A Streptomyces recombinant engineering strain producing mycosporin-like metabolites and its construction method and application

Abstract

The invention discloses a Streptomyces recombinant engineering strain for producing mycosporin-like metabolites, wherein a gene expression cassette is integrated into the genome of the Streptomyces recombinant engineering strain, and the gene expression cassette is recombined into the chromosome of the Streptomyces chassis strain. attB The gene expression cassette is an operon controlled by a single promoter, and the operon includes a mycosporin-like metabolite synthesis pathway. MysA , MysB , MysC and MysD or mysD p334 The present invention also discloses a method for constructing the above-mentioned Streptomyces recombinant engineering strain and its application in preparing mycosporin-like metabolites; mycosporin-like metabolites are efficiently produced by heterologously expressing mycosporin-like metabolite synthesis gene elements in Streptomyces, and four mycosporin-like metabolites with high yield and strong ultraviolet absorption activity can be obtained by reconstructing the biosynthetic gene elements and recombinantly expressing them in Streptomyces lividans.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and metabolic engineering, and specifically relates to a Streptomyces recombinant engineering strain for producing mycosporin-like metabolites, and a construction method and application thereof. Background Art

[0002] Mycosporine-like amino acids (MAAs) are a class of small molecule compounds with photoprotective activity produced by marine plankton and red algae. FEMS Microbiol. Lett . 2007,269, 1-10.). They can effectively absorb ultraviolet (UV) radiation and protect the DNA of organisms from UV damage. MAAs are the strongest known water-soluble natural UV absorbers and have antioxidant and anti-inflammatory effects. This makes MAAs have important application potential in the development of medicine, cosmetics and as natural sunscreens.

[0003] At present, the crude materials of commercialized MAAs are derived from the extraction of large algae. For example, crude extracts of red algae are used in the development of sunscreen products. However, due to the very low natural content of MAAs in organisms, expensive raw materials, and low yield of extraction methods, the cost of crude extracts of MAAs is high, which seriously limits the development of applied products for MAAs. In addition, given that existing commercial sunscreen products mainly rely on chemically synthesized UV absorbers, such as inorganic protective agents such as TiO2 and ZnO, and organic synthetic protective agents such as oxybenzone, these substances not only have potential pollution risks to the environment, but also may have adverse effects on the human body if used for a long time ( Marine Drugs . 2017, 15).

[0004] Synthetic biology uses genetic engineering to transfer genes that control the biosynthesis of specific compounds into suitable hosts, and uses the host's metabolic system to produce target compounds. This method can not only increase the yield of target compounds, but also provide new ways to study their biosynthetic pathways.

[0005] The present invention intends to construct an engineered strain for efficiently producing MAAs metabolites by heterologously expressing MAAs biosynthetic gene elements in Streptomyces, thereby achieving efficient production of MAA metabolites with ultraviolet light protection activity such as mycosporine-like amino acids 4-deoxygadusol (4-DG), mycosporine-glycine (MG), shinorine and porphyra-334. Summary of the invention

[0006] The primary purpose of the present invention is to provide a recombinant engineered strain of Streptomyces for producing mycosporine-like metabolites.

[0007] Another object of the present invention is to provide a method for constructing the above-mentioned Streptomyces recombinant engineering strain that produces mycosporine-like metabolites.

[0008] Another object of the present application is to provide the use of the above-mentioned Streptomyces recombinant engineered strain for producing mycosporine-like metabolites in the preparation of mycosporine-like metabolites.

[0009] The first object of the present invention can be achieved by the following technical scheme: a recombinant engineering strain of Streptomyces that produces mycosporin-like metabolites, wherein a gene expression cassette is integrated into the genome of the recombinant engineering strain of Streptomyces, and the gene expression cassette is recombined into the chromosome of the chassis strain of Streptomyces. attB The gene expression cassette is an operon controlled by a single promoter, and the operon includes a mycosporin-like metabolite synthesis pathway. MysA , MysB , MysC and MysD or mysD p334 2 to 4 biosynthetic gene elements in the.

[0010] like Figure 1 As shown in Figure 2, mycosporin-like amino acid metabolites MAAs can be synthesized from sedoheptulose 7-phosphate (S7P), an intermediate in the pentose phosphate pathway, or 3-dehydroquinate synthase (DHQS), an intermediate in the shikimate pathway. -epi- 5 -epi- valiolone synthase (EEVS) (MysA) / desmethyl-4-deoxygadusol synthase (DDGS) (MysA), O -methyltransferase (OMT) (MysB) catalyzes the production of 4-deoxygadusol (4-DG) to form the parent core structure. All MAAs gene clusters encode an ATPgrasp-like enzyme (MysC) that introduces glycine onto 4-DG to form MG. Finally, the fourth nonribosomal peptide synthetase (NRPS) or D-Ala-D-Ala ligase (MysD) introduces the second amino acid to form disubstituted amino acid MAAs, such as shinorine, porphyra-334, etc. Therefore:

[0011] Optionally, the operon must contain two upstream biosynthetic gene elements mysA Genes and mysB Gene; when the operon has no downstream gene elements, it only containsmysA Genes and mysB When the gene is expressed, the metabolite of the Streptomyces recombinant engineering strain is 4-deoxygadusol, abbreviated as 4-DG ( Figure 1 ).

[0012] The present invention mysA The gene function is demethyl-4-deoxygadusol synthase or 2 -epi- 5 -epi- valiolone synthase, abbreviated as DDGS and EEVS, respectively, mysB Oxygen methyltransferase O -methyltransferase, abbreviated as O -MT.

[0013] Furthermore, the operon also includes downstream biosynthetic gene elements mysC , mysC and mysD or mysC and mysD p334 ; When the operon contains upstream genes mysA and mysB Under the premise of only containing downstream gene elements mysC When the metabolite of the Streptomyces recombinant engineering strain is mycosporine-glycine, abbreviated as MG; when the operon contains the upstream gene mysA and mysB Under the premise of containing downstream gene elements mysC and mysD or mysC and mysD p334 When the metabolite of the Streptomyces recombinant engineered strain is a mycosporin-like metabolite shinorine or porphyra-334 represented by formula (I):

[0014] In formula (I), when the mycosporin metabolite is shinorine, R = -CH2OH; when the mycosporin metabolite is porphyra-334, R = -CH(OH)CH3 ( Figure 1 ).

[0015] In the present invention mysC The function of the gene is ATP-grasp enzyme. mysD The function of the gene is NRPS-like enzyme or d-Ala-d-Ala-ligase.

[0016] Optionally, the Streptomyces chassis strain is Streptomyces lividans Streptomyces lividans TK24.

[0017] Alternatively, the promoter of the operon may be kasO p. tac p or tet p initiates transcription of the structural gene element, and the transcription is terminated by the terminator. More preferably, the promoter is kasO p. tac p or tet p.

[0018] The second object of the present invention can be achieved by the following technical scheme: The method for constructing the Streptomyces recombinant engineering strain producing mycosporin-like metabolites comprises the following steps: constructing a recombinant plasmid pSET- mysAB 、pSET- mysABC 、pSET- mysABCD or pSET- mysABCD p334 , the recombinant plasmid pSET- mysAB 、pSET- mysABC 、pSET- mysABCD or pSET- mysABCD p334 Integration into the chromosome of the Streptomyces chassis strain attB The site is used to realize the integrated expression of the gene expression cassette and the production of different target metabolites, which specifically includes the following steps:

[0019] (1) Donor bacteria: Construction of recombinant plasmid pSET- mysAB 、pSET- mysABC 、pSET- mysABCD or pSET- mysABCD p334 And transform competent Escherichia coli E. coli ET12567, transformants were picked as donor strains for conjugative transfer;

[0020] (2) Receptor bacteria: Select Streptomyces lividans Streptomyces lividans TK24 is the recipient bacterium for conjugative transfer, and the spores of the recipient bacterium are collected and prepared into a spore suspension for conjugative transfer;

[0021] (3) Conjugation transfer: Transform Streptomyces lividans Streptomyces lividans The spore suspension of TK24 and E. coli carrying the recombinant plasmid were conjugated and transferred, and the conjugants were picked;

[0022] (4) Conjugate genotype verification: The conjugate is genotyped and the recombinant engineered strain of Streptomyces that produces mycosporine-like metabolites is obtained.

