CN113604413B - Recombinant strain, preparation method and application - Google Patents

Abstract

The application discloses a recombinant strain, a preparation method and application thereof. The recombinant strain integrates a fragment I on the genome of a host; the fragment I contains a styrene monooxygenase gene styA and a styrene monooxygenase gene styB. The genome of the recombinant strain is integrated with a styrene monooxygenase gene styA and a styrene monooxygenase gene styB, so that the recombinant strain has good stability and higher yield of indigo pigment.

Description

Recombinant strain, preparation method and application

Technical Field

The application relates to a recombinant strain, a preparation method and application thereof, and belongs to the technical field of genetic engineering.

Background

The present indigo production comprises natural extraction, chemical synthesis, microbial synthesis and the like. The synthesis of indigo pigment by using colibacillus, saccharomycetes, bacillus subtilis and the like is an important research direction in the field, however, a plasmid system is still needed at present, and the yield is unstable. Moreover, in order to improve the yield of indigo fermentation and perfect fermentation conditions, a fermenter is currently used for batch fermentation, but the batch fermentation has the conditions that nutrients and substrate concentration are insufficient to cause strain death and finally the indigo production is unstable and not continuous.

Disclosure of Invention

According to one aspect of the present application, there is provided a recombinant strain having a styrene monooxygenase gene styA and a styrene monooxygenase gene styB integrated on the genome, having good stability, and a high yield of indigo pigment.

A recombinant strain having fragment I integrated on the genome of a host;

the fragment I contains a styrene monooxygenase gene styA and a styrene monooxygenase gene styB.

Alternatively, the styrene monooxygenase gene styra is located upstream of the styrene monooxygenase gene styrb.

Alternatively, the host is selected from any one of E.coli;

alternatively, the E.coli is E.coli DH 5. Alpha.

Alternatively, the integration is by replacing the nucleotide sequence of the host integration site with fragment I;

alternatively, the integration site is selected from any one of trpR, trpB, hisD.

Optionally, the coding sequence of the styrene monooxygenase gene styra is shown as SEQ ID NO. 1;

the coding sequence of the styrene monooxygenase gene styrb is shown as SEQ ID NO. 2;

optionally, the fragment I further comprises an inducible promoter, an operator;

sequentially arranging an inducible promoter, an operon and a styrene monooxygenase gene styA and a styrene monooxygenase gene styB;

optionally, the inducible promoter is an inducible promoter cat;

the operon is lactose operon lac.

According to another aspect of the present application, there is provided a method for producing a recombinant strain according to any one of the above, the method comprising the steps of:

integrating the fragment I into the genome of the host to obtain the recombinant strain.

Optionally, the preparation method comprises the following steps:

(S1) amplifying the fragment II by using a recombinant vector containing the fragment II as a template;

(S2) transferring the fragment II into a host competent cell expressing a recombinase to obtain the recombinant strain;

the fragment II sequentially comprises an integration site left homology arm ', a fragment I and an integration site right homology arm';

preferably, the nucleotide sequence of the left homology arm' of the integration site is shown as 173 th to 358 th bases shown in SEQ ID NO.3, 104 th to 353 th bases shown in nucleoside SEQ ID NO.5 or 164 th to 346 th bases shown in SEQ ID NO. 7;

the nucleotide sequence of the right homology arm' of the integration site is shown as 1 st to 337 th bases shown in SEQ ID NO.4, 1 st to 311 st bases shown in SEQ ID NO.6 or 1 st to 180 th bases shown in SEQ ID NO. 8;

optionally, the backbone vector of the recombinant vector is pHS-AVC-LW;

optionally, the fragment II comprises, in order, an integration site left homology arm ', a resistance gene, a fragment I, an integration site right homology arm';

transferring the fragment II into a host competent cell expressing the recombinase, and knocking out a resistance gene to obtain the recombinant strain;

alternatively, the resistance gene is selected from at least one of chloramphenicol resistance gene, kanamycin resistance gene;

alternatively, the host competent cell expressing the recombinase is obtained by:

transferring the pKD46 plasmid into a host cell, and culturing in a medium containing L-arabinose to obtain the host competent cell expressing the recombinase.

According to another aspect of the present application, there is provided a method of producing indigo pigment, the method comprising the steps of:

fermenting the recombinant strain in a culture medium to obtain the indigo pigment;

the recombinant strain comprises at least one of the recombinant strain described in any one of the above, and the recombinant strain prepared by the preparation method described in any one of the above.

Optionally, the culture medium contains a fermentation substrate comprising at least one of indole, tryptophan;

optionally, the concentration of the indole is 0.5-5 g/L;

the concentration of the tryptophan is 0.5-10 g/L.

Optionally, the concentration of the indole is 0.5-3 g/L;

the concentration of the tryptophan is 0.5-3 g/L.

Optionally, the concentration of the indole is 1-2 g/L;

the concentration of the tryptophan is 1-2 g/L.

Optionally, the conditions of the fermentation include:

the temperature is 30-40 ℃;

the revolution is 50-400 rpm;

preferably, the conditions of the fermentation further comprise:

the ventilation ratio is 0.2-1.0 vvm;

the pH value is 6.0-8.0;

the dissolved oxygen correction is 80-100%.

Supplementing a substrate solution in the fermentation broth;

the concentration of the substrate solution is 0-8 g/L;

the volume ratio of the fermentation liquor to the substrate solution is 1L: 20-160 ml.

Optionally, the interval time of the substrate replenishing solution is 4-20 h.

Optionally, the flow rate of the supplementary substrate solution is 1-5 mL/min.

Optionally, the number of times the substrate solution is replenished is 1 to 4.

Alternatively, the upper concentration limit of the substrate solution is selected from 1, 2, 3, 4, 5, 6, 7, 8g/L, and the lower concentration limit is selected from 0, 1, 2, 3, 4, 5, 6, 7g/L.

Optionally, the upper limit of the volume ratio of the fermentation broth to the substrate solution is selected from 1L:20ml, 1L:40ml, 1L:80ml, 1L:100ml, 1L:120ml; the lower limit is selected from 1L:40ml, 1L:80ml, 1L:100ml, 1L:120ml, 1L:160ml.

As one embodiment, the present application has studied the chromosomal expression of the gene styrAB related to indigo pigment synthesis in E.coli in order to improve the yield of indigo pigment by expressing the chromosome of the gene and to stably realize the synthesis of indigo pigment using the chromosome as a vector, in order to solve the above-mentioned problems. Integrating indigo pigment synthesis genes styrb into escherichia coli chromosomes by utilizing a homologous recombination method, selecting trpR, trpB, hisD genes at the left and right ends of different chromosome integration sites as homologous left and right arms, integrating target gene fragments by using an inducible cat+lac promoter and a lactose operon, comparing and screening engineering bacteria at three different sites, integrating the target genes into a framework vector plasmid for recombination to store, obtaining pHS-AVC-01 and pHS-AVC-02, pHS-AVC-03, obtaining a target targeting fragment, preparing electrotransformation E.coli DH5 alpha competent by using a temperature-sensitive plasmid pKD46, then integrating the left and right homologous arms into E.coli DH5 alpha, finally screening and selecting positive monoclonal by using a resistance plate, and performing PCR verification to obtain correct engineering bacteria, and fermenting to produce indigo by using a fermentation tank.

