses folic acid as a substrate to reduce the folic acid into Tetrahydrofolate (THF), optically pure (6S) -Tetrahydrofolate ((6S) -THF) is obtained through resolution of chiral auxiliary agents such as toluenesulfonic acid or benzenesulfonic acid, formylation reaction is carried out on the optically pure (6S) -Tetrahydrofolate ((6S) -THF) and formaldehyde under alkaline conditions, then borohydride is used for reduction, and finally the optically pure (6S) -Tetrahydrofolate is salified with calcium chloride to obtain white solid (6S) -5-methyltetrahydrofolate calcium; or synthesizing a final product (6R, 6S) -5-MTHF and then splitting the final product into (6S) -5-MTHF; the chemical resolution method has the main problems that the synthesized intermediate product THF and the final product 5-MTHF are easy to oxidize, the yield of the multi-step reaction product is not high, and the theoretical yield is halved after resolution. The asymmetric hydrogenation method takes folic acid as a starting material, takes Rh/BPPM complex and the like as a catalyst, synthesizes (6S) -THF through selective catalytic hydrogenation, and carries out formylation, reduction and salification subsequently, and the method has the biggest problem that the current optical purity is up to 90 percent.
The dihydrofolate reductase method has been published in 1971 as early as possible, and has been industrialized in recent years. Folic acid is reduced into dihydrofolic acid by zinc powder, then dihydrofolate reductase (Dihydrofolate reductase, DHFR) is coupled with gluconic acid dehydrogenase (Gluconate dehydrogenase, GDH) or hydrogenase to construct an NAD (P) H coenzyme circulation system, natural (6S) -THF is synthesized by utilizing the specificity of the enzyme, and then (6S) -5-MTHF is synthesized by formylation and reduction. Compared with the pure chemical method, the method has high chiral purity and considerable yield.
The metabolic engineering method relies on the metabolism of thalli in vivo, and the (6S) -5-MTHF can be synthesized by using glucose, and researches mainly focus on the transformation of one-carbon metabolic pathways of escherichia coli, bacillus subtilis and lactococcus lactis so as to improve the yield of the (6S) -5-MTHF. The thallus is characterized in that folic acid is utilized, but the product (6S) -5-MTHF thallus cannot be actively discharged, so that the product is generally characterized by intracellular accumulation, the thallus needs to be broken for extraction, an intermediate THF is a biological coenzyme, a plurality of amino acids are associated for synthesis and metabolism, folic acid circulation is an important way of one-carbon metabolism in organisms, and the metabolic pathway is complex and various in regulation and control, and is still in a laboratory stage at present, and the yield is very low. Team Liu Yanfeng, 2022, (J. Agric. Food chem.2022,70,19,5849-5859) integrated the critical gene, comGC, for improved biosynthesis of 5-MTHF into the genome of the modular engineered Bacillus subtilis strain B89, 5-MTHF synthesis reached 3.41.+ -. 0.10mg/L with a productivity of 0.21mg/L/h, which is the highest level of current microbial synthesis. The requirement of industrial production is met through metabolic modification, and a great deal of research is still needed.
Although the industrialization of (6S) -5-MTHF synthesized by the chemical method and the dihydrofolate reductase method is realized at present, formaldehyde or high-pressure process conditions are inevitably used, and chiral auxiliary agents such as benzenesulfonic acid and the like required by chemical resolution have residual hidden dangers. There is therefore a need in the art to develop new processes that create a milder, safer (6S) -5-MTHF synthesis process.
Disclosure of Invention
Aiming at the problems of the prior art, the invention provides a recombinant genetic engineering bacterium and a preparation method of (6S) -5-methyltetrahydrofolate, and aims to couple key reaction enzymes in a natural (6S) -5-MTHF synthesis path with a glucose dehydrogenase coenzyme circulation system, construct a process route for synthesizing (6S) -5-MTHF by an enzymatic method in one pot, and realize a (6S) -5-MTHF synthesis process with mild, safe and efficient conditions.
