CN118995553A

CN118995553A - Shinorine high-yield engineering strain, construction method and application thereof

Info

Publication number: CN118995553A
Application number: CN202411229061.5A
Authority: CN
Inventors: 张芳
Original assignee: Shandong Hecheng Biotechnology Co ltd
Current assignee: Shandong Hecheng Biotechnology Co ltd
Priority date: 2024-09-03
Filing date: 2024-09-03
Publication date: 2024-11-22

Abstract

The invention provides a Shinorine high-yield engineering strain, a construction method and application thereof, belongs to the technical field of biology, and aims at carrying out codon optimization on 4 key genes mysA, mysB, mysC and mysD in three sets of Shinorine synthetic gene clusters derived from algae and constructing different recombinant escherichia coli strains, wherein the highest Shinorine yield of recombinant escherichia coli constructed by 4 key genes mysA ₃、mysB₃、mysC₃ and mysD ₃ in the Shinorine synthetic gene clusters from Nostoc LINKIA NIES-25 is found; after screening recombinant escherichia coli with highest Shinorine yield, further introducing a T7 promoter into the recombinant escherichia coli, and optimizing fermentation conditions, so that the Shinorine yield is improved.

Description

Shinorine high-yield engineering strain, construction method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a Shinorine high-yield engineering strain, a construction method and application thereof.

Background

The class of bacteriocin amino acids (Mycosporine-like amino acids, MAAs) widely exist in large red algae, and are active substances with good application prospects. The Shinorine belongs to a biodegradable ultraviolet absorbing substance in MAAs, is generally naturally produced by blue algae and seaweed, is difficult to artificially synthesize, and has a structural formula shown in formula (1):

currently, commercially available Shinorine is generally derived from red algae harvested from the sea, but the efficiency of extracting Shinorine from red algae is very low, and harvesting red algae by aquaculture increases the production cycle and production costs, so that the yield of Shinorine may vary with season and geographic location, and supply is limited.

The Shinorine can be synthesized manually through a chemical method besides natural production, but the chemical method has long production period and extremely high price, and is not economically feasible if the Shinorine is produced in large quantities; in addition, chemical synthesis still has a certain technical limit, and Shinorine is difficult to obtain in a sustainable manner.

The development of the natural product biosynthesis pathway is combined with advanced molecular biology technology, so that the method has multiple benefits of shortening the development period, reducing the production cost, improving the product output and the like, and therefore, the production of Shinorine by means of a heterologous host is greatly concerned, and compared with natural production and chemical synthesis, the production of Shinorine by using biotechnology has a strong competitive advantage.

The research and development of dominant engineering bacteria for producing Shinorine has important significance for producing Shinorine by using biotechnology.

Disclosure of Invention

The invention provides a Shinorine high-yield engineering strain and a construction method and application thereof, and the invention discovers for the first time that compared with 4 key enzyme genes mysA, mysB, mysC and mysD in Shinorine synthetic gene clusters derived from other algae strains (Nostoc punctiforme ATCC 29133 and Anabaena variabilis ATCC 29413), the effect of producing Shinorine by using the engineering strain constructed by 4 key enzyme genes mysA ₃、mysB₃、mysC₃ and mysD ₃ in the Shinorine synthetic gene clusters from the algae strains Nostoc LINKIA NIES-25 is better; in addition, the T7 promoter is added to the engineering strain obtained by screening, and fermentation conditions are optimized, so that the yield of Shinorine is further improved.

The technical scheme of the invention is as follows:

A high-yield engineering strain of Shinorine is obtained by introducing an AB gene fragment and a CD gene fragment into escherichia coli through a vector, wherein the nucleotide sequence of the AB gene fragment is shown as SEQ ID NO.4, and the nucleotide sequence of the CD gene fragment is shown as SEQ ID NO. 5.

Preferably, the vector is a pETDuet1 plasmid vector.

Further preferably, the introduction positions of the AB gene fragment and the CD gene fragment in the pETDuet1 plasmid vector are as follows: the AB gene fragment was seamlessly cloned into the PstI cleavage site of the pETDuet1 plasmid vector, and the CD gene fragment was introduced between the NdeI-XhoI cleavage sites of the pETDuet1 plasmid vector.

Preferably, the escherichia coli is E.coli BL21.

Preferably, the Shinorine high-yield engineering strain also comprises a T7 promoter, and the nucleotide sequence of the T7 promoter is shown as SEQ ID NO. 28.

Further preferably, the construction method of the Shinorine high-yield engineering strain containing the T7 promoter comprises the following steps:

(1) The AB gene segment is used as a template, and primers mysA ₃ -F and mysA ₃ -R are used for PCR amplification to obtain a gene segment mysA ₃, and primer sequences of mysA ₃ -F and mysA ₃ -R are shown as SEQ ID NOs.18-19;

The AB gene segment is used as a template, and primers mysB ₃ -F and mysB ₃ -R are used for PCR amplification to obtain a gene segment mysB ₃, and primer sequences of mysB ₃ -F and mysB ₃ -R are shown as SEQ ID NOs.20-21;

Carrying out double enzyme digestion treatment on plasmid pRSFDuet by using NcoI and NotI, purifying and recovering to obtain pRSFDuet1 vector fragment;

