CN114045219A

CN114045219A - Method for rapidly screening high-yield caffeic acid strains in high flux

Info

Publication number: CN114045219A
Application number: CN202111362147.1A
Authority: CN
Inventors: 刘人铭; 孙敬方
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-11-17
Filing date: 2021-11-17
Publication date: 2022-02-15
Anticipated expiration: 2041-11-17
Also published as: CN114045219B

Abstract

The invention discloses a method for rapidly screening high-yield caffeic acid strains in a high throughput manner, and belongs to the technical field of biological engineering. The method comprises the steps of fermenting the strains to be screened to obtain fermentation liquor; centrifuging the obtained fermentation liquor to obtain fermentation supernatant; placing the fermentation supernatant in a perforated plate; adding ferric chloride reaction liquid into the fermentation supernatant for color reaction, and detecting the OD value under 680nm by using a full-wavelength microplate reader; selecting caffeic acid producing strain with high OD value. The high-throughput screening method can greatly improve the screening efficiency of the strains, greatly increase the number of the strains screened in a single batch, and shorten the screening experiment period of the caffeic acid high-yield strains.

Description

Method for rapidly screening high-yield caffeic acid strains in high flux

Technical Field

The invention discloses a method for rapidly screening high-yield caffeic acid strains in a high throughput manner, and belongs to the technical field of biological engineering.

Background

Caffeic acid is a natural phenolic acid compound, and is derived from phenylalanine pathway in plants. Phenylpropionic acids, particularly caffeine, are receiving increasing attention due to their important pharmacological effects, such as antioxidant, anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressant properties.

However, since caffeic acid is difficult to extract from plants, the cost is high, and the requirement of industrial production cannot be met. In order to meet the market demand of caffeic acid and realize high-level production, researchers use metabolic engineering and other means to make escherichia coli ferment to produce caffeic acid, in the process of fermenting caffeic acid, the acid production capacity of a production strain is a key ring for determining production cost, and it is very important to obtain high-yield caffeic acid strains.

In order to overcome the defects of the prior art, a method for quickly screening the caffeic acid high-yield strains in a large batch of strains to be tested with high efficiency and low cost is urgently needed.

Disclosure of Invention

The invention provides a method for rapidly screening high-yield caffeic acid strains in a high throughput manner, which can greatly improve the screening efficiency of strains, greatly increase the number of strains screened in a single batch, and shorten the screening experiment period of the high-yield caffeic acid strains.

The technical scheme adopted by the invention is as follows:

the invention provides a method for rapidly screening high-yield caffeic acid strains in a high-throughput manner, which specifically comprises the following steps:

carrying out fermentation culture on a strain to be screened to obtain fermentation liquor, and centrifuging to obtain fermentation supernatant; and transferring the fermentation supernatant into a porous plate, wherein the porous plate can be a conventional 96 porous plate or 384 porous plate, adding 3-valent iron ion reaction liquid into the fermentation supernatant for color development reaction, then placing the fermentation supernatant into a full-wavelength microplate reader for detection, detecting the OD value of the fermentation supernatant corresponding to the strain at 680nm, and reflecting the caffeic acid production capacity of the corresponding strain by using the obtained OD value, wherein the color development reaction and the OD value detection are carried out in the porous plate without carrying out the transfer and repeated sampling of the fermentation supernatant.

As an implementation mode of the invention, the method for rapidly screening the high-yield caffeic acid strains at high flux of the invention takes escherichia coli as an engineering chassis strain, uses coumaric acid as a substrate to carry out fermentation to produce caffeic acid, and needs to be noted that components in the fermentation substrate do not interfere with detection results, the p-coumaric acid in the fermentation substrate can also react with ferric chloride, but the p-coumaric acid reacts with ferric chloride to form yellow brown, the caffeic acid reacts with ferric chloride to form dark green, the caffeic acid-producing strain in the strain to be screened can be preliminarily judged according to the color of reaction products, the yield of the caffeic acid can be preliminarily judged according to the depth of the presented dark green, and the absorbance of the reaction products of the p-coumaric acid and the ferric chloride is extremely low at 680nm, therefore, the p-coumaric acid and the caffeic acid can both react with the ferric chloride to develop color, but the maximum absorption wavelengths of the two are different after the reaction, therefore, the interference to the coumaric acid can be completely eliminated under 680 nm; in addition, the glucose concentration in the fermentation liquor is low, no obvious color reaction is caused after the color developing agent is added, and even if 50 percent of high-concentration glucose solution reacts with ferric chloride to be light yellow, the OD value of the glucose solution at 680nm is extremely low; the other intermediate metabolite tyrosine does not react with ferric chloride.