A recombinant genetically engineered bacterium is recombinant escherichia coli containing one or more genes corresponding to dihydrofolate reductase, serine hydroxymethyl transferase, methylene tetrahydrofolate reductase and glucose dehydrogenase;
or a combination of a plurality of recombinant E.coli comprising the corresponding genes for dihydrofolate reductase, serine hydroxymethyltransferase, methylene tetrahydrofolate reductase and glucose dehydrogenase, respectively.
Preferably, the gene corresponding to the dihydrofolate reductase is derived from bacillus subtilis, escherichia coli, bacillus licheniformis, bacillus amyloliquefaciens, bacillus megaterium, bacillus coagulans, corynebacterium glutamicum, pseudomonas aeruginosa or Brevibacterium deficiency; preferably from the Bacillus subtilis168 dihydrofolate reductase gene.
Preferably, the serine hydroxymethyltransferase corresponding gene is derived from bacillus subtilis, escherichia coli, bacillus licheniformis, bacillus amyloliquefaciens, bacillus megaterium, bacillus coagulans, corynebacterium glutamicum, pseudomonas aeruginosa or Brevundimonas deficiency; serine hydroxymethyltransferase gene derived from Bacillus subtilis is preferred.
Preferably, the gene corresponding to the methylene tetrahydrofolate reductase is derived from Brevibacterium deficiency, escherichia coli, corynebacterium glutamicum or Pseudomonas aeruginosa; preferably a methylene tetrahydrofolate reductase gene derived from Brevundimonas diminuta ATCC 19146.
Preferably, the gene corresponding to the glucose dehydrogenase is derived from bacillus subtilis or bacillus megaterium; preferably a glucose dehydrogenase gene derived from Bacillus subtilis.
Preferably, the recombinant E.coli is Escherichia coli BL (DE 3).
Preferably, the recombinant genetically engineered bacterium is escherichia coli containing a recombinant plasmid pET-26 b; the recombinant plasmid pET-26b is a pET-26b plasmid containing a corresponding gene of at least one enzyme of dihydrofolate reductase, serine hydroxymethyl transferase, methylene tetrahydrofolate reductase and glucose dehydrogenase.
The invention also provides the application of the recombinant genetically engineered bacterium in preparing (6S) -5-methyltetrahydrofolate or a preparation thereof.
The invention also provides a preparation method of the (6S) -5-methyltetrahydrofolate, which comprises the following steps:
step 1, preparing crude enzyme liquid containing dihydrofolate reductase, serine hydroxymethyl transferase, methylene tetrahydrofolate reductase and glucose dehydrogenase;
step 2, feeding raw materials, cofactors, crude enzyme liquid and antioxidants for reaction to obtain (6S) -5-methyltetrahydrofolate;
the raw materials comprise folic acid, glucose and L-serine; the cofactor comprises NADP + 、NAD + 、PLP、Zn 2+ And FAD 2+ The method comprises the steps of carrying out a first treatment on the surface of the The antioxidant is vitamin C; the reaction conditions are light-proof and deoxidized, and the reaction temperature is 10-30 ℃; the pH of the reaction solution is controlled within the range of 7.0-7.5.
Preferably, the crude enzyme solutions of the dihydrofolate reductase, serine hydroxymethyltransferase, methylene tetrahydrofolate reductase and glucose dehydrogenase in the step 1 are bacterial disrupted solutions obtained by fermenting the recombinant genetically engineered bacteria according to any one of claims 1 to 8;
the thallus crushing liquid is prepared by crushing wet thallus containing enzyme and suspended in 20-50% concentration W/V in sterile water or neutral buffer solution through a high pressure homogenizer and centrifuging to remove cell fragments.
Preferably, in the step 2, the feeding molar ratio of folic acid, glucose and L-serine is1 (3-3.5): 2-2.5; the substrate concentration of folic acid is 0.1-5wt.%.
Preferably, in step 2, the cofactor addition amount is: NADP (NADP) + 0.1-1wt.‰，NAD + 0.1-1 wt.% > of PLP0.1-5wt.‰，Zn 2+ 1-5mM，FAD 2+ 0.1-1wt.‰；
And/or the antioxidant is added in an amount of 1-10 wt-%.
