CN105861534A - Biological method for improving yield of L-5-methyltetrahydrofolate by virtue of two-plasmid engineering bacteria - Google Patents
Biological method for improving yield of L-5-methyltetrahydrofolate by virtue of two-plasmid engineering bacteria Download PDFInfo
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Abstract
The invention provides a biological synthesis method for increasing the accumulation of L-5-methyltetrahydrofolate, an L-5-methyltetrahydrofolate synthetase system co-expressed recombinant plasmid and a construction method and application thereof. The L-5-methyltetrahydrofolate synthetase system co-expressed recombinant plasmid comprises serine hydroxymethyltransferase (SHMT) gene GlyA and methylenetetrahydrofolate reductase (MTHFR) gene MetF sequences. The biological synthesis method for increasing the accumulation of the L-5-methyltetrahydrofolate comprises the following steps: converting for accumulating an original strain of the L-5-methyltetrahydrofolate to obtain a recombinant strain by virtue of the L-5-methyltetrahydrofolate synthetase system co-expressed recombinant plasmid, and fermenting the recombinant strain. The accumulation of the L-5-methyltetrahydrofolate in the recombinant strain in a final fermentation product is remarkably higher than that of the original strain, the utilization rate of the raw material is increased, and production cost and energy consumption are reduced.
Description
Technical field
The present invention relates to a kind of metabolic engineering technical field synthetic method, a kind of L-5-methyl tetrahydrofolate yield that improves
Biological synthesis method.
Background technology
L-5-methyl tetrahydrofolate has another name called (6S)-5-methyltetrahydrofolate, chemical name be (6S)-N-[4-[[(2-amino-Isosorbide-5-Nitrae,
5,6,7,8-hexahydro-4-oxygen-5-methyl-6-pteridine radicals) methyl] amino] benzoyl]-Pidolidone.
L-5-methyl tetrahydrofolate is the form that folic acid is the most active, is also the folic acid form utilized that can directly be absorbed by the body.General
Logical folic acid only has metabolism to be that L-5-methyl tetrahydrofolate could participate in methylation procedure and DNA synthesizes, and L-5-methyl tetrahydrochysene
Folic acid biological availability is apparently higher than common folic acid.
L-5-methyl tetrahydrofolate is blood plasma and the unique activity form of myelencephalon Folic Acid, participates in many one carbon backs in organism
Group's transfer reaction, such as: recombining of purine and thymus gland glycosides.The DNA damage that cell is spontaneous can also be reduced, increase it
Appreciation rate, is the co-factor of the methionine synthases that catalysis homocysteine changes to methionine simultaneously, is again active
Methyl group donor S-adenosyl methionine (for multiple methylation reaction, such as DNA, protein, the methylating of phosphatide) before
Body.
As uniquely penetrating the folic acid class medicine of blood-brain barrier, L-5-methyl tetrahydrofolate has certain treatment to Alzheimer's disease
Effect.Additionally, L-5-methyl tetrahydrofolate has effect, such as rheumatic arthritis, cardiovascular disease in the treatment of numerous disease
Disease, depression in old age, embryo methylate, old macrocytic anemia and tumour etc..
European Union has been approved by a L-5-methyl tetrahydrofolate and adds in food as food additives, and this makes its demand drastically
Increase.At present, external manufacturing enterprise only has Merck company, and synthesis technique is complicated, the L-5-methyl tetrahydrofolate of production
Based on raceme, it is difficult to split, has proven to D-5-methyl tetrahydrofolate and do not treat or health-care effect, and can not be by people
Body absorbs.Domestic manufacturer is also few, and also mostly is the method using chemical synthesis.
The synthesis technique complex process of chemical synthesis, Optical Instruments Industry difficulty, production cost is the highest.Need a kind of new
Method produces the L-5-methyl tetrahydrofolate that optical purity is higher, utilizes technique for gene engineering to build engineering bacteria and synthesizes for bioanalysis
L-5-methyl tetrahydrofolate provides a kind of new approaches.
The growing environment of microorganism is simple, and growth and breeding speed is fast, and toxigenic capacity is the lowest.The metabolism of microorganism is to be sent out by enzymatic
Raw reaction, enzyme has high specificity with specific, and the most specific catalysis of a kind of enzyme is a kind of or class substrate generation is unique
Product, does not have the generation of isomers.Hence with the metabolic response of organism, organism synthesizes L-5-methyl tetrahydrochysene leaf
Acid, can solve the problem that chemical synthesis produces raceme.