In a preferred embodiment of the invention, the fermentation culture adopts a P-5052 automatic induction culture medium, and the shaking fermentation culture of a 96-deep-hole plate is carried out in a shaking table at the temperature of 32-38 ℃ and the rpm of 100-240, wherein each liter of the P-5052 automatic induction culture medium contains Na₂HPO₄ 50～60mM，KH₂PO₄ 50～60mM，(NH₄)₂SO₄ 20～30mM，MgSO₄2 to 3mM, 0.2 to 0.5 percent of glycerol, 0.1 to 0.2 percent of lactose, 0.04 to 0.05 percent of glucose, 0.1 to 0.4mM of p-coumaric acid and pH of 7.0 to 7.4.

In a preferred embodiment of the present invention, the P-5052 auto-induction medium contains Na₂HPO₄ 50mM，KH₂PO₄ 50mM，(NH₄)₂SO₄ 25mM，MgSO₄2mM, 0.5% of glycerol, 0.2% of lactose, 0.05% of glucose and 0.3mM of p-coumaric acid, wherein the pH value is 7.0-7.4; go toPreferably, the pH of the P-5052 auto-induction medium is 7.2.

In a preferred embodiment of the invention, the fermentation culture adopts a P-5052 automatic induction culture medium, and the shaking fermentation culture is carried out in a shaking table at 37 ℃ and 220rpm in a 96 deep-well plate, wherein the culture time is 18-24 h.

In a preferred embodiment of the invention, the fermentation supernatant is obtained after centrifugation at 14,000rpm for 2 min.

In a preferred embodiment of the invention, the strain screening is performed in a 96-well plate, and the volume of the absorbed fermentation supernatant is 100-250 μ l; preferably, the volume of the aspirated fermentation supernatant is 200. mu.l.

In one embodiment of the present invention, the reaction solution of iron ions with 3 valence is an aqueous solution of ferric chloride; further, the concentration of the ferric chloride aqueous solution is 25-28 g/L; preferably, the concentration of the ferric chloride aqueous solution is 27.03 g/L.

In one embodiment of the invention, the addition amount of the 3-valent iron ion reaction solution is 1/5-1/20 of the volume of the fermentation supernatant; preferably, the amount of the added 3-valent iron ion reaction solution is 1/10 of the volume of the fermentation supernatant.

In one embodiment of the present invention, in order to quantify the caffeic acid production capability of a strain to be screened more rapidly and conveniently by an OD value, the method for rapidly screening a caffeic acid producing strain with high throughput further comprises the establishment of a standard curve of the corresponding relationship between the caffeic acid yield and the OD value, the caffeic acid yield value of a corresponding strain can be obtained by substituting the OD value measured by fermentation supernatant of the strain to be screened into the standard curve, and the standard curve establishment process comprises: weighing a proper amount of caffeic acid standard substance powder, adding a proper amount of methanol to prepare caffeic acid mother liquor with a proper concentration, diluting the caffeic acid mother liquor with ultrapure water to obtain gradient solutions of 1mg/ml, 800 mu g/ml, 600 mu g/ml, 300 mu g/ml, 150 mu g/ml, 100 mu g/ml, 50 mu g/ml and 25 mu g/ml, and using the ultrapure water as a blank control; respectively and equivalently absorbing two thirds of the volume of the porous plate with the caffeic acid diluted solution, the further diluted solution and the blank control solution to the porous plate, adding a proper amount of 3-valent iron ion reaction solution into each hole, standing at room temperature for at least 1min, putting the porous plate into a multifunctional microplate reader, shaking for 3s, reading the OD value at 680nm, and taking the caffeic acid concentration as the abscissa and the OD value at 680nm as the ordinate as a standard curve.

