CN119193457B

CN119193457B - A Streptomyces recombinant engineering strain producing mycosporin-like metabolites and its construction method and application

Info

Publication number: CN119193457B
Application number: CN202411738807.5A
Authority: CN
Inventors: 王楠; 冯舒婷
Original assignee: Hainan Research Institute Of Zhejiang University
Current assignee: Hainan Research Institute Of Zhejiang University
Priority date: 2024-11-29
Filing date: 2024-11-29
Publication date: 2025-03-18
Anticipated expiration: 2044-11-29
Also published as: CN119193457A

Abstract

The invention discloses a streptomycete recombinant engineering strain for producing a mycosporine-like metabolite, wherein a gene expression cassette is integrated in the genome of the streptomycete recombinant engineering strain, the gene expression cassette is recombined to attB sites of chromosomes in a streptomycete chassis strain, the gene expression cassette is an operon controlled by a single promoter, the operon comprises MysA, mysB, mysC and MysD or 2-4 biosynthesis gene elements in mysD _p334 in a mycosporine-like metabolite synthesis pathway, the invention also discloses a construction method of the streptomycete recombinant engineering strain and application of the streptomycete recombinant engineering strain in preparation of the mycosporine-like metabolite, the mycosporine-like metabolite is efficiently produced by heterologously expressing the mycosporine-like metabolite synthesis gene element in streptomycete, and four mycosporine-like metabolites with high yield and strong ultraviolet absorption activity can be obtained by reconstructing the biosynthesis gene element and recombining expression in the streptomycete-like.

Description

Streptomyces recombinant engineering strain for producing mycosporine-like metabolites and construction method and application thereof

Technical Field

The invention belongs to the technical fields of genetic engineering and metabolic engineering, and particularly relates to a streptomycete recombinant engineering strain for producing a mycosporine-like metabolite, and a construction method and application thereof.

Background

The class of bacteriocin amino acids (mycosporine-like amino acids, MAAs for short) is a class of small molecule compounds with photoprotective activity produced by marine plankton, red algae, etc. (FEMS Microbiol. Lett. 2007, 269, 1-10.). They are effective in absorbing Ultraviolet (UV) radiation and protecting 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 of great potential for use in medicine, cosmetics and as natural sunscreens.

Currently, crude MAAs are commercially available from extraction of macroalgae. Crude extracts such as red algae are used for the development of sun protection products and the like. However, because of the low natural content of MAAs in organisms, the crude extract of MAAs has high cost due to the high raw materials and low yield of the extraction method, and the like, the development of the applicable product of the MAAs is severely limited. In addition, in view of the fact that existing commercial sunscreen products mainly rely on chemically synthesized uv absorbers, such as inorganic protective agents like TiO ₂ and ZnO, and organic synthetic protective agents like oxybenzone, these substances not only have potential pollution risks to the environment, but also may have adverse effects on the human body after long-term use (Marine drugs 2017, 15).

Synthetic biology is the production of a target compound by transferring genes controlling biosynthesis of a specific compound into an appropriate host by genetic engineering means and using the host's metabolic system. The method not only can improve the yield of the target compound, but also can provide a new way for researching the biosynthesis way of the target compound.

The invention aims to construct an engineering strain for efficiently producing MAAs metabolites by a method for heterologously expressing MAAs biosynthesis gene elements in streptomycete, thereby realizing the efficient production of MAA metabolites with ultraviolet light protection activities on the mycosporine-like amino acids 4-deoxygadusol (4-DG), mycosporine-glycine (MG), shinorine, porphyra-334 and the like.

Disclosure of Invention

The primary aim of the invention is to provide a recombinant engineering strain of streptomycete for producing a mycosporine-like metabolite.

The invention also aims to provide a construction method of the streptomyces recombinant engineering strain for producing the mycosporine-like metabolites.

It is still another object of the present application to provide the use of the recombinant engineering strain of Streptomyces producing a retinoid metabolite as described above for the preparation of a retinoid metabolite.

The first object of the invention can be achieved by a recombinant engineering strain of Streptomyces producing a retinoid metabolite, wherein a gene expression cassette is integrated in the genome of the recombinant engineering strain of Streptomyces, the gene expression cassette is recombined to attB site of chromosome in Streptomyces chassis strain, the gene expression cassette is an operon controlled by a single promoter, and the operon comprises MysA, mysB, mysC and 2-4 biosynthesis gene elements in MysD or mysD _p334 in the retinoid metabolite synthesis pathway.

As shown in FIG. 1, the retinoid amino acid metabolite MAAs can be synthesized from the intermediate sedoheptulose-phosphopentose pathway, 7-phosphoate (S7P), or the intermediate 3-dehydroquinate synthase of shikimate pathway (DHQS). First, SH-7P is catalyzed by 2-epi-5-epi-valiolone synthase（EEVS）（MysA）/desmethyl-4-deoxygadusol synthase（DDGS）（MysA）、O-methyltransferase（OMT）（MysB） to form 4-deoxygadusol (4-DG) to form the parent core structure. All MAAs gene clusters encode a ATP grasp-like enzyme (MysC) that introduces glycine into 4-DG to form MG. Finally, a fourth non-ribosomal peptide synthetase (NRPS) or D-Ala-D-Ala ligase (MysD) is introduced to the second amino acid to form a di-substituted amino acid MAAs, e.g., shinorine, porphyra-334, etc. Thus:

Alternatively, the operon must contain two upstream biosynthetic gene elements mysA and mysB, and the metabolic product of the recombinant engineering strain of Streptomyces is 4-deoxygadusol, abbreviated 4-DG (FIG. 1), when the operon does not contain downstream gene elements, i.e., only mysA and mysB.