Summary of the invention
It is an object of the invention to provide a kind of L-5-methyl tetrahydrofolate synthetase series coexpression recombinant plasmid, raw for building
Produce and accumulate the bacterial strain of L-5-methyl tetrahydrofolate.
A further object of the invention is, it is provided that the structure of a kind of L-5-methyl tetrahydrofolate synthetase series coexpression recombinant plasmid
Method.
A further object of the invention is, it is provided that the application of a kind of L-5-methyl tetrahydrofolate synthetase series coexpression recombinant plasmid.
A further object of the invention is, it is provided that a kind of method improving L-5-methyl tetrahydrofolate cumulant.
The L-5-methyl tetrahydrofolate synthetase series coexpression recombinant plasmid that the present invention provides, including serine hydroxymethylase base
Because of GlyA sequence, Methylene tetrahydrofolate reductase gene MetF sequence and a suitable carrier segments;Described GlyA
Sequence and MetF sequence respectively as on NCBI Gene ID be shown in the sequence of 947022 and 948432.
According to one preferred example of the present invention, in L-5-methyl tetrahydrofolate synthetase series coexpression recombinant plasmid, GlyA and
MetF code area is placed between T7 promoter and 1ac operon.
According to one preferred example of the present invention, one is also included at L-5-methyl tetrahydrofolate synthetase series coexpression recombinant plasmid
Kalamycin resistance gene (nptII), in order to screen recombinant bacterium.
The construction method of the L-5-methyl tetrahydrofolate synthetase series coexpression recombinant plasmid that the present invention provides comprises the following steps:
A) PCR amplification obtains GlyA and MetF sequence;
B) GlyA and MetF sequence is cloned into suitable expression vector jointly.
The L-5-methyl tetrahydrofolate synthetase series coexpression recombinant plasmid that the present invention provides may be used for converting accumulation L-5-methyl four
The original strain of hydrogen folic acid.
The biological synthesis method of the L-5-methyl tetrahydrofolate cumulant that the present invention provides includes the L-5-first by the present invention being provided
The step of base tetrahydrofolic acid synthetase series coexpression recombinant plasmid transformed accumulation L-5-methyl tetrahydrofolate original bacteria, it is thus achieved that recombinant bacterium.
According to a preferred embodiment of the present invention, by this recombinant bacterium of fermented and cultured, improve L-5-methyl tetrahydrofolate cumulant.
According to another preferred embodiment of the present invention, this recombinant bacterium is E. coli BL21 (DE3).
According to another preferred embodiment of the present invention, improve the biological synthesis method of L-5-methyl tetrahydrofolate cumulant also by,
In fermented and cultured to OD value to time between 0.6~0.8, by the key in derivant induction L-5-methyl tetrahydrofolate metabolic pathway
The expression of enzyme gene GlyA and MetF.
The L-5-methyl tetrahydrofolate synthetase series coexpression recombinant plasmid using the present invention to provide can convert accumulation L-5-methyl
The original strain of tetrahydrofolic acid, it is thus achieved that recombinant bacterium fermented and cultured, in tunning, L-5-methyl tetrahydrofolate cumulant is significantly high
Yield in original strain E.coli BL21 (DE3).The raising L-5-methyl tetrahydrofolate cumulant of employing present invention offer is described
Method, improves raw material availability, reduces production cost and energy consumption, for research bioanalysis synthesis L-5-methyl tetrahydrochysene further
Folic acid sets up a kind of method, produces for its industrialization and establishes a kind of basis.
Accompanying drawing explanation
Fig. 1 is the testing result that PCR amplification obtains GlyA, and wherein swimming lane 1 is GlyA.
Fig. 2 is the testing result that PCR amplification obtains MetF, and wherein swimming lane 1 is MetF.
Fig. 3 is the double digestion detected through gel electrophoresis result of plasmid pET28a-GlyA, pMD19T-MetF plasmid, wherein swimming lane 1
Being plasmid pET28a-GlyA and plasmid pMD19T-MetF respectively with 3, swimming lane 0 and 2 is to be digested rear matter pET28a-GlyA respectively
Being the DNA fragmentation of gene GlyA with the band of plasmid pMD19T-MetF, about 1200bp, the band of about 900bp is
The DNA fragmentation of gene M etF.
