CN109295123A

CN109295123A - A kind of biological production method of beta-flavin

Info

Publication number: CN109295123A
Application number: CN201811064631.4A
Authority: CN
Inventors: 赵广荣; 侯亚男; 于思礼
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2018-09-12
Filing date: 2018-09-12
Publication date: 2019-02-01

Abstract

本发明公开了一种甜菜黄素的生物生产方法，包括如下步骤：合成具有两个P_trc启动子的高拷贝表达载体pHTrc，合成4,5‑多巴雌二醇双加氧酶基因syndoda，从大肠杆菌BL2l(DE3)中克隆得到內源酪氨酸羟化酶基因HpaBC；将基因syndoda和HpaBC与表达载体连接，得到重组表达载体pHTDH，并转化到高产L‑酪氨酸的菌株SyBE‑002444中得到重组大肠杆菌BTX1；将活化的BTX1接种到添加卡那霉素的LB培养基中培养，加入IPTG诱导，饲喂组氨酸、赖氨酸或精氨酸，继续发酵培养，得到甜菜黄素。本发明利用游离诱导表达进行发酵生产不同种类甜菜黄素，解决单一种类的甜菜黄素难以获得的问题。The invention discloses a biological production method of betaflavin, comprising the following steps: synthesizing a high-copy expression vector pHTrc with two P _trc promoters, synthesizing a 4,5-dopaestradiol dioxygenase gene syndoda, The endogenous tyrosine hydroxylase gene HpaBC was cloned from Escherichia coli BL21 (DE3); the genes syndoda and HpaBC were connected with the expression vector to obtain the recombinant expression vector pHTDH, and transformed into the high-producing strain SyBE- of L-tyrosine Recombinant Escherichia coli BTX1 was obtained in 002444; the activated BTX1 was inoculated into LB medium supplemented with kanamycin for culture, induced by adding IPTG, fed with histidine, lysine or arginine, and continued to ferment and culture to obtain sugar beet flavin. The invention utilizes free inducible expression to ferment and produce different kinds of betaaxanthin, and solves the problem that a single type of betaaxanthin is difficult to obtain.

Description

A kind of biological production of betaxanthin

Technical field

The invention belongs to biomedicine technical field, it is related to producing the recombinant bacterium of betaxanthin and construction method and purposes.

Background technique

Betaxanthin (betaxanthin) is a kind of yellow form of betanidin (betAin), is typically used as food addition Agent, cosmetic colorant, while having both the bioactivity such as anti-oxidant, antitumor, liver protection and potential health care medical value.

Up to the present the industrial betanidin product overwhelming majority remains in plant extract, edible natural garden beet element Source is even more to be limited in beet and three-coloured amaranth, this also results in the betanidin type scarcity of commercialization and purity is not high.Now can The betanidin product enough obtained is mostly red beet root extract, and different types of betaxanthin there is no realization commodity Change, be merely present in laboratory and prepare on a small scale, this is unfavorable for the big rule for promoting betanidin class natural drug and food additives Mould innovative development and application.

Most countries food additives are all by strictly control, in food and pharmaceutical industry, the benefit of natural pigment With causing more and more to pay close attention to.A kind of natural pigment of the betanidin as safe and non-toxic, be commonly used for food additives come for Food provides color, and as anthocyanidin substitute.Its anti-oxidant, pre- preventing tumor reduces the works such as blood lipid, alleviation muscular fatigue With with important health value.

Summary of the invention

The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of biological production of betaxanthin.

Technical solution of the present invention is summarized as follows:

A kind of biological production of betaxanthin, includes the following steps:

(1) design and fully synthetic tool is there are two P_trcThe high copy expression carrier pHTrc of promoter, the nucleosides of the pHTrc Acid sequence is as shown in SEQ ID No.3；Artificial fully synthetic 4,5- DOPA estradiol dioxygenase gene syndoda, the gene The nucleotide sequence of syndoda is as shown in SEQ ID No.4；Endogenous tyrosine is obtained from clone in Escherichia coli BL2l (DE3) The nucleotide sequence of '-hydroxylase gene HpaBC, the gene HpaBC are as shown in SEQ ID No.5；

(2) the gene syndoda is connected by digestion connection type and the first set promoter of expression vector pHTrc It connects, obtains recombinant expression carrier pHTD；The gene HpaBC is passed through into digestion connection type and second set of expression vector pHTD Promoter connection, obtains recombinant expression carrier pHTDH；