Preferably, in step 2, the crude enzyme solution is added in an amount of: 2-10% of dihydrofolate reductase, 0.5-5% of serine hydroxymethyltransferase, 2-10% of methylene tetrahydrofolate reductase and 1-5% of glucose dehydrogenase.
The invention constructs the technological route of the enzymatic one-pot synthesis of (6S) -5-MTHF by coupling key reaction enzyme in the natural (6S) -5-MTHF synthesis route with a glucose dehydrogenase coenzyme circulation system (figure 1). The reaction system mainly comprises vitamins and amino acids, and is safe and harmless; the natural (6S) -5-MTHF synthesis path is adopted, so that not only is the (6S) -5-MTHF specifically synthesized, but also the problems of oxidative decomposition and the like generated by separating and extracting intermediate products in the step-by-step synthesis process are avoided by a one-pot method.
In a preferred scheme, enzymes of different sources are optimized for each step in a process route, recombinant genetic engineering bacteria containing expressed genes of the enzymes can be constructed, high-efficiency preparation of crude enzyme liquid is realized, and the efficiency of a (6S) -5-MTHF synthesis process is further improved.
Therefore, the invention has the advantages of mild condition, safety and high efficiency, and has good application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a schematic diagram of the enzymatic one-pot synthesis of (6S) -5-methyltetrahydrofolate;
FIG. 2 is a liquid chromatography-mass spectrometry (LC-MS), TIC full scan (B) and mass spectrum (C) of the enzymatic reaction solution of example 1;
FIG. 3 is a liquid chromatogram of the enzymatic reaction results in example 6.
Detailed Description
In the following examples, reagents and raw materials not specifically described were commercially available, and the purity was analytical grade.
Example 1 preparation of (6S) -5-methyltetrahydrofolate
The overall reaction scheme for the one-pot synthesis of (6S) -5-MTHF in this example is shown in FIG. 1.
Under the condition of oxygen-free light shielding, one molecular (6S) -THF and two molecular NADP are synthesized by adding two molecules of H to one molecular of folacin and two molecules of NADPH under the slow catalysis of dihydrofolate reductase DHFR + The method comprises the steps of carrying out a first treatment on the surface of the Serine hydroxymethyltransferase (Serine hydroxymethyltransferase, SHMT) reversibly converts (6S) -THF and L-serine upon pyridoxal phosphate (PLP) activation
(6R) -5,10-MTHF and glycine, removing one molecule of water; methylene tetrahydrofolate reductase (Methylenetetrahydrofolate reductase, MTHFR) in FAD 2+ In the auxiliary electron transfer process, (6R) -5,10-MTHF is converted into a final product, (6S) -5-MTHF), VC is added into the reaction system to prevent the oxidation of active folic acid, and the reduction state of the reaction solution is maintained.
Specific:
reaction stage one: synthesis of (6S) -tetrahydrofolic acid. Glucose (2 eq folic acid) in the reaction vessel is catalyzed by GDH to form glucono-1, 5-lactone and is immediately hydrolyzed to gluconic acid (GluA) with NADP + Energy conversion to synthesize NADPH; NADPH and H produced + (2 eq folic acid) provides energy for DHFR, which catalyzes the removal of folic acid to synthesize (6S) -THF through four molecules of H; DHFR is a multifunctional enzyme that can first synthesize DHF using the substrate folic acid, and then reduce DHF to (6S) -THF with two molecules of H removed; it is also possible to directly reduce folic acid to (6S) -THF, which is generally expressed as one molecule of folic acid converted to (6S) -THF, requiring consumption of two molecules of NADPH and two molecules of H + 。
And a second reaction stage: synthesis of (6R) -5, 10-MTHF. Cofactor PLP in the solution enters into the SHMT active center, zn 2+ Binding to the metal ion binding site activates SHMT catalytic activity, reversing (6S) -THF and L-Ser into (6R) -5,10-MTHF, glycine and water. Since the entire reaction is reversible, an excess of L-Ser (> 2eq folic acid) and immediate conversion of the product (6R) -5,10-MTHF is required to drive the reaction forward.