In one embodiment of the invention, Escherichia coli is used as an engineering chassis strain, the strain to be screened is selected from the caffeic acid producing mutant strain library, and the caffeic acid producing mutant library establishment process comprises the following steps: and (3) designing a primer to carry out error-prone PCR amplification by taking the hydroxylase gene as a template to obtain a target gene fragment, recombining a plasmid, and then loading the plasmid into escherichia coli to construct a mutant strain library.

In a preferred embodiment of the present invention, the hydroxylase gene is a complex 4-hydroxyphenylacetic acid-3-hydroxylase gene, hereinafter referred to as 4HPA3H gene. Further, the 4HPA3H gene sequence is shown in SEQ ID NO. 1.

Compared with the prior art, the invention has the beneficial effects that:

in a specific bacterial population, in the process of obtaining a specific product strain with high yield, high conversion efficiency and short fermentation time by screening, the yield detection of the specific product is a key link, at present, the yield detection of the caffeic acid high-yield strain generally uses a High Performance Liquid Chromatography (HPLC) detection method, and the method is practical under the condition of less strain quantity to be screened, but for a large-batch series strain library established by the invention, the problems of low efficiency, complicated processing steps and high cost exist when the method is used for screening the high-yield strain, so the traditional screening method is not suitable.

The invention directly reacts and develops the fermentation supernatant in a 96-well plate or other multi-well plates, which not only greatly reduces the sampling amount and the usage amount of ferric chloride reaction liquid; but also greatly shortens the color reaction time of the fermentation supernatant and the speed of screening the caffeic acid production strains. Therefore, the method of the invention can efficiently and quickly screen the caffeic acid high-yield strains.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1a is a photograph of a caffeic acid standard solution after addition of a chromogenic solution in accordance with an embodiment;

FIG. 1b is a wavelength scan of the chromogenic solution of FIG. 1 a;

FIG. 2 is a map of the recombinant plasmid pET24a (+) -4HPA3H in accordance with an embodiment;

FIG. 3 is a graph of absorbance of caffeic acid after addition of a chromogenic solution at a fixed wavelength;

FIG. 4 is a graph of a fitted curve over a concentration range of 0-0.3 mg/ml.

Detailed Description

The currently adopted engineering chassis strain for biologically synthesizing caffeic acid is mainly escherichia coli, while experiments prove that a catalytic path with higher titer still adopts one step of catalyzing P-coumaric acid as a target product caffeic acid, and three isoenzymes in the step can be used, namely 4-coumaric acid-3-hydroxylase (Coum3H), cytochrome enzyme P450 CYP199A2 and hydroxylase compound 4-hydroxyphenylacetic acid-3-hydroxylase (4HPA 3H).

In the following examples, Escherichia coli is used as an engineering chassis Strain, coumaric acid is used as a substrate for fermentation to produce caffeic acid, specifically E.coli is E.coli BL21(DE3) Strain, and the biological enzyme is a hydroxylase complex 4-hydroxyphenylacetic acid-3-hydroxylase (4HPA3H), and the invention is further explained by combining specific examples.

FeCl with a valence-3 iron ion reaction solution of 27.03g/L used in the following examples₃·6H₂O; the culture medium is P-5052 auto-induction culture medium, the rest is purchased from TAKARA except template, and each liter of culture medium contains Na₂HPO₄ 50mM，KH₂PO₄ 50mM，(NH₄)₂SO₄ 25mM，MgSO₄2mM, 0.5% of glycerol, 0.2% of lactose and glucose0.05% glucose, 0.3mM p-coumaric acid, pH 7.2; the model of the multifunctional microplate reader is BioTek, Winooski, VT, USA.

Example 1: method for rapidly screening high-yield caffeic acid strains in high flux

The method comprises the following specific steps:

step (1): inoculating the strain to be screened into a 96 deep-well plate containing a P-5052 culture medium, and culturing for 24 hours at 37 ℃ by a shaking table at 220 rpm; p-5052 medium without inoculated strain was additionally used as a blank control;

step (2): centrifuging the 96 deep-hole plate fermentation liquor obtained by culturing the strain in the step (1) for 2min at 14000rpm to obtain fermentation supernatant;

and (3): respectively sucking 200 mul of the obtained fermentation supernatant of different strains and a blank control P-5052 culture medium, transferring the fermentation supernatant and the blank control P-5052 culture medium into a 96-micropore plate, adding 10 mul of 3-valent iron ion reaction solution into each micropore, scanning an OD value under 680nm by using an enzyme-labeling instrument, wherein the strain corresponding to the reaction solution with the high OD value is the strain with the high caffeic acid yield.