(3) recombinant expression carrier pHTDH is transformed into the bacterial strain SyBE-002444 of high yield l-tyrosine, to monoclonal Screening obtains the recombination bacillus coli BTX1 for producing betaxanthin；

(4) the recombination bacillus coli BTX1 for producing betaxanthin is activated into 10-12 in the LB culture medium of addition kanamycins Hour；

(5) the recombination bacillus coli BTX1 of activation is cultivated by the LB that 1%-2% volume ratio is inoculated into addition kanamycins In base, culture to OD is 4-6, is transferred in M9 culture medium, continues culture to logarithmic phase；IPTG is added, continues to cultivate, feeding group Propylhomoserin, lysine or arginine continue fermented and cultured, obtain betaxanthin, and the IPTG is isopropyl-β-D-thiogalactose The abbreviation of glycosides.

Step (5) is preferred are as follows: the recombination bacillus coli BTX1 of activation is inoculated into 100mL addition by 1%-2% volume ratio In the LB culture medium of kanamycins, 37 DEG C, 220rpm cultivate to OD be 4-6, be transferred in 50mL M9 culture medium, initial OD is 1.0,30 DEG C, 200rpm continue culture to logarithmic phase；Be added final concentration 0.1mM IPTG, 30 DEG C, 200rpm continue to cultivate, feed Histidine, lysine or the arginine of final concentration 6mM continues fermented and cultured, obtains betaxanthin, and the IPTG is isopropyl- The abbreviation of β-D- thiogalactoside.

Advantages of the present invention:

The recombination bacillus coli of production betaxanthin of the invention, carries out fermenting and producing variety classes using free inducing expression Betaxanthin can solve the problems, such as that the betaxanthin of single kind is difficult to obtain, and provide method to synthesize different betaxanthins.

Detailed description of the invention

Fig. 1 is the synthetic route of betaxanthin.

Fig. 2 is the fully synthetic high copy expression carrier pHTDH schematic diagram of design.

Fig. 3 is the histidine-betaxanthin LC-MS map to be fermented with the recombinant bacterial strain.

Fig. 4 is the HPLC map of the three kinds of betaxanthins in total to be fermented with the recombinant bacterial strain.

Specific embodiment

Utilize artificial fully synthetic 4,5- DOPA estradiol dioxygenase gene syndoda and Escherichia coli endogenous tyrosine Hydroxylase HpaBC gene, realizes de novo formation betalamic acid in engineering colon bacillus.Betalamic acid synthetic quantity most The big stage adds the generation that different amino acid realizes variety classes betaxanthin.

Coli strain E.coli BL21 (DE3) used in the present invention is bought from the complete limited public affairs of formula gold biotechnology in Beijing Department.

Bacillus coli DH 5 alpha competent cell used in the present invention is bought from Beijing Bo Maide gene technology Co., Ltd.

Escherichia coli used in the present invention (escherichia coli) (Escherichia coli) be SyBE_002444, now in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, collection number of registering on the books is CGMCC No.7961.The preservation time is on July 22nd, 2013, and address is the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences Institute of microbiology, postcode 100101.

LB culture medium composition are as follows: 10g/L NaCl, 10g/L tryptone and 5g/L yeast powder, when configuring solid medium 1.5g agar powder is added in every 100mL, sterilize 20min at 121 DEG C of 0.1Mpa pressure.

M9 fermentation medium composition are as follows: 0.5g/L NaCl, 1g/L NH₄Cl 3g/L KH₂PO₄、17.1g/L Na₂HPO₄· 12H₂O, culture medium sterilize 20min at 121 DEG C of 0.1Mpa pressure, and the final concentration 5mM MgSO of sterilizing is then added₄、0.1mM CaCl₂With 5g/L glucose.

Added kanamycins concentration is 30 μ g/mL in thallus incubation.

IPTG is the abbreviation of isopropyl-β-D-thiogalactoside.

The biosynthesis pathway of betaxanthin of the present invention as shown in Figure 1: glucose through l-tyrosine in endogenous junket L-3,4 dihydroxyphenylalanine is generated under the action of propylhomoserin hydroxylase, L-3,4 dihydroxyphenylalanine generates betaine aldehyde under the catalysis of 4,5- DOPA estradiol dioxygenase Propylhomoserin.The betalamic acid production quantity maximum stage individually adds different types of amino acid or amino acid derivativges, such as when R base point Not Wei 4-methylimidazole, 1- butylamine and when guanidine propyl, addition is respectively histidine, lysine and arginine, continues to close At corresponding betaxanthin.