[0023] In the above-mentioned method for constructing a recombinant engineered strain of Streptomyces that produces mycosporine-like metabolites:

[0024] Optionally, the recombinant plasmid pSET- mysAB 、pSET- mysABC 、pSET- mysABCD or pSET- mysABCD p334 The recombinant plasmid for constructing mycosporin-like metabolites using the integrative vector pSET152 specifically comprises:

[0025] S1) Plasmid backbone: Select the integrative expression vector pSET152, linearize the plasmid by double restriction digestion, and recover the linearized plasmid backbone fragment;

[0026] S2) Insert fragments: Three DNA insert fragments were synthesized separately. The first one contained only mysA and mysB DNA insert mysAB , the second type includes mysA , mysB and mysC DNA inserts of three genetic elements mysABC , the third type includes mysA , mysB , mysC and mysD or mysD p334 DNA inserts of all four genetic elements mysABCD or mysABCD p334 , each gene element is connected by RBS sequence;

[0027] S3) Ligation: Linearized pSET152 vector was mixed with the three insert fragments in step S2) respectively and ligated overnight with ligase;

[0028] S4) Transformation: The ligation reaction mixture is transformed into competent E. coli and transformants are screened;

[0029] S5) Plasmid verification: Extract the plasmids of the transformants and verify the sequence correctness to obtain the recombinant expression plasmid pSET- mysAB , pSET- mysABC and pSET- mysABCD or pSET- mysABCD p334 .

[0030] The inserted fragment in step S2) of the present invention mysABThe sequence is shown in SEQ ID NO.1, and the inserted fragment mysABC The sequence is shown in SEQ ID NO.2, and the inserted fragment mysABCD The sequence is shown in SEQ ID NO.3, the inserted fragment mysABCD p334 The sequence is shown in SEQ ID NO.4.

[0031] The pSET- mysAB Contains and expresses two upstream gene elements mysA and mysB, The pSET- mysABC Contains and expresses two upstream gene elements mysA and mysB and a downstream gene element mysC , the pSET- mysABCD or pSET- mysABCD p334 Contains and expresses two upstream gene elements mysA and mysB and two downstream gene elements mysC and mysD or mysC and mysD p334 .

[0032] Preferably, the Streptomyces engineered strains in step (4) are named Streptomyces lividans according to the differences of the four recombinant plasmids in step (1). S. lividans TKmysAB, S. lividans TKmysABC, S. lividans TKmysABCD and S. lividans TKmysABCD p334 The four engineered strains are capable of producing 4-deoxygadusol, mycosporine-glycine, and mycosporine-like metabolites shinorine or porphyra-334 represented by the following formula (I):

[0033] In formula (I), when the mycosporin-like metabolite is shinorine, R = -CH2OH; when the mycosporin-like metabolite is porphyra-334, R = -CH(OH)CH3.

[0034] Preferably, as a specific embodiment of the present invention, the method for constructing an engineered strain for efficiently producing mycosporin-like metabolites using the above-mentioned recombinant plasmid and Streptomyces chassis strain can be achieved by the following steps:

[0035] S1. Donor bacteria: The above recombinant plasmid pSET- mysAB 、pSET- mysABC 、pSET- mysABCD or pSET- mysABCD p334 Transform competent E. coli E. coli ET12567, transformants were picked as donor strains for conjugative transfer;

[0036] S2. Receptor bacteria: Select Streptomyces and preferably Streptomyces lividans S. lividans TK24 is the recipient bacterium for conjugation transfer; spores of the recipient bacterium are collected and prepared into a spore suspension for conjugation transfer;

[0037] S3. Conjugative transfer: Streptomyces lividans S. lividans The spore suspension of TK24 and the Escherichia coli donor bacteria carrying the recombinant plasmid were mixed at room temperature, centrifuged to remove the supernatant, and then coated on MS medium plates; after culturing at 30 °C for 16 h, the plates were covered with a drug solution containing nalidixic acid with a final concentration of 500 μg / mL and apramycin with a final concentration of 1250 μg / mL; after continuing to culture for 2-5 days, the conjugates were picked; the conjugates were subcultured three times on a resistant MS medium containing apramycin (final concentration of 50 μg / mL) to remove Escherichia coli to obtain purified conjugates;

[0038] S4. Confirmation of zygote genotype: Colony PCR was used to verify whether the expression cassette was correctly integrated into the chromosome of Streptomyces. attB The obtained conjugate with the correct genotype is an engineered strain of Streptomyces that can efficiently produce mycosporin-like metabolites.

[0039] Preferably, the MS solid culture medium comprises: 20.0 g / L soybean powder, 20.0 g / L mannitol, 3.0 g / L CaCO3, and 20.0 g / L agar.

[0040] The last object of the present invention can be achieved by the following technical solution: use of the above-mentioned Streptomyces recombinant engineering strain in the preparation of mycosporin-like metabolites.

[0041] Preferably, the application comprises the following steps: inoculating a recombinant engineered strain of Streptomyces into a fermentation medium for cultivation, and obtaining mycosporine-like metabolites after enrichment and purification, wherein the mycosporine-like metabolites include 4-deoxygadusol, mycosporine-glycine, and mycosporine-like metabolites shinorine or porphyra-334 represented by the following formula (I):

[0042] In formula (I), when the mycosporin-like metabolite is shinorine, R = -CH2OH; when the mycosporin-like metabolite is porphyra-334, R = -CH(OH)CH3.

[0043] Furthermore, the method for fermenting and preparing mycosporin-like metabolites using the above-mentioned Streptomyces recombinant engineered strain comprises the following steps:

[0044] (a) Seed solution: After the recombinant engineered strain of Streptomyces is activated in MS solid medium, it is inoculated into TSB liquid medium for cultivation to obtain seed solution;

[0045] (b) Expanding the fermentation: inoculating the seed solution obtained in step (a) into a liquid synthetic culture medium, culturing in a shake flask or a fermenter, stopping the fermentation, centrifuging, collecting the bacterial cells and the fermentation supernatant, washing the bacterial cells and setting aside;

[0046] (c) Product enrichment: The bacterial cells obtained in step (b) are ultrasonically extracted with methanol, centrifuged, and the supernatant is collected. After concentration, a crude intracellular extract is obtained. The crude intracellular extract is enriched by passing it through a dextran gel chromatography column, firstly eluted with 5 column volumes of pure water to remove impurities, then eluted with 5 column volumes of pure water and the eluate is collected, and concentrated under reduced pressure to obtain a crude extract of the target compound;

[0047] (d) Product purification: Use an ODS column and then isocratically elute with methanol (MeOH) to collect the target chromatographic peaks. The product recovered under reduced pressure is the purified metabolite.

[0048] As a specific embodiment of the present invention, a method for fermenting and preparing mycosporin-like metabolites using the above-mentioned Streptomyces recombinant engineering strain is as follows:

[0049] 1) Seed solution: After activating the above Streptomyces recombinant engineered strain in MS solid medium, inoculate it into TSB liquid medium containing apramycin and culture it at 28 °C for 2 days (48 h);

[0050] 2) Expand the fermentation: Inoculate the seed liquid into the liquid synthetic medium at an inoculation ratio of 5-10% (v / v). Under the condition of 28 °C, stop the fermentation after 5 days of shaking flask culture or fermentation tank culture. Collect the bacteria and fermentation supernatant by centrifugation at 4000 rpm for 20 min. Wash the bacteria with pure water for 2-3 times for later use.

[0051] 3) Product enrichment: The cells were ultrasonically extracted with 50% methanol for 20 min, and the supernatant was collected after centrifugation at 4000 rpm for 10 min. The intracellular crude extract was obtained after concentration. The fermentation supernatant was enriched by a dextran gel ODS column. First, impurities were removed by elution with 5 column volumes (5 CV) of pure water, and then eluted with 5 CV of pure water and the eluate was collected. The crude extract of the target compound was obtained by concentration under reduced pressure.

[0052] 4) Product purification: Use an ODS column that can be applied to a pure water system, isocratically elute with 2% MeOH (v / v), collect the target chromatographic peak, and recover the product under reduced pressure to obtain the purified metabolite.

[0053] Preferably, the liquid culture medium TSB is composed of: 17.0 g / L trypticase, 3.0 g / L soybean papain hydrolysate, 2.5 g / L glucose, 5.0 g / L sodium chloride, 2.5 g / L potassium hydrogen phosphate, and a pH of 7.3.