Meanwhile, in the optimization process of the indigo pigment synthesis engineering bacteria, the fact that the bacteria are not grown after long-time fermentation is found, the yield of the indigo is not increased after fermentation for 32 hours in batch fermentation is not increased, and the fact that the bacteria are inactivated or plasmids are lost due to insufficient nutrient content of a fermentation medium is considered at the moment, and batch fed-batch fermentation is carried out for the insufficient nutrient content. The work provides theoretical basis and technical guidance for further research on chromosome expression of subsequent natural indigo pigment biosynthesis genes.

The beneficial effects that this application can produce include:

(1) The genome of the recombinant strain provided by the application is integrated with the styrene monooxygenase gene styA and the styrene monooxygenase gene styB, so that the stability of the recombinant strain can be improved, and the yield of the indigo pigment can be improved.

(2) The recombinant strain provided by the application integrates the styrene monooxygenase gene styA and the styrene monooxygenase gene styB at specific sites of a chromosome, and has better biological activity.

(3) The indigo pigment provided by the application is beneficial to improving the yield of the indigo pigment through the selection of substrates, the control of feeding conditions and the like.

Drawings

FIG. 1 is a diagram showing construction of integrated recombinant plasmids pHS-AVC-01 (A), pHS-AVC-02 (B) and pHS-AVC-03 (C);

FIG. 2 is an electrophoresis chart of the integrated recombinant plasmid pHS-AVC-01 and the enzyme digestion verification;

FIG. 3 is an electrophoresis chart of the integrated recombinant plasmid pHS-AVC-02 and the enzyme digestion verification;

FIG. 4 is an electrophoresis chart of the integrated recombinant plasmid pHS-AVC-03 and the enzyme digestion verification;

FIG. 5 is a colony PCR electrophoretogram of the integrated recombinant plasmids pHS-AVC-01 (A), pHS-AVC-02 (B), pHS-AVC-03 (C);

FIG. 6 is a map of the integration and sequencing of pHS-AVC-01 (A), pHS-AVC-02 (B) and pHS-AVC-03 (C); wherein, left arm is homologous left arm, right arm is homologous right arm, promoter is Promoter, repeat region is repeat region;

FIG. 7 is an electrophoresis diagram of pHS-AVC-01 targeting fragment;

FIG. 8 is an electrophoresis diagram of pHS-AVC-02 and pHS-AVC-03 targeting fragments;

FIG. 9 is a PCR identification chart of engineering bacteria E.coli AB-01 colonies;

FIG. 10 is a PCR identification chart of the colony of the engineering bacterium E.coli AB-02 and E.coli AB-03;

FIG. 11 is a graph showing the measurement of the growth curve of engineering bacteria;

FIG. 12 shows the expression levels of the styrA and styrB genes of engineering bacteria E.coli AB-01 and E.coli AB-02 with tryptophan as a substrate at different fermentation times;

FIG. 13 shows the expression levels of the styrA and styrB genes of engineering bacteria E.coli AB-01 and E.coli AB-02 with indole as a substrate at different fermentation times;

FIG. 14 is the effect of feed concentration on indigo pigment production by engineering bacteria fermentation;

FIG. 15 is the effect of feed rate on indigo pigment production by engineering bacteria fermentation;

FIG. 16 is the effect of feed gap time on indigo pigment production by engineering bacteria fermentation;

FIG. 17 is a graph showing the effect of feed flow rate on indigo pigment production by engineering bacteria fermentation;

FIG. 18 is a graph showing the effect of passage times on the stability of different types of engineered bacteria.

Detailed Description

The present application is described in detail below with reference to examples, but the present application is not limited to these examples.

Unless otherwise indicated, all starting materials in the examples of the present application were purchased commercially.

Wherein:

(1) Materials and reagents

Indigo pigment standard was purchased from Sigma-Aldrich, usa, tryptophan, indole, kanamycin, chloramphenicol, sodium hydroxide, N-dimethylformamide, dodecanol and disodium hydrogen phosphate, monopotassium phosphate, ammonium chloride, sodium chloride, magnesium sulfate, yeast extract, tryptone, soyase peptone, kanamycin, etc. were all purchased from pinellia biotechnology limited; primeSTAR HS (Premix), 10000DL DNA Marker, available from Takara Bio-engineering (Dalian) Inc.; centrifugal column type ultra-pure total RNA rapid extraction Kit, fastKing RT Kit (With gDNase) reverse transcription Kit, superReal PreMix Plus (SYBR Green) Kit, high-purity plasmid miniprep Kit, multifunctional DNA purification recovery Kit and the like, which are purchased from Beijing Tian Gen Biochemical technology Co.

(2) Apparatus and device

C1000 A Touch PCR instrument, a PowerPac Basic electrophoresis instrument (provided with a horizontal electrophoresis tank), and a CFX96 Touch fluorescence quantitative PCR detection system, BIO-RAD company, U.S.A.; 3K15 high speed centrifuges, USA SIGMA, inc.; SPX-150B biochemical incubator, shanghai Boqing medical biological instruments Co., ltd; bioSpectrum gel imager, UVP company, USA; small high-speed centrifuges, SIGMA company, usa; vortex oscillator, IKA company, germany; electronic balance (0.01 g), shanghai Pu Chun metering instruments limited; continuous wavelength multifunctional microplate reader SpectraMax 13, us Molecular Devices; constant temperature cradle, shanghai Boqun medical biological instruments Co., ltd; 10-1L mini-fermentor, shanghai Bailun Bio Inc.

(3) Used strain and plasmid and constructed strain and plasmid

The strain and plasmid used and the strain and plasmid constructed are shown in Table 1

TABLE 1 bacterial species and plasmids

It should be noted that:

in the examples of the present application, the wild-type strain pseudomonas (Pseudomonas putida) B3 stored in the present experiment is used as the indigo pigment synthesis related gene (styrene monooxygenase gene) styrab, but the scheme provided in the present application is not implemented by the present application, but the present application can be applied to the present application as long as the pseudomonas carrying the indigo pigment synthesis related genes styra and styrb can be used as the sources of the indigo pigment synthesis related genes styra and styrb, for example, the strain with the preservation number of cgmccno.12202 disclosed in the patent application publication No. CN105802986 a; alternatively, the nucleotide sequence of the indigo pigment synthesis-related gene styrab disclosed in the present application may be used for the present application, by synthesizing the indigo pigment synthesis-related gene styrab by an existing method.