The mysA gene has the functions of demethyl-4-deoxygadusol synthase or 2-epi-5-epi-valiolone synthase, which are abbreviated as DDGS and EEVS respectively, and the mysB gene has the functions of oxymethyl transferase O-METHYLTRANSFERASE, which is abbreviated as O-MT.

Further, the operon further comprises downstream biosynthesis gene elements mysC, mysC and mysD or mysC and mysD _p334, when the operon only comprises downstream gene elements mysC on the premise of comprising upstream genes mysA and mysB, the metabolic product of the recombinant engineering strain of streptomycete is the cephalosporin glycine, mycosporine-glycine, abbreviated as MG, and when the operon simultaneously comprises downstream gene elements mysC and mysD or mysC and mysD _p334 on the premise of comprising upstream genes mysA and mysB, the metabolic product of the recombinant engineering strain of streptomycete is the cephalosporin-like metabolic product shinorine or porphyra-334 represented by the formula (I):

R= -CH ₂ OH when the cephalosporin metabolite in formula (I) is shinorine, R= -CH (OH) CH ₃ when the cephalosporin metabolite is porphyra-334 (FIG. 1).

The mysC gene in the invention has the function of ATP-grasp enzyme, and the mysD gene has the function of non-ribosome-like synthetase NRPS-like enzyme or d-Ala-d-Ala-ligase.

Optionally, the Streptomyces chassis strain is Streptomyces lividans LIVIDANS TK.

Alternatively, the promoter of the operon may be a constitutive strong promoter such as kasOp, tacp or tetp, which initiates transcription of the structural gene element, and the transcription is terminated by a terminator. More preferably, the promoter is kasOp, tacp or tetp.

The second object of the invention can be achieved by the construction method of the recombinant engineering strain of Streptomyces producing a retinoid metabolite, comprising the steps of constructing recombinant plasmids pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD _p334, integrating the recombinant plasmids pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD _p334 into attB sites of chromosomes in a Streptomyces chassis strain by combining and transferring, realizing the integrated expression of gene expression cassettes and the production of different target metabolites, and specifically comprising the steps of:

(1) Constructing recombinant plasmids pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD _p334 and respectively converting competent E.coli ET12567, and selecting the transformant as a donor strain for conjugal transfer;

(2) The recipient bacteria are Streptomyces lividans Streptomyces LIVIDANS TK as conjunctive transfer recipient bacteria, and spores of the recipient bacteria are collected to prepare spore suspension for conjunctive transfer;

(3) Performing joint transfer, namely performing joint transfer on spore suspension of Streptomyces lividans Streptomyces lividansTK and escherichia coli carrying recombinant plasmids, and picking a zygote;

(4) And (3) genotype verification of the zygote, namely, carrying out genotype verification on the zygote to obtain the streptomycete recombinant engineering strain for producing the mycosporine-like metabolite.

The construction method of the streptomycete recombinant engineering strain for producing the mycosporine-like metabolite comprises the following steps:

alternatively, the recombinant plasmid pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD _p334 is a recombinant plasmid for constructing a mycosporine-like metabolite by using an integrative vector pSET152, and specifically comprises:

s1) selecting an integrated expression vector pSET152, carrying out tangential linearization on a plasmid by double enzymes, and recovering a linearized plasmid skeleton fragment;

S2) the inserts were synthesized, three DNA inserts were synthesized, the first DNA insert mysAB comprising only mysA and mysB, the second DNA insert mysABC comprising three gene elements mysA, mysB and mysC, the third DNA insert mysABCD or mysABCD _p334 comprising all four gene elements mysA, mysB, mysC and mysD or mysD _p334, the individual gene elements being joined by RBS sequences;

s3) ligation, wherein the linearized pSET152 vector is mixed with the three inserts in the step S2) respectively and is ligated by ligase overnight;

s4) transformation, namely respectively transforming competent escherichia coli into the ligation reaction mixtures, and screening transformants;

S5) plasmid verification, namely respectively extracting plasmids of the transformants, and obtaining recombinant expression plasmids pSET-mysAB, pSET-mysABC and pSET-mysABCD or pSET-mysABCD _p334 after verifying sequence correctness.

The sequence of the insert mysAB in the step S2) is shown as SEQ ID NO.1, the sequence of the insert mysABC is shown as SEQ ID NO.2, the sequence of the insert mysABCD is shown as SEQ ID NO.3, and the sequence of the insert mysABCD _p334 is shown as SEQ ID NO. 4.

The pSET-mysAB in step S5) of the present invention comprises and expresses two upstream gene elements mysA and mysB, the pSET-mysABC comprises and expresses two upstream gene elements mysA and mysB and one downstream gene element mysC, and the pSET-mysABCD or pSET-mysABCD _p334 comprises and expresses two upstream gene elements mysA and mysB and two downstream gene elements mysC and mysD or mysC and mysD _p334.

Preferably, the Streptomyces engineering strain in step (4) is named S.lividans S. lividans TKmysAB, S. lividans TKmysABC, S. lividans TKmysABCD and S. lividans TKmysABCD _p334, respectively, according to the differences of the four recombinant plasmids in step (1), and the four engineering strains are capable of producing 4-deoxygadusol, cephalosporin glycine mycosporine-glycine and cephalosporin metabolites shinorine or porphyra-334 represented by the following formula (I), respectively:

R= -CH ₂ OH when the cephalosporin metabolite is shinorine and R= -CH (OH) CH ₃ when the cephalosporin metabolite is porphyra-334 in the formula (I).