The present invention is further illustrated combined with specific embodiments below.

Embodiment 1

The acquisition of 4,5- DOPA estradiol dioxygenase gene syndoda and endogenous tyrosine '-hydroxylase gene HpaBC:

The amino acid sequence such as sequence of the 4,5- DOPA estradiol dioxygenase in the source four-o'clock (Mirabilis jApa) In table shown in SEQ ID No.1, in the amino acid sequence of Escherichia coli BL2l (DE3) endogenous tyrosine hydroxylase such as sequence table Shown in SEQ ID No.2.

Using the online codon optimization tool (http://genomes.urv.es/OPTIMIZER/) of OPTIMIZER, with Escherichia coli optimize the Preference of codon, and the full-length gene of design coding MjDODA is named as syndoda, core Nucleotide sequence is as shown in SEQ ID No.4.By the fully synthetic obtained syndoda gene of chemistry, two sides have NcoI and BamHI enzyme Enzyme site.Using e. coli bl21 (DE3) bacterium solution as template, primers F-HPNd, R- containing KpnI and HindIII restriction enzyme site HPKp carries out PCR amplification, obtains overall length HpaBC gene, nucleotide sequence is as shown in SEQ ID No.5.

The primer for cloning HpaBC full-length gene is as follows:

F-HPNd:CGGGGTACCATGAAACCAGAAGATTTCCGC (SEQ ID No.6)

R-HPKp:CCCAAGCTTTTATTAAATCGCAGCTTCCA (SEQ ID No.7)

Embodiment 2

The building of recombinant expression carrier pHTDH:

For having the syndoda genetic fragment of restriction enzyme site, carried out using FastDigest restriction endonuclease NcoI, BamHI Double digestion cuts the pHTrc expression vector (nucleotide sequence of the pHTrc such as SEQ ID No.3 institute with identical restriction endonuclease Show).Reaction system are as follows: 5 μ L 10 × FastDigest Buffer, 2 μ L restriction endonucleases 1,2 μ L restriction endonucleases 2,41 μ L genetic fragments. Reaction condition are as follows: 37 DEG C, 1h.The plasmid vector and target gene fragment obtained after endonuclease reaction, passes through PCR product purification and recovery Operating method in kit carries out purification and recovery.Product after purification is by connection reaction forming at pHTD.Linked system are as follows: 6 μ L digestion genetic fragments, 2 μ L digestion carrier segments, 1 μ L10 × T4 ligase buffer, 1 μ L T4DNA ligase.React item Part are as follows: 22 DEG C, 30min.Reaction system after connection needs conversion in time into competent cell, the specific steps are as follows: super Connection product is added to the DH5 α competent cell of 200 μ L in net workbench, 30min on ice is placed after mixing well.Ice bath The heat shock 90s in 42 DEG C of thermostat water baths of system afterwards is further continued for ice bath 2min, then adds in superclean bench to centrifuge tube Enter 600 μ L LB culture mediums, 37 DEG C, recovery 1h under conditions of 160rpm, after bacterium solution 4500rpm is centrifuged 1min, removal supernatant is trained Base is supported, thallus is resuspended with the LB culture medium of the centrifugation remaining about 50 μ L of bottom of the tube, and be uniformly coated to kalamycin resistance LB solid plate on, plate is placed in 37 DEG C of incubator and cultivates 12h or so and grow single colonie.Drawn with general on carrier Object F-pHTrctest, R-pHTrctest carry out bacterium colony PCR verifying, filter out the correct positive colony of conversion and sequence verification. Make in the same way, double enzymes are carried out to gene HpaBC and plasmid pHTD with FastDigest restriction endonuclease KpnI, HindIII It cuts, is attached after purification, carry out bacterium colony PCR verifying with universal primer F-pHTrctest, R-pHTrctest on carrier, sieve Correct positive colony and sequence verification are selected, plasmid pHTDH is obtained.

Successful pHTDH recombinant expression carrier is constructed for testing in next step.

PTrc universal primer is as follows:

F-pHTrctest:TTTGCGCCGACATCATAACG(SEQ ID No.8)

R-pHTrctest:CAACGTTCAAATCCGCTCCC(SEQ ID No.9)

Embodiment 3

By in the electroporated SyBE-002444 bacterial strain to high yield l-tyrosine of pHTDH recombinant expression carrier, BTX1 is obtained Recombinant bacterial strain.