E.coli DH 5. Alpha., pHS-AVC-LW, pKD46, pCP20 are commercially available.

The recombinant vectors pHS-AVC-01, pHS-AVC-02, pHS-AVC-03, recombinant strains E.coli AB-01 and E.coli AB-02 disclosed in the application can be obtained by repeating the method disclosed in the application.

(4) Culture medium

The culture medium is prepared according to the following method:

LB liquid medium (g/L): yeast extract 5.0, tryptone 10.0,NaCI 10.0,pH 7.0, 121.0 ℃ high temperature sterilization 15.0min.

LB solid medium (g/L): yeast extract 5.0, tryptone 10.0, naci 10.0, agar 15.0, ph 7.0, and high temperature sterilization at 121.0 ℃ for 15.0min.

Tryptophan fermentation medium (g/L): na (Na) ₂ HPO ₄ ·12H ₂ O 17.0，KH ₂ PO ₄ 3.0，NH ₄ Cl 1.0，NaCl 0.5，MgSO ₄ 0.1, yeast extract powder 3.0, substrate tryptophan 1.6, lactose 3.0.121.0deg.C, and sterilizing at high temperature for 15.0min.

Indole fermentation medium (g/L): na (Na) ₂ HPO ₄ ·12H ₂ O 17.0，KH ₂ PO ₄ 3.0，NH ₄ Cl 1.0，NaCl 0.5，MgSO ₄ 0.1, yeast extract 3.0, soytone 3.0, substrate indole 1.6, and high temperature sterilization at 121.0 ℃ for 15.0min.

Preparing antibiotics:

kanamycin (Kan): the stock solution was prepared at a concentration of 10.0mg/mL, and kanamycin at a concentration of 50.0. Mu.g/mL was used and stored at 4℃for further use.

Chloramphenicol (Chl): the stock solution was prepared at a concentration of 10.0mg/mL, and kanamycin at a concentration of 100.0. Mu.g/mL was used and stored at 4℃for further use.

Ampicillin (Amp): the stock solution was prepared at a concentration of 10.0mg/mL, and kanamycin at a concentration of 50.0. Mu.g/mL was used and stored at 4℃for further use.

The inoculum size in this application refers to the ratio of the volume of the seed liquid to the volume of the fermentation liquid (i.e. the total volume of the seed liquid and the medium), for example, an inoculum size of 1.2% refers to the volume of the seed liquid being 1.2 and the volume of the fermentation liquid (i.e. the total volume of the seed liquid and the medium) being 100.

In the present application, styrenemonooxygenase gene styA and styrenemonooxygenase gene styB are linked together in the order of styrenemonooxygenase gene styA and styrenemonooxygenase gene styB along the expression direction.

EXAMPLE 1 construction of recombinant vector

1.1 primer design

Some of the primers of this application are shown in Table 2.

TABLE 2 primer list

1.2 PCR amplification of homologous arm, indigo pigment synthesis related genes styA and styB

Amplifying a left arm and a right arm by taking E.coli DH5 alpha trpR as an integration site, wherein the size of the left arm is 358bp (the sequence is shown as SEQ ID NO. 3), and the size of the right arm is 357bp (the sequence is shown as SEQ ID NO. 4); amplifying a left arm and a right arm by taking E.coli DH5 alpha trpB as an integration site, wherein the size of the left arm is 353bp (the sequence is shown as SEQ ID NO. 5), and the size of the right arm is 351bp (the sequence is shown as SEQ ID NO. 6); e.coli DH 5. Alpha. HisD is used as an integration site to amplify a left arm and a right arm, wherein the size of the left arm is 346bp (the sequence is shown as SEQ ID NO. 7), and the size of the right arm is 369bp (the sequence is shown as SEQ ID NO. 8).

The wild indigo synthetic strain Pseudomonas (Pseudomonas putida) B3 is extracted as a whole genome template, and Primer design is carried out by utilizing Primer Premier 5.0 according to a styrene monooxygenase gene sequence (GenBank accession No. DQ 177365.1) in GeneBank, and the sequences of the styrene monooxygenase gene styA and the styrene monooxygenase gene styB are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

1.3 Construction of styAB Gene integration recombinant plasmid

According to the principle of constructing a homologous recombination vector by a one-step method, respectively taking gene fragments at two sides of trpR, trpB, hisD genes of three integration sites of escherichia coli as homologous fragments, connecting a promoter and a resistance gene with homologous arms and target gene fragments, and finally constructing integrated recombinant plasmids pHS-AVC-01, pHS-AVC-02 and pHS-AVC-03 by homologous recombination by taking plasmid pHS-AVC-LW as a template.

1.4 Transformation of the styAB Gene and recombinant plasmid ligation products into E.coli competence

E.coli DH5 alpha electrotransformation competence is prepared, 3 integrated recombinant plasmids constructed in 1.3 are respectively electrotransformed, three resistance screening is carried out corresponding to a Chl culture medium, a Kan culture medium and a Kan culture medium, positive monoclonal is obtained, and an insertion sequence is verified by sequencing.

Example 2 targeting fragment acquisition

2.1 plasmid extraction

1.4, the genomic DNA of positive clones verified to be error-free was extracted using the bacterial genomic DNA extraction kit (centrifugal column type) from Baitaike company, and the specific operation is described in the kit specification.

2.2 PCR amplified targeting fragment

The DNA concentrations of three integrated recombinant plasmids of pHS-AVC-01, pHS-AVC-02 and pHS-AVC-03 extracted in 2.1 were measured, and the extraction concentration was about 200 ng/. Mu.L, and the PCR reaction system using pHS-AVC-01 as a template was as shown in Table 3:

TABLE 3 PCR reaction System for targeting Gene fragments

The PCR reaction with pHS-AVC-01 as template was performed as follows:

the PCR reaction system using pHS-AVC-02 as a template is shown in Table 4:

TABLE 4 PCR reaction System for targeting Gene fragments

The PCR reaction of pHS-AVC-02 was performed as follows:

the PCR reaction system of pHS-AVC-03 is shown in Table 5:

TABLE 5PCR reaction System for targeting Gene fragments

The PCR reaction of pHS-AVC-03 was performed as follows:

2.3 Gene fragment gel electrophoresis and product recovery

Specific procedures for agarose gel electrophoresis are described in standard specifications.

The gel electrophoresis product is recovered by adopting a centrifugal column type multifunctional DNA purification recovery kit of Beijing Baitaike company, and the specific operation is shown in the specification of the kit.

EXAMPLE 3 construction of recombinant strains

3.1 Inducible expression of Red Gene

Transformation of pKD46 plasmid into E.coli DH 5. Alpha. Competent, recovery culture at 30℃for 1h, ampicillin (Amp ⁺ ) Plates were incubated overnight at 30 ℃. The monoclonal was picked up and cultured overnight in LB tubes containing Amp.