Preferably, as a specific embodiment of the present invention, the method for constructing an engineering strain for efficiently producing a cephalosporin metabolite by using the recombinant plasmid and the Streptomyces chassis strain can be realized by the following steps:

S1, donor bacteria, namely respectively converting the recombinant plasmids pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD _p334 into competent E.coli ET12567, and selecting transformants as donor strains for joint transfer;

s2, recipient bacteria, namely selecting streptomycete and preferably selecting Streptomyces lividans S. LIVIDANS TK24 as joint transfer recipient bacteria, collecting spores of the recipient bacteria, and preparing spore suspension for joint transfer;

S3, joint transfer, namely mixing spore suspension of the Streptomyces lividans S. LIVIDANSTK24 < 24 > and escherichia coli donor bacteria carrying recombinant plasmids at normal temperature, centrifuging to remove supernatant, coating on an MS culture medium plate, culturing at 30 ℃ for 16 < h >, covering with a pesticide solution containing nalidixic acid with the final concentration of 500 mu g/mL and apramycin with the final concentration of 1250 mu g/mL, continuously culturing for 2-5 days, picking the zygote, and passaging the zygote on a resistance MS culture medium (with the final concentration of 50 mu g/mL) containing apramycin for three times to remove escherichia coli to obtain the purified zygote;

s4, verifying the genotype of the zygote, namely verifying whether the expression cassette is correctly integrated at the attB site of the streptomycete chromosome by adopting a colony PCR method, wherein the obtained zygote with the correct genotype is the streptomycete engineering strain capable of efficiently producing the cephalosporin metabolite.

Preferably, the MS solid culture medium is soybean powder 20.0 g/L, mannitol 20.0 g/L, caCO ₃ 3.0.0 g/L and agar 20.0 g/L.

The last aim of the invention can be achieved by the following technical scheme that the streptomycete recombinant engineering strain is applied to the preparation of the mycosporine-like metabolites.

Preferably, the application comprises the steps of inoculating the recombinant engineering strain of streptomycete into a fermentation medium for culture, and enriching and purifying to obtain a retinoid metabolite, wherein the retinoid metabolite comprises 4-deoxygadusol, the cephalosporin glycine mycosporine-glycine and the retinoid metabolite shinorine or porphyra-334 shown in the following formula (I):

Further, the method for producing and preparing the mycosporine-like metabolite by fermenting the streptomycete recombinant engineering strain comprises the following steps:

(a) Activating the streptomycete recombinant engineering strain in an MS solid culture medium, and inoculating the streptomycete recombinant engineering strain in a TSB liquid culture medium for culture to obtain seed liquid;

(b) Inoculating the seed liquid obtained in the step (a) into a liquid synthetic culture medium, stopping fermentation after shaking culture or fermentation tank culture, centrifuging, respectively collecting thalli and fermentation supernatant, and washing thalli for later use;

(c) The product enrichment, namely ultrasonically extracting thalli obtained in the step (b) by using methanol, centrifuging, collecting supernatant, concentrating to obtain an intracellular crude extract, enriching the intracellular crude extract by using a sephadex chromatographic column, eluting by using pure water with the volume of 5 times of the column to remove impurities, eluting by using pure water with the volume of 5 times of the column, collecting eluent, and concentrating under reduced pressure to obtain a crude extract of a target compound;

(d) Purifying the product by using ODS chromatographic column, eluting with methanol MeOH at equal degree, collecting target chromatographic peak, and recovering the product under reduced pressure to obtain purified metabolite.

As a specific embodiment of the invention, the method for producing and preparing the mycosporine-like metabolite by fermenting the streptomycete recombinant engineering strain comprises the following steps:

1) Activating the recombinant engineering strain of streptomyces in a solid culture medium containing MS, inoculating the recombinant engineering strain into a TSB liquid culture medium containing apramycin, and culturing for 2 days at 28 ℃ (48 h);

2) The method comprises the steps of (1) inoculating seed liquid into a liquid synthetic culture medium according to an inoculation proportion of 5-10% (v/v), stopping fermentation after 5 days of shaking culture or fermentation tank culture at 28 ℃, respectively collecting thalli and fermentation supernatant after 20-min centrifugation by 4000 rpm, and performing suspension washing on the thalli for 2-3 times by pure water for later use;

3) The enrichment of the product, namely, extracting thalli with 50% methanol for 20 min by ultrasonic, centrifuging at 4000 rpm for 10min min, collecting supernatant, concentrating to obtain intracellular crude extract, enriching fermentation supernatant by a sephadex ODS chromatographic column, eluting with 5 times of pure water with the volume of the column (5 CV) to remove impurities, eluting with 5 CV pure water, collecting eluent, and concentrating under reduced pressure to obtain crude extract of target compound;

4) Purifying the product by using ODS chromatographic column which can be applied to pure water system, 2% MeOH (v/v) isocratic eluting, collecting target chromatographic peak, and recovering the product under reduced pressure to obtain purified metabolite.

Preferably, the composition of the liquid culture medium TSB is tryptone 17.0 g/L, soybean papain hydrolysate 3.0 g/L, glucose 2.5 g/L, sodium chloride 5.0 g/L, dipotassium hydrogen phosphate 2.5 g/L and pH value of 7.3.

Preferably, the liquid synthesis medium comprises glucose 60 g/L, ammonium sulfate 2 g/L, mgSO ₄·7H₂ O0.1 g/L, dipotassium hydrogen phosphate 0.5 g/L, sodium chloride 2 g/L,FeSO₄·7H₂O 0.05 g/L,ZnSO₄·7H₂O 0.05 g/L,MnCl₄·4H₂O 0.05 g/L, calcium carbonate 5 g/L, yeast extract 2 g/L and pH 7.0.