It operates as follows:

1, the Escherichia coli SyBE-002444 frozen is connected in 5mL LB liquid medium by 1% volume ratio, and 37 DEG C, 220rpm is activated overnight culture.Activated bacterium solution continues to access in 10mL LB liquid medium with 1% volume ratio, 37 DEG C, 220rpm cultivates about 4h.

2, bacterium solution is all poured into the centrifuge tube of pre-cooling, 4000rpm, 4 DEG C of centrifugation 5min, discards the collection of supernatant culture medium Thallus.10% glycerol of 5mL of thallus pre-cooling is resuspended, and continues 4500rpm after mixing, and 4 DEG C of centrifugation 5min are discarded supernatant, This operation is repeated, is cleaned thallus 3 times with glycerol in total.Then 200 μ L, 10% glycerol is added thallus to be resuspended that competence is made is thin Born of the same parents.

3,100 μ L competent cells are drawn, are gone in sterilized and pre-cooling centrifuge tube, and 2.5 μ L structures are added thereto Successful pHTDH expression vector plasmid is built to be uniformly mixed.

4, the mixture of thallus and plasmid is all added in the electric revolving cup of pre-cooling, is put into electroporation, voltage is set 2500V, electroporated 5ms.The cell after electricity turns is sucked out into 1.5mL centrifuge tube, the liquid LB culture of 1mL non-resistant is added Base, piping and druming mixing, 37 DEG C, recovery culture 2h under the conditions of 160rpm.

6, the bacterium solution after drawing recovery in super-clean bench is inhaled 50 μ L and is evenly coated on the LB solid plate of kalamycin resistance, Cultivate 12h in 37 DEG C of constant incubator, the single colonie that picking is grown, until in LB liquid medium, 37 DEG C, 220rpm constant temperature Bacterium is deposited after culture 12h, obtains BTX1 recombinant bacterial strain, cryopreservation.

Embodiment 4

The recombination bacillus coli BTX1 for producing betaxanthin is activated to 10 hours in the LB culture medium of addition kanamycins (in fact It verifies bright, 11 hours or 12 hours can also be activated)；

Recombinant bacterium BTX1 fermentation detection:

The recombination bacillus coli BTX1 of activation is inoculated into 100mL by 1% volume ratio (being also possible to by 2% volume ratio) to add Add in the LB culture medium of kanamycins, 37 DEG C, 220rpm cultivate to OD be 5 (being also possible to 4 or 6).It is transferred to 50mL M9 culture In base, 1.0,30 DEG C of initial OD, 200rpm continue culture to logarithmic phase.Final concentration 0.1mM IPTG, 30 DEG C, 200rpm are added Continue to cultivate, feed final concentration 6mM histidine, continues fermented and cultured, obtain betaxanthin.The IPTG is isopropyl-β-D- The abbreviation of thiogalactoside.

High performance liquid chromatography-mass spectrometry (LC-MS) system is carried out after taking 0.22 μm of filtering with microporous membrane of supernatant Detection.Chromatographic condition is as follows: Alltima^TMC18 (5u 250mm × 4.6mm) chromatographic column, 25 DEG C of column temperature；0-25min is linearly terraced Degree, methanol: 1.5% phosphoric acid water=5:95 to methanol: 1.5% phosphoric acid water=60:40,25-30min linear gradient, methanol: 1.5% phosphoric acid water=60:40 is to methanol: 1.5% phosphoric acid water=5:95, flow velocity 1mL/min；UV detector, Detection wavelength 470nm.Mass Spectrometry Conditions are as follows: electron spray positively ionized (ESI+) detection, and scanning range is m/z 50-m/z 1000, spraying electricity Press 4500V, atomization air pressure 0.8Bar, dry 180 DEG C of temperature degree, flow velocity 6L/min；Collision cell radio-frequency voltage: 150.0Vpp.

As shown in figure 3, LC-MS map, which is shown, successfully synthesizes histidine-betaxanthin.

It is demonstrated experimentally that substituting the histidine in the present embodiment with lysine and arginine, it is also possible to obtain corresponding beet Flavine, as shown in figure 3, in addition to generate histidine-betaxanthin, efficient liquid phase chromatic graph also show lysine-betaxanthin and The generation of arginine-betaxanthin.