Inoculating into LB medium with Amp at 1:100, adding L-arabinose to final concentration of 1mM, culturing at 30deg.C, and OD _600nm No more than 0.6, and preparing electric conversion competence.

3.2 electric rotating targeting fragment

The target DNA fragment obtained in 2.2 was mixed with electrotransformation competent cells (100. Mu.L competent cells+about 200ng of target DNA fragment, transferred into a pre-chilled electrotransformation cup, three monoclonal electric shocks were taken from each electrotransformation competent plate prepared in step 3.1, LB medium was added after the electric shocks, and the mixture was placed at 30℃for resuscitation for 2 hours, and the mixture was spread on corresponding double-resistant (chl+Amp or Kan+Amp) plates, and incubated overnight at 30 ℃.

3.3 engineering bacteria identification

From the double-resistant plates after the culture in step 3.2, 10. Mu.L of ddH was picked up, respectively ₂ In O, preparing bacterial suspension, taking 1 mu L of bacterial suspension as a template, and taking F-0151/R-0151, F-0152/R-0152 and F-0153/R-0153 as primers to carry out PCR, wherein the system is shown in Table 6:

TABLE 6 identification of strains PCR reaction System

The PCR reaction conditions were the same as described in example 2, respectively. The results were detected and recorded by gel imager using 1% agarose gel electrophoresis for 30 min.

The remaining 9. Mu.L of the bacterial suspension was subjected to a strain which was resistant to the corresponding strain (Kan ⁺ Or Chl ⁺ ) LB medium was cultured at 42℃to remove plasmid pKD46, and then the LB plate was streaked (Kan ⁺ Or Chl ⁺ ) The single colonies obtained were then subjected to Amp ⁺ And detecting sensitivity, namely removing the plasmid pKD46 strain from the strain sensitive to the Amp. Extracting strain DNA, preserving strain in glycerol, and sending to company for sequencing.

3.4 removal of Kan+ or Chl+ resistance Gene by knockout

Electrotransformation of pCP20 plasmid in 3.3 identified error-free recombinant Strain and Amp coating ⁺ Plates were incubated overnight at 30 ℃.

Picking up the monoclonal antibody into a test tube added with Amp, and culturing at 30 ℃ until OD ₆₀₀ About 0.4-0.6, heat shock at 37℃for 1h (heat activated protein expression), overnight incubation at 30℃and PCR validation of Kan ⁺ Or Chl ⁺ Whether to eliminate (or directly to test tubes with kanamycin or chloramphenicol for verification).

After verifying correct streak isolation of the monoclonal, culturing at 42℃and then at Amp ⁺ The pCP20 plasmid was verified for elimination in a plate-on-fire tube.

EXAMPLE 4 analysis of target Gene expression

4.1 Total RNA extraction of engineering bacteria

The extraction of total RNA in thalli adopts a centrifugal column type ultra-pure total RNA rapid extraction kit of Beijing Baitaike company, and specific operation steps are shown in a kit instruction book.

4.2 reverse transcription of Total RNA into cDNA

The cDNA is synthesized by using ReverTra Ace qPCR RT Master Mix reverse transcription kit of TOYOBO (Shanghai) company, and the specific operation method is shown in the specification.

4.3 RT-PCR analysis

And (3) designing primers and evaluating the specificity by Beacon Design software according to the styA and styB gene sequence information. The primer sequences are shown in Table 7.

TABLE 7 RT-PCR primers

Gene	Primer name	Numbering in the sequence listing	Sequence (5 '-3')
				styA	F-A	SEQ ID NO.20	GGCGAGCTGATTGAGATTC
styA	R-A	SEQ ID NO.21	TTTTGCCGTTATTGAGGGT
				styB	F-B	SEQ ID NO.22	AAAAGATGTGGTGGTGGAT
styB	R-B	SEQ ID NO.23	TGCTGAAGAATGCCGATAA
				16s	F-16s	SEQ ID NO.24	CCACCTGGACTGATACT
16s	R-16s	SEQ ID NO.25	GCACCTGTCTCAATGTT

The test uses SYBR Green Realtime PCR Master Mix kit of TOYOBO (Shanghai) of Toyobo, and uses SYBR Green method to make fluorescence quantification, and the reaction system is shown in Table 8.

TABLE 8 RT-PCR reaction System

The RT-qPCR reaction performed the following procedure:

calculating the relative expression quantity: real-time fluorescence quantification Using 16s gene as an internal control and different fermentation times E.coli-styAB-01 strain at tryptophan concentration of 1.2mg/mL or indole of 0.01wt% as a control and 2 ^-ΔΔCt The method performs relative quantitative calculations. The numerical value represents the change multiple of the expression level of the objective gene of the experimental group relative to the control group. Delta Ct value calculation formula:

△△Ct＝(Ct _{target gene} -Ct _{Reference gene} ) Experimental group- (Ct) _{Target gene} -Ct _{Reference gene} ) Control group

EXAMPLE 5 recombinant strain growth Curve determination

5.1 determination of growth curves

The recombinant strain was cultured in LB medium at 37℃and 400rpm, and purchased from laboratory at various time points

The type I microbial reactor detects the strain seed liquid in real time and records the growth curve. Biomass was detected every hour for the first 12 hours and every 4 hours after 12 hours.

Example 6 indigo pigment yield determination

6.1 indigo pigment yield determination

Making indigo standard curve: dissolving indigo standard substance with DMF to constant volume to 50.0 μg/mL standard solution, making standard curve, measuring with enzyme-labeled instrument, and measuring at OD _{610 nm} The absorbance is detected.

Indigo concentration determination: 1% seed solution was inoculated into 40mL of LB liquid medium, and the bacterial solution was obtained at 37℃overnight. Adding 1mL of bacterial liquid into 40mL of LB liquid culture medium, culturing for 3 hours at 37 ℃, adding IPTG to a final concentration of 1mM, adjusting the temperature to 30 ℃, adding indole (the final concentration of 1.0 g/L) or tryptophan (the final concentration of 1.6 g/L) into the LB liquid culture medium after shake culturing for 3 hours, adding 50mL of fermentation medium into the LB liquid culture medium after shake culturing for 160r/min at 31 ℃, culturing for 48 hours at 200r/min, taking the fermentation liquid 9000r/min, centrifuging for 20 minutes, adding 10mLN, N-dimethylformamide solution after removing supernatant, carrying out ultrasonic treatment until the indigo is completely dissolved, diluting, and adopting an enzyme-labeling instrument at OD _{610 nm} Detecting the light absorption value, substituting the light absorption value into the standard curve, and calculating to obtain the indigo concentration.

EXAMPLE 7 plasmid stability analysis

7.1 stability analysis

Respectively preparing an antibiotic LB solid plate with Kan resistance and a LB solid plate without resistance, taking fermentation liquor (obtained by culturing recombinant strain strains which are identified in 3.3 and have not been knocked out of resistance in LB liquid culture medium, wherein the culture condition is 37 ℃,400 rpm) at different time points, respectively diluting and coating the fermentation liquor on the corresponding resistant plates, and counting the number of colonies by a colony counter after culturing in an incubator for 12 hours.