The invention has the following advantages:

(1) The invention efficiently produces the retinoid metabolite by heterologously expressing the retinoid metabolite synthetic gene element in streptomycete, and the engineering strain obtained by reconstructing the biosynthesis gene element and recombining and expressing in streptomycete LIVIDANS TK can efficiently produce MAAs compounds with strong ultraviolet absorption activity such as 4-deoxygadusol (4-DG), mycosporine-glycine (MG), shinorine, porphyra-334 and the like, which are applied to product development;

(2) Of the four metabolites produced by the present invention, 4-DG had a yield titer of 234.3 MG/L, MG had a yield titer of 634.4 MG/L, shinorine had a yield titer of 342.6 MG/L, porphyra-334 had a yield titer of 394.3 MG/L, all significantly higher than the yield of about 150 MG/L in the currently reported Streptomyces system.

Drawings

The invention will be further described with reference to the accompanying drawings, in conjunction with examples.

FIG. 1 is a schematic representation of the biosynthetic pathway of a cephalosporin metabolite, part of the invention;

FIG. 2 shows the result of agarose gel electrophoresis analysis of the pSET-mysABCD plasmid by PCR verification of bacterial liquid of E.coli DH5 alpha-transformants in example 1, lanes 1-8 are the result of PCR amplification of 8 transformants, wherein lanes 1-8 are the Marker of Trans2K ^® Plus, lanes 1-8 are 8 E.coli DH5 alpha-transformants, wherein lanes 3, 4, 7,8 with amplified bands are correct transformants at 5000 bp;

FIG. 3 is a diagram of the recombinant expression plasmids pSET-mysABCD in example 1, in which MSEDJ _27540, MSEDJ _27550, MSEDJ _27560 and MSEDJ _27570 represent MysA, mysB, mysC and MysD, respectively, and the same applies below;

FIG. 4 shows the result of agarose gel electrophoresis analysis of the recombinant expression plasmid pSET-mysABCD by bacterial liquid PCR verification of E.coli ET12567 transformants in example 1, lanes Marker: trans2K ^® Plus, lanes 1-8:8 E.coli ET12567 transformants, wherein lane 1 with amplified band at 5000 bp is the correct transformant;

FIG. 5 shows the result of PCR verification of the genotype of Streptomyces engineering strain Streptomyces lividans TKmysABCD in example 1. Lanes 1-16, wherein the lanes 1-16 are splicers, all splicers have bands at 1000 bp and are correct splicers, the lanes 2K ^® Plus are lanes markers at the upstream interface, and the lanes 1-16 are splicers, all splicers have bands at 1000 bp and are correct splicers;

FIG. 6 is an HPLC analysis of the extracellular metabolic phenotype of Streptomyces engineering strain Streptomyces lividans TKmysABCD of example 1, which produced the chromatographic peak of the metabolite shinorine of interest extracellular compared to Streptomyces LIVIDANS TK;

FIG. 7 is an HPLC analysis of the metabolic phenotype of Streptomyces engineering strain Streptomyces lividans TKmysABCD of example 1, which produces a chromatographic peak of target metabolite shinorine in cells compared to Streptomyces LIVIDANS TK;

FIG. 8 is a UV spectrum of the metabolite shinorine of Streptomyces engineering strain Streptomyces lividans TKmysABCD in example 1;

FIG. 9 is a high resolution mass spectrum of the metabolite shinorine of Streptomyces engineering strain Streptomyces lividans TKmysABCD in example 1;

FIG. 10 is a standard curve of the metabolite shinorine of the Streptomyces engineering strain of example 1;

FIG. 11 shows the result of agarose gel electrophoresis analysis of the pSET-mysABC plasmid by PCR verification of the bacterial liquid of the E.coli DH5 alpha-transformant in example 2, lanes Marker: trans2K ^® Plus, lanes 1-16:16 E.coli DH5 alpha-transformants, lane 14 in which the correct genotype band appears at 3700 bp being the correct transformant;

FIG. 12 is a recombinant expression plasmid map of pSET-mysABC in example 2;

FIG. 13 shows the result of agarose gel electrophoresis analysis of the recombinant expression plasmid pSET-mysABC by bacterial liquid PCR verification of E.coli ET12567 transformants in example 2, lanes Marker: trans2K ^® Plus, lanes 1-16:16 E.coli ET12567 transformants, the lanes 3700-bp are the lanes of the correct transformants, and the result shows that lanes 1-8, 10-12, 14-16 are the correct transformants;

FIG. 14 is a result of PCR verification of the genotype of Streptomyces engineering strain Streptomyces lividans TKmysABC in example 2;

FIG. 15 is an HPLC analysis of the extracellular metabolic phenotype of Streptomyces engineering strain Streptomyces lividans TKmysABC of example 2, which produced the chromatographic peak of the metabolite mycosporine glycine of interest extracellular compared to Streptomyces LIVIDANS TK;

FIG. 16 is an HPLC analysis of the metabolic phenotype of Streptomyces engineering strain Streptomyces lividans TKmysABC of example 2, which produces a chromatographic peak of target metabolite mycosporine glycine in cells compared to Streptomyces LIVIDANS TK;

FIG. 17 is a UV spectrum of metabolite mycosporine glycine of Streptomyces engineering strain Streptomyces lividans TKmysABC in example 2;

FIG. 18 is a high resolution mass spectrum of the metabolite mycosporine glycine of Streptomyces engineering strain Streptomyces lividans TKmysABC in example 2;

FIG. 19 is a standard curve of the metabolite mycosporine glycine of the Streptomyces engineering strain of example 2;

FIG. 20 shows the result of agarose gel electrophoresis analysis of the pSET-mysAB plasmid by PCR of the bacterial liquid of the E.coli DH5 alpha-transformant in example 3, lanes Marker: trans2K ^® Plus, lanes 1-16:16 E.coli DH5 alpha-transformants, the bands of 2400 bp being the bands of the correct transformants, and the result showing that lanes 8, 9, 14 are the correct transformants;

FIG. 21 is a recombinant expression plasmid map of pSET-mysAB in example 3;