Example 2: establishment of standard curve of corresponding relation between caffeic acid yield and OD value

Step (1): weighing a proper amount of caffeic acid standard substance powder, and adding a proper volume of methanol to prepare caffeic acid mother liquor of 10 mg/ml.

Step (2): diluting 10mg/ml caffeic acid mother liquor with ultrapure water to obtain caffeic acid diluted solutions with concentration gradient of 1mg/ml, 800 μ g/ml, 600 μ g/ml, 300 μ g/ml, 150 μ g/ml, 100 μ g/ml, 50 μ g/ml and 25 μ g/ml, respectively, using ultrapure water as blank control, namely 0 μ g/ml caffeic acid diluted solution, and using caffeic acid diluted solution as standard solution;

and (3): selecting the maximum absorption wavelength of the reaction product: respectively sucking 200 mul of the standard solution, transferring the standard solution into a 96-microporous plate, adding 20 mul of 3-valent iron ion reaction solution into each hole, standing at room temperature for 1min, performing color reaction, and then as shown in figure 1a, scanning an OD value under 500-850 nm by using an enzyme reader, and as shown in figure 1b, obtaining a product after detection reaction through data statistics, wherein the maximum absorption wavelength of the product is 680nm, and the linear range of the caffeic acid concentration, which can be detected by the detection method through linear fitting, is 0-0.3 mg/ml;

and (4): respectively sucking 200 μ l of caffeic acid diluted solution including blank control solution into 96 multi-well plate, adding 10 μ l of 3-valent iron ion reaction solution into each well, standing at room temperature for 1min, placing 96 multi-well plate into multifunctional microplate reader after color reaction, shaking for 3s, reading OD value at 680nm, as shown in FIG. 3, with caffeic acid concentration as abscissa and OD value at 680nm as ordinate, and when caffeic acid concentration is higher than 0.3mg/ml, OD value is higher than OD value₆₈₀The numerical value changes slowly and approaches the plateau period; when the caffeic acid concentration is higher than 1mg/ml, the obtained OD₆₈₀If the caffeic acid yield of the production strain is higher than 0.3mg/ml, adding 3-valent iron ion reaction liquid and detecting the concentration of the caffeic acid which cannot reflect the exact production of the strain at 680nm, but quickly screening out high-yield strains and low-yield strains in a short time, and further diluting the measured OD value to judge the caffeic acid yield;

in the concentration range of 0.3mg/ml, 3 independent replicates were tested, as shown in FIG. 4, to obtain a linear fit equation:

y＝4.085C-0.04414，

wherein C is caffeic acid concentration (mg/ml) and y is OD₆₈₀Linear fitting constant R of linear fitting equation²0.9618, the detection limit under 3 times of signal-to-noise ratio is 0.041mg/ml, as shown in the following table 1, the SD values in the linear range are all lower than 0.01, the precision and the repeatability are good, and the standard curve can be used;

table 1: OD in the concentration range of 0-0.3 mg/ml₆₈₀Measured value and SD value correspondence table

Adding 100 mul of 0.2mg/ml caffeic acid standard solution into 100 mul of 0.1mg/ml caffeic acid standard solution and 0.4mg/ml caffeic acid standard solution respectively, mixing uniformly, adding 10 mul of 3-valent iron ion reaction solution respectively, scanning OD value under 680nm with enzyme-labeling instrument, calculating recovery rates to obtain 106% and 97.2% respectively, and the recovery rate is good.