Connecting the gene fragment mysA ₃, the gene fragment mysB ₃ and the pRSFDuet1 vector fragment, and converting the connection product into E.coli XL1-Blue to obtain recombinant plasmids pRSFDuet-mysA ₃B₃;

(2) Carrying out PCR amplification by using pRSFDuet as a template and using primers pRSF-T7-Not-F and pRSF-T7-Nde-R to obtain a T7 promoter, wherein the primer sequences of pRSF-T7-Not-F and pRSF-T7-Nde-R are shown as SEQ ID NOs.22-23;

performing PCR amplification by using the CD gene fragment as a template and using primers mysC ₃ -F and mysC ₃ -R to obtain a gene fragment mysC ₃, wherein primer sequences of mysC ₃ -F and mysC ₃ -R are shown as SEQ ID NOs.24-25;

performing PCR amplification by using the CD gene fragment as a template and using primers mysD ₃ -F and mysD ₃ -R to obtain a gene fragment mysD ₃, wherein primer sequences of mysD ₃ -F and mysD ₃ -R are shown as SEQ ID NOs.26-27;

(3) Performing double enzyme digestion treatment on the recombinant plasmids pRSFDuet-mysA ₃B₃ obtained in the step (1) by using enzymes NdeI and XhoI, purifying and recovering to obtain pRSFDuet1-mysA ₃B₃ vector fragments;

(4) Connecting a T7 promoter, a gene fragment mysC ₃ and a gene fragment mysD ₃、pRSFDuet1-mysA₃B₃ carrier fragment, and converting the connection product into E.coli XL1-Blue to obtain a recombinant plasmid pRSFDuet-T7X 4-mysA ₃B₃C₃D₃;

(5) And (3) transforming the recombinant plasmid pRSFDuet-T7 multiplied by 4-mysA ₃B₃C₃D₃ into E.coli BL21 to obtain the Shinorine high-yield engineering strain containing the T7 promoter.

According to the fermentation method of the Shinorine high-yield engineering strain, bacterial liquid of the Shinorine high-yield engineering strain is inoculated into an LB liquid culture medium for culture, then IPTG with the final concentration of 0.1-0.5 mM is added, and the culture is induced for 20-36 h at the culture temperature of 25-30 ℃ to obtain fermentation liquor.

Preferably, the final concentration of IPTG is 0.2mM.

Preferably, the culture temperature is 30 ℃.

Preferably, the time of the induction culture is 24 hours.

Preferably, the LB liquid medium contains xylose with a final concentration of 7.5g/L to 20 g/L.

Further preferably, the LB liquid medium contains xylose at a final concentration of 7.5 g/L.

The nucleotide sequence of the AB gene fragment is shown as SEQ ID NO.4, and the nucleotide sequence of the CD gene fragment is shown as SEQ ID NO. 5.

The application of the Shinorine high-yield engineering strain in the production of Shinorine.

The beneficial effects are that:

The invention optimizes codons for 4 key genes mysA, mysB, mysC and mysD in three sets of algae-derived Shinorine synthesis gene clusters and constructs different recombinant escherichia coli strains, and discovers that the yield of the recombinant escherichia coli constructed by 4 key genes mysA ₃、mysB₃、mysC₃ and mysD ₃ in the Shinorine synthesis gene clusters from Nostoc LINKIA NIES-25 is higher; after screening recombinant escherichia coli with high-yield Shinorine advantage, further introducing a T7 promoter into the recombinant escherichia coli, and optimizing fermentation conditions, so that the optimal yield of Shinorine is obtained.

Drawings

FIG. 1 is a diagram showing the protein expression of E.coli BL 21-I, E.coli BL 21-II and E.coli BL 21-III.

FIG. 2 is a graph showing the protein expression of E.coli BL 21-III and E.coli BL21/pRSFDuet 1-T7X 4-mysA ₃B₃C₃D₃.

FIG. 3 is a graph showing the results of HPLC detection of E.coli BL 21-I, E.coli BL 21-II, and E.coli BL 21-III.

FIG. 4 is an ultraviolet absorption spectrum of E.coli BL 21-I, E.coli BL 21-II, E.coli BL 21-III.

FIG. 5 is a graph of LC-MS detection results of E.coli BL 21-I, E.coli BL 21-II, and E.coli BL 21-III.

FIG. 6 is a graph of HPLC and LC-MS detection results of E.coli BL 21-III, E.coli BL21/pRSFDuet 1-T7X 4-mysA ₃B₃C₃D₃.

FIG. 7 is a graph of the results of optimization of fermentation conditions for E.coli BL 21/pRSFDuet-T7X 4-mysA ₃B₃C₃D₃.

Detailed Description

The following description is made in connection with specific embodiments:

(1) Plasmid and strain sources:

pRSFDuet1 plasmid: purchased from pesen nuo biosciences, inc.

PET22b, pETDuet1, E.coli XL1-Blue, E.coli BL21: the products are all conventional commercial products from the biological laboratories of the universities of Qilu industry.

(2) The following examples were used to formulate the media and primary reagents:

LB liquid medium: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, and the balance water.

LB solid medium: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 20g/L agar, and the balance water.