FIG. 22 shows the result of agarose gel electrophoresis analysis of the recombinant expression plasmid pSET-mysAB verified by bacterial liquid PCR of the E.coli ET12567 transformant in example 3, lanes Marker: trans2K ^® Plus, lanes 1-16:16 E.coli ET12567 transformants, the bands of 2400 bp are the bands of correct transformants, and the result shows that lanes 1-16 are all correct transformants;

FIG. 23 shows the results of PCR verification of the genotype of Streptomyces engineering strain Streptomyces lividans TKmysAB in example 3, lanes Marker: trans2K ^® Plus at the upstream interface, lanes 1-16:16 of the spliceosome containing pSET_ mysAB, lanes 1000 bp of the spliceosome indicating that the correct genotype was obtained, lanes Marker: trans2K ^® Plus at the downstream interface, lanes 1-16:16 of the spliceosome containing pSET_ mysAB, lanes 1000 bp of the spliceosome of the correct genotype, and the results indicate that lanes 1-16 of the spliceosome of the correct genotype was obtained;

FIG. 24 is an HPLC analysis of the extracellular metabolic phenotype of Streptomyces engineering strain Streptomyces lividans TKmysAB of example 3, which produced the chromatographic peak of the target metabolite 4-deoxygadusol extracellular compared to Streptomyces LIVIDANS TK;

FIG. 25 is a UV spectrum of metabolite 4-deoxygadusol of Streptomyces engineering strain Streptomyces lividans TKmysAB in example 3;

FIG. 26 is a high resolution mass spectrum of metabolite 4-deoxygadusol of Streptomyces engineering strain Streptomyces lividans TKmysAB in example 3;

FIG. 27 is a standard curve of the metabolite 4-deoxygadusol of the Streptomyces engineering strain of example 3;

FIG. 28 shows the result of agarose gel electrophoresis analysis of the pSET-mysABCD _p334 plasmid verified by bacterial liquid PCR of the E.coli DH5 alpha transformant in example 4, lanes Marker: trans2K ^® Plus, lanes 1-4:4 E.coli DH5 alpha transformants. 4800 The bp band is the band of the correct transformant, and the result shows that lanes 2-4 are the correct transformant;

FIG. 29 is a diagram of a recombinant expression plasmid for pSET-mysABCD _p334 in example 4, in which Nostoc linckiaNIES-25BAY_79928 is MysD _p334;

FIG. 30 shows the result of PCR verification of the genotype of Streptomyces engineering strain Streptomyces lividans TKmysABCD _p334 in example 4, lanes Marker: trans2K Plus, lanes 1-12:12 E.coli ET12567 transformants, lanes 4800 bp as the correct transformants, and the result shows that lanes 8, 11 are the correct transformants;

FIG. 31 shows the result of agarose gel electrophoresis analysis of recombinant expression plasmid pSET-mysABCD _p334 by bacterial liquid PCR verification of E.coli ET12567 transformant in example 4, upstream interface, lane Marker: trans2K ^® Plus, lanes 1-8:8 of the spliceosome containing pSET_ mysABCDP, 1000 bp of the spliceosome indicating that the correct genotype was obtained, downstream interface, lane Marker: trans2K ^® Plus, lanes 1-8:8 of the spliceosome containing pSET_ mysABCDP334, and lanes 1000 bp of the spliceosome containing the correct genotype, indicating that lanes 1-8 of the spliceosome containing the correct genotype were all obtained;

FIG. 32 is an HPLC analysis of the extracellular metabolic phenotype of Streptomyces engineering strain Streptomyces lividans TKmysABCD _p334 of example 4, which produced the chromatographic peaks of the target metabolites porphyra-334 extracellular compared to Streptomyces LIVIDANS TK strain;

FIG. 33 is a UV spectrum of the metabolite porphyra-334 of Streptomyces engineering strain Streptomyces lividans TKmysABCD _p334 in example 4;

FIG. 34 is a high resolution mass spectrum of the metabolite porphyra-334 of Streptomyces engineering strain Streptomyces lividans TKmysABCD _p334 in example 4;

FIG. 35 is a standard curve of the metabolite porphyra-334 of the Streptomyces engineering strain in example 4.

Detailed Description

The reagents or materials used in the examples below, unless otherwise specified, were all commercially available. Unless otherwise indicated, all laboratory instruments used are laboratory conventional.

In the following examples, the nucleic acid sequence of upstream gene element mysA is described in GenBank BBY28658, the nucleic acid sequence of mysB is described in GenBank BBY28659, the nucleic acid sequence of downstream gene element mysC is described in GenBank BBY28660, the nucleic acid sequence of mysD is described in GenBank BBY28661, and the nucleic acid sequence of mysD _P334 is described in GenBank BAY79928.

The nucleic acid sequence of the promoter tacp is shown in a part fragment of GenBank FJ389173, 2926-2884, the nucleic acid sequence of the promoter tetp is shown in a part fragment of GenBank MZ476912, 1226-1296, the nucleic acid sequence of the terminator lambda t ₀ is shown in a part fragment of GenBank JX560387, 2163-2197, and the nucleotide sequence of the RBS is shown in a part fragment of GenBank MN306530, 11376-11395.

The nucleotide sequence of the kasOp promoter is shown as SEQ ID NO.11, the promoter is an improved promoter, the promoter has a much higher starting efficiency than a wild kasO type promoter, and a public database has no consistent sequence, so that the high yield of the invention has strong correlation with the promoter.