Example 3: mutant library construction for caffeic acid production

Step (1): taking the hydroxylase gene shown in SEQ ID NO.1 as a template, designing a PCR primer pair to carry out error-prone PCR amplification, and carrying out agarose gel electrophoresis, recovering and purifying to obtain a target gene fragment;

wherein restriction enzyme Nde I and Not I restriction sites are added at the front and back ends of the target gene, as shown below:

nde I: enzyme digestion sequence and protected base: GGGAATTC

Not I: enzyme digestion sequence and protected base: AAGGAAAAAA

Error-prone PCR amplification system (50. mu.l) included: the PCR primer pair comprises upstream primer F-4HPA3H 0.2.2 μ l shown in SEQ ID NO.2, downstream primer R-4HPA3H 0.2.2 μ l shown in SEQ ID NO.3, and 10mM Mn²⁺0.6. mu.l dNTP (25mM), 4. mu.l dTTP (100mM), 4. mu.l dCTP (100mM), 0.4. mu.l template, 5. mu.l 10 XPCR buffer, 1. mu.l rTaq enzyme, 25mM MgCl₂5 μ l with ddH₂O to 50. mu.l volume;

step (2): digesting the target gene and an expression vector pET24a (+) for 24h at 37 ℃ by using restriction enzymes Nde I and Not I, and recovering and purifying the digested fragments by agarose gel electrophoresis;

and (3): as shown in FIG. 2, the purified target gene fragment and linearized pET24a (+) were ligated with T4 DNA ligase at 16 ℃ to obtain a recombinant plasmid containing the target gene, designated pET24a (+) -4HPA3H, which was transformed into E.coli BL21(DE3), plated with LB plate containing 50. mu.g/ml kanamycin, and cultured by inversion at 37 ℃ overnight;

and (4): single colony clones grown on the plates were inoculated into 96-deep-well plates containing P-5052 medium (containing 0.3mM P-coumaric acid) for 24h at 37 ℃ with shaking at 220rpm, together with an empty plasmid pET24a (+) strain (as a negative control strain) not containing the gene of interest. P-5052 medium without inoculated strain was additionally used as a blank.

Test example: verification of high-yield caffeic acid bacterial strain

Step (1): the mutant strain and the original strain which are screened from the mutant library in the embodiment 3 by the method in the embodiment 1 and have the highest OD680 value are respectively inoculated into a seed culture medium, 50ml of the mutant strain and the original strain are shake-bottled with 500ml, cultured for 16h at 37 ℃ and 220rpm, and transferred into a 5L fermentation tank containing 2L of fermentation medium for fermentation by 5 percent of inoculum size, wherein the initial fermentation condition is that the initial sugar concentration is 50g/L, the rotation speed is 400rpm, the temperature is 37 ℃, and the aeration rate is 1 vvm;

step (2): when the concentration of reducing sugar is below 20g/L in the fermentation process, adding a certain amount of 500g/L glucose feeding liquid;

and (3): monitoring the concentration of reducing sugar every 4h, and continuously adding a certain amount of feed supplement liquid to maintain the concentration of the reducing sugar;

and (4): after fermentation for 36h, the caffeic acid titer of the original strain and the mutant strain is respectively detected.

The detection result is as follows: the caffeic acid yield of the original strain is 1.4g/L, the caffeic acid yield of the mutant strain with the highest yield is 4.2g/L, the hydroxylase gene sequence of the high-yield mutant strain is detailed in SEQ ID NO.4, and the amino acid sequence of the high-yield mutant strain is detailed in SEQ ID NO. 5.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> university of Chinese pharmacy

<120> method for rapidly screening high-yield caffeic acid strains in high flux

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1479

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgattaaaa atggaacaca acacatagct tcgctgcgcg acggccgtca ggtgtacctg 60