LB liquid medium with 0.75%, 1%, 2% xylose: xylose was added to LB liquid medium at 7.5g/L, 10g/L and 20g/L, respectively.

PBS buffer: 8.0g NaCl,0.2g KCl,1.44g Na ₂HPO₄,0.24g KH₂PO₄, 1000mL of distilled water, HCl was adjusted to pH 7.4.

SDS PAGE loading buffer: 1.25mL of 1M Tris-HCl buffer, 0.5g of sodium dodecyl sulfate, 0.025g of bromophenol blue, 2.5mL of glycerol, 0.25mL of beta-mercaptoethanol, and ddH ₂ O to 25mL were taken.

Example 1: construction of recombinant E.coli BL 21-I, E.coli BL 21-II and E.coli BL 21-III

1. Excavating, optimizing and synthesizing of Shinorine synthetic gene

The inventor selects 3 Shinorine synthesis gene clusters from Nostoc punctiforme ATCC 29133, anabaena variabilis ATCC 29413 and Nostoc LINKIA NIES-25 respectively, and sends 4 key enzyme genes mysA, mysB, mysC and mysD in the Shinorine synthesis gene clusters to the personii biotechnology company for codon optimization and gene synthesis.

Wherein:

The Shinorine synthesis gene cluster from Nostoc punctiforme ATCC 29133 is a gene cluster 1, wherein the gene cluster 1 comprises an ABC gene fragment and a D gene fragment, the nucleotide sequence of the ABC gene fragment is shown as SEQ ID NO.1, and the nucleotide sequence of the D gene fragment is shown as SEQ ID NO. 2; the 4 key enzyme genes in gene cluster 1 are referred to as "mysA ₁、mysB₁、mysC₁ and mysD ₁", respectively;

The Shinorine synthetic gene cluster from Anabaena variabilis ATCC 29413 is a gene cluster 2, the nucleotide sequence of which is shown in SEQ ID NO.3, wherein 4 key enzyme genes are respectively called 'mysA ₂、mysB₂、mysC₂ and mysD ₂';

The Shinorine synthesized gene cluster from Nostoc LINKIA NIES-25 is a gene cluster 3, wherein the gene cluster 3 comprises an AB gene fragment and a CD gene fragment, the nucleotide sequence of the AB gene fragment is shown as SEQ ID NO.4, and the nucleotide sequence of the CD gene fragment is shown as SEQ ID NO. 5; the 4 key enzyme genes in gene cluster 3 are referred to as "mysA ₃、mysB₃、mysC₃ and mysD ₃", respectively.

2. Construction of recombinant plasmids

(1) Construction of recombinant plasmid pETDuet1-mysA ₁B₁

① The ABC gene fragment in the gene cluster 1 is used as a template, and a primer mysA ₁-F(SEQ ID NO.6)、mysA₁ -R (SEQ ID NO. 7) is used for PCR amplification to obtain a gene fragment mysA ₁.

② The pETDuet1 plasmid was subjected to double cleavage treatment using NcoI and NotI, the double cleavage system is shown in Table 1 below:

TABLE 1 double enzyme digestion System

Total system	10μL
		NcoI	1μL
NotI	1μL
		10×Buffer	1μL
PETDuet1 plasmid	7μL

③ The digested product (about 5400 bp) was purified and recovered using a DNA purification recovery kit to obtain a vector fragment.

④ And (3) connecting the gene fragment mysA ₁ and the vector fragment by using a ClonExpress II one-step cloning kit to obtain a recombinant plasmid pETDuet1-mysA ₁.

⑤ Subjecting the recombinant plasmid pETDuet1-mysA ₁ to double digestion treatment by NdeI and XhoI, and recovering digestion products (about 6600 bp); the ABC gene fragment in the gene cluster 1 is used as a template, and a primer mysB ₁-F(SEQ ID NO.8)、mysB₁ -R (SEQ ID NO. 9) is used for PCR amplification to obtain a gene fragment mysB ₁.

⑥ And (3) connecting the gene fragment mysB ₁ in the step ⑤ with an enzyme digestion product by using ClonExpress II one-step cloning kit to obtain a recombinant plasmid pETDuet1-mysA ₁B₁.

(2) Construction of recombinant plasmid pRSFDuet A-mysC ₁D₁

① The ABC gene fragment in the gene cluster 1 is used as a template, and a primer mysC ₁-F(SEQ ID NO.10)、mysC₁ -R (SEQ ID NO. 11) is used for PCR amplification to obtain a gene fragment mysC ₁.

② The pRSFDuet.sup.1 plasmid was subjected to double cleavage with NcoI and NotI, and the double cleavage system was as above.

③ And (3) purifying and recovering the enzyme digestion product (about 3800 bp) by using a DNA purification and recovery kit to obtain a carrier fragment.

④ The gene fragment mysC ₁ and the vector fragment were ligated using ClonExpress II one-step cloning kit to give recombinant plasmids pRSFDuet-mysC ₁.

⑤ Subjecting the recombinant plasmids pRSFDuet-mysC ₁ to double digestion with NdeI and XhoI to recover digested products (about 5200 bp); the D gene fragment in the gene cluster 1 is used as a template, and a primer mysD ₁-F(SEQ ID NO.12)、mysD₁ -R (SEQ ID NO. 13) is used for PCR amplification to obtain a gene fragment mysD ₁.