Fig. 4 is the structural representation of plasmid pET28a-GlyA-MetF.
Fig. 5 is the soluble protein SDS-PAGE of the bacterial strain of the plasmid containing pET28a-GlyA-MetF, pET28a-GlyA
Testing result.Wherein swimming lane M is albumen Marker, and swimming lane 0 is the bacterial strain containing pET28a-GlyA plasmid, and swimming lane 1 is for containing
PET-28a (+) the blank bacterial strain of plasmid, swimming lane 2 is the bacterial strain containing pET28a-GlyA-MetF plasmid.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following example are merely to illustrate the present invention
Not for limiting the scope of the present invention.
For indicating the experimental technique of actual conditions, generally according to normal condition, such as " molecular cloning: laboratory in following example
Handbook " condition described in (New York:CoLd Spring Harbor Laboratory Press, 1989) or manufacturer provide
Scheme is carried out.
In the following embodiment of the present invention, the glue of use reclaims kit, plasmid extraction kit, genome extraction kit
It is purchased from Sheng Gong biotech firm.
In the following embodiment of the present invention, the pMD-19T SimpLe Vector of use, Hind III, EcoR I, Taq DNA
Polymerase, T4 DNA ligase, DNA Marker, it is purchased from TAKARA company.
In the following embodiment of the present invention, the expression vector pET28a of use, bacterial classification E.coli BL21 (DE3), bacterial classification E.coli
K12, E.coli DH5 α, preserves for life central laboratory of China Medicine University, the wherein ATCC of bacterial classification E.coli BL21 (DE3)
Numbered BAA-1025TM.
In the following embodiment of the present invention, E.coli BL21 (DE3) competent cell of use, E.coli DH5 α competence are thin
Born of the same parents, are carried out according to the method provided in Invitrogen company Pichia Expression Kit handbook.
In the following embodiment of the present invention, the formula of the LB culture medium of use is: 1% tryptose, 0.5% yeast leaching powder,
1%NaCl;The formula of the Medium of shaking flask fermentation used is: 1% tryptose, 0.5% yeast leaching powder, 1%NaCl.Configuration is solid
During body LB plating medium, add 2% agar powder according to above-mentioned formula.
In the following embodiment of the present invention, the preparation of competent cell and conversion, according to " molecular cloning: laboratory manual "
The method provided is carried out.
In the lower book example of the present invention, the detection reference of L-5-methyl tetrahydrofolate accumulation: Jastrebova J,C,
Grahn A, et al.HPLC determination of folates in raw and processed beetroots [J] .Food Chemistry,
2003,80 (4): 579-588.
The structure of embodiment 1 co-expression plasmid
1.1 design of primers
According to GlyA and the MetF sequence of NCBI report, following 4 primers of design:
GlyAUP:AAGCTTATGTTAAAGCGTGAAATGAACA
GlyADOWN:GAATTCTTATGCTGAAACCGGGTAAC
MetFUP:GAGCTCATGAGCTTTTTTCACGCCAG
MetFDOWN:CTCGAGTTATAAACCAGGTCGAACCCC
Wherein GlyAUP and GlyADOWN is used for expanding GlyA code area;MetFUP and MetFDOWN is used for expanding
MetFDE code area;Introduce HindIII on GlyAUP, GlyADOWN, MetFUP, MetFDOWN respectively and be digested position
Point, EcoRI restriction enzyme site, SacI restriction enzyme site, XhoI restriction enzyme site.
1.2PCR expands GlyA sequence
Extracting obtains the genome of E. coli BL21.
With E. coli BL21 genome as template, respectively with GlyAUP and GlyADOWN of design in step 1.1
For primer pair, carry out PCR amplification, specific as follows:
The reaction system of amplification GlyA is: Premix Taq archaeal dna polymerase 25 μ L, E.coli BL21 (DE3) genomic DNA
1 μ L, each 1 μ L of upstream and downstream primer (10 μMs), add deionized water 20 μ l, be 50 μ L to system cumulative volume.