aacggtcaac cggtgggcga cgttacggcc cacgccgcgt ttcgtaacag cattcgtagc 120

tacgcgtcac tgtacgacta ccaagcgcgt gacgagaacg tggagaaaat gaccttcgtt 180

tccccgggtt ctggtaatcg tgtctcccgg atctggcagc tgcctactag ctacgacgag 240

ttggtcgagc gccgcgctgc cttggaagct tggtcagagc tgcattatgg cttcatgggt 300

cgtagcccgg accacgttgc cagttgcctg tccggtatgt atatgggtgc cgatgtgttc 360

gaacagtatg acccggcacg tgccggtgcg ctgagggatt attaccgcta tgtacgtgac 420

aatgatttat ttctcaccta tgttattgtg aacccgcagg caaatcaaag caaagcggcg 480

cacgaacaag aagataaata tctcgcggta ggcatcgtcg atcaggatgc agaaggtatt 540

accgtccgcg gtgctaagat gttggcgact agcgggatca tggcaaatga agttttctgc 600

tcttgcattc agccgctgcg tgagggcgat gagatgtacg cgctcacctt tgcggttccg 660

atgaatgcga aaggtatgaa gatcatgagc cgtaaaagct atgaggaaaa cgccaccagc 720

gttttcgaca acccgctgag ctctagattc gacgagaacg acgcggttct gtacttcgat 780

gatgttaagg tgccgtggga acgcatcttt gtggcgggta acaccgcaat gtgcgcaaag 840

cagtttcatg cgaccccggc acacgtgtac caaaactatc aatgtcaggt gcgtttaatg 900

accaagctgc gcttcctggt gggtctgggc ctgaaaatca gcgagatcaa cggcacgaac 960

acgtttccgc aggtgcgcga gacactgggt cagctggctg cggaagctag catggtggaa 1020

gcgtgggttt acggcatgga agcgaagggc accgtggtga atggcttcta cgtcccagat 1080

cgtaatatgc tgtacggctc tcaggttgtt acgcagcagt tgtactccaa ggtgcttaac 1140

accctgcgcg agctggcggg tggtggcatg attgttttgc caagcagcgt tcgtgacttc 1200

gagaacccgg acctgctgcg tatcattgaa aaaacccaaa aaagcccggt ttgttcctcc 1260

gaggaacgtg tgaagttctt taagttggct tgggatgcgg tgggtagcga gttcgcgtcg 1320

cgccacaatc aatacgaatt gttttatgca ggtgcaagct ttgtcaccaa aggtcatgcg 1380

tatcgtacct atgattggca acgtgcgagc catctggttg acagcatgct tggcagctac 1440

agcttgaacc aagagctgtc gaccccgcgt gctgcttaa 1479

<210> 2

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gggaattcca tatgattaaa acaggatcag cac 33

<210> 3

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aaggaaaaaa gcggccgctt actcagcagc acgcgccgcg gttggcgcc 49

<210> 4

<211> 1485

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgattaaaa caggatcagc acacatagct tccctgcgcg acggtcgcga gatctttcta 60