⑥ And (3) connecting the gene fragment mysD ₁ in the step ⑤ with an enzyme digestion product by using ClonExpress II one-step cloning kit to obtain recombinant plasmids pRSFDuet-mysC ₁D₁.

(3) Construction of recombinant plasmid pET22b-mysA ₂B₂C₂

① PCR amplification is carried out by using the gene cluster 2 as a template and using a primer mysA ₂B₂C₂-F(SEQ ID NO.14)、mysA₂B₂C₂ -R (SEQ ID NO. 15) to obtain a gene fragment mysA ₂B₂C₂.

② The pET22b plasmid was subjected to double digestion with NdeI and XhoI, and the double digestion system was the same as above.

③ And (3) purifying and recovering the enzyme digestion product (about 5500 bp) by using a DNA purification and recovery kit to obtain a carrier fragment.

④ And (3) connecting the gene fragment mysA ₂B₂C₂ with the vector fragment by using ClonExpress II one-step cloning kit to obtain a recombinant plasmid pET22b-mysA ₂B₂C₂.

(4) Construction of recombinant plasmid pRSFDuet A-mysD ₂

① PCR amplification is carried out by using the gene cluster 2 as a template and using a primer mysD ₂-F(SEQ ID NO.16)、mysD₂ -R (SEQ ID NO. 17) to obtain a gene fragment mysD ₂.

② The pRSFDuet plasmid was subjected to double cleavage with NdeI and XhoI, and the double cleavage system was as above.

④ The gene fragment mysD ₂ and the vector fragment were ligated using ClonExpress II one-step cloning kit to give recombinant plasmids pRSFDuet-mysD ₂.

(5) Construction of recombinant plasmid pETDuet1-mysA ₃B₃C₃D₃

The construction process of the recombinant plasmid pETDuet1-mysA ₃B₃C₃D₃ is directly completed by Nanjinouzan company, and the specific construction method is as follows: the AB gene segment shown in SEQ ID NO.4 is cloned to PstI restriction enzyme cutting site of pETDuet1 plasmid vector in a seamless way, and the CD gene segment shown in SEQ ID NO.5 is led into between NdeI-XhoI restriction enzyme cutting sites of pETDuet1 plasmid vector, thus obtaining recombinant plasmid pETDuet1-mysA ₃B₃C₃D₃.

The PCR amplification systems and the PCR amplification procedures in the above steps (1) to (3) are shown in tables 2 and 3, respectively, below:

TABLE 2 PCR amplification System

TABLE 3 PCR amplification procedure

PCR program	Temperature (temperature)	Time of	Circulation
				Pre-denaturation	98℃	30s	1
Denaturation (denaturation)	98℃	10s	30
				Annealing	50℃	5s	30
Extension of	72℃	10s	30
				Final extension	72℃	1min	1

3. Construction of recombinant E.coli BL 21-I, E.coli BL 21-II and E.coli BL 21-III

Taking out competent cells E.coli XL1-Blue from the ultra-low temperature refrigerator, and placing the competent cells E.coli XL1-Blue on ice for standby; and (2) respectively adding 10 mu L of each recombinant plasmid in the step (II) into competent cells, slightly blowing and uniformly mixing, then placing the mixture on ice for 30min, then performing heat shock on the mixture for 90s in a water bath kettle at 42 ℃, and immediately placing the mixture on ice for 2min to obtain a conversion solution. Adding 900 mu L of LB liquid medium into the transformation solution, recovering at 37 ℃ for 1h by a shaking table, centrifuging at 600 rpm for 3min, sucking 500 mu L of supernatant, lightly mixing the rest supernatant and thalli to obtain a bacterial suspension, sucking 100 mu L of bacterial suspension, coating the bacterial suspension on LB solid medium containing 50 mu g/mL Kan and 50 mu g/mL Amp, and culturing at 37 ℃ for 12h until single colony grows;

Single colony is selected for preliminary verification of colony PCR, single colony with successful preliminary verification of PCR is selected, bacterial liquid culture and plasmid extraction and sequencing are carried out, and plasmids with correct sequencing results are respectively transformed into E.coli BL21 to construct recombinant escherichia coli, wherein the specific transformation process is as follows (the transformation method is the same as above):

Co-transforming pETDuet1-mysA ₁B₁ and pRSFDuet-mysC ₁D₁ into E.coli BL21 to obtain recombinant E.coli BL 21-I;

Co-transforming pET22b-mysA ₂B₂C₂ and pRSFDuet-mysD ₂ into E.coli BL21 to obtain recombinant E.coli BL 21-II;

And (3) converting pETDuet1-mysA ₃B₃C₃D₃ into E.coli BL21 to obtain recombinant E.coli BL 21-III.

The recombinant E.coli BL 21-I and E.coli BL 21-II bacterial solutions are respectively coated on an LB solid medium containing 50 mug/mL Kan and 50 mug/mL Amp, and the recombinant E.coli BL 21-III bacterial solution is coated on an LB solid medium containing 50 mug/mL Amp; the above culture was incubated at 37℃for 12 hours until single colonies were grown.