[0054] Preferably, the composition of the liquid synthetic culture medium is: 60 g / L glucose, 2 g / L ammonium sulfate, 0.1 g / L MgSO4·7H2O, 0.5 g / L dipotassium hydrogen phosphate, 2 g / L sodium chloride, 0.05 g / L FeSO4·7H2O, 0.05 g / L ZnSO4·7H2O, 0.05 g / L MnCl4·4H2O, 5 g / L calcium carbonate, 2 g / L yeast extract, and pH is 7.0.

[0055] The present invention has the following advantages:

[0056] (1) The present invention efficiently produces mycosporin-like metabolites by heterologously expressing mycosporin-like metabolite synthesis gene elements in Streptomyces, and by reconstructing the biosynthetic gene elements and inducing the production of mycosporin-like metabolites in Streptomyces lividans. Streptomyces lividans The engineered strain obtained by recombinant expression in TK24 can efficiently produce MAAs compounds with strong UV absorption activity such as 4-deoxygadusol (4-DG), mycosporine-glycine (MG), shinorine and porphyra-334 for use in product development;

[0057] (2) Among the four metabolites prepared in the present invention, the production titer of 4-DG was 234.3 mg / L, the production titer of MG was 634.4 mg / L, the production titer of shinorine was 342.6 mg / L, and the production titer of porphyra-334 was 394.3 mg / L. All the yields were much higher than the yield of about 150 mg / L in the currently reported Streptomyces system. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0059] Figure 1 It is a schematic diagram of the biosynthetic pathway of some mycosporin-like metabolites in the content of the invention;

[0060] Figure 2 The bacterial solution PCR of E. coli DH5ɑ transformant in Example 1 verified pSET- mysABCD Agarose gel electrophoresis analysis results of plasmids; Lanes 1 to 8 are the PCR amplification results of 8 transformants, where lane Marker: Trans2K ® Plus; Lanes 1-8: 8 E. coli DH5ɑ transformants, among which lanes 3, 4, 7, and 8 with amplified bands at 5000 bp are correct transformants;

[0061] Figure 3 is pSET- mysABCD Map of recombinant expression plasmids, where MSEDJ_27540, MSEDJ_27550, MSEDJ_27560 and MSEDJ_27570 represent MysA, MysB, MysC and MysD, respectively, the same below;

[0062] Figure 4 The recombinant expression plasmid pSET- mysABCD Agarose gel electrophoresis analysis results; Lane Marker: Trans2K ® Plus; Lanes 1-8: 8 E. coli ET12567 transformants, among which Lane 1 with an amplified band at 5000 bp is the correct transformant;

[0063] Figure 5 The Streptomyces engineering strain in Example 1 Streptomyces lividans TK mysABCD PCR verification results of genotype. Upstream interface, lane marker: Trans2K ® Plus; Lanes 1-16: conjugates, all conjugates have a band at 1000 bp, which are correct conjugates, downstream interface, lane marker: Trans2K ® Plus; Lanes 1-16: conjugates, all conjugates have a band at 1000 bp, and are correct conjugates;

[0064] Figure 6 The Streptomyces engineering strain in Example 1 Streptomyces lividansHPLC analysis of the extracellular metabolic phenotype of TKmysABCD; and chassis strains Streptomyces lividans Compared with TK24, the engineered strain of Streptomyces produced the chromatographic peak of the target metabolite shinorine extracellularly;

[0065] Figure 7 The Streptomyces engineering strain in Example 1 Streptomyces lividans HPLC analysis of the intracellular metabolic phenotype of TKmysABCD; and chassis strains Streptomyces lividans Compared with TK24, the engineered strain of Streptomyces produced the chromatographic peak of the target metabolite shinorine intracellularly;

[0066] Figure 8 The Streptomyces engineering strain in Example 1 Streptomyces lividans UV spectrum of shinorine, a metabolite of TKmysABCD;

[0067] Figure 9 The Streptomyces engineering strain in Example 1 Streptomyces lividans High-resolution mass spectrum of shinorine, a metabolite of TKmysABCD;

[0068] Figure 10 is the standard curve of shinorine, a metabolite of the Streptomyces engineered strain in Example 1;

[0069] Figure 11 This is the agarose gel electrophoresis analysis result of the bacterial solution PCR of Escherichia coli DH5ɑ transformant in Example 2 to verify the pSET-mysABC plasmid; Lane Marker: Trans2K ® Plus; Lanes 1-16: 16 E. coli DH5α transformants, lane 14 with a correct genotype band at 3700 bp is the correct transformant;

[0070] Figure 12 is pSET- mysABC Recombinant expression plasmid map;

[0071] Figure 13 The recombinant expression plasmid pSET- mysABC Agarose gel electrophoresis analysis results; Lane Marker: Trans2K ® Plus; Lanes 1-16: 16 E. coli ET12567 transformants, the 3700 bp band is the correct transformant band, and the results show that lanes 1-8, 10-12, and 14-16 are correct transformants;

[0072] Figure 14The Streptomyces engineering strain in Example 2 Streptomyces lividans PCR verification results of TKmysABC genotype;

[0073] Figure 15 The Streptomyces engineering strain in Example 2 Streptomyces lividans HPLC analysis of the extracellular metabolic phenotype of TKmysABC; and chassis strains Streptomyces lividans Compared with TK24, the engineered strain of Streptomyces produced a chromatographic peak of the target metabolite mycosporine glycine extracellularly;

[0074] Figure 16 The Streptomyces engineering strain in Example 2 Streptomyces lividans HPLC analysis of the intracellular metabolic phenotype of TKmysABC; and chassis strains Streptomyces lividans Compared with TK24, the engineered strain of Streptomyces produced a chromatographic peak of the target metabolite mycosporine glycine intracellularly;

[0075] Fig.17 The Streptomyces engineering strain in Example 2 Streptomyces lividans UV spectrum of mycosporine glycine, a metabolite of TKmysABC;

[0076] Fig.18 The Streptomyces engineering strain in Example 2 Streptomyces lividans High-resolution mass spectrum of mycosporine glycine, a metabolite of TKmysABC;

[0077] Fig.19 is the standard curve of mycosporine glycine, a metabolite of the Streptomyces engineered strain in Example 2;

[0078] Fig. 20 This is the agarose gel electrophoresis analysis result of the bacterial solution PCR of E. coli DH5ɑ transformant to verify the pSET-mysAB plasmid in Example 3; Lane Marker: Trans2K ® Plus; Lanes 1-16: 16 E. coli DH5ɑ transformants, the 2400 bp band is the band of the correct transformant, and the results show that lanes 8, 9, and 14 are the correct transformants;

[0079] Fig.21 is pSET- mysAB Recombinant expression plasmid map;

[0080] Fig. 22 The recombinant expression plasmid pSET- mysABAgarose gel electrophoresis analysis results; Lane Marker: Trans2K ® Plus; Lanes 1-16: 16 E. coli ET12567 transformants, the 2400 bp band is the band of the correct transformant, and the results show that lanes 1-16 are all correct transformants;

[0081] Fig.23 The Streptomyces engineering strain in Example 3 Streptomyces lividans PCR verification results of TKmysAB genotype; upstream interface, lane marker: Trans2K ® Plus; Lanes 1-16: 16 conjugates containing pSET_mysAB, the 1000 bp band indicates the correct genotype, downstream interface, lane marker: Trans2K ® Plus; Lanes 1-16: 16 conjugates containing pSET_mysAB, the 1000 bp band is the band of the correct genotype, and the results show that lanes 1-16 are conjugates with the correct genotype;

[0082] Fig.24 The Streptomyces engineering strain in Example 3 Streptomyces lividans HPLC analysis of the extracellular metabolic phenotype of TKmysAB; and chassis strains Streptomyces lividans Compared with TK24, the engineered strain of Streptomyces produced a chromatographic peak of the target metabolite 4-deoxygadusol extracellularly;

[0083] Fig.25 The Streptomyces engineering strain in Example 3 Streptomyces lividans UV spectrum of TKmysAB metabolite 4-deoxygadusol;

[0084] Fig.26 The Streptomyces engineering strain in Example 3 Streptomyces lividans High-resolution mass spectrum of 4-deoxygadusol, a metabolite of TKmysAB;

[0085] Fig. 27 is the standard curve of 4-deoxygadusol, a metabolite of the Streptomyces engineered strain in Example 3;