Plasmid stability (%) = number of resistant plate colonies/number of non-resistant plate colonies x 100%

EXAMPLE 8 preparation of indigo fermentation broth

Preparing seed liquid: 200 mu L of indigo engineering bacteria liquid preserved at-20 ℃ is taken in an ultra-clean workbench, is inoculated into 20mL of LB liquid medium containing 50.0 mu g/mL of kanamycin, is put into a shaking table for activation for 12h at 37 ℃ and 200r/min, and is transferred for two times for standby.

Preparation of fermentation liquor: inoculating the prepared seed liquid into 500mL of fermentation medium with an inoculum size of 1.2%, setting the temperature of a fermentation tank to be 31 ℃, the rotating speed to be 200r/min, the aeration ratio to be 0.5vvm, the pH value to be 7.0, correcting dissolved oxygen to be 100%, and culturing for 48 hours to obtain the indigo fermentation liquid.

Example 9 fed-batch (tryptophan-supplemented) fermentation process

And selecting the feeding concentration, feeding time, feeding flow rate, feeding times and fermentation time as main factors influencing the stability of the fed-batch fermented indigo engineering bacteria, and measuring the stability of the plasmid engineering bacteria under different conditions.

9.1 Effect of the concentration of the feed on the production of indigo pigment by fermentation of engineering bacteria

In 1L fermentation broth, tryptophan aqueous solutions (i.e., tryptophan aqueous solutions with concentrations of 1.6 x 0, 1.6 x 1, 1.6 x 2, 1.6 x 3, 1.6 x 4 and 1.6 x 5 g/L) with concentrations of 0 (corresponding to the "original concentration" in FIG. 14) of the original fermentation medium (tryptophan concentration in the original medium) and 1, 2, 3, 4 and 5 times were respectively supplemented, and the total feeding was performed once every 8h, 80mL each time, the feeding flow rate was 5mL/min, and samples were taken after 48h of fermentation, so as to determine the indigo pigment yield.

9.2 influence of the number of feeding times on the production of indigo pigment by fermentation of engineering bacteria

In 1L fermentation broth, a tryptophan aqueous solution with the concentration being 2 times that of an original fermentation medium (tryptophan concentration in the original medium is 1.6 g/L) is supplemented, feeding is carried out once every 8 hours, feeding times are set to be 0, 1, 2, 3 and 4 times, 80mL of each time are set, the feeding flow rate is 5mL/min, sampling is carried out after 48 hours of fermentation, and the yield of the indigo pigment is measured.

9.3 Effect of feeding Interval time on indigo pigment production by engineering bacterium fermentation

In 1L fermentation broth, tryptophan aqueous solution with the concentration being 2 times that of the original fermentation medium (tryptophan concentration in the original medium is 1.6 g/L) is supplemented, feeding time intervals are set to be 4, 8, 12, 16 and 20h, 80mL of each time, the feeding flow rate is 5mL/min, sampling is carried out after 48h of fermentation, and the indigo pigment yield is measured.

9.4 Effect of feed flow Rate on indigo pigment production by engineering bacteria fermentation

In 1L fermentation broth, a tryptophan aqueous solution with the concentration being 2 times that of an original fermentation medium (tryptophan concentration in the original medium is 1.6 g/L) is supplemented, 80mL of the raw material is supplemented every 16 hours, the total feeding is carried out twice, the feeding flow rates are set to be 1, 2, 3, 4 and 5mL/min, sampling is carried out after 48 hours of fermentation, and the yield of the indigo pigment is measured.

Example 10 analysis of experimental results

All experiments were repeated 3 times, and the significance analysis and mapping of the data was performed using the originPro 2018 software, with 0.01 < p < 0.05 being significant, p < 0.01 being extremely significant, and p > 0.05 being insignificant.

10.1 construction of recombinant plasmid map

According to the analysis design of SnapGene software, the plasmid construction map of the integrated engineering bacterium is shown in figure 1, wherein A is pHS-AVC-01, B is pHS-AVC-02, C is pHS-AVC-03, and subsequent experiments are carried out according to the map.

10.2 construction and restriction enzyme digestion verification of styrene monooxygenase Gene styAB integration recombinant plasmid

Enzyme digestion verification principle: the pHS-AVC-LW skeleton carrier has 1 EcoRV restriction enzyme site, tyB gene has 1 EcoRV restriction enzyme site, so if the recombinant plasmid is constructed successfully, it has two EcoRV restriction enzyme sites, and two strips are obtained after EcoRV restriction enzyme and electrophoresis.

The gene fragments at two sides of the trpR gene of the escherichia coli are used as homologous fragments, a promoter and a resistance gene are amplified by PCR, and the homologous arm and the target gene fragment are connected to construct an integrated recombinant plasmid pHS-AVC-01 of the trpR arm-Chl-lac promoter-styAB-trpR right arm. As can be seen from the total plasmid size of 6692bp, the recombinant plasmid gel electrophoresis chart and the restriction enzyme electrophoresis chart (FIG. 2), lane 1 is the plasmid pHS-AVC-01 constructed gene band, and the EcoRV restriction enzyme is utilized to verify that lane 2 presents 4170bp and 2522bp bands, which proves that the recombinant plasmid can be cut, and the band sizes are consistent, which indicates that the recombinant plasmid pHS-AVC-01 has been constructed.

The gene fragments at two sides of the trpB gene of the escherichia coli are used as homologous fragments, a promoter and a resistance gene are amplified by PCR, and the homologous arm and the target gene fragment are connected to construct an integrated recombinant plasmid pHS-AVC-02 of the trpB left arm-Kan-lac promoter-styAB-trpB right arm. As can be seen from the total plasmid size of 6742bp, the recombinant plasmid gel electrophoresis chart and the restriction enzyme electrophoresis chart (FIG. 3), lane 1 is the plasmid pHS-AVC-02 constructed gene band, and by using EcoRV restriction enzyme digestion to verify, lane 2 presents two bands of 4255bp and 2487bp, which proves that the recombinant plasmid can be cut and is consistent with the expected band size, indicating that the recombinant plasmid pHS-AVC-02 has been constructed.

The gene fragments at two sides of the escherichia coli hisD gene are used as homologous fragments, a promoter and a resistance gene are amplified by PCR, and the homologous arm and the target gene fragment are connected to construct the integrated recombinant plasmid pHS-AVC-03 of hisD left arm-Kan-lac promoter-styrAB-hisD right arm. As can be seen from the total plasmid size of 6763bp, the gel electrophoresis diagram and the restriction enzyme electrophoresis diagram (FIG. 4) of the recombinant plasmid, lane 1 is the plasmid pHS-AVC-03 constructed gene band, and by using EcoRV restriction enzyme digestion to verify, lane 2 presents 4258bp and 2505bp bands, which proves that the recombinant plasmid can be cut and is consistent with the expected band size, thus indicating that the recombinant plasmid pHS-AVC-03 has been constructed.