Selecting single bacterial colony for colony PCR verification and sequencing, and inoculating bacterial solutions of recombinant strains E.coli BL 21-I and E.coli BL 21-II with correct sequencing results into LB liquid culture medium simultaneously containing 50 mug/mL Kan and 50 mug/mL Amp at a ratio of 1% (v/v) respectively; inoculating the bacterial liquid of the recombinant strain E.coli BL 21-III with the correct sequencing result into LB liquid medium containing 50 mug/mL Amp at a ratio of 1% (v/v); shake culturing at 37deg.C for 1.5 hr until OD ₆₀₀ is about 0.6; IPTG was added at a final concentration of 0.2mM, and the culture was induced at 25℃for 20 hours.

After the induction culture was completed, the culture broth was centrifuged at 10000rpm for 5min, the bacterial pellet was collected, washed 2 times with PBS buffer and resuspended, and the resuspension was broken (4 s suspension by ultrasound) using an ultrasonic breaker at 400w for 20min.

After the ultrasonic treatment is finished, the liquid after the ultrasonic treatment is absorbed and then SDS PAGE Loading buffer is added to prepare a whole cell sample; centrifuging the liquid after ultrasonic treatment at 10000rpm for 10min, and adding SDS PAGE Loading buffer into the supernatant to obtain the whole intracellular sample.

The protein expression in the whole cell sample and the whole intracellular sample is analyzed by 12% SDS-PAGE, and the results are shown in figure 1, wherein the A diagram is a schematic diagram of the construction and gene cluster of E.coli BL 21-I, E.coli BL 21-II and E.coli BL 21-III; panel B shows the results of SDS-PAGE analysis of E.coli BL 21-I, E.coli BL 21-II and E.coli BL 21-III, M:180kDa Prestained Protein Marker, lanes 1-2: full cell and intracellular protein expression schematic of the coll BL21 blank, lanes 3-4: full cell and intracellular protein expression schematic of BL 21-II, lanes 5-6: full cell and intracellular protein expression schematic of BL 21-I, lanes 7-8: schematic representation of BL 21-III whole cell and intracellular protein expression.

As can be seen from FIG. 1, the protein expression of E.coli BL 21-I, E.coli BL 21-II, E.coli BL 21-III is as follows: mysA (MysA ₁、MysA₂、MysA₃) has a protein band size of about 45 kD; mysB (MysB ₁、MysB₂、MysB₃) has a protein band size of about 31 kD; mysC (MysC ₁、MysC₂、MysC₃) has a protein band size of about 51 kD; mysD ₁ and MysD ₃ are 38kD in size and MysD ₂ is about 97kD; all proteins were soluble proteins, and did not produce inclusion bodies, indicating that the induction conditions selected were appropriate; in terms of protein expression levels, mysA is the highest among all enzymes, mysC is far lower in protein expression levels than other proteins, and thus Shinorine synthesis is limited by MysC expression; mysB has a protein expression level slightly higher than MysD ₂,MysD₂ and slightly higher than MysC; in addition, the E.coli BL 21-III gene cluster is expressed at the highest level compared to E.coli BL 21-I and E.coli BL 21-II.

Example 2: construction of recombinant Strain E.coli BL21/pRSFDuet 1-T7X 4-mysA ₃B₃C₃D₃ containing a Cascade T7 promoter

Based on recombinant E.coli BL 21-III, a cascade T7 promoter strategy is adopted, and the increase of the synthesis amount of Shinorine is promoted by enhancing the expression level of protein, wherein the nucleotide sequence of the T7 promoter is shown as SEQ ID NO. 28. The specific operation steps are as follows:

① The AB gene fragment was used as a template, and PCR amplification was performed using primers mysA ₃ -F (SEQ ID NO. 18) and mysA ₃ -R (SEQ ID NO. 19) to obtain a gene fragment mysA ₃.

② The AB gene fragment was used as a template, and PCR amplification was performed using primers mysB ₃ -F (SEQ ID NO. 20) and mysB ₃ -R (SEQ ID NO. 21) to obtain a gene fragment mysB ₃.

③ Plasmid pRSFDuet was subjected to double cleavage with NcoI and NotI, and purified to obtain pRSFDuet vector fragment.

④ Through ClonExpress II one-step cloning kit, gene fragment mysA ₃, gene fragment mysB ₃ and pRSFDuet1 carrier fragment are connected, the connection product is transformed into E.coli XL1-Blue, the transformant is selected for colony PCR preliminary verification, then plasmid verification is extracted and sequencing is carried out, and recombinant plasmid pRSFDuet1-mysA ₃B₃ is obtained.