[0086] Fig.28 The bacterial solution PCR of E. coli DH5ɑ transformant in Example 4 verified pSET- mysABCD p334 Agarose gel electrophoresis analysis results of plasmid; Lane Marker: Trans2K ®Plus; Lanes 1-4: 4 E. coli DH5ɑ transformants. The 4800 bp band is the correct transformant band, and the results show that lanes 2-4 are correct transformants;

[0087] Fig.29 is pSET- mysABCD p334 Recombinant expression plasmid map, including Nostoc linckia NIES-25BAY_79928 for MysD p334 ;

[0088] Fig.30 The Streptomyces engineering strain in Example 4 Streptomyces lividans TKmysABCD p334 PCR verification results of genotype; Lane Marker: Trans2K® Plus; Lanes 1-12: 12 E. coli ET12567 transformants, the 4800 bp band is the band of the correct transformant, and the results show that lanes 8 and 11 are the correct transformants;

[0089] Fig.31 The recombinant expression plasmid pSET- mysABCD p334 Agarose gel electrophoresis analysis results; at the upstream interface, lane marker: Trans2K ® Plus; Lanes 1-8: 8 conjugates containing pSET_mysABCDP334, the 1000 bp band indicates the correct genotype, at the downstream interface, lane marker: Trans2K ® Plus; Lanes 1-8: 8 conjugates containing pSET_mysABCDP334, the 1000 bp band is the band of the correct genotype, and the results show that lanes 1-8 are all conjugates with the correct genotype;

[0090] Fig.32 The Streptomyces engineering strain in Example 4 Streptomyces lividans TKmysABCD p334 HPLC analysis of the metabolic phenotype of extracellular and chassis strains Streptomyces lividans Compared with TK24, the engineered strain of Streptomyces produced the chromatographic peak of the target metabolite porphyra-334 extracellularly;

[0091] Fig.33 The Streptomyces engineering strain in Example 4 Streptomyces lividans TKmysABCD p334 UV spectrum of the metabolite porphyra-334;

[0092] Fig.34 The Streptomyces engineering strain in Example 4 Streptomyces lividans TKmysABCD p334 High-resolution mass spectrum of the metabolite porphyra-334;

[0093] Fig.35 This is the standard curve of the metabolite porphyra-334 of the Streptomyces engineered strain in Example 4. DETAILED DESCRIPTION

[0094] The reagents and materials used in the following examples, unless otherwise specified, are from commercial sources. Unless otherwise specified, the experimental instruments used are all conventional laboratory instruments.

[0095] In the following examples: upstream gene element sA The nucleic acid sequence is available in GenBank: BBY28658; mysB The nucleic acid sequence of sC The nucleic acid sequence is available in GenBank: BBY28660; sD The nucleic acid sequence is available in GenBank: BBY28661; sD P334 The nucleic acid sequence of is shown in GenBank: BAY79928.

[0096] The promoter is tacp The nucleotide sequence of the promoter is shown in GenBank: FJ389173: 2926~2884; the promoter is tetp The nucleotide sequence of the promoter is shown in GenBank: partial fragment of MZ476912: 1226~1296; the terminator is lambda t For the nucleic acid sequence of the 0 terminator, see GenBank: partial fragment of JX560387: 2163~2197); RBS For the nucleotide sequence of the sequence, see GenBank: MN306530 (partial fragment: 11376~11395).

[0097] The promoter is kasOp The nucleotide sequence of the promoter is shown in SEQ ID NO.11. The promoter is a modified promoter with a much higher promoter efficiency than the wild type. kasO , there is no consistent sequence in the public database; the high yield of the present invention is strongly correlated with it.

[0098] Example 1 A method for constructing a recombinant engineered strain of Streptomyces for producing shinorine

[0099] 1. Construction of recombinant plasmid pSET- mysABCD

[0100] 1.1 Artificial sequence synthesis: MAA recombinant plasmid contains a MAA biosynthetic operon, which is driven by a promoter kas Op and a lambdat 0 terminator control, inserted between the two sA , mysB、mysC、 sD All four genes (abbreviated mysABCD ), there are 3 RBS sequences between the 4 gene coding sequences, and there are restriction sites XbaI / NdeI before and after the MAA biosynthesis operon. The MAA biosynthesis operon was first constructed on pUC57 to obtain pUC57- mysABCD Plasmid. The complete sequence of the operon is shown in SEQ ID NO.3.

[0101] 1.2 Operon cloning:

[0102] Enzyme digestion: Select the integrative vector pSET152 as the plasmid backbone, and select appropriate restriction sites according to the vector sequence and the target gene sequence. In this example, XbaI and NdeI restriction endonucleases are selected to digest pSET152 and pUC57- mysABCD Double enzyme digestion was performed, and the enzyme digestion reaction system (50 μL) was as follows: 1 μL restriction endonuclease, 1 μg plasmid / vector DNA, 5 μL 10×NEBuffer, and ultrapure H2O was added to 50 μL. After gently pipetting and mixing, the reaction was incubated at 37°C for 3 h; the reaction was heat-inactivated at 65°C for 20 min, and cooled on ice.

[0103] Fragment recovery: The reaction mixture was separated by gel electrophoresis using 1.0% agarose gel, and the linearized pSET152 vector and mysABCD Insert fragment.

[0104] Ligation: Linearized pSET152 vector and mysABCDThe insert fragments were mixed at a molar ratio of 1:3 and ligated with T4 ligase. The recommended amount of vector added (ng): 0.02 × fragment length bp; the recommended amount of insert fragment added (ng): 0.04 × insert fragment length bp. The ligation system is as follows (total reaction system 20 mL): 1 μL of T4 ligase, 4 μL of linearized vector (105 ng), 1 μL of target fragment (258 ng), 2 μL of 10×T4 DNA Ligase Reaction Buffer (1×), and 20 μL of H2O. After gently pipetting and mixing, react in a water bath at 16 °C overnight for 12 h and cool on ice.

[0105] Transformation: Take 10 μL of the recombinant product and add it to 100 μL of DH5ɑ cloning competent medium, flick the tube wall to mix, let it stand on ice for 10 min, and heat shock at 42°C for 90 s. Immediately cool on ice for 2-3 min; add 900 μL of LB medium to the transformation product in a clean bench, culture at 37 °C and 200 rpm for 1 h, and then concentrate by centrifugation to 100 μL. Take 100 μL of bacterial solution and evenly spread it on a plate of LB medium supplemented with apramycin (final antibiotic concentration is 50 mg / mL), and culture the plate upside down at 37°C for 12-16 h until transformants appear.

[0106] Verification: Transformants were picked from the plate and transferred to 1 mL of LB liquid medium containing 50 mg / mL apramycin. The culture was shaken at 37 °C and 200 rpm for 3-6 h. The bacterial solution was used as a template and 0.5 μL of the primer pair pSET-ABCDyz-F / R was used to amplify the target fragment. The total PCR system (10 μL) was: 1 μL of bacterial solution, 5 μL of 2× PrimeSTAR MAX DNA Polymerase, and 10 μL of H2O. The PCR program was: 98 °C pre-denaturation for 3 min; then 35 cycles, each cycle including 98 °C for 10 s, 60 °C for 5 s, 72 °C for 25 s, and finally 72 °C for 5 min. The electrophoretic diagram of bacterial solution PCR detection of 8 E. coli DH5α transformants is shown in the figure below. Figure 2 As shown in the figure, among the transformants that amplified the 5000 bp band, 3, 4, 7, and 8 were correct transformants. The E. coli DH5α strain containing the correct plasmid was extracted to obtain the recombinant plasmid pSET- mysAB CD, such as Figure 3 shown.

[0107] The primer sequences are:

[0108] pSET-ABCDyz-F: 5′-GTAAAACGACGGCCAGTGCCAAGCT-3′ (SEQ ID NO.5);

[0109] pSET-ABCDyz-R: 5′-CTTTATGCTTCCGGCTCGTATGTTGTGTGG 3′ (SEQ ID NO. 6).

[0110] 2. Construction of Streptomyces engineered strains producing shinorine

[0111] 2.1 Transformation of donor bacteria: Transform the recombinant plasmid pSET- mysABCD The cells were transformed into competent E. coli ET12567 to obtain donor E. coli ET12567. The transformants were verified by bacterial liquid PCR. The specific operation steps were the same as those in 1.2. The results of agarose gel electrophoresis analysis were as follows: Figure 4 As shown in Figure 1, the 5000 bp band amplified is the correct transformant in lane 1. The verified monoclonal bacteria were resuspended in 1 mL LB medium for later use.