Reaction stage three: (6S) -5-methyltetrahydrofolate synthesis. The 5, 10-methylene tetrahydrofolate reductase belongs to NAD (P) H-dependent reductase, eukaryotic MTHFR cofactor is NADPH, prokaryotic is NADH, and is generally tetrad protein and each subunit contains a non-covalently bound flavin adenine dinucleotide molecule (FAD 2+ ) Assisting electron transfer. The MTHFR constructed by the invention is NADH-dependent 5, 10-methylene tetrahydrofolate reductase, and can also catalyze the reaction by combining with NADPH, but the speed is lower; the phase three reaction still requires a cycle of coupled GDH coenzyme. Glucose (1 eq folic acid) in the reaction vessel is catalyzed by GDH to form glucono-1, 5-lactone (GDL) and is immediately hydrolyzed to gluconic acid (GluA) with NAD + Energy conversion of the synthesized NADH; NADH and H produced + Providing energy for reduction of MTHFR at the cofactor FAD 2+ Assisted reduction of (6R) -5,10-MTHF to (6S) -5-MTHF and NADH to NAD + Re-enter the GDH coenzyme circulation system.
The enzymes used in each step are preferably selected according to the above reaction scheme, and the specific steps and results are as follows:
1. liquid phase detection conditions of the reaction liquid
Specific conditions for the HPLC analysis in this example: detection wavelength 280nm; the column packing material was octadecylsilane chemically bonded silica (250X 4.6mm,5 μm); mobile phase 10% acetonitrile (containing 1%o trifluoroacetic acid); the flow rate is 1.0mL/min; sample injection amount is 10 μl; the detection sample is diluted in a Tris-HCl buffer solution with pH of 8.0 and added with a DTT antioxidant with the final concentration of 1mM to keep the components stable; the mass-charge ratio (m/z) -1 of the compound in the graph C corresponds to the molecular weight of each peak in the graph A, and each substance is judged by the molecular weight.
2. Plasmid construction
Searching related genes of DHFR, SHMT and MTHFR in a laboratory strain gene library, designing corresponding primers to acquire target genes, carrying out double digestion of a vector plasmid pET-26b by using restriction endonucleases NdeI and XhoI, connecting a vector fragment and the target fragment by using a commercial one-step cloning kit, immediately transferring into Escherichia coli DH alpha chemistry competence to carry out plasmid repair and replication, coating a Kana resistance flat plate, selecting single colony for sequencing verification, and finally extracting plasmids for preservation at the temperature of minus 20 ℃. The folate cycle is a common metabolic pathway of microorganisms, and considering the problem of protein solubility of heterologous expression of E.coli, this example only excavates part of genes derived from E.coli, bacillus, corynebacterium glutamicum and Pseudomonas for construction of minienzyme libraries.
Glucose dehydrogenase widely exists in bacillus and extremely thermophilic archaea, such as bacillus subtilis, bacillus megaterium, bacillus cereus, thermophilus and the like, and BsGDH (Bacillus subtilis) and BmGDH (Bacillus megaterium IWG 3) are the most commonly used GDH for constructing coenzyme regeneration at present due to high enzyme activity; the present invention employs glucose dehydrogenase derived from Bacillus subtilis168 for the construction of coenzyme cycles. Inquiring BsGDH gene, designing corresponding primer to obtain target gene, double-enzyme cutting carrier plasmid pET-26b by using restriction endonuclease NdeI and XhoI, connecting carrier fragment and target fragment by using one-step cloning kit, immediately transferring into Escherichia coli DH alpha chemistry competence to make plasmid repair and replication, coating a Carna resistance flat plate, selecting single colony to sequence and verify, finally extracting plasmid and preserving at-20 ℃.