⑤ Carrying out PCR amplification by using pRSFDuet as a template and using primers pRSF-T7-Not-F (SEQ ID NO. 22) and pRSF-T7-Nde-R (SEQ ID NO. 23) to obtain a T7 promoter;

PCR amplification is carried out by using the CD gene fragment as a template and using primers mysC ₃ -F (SEQ ID NO. 24) and mysC ₃ -R (SEQ ID NO. 25) to obtain a gene fragment mysC ₃;

PCR amplification is carried out by using the CD gene fragment as a template and using primers mysD ₃ -F (SEQ ID NO. 26) and mysD ₃ -R (SEQ ID NO. 27) to obtain a gene fragment mysD ₃;

⑥ Performing double enzyme digestion on recombinant plasmids pRSFDuet-mysA ₃B₃ by using enzymes NdeI and XhoI, purifying and recovering to obtain pRSFDuet1-mysA ₃B₃ vector fragments;

⑦ And (3) connecting a T7 promoter, a gene fragment mysC ₃ and a gene fragment mysD ₃、pRSFDuet1-mysA₃B₃ carrier fragment through a ClonExpress II one-step cloning kit, converting the connection product into E.coli XL1-Blue, picking the transformant for colony PCR preliminary verification, extracting plasmid verification and sequencing to obtain a recombinant plasmid pRSFDuet-T7 multiplied by 4-mysA ₃B₃C₃D₃.

Recombinant plasmid pRSFDuet-T7X 4-mysA ₃B₃C₃D₃ was transformed into E.coli BL21, the transformation method was the same. 100 mu L of the transformation solution is absorbed and coated on LB solid medium containing 50 mu g/mL Kan, and the transformation solution is cultured for 12 hours at 37 ℃ until single colony is grown; and selecting a single colony for PCR verification and sequencing screening to obtain a positive transformant, namely a recombinant strain E.coli BL21/pRSFDuet1-T7 multiplied by 4-mysA ₃B₃C₃D₃.

Inoculating the recombinant strain E.coli BL 21/pRSFDuet-T7X 4-mysA ₃B₃C₃D₃ bacterial liquid into LB liquid culture medium containing 50 mug/mL Kan according to the proportion of 1% (v/v), and culturing until OD ₆₀₀ reaches about 0.6; IPTG was added at a final concentration of 0.2mM, and the culture was induced at 25℃for 25 hours.

After the induction culture was completed, the culture broth was centrifuged at 10000rpm for 5min, the bacterial pellet was collected, washed 2 times with PBS buffer and resuspended, and the resuspension was broken (4 s suspension by ultrasound) using an ultrasonic breaker at 400w for 20min. Whole cell samples and whole intracellular samples were prepared (preparation method is the same as above) and protein expression was analyzed by 12% SDS-PAGE.

The results are shown in FIG. 2, wherein FIG. A is a schematic diagram of the construction and gene cluster of E.coli BL21/pRSFDuet 1-T7X 4-mysA ₃B₃C₃D₃; FIG. B is a schematic representation of E.coli BL21 and E.coli BL21/pRSFDuet 1-T7X 4-mysA ₃B₃C₃D₃ protein expression, M:180kDa Prestained Protein Marker, lanes 1-2: full cell and intracellular protein expression schematic of the coll BL21 blank, lanes 3-4: e. Whole cell and intracellular protein expression schematic of coli BL 21-III, lanes 5-6: schematic representation of whole cell and intracellular protein expression of coll BL21/pRSFDuet 1-T7X 4-mysA ₃B₃C₃D₃.

As can be seen from fig. 2, after the cascade promoter T7 was introduced, the expression level of MysA, mysB, mysC, mysD protein in e.coll BL21/pRSFDuet1-T7 x 4-mysA ₃B₃C₃D₃ was significantly improved, and although the expression level of MysC protein was still relatively low in all target proteins, the expression level was still slightly increased compared to the case where the cascade promoter T7 was not used. The results show that the introduction of the cascade promoter T7 can effectively promote the expression of target proteins, thereby improving the synthesis efficiency of Shinorine.

Example 3: extraction and detection of Shinorine in engineering strains E.coli BL 21-I, E.coli BL 21-II and E.coli BL 21-III

1. Extraction of Shinorine

Activating engineering strains E.coli BL 21-I, E.coli BL 21-II and E.coli BL 21-III by using an LB liquid medium; inoculating the activated bacterial liquid into LB liquid culture medium according to the proportion of 2% (v/v), and culturing the bacterial concentration to OD ₆₀₀ of about 0.6; IPTG was added at a final concentration of 0.2mM and the culture was induced at 25℃for 20 hours.

After the induction culture was completed, the culture broth was centrifuged at 10000rpm for 5min, the bacterial pellet was collected, washed 2 times with PBS buffer and resuspended, and the resuspension was broken (4 s suspension by ultrasound) using an ultrasonic breaker at 400w for 20min. Centrifuging the liquid after ultrasonic treatment at 10000rpm for 20min, collecting supernatant, adding equal volume of methanol into the supernatant, leaching at 37deg.C for 2h, then ultrasonic treating in ultrasonic bath (100 w) for 1h, centrifuging the mixed liquid after ultrasonic treatment at 4deg.C for 20min at 10000rpm to remove polysaccharide, protein, nucleic acid, etc., subjecting the supernatant to rotary steaming at low temperature, lyophilizing to obtain lyophilized sample containing Shinorine, and storing at-80deg.C.

2. Detection of Shinorine

(1) HPLC detection: the extracted lyophilized sample was dissolved in ultrapure water, and after filtering the impurities with a 0.22 μm filter, HPLC detection was performed with E.coli BL21 as a blank.