[0112] 2.2 Pretreatment of donor bacteria: Take the bacterial solution in 2.1 and inoculate it into 5 mL of LB medium containing apramycin (final antibiotic concentration is 50 mg / mL) at an inoculation ratio of 5-10% (v / v). Place the activated tube after inoculation in a 37°C shaker at 180 rpm for overnight culture. Inoculate the overnight activated bacterial solution into 50 mL of LB medium containing apramycin (final antibiotic concentration is 50 mg / mL) at an inoculation volume of 4%. Incubate in a 37°C, 180 rpm shaker until the OD value is 0.5-0.8. Pour the cultured bacterial solution into a 50 mL centrifuge tube in a clean bench and centrifuge at 4°C, 3500 rpm for 5 min. Discard the supernatant, add an equal volume of LB to resuspend the bacteria, centrifuge at 4°C, 3500 rpm for 5 min, and repeat this step once. Finally, resuspend the bacteria in 1 mL LB for later use.

[0113] 2.3 Recipient bacteria pretreatment

[0114] Spread the bacterial liquid of Streptomyces lividans TK24 on MS plates and culture at 28 °C for about 7 days. Collect spores, suspend them in 20 mL ultrapure water, centrifuge at 3500 rpm for 10 min, and discard the supernatant. Resuspend the spores in 10 mL of 2×YTbroth and heat shock at 50°C for 10 min. After the heat-shocked spore liquid is naturally cooled to room temperature, add 10 mL of 2×YT broth to the system, and finally place it in a 37°C shaker at 180 rpm for germination for 3 h. After germination, centrifuge the spores at 3500 rpm for 5 min, discard the supernatant, and resuspend them in LB for later use.

[0115] 2.4 Joint transfer

[0116] Take 100 μL of spore solution in 2.3 and 100 μL of E. coli solution in 2.2, mix them evenly, and centrifuge them at room temperature at 3500 rpm for 5 min. Discard part of the supernatant and keep 100 μL of the resuspended mixed bacteria. Take 10 μL from the 100 μL resuspended mixed bacteria into 90 μL ultrapure water and dilute them in 3 gradients (10 -1 / 10 -2 / 10 -3 Number level), 10 -0 / 10 -1 / 10 -2 / 10 -3 Four orders of magnitude of mixed bacteria were spread on MS plates containing 10 mM MgCl2. 90 μL of spore liquid was taken from each plate and spread on MS plates containing a final concentration of 10 mM MgCl2. 2+ , as negative and positive controls, respectively. All coated plates were placed in a 30°C incubator for incubation, and the experimental group and negative control group were covered with drugs after 16 h of incubation. 1 mL of the above antibiotic mixture was added to each plate, so that the final concentrations of nalidixic acid and apramycin were 500 mg / mL and 1250 mg / mL, respectively. The drug solution was evenly spread over the entire plate by rolling the plate or with the help of a gun tip. The plate was blown dry in the clean bench and returned to the incubator for continued incubation for about 5 days to observe the conjugants. Single conjugant colonies were picked from the plate and placed in 30 μL 10mM NaOH solution. The genome was extracted by repeated freezing and thawing for subsequent genotype PCR verification; at the same time, the conjugants were cultured on MS medium containing 50 mg / mL apramycin and purified by continuous subculture.

[0117] 2.5 PCR verification of the zygote genotype: Using the zygote as a template, primer pairs SL-1-F / R and SL-3-F / R were used to amplify the operon in the Streptomyces chromosome DNA. atBThe interface upstream and downstream of the integration site. The 10 μL PCR reaction system contains: 1 μL bacterial solution, 0.5 μL of each primer, 5 μL of 2× PrimeSTAR MAX DNA Polymerase, and 10 μL of H2O; the PCR program is: 98 °C for 3 min, then 35 cycles, each cycle includes 98 °C for 10 s, 60 °C for 5 s, 72 °C for 25 s, and finally 72 °C for 5 min; PCR detection bands are as follows Figure 5 As shown, the 1000 bp band amplified indicates that the correct recombinant strain was obtained. Lanes 1-16 are the correct recombinant Streptomyces engineering strains, named Streptomyces lividans Streptomyces lividans TKmysABCD.

[0118] The primer sequences are:

[0119] SL-1-F: 5′-GGCTTCGTGGTTCCAGGTGG-3′ (SEQ ID NO.7);

[0120] SL-1-R: 5′-CGGTGAGCCAGAGTTTCAGCAG-3′ (SEQ ID NO. 8);

[0121] SL-3-F: 5′-GGTCGCGCTGAATGCGGTAAC-3′ (SEQ ID NO.9);

[0122] SL-3-R: 5′-CCGTTGGAGTCCTGGTGGTCG-3′ (SEQ ID NO. 10).

[0123] 2.6 Product detection and separation: The recombinant engineered Streptomyces with successfully verified genotypes were inoculated into 5 mL of TSB liquid culture medium containing 50 mg / mL apramycin and cultured at 28 °C, 220 rpm for two days. The seed liquid was inoculated into the fermentation medium at a 10% inoculation rate and fermented at 28 °C, 220 rpm for 5 days. After the fermentation was completed, the bacteria and the fermentation supernatant were collected by centrifugation at 4000 rpm for 20 min, and the bacteria were ultrasonicated with 50% methanol for 20 min. After the ultrasonication, the supernatant was centrifuged at 4000 rpm for 20 min to obtain the intracellular extract. The fermentation supernatant and intracellular extract were analyzed by HPLC and HPLC-MS, respectively. The analysis results are as follows Figures 6-8 As shown, it can be seen that the target product shinorine was successfully produced both inside and outside the cell.

[0124] The HPLC-MS analysis method is as follows:

[0125] Analytical instruments: Agilent Technologies 1260 Infinity, 6230 TOF LC / MS.

[0126] Chromatographic column: analytical column YMC-Pack-ODS-A (5 μm, 4.6×250 mm);

[0127] Mobile phase: pure water containing 0.2% formic acid, isocratic elution at a flow rate of 0.5 mL / min for 20 min;

[0128] Depend on Figure 6 and Figure 7 The results showed that compared with the chassis strain Streptomyces lividans TK24, a new chromatographic peak appeared both inside and outside the recombinant strain. Figure 8 As shown in FIG. 1 , the chromatographic peak has a maximum absorption at 330 nm, showing the typical ultraviolet absorption of mycosporin-like amino acids in the UVA band. Fig. 9 As shown, according to its high-resolution mass spectrum, it is determined to be the target product shinorine. Through semi-preparative or preparative chromatography, the chromatographic peak is collected to obtain pure shinorine.

[0129] Fermentation titer determination: The standard curve was determined by analytical HPLC using pure shinorine. Accurately weigh 5.04 mg of shinorine standard and dilute to 5 mL volumetric flask with 0.2% (v / v) formic acid water to obtain a 1.01 mg / mL standard stock solution. Dilute with 0.2% formic acid water to prepare 0.01, 0.02, 0.04, 0.08, 0.1, 0.15, 0.18, and 0.2 mg / mL standard solutions. Take 200 μL of each concentration of standard and add it to a liquid phase injection bottle, and set up three parallels for each concentration. Using the above-mentioned HPLC determination method, determine the peak area of ​​shinorine at the corresponding concentration. Draw a standard curve with the concentration of the standard as the horizontal axis and the peak area of ​​shinorine as the vertical axis (such as Fig.10 shown).

[0130] Sample concentration determination: Take 1.5 mL of each of the three fermentation broth samples, centrifuge at 10,000 rpm, take the supernatant, dilute 50 times, filter through the membrane, and transfer 200 μL to the injection sample bottle. Inject three times and calculate the peak area. According to the standard curve, the fermentation titer of shinorine was 342.6 mg / L.

[0131] Example 2 Construction method and application of an engineered Streptomyces strain producing mycosporine glycine

[0132] 1. Construction of recombinant plasmid pSET- mysABC

[0133] 1.1 Artificial sequence synthesis: MAA recombinant plasmid contains a MAA biosynthetic operon, which is driven by a promoter kas Op and a lambda t0 terminator control. Insert between the two mysA、mysB and sC (Abbreviation mysABC ) three genes, and two RBS sequences are contained between the coding sequences of the three genes. The MAA biosynthetic operon has restriction sites XbaI / NdeI before and after. The MAA biosynthetic operon was first constructed on pUC57 to obtain pUC57- mysABC Plasmid. The complete sequence of the operon is shown in Seq ID No.2.