3. Enzyme library construction
Transferring the plasmid constructed in the 2 nd part into Escherichia coli BL (DE 3) competent cells, selecting single colony, culturing overnight in LB liquid medium, inoculating LB culture broth into TB medium containing Canna antibiotic at 2% inoculum size, culturing at 37deg.C for 1-2 hr to obtain bacterial OD 600 When the strain grows to 0.6-0.8, 0.2mM IPTG inducer is added, the strain is induced for 16-20h at 20 ℃ and 200rpm, and the strain is collected by centrifugation. Adding 40% (W/V) wet thalli into a Tris-HCl buffer solution with the pH of 7.2 and 50mM, fully suspending, and crushing the thalli by using a high-pressure homogenizer; taking 20ul of broken liquid for SDS-PAGE polyacrylamide gel electrophoresis protein solubility analysis, centrifuging the rest broken liquid at 4000rpm for 20min to remove cell fragments, obtaining a lower clear liquid which is crude enzyme liquid, registering and warehousing, and preserving at 4 ℃ for later use, wherein the information of the enzyme library is shown in Table 1.
Table 1 enzyme library catalogue and solubility analysis table
4. Screening of enzymes
(1) Dihydrofolate reductase screening:
in a 2mL centrifuge tube, 2mL of the reaction system was prepared. Adding folic acid with a final concentration of 2mM, glucose with a final concentration of 4.1mM and NADP with a final concentration of 0.1 wt.% + 1 wt..mu.L of VC, 50. Mu.L of GDH crude enzyme solution and 100. Mu.L of DHFR crude enzyme solution from different sources are dissolved in 50mM of pH7.5Tris-HCl buffer solution, reacted overnight at 600rpm in a metal bath at 20 ℃, detected in a liquid phase, and the conversion rate is calculated to screen the DHFR with the highest conversion rate. As shown in Table 2, bsDHFR conversion was highest, and (6S) -THF conversion was 95.25% for overnight reaction, which was used in the subsequent reaction.
TABLE 2 enzymatic Activity screening of dihydrofolate reductase
(2) Screening of serine hydroxymethyltransferase:
because of the reversible reaction of the SHMT, (6R) -5,10-MTHF is difficult to accumulate, the activity of the SHMT cannot be intuitively judged, the SHMT activity is judged by combining MTHFR screening and the yield of the final product (6S) -5-MTHF, and more ECMTHFR is reported to participate in cascade reaction by adopting literature. In a 2mL centrifuge tube, 2mL of the reaction system was prepared. Adding folic acid with a final concentration of 2mM, glucose with a final concentration of 6.1mM and NADP with a final concentration of 0.1 wt.% + ，0.1wt.‰NAD + 4.2mM L-serine, 1 wt..permillage PLP,1mM ZnCl 2 ，0.1wt.‰FAD 2+ 1 wt..mu.L of VC, 50. Mu.L of GDH crude enzyme solution, 100. Mu.L of BsDHFR, 100. Mu.L of LEcMTTHFR and 100. Mu.L of SHMT from the above sources were dissolved in 50mM pH7.5Tris-HCl buffer, reacted overnight at 600rpm in a metal bath at 20℃and subjected to liquid phase detection, and the conversion was calculated to select the SHMT with the highest conversion. As shown in Table 3, the effect of SHMT on the final product conversion is small, the conversion rate of (6S) -MTHF of 9 SHMTs is about 70%, wherein the final product conversion rate of BsSHMT is high and can reach 76.05%, and the method can be used for subsequent reactions.
TABLE 3 enzymatic screening of serine hydroxymethyltransferase
Serine hydroxymethyltransferase (6S) -MTHF conversion/%
EcSHMT 67.91
BsSHMT 76.05
BlSHMT 66.81
BaSHMT 66.81
BmSHMT 73.82
BcSHMT 72.25
CgSHMT 63.29
PaSHMT 68.47
BdSHMT 69.38
(3) Screening of methylene tetrahydrofolate reductase:
in a 2mL centrifuge tube, 2mL of the reaction system was prepared. Adding folic acid with a final concentration of 2mM, glucose with a final concentration of 6.1mM and NADP with a final concentration of 0.1 wt.% + ，0.1wt.‰NAD + 4.2mM L-serine, 1 wt..permillage PLP,1mM ZnCl 2 ，0.1wt.‰FAD 2+ 100. Mu.L of BsDHFR, 50. Mu.L of BsSHMT and MTHFR from the above sources were dissolved in 50mM pH7.5Tris-HCl buffer, reacted overnight at 600rpm in a metal bath at 20℃and detected in a liquid phase, and the conversion was calculated to select the MTHFR having the highest conversion. As shown in Table 4, the activity of MTHFR has a very large influence on the conversion rate of the final product (6S) -MTHF, and the conversion rate of MTHFR of Bacillus series is almost zero, and the molecular weight of the protein may be too largeHeterologous expression is carried out by using escherichia coli, the polypeptide chain is folded incorrectly, and the enzyme molecule has no biological activity; the highest BdMTHFR conversion rate in all soluble expressed reductases can reach 89.32%, and can be used for subsequent optimization and amplification.