Example 1 construction method of recombinant engineering Strain of Streptomyces for producing shinorine

1. Construction of recombinant plasmid pSET-mysABCD

1.1 Synthesis of artificial sequence, wherein the MAA recombinant plasmid comprises a MAA biosynthesis operon which is controlled by a promoter kasOp and a lambdat ₀ terminator, wherein all four genes (mysABCD for short) of mysA, mysB, mysC, mysD are inserted between the two, 3 RBS sequences are contained between 4 gene coding sequences, and enzyme cutting sites XbaI/NdeI are respectively arranged before and after the MAA biosynthesis operon, and the MAA biosynthesis operon is firstly constructed on pUC57 to obtain the pUC57-mysABCD plasmid. The complete sequence of the operon is shown as SEQ ID NO. 3.

1.2 Cloning of operon:

The integrated vector pSET152 was selected as a plasmid backbone, and appropriate cleavage sites were selected according to the vector sequence and the target gene sequence, and in this example, xbaI and NdeI restriction enzymes were selected to double-cleave pSET152 and pUC57-mysABCD, respectively, and the cleavage reaction system (50. Mu.L) was 1. Mu.L of restriction enzyme, 1. Mu.g of plasmid/vector DNA, 10 XNEBuffer 5. Mu.L, and ultra-pure H ₂ O was used to make up 50. Mu.L. After gentle blowing and mixing, the mixture is reacted for 3 hours at 37 ℃ and then is thermally deactivated for 20min at 65 ℃ and is cooled on ice.

Fragment recovery the reaction mixtures were separated by gel electrophoresis using 1.0% agarose, and the linearized pSET152 vector and mysABCD insert were recovered by gel cutting.

Ligation linearized pSET152 vector and mysABCD insert were mixed in a 1:3 molar ratio and ligation was performed using T ₄ ligase. Vector recommended addition (ng): 0.02×fragment length bp number, insert recommended addition (ng): 0.04×insert length bp number. The ligation system (total reaction system 20 mL) was 1. Mu.L of T ₄ ligase, 4. Mu.L of linearized vector (105 ng), 1. Mu.L of target fragment (258 ng), and 20. Mu.L of 10 XT ₄ DNA Ligase Reaction Buffer 2 μL（1×）,H₂ O. After gentle blowing and mixing, the mixture was reacted in a water bath at 16 ℃ overnight for 12 h and cooled on ice.

Conversion, namely adding 10 mu L of recombinant product into 100 mu L of DH5 alpha clone competence, uniformly mixing the light elastic tube wall, standing for 10min on ice, and carrying out heat shock at 42 ℃ for 90 s. Immediately cooling 2-3 min on ice, adding 900 μl of LB medium into the transformed product in a super clean bench, culturing 1h at 37deg.C under 200 rpm, and centrifuging to concentrate to 100 μl. 100. Mu.L of the bacterial liquid is evenly coated on a flat plate of LB culture medium (the final concentration of antibiotics is 50 mg/mL) added with apramycin, and the flat plate is reversely cultured at 37 ℃ for 12-16 h until the transformant appears.

The verification is that transformants are picked from the flat plate and transferred into 1 mL LB liquid medium containing 50 mg/mL apramycin, the culture is carried out under shaking at 37 ℃ and 200 rpm for 3-6 h, bacterial liquid is used as a template, and a primer pair pSET-ABCDyz-F/R is used for amplifying target fragments with 0.5 mu L each. The total PCR system (10. Mu.L) comprises 1. Mu.L of bacterial liquid, 2X PRIMESTAR MAX DNA POLYMERASE. Mu.L and 10. Mu.L of H ₂ O, the PCR program comprises 3 min of 98 ℃ pre-denaturation, then 35 cycles, each cycle comprises 10 s of 98 ℃ duration, 5 s of 60 ℃ duration, 25 s of 72 ℃ duration and 5min of 72 ℃ duration, the electrophoresis diagram of bacterial liquid PCR detection of 8 E.coli DH5 alpha transformants is shown in FIG. 2, and 3, 4, 7 and 8 of the transformants with the band of 5000 bp are correct transformants. The recombinant plasmid pSET-mysABCD is obtained by extracting the DH5 alpha strain of the escherichia coli containing the correct plasmid, and is shown in figure 3.

The primer sequence is as follows:

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

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

2. construction and production of shinorine Streptomyces engineering strain

2.1 Transforming donor bacteria, namely transferring the recombinant plasmid pSET-mysABCD into competent escherichia coli ET12567 to obtain escherichia coli ET12567 donor bacteria, verifying the transformant by using bacterial liquid PCR, wherein the specific operation steps are the same as those of the transformation step in 1.2. As a result of agarose gel electrophoresis, as shown in FIG. 4, lane 1, in which a band of 5000 bp was amplified, was the correct transformant. The verified monoclonal bacteria were resuspended in 1 mL LB medium for use.

2.2 Pretreating donor bacteria, namely inoculating the bacterial liquid in 2.1 into 5-mL LB culture medium (the final antibiotic concentration is 50 mg/mL) containing apramycin according to an inoculation proportion of 5-10% (v/v), placing an activated tube after inoculation into a shaking table at 37 ℃, and culturing 180 rpm overnight. The overnight activated broth was inoculated into 50 mL apramycin-containing LB medium (final antibiotic concentration 50 mg/mL) at an inoculum size of 4%. Shaking culture is carried out at 37 ℃ and 180 rpm until the OD value is 0.5-0.8. The cultured bacterial liquid is poured into a 50 mL centrifuge tube in an ultra clean bench, and centrifuged at 4 ℃ and 3500 rpm and 5 min. The supernatant was discarded, an equal volume of LB resuspended cells at 4℃was added, 3500 rpm, and centrifuged 5.5 min, and the procedure was repeated once. Finally, the cells were resuspended in 1mL LB for later use.