[0134] 1.2 Operon cloning:

[0135] Enzyme digestion: Select the integrative vector pSET152 as the plasmid backbone, and select appropriate restriction sites according to the vector sequence and the target gene sequence. In this example, XbaI and NdeI restriction endonucleases are selected to digest pSET152 and pUC57- mysABC Double enzyme digestion was performed, and the specific operation steps were the same as the enzyme digestion steps in 1.2 of Example 1.

[0136] Fragment recovery: The reaction mixture was separated by gel electrophoresis using 1.0% agarose gel, and the linearized pSET152 vector and mysABC Insert fragment.

[0137] Ligation: Linearized pSET152 vector and mysABC The insert fragments were mixed at a molar ratio of 1:3 and ligated using T4 ligase. The specific operation steps were the same as those in 1.2 of Example 1.

[0138] Transformation: The specific operation steps are the same as the transformation steps in 1.2 of Example 1.

[0139] Verification: The specific operation steps are the same as those in 1.2 of Example 1. The PCR detection bands are as follows Fig.11 As shown, a band of 3700 bp was amplified in lane 14, indicating that it was the correct recombinant plasmid. The recombinant plasmid pSET_ mysABC , its plasmid map is as follows Fig.12 The primer sequence is the same as the primer sequence in 1.2 of Example 1.

[0140] 2. Construction of an engineered Streptomyces strain producing mycosporine glycine

[0141] 2.1 Transformation of donor bacteria: Transform the recombinant plasmid pSET_ mysABC The cells were transformed into competent E. coli ET12567 to obtain donor E. coli ET12567. The transformants were verified by bacterial liquid PCR. The specific experimental operation was the same as the transformation step 1.2 of Example 1. The results of agarose gel electrophoresis analysis were as follows: Fig.13 As shown in the figure, the amplified 3700 bp band indicated that the correct transformant was obtained (lanes 1-8, 10-12, 14-16). The verified monoclonal bacteria were resuspended in 1 mL LB medium for later use.

[0142] 2.2 Pretreatment of donor bacteria: The specific experimental operation is the same as the pretreatment step 2.2 of Example 1.

[0143] 2.3 Pretreatment of recipient bacteria: The specific experimental operation is the same as the pretreatment step 2.3 of Example 1.

[0144] 2.4 Conjugation transfer: The specific experimental operation is the same as the pretreatment step 2.4 of Example 1.

[0145] 2.5 PCR verification of the zygote genotype: Using the zygote as a template, primers (SL-1-F / R and SL-3-F / R) were used to amplify the operon in the Streptomyces chromosome DNA. atB The specific experimental operation is the same as the verification step 2.2.5 of Example 1. The results of PCR genotype detection are as follows Fig.14 As shown, the 1000 bp band amplified indicates that the correct recombinant strain has been obtained. Lanes 1-16 are all correct recombinant Streptomyces engineering strains, named Streptomyces lividans The primer sequence is the same as the primer sequence in 2.5 of Example 1.

[0146] 2.6 Product detection and separation: The specific experimental operation was the same as the detection and separation step of 2.6 of Example 1. The fermentation supernatant and intracellular extract were analyzed by HPLC and HPLC-MS, respectively. The analysis results were as follows: Figures 15 to 18 The engineered strain successfully produced the target metabolite mycosporine glycine.

[0147] The HPLC-MS analysis method is as follows:

[0148] Analytical instruments: Agilent Technologies 1260 Infinity, 6230 TOF LC / MS.

[0149] Chromatographic column: liquid phase analysis column YMC-Pack-ODS-A (5 μm, 4.6×250 mm);

[0150] Mobile phase: methanol-pure water containing 0.2% formic acid, the ratio is 2:98 (v / v), isocratic elution at a flow rate of 0.5 mL / min for 30 min;

[0151] Depend on Fig.15 and Fig.16 The results showed that the recombinant strain produced a new chromatographic peak both inside and outside the cell. Fig.17 As shown in Figure 1, the chromatographic peak has the maximum absorption in the UVA band at 310 nm. Further high-resolution mass spectrometry data confirmed that it is mycosporine glycine. The high-resolution mass spectrum is shown in Figure 1. Fig.18 By semi-preparative or preparative chromatography, the chromatogram is collected to obtain pure mycosporine glycine.

[0152] Fermentation titer determination: The standard curve was determined by analytical HPLC using pure mycosporine glycine. Accurately weigh 5.12 mg of the mycosporine glycine standard and dilute it to a 5 mL volumetric flask with 0.2% (v / v) formic acid water to obtain a 1.02 mg / mL standard stock solution. Dilute with 0.2% formic acid water to prepare standard solutions of 0.01, 0.02, 0.04, 0.08, 0.1, 0.15, 0.18, and 0.2 mg / mL. Take 200 μL of each concentration of the standard and add it to a liquid injection bottle, and set up three parallels for each concentration. Using the above-mentioned HPLC determination method, determine the peak area of ​​mycosporine glycine at the corresponding concentration. Draw a standard curve with the concentration of the standard as the horizontal axis and the peak area of ​​mycosporine glycine as the vertical axis (such as Fig.19 shown).

[0153] Sample concentration determination: Take 1.5 mL of each of the three fermentation broth samples, centrifuge at 10,000 rpm, take the supernatant, dilute 50 times, filter through the membrane, and transfer 200 μL to the injection sample bottle. Inject three times and calculate the peak area. The fermentation titer of mycosporine glycine was 634.4 mg / L according to the standard curve.

[0154] Example 3. Construction method and application of an engineered Streptomyces strain for producing 4-deoxygadusol

[0155] 1. Construction of recombinant plasmid pSET- mysAB

[0156] Artificial sequence synthesis: MAA recombinant plasmid contains a MAA biosynthetic operon, which is driven by a promoter kas Op and a lambda t0 terminator control. Insert between the two mysA、mysB Two genes (abbreviated mysAB ), and an RBS sequence is included between the two gene coding sequences. The MAA biosynthetic operon has restriction sites XbaI / NdeI before and after. The MAA biosynthetic operon was first constructed on pUC57 to obtain pUC57- mysAB Plasmid. The complete sequence of the operon is shown in Seq ID No.1.

[0157] Operon cloning:

[0158] Enzyme digestion: Select the integration vector pSET152 as the plasmid backbone. Select appropriate restriction sites based on the vector sequence and target gene sequence. In this example, XbaI and NdeI restriction endonucleases were selected to digest pSET152 and pUC57- mysAB The specific operation steps are the same as the enzyme digestion step in 1.2 of Example 1.

[0159] Fragment recovery: The reaction mixture was separated by gel electrophoresis using 1.0% agarose gel, and the linearized pSET152 vector and mysAB Insert fragment.

[0160] Ligation: Linearized pSET152 vector and mysAB The insert fragments were mixed at a molar ratio of 1:3 and ligated using T4 ligase. The specific operation steps were the same as those in 1.2 of Example 1.

[0161] Transformation: The specific operation steps are the same as the transformation steps in 1.2 of Example 1.

[0162] Verification: The specific operation steps are the same as those in 1.2 of Example 1. The PCR detection bands are as follows Fig. 20 As shown in Figure 1, lanes 8, 9, and 14 that can amplify 2400 bp bands are the correct recombinant plasmids. The E. coli DH5α strain containing the correct plasmid was extracted to obtain the recombinant plasmid pSET- mysAB ,like Fig.21 shown.

[0163] The primer sequence is the same as the primer sequence in 1.2 of Example 1.

[0164] 2. Construction of an engineered Streptomyces strain producing 4-deoxygadusol

[0165] 2.1 Transformation of donor bacteria: Transform the recombinant plasmid pSET_ mysABC The cells were transformed into competent E. coli ET12567 to obtain donor E. coli ET12567. The transformants were verified by bacterial liquid PCR. The specific experimental operation was the same as the transformation step 1.1.2 of Example 1. The results of agarose gel electrophoresis analysis were as follows: Fig. 22 As shown, the 2400 bp bands were amplified in lanes 1 to 16, indicating that they were all correct transformants. The verified monoclonal bacteria were resuspended in 1 mL LB medium for later use.