Illustrative description has been done to the present invention above, it should explanation, in the case where not departing from core of the present invention, Any simple deformation, modification or other skilled in the art can not spend the equivalent replacement of creative work to each fall within Protection scope of the present invention.

Sequence table

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gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 2640

gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa 2700

ttcgcgcgcg aaggcgaagc ggcatgcatt tacgttgcgc aacgcaatta atgtgagtta 2760

gcgcgaattg atctg 2775

<210> 4

<211> 825

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

catgccatgg gcatgaaagg gacatactat attaatcacg gcgaccctct gatgtacctg 60

aaaaagcaca ttaaattgcg tcagttcctg gagggttggc aggagaacgt tgtgatcgaa 120

aagccgaagt ccattctgat catttccgcc cattgggaca ctaacgtgcc gactgtgaac 180

ttcgtggaac attgcgacac catccacgat ttcgacgact acccggaccc actgtatcag 240

atccagtatc gcgcaccggg tgctcctaac ctggctaaaa aagttgagga gctgctgaaa 300

gagtctggta tggagtgcga aattgatacc aaacgtggtc tggatcacgc agcgtggttc 360

ccgctgatgt tcatgtatcc ggaagctaat attcctattt gtgaactctc tgttcagccg 420

agcaaagacg gtattcacca ttacaacgtg ggcaaggcac tgtcaccgct gctccaacag 480

ggtgttctga tcatcggttc gggcggcacc gttcacccga gcgacgacac cccgcactgc 540

ccgaacggtg ttgcgccctg ggctatcgag tttgataact ggctggaaga tgccctgctg 600

tctggtcgtt acgaagatgt taacaacttt aaaaagctgg caccgaactg ggaaatctcc 660

cacccgggcc aggagcacct gtatccgctg cacgttgcac tgggcgcggc tggtaaaaac 720

ccgaaaaccc aactgattca tcgttcctgg gcggccaacg gcgtattcgg ttatagcact 780

tacaacttca cccctactac tcaaaaaacg gactaaggat ccgcg 825

<210> 5

<211> 2114

<212> DNA

<213>Escherichia coli (Escherichia coli)