2.3 Pretreatment of recipient bacteria

The Streptomyces lividans TK24 bacterial liquid is coated on an MS flat plate, cultured for about 7 days at 28 ℃, spores are collected, the spores are suspended in 20 mL ultrapure water at 3500rpm, the centrifugation is carried out for 10 min, and the supernatant is discarded. Spores were resuspended in 2 XYT broth of 10mL and heat-shocked at 50℃10 min. After naturally cooling the spore liquid after heat shock to room temperature, adding 10mL XYT broth into the system, and finally placing in a shaking table at 37 ℃ for 180: 180 rpm germination 3: 3 h. The germinated spores were centrifuged at 3500rpm a for 5a min a, the supernatant was discarded and resuspended in LB for further use.

2.4 Transfer of engagement

Mixing 100 μl of spore liquid in 2.3 and 100 μl of Escherichia coli bacterial liquid in 2.2, and centrifuging at normal temperature 3500 rpm: 5 min. Part of the supernatant was discarded, and 100. Mu.L of the resuspended mixed cells was left. From 100. Mu.L of the resuspended mixed cells, 10. Mu.L to 90. Mu.L of ultrapure water were taken, and 3 gradients (10 ^-1/10^-2/10^-3 orders) were sequentially diluted, and 10 ^-0/10^-1/10^-2/10^-3 four orders of magnitude of the mixed cells were applied to MS plates containing 10mM MgCl ₂, respectively. 90. Mu.L of each spore solution was plated on MS plates with final concentration of 10mM Mg ²⁺ as negative and positive controls, respectively. All coated plates were placed in a 30 ℃ incubator for incubation, wherein the experimental and negative control groups were capped after 16 h cultures. 1mL of the mixed antibiotic solution is added into each plate, so that the final concentration of nalidixic acid and apramycin is 500 mg/mL and 1250 mg/mL respectively, and the whole plate is uniformly distributed with the liquid medicine by rolling the plate or with the aid of a gun head. The plate was dried in an ultra clean bench and returned to the incubator for 5 days to allow observation of the zygote. The zygote single colony was picked from the plate in 30. Mu.L of 10mM NaOH solution, and the genome was repeatedly freeze-thawed to perform the subsequent genotyping PCR verification, while the zygote was picked onto MS medium containing 50 mg/mL apramycin and purified by successive subcultures.

2.5 PCR verification of the zygote genotype the interface of the operon upstream and downstream of the attB integration site of the chromosomal DNA of Streptomyces using the zygote cell as template and the primer pairs SL-1-F/R and SL-3-F/R, respectively. 10. The PCR reaction system of the mu L comprises 1 mu L of bacterial liquid and 0.5 mu L of primer respectively, wherein 10 mu L of primer is complemented by 2X PRIMESTAR MAX DNA POLYMERASE mu L, H ₂ O, the PCR process comprises the steps of lasting for 3 min at 98 ℃, then 35 cycles, wherein each cycle comprises lasting for 10 s at 98 ℃, lasting for 5 s at 60 ℃, lasting for 25 s at 72 ℃, lasting for 5min at 72 ℃, the PCR detection strip is shown in figure 5, the amplification of the PCR detection strip to obtain a 1000 bp strip shows that the correct recombinant strain is obtained, and lanes 1-16 are the correct recombinant Streptomyces lividans engineering strain which is named as Streptomyces lividans Streptomyces lividans TKmysABCD.

The primer sequences are respectively as follows:

SL-1-F:5′-GGCTTTCGTGGTTCCAGGTGG-3′(SEQ ID NO.7);

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

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

SL-3-R:5′-CCGTTGGAGTCCTGGTGGTCG-3′(SEQ ID NO.10)。

2.6 Product detection and isolation recombinant Streptomyces engineering with successful genotype verification is inoculated into 5mL TSB liquid culture medium containing 50 mg/mL apramycin, and cultured for two days at 28 ℃ and 220 rpm. The seed solution was inoculated into a fermentation medium at an inoculum size of 10%, at 28 ℃,220 rpm, and fermented for 5 days. After fermentation, 4000rpm and 20 min centrifugation are carried out to collect thalli and fermentation supernatant respectively, the thalli are subjected to ultrasonic treatment by 50% methanol for 20 min, and after ultrasonic treatment, 4000rpm and 20 min centrifugation are carried out to obtain intracellular extract of the supernatant. HPLC and HPLC-MS analysis were performed on the fermentation supernatant and intracellular extracts, respectively. The analysis results are shown in fig. 6 to 8. It can be seen that both intracellular and extracellular success produced the target product shinorine.

The HPLC-MS analysis method is as follows:

Analytical instrument Agilent Technologies 1260 Infinity,6230 TOF LC/MS.

Analytical column YMC-Pack-ODS-A (5 μm, 4.6X1250 mm);

mobile phase, namely pure water containing 0.2% formic acid, and eluting 20min at equal temperature at a flow rate of 0.5 mL/min;

From the results of FIGS. 6 and 7, it was shown that a new chromatographic peak appears both intra-and extracellular in the recombinant strain compared to Streptomyces lividans TK24 of the Chassis strain. As shown in fig. 8, this chromatographic peak gives a maximum absorption at 330 nm, showing the uv absorption of typical retinoid amino acids in the UVA band. As shown in fig. 9, it is determined as the target product shinorine from its high-resolution mass spectrum. Collecting the chromatographic peak by semi-preparation or preparative chromatography to obtain shinorine pure product.

Determination of fermentation titres standard curves were determined by analytical HPLC using shinorine pure products. Shinorine standard 5.04 mg was prepared by precise weighing, and the volume was fixed with 0.2% (v/v) formic acid water into a 5mL volumetric flask to give 1.01mg/mL standard stock solution. The solution was diluted 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. 200 mu L of standard substances with each concentration are respectively taken and added into a liquid phase sample injection bottle, and three parallel concentrations are respectively arranged. Peak areas of shinorine at the corresponding concentrations were determined using the HPLC assay methods described above. A standard curve (as shown in fig. 10) was plotted with the standard concentration on the abscissa and the shinorine peak area on the ordinate.