[0166] 2.2 Pretreatment of donor bacteria: The specific experimental operation is the same as the pretreatment step of 2.2.2 of Example 1.

[0167] 2.3 Pretreatment of recipient bacteria: The specific experimental operation is the same as the pretreatment step 2.2.3 of Example 1.

[0168] 2.4 Conjugation transfer: The specific experimental operation is the same as the pretreatment step of 2.2.4 of Example 1.

[0169] 2.5 PCR verification of the zygote genotype: Using the zygote as a template, primers (SL-1-F / R and SL-3-F / R) were used to amplify the operon in the Streptomyces chromosome DNA. atB The interface upstream and downstream of the integration site. The specific experimental operation is the same as the verification step 2.2.5 of Example 1. PCR detection band Fig.23 As shown, the 1000 bp band amplified indicates that the correct recombinant strain has been obtained. Lanes 1-16 are all correct recombinant Streptomyces engineering strains, named Streptomyces lividans TKmysAB.

[0170] The primer sequence is the same as the primer sequence in 2.5 of Example 1.

[0171] 2.6 Product detection and separation: The specific experimental operation is the same as the detection and separation step of 2.2.6 of Example 1. The fermentation supernatant and intracellular extract were analyzed by HPLC and HPLC-MS, respectively. The analysis results are as follows: Figures 24 to 26 The engineered strain successfully produced the target metabolite 4-deoxygadusol.

[0172] The HPLC-MS analysis method is as follows:

[0173] Analytical instruments: Agilent Technologies 1260 Infinity, 6230 TOF LC / MS.

[0174] Chromatographic column: liquid phase analysis column YMC-Pack-ODS-A (5 μm, 4.6×250 mm);

[0175] Mobile phase: methanol-pure water containing 0.2% formic acid, the ratio is 2:98 (v / v), isocratic elution at a flow rate of 0.5 mL / min for 30 min;

[0176] Depend on Fig.24 The results showed that a new chromatographic peak appeared in the fermentation broth of the recombinant strain, while no signal was detected inside the cell. Fig.25 As shown, the chromatographic peak gives a maximum absorption at 268 nm, showing the typical ultraviolet absorption of mycosporin amino acids in the UVB band. Fig.26 As shown, according to further high-resolution mass spectrometry data, it is determined to be 4-deoxygadusol. Through semi-preparative or preparative chromatography, the chromatogram is collected to obtain pure 4-deoxygadusol.

[0177] Fermentation titer determination: The standard curve was determined by analytical HPLC using pure 4-deoxygadusol. Accurately weigh 4.97 mg of 4-deoxygadusol standard and dilute to a 5 mL volumetric flask with 0.2% (v / v) formic acid water to obtain a 1.00 mg / mL standard stock solution. Dilute with 0.2% formic acid water to prepare standard solutions of 0.01, 0.02, 0.04, 0.08, 0.1, 0.15, 0.18, and 0.2 mg / mL. Take 200 μL of each concentration of standard and add it to a liquid injection bottle, and set up three parallels for each concentration. Using the above-mentioned HPLC determination method, determine the peak area of ​​4-deoxygadusol at the corresponding concentration. Draw a standard curve with the concentration of the standard as the horizontal axis and the peak area of ​​4-deoxygadusol as the vertical axis (such as Fig. 27 shown).

[0178] Sample concentration determination: Take 1.5 mL of each of the three fermentation broth samples, centrifuge at 10,000 rpm, take the supernatant, dilute 50 times, filter through the membrane, and transfer 200 μL to the injection sample bottle. Inject three times and calculate the peak area. According to the standard curve, the fermentation titer of 4-deoxygadusol is 234.3 mg / L.

[0179] Example 4. Construction method and application of an engineered Streptomyces strain for producing porphyra-334

[0180] 1. Construction of recombinant plasmid pSET- mysABCD P334

[0181] Artificial sequence synthesis: MAA recombinant plasmid contains a MAA biosynthetic operon, which is driven by a promoter kas Op and a lambda t0 terminator control. Insert between the two mysA, mysB, mysC, sD p334 All four genes (abbreviated mysABCD P334 ), and three RBS sequences are contained between the four gene coding sequences. The MAA biosynthetic operon has restriction sites XbaI / NdeI before and after. The MAA biosynthetic operon was first constructed on pUC57 to obtain pUC57- mysABCD P334 Plasmid. Among them, sD p334 The sequence is different from that in Example 1 sD .Include mysABCD p334 The complete sequence of the operon is shown in SEQ ID NO.4.

[0182] Operon cloning:

[0183] Enzyme digestion: Select the integration vector pSET152 as the plasmid backbone. Select appropriate restriction sites based on the vector sequence and target gene sequence. In this example, XbaI and NdeI restriction endonucleases were selected to digest pSET152 and pUC57- mysABCD P334 The specific operation steps are the same as the enzyme digestion step in 1.1.2 of Example 1.

[0184] Fragment recovery: The reaction mixture was separated by gel electrophoresis using 1.0% agarose gel, and the linearized pSET152 vector and mysABCD P334 Insert fragment.

[0185] Ligation: Linearized pSET152 vector and mysABCD P334 The insert fragments were mixed at a molar ratio of 1:3 and ligated using T4 ligase. The specific operation steps were the same as those in 1.1.2 of Example 1.

[0186] Transformation: The specific operation steps are the same as the transformation steps in 1.1.2 of Example 1.

[0187] Verification: The specific operation steps are the same as those in 1.1.2 of Example 1. The PCR detection bands are as follows Fig.28As shown in the figure, the 4800bp band amplified indicated that the correct recombinant plasmid was obtained, and the results showed that lanes 2 to 4 were correct transformants. The E. coli DH5α strain containing the correct plasmid was extracted to obtain the recombinant plasmid pSET- mysABCD P334 . Plasmid map Fig.29 shown.

[0188] The primer sequence is the same as the primer sequence in 1.2 of Example 1.

[0189] 2. Construction of an engineered Streptomyces strain producing porphyra-334

[0190] 2.1 Transformation of donor bacteria: Transform the recombinant plasmid pSET- mysABCD P334 The cells were transformed into competent E. coli ET12567 to obtain donor E. coli ET12567. The transformants were verified by bacterial liquid PCR. The specific experimental operation was the same as the transformation step 1.1.2 of Example 1. The results of agarose gel electrophoresis analysis were as follows: Fig.30 As shown, the amplified 4800 bp band indicated that the correct transformant was obtained, and the results showed that lanes 8 and 11 were correct transformations. The verified monoclonal bacteria were resuspended in 1 mL LB medium for later use.

[0191] 2.2 Pretreatment of donor bacteria: The specific experimental operation is the same as the pretreatment step of 2.2.2 of Example 1.

[0192] 2.3 Pretreatment of recipient bacteria: The specific experimental operation is the same as the pretreatment step 2.2.3 of Example 1.

[0193] 2.4 Conjugation transfer: The specific experimental operation is the same as the pretreatment step of 2.2.4 of Example 1.

[0194] 2.5 PCR verification of the zygote genotype: Using the zygote as a template, primers (SL-1-F / R and SL-3-F / R) were used to amplify the operon in the Streptomyces chromosome DNA. atB The specific experimental operation is the same as the verification step 2.2.5 of Example 1. The PCR detection band is as follows Fig.31 As shown, the 1000 bp band amplified indicates that the correct recombinant strain has been obtained. Lanes 1-8 are all correct recombinant Streptomyces engineering strains, named Streptomyces lividans TKmysABCD P334 .

[0195] The primer sequence is the same as the primer sequence in 2.5 of Example 1.

[0196] 2.6 Product detection and separation: The specific experimental operation is the same as the detection and separation step of 2.2.6 of Example 1. The fermentation supernatant was analyzed by HPLC and HPLC-MS. The analysis results are shown in Figure 32-33 The engineered strain successfully produced the target metabolite porphyra-334.

[0197] The HPLC-MS analysis method is as follows:

[0198] Analytical instruments: Agilent Technologies 1260 Infinity, 6230 TOF LC / MS.