<400> 5

cggggtacca tgaaaccaga agatttccgc gccagtaccc aacgtccttt caccggggaa 60

gagtatctga aaagcctgca ggatggtcgc gagatctata tctatggcga gcgagtgaaa 120

gacgtcacca ctcatccggc atttcgtaat gcggcagcgt ctgttgccca gctgtacgac 180

gcactgcaca aaccggagat gcaggactct ctgtgttgga acaccgacac cggcagcggc 240

ggctataccc ataaattctt ccgcgtggcg aaaagtgccg acgacctgcg ccagcaacgc 300

gacgccatcg ctgagtggtc acgcctgagc tatggctgga tgggccgtac cccagactac 360

aaagccgctt tcggttgcgc actgggcgcg aatccgggct tttacggtca gttcgagcag 420

aacgcccgta actggtacac ccgtattcag gaaactggcc tctactttaa ccacgcgatt 480

gttaacccac cgatcgatcg tcatttgccg accgataaag tgaaagacgt ttacatcaag 540

ctggaaaaag agactgacgc cgggattatc gtcagcggtg cgaaagtggt tgccaccaac 600

tcggcgctga ctcactacaa catgattggc ttcggctcgg cacaagtgat gggcgaaaac 660

ccggacttcg cactgatgtt cgttgcgcca atggatgccg atggcgtgaa attaatctcc 720

cgcgcctctt atgagatggt cgcgggtgct accggctcgc catacgacta cccgctctcc 780

agccgcttcg atgagaacga tgcgattctg gtgatggata acgtgctgat tccatgggaa 840

aacgtgctga tctaccgcga ttttgatcgc tgccgtcgct ggacgatgga aggcggtttt 900

gcccgtatgt atccgctgca agcctgtgtg cgcctggcag tgaaattaga cttcattacg 960

gcactgctga aaaaatcact cgaatgtacc ggcaccctgg agttccgtgg tgtgcaggcc 1020

gatctcggtg aagtggtagc gtggcgcaac accttctggg cattgagtga ctcgatgtgt 1080

tcagaagcaa cgccgtgggt caacggggct tatttaccgg atcatgccgc actgcaaacc 1140

tatcgcgtac tggcaccaat ggcctacgcg aagatcaaaa acattatcga acgcaacgtt 1200

accagtggcc tgatctatct cccttccagt gcccgtgacc tgaataatcc gcagatcgac 1260

cagtatctgg cgaagtatgt gcgcggttcg aacggtatgg atcacgtcca gcgcatcaag 1320

atcctcaaac tgatgtggga tgctattggc agcgaatttg gtggtcgtca cgaactgtat 1380

gaaatcaact actccggtag ccaggatgag attcgcctgc agtgtctgcg ccaggcacaa 1440

aactccggca atatggacaa gatgatggcg atggttgatc gctgcctgtc ggaatacgac 1500

caggacggct ggactgtgcc gcacctgcac aacaacgacg atatcaacat gctggataag 1560

ctgctgaaat aacgcagcag gaggttaaga tgcaattaga tgaacaacgc ctgcgctttc 1620

gtgacgcgat ggccagcctg tcggcagcgg taaatattat caccaccgag ggcgacgccg 1680

gacaatgcgg gattacggca acggccgtct gctcggtcac ggatacacca ccgtcgctga 1740

tggtgtgcat taacgccaac agtgcgatga acccggtttt tcagggcaac ggcaagttgt 1800

gcgtcaacgt cctcaaccat gagcaggaac tgatggcacg ccacttcgcg ggcatgacag 1860

gcatggcgat ggaagagcgt tttagcctct catgctggca aaaaggtccg ctggcgcagc 1920

cggtgctaaa aggttcgctg gccagtcttg aaggtgagat ccgcgatgtg caggcaattg 1980

gcacacatct ggtgtatctg gtggagatta aaaacatcat cctcagtgca gaaggtcatg 2040

gacttatcta ctttaaacgc cgtttccatc cggtgatgct ggaaatggaa gctgcgattt 2100

aataaaagct tggg 2114

<210> 6

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

cggggtacca tgaaaccaga agatttccgc 30

<210> 7

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

cccaagcttt tattaaatcg cagcttcca 29

<210> 8

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

tttgcgccga catcataacg 20

<210> 9

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

caacgttcaa atccgctccc 20

Claims

1. a biological production method of beta-flavin, is characterized in that comprising the steps:

(1) Design and fully synthesize a high-copy expression vector pHTrc with two P _trc promoters, the nucleotide sequence of pHTrc is shown in SEQ ID No. 3; artificial total synthesis of 4,5-dopaestradiol The dioxygenase gene syndoda, the nucleotide sequence of the gene syndoda is shown in SEQ ID No. 4; the endogenous tyrosine hydroxylase gene HpaBC was cloned from Escherichia coli BL21 (DE3), and the gene HpaBC The nucleotide sequence is shown in SEQ ID No.5;

(2) connecting the gene syndoda with the first set of promoters of the expression vector pHTrc by enzymatic cleavage to obtain a recombinant expression vector pHTD; connecting the gene HpaBC with the second set of the expression vector pHTD through enzymatic cleavage sub-connected to obtain the recombinant expression vector pHTDH;

(3) The recombinant expression vector pHTDH was transformed into the high L-tyrosine-producing strain SyBE-002444, and the single clone was screened to obtain a beta-flavin-producing recombinant Escherichia coli BTX1;

(4) activating beta-flavin-producing recombinant Escherichia coli BTX1 in LB medium supplemented with kanamycin for 10-12 hours;

(5) Inoculate the activated recombinant Escherichia coli BTX1 into the LB medium supplemented with kanamycin at a volume ratio of 1%-2%, cultivate to an OD of 4-6, transfer it to the M9 medium, and continue to cultivate until Logarithmic phase; add IPTG, continue to culture, feed histidine, lysine or arginine, continue to ferment and culture to obtain betaflavin, the IPTG is isopropyl-β-D-thiogalactoside abbreviation of.

2. method according to claim 1, is characterized in that described step (5) is: by 1%-2% volume ratio inoculate in the LB substratum of 100mL adding kanamycin by the activated recombinant Escherichia coli BTX1 , cultivated at 37°C, 220rpm until the OD is 4-6, transferred to 50mL M9 medium, the initial OD was 1.0, continued to cultivate to log phase at 30°C, 200rpm; added final concentration of 0.1mM IPTG, continued to culture at 30°C, 200rpm , feeding histidine, lysine or arginine with a final concentration of 6 mM, and continuing to ferment and culture to obtain betaflavin, the IPTG is the abbreviation of isopropyl-β-D-thiogalactoside.