And (3) measuring the concentration of the sample, namely taking supernatant after carrying out 1.5 mL,10,000 rpm centrifugation on each of 3 fermentation liquor samples, diluting the supernatant by 50 times, filtering the supernatant by a filter membrane, taking 200 mu L of the filtered supernatant, and transferring the filtered supernatant into a sample injection sample bottle. The peak areas were calculated by three separate injections. The fermentation titer was 342.6 mg/L as determined according to the standard curve shinorine.

Example 2 construction method and application of Streptomyces engineering bacteria for producing mycosporine glycine

1. Construction of recombinant plasmid pSET-mysABC

1.1 Synthesis of artificial sequence the MAA recombinant plasmid contains a MAA biosynthetic operon controlled by a promoter kasOp and a lambda t ₀ terminator. Three genes mysA, mysB and mysC (mysABC for short) are inserted between the two genes, and 2 RBS sequences are contained between the three gene coding sequences. The MAA biosynthesis operon is provided with enzyme cutting sites XbaI/NdeI respectively before and after. MAA biosynthetic operon was constructed on pUC57 to give pUC57-mysABC plasmid. The complete sequence of the operon is shown as Seq ID No. 2.

1.2 Cloning of operon:

The procedure of this example was the same as that of example 1.2, except that the integrated vector pSET152 was selected as the plasmid backbone, and the appropriate cleavage sites were selected based on the vector sequence and the target gene sequence, and XbaI and NdeI restriction enzymes were selected to double-cleave pSET152 and pUC57-mysABC, respectively.

Fragment recovery the reaction mixtures were separated by gel electrophoresis using 1.0% agarose, cut to linearize the pSET152 vector and mysABC insert, respectively.

Ligation linearized pSET152 vector and mysABC insert were mixed in a 1:3 molar ratio and ligation was performed using T ₄ ligase. The specific procedure was the same as the ligation procedure in 1.2 of example 1.

The specific procedure for the transformation was the same as in 1.2 of example 1.

Verification the specific procedure was the same as that in 1.2 of example 1. The PCR detection band is shown in FIG. 11, and lane 14 is amplified to obtain 3700bp band, which shows that the recombinant plasmid is the correct recombinant plasmid. The recombinant plasmid pSET_ mysABC is obtained by extracting the DH5 alpha strain of the escherichia coli containing the correct plasmid, and the plasmid map is shown in figure 12. The primer sequence was identical to the primer sequence of example 1.2.

2. Construction and production of mycosporine glycine Streptomyces engineering strain

2.1 Transformation of donor bacteria the recombinant plasmid pSET_ mysABC was transformed into competent E.coli ET12567 to give E.coli ET12567 donor bacteria, and the transformant was verified by bacterial liquid PCR, the specific experimental procedure was the same as the transformation procedure of 1.2 of example 1. As shown in FIG. 13, the agarose gel electrophoresis analysis results show that the amplified 3700 bp bands indicate that the correct transformants were obtained (lanes 1-8, 10-12, 14-16). The verified monoclonal bacteria were resuspended in 1 mL LB medium for use.

2.2 Pretreatment of donor bacteria the specific experimental procedure was the same as the pretreatment procedure of example 1, 2.2.

2.3 Pretreatment of recipient bacteria the specific experimental procedure was the same as the pretreatment procedure of example 1, 2.3.

2.4 Bond transfer specific experimental procedure was the same as the pretreatment procedure of example 1, 2.4.

2.5 PCR verification of the zygote genotype the interface of the operon upstream and downstream of the attB integration site of the chromosomal DNA of Streptomyces using the zygote cell as template and primers (SL-1-F/R and SL-3-F/R) respectively. The specific experimental procedure was the same as the validation procedure of example 1, 2.2.5. The result of PCR detection of the genotype is shown in FIG. 14, and the amplification of the band at 1000 bp indicates that the correct recombinant strain was obtained, and lanes 1-16 were all the correct recombinant Streptomyces engineering strain designated Streptomyces lividans TKmysABC. The primer sequence was identical to the primer sequence in example 1, 2.5.

2.6 Product detection and isolation the specific experimental procedure was the same as the detection and isolation procedure of example 1, 2.6. HPLC and HPLC-MS analysis are respectively carried out on the fermentation supernatant and the intracellular extract, and analysis results are respectively shown in figures 15-18. The engineered strain successfully produces the target metabolite mycosporine glycine.

The HPLC-MS analysis method is as follows:

Analytical instrument Agilent Technologies 1260 Infinity,6230 TOF LC/MS.

Chromatographic column YMC-Pack-ODS-A (5 μm, 4.6X1250 mm);

Methanol-pure water containing 0.2% formic acid at a ratio of 2:98 (v/v), and isocratically eluting at a flow rate of 0.5 mL/min for 30: 30 min;

The results in FIGS. 15 and 16 show that both the intracellular and extracellular production of the recombinant strain produced a new chromatographic peak. As shown in fig. 17, this chromatographic peak gives a UVA band maximum absorption at 310 nm. Further, by high-resolution mass spectrum data, mycosporine glycine was determined, and the high-resolution mass spectrum is shown in fig. 18. Collecting the pure mycosporine glycine product by semi-preparation or preparative chromatography.

Determination of fermentation titres standard curves were determined by analytical HPLC using mycosporine glycine pure products. The mycosporine glycine standard 5.12 is precisely weighed and prepared mg, and 0.2% (v/v) formic acid water is used for metering the volume into a 5 mL volumetric flask, so that 1.02 mg/mL standard stock solution is obtained. The