[0199] Chromatographic column: liquid phase analysis column YMC-Pack-ODS-A (5 μm, 4.6×250 mm);

[0200] Mobile phase: pure water containing 0.2% formic acid, isocratic elution at a flow rate of 0.5 mL / min for 20 min;

[0201] like Fig.32 As shown in Figure 2, a new chromatographic peak appeared in the fermentation broth of the recombinant strain. Fig.33 As shown in FIG. 1 , the chromatographic peak has a maximum absorption at 334 nm, showing typical ultraviolet absorption of mycosporin-like amino acids in the UVA band. Fig.34 As shown, it is further confirmed to be porphyra-334 by high-resolution mass spectrometry data. Pure porphyra-334 is obtained by semi-preparative or preparative chromatography and collection of the chromatogram.

[0202] Fermentation titer determination: The standard curve was determined by analytical HPLC using pure porphyra-334. Accurately weigh 5.05 mg of the porphyra-334 standard and dilute it to a 5 mL volumetric flask with 0.2% (v / v) formic acid water to obtain a 1.01 mg / mL standard stock solution. Dilute it with 0.2% formic acid water to prepare standard solutions of 0.01, 0.02, 0.04, 0.08, 0.1, 0.15, 0.18, and 0.2 mg / mL. Take 200 μL of each concentration of the standard and add it to a liquid phase injection bottle, and set up three parallels for each concentration. Using the above-mentioned HPLC determination method, determine the peak area of ​​porphyra-334 at the corresponding concentration. Draw a standard curve with the concentration of the standard as the horizontal axis and the peak area of ​​porphyra-334 as the vertical axis (such as Fig.35 shown).

[0203] Sample concentration determination: Take 1.5 mL of each of the three fermentation broth samples, centrifuge at 10,000 rpm, take the supernatant, dilute 50 times, filter through the membrane, and transfer 200 μL to the injection sample bottle. Inject three times and calculate the peak area. According to the standard curve, the fermentation titer of porphyra-334 was 394.3 mg / L.

[0204] The above embodiments are merely examples for demonstrating the technical solutions of the present invention and are not intended to be limitative descriptions of the invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A recombinant engineered strain of Streptomyces for producing mycosporin-like metabolites, characterized in that: A gene expression cassette is integrated into the genome of the Streptomyces recombinant engineering strain, and the gene expression cassette is recombined into the attB site of the chromosome in the Streptomyces chassis strain, and the gene expression cassette is an operon controlled by a single promoter; The operon includes MysA and MysB, or MysA, MysB and MysC, or MysA, MysB, MysC and MysD, or MysA, MysB, MysC and mysD in the mycosporin-like metabolite synthesis pathway. p334 biosynthetic gene elements; When the operon includes MysA and MysB biosynthetic gene elements in the mycosporin-like metabolite synthesis pathway, the nucleic acid sequence of the operon is shown in SEQ ID NO.1, and the metabolite of the corresponding Streptomyces recombinant engineered strain is 4-deoxygadusol; When the operon includes MysA, MysB and MysC biosynthetic gene elements in the mycosporine-like metabolite synthesis pathway, the nucleic acid sequence of the operon is shown in SEQ ID NO. 2, and the metabolite of the corresponding Streptomyces recombinant engineered strain is mycosporine-glycine; When the operon includes MysA, MysB, MysC and MysD biosynthetic gene elements in the mycosporin-like metabolite synthesis pathway, the nucleic acid sequence of the operon is shown in SEQ ID NO.3, and the metabolite of the corresponding Streptomyces recombinant engineered strain is shinorine; When the operon includes MysA, MysB, MysC and mysD in the mycosporin-like metabolite synthesis pathway p334 When the biosynthetic gene element is used, the nucleic acid sequence of the operon is shown in SEQ ID NO. 4, and the metabolite of the corresponding recombinant engineered strain of Streptomyces is porphyra-334; The Streptomyces chassis strain is Streptomyces lividans TK24; The promoter is kasOp, and the nucleic acid sequence of the kasOp promoter is shown in SEQ ID NO.11; The nucleic acid sequence of the mysA is referred to GenBank: BBY28658, the nucleic acid sequence of the mysB is referred to GenBank: BBY28659, the nucleic acid sequence of the mysC is referred to GenBank: BBY28660, the nucleic acid sequence of the mysD is referred to GenBank: BBY28661, and the nucleic acid sequence of the mysDP334 is referred to GenBank: BAY79928.

2. The method for constructing a recombinant engineered strain of Streptomyces for producing mycosporine-like metabolites according to claim 1, characterized in that: The following steps are involved: Construction of recombinant plasmid pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD p334 The recombinant plasmid pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD is transferred by conjugation. p334 Integrate into the attB site of the chromosome in the Streptomyces chassis strain to achieve integrated expression of the gene expression cassette and production of different target metabolites, specifically including the following steps: (1) Donor bacteria: Construction of recombinant plasmids pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD p334 Competent E. coli ET12567 were transformed respectively, and transformants were selected as donor strains for conjugative transfer; (2) Recipient bacteria: Streptomyces lividans TK24 is selected as the recipient bacteria for conjugation transfer, and the spores of the recipient bacteria are collected to prepare a spore suspension for conjugation transfer; (3) Conjugative transfer: Conjugate and transfer the spore suspension of Streptomyces lividans TK24 and the Escherichia coli carrying the recombinant plasmid, and pick out the conjugants; (4) Conjugate genotype verification: The conjugate is genotyped and the recombinant engineered strain of Streptomyces that produces mycosporin-like metabolites is obtained.

3. The method for constructing a recombinant engineered strain of Streptomyces for producing mycosporin-like metabolites according to claim 2, characterized in that: The recombinant plasmid pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD p334 The recombinant plasmid for constructing mycosporin-like metabolites using the integrative vector pSET152 specifically comprises: S1) Plasmid backbone: Select the integrative expression vector pSET152, linearize the plasmid by double restriction digestion, and recover the linearized plasmid backbone fragment; S2) Insert fragments: Three DNA insert fragments were synthesized, the first one was a DNA insert fragment mysAB containing only mysA and mysB, the second one was a DNA insert fragment mysABC containing three gene elements of mysA, mysB and mysC, and the third one was a DNA insert fragment containing mysA, mysB, mysC and mysD or mysD p334 DNA inserts of all four genetic elements mysABCD or mysABCD p334 , each gene element is connected by RBS sequence; S3) Ligation: The linearized pSET152 vector was mixed with the three insert fragments in step S2) respectively, and ligated overnight using ligase; S4) transformation: the ligation reaction mixture is transformed into competent Escherichia coli, and transformants are screened; S5) Plasmid verification: Extract the plasmids of the transformants respectively, and verify the sequence correctness to obtain the recombinant plasmids pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD p334 .

4. The method for constructing a recombinant engineered strain of Streptomyces for producing mycosporin-like metabolites according to claim 3, characterized in that: The sequence of the inserted fragment mysAB in step S2) is shown in SEQ ID NO.1, the sequence of the inserted fragment mysABC is shown in SEQ ID NO.2, the sequence of the inserted fragment mysABCD is shown in SEQ ID NO.3, and the sequence of the inserted fragment mysABCD is shown in SEQ ID NO.

4. p334 The sequence is shown in SEQ ID NO.4; in step S5), the pSET-mysAB contains and expresses two upstream gene elements mysA and mysB, the pSET-mysABC contains and expresses two upstream gene elements mysA and mysB and one downstream gene element mysC, the pSET-mysABCD or pSET-mysABCD p334 Contains and expresses two upstream gene elements mysA and mysB and two downstream gene elements mysC and mysD or mysC and mysD p334 .

5. The method for constructing a recombinant engineered strain of Streptomyces for producing mycosporin-like metabolites according to claim 2, characterized in that: The Streptomyces engineering strains in step (4) are named Streptomyces lividans TKmysAB, S.lividans TKmysABC, S.lividans TKmysABCD and S.lividans TKmysABCD, respectively, according to the differences of the four recombinant plasmids in step (1). p334 The four engineered strains were able to produce 4-deoxygadusol, mycosporine-glycine, shinorine or porphyra-334, respectively.

6. Use of the Streptomyces recombinant engineered strain of claim 1 or the Streptomyces recombinant engineered strain constructed by any one of claims 2 to 5 in the preparation of mycosporin-like metabolites, characterized in that: The method comprises the following steps: inoculating a recombinant engineering strain of Streptomyces into a fermentation medium for cultivation, and obtaining mycosporine-like metabolites after enrichment and purification. The mycosporine-like metabolites include 4-deoxygadusol, mycosporine-glycine, shinorine or porphyra-334.

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