10.3 colony PCR verification

The three recombinant plasmids of pHS-AVC-01, HS-C-0151-2 and HS-C-KAN to pHS-AVC-02, HS-C-KAN and HS-C-0151-3 to pHS-AVC-03 are respectively subjected to PCR verification by using identification primers HS-C-0151-1 and HS-AVC-0063-7, and the three recombinant plasmids have bright bands, so that the successful construction of the plasmid is proved; wherein A is pHS-AVC-01, B is pHS-AVC-02, and C is pHS-AVC-03.

10.4 sequencing verification of plasmid integration sequences

The sequencing verification of the three recombinant plasmids respectively shows that the left arm-styrab-right arm fragments of the three plasmids are integrated correctly, and the plasmid integration sequence is proved to be correct again, so that the plasmid construction is successful, wherein a is pHS-AVC-01, b is pHS-AVC-02, and c is pHS-AVC-03.

10.5PCR amplified targeting fragment

The primers F-0151/R-0151, F-0152/R-0152 and F-0153/R-0153 are used for respectively carrying out PCR amplification on three plasmids, and the base numbers of pHS-AVC-01, pHS-AVC-02 and pHS-AVC-03 targeting fragments are 3683bp, 3740bp and 3761bp in sequence. FIG. 7 is an electrophoretogram of pHS-AVC-01 targeting fragment, which was subjected to 6 groups of parallel experiments, lanes 1-6 each have a bright band between 2000-4000bp, demonstrating that pHS-AVC-01 targeting fragment was successfully obtained; lanes 1-6 in FIG. 8 are the electrophoresis patterns of the targeting fragment of pHS-AVC-02, lanes 7-12 are the electrophoresis patterns of the targeting fragment of pHS-AVC-03, all of which are 6 sets of parallel, and it can be seen from the figure that there is a bright band between 2000-4000bp, which is the targeting fragment, and the target fragment is recovered by cutting gel for the next step of the test.

10.6 engineering bacteria identification

And (3) carrying out plating culture on the strains subjected to electric transfer, and selecting single bacterial colonies to identify engineering bacteria. In FIG. 9, lane 1 is a blank control group, lanes 2-4 are different single colonies on the same plate after the pHS-AVC-01 targeting fragment is electroblotted, the colony template is identified by using a primer F-0151F/R-0151, lane 2 has obvious bands at 2000-4000bp, and lanes 3 and 4 are free from the required fragment, so that the single colony of lane 2 is a successfully constructed strain, and subsequent experiments can be continued, so E.coli AB-01 construction is successful.

In FIG. 10, lanes 1 and 5 are blank control groups, lanes 2 to 4 are different single colonies on the same plate after the pHS-AVC-02 targeting fragment is electroblotted, the colony templates are identified by using a primer F-0152/R-0152, lanes 2 have obvious bands between 2000 and 4000bp, lanes 3 and 4 have no obvious bands, the colony of lane 2 is proved to be a required strain, lanes 3 and 4 are false positive strains, so the strain of lane 6 is further cultured, and E.coli AB-02 strain is constructed; lanes 6-7 are different single colonies on the same plate after the pHS-AVC-03 targeting fragment is electroblotted, the colony templates are identified by using the primer F-0153/R-0153, no obvious band is formed at the position of 2000-4000bp, the colony grows out on a resistance plate, the gene fragment is not integrated completely, and the gene fragment is false positive, and the E.coli AB-03 is not constructed successfully considering that the integration efficiency of the integration site is not high.

In conclusion, E.coli AB-01 and E.coli AB-02 strains are successfully constructed, and subsequent experiments can be carried out.

10.7 determination of growth curves

The growth curves of Pseudomonas B3, plasmid engineering bacteria E.coli-styrAB (prepared according to the method disclosed in the following academic paper: cheng Lei. Pseudomonas putida B4, indigo pigment anabolism regulating mechanism research [ D ]. Chinese university of agriculture, 2016.), E.coli AB-01, E.coli AB-02 are shown in FIG. 11. The E.coli AB-01 and E.coli AB-02 have the same growth trend, have a first growth peak period in 8 hours, continue to increase but have a slow rate, the E.coli AB-02 has higher biomass than E.coli AB-01 before 28 hours, which means that the E.coli AB-02 has stronger activity than E.coli AB-01, and the E.coli AB-01 has higher biomass than E.coli AB-02 after 28 hours, which means that the E.coli AB-01 has increased activity over 28 hours. The whole growth trend of the plasmid engineering bacteria is similar to that of the integrated engineering bacteria, but the biomass of the plasmid engineering bacteria is lower than that of the integrated engineering bacteria. The growth curve of the wild pseudomonas B3 reaches a peak value at 10 hours, gradually descends and improves to 40 hours, so that the death speed of the wild strain is high, and no engineering strain has strong activity.

10.8 Expression level of styA and styB genes in E.coli AB-01 and E.col AB-02

The mRNA expression levels of the styrA and styrB genes were quantitatively analyzed at the transcription level by RT-PCR technique using tryptophan as substrate to ferment engineering bacteria E.coli AB-01 and E.coli AB-02, and the results are shown in FIG. 12. As can be seen from the graph, the different fermentation times have a significant effect on the expression of the styA and styB genes in the anabolism of indigo pigments (0.01 < p < 0.05), and a trend of increasing and then decreasing is shown. The gene expression quantity is lower when the fermentation time is 12h and 36 h; when the fermentation time reaches 24 hours, gene expression increases sharply. The expression level of the styra gene of the E.coli AB-02 is obviously higher than that of the E.coli AB-01 and is 67.9 percent higher than that of the E.coli AB-01 in 24 hours, the expression level of the styrb gene is only 19.1 percent lower than that of the E.coli AB-01, and the total expression level of the styra gene and the styrb gene is higher than that of the E.coli AB-01 and is higher than that of the E.coli AB-01 by 31.4 percent in total, so that the E.coli AB-02 has better gene expression level than that of the E.coli AB-01.

Indole is used as a substrate to ferment engineering bacteria E.coli AB-01 and E.coli AB-02, and mRNA expression levels of the styrA and styrB genes are quantitatively analyzed at a transcription level by an RT-PCR technology, and the result is shown in FIG. 13. As can be seen from the graph, the different fermentation times have a remarkable effect on the expression of the styrA and styrB genes in the anabolism of indigo pigments (0.01 < p < 0.05), and show a trend of increasing first and then decreasing, and only the styrB gene of E.coli AB-02 shows a trend of continuously increasing. When the fermentation time is 12 hours, the gene expression quantity is low; when the fermentation time reaches 24 hours, the gene expression increases sharply; when the fermentation time reaches 36h, the gene expression level slowly decreases, and the styrb gene of E.coli AB-02 continues to increase. And the total expression level of E.coli AB-02 is higher than that of E.coli AB-01 by 43.3% at 24 hours whenever E.coli AB-02 is higher than that of E.coli AB-01.