The reaction condition of amplification GlyA: 94 DEG C of 4min;94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 34 circulations;72℃
10min;4 ° of ∞, 30 circulations.
The reaction condition of amplification MetF: 94 DEG C of 4min;94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 34 circulations;72℃
10min;4 ° of ∞, 30 circulations.
Pcr amplification product carries out detected through gel electrophoresis, and testing result is as depicted in figs. 1 and 2.The size of the product obtained is respectively
For 1254bp and 891bp, meet the expection size of GlyA and MetF product.
The structure of 1.3 expression vectors
1.3.1 pretreatment is built
Use glue to reclaim the PCR primer obtained in kits step 1.2, then with pMD-19T SimpLe carrier, use
T4 DNA ligase, connects overnight in 16 DEG C, and reaction system is as follows:
The PCR primer of 4.6 μ L, 0.4 μ LpMD-19T SimpLe carrier, 5 μ L Solution I.
Connect product convert bacillus coli DH 5 alpha and be coated with the solid LB flat board containing 50 μ g/mL ampicillins, 37 DEG C of trainings
Support to transformant and grow.Wherein E.coli DH5 α is without ampicillin resistant strain, it is impossible at the solid containing ampicillin
Growing on LB flat board, therefore, the transformant that flat board grows is and has converted pMD19T-GlyA plasmid or pMD19T-MetF
The Escherichia coli of plasmid.Picking transformant is identified, according to qualification result, final obtain plasmid pMD19T-GlyA and
pMD19T-MetF。
Doing bacterium solution PCR with the bacterial strain containing plasmid pMD19T-GlyA and pMD19T-MetF, system is: Premix Taq enzyme
5 μ l, deionized water 3.7 μ l, template 0.5 μ l, upstream primer and downstream primer are respectively 0.4 μ l, are 10 μ l to system cumulative volume.
The bacterial strain amplifying purpose band does order-checking.
Plasmid pMD-GlyA and pMD19T-MetF, through sequence verification, comprises GlyA and MetF code area (GlyA and MetF
Coding region sequence is respectively as shown in SQ ID NO.1 and SQ ID NO.2), and code area suddenlys change without amino acid residue.
1.3.2 the structure of expression vector
By double digestion product GlyA in 1.3.1 and MetF gel purification, respectively with the table of Hind III/EcoR I double digestion
Reach carrier pET28a to connect, connection product is transformed into respectively Host Strains E.coli DH5 α, and is coated with containing 100 μ g/mL cards
That mycin solid LB flat board, 37 DEG C of cultivation to transformants grow.Wherein E.coli DH5 α is without kalamycin resistance bacterial strain, no
Can grow on the solid LB flat board containing kanamycins, therefore, the transformant that flat board grows is conversion
The Escherichia coli of pET28a-GlyA plasmid.Picking transformant is identified, according to qualification result, and final acquisition plasmid pET28a-GlyA
And pET28a-MetF.
Doing bacterium solution PCR with the bacterial strain containing plasmid pMD19T-GlyA and pMD19T-MetF, system is: Premix Taq enzyme
5 μ l, deionized water 3.7 μ l, template 0.5 μ l, upstream primer and downstream primer are respectively 0.4 μ l, are 10 μ l to system cumulative volume.
The bacterial strain amplifying purpose band does order-checking.
Plasmid pET28a-GlyA and pET28a-MetF, through order-checking, comprises GlyA code area (GlyA and MetF in this plasmid
Coding region sequence is respectively as shown in SQ ID NO.1 and SQ ID NO.2), wherein GlyA and MetF code area is placed in T7 and opens
Under mover, Lac operon, and code area suddenlys change without amino acid residue.
Owing to T7 promoter is strong transcription and translation signal, therefore, under the derivant of suitable concn, the two plasmid is permissible
High-caliber expression GlyA and MetF respectively.
The structure of 1.4 co-expression plasmids
SacI/XhoI digested plasmid pMD19T-MetF, it is thus achieved that MetF expression cassette, is then connected into use HindIII and EcoRI
The plasmid pET28a-GlyA being digested.