TABLE 4 enzymatic Activity screening of methylene tetrahydrofolate reductase
Methylene tetrahydrofolate dehydrogenase (6S) -MTHF conversion/%
EcMTHFR 68.19
BsMTHFR 2.50
BlMTHFR 2.11
BaMTHFR 2.61
BmMTHFR 1.87
BcMTHFR 1.41
CgMTHFR 30.86
PaMTHFR 75.24
BdMTHFR 89.32
In summary, the preferred choice of dihydrofolate reductase is BsDHFR (from Bacillus subtilis) and the preferred choice of serine hydroxymethyltransferase is BsSHMT (from Bacillus subtilis), and the preferred choice of methylene tetrahydrofolate reductase is BdMTHFR (from Brevundimonas diminuta ATCC 19146).
More specific examples of the reaction scheme and specific reaction conditions are shown in examples 2 to 4.
Example 2 preparation of (6S) -5-methyltetrahydrofolate
The reaction principle is shown in example 1. Adding 0.1g folic acid, 0.13g glucose, 0.06g L-serine and 1mg NADP into the reaction kettle + ，1mg NAD + Pyridoxal phosphate (PLP) 10mg, znCl 2.7mg 2 ，1mg FAD 2+ 0.1g VC, pH of the solution was adjusted to 7.0 using 10% (W/V) NaOH; then 250. Mu.L of BsDHFR, 50. Mu.L of BsSHMT, 200. Mu.L of BdMTHFR and 150. Mu.L of crude enzyme disruption solution were added; supplementing water to 10mL; under the condition of avoiding light, nitrogen is continuously introduced into a reaction kettle, the mixture is slowly stirred, the pH is regulated to 7.0-7.2 by saturated sodium carbonate aqueous solution, the reaction is carried out for 10 hours at room temperature, the concentration of the final product (6S) -5-MTHF is 9.78g/L, and the conversion rate is 93.99 percent.
Example 3 preparation of (6S) -5-methyltetrahydrofolate
The reaction principle is shown in example 1.
5g folic acid, 6.3g glucose, 2.6g L-serine and 20mg NADP are added into a reaction kettle at one time + ，20mg NAD + Pyridoxal phosphate (PLP) 0.2g, znCl 54mg 2 ，10mg FAD 2+ 2g VC, pH of the solution was adjusted to 7.0 using 20% NaOH; then, 5mL of BsDHFR, 1mL of BsSHMT, 4mL of BdMTHFR crude enzyme solution and 2mL of GDH were added, and the mixture was supplemented with water to 200mL; continuously introducing nitrogen into the reaction kettle under the condition of light shielding, slowly stirring, regulating the pH to 7.0-7.2 by 10% (W/V) NaOH solution, reacting for 17h at room temperature, and detecting by liquid phaseThe liquid chromatogram of the reaction result of this example is shown in FIG. 3, and the concentration of the final product (6S) -5-MTHF was 22.85g/L, and the conversion was 95.08%.
Example 4 preparation of (6S) -5-methyltetrahydrofolate
The reaction principle is shown in example 1.