The detection result is shown in FIG. 3, wherein the A graph is E.coli BL21 blank, the B graph is E.coli BL21-I, the C graph is E.coli BL21-II, and the D graph is E.coli BL 21-III. From FIG. 3, it can be seen that two significant absorption peaks appear at about 7.4min and 7.6min in each of panels B-D, as compared to panel A, which is consistent with the expected retention times of Shinorine and Mycosporine-glycine, thus confirming that the engineering strains E.coli BL21-I, E.coli BL21-II, E.coli BL21-III are capable of producing Shinorine. Then, by comparing the peak heights with the peak areas, the engineering strain E.coli BL21-III has the highest efficiency in the aspect of producing Shinorine compared with E.coli BL21-I and E.coli BL 21-II.

(2) Ultraviolet absorption spectrometry detection: the extracted lyophilized sample was dissolved in ultrapure water, and after filtering the impurities with a 0.22 μm filter, detection by ultraviolet absorption spectrometry was performed with E.coli BL21 as a blank control.

The test results are shown in FIG. 4, wherein the A graph is E.coli BL21 blank, the B graph is E.coli BL 21-I, the C graph is E.coli BL 21-II, and the D graph is E.coli BL 21-III. As can be taken from fig. 4, there is no characteristic absorption peak in e.coli BL 21; e.coli BL 21-I and E.coli BL 21-III both have obvious absorption peaks at 334nm, which indicates that the generation of Shinorine is shown, and smaller absorption peaks appear at 310nm, and the corresponding products are Mycosporine-glycine; the absorption peak of the E.coli BL 21-II is wide, and the absorption peak is at 310mn-340nm, which indicates that Shinorine and Mycosporine-glycine are also present.

(3) LC-MS detection: the extracted lyophilized sample was dissolved in ultrapure water, and after filtering the impurities with a 0.22 μm filter, LC-MS detection was performed.

The detection results are shown in FIG. 5, wherein the A diagram is Mycosporine-glycine secondary mass spectrum and structure of E.coli BL 21-I, the B diagram is Shinorine secondary mass spectrum and structure of E.coli BL 21-I, the C diagram is Mycosporine-glycine secondary mass spectrum of E.coli BL 21-II, the D diagram is Shinorine secondary mass spectrum of E.coli BL 21-II, the E diagram is Mycosporine-glycine secondary mass spectrum of E.coli BL 21-III, and the F diagram is Shinorine secondary mass spectrum of E.coli BL 21-III.

Molecular masses of Mycosporine-glycine and Shinorine are determined to be 245 and 332 respectively, and in a positive ion scanning mode, mass-to-charge ratios of three engineering strains of E.coli BL21-I, E.coli BL21-II and E.coli BL21-III are detected and compared with theoretical values. As can be taken from fig. 5, the detected data are highly consistent with the expected molecular mass, and based on the above results, the presence of Shinorine and Mycosporine-glycine was further confirmed.

Example 4: extraction and detection of Shinorine in engineering strain E.coli BL 21/pRSFDuet-T7X 4-mysA ₃B₃C₃D₃

1. Extraction of Shinorine

The extraction method was the same as in example 3 to obtain a lyophilized sample containing Shinorine.

2. Detection of Shinorine

The extracted lyophilized sample was dissolved in ultrapure water, and after filtering the impurities with a 0.22 μm filter membrane, HPLC and LC-MS detection were performed, with the engineering strain E.coli BL 21-III as a control.

The results of the assay are shown in FIG. 6, wherein Panel A is an HPLC plot of Shinorine generated in E.coli BL21/pRSFDuet 1-T7X 4-mysA ₃B₃C₃D₃; panel B shows a comparison of the yields of Shinorine and Mycosporine-glycine for E.coli BL 21-III and E.coli BL21/pRSFDuet 1-T7X 4-mysA ₃B₃C₃D₃; panel C shows the secondary mass spectrum and structure of Mycosporine-glycine; panel D shows the secondary mass spectrum and structure of Shinorine.

As can be seen from the B graph in FIG. 6, the peak area comparison method was used to estimate the production of Shinorine and Mycosporine-glycine of different strains, and the yield in BL21-III (T7) (E.coli BL21/pRSFDuet 1-T7X 4-mysA ₃B₃C₃D₃) was evaluated with the peak areas of Shinorine and Mycosporine-glycine in BL21-III as a reference standard, and the results showed that the yields of Shinorine and Mycosporine-glycine in BL21-III (T7) were increased by 1.56 times and 1.22 times, respectively, as compared with BL 21-III. The results show that the engineering strain E.coli BL21/pRSFDuet 1-T7X 4-mysA ₃B₃C₃D₃ modified by the cascade T7 promoter can effectively enhance the biosynthesis efficiency of Shinorine and Mycosporine-glycine compared with E.coli BL21-III, and improves the yield.

Example 5: optimization of fermentation conditions

S7P (sedoheptulose-7-phosphate) is used as an initial substrate for the action of DDG synthase in the Shinorine biosynthesis pathway, is crucial to the whole synthesis process, and is a key intermediate product of the pentose phosphate pathway, so that the increase of carbon flux in the pentose phosphate pathway is expected to further improve the Shinorine production efficiency. Based on the theory, we want to promote the accumulation of S7P by adding xylose as a co-substrate in a culture medium, so that the engineering strain has the potential of efficiently producing Shinorine as a chassis cell, and the specific experimental steps are as follows:

(1) Optimization of IPTG optimal induction concentration: culturing engineering strain E.coli BL 21/pRSFDuet-T7X 4-mysA ₃B₃C₃D₃ in LB liquid medium until the concentration of cultured bacterial cells reaches about OD ₆₀₀ of 0.6; IPTG was added at final concentrations of 0.1mM, 0.2mM, 0.3mM and 0.5mM, respectively, and the culture was induced at 30℃for 24 hours. After the induction culture was completed, shinorine extraction and content detection were performed according to the method in example 3.