Through order-checking, and sequencing result is analyzed obtaining the structural representation of pET28a-GlyA-MetF plasmid, result such as figure
Shown in 4.In this plasmid, (GlyA and MetF coding region sequence is respectively such as SQ ID NO.1 and SQ in GlyA and MetF code area
Shown in ID NO.2) it is arranged in series under T7 promoter and the 1ac operon of plasmid pET28a-GlyA-MetF, Er Qiebian
Code district is without gene mutation.Testing result according to Fig. 4, SDS-PAGE and the strong transcription and translation signal of T7 promoter,
Suitable concentration lactose-induced under, plasmid pET28a-GlyA-MetF can high level coexpression GlyA and MetF.
Embodiment 2
By plasmid pET28a-GlyA-MetF, pET28a-GlyA and pET28a (+) be transformed into Host Strains E.coli respectively
BL21 (DE3), each names PGM01, PGM02 and P03, and is coated with the solid LB flat board containing 50 μ g/mL kanamycins,
37 DEG C of cultivation to transformants grow.Wherein E.coli BL21 (DE3) is without kalamycin resistance bacterial strain, it is impossible to containing kanamycins
Solid LB flat board on grow, therefore, the transformant that flat board grows be converted pET28a-GlyA-MetF,
PET28a-GlyA and pET28a (+) Escherichia coli of plasmid.
Respectively random picking some containing pET28a-GlyA-MetF, pET28a-GlyA and pET28a (+) plasmid conversion
Son, shake flask fermentation is cultivated, and grows into and adds derivant when OD value is between 0.6~0.8, induction 4~10h, sonicated cells,
Soluble protein is carried out SDS-PAGE, result such as Fig. 5.Result according to Fig. 5 shows PGM01 and PGM02 height respectively
Level have expressed GlyA and MetF and GlyA, meet the expection size of GlyA and MetF product;And P03 is i.e. the biggest
Scale reaches GlyA, does not also have great expression MetF.
The DNA of extracting PGM01 and PGM02, respectively with primer GlyAUP and GlyADOWN, MetFUP and
MetFDOWN, as primer pair, carries out PCR amplification to above-mentioned extract, it is thus achieved that a length of 1200bp's and 900bp
DNA fragmentation, the fragment of a length of 1200bp.Through sequence verification, GlyA and MetF expression cassette inserts the expression cassette of plasmid
Having inserted plasmid pET28a-GlyA-MetF, the expression cassette of GlyA inserts plasmid pET28a-GlyA.
According to the above results, plasmid pET28a-GlyA-MetF and pET28a-GlyA successful conversion enters Host Strains E.coli
BL21 (DE3), is respectively designated as PGM01 and PGM02.
Embodiment 3PGM01 and PGM02 and E.coli BL21 (DE3) shake flask fermentation L-5-methyl tetrahydrofolate
By grow on 37 DEG C of solid LB flat boards PGM01, PGM02 and E.coli BL21 (DE3) overnight (original bacteria,
Named BL21) single bacterium colony, be respectively connected in LB liquid medium, Shaking culture is overnight.
Take appropriate bacterium solution respectively and transfer into Medium of shaking flask fermentation, cultivate 11h.Then 1% inoculation, cultivate OD value 0.6~
Derivant is added time between 0.8;Add precursor folic acid 0.05g and glycerine 8% in good time, and take in fermentation 4h, 6h, 8h, 10h
Sample, the cumulant of L-5-methyl tetrahydrofolate in detection tunning, testing result is as shown in Figure 1.
According to the result of table 1, the cumulant of the L-5-methyl tetrahydrofolate in PGM01, PGM02 and BL21, in fermentation
Reach the highest during 10h, respectively reach about 12 times and 5.5 times of original bacteria, and the cumulant of bacterial strain PGM01 is maximum.Cause
This, in next example, the PGM01 only choosing shake flask fermentation later stage cumulant the highest carries out ferment tank.
The cumulant of the L-5-methyl tetrahydrofolate in table 1 PGM01, PGM02 and BL21 is over time
Embodiment 4PGM01 ferment tank L-5-methyl tetrahydrofolate
The mono-bacterium colony of PGM01 that will grow on solid LB (containing 50 μ g/mL kanamycins) flat board, is seeded to fermented and cultured
Base, shaking flask is overnight;1mL is gone to be seeded to 100mL ferment tank culture medium, Shaking culture about 3h;Then 1L is accessed
Ferment tank culture medium in (2.5L fermentation tank), and add defoamer polyoxypropylene polyethylene glycols ether (i.e. bubble enemy GPE),
Ferment tank is cultivated, and in time adds the derivant of debita spissitudo, and stream fills it up with the sweet of sertoli cell growth needs in sweat
Oil, meanwhile, by Feeding ammonia water, controlling pH is 6.4;Sweat adds precursor substance folic acid.Fermentation 10h stops sending out
Ferment.