gacggccaaa aaattagtga tgcgaccacg catccggcgt ttcgtcgtgc ggtggcgtcc 120

gtgggccgta tgttcgattt tcacagcgcc ccggacaacc gtgcgctgat gacctttgag 180

acggataccg gtacgctggc gaatcgtatc tggcagctgc cgaccagcta cgacgagctc 240

aagacgcgtc gtcgtggttt ggaggcctgg accgaactcc atgcgggttt tttgggtaga 300

gcaccggacc acgttgcttc ttgtattagc ggcatgtata tgggtttgga ccagttcgaa 360

ctgtatgacc cagcgcgcgc gcgtgcgctc ggcgattact atcgtcacgc ccgtgacaat 420

gacctgtacc tgacctacgt gatcatcaac ccgcaagctg accgcagcaa aaacgcagcc 480

gaacaggcgg acccgttttt gtctgcggcg atcgttgatg aagatacgtc tggtattact 540

gtacgcggtg cgaaaatgct tgctaccggt ggcattatgg ctaacgaggt gtttgtcacc 600

tgcatccagc cactgcaaaa aggtgaggaa gcgtacgcgc tttccttcgc aattccgatg 660

aacacgagag gcctgaagat cctgtcgcgt aaaagctacg aagcgggtgc gccgagcgtg 720

ttcgacaatc cgttgagctc tcgctttgac gagaacgatg cggtgctgta cttcgatgat 780

gtgaaggttc cgtgggatcg tgttttcatc gcgggcgata tcgcaatgac cggccgccag 840

ttccacgcga ccccggccca tgtttatcaa aactatcagg ctcagattcg tctgagcgtg 900

aagctgaaat ttctcctggc gatcgcccgc cgtactgccg aggtcaatgg caccactggt 960

ttcccgcagg ttagagagac cctgggtcag ttggctgctg aggcggctat ggttgatgcc 1020

tttgttgcgg ctatggaagc aaagggcagc ttctacggcc cttattttgt cccggaccgt 1080

cacaccttgt acgcagacca aaccctgacc cagcagctgt acggcaaatt catcacctcc 1140

ctgcgtgaac tggcaggagg cggtatgatt atgcttccga gcagcgtgca tgattaccaa 1200

aatccggaat tagcggctct gattgccaag actcaacaga gcccggcagc tagcgcggag 1260

gaaaaagtta agttctacaa gctggcatgg gatgcggtgg gttcggagtt tgcttcccgc 1320

cacgcccaat atgaaatgtt ctatgcgggt gcgggtttcg ttgttaagaa ccactctttc 1380

cggacctacg actgggcagg cgcggagcgc ttgctggaca tgatgttgtc gtcctacagc 1440

ctggctgacg aagtggcgcc aaccgcggcg cgtgctgctg agtaa 1485

<210> 5

<211> 478

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ile Lys Thr Gly Ser Ala His Ile Ala Ser Leu Arg Asp Gly Arg Glu