50g folic acid, 63g glucose, 26g L-serine and 0.1g NADP are added into a reaction kettle at one time + ，0.1g NAD + Pyridoxal phosphate (PLP) 1g, znCl 0.27g 2 ，40mg FAD 2+ 10g of VC, the pH of the solution was adjusted to 7.0 using 20% (W/V) NaOH; then, 25mL of BsDHFR, 5mL of BsSHMT, 20mL of BdMTHFR crude enzyme solution and 10mL of GDH were added, and the mixture was made up to 1L; under the condition of avoiding light, nitrogen is continuously introduced into a reaction kettle, the mixture is slowly stirred, the pH is regulated to 7.0-7.2 by 20% NaOH solution, the reaction is carried out for 41 hours at room temperature, the concentration of the final product (6S) -5-MTHF is 46.97g/L, and the conversion rate is 90.11%.
EXAMPLE 5 recombinant genetically engineered bacteria
This example provides a recombinant genetically engineered bacterium that can be used to prepare a crude enzyme solution for use in examples 1-4.
The recombinant genetically engineered bacterium of the embodiment is constructed according to the following method:
the following genes were obtained using primers:
1. a gene derived from Bacillus subtilis, 168, expressing a dihydrofolate reductase;
2. a gene derived from Bacillus subtilis, 168 expressing serine hydroxymethyltransferase;
3. a gene derived from Brevundimonas diminuta ATCC 19146 expressing methylene tetrahydrofolate reductase;
4. a gene derived from Bacillus subtilis and expressing glucose dehydrogenase.
Double digestion of vector plasmid pET-26b was performed using restriction endonucleases NdeI and XhoI, ligation of the vector fragment and the above-described fragment of interest was performed by a commercially available one-step cloning kit, immediately transferred into Escherichia coli DH 5. Alpha. Chemistry competence for plasmid repair and replication, and finally plasmid was extracted and stored at-20 ℃. Transferring the plasmid into Escherichia coli BL (DE 3) chemical competence to obtain the final product.
The method for preparing the crude enzyme solution by adopting the recombinant genetically engineered bacteria comprises the following steps:
selecting single colony, culturing overnight in LB liquid medium, inoculating LB culture solution into TB medium containing kanna antibiotic at 2% inoculum size, culturing at 37deg.C for 1-2 hr, and culturing to obtain thallus OD 600 When the strain grows to 0.6-0.8, 0.2mM IPTG inducer is added, the strain is induced for 16-20h at 20 ℃ and 200rpm, and the strain is collected by centrifugation. Adding 40% (W/V) wet thalli into a Tris-HCl buffer solution with the pH of 7.2 and 50mM, fully suspending, and crushing the thalli by using a high-pressure homogenizer; and (3) carrying out SDS-PAGE polyacrylamide gel electrophoresis protein solubility analysis on 20ul of the crushed solution, centrifuging the rest crushed solution at 4000rpm for 20min to remove cell fragments, and obtaining the supernatant as crude enzyme solution.
From the above examples, it can be seen that the present invention combines the key reaction enzymes in the natural (6S) -5-MTHF synthesis pathway with the glucose dehydrogenase coenzyme circulation system to construct a new enzymatic one-pot synthesis process route for (6S) -5-MTHF. The method has the advantages of mild conditions, safety and high efficiency, and has good application prospect.

Claims (13)

1. A recombinant genetically engineered bacterium is characterized in that: it is a recombinant E.coli comprising one or more genes corresponding to dihydrofolate reductase, serine hydroxymethyltransferase, methylene tetrahydrofolate reductase and glucose dehydrogenase;
or a combination of a plurality of recombinant E.coli comprising the corresponding genes for dihydrofolate reductase, serine hydroxymethyltransferase, methylene tetrahydrofolate reductase and glucose dehydrogenase, respectively.
2. The recombinant genetically engineered bacterium of claim 1, wherein: the gene corresponding to the dihydrofolate reductase is derived from bacillus subtilis, escherichia coli, bacillus licheniformis, bacillus amyloliquefaciens, bacillus megaterium, bacillus coagulans, corynebacterium glutamicum, pseudomonas aeruginosa or Brevibacterium deficiency; preferably from the Bacillus subtilis168 dihydrofolate reductase gene.