Use said method, carry out the L-5-methyl tetrahydrofolate cumulant in parallel laboratory test, and separately sampled detection tunning,
The recombinant bacterium fermented and cultured obtained, the accumulation of L-5-methyl tetrahydrofolate in L-5-methyl tetrahydrofolate original strain in tunning
Amount, less than the accumulation L-5-methyl tetrahydrofolate original strain of conversion pET28a-GlyA-MetF plasmid, but by by matter
PET28a-GlyA converts accumulation L-5-methyl tetrahydrofolate original strain, it is thus achieved that recombinant bacterium, ferment this recombinant bacterium, applicable
Time adds derivant lactose, the method improving L-5-methyl tetrahydrofolate cumulant, also belongs to the scope of the present invention.
Claims (10)
1. a L-5-methyl tetrahydrofolate synthetase series coexpression recombinant plasmid, it is characterised in that include that serine hydroxymethyl turns
Move enzyme gene GlyA sequence, Methylene tetrahydrofolate reductase gene MetF sequence and carrier, the sequence of described GlyA and MetF
Respectively as on NCBI Gene ID be shown in the sequence of 947022 and 948432.
2. coexpression recombinant plasmid as claimed in claim 1, it is characterised in that described GlyA and MetF sequence is placed in T7 and opens
Under mover and lac operon.
3. coexpression recombinant plasmid as claimed in claim 1 or 2, it is characterised in that described coexpression recombinant plasmid also includes resisting
Property genetic fragment, described resistance gene fragment is kalamycin resistance gene (nptII).
4. coexpression recombinant plasmid as claimed in claim 1 or 2, it is characterised in that described GlyA sequence and MetF sequence are
Derive from the gene of E.coli BL21 (DE3).
5. the construction method of the coexpression recombinant plasmid according to any one of claim 1-4, it is characterised in that described method bag
Include following steps:
A) PCR amplification obtains GlyA and MetF sequence;
B) GlyA and MetF sequence is cloned into expression vector jointly.
6. the coexpression recombinant plasmid according to any one of claim 1-4 is converting the original bacteria of accumulation L-5-methyl tetrahydrofolate
Application in strain.
7. the biological synthesis method improving L-5-methyl tetrahydrofolate cumulant, it is characterised in that described method includes following
Coexpression recombinant plasmid transformed according to any one of claim 1-3 is accumulated L-5-methyl tetrahydrofolate original strain by step (1),
Obtain recombinant bacterium;(2) recombinant bacterium fermented and cultured step (1) obtained, induces L-5-methyl tetrahydrofolate metabolism with derivant
The expression of two key enzyme GlyA and MetF in approach, obtains L-5-methyl tetrahydrofolate.
8. method as claimed in claim 7, it is characterised in that the original strain of described accumulation L-5-methyl tetrahydrofolate is big
Enterobacteria.
9. application as claimed in claim 8 and method, it is characterised in that described Escherichia coli are E.coli BL21 (DE3).
10. method as claimed in claim 8, it is characterised in that derivant used is lactose.
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CN113403355A (en) * | 2021-06-17 | 2021-09-17 | 山东大学 | Method for producing L-5-methyltetrahydrofolic acid by biological method |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109536430A (en) * | 2018-12-18 | 2019-03-29 | 江南大学 | A kind of accumulation L-5- methyl tetrahydrofolate Lactococcus lactis and its construction method |
CN109652351A (en) * | 2018-12-18 | 2019-04-19 | 江南大学 | A kind of high yield 5-methyltetrahydrofolate recombined bacillus subtilis and its application |
CN113403355A (en) * | 2021-06-17 | 2021-09-17 | 山东大学 | Method for producing L-5-methyltetrahydrofolic acid by biological method |
CN113403355B (en) * | 2021-06-17 | 2022-05-31 | 山东大学 | Method for producing L-5-methyltetrahydrofolic acid by biological method |
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