In conclusion, the indole is used as a substrate to ferment engineering bacteria E.coli AB-01 and E.coli AB-02, so that the gene expression level is more stable, and the gene expression level of E.coli AB-02 is higher.

10.9 Effect of the feed concentration on the production of indigo pigment by engineering bacteria fermentation

9.1 the results of the test performed after 48h fermentation are shown in FIG. 14. As can be seen from the figure, the most indigo is produced at a feed concentration of twice the concentration of the primary fermentation medium, which can be up to 1.15g/L. The low nutrient concentration of the feed is insufficient to support the growth of strains, so that the strains grow too slowly; too high concentration of the feed supplement can easily cause too fast metabolism of the strain and too high osmotic pressure to cause the strain to die due to water loss. So that an appropriate amount of feed medium should be selected to continue the subsequent optimization experiments.

10.10 influence of the number of feeding times on the production of indigo pigment by fermentation of engineering bacteria

9.2 the results of the test performed after 48h fermentation are shown in FIG. 15. The yield of the indigo is lower when the feed is not fed and only fed once, and too little nutrient substances in the fermentation liquor cause too slow growth of strains and low yield of the indigo; the increasing trend of the indigo concentration is not great when the feeding times are two or more, the indigo yield is basically kept at about 1.15g/L along with the increase of the feeding times, and the situation of losing plasmids for later strains is considered to be more at the moment, so that the indigo pigment is not produced any more. So two feeds per fermentation were chosen to continue the subsequent experiments.

10.11 Effect of feed Interval time on indigo pigment production by engineering bacterium fermentation

9.3 the results of the tests performed are shown in FIG. 16. According to the graph, the indigo production trend is different in different feeding times, the indigo concentration is highest when the feeding interval time is 16h during 20h fermentation, at the moment, the indigo concentration after 48h fermentation of the indigo engineering bacteria can reach 1.189g/L, and the concentration of fed materials is lowest every 4 h. The method is characterized in that nutrients in the feed medium are rich in the early stage, the strains can not grow due to the fact that the nutrients are added more, the strains die due to the fact that the nutrients are too much, and the situation of insufficient nutrition can occur in the later stage along with the consumption of the nutrients in the earlier stage, so that the yield is low; the effect of supplementing the material once every 16 hours is optimal, and when the material supplementing time exceeds 16 hours, the strain has excessive nutrient substance consumption in the earlier stage, the growth is slow, and the plasmid loss is too fast. So that proper feeding time should be selected to perform feeding so as to achieve optimal feeding fermentation effect.

10.12 Effect of feed flow Rate on indigo pigment production by engineering bacteria fermentation

9.4, the feeding flow rates are set to be 1, 2, 3, 4 and 5mL/min, and the influence of different feeding flow rates on the indigo pigment produced by the fermentation of the indigo engineering bacteria is shown in figure 17. The best effect of the feeding flow rate is 3mL/min, and the indigo yield after 48h fermentation can reach 1.212g/L. Too low a feeding flow rate can not keep up with nutrient substances in the growth period of the strain, so that partial strain plasmids are lost or dead, and too high a feeding flow rate can also cause too high a concentration of a culture medium to cause partial strain to lose water and die. Therefore, the flow rate of 3mL/min is finally selected for feeding.

Influence of the number of passages 10.13 on the stability of engineering bacteria

As can be seen from fig. 18, the stability of the plasmid type engineering bacteria is gradually reduced along with the increase of the passage times, the metabolic burden is increased as the passage times are increased, the plasmid is lost faster, the plasmid stability is only 57.11% when the passage times are 20 times, and finally the yield of the plasmid type engineering bacteria is greatly inferior along with the loss of the plasmid; the stability trend of the two integrated engineering bacteria is the same, the stability of the engineering bacteria is basically kept unchanged along with the increase of passage times, the stability decline trend is extremely gentle, and the plasmid stability of the engineering bacteria is kept at about 94%. Therefore, the yield of the integrated engineering bacteria is not as high as that of the plasmid, but the stability is about 37% higher than that of the integrated engineering bacteria.

The comparison analysis of two different engineering bacteria is synthesized, the plasmid engineering bacteria have high indigo yield compared with the integrated engineering bacteria due to multiple copies, but the stability is not strong, the stable indigo yield cannot be ensured by fermentation after multiple passages, and the problem of unstable strains after the passages can be solved by the integrated engineering bacteria.

The integrated engineering bacteria E.coli AB-01 and E.coli AB-02 are constructed by the homologous recombination technology. Comparing and analyzing the gene expression quantity of the chromosome engineering bacteria at different sites, when tryptophan is used as a substrate, the gene expression quantity of fermentation for 24 hours is highest, and the gene expression level of engineering bacteria E.coli AB-02 with trpB as an integration site is higher and is higher by 31.4% in total. Comparing and analyzing the indigo blue output of the chromosome engineering bacteria at different sites: the yield of the indigo pigment of engineering bacteria E.coli AB-02 with trpB as an integration site is higher when two different substrates are fermented. The concentration of indigo is 83.3% higher than E.coli AB-01 when E.coli AB-02 is fermented with tryptophan as substrate.

Therefore, in order to improve the yield of engineering bacteria, the constructed integrated engineering bacteria E.coli AB-02 is fed and fermented in batches by a fermentation tank, the feeding conditions are designed and optimized, and the conditions most suitable for the feeding and fermentation of the indigo engineering bacteria are researched and determined: the concentration of the fed material is 2 times of that of the original fermentation medium, the fed material is fed every 16 hours, the total fed material is fed for 2 times, the feeding flow rate is 3mL/min, the total fermentation is carried out for 48 hours, the final fermentation yield is 3.5g/L, and the fermentation yield is improved by 5 times compared with the fermentation yield without fed material.

In conclusion, the engineering bacterium E.coli AB-02 taking tryptophan as a substrate and fermenting trpB as an integration site has stable gene expression level and stable and high-yield indigo pigment, and after fermentation culture optimization and feed supplement culture, the final fermentation yield is 3.5g/L, so that the current biosynthesis level of the indigo pigment at home and abroad is higher.

The foregoing description is only a few examples of the present application and is not intended to limit the present application in any way, and although the present application is disclosed in the preferred examples, it is not intended to limit the present application, and any person skilled in the art may make some changes or modifications to the disclosed technology without departing from the scope of the technical solution of the present application, and the technical solution is equivalent to the equivalent embodiments.