1 5 10 15

Ile Phe Leu Asp Gly Gln Lys Ile Ser Asp Ala Thr Thr His Pro Ala

20 25 30

Phe Arg Arg Ala Val Ala Ser Val Gly Arg Phe Asp Phe His Ser Ala

35 40 45

Pro Asp Asn Arg Ala Leu Thr Phe Glu Thr Asp Thr Gly Thr Leu Ala

50 55 60

Asn Arg Ile Trp Gln Leu Pro Thr Ser Tyr Asp Glu Leu Lys Thr Arg

65 70 75 80

Arg Arg Gly Leu Glu Ala Trp Thr Glu Leu His Ala Gly Phe Leu Gly

85 90 95

Arg Ala Pro Asp His Val Ala Ser Cys Ile Ser Gly Tyr Gly Leu Asp

100 105 110

Gln Phe Glu Leu Tyr Asp Pro Ala Arg Ala Arg Ala Leu Gly Asp Tyr

115 120 125

Tyr Arg His Ala Arg Asp Asn Asp Leu Tyr Leu Thr Tyr Val Ile Ile

130 135 140

Asn Pro Gln Ala Asp Arg Ser Lys Asn Ala Ala Glu Gln Ala Asp Pro

145 150 155 160

Phe Leu Ser Ala Ala Ile Val Asp Glu Asp Thr Ser Gly Ile Thr Val

165 170 175

Arg Gly Ala Lys Leu Ala Thr Gly Gly Ile Ala Asn Glu Val Phe Val

180 185 190

Thr Cys Ile Gln Pro Leu Gln Lys Gly Glu Glu Ala Tyr Ala Leu Ser

195 200 205

Phe Ala Ile Pro Asn Thr Arg Gly Leu Lys Ile Leu Ser Arg Lys Ser

210 215 220

Tyr Glu Ala Gly Ala Pro Ser Val Phe Asp Asn Pro Leu Ser Ser Arg

225 230 235 240

Phe Asp Glu Asn Asp Ala Val Leu Tyr Phe Asp Asp Val Lys Val Pro

245 250 255

Trp Asp Arg Val Phe Ile Ala Gly Asp Ile Ala Thr Gly Arg Gln Phe

260 265 270

His Ala Thr Pro Ala His Val Tyr Gln Asn Tyr Gln Ala Gln Ile Arg

275 280 285

Leu Ser Val Lys Leu Lys Phe Leu Leu Ala Ile Ala Arg Arg Thr Ala

290 295 300

Glu Val Asn Gly Thr Thr Gly Phe Pro Gln Val Arg Glu Thr Leu Gly

305 310 315 320

Gln Leu Ala Ala Glu Ala Ala Val Asp Ala Phe Val Ala Ala Glu Ala

325 330 335

Lys Gly Ser Phe Tyr Gly Pro Tyr Phe Val Pro Asp Arg His Thr Leu

340 345 350

Tyr Ala Asp Gln Thr Leu Thr Gln Gln Leu Tyr Gly Lys Phe Ile Thr

355 360 365

Ser Leu Arg Glu Leu Ala Gly Gly Gly Ile Leu Pro Ser Ser Val His

370 375 380

Asp Tyr Gln Asn Pro Glu Leu Ala Ala Leu Ile Ala Lys Thr Gln Gln

385 390 395 400

Ser Pro Ala Ala Ser Ala Glu Glu Lys Val Lys Phe Tyr Lys Leu Ala

405 410 415

Trp Asp Ala Val Gly Ser Glu Phe Ala Ser Arg His Ala Gln Tyr Glu

420 425 430

Phe Tyr Ala Gly Ala Gly Phe Val Val Lys Asn His Ser Phe Arg Thr

435 440 445

Tyr Asp Trp Ala Gly Ala Glu Arg Leu Leu Asp Leu Ser Ser Tyr Ser

450 455 460

Leu Ala Asp Glu Val Ala Pro Thr Ala Ala Arg Ala Ala Glu

465 470 475

Claims

1. A method for rapidly screening high-yield caffeic acid strains in a high throughput manner is characterized by comprising the following steps: carrying out fermentation culture on a strain to be screened to obtain fermentation liquor; centrifuging the obtained fermentation liquor to obtain fermentation supernatant; and (3) placing the fermented supernatant into a microporous plate, adding a 3-valent iron ion reaction solution into the fermented supernatant for color reaction, then placing the fermented supernatant into a full-wavelength microplate reader for detection, detecting the OD value of the fermented supernatant corresponding to the strain at 680nm, wherein the obtained OD value can reflect the caffeic acid production capacity of the corresponding strain, and selecting the corresponding strain with a high OD value as a high-yield caffeic acid strain for subsequent production.

2. The method for high-throughput rapid screening of high-yield caffeic acid bacteria according to claim 1, wherein the fermentation culture adopts a P-5052 auto-induction culture medium, the deep-well plate shaking fermentation culture is carried out in a shaker at 100-240 rpm at 32-38 ℃ for 18-24 h, and each liter of the P-5052 auto-induction culture medium contains Na₂HPO₄ 50～60mM，KH₂PO₄ 50～60mM，(NH₄)₂SO₄ 20～30mM，MgSO₄2 to 3mM, 0.2 to 0.5 percent of glycerol, 0.1 to 0.2 percent of lactose, 0.04 to 0.05 percent of glucose, 0.1 to 0.4mM of p-coumaric acid and pH of 7.0 to 7.4.

3. The method for high-throughput rapid screening of high-yield caffeic acid bacteria according to claim 1, wherein: the fermentation supernatant is obtained after the fermentation broth is centrifuged for 2min at 14000 rpm.

4. The method for high-throughput rapid screening of high-yield caffeic acid bacteria according to claim 1, wherein the 3-valent iron ion reaction solution is ferric chloride aqueous solution.

5. The method for high-throughput rapid screening of high-yield caffeic acid bacteria according to claim 4, wherein: the concentration of the ferric chloride aqueous solution is 25-28 g/L.

6. The method for high-throughput rapid screening of high-yield caffeic acid bacteria according to claim 1, wherein the addition amount of the 3-valent iron ion reaction solution is 1/5-1/20 of the volume of the fermentation supernatant.

7. The method for high-throughput rapid screening of high-yield caffeic acid bacteria according to any one of claims 1-6, wherein the strain to be screened is selected from mutant strain library, the mutant strain library construction method comprises: adding PCR primer pair to carry out error-prone PCR amplification by taking hydroxylase gene as a template to obtain a target gene fragment, and loading recombinant plasmid into escherichia coli to construct a mutant strain library.

8. The method for high-throughput rapid screening of high-yield caffeic acid bacteria according to claim 7, wherein the hydroxylase gene sequence is shown as SEQ ID No.1, restriction enzymes NdeI and NotI restriction sites are added at the front and back ends of the hydroxylase gene, and the method is specifically shown as follows:

NdeI: enzyme digestion sequence and protected base:

NotI: enzyme digestion sequence and protected base:

9. the method for high-throughput rapid screening of high-yielding caffeic acid bacteria according to claim 8, wherein the upstream primer of said PCR primer pair has a nucleotide sequence shown as SEQ ID No.2, and the downstream primer of said PCR primer pair has a nucleotide sequence shown as SEQ ID No. 3.

10. The method for high-throughput rapid screening of high-yield caffeic acid bacteria according to any one of claims 1-7, wherein said method further comprises the steps of: and establishing a standard curve of the corresponding relation between the caffeic acid yield and the OD value, and substituting the OD value measured by the fermentation supernatant of the strain to be screened into the standard curve to obtain the caffeic acid yield value of the corresponding strain.