(2) Optimization of optimal induction temperature: culturing engineering strain E.coli BL 21/pRSFDuet-T7X 4-mysA ₃B₃C₃D₃ in LB liquid medium until the concentration of cultured bacterial cells reaches about OD ₆₀₀ of 0.6; IPTG was added at a final concentration of 0.2mM and the culture was induced at 16℃and 25℃and 30℃and 37℃for 24 hours, respectively. After the induction culture was completed, shinorine extraction and content detection were performed according to the method in example 3.

(3) Optimization of the optimal induction time: culturing engineering strain E.coli BL 21/pRSFDuet-T7X 4-mysA ₃B₃C₃D₃ in LB liquid medium until the concentration of cultured bacterial cells reaches about OD ₆₀₀ of 0.6; IPTG was added at a final concentration of 0.2mM and the culture was induced at 30℃for 20h, 24h, 30h, 36h, respectively. After the induction culture was completed, shinorine extraction and content detection were performed according to the method in example 3.

(4) Optimization of the optimal xylose content: culturing engineering strain E.coli BL 21/pRSFDuet-T7X 4-mysA ₃B₃C₃D₃ in LB liquid medium containing 0%, 0.75%, 1% and 2% xylose respectively, and culturing to obtain bacterial concentration of about OD ₆₀₀ of 0.6; IPTG was added at a final concentration of 0.2mM and the culture was induced at 30℃for 24 hours. After the induction culture was completed, shinorine extraction and content detection were performed according to the method in example 3.

The experimental results are shown in fig. 7, wherein graph a shows the optimized result of the optimal xylose content, graph B shows the optimized result of the optimal induction concentration of IPTG, graph C shows the optimized result of the optimal induction temperature, and graph D shows the optimized result of the optimal induction time. As can be seen from FIG. 7, by optimizing the fermentation conditions of the engineering strain E.coli BL21/pRSFDuet 1-T7X 4-mysA ₃B₃C₃D₃, it was found that the yields of the engineering strain E.coli BL21/pRSFDuet 1-T7X 4-mysA ₃B₃C₃D₃ by 1.47 and Mycosporine-glycine were increased by 1.47-fold and 1.30-fold, respectively, when 0.75% of xylose was added to the medium under the conditions of optimal induction IPTG concentration (0.2 mM), optimal induction temperature (30 ℃) and optimal induction time (24 hours), as compared with when xylose was not added.

Claims

1. A Shinorine high-yield engineered strain, characterized in that it is obtained by introducing an AB gene fragment and a CD gene fragment into Escherichia coli via a vector, wherein the nucleotide sequence of the AB gene fragment is shown in SEQ ID NO.4, and the nucleotide sequence of the CD gene fragment is shown in SEQ ID NO.5.

2. The Shinorine high-yield engineered strain according to claim 1, characterized in that the vector is a pETDuet1 plasmid vector.

3. The Shinorine high-yield engineered strain as described in claim 2 is characterized in that the introduction positions of the AB gene fragment and the CD gene fragment in the pETDuet1 plasmid vector are: the AB gene fragment is seamlessly cloned into the PstI restriction site of the pETDuet1 plasmid vector, and the CD gene fragment is introduced between the NdeI-XhoI restriction sites of the pETDuet1 plasmid vector.

4. The Shinorine high-yield engineered strain according to claim 1, wherein the Escherichia coli is E. coli BL21.

5. The Shinorine high-yield engineered strain according to claim 1, characterized in that the Shinorine high-yield engineered strain further contains a T7 promoter, and the nucleotide sequence of the T7 promoter is shown in SEQ ID NO.28.

6. The fermentation method of the Shinorine high-yield engineered strain according to claim 1 is characterized in that the bacterial liquid of the Shinorine high-yield engineered strain is inoculated into LB liquid culture medium for cultivation, and then IPTG with a final concentration of 0.1 to 0.5 mM is added, and the induction culture is carried out at a culture temperature of 25 to 30° C. for 20 to 36 hours to obtain a fermentation liquid.

7. The fermentation method according to claim 6, wherein the final concentration of IPTG is 0.2 mM.

8. The fermentation method according to claim 6, characterized in that the LB liquid culture medium contains xylose at a final concentration of 7.5 g/L to 20 g/L; preferably, the LB liquid culture medium contains xylose at a final concentration of 7.5 g/L.

9. Application of AB gene fragment and CD gene fragment in preparing Shinorine high-yield engineered strain, characterized in that the nucleotide sequence of the AB gene fragment is shown in SEQ ID NO.4, and the nucleotide sequence of the CD gene fragment is shown in SEQ ID NO.5.

10. Use of the Shinorine high-yield engineered strain according to claim 1 in producing Shinorine.