3. The recombinant genetically engineered bacterium of claim 1, wherein: the serine hydroxymethyl transferase corresponding genes are derived from bacillus subtilis, escherichia coli, bacillus licheniformis, bacillus amyloliquefaciens, bacillus megaterium, bacillus coagulans, corynebacterium glutamicum, pseudomonas aeruginosa or Brevibacterium deficiency; serine hydroxymethyltransferase gene derived from Bacillus subtilis is preferred.
4. The recombinant genetically engineered bacterium of claim 1, wherein: the genes corresponding to the methylene tetrahydrofolate reductase are derived from Brevundimonas deficiency, escherichia coli, corynebacterium glutamicum or pseudomonas aeruginosa; preferably a methylene tetrahydrofolate reductase gene derived from Brevundimonas diminuta ATCC 19146.
5. The recombinant genetically engineered bacterium of claim 1, wherein: the gene corresponding to the glucose dehydrogenase is derived from bacillus subtilis or bacillus megatherium; preferably a glucose dehydrogenase gene derived from Bacillus subtilis.
6. The recombinant genetically engineered bacterium of claim 1, wherein: the recombinant E.coli was Escherichia coli BL (DE 3).
7. The recombinant genetically engineered bacterium of claim 1, wherein: the recombinant genetically engineered bacterium is coliform bacterium containing recombinant plasmid pET-26 b; the recombinant plasmid pET-26b is a pET-26b plasmid containing a corresponding gene of at least one enzyme of dihydrofolate reductase, serine hydroxymethyl transferase, methylene tetrahydrofolate reductase and glucose dehydrogenase.
8. Use of the recombinant genetically engineered bacterium of any one of claims 1-7 in the preparation of (6S) -5-methyltetrahydrofolate or a formulation thereof.
9. A method for preparing (6S) -5-methyltetrahydrofolate, which is characterized by comprising the following steps:
step 1, preparing crude enzyme liquid containing dihydrofolate reductase, serine hydroxymethyl transferase, methylene tetrahydrofolate reductase and glucose dehydrogenase;
step 2, feeding raw materials, cofactors, crude enzyme liquid and antioxidants for reaction to obtain (6S) -5-methyltetrahydrofolate;
the raw materials comprise folic acid, glucose and L-serine; the cofactor comprises NADP + 、NAD + 、PLP、Zn 2+ And FAD 2+ The method comprises the steps of carrying out a first treatment on the surface of the The antioxidant is vitamin C; the reaction conditions are light-proof and deoxidized, and the reaction temperature is 10-30 ℃; the pH of the reaction solution is controlled within the range of 7.0-7.5.
10. The method of preparing as claimed in claim 9, wherein: the crude enzyme liquid of the dihydrofolate reductase, serine hydroxymethyl transferase, methylene tetrahydrofolate reductase and glucose dehydrogenase in the step 1 is the bacterial crushing liquid obtained by fermenting the recombinant genetically engineered bacteria in any one of claims 1-8; the thallus crushing liquid is prepared by crushing wet thallus containing enzyme and suspended in 20-50% concentration W/V in sterile water or neutral buffer solution through a high pressure homogenizer and centrifuging to remove cell fragments.
11. The method of preparing as claimed in claim 9, wherein: in the step 2, the feeding mole ratio of folic acid, glucose and L-serine is1 (3-3.5): 2-2.5; the substrate concentration of folic acid is 0.1-5wt.%.
12. The method of preparing as claimed in claim 9, wherein: in the step 2, the addition amount of the cofactor is as follows: NADP (NADP) + 0.1-1wt.‰，NAD + 0.1-1wt. -%, PLP addition 0.1-5wt. -%, zn 2+ 1-5mM，FAD 2+ 0.1-1wt.‰；
And/or the antioxidant is added in an amount of 1-10 wt-%.
13. The method of preparing as claimed in claim 9, wherein: in the step 2, the addition amount of the crude enzyme solution is as follows: 2-10% of dihydrofolate reductase, 0.5-5% of serine hydroxymethyltransferase, 2-10% of methylene tetrahydrofolate reductase and 1-5% of glucose dehydrogenase.
CN202311351532.5A 2023-10-18 2023-10-18 Recombinant genetic engineering bacterium and preparation method of (6S) -5-methyltetrahydrofolate Pending CN117448245A (en)

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