Sequence listing

<110> university of Beijing Industrial and commercial university

<120> recombinant strain, preparation method and application thereof

<160> 25

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1248

<212> DNA

<213> Pseudomonas putida

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ggactgcggc tcctgaatac cgttgctcac aacgcggtga cggtgcagcg ggaggttgcc 180

ctcgacgtca atgagtggcc gtctgaggag tttggctatt tcggccacta ctactacgta 240

ggtgggccgc agcccatgcg tttctacggt gatctcaagg ctcccagccg tgcagtggac 300

taccgtctct acctgccgat gctgatgcgt gcactggaag ccaggggcgg caagttctgc 360

tacgacgccg tgtctgccga agatctggaa gggctgtcgg agcagtatga tctgctggtt 420

gtgtgcactg gtaaatacgc cctcggcaag gtgttcgaga agcagtccga aaactcgccc 480

ttcgagaagc cgcaacgggc actgtgcgtt ggtctcttca agggcatcaa ggaagcaccg 540

attcgcgcgg tgactatgtc cttctcgcca gggcatggcg agctgattga gattccaacc 600

ctgtcgttca atggcatgag cacagcgctg gtgctcgaaa accatattgg tagcgatctg 660

gaagtcctcg cccacaccaa gtatgacgat gacccgcgtg cgttcctcga tctgatgctg 720

gagaagctgc gtaagcatca tccttccgtt gccgagcgca tcgatccggc tgagttcgac 780

ctggccaaca gttctctgga catcctccag ggcggtgttg tgccagtatt ccgcgacggt 840

catgcgaccc tcaataacgg caaaaccatc atcgggctgg gcgacatcca ggcaactgtc 900

gatccggtct tgggccaggg cgcgaacatg gcgtcctatg cggcatggat tctgggcgag 960

gaaatccttg cgcactctgt ctacgacctg cgcttcagcg aacacctgga gcgtcgccgc 1020

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cttccgccgg aattcctcac cttccttcag atcctgagcc agagccgtga aatggctgat 1140

gagttcacgg acaacttcaa ctatccggaa cttcagtggg atcgcttctc cagcccggaa 1200

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<210> 2

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<213> Pseudomonas putida

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gttagcgcct gtggagtgcc ggaggcgaat gccccccagc cgctgctgtt ctttgccagc 480

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ggcttatgac gcttactacc gctatttcat gggggataaa ccgacgttga tgagcgccac 240

ggaatgggga cgtcgttact gatccgcacg tttatgatat gctatcgtac tctttagcga 300

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agacgcggca gcgtcgcatc aggcgcttaa tacacggcat tatgaaacgg actcagcgcc 180

aggatcaccg cctggtgata gacgctggcg cgagtgagtt tcccggcggt aaacacgccg 240

atcgcccctt ccttacgacc aatctcatca ataccggtat aacgcgacat cacgggacca 300

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cgttctgctg gcggggggct taggcgcaga taactgcgtg gaagcggcac aaaccggctg 240

cgccggactt gattttaatt ctgctgtaga gtcgcaaccg ggcatcaaag acgcacgtct 300

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<213> Escherichia coli

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ccgacccact ggcggatggc ccgacgattc aaaacgccac tctgcgcgcc tttgcggcag 240

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<210> 7

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<210> 8

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acccggaatg ccagccgaaa gcggtgattg aaaattacgc gcaatatgca ggcgtaaaac 240

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<210> 9

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<212> DNA

<213> Artificial sequence (Artificial Sequence)

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cgcaagatgt ggcgtgttac 20

<210> 10

<211> 20

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<213> Artificial sequence (Artificial Sequence)

<400> 10

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<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

cgcatcgagc gagcacgtac 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

cggcattctt cagcaagcgt 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

tcaggccgca atagtgggtg 20

<210> 14

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<213> Artificial sequence (Artificial Sequence)

<400> 14

aacgtgctgg cttatgacgc ttact 25

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

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cggtgattct ggaaaaagtg cgtga 25

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

tatgttttag acaacggcca gggtg 25

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

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<210> 18

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<213> Artificial sequence (Artificial Sequence)

<400> 18

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<210> 19

<211> 25

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<213> Artificial sequence (Artificial Sequence)

<400> 19

agcggttgag cgtttgctga gtaag 25

<210> 20

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

ggcgagctga ttgagattc 19

<210> 21

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

ttttgccgtt attgagggt 19

<210> 22

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

aaaagatgtg gtggtggat 19

<210> 23

<211> 19

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<213> Artificial sequence (Artificial Sequence)

<400> 23

tgctgaagaa tgccgataa 19

<210> 24

<211> 17

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

ccacctggac tgatact 17

<210> 25

<211> 17

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

gcacctgtct caatgtt 17

Claims (8)

1. A recombinant strain, characterized in that it has fragment I integrated on the genome of the host;

the fragment I contains a styrene monooxygenase genestyAAnd styrene monooxygenase genesstyB；

The styrene monooxygenase genestyAThe coding sequence of (2) is shown as SEQ ID NO. 1;

the styrene monooxygenase genestyBThe coding sequence of (2) is shown as SEQ ID NO. 2;

the integration is by replacing the nucleotide sequence of the host integration site with fragment I;

the integration site is selected fromtrpR、trpB、hisDAny one of them.

2. The recombinant strain according to claim 1, wherein the host is selected from any one of e.

3. The recombinant strain according to claim 2, wherein the escherichia coli isE.coliDH5α。

4. The recombinant strain according to claim 1, wherein the fragment I further comprises an inducible promoter, an operator;

according to inducible promoter, operon and styrene monooxygenase genestyA、Styrene monooxygenase genesstyBSequentially arranged.

5. The recombinant strain according to claim 4, wherein the inducible promoter is an inducible promoter cat;

the operon is lactose operon lac.

6. The method for preparing a recombinant strain according to any one of claims 1 to 5, comprising the steps of:

integrating the fragment I into the genome of a host to obtain the recombinant strain; in particular, the method comprises the steps of,

(S1) amplifying the fragment II by using a recombinant vector containing the fragment II as a template;

(S2) transferring the fragment II into a host competent cell expressing a recombinase to obtain the recombinant strain;

wherein, the fragment II sequentially comprises an integration site left homology arm ', a fragment I and an integration site right homology arm';

transferring the pKD46 plasmid into a host cell, and culturing in a medium containing L-arabinose to obtain the host competent cell expressing the recombinase.

7. A method for producing indigo pigment, characterized in that the method comprises the steps of:

fermenting the recombinant strain in a culture medium to obtain the indigo pigment;

the recombinant strain comprises at least one of the recombinant strain according to any one of claims 1-5 and the recombinant strain prepared by the preparation method according to claim 6.

8. The method of claim 7, wherein the medium comprises a fermentation substrate comprising at least one of indole, tryptophan; the concentration of the indole is 0.5-5 g/L; the concentration of the tryptophan is 0.5-10 g/L; the temperature is 30-40 ℃; the revolution is 50-400 rpm; the ventilation ratio is 0.2-1.0 vvm; the pH value is 6.0-8.0; the dissolved oxygen correction is 80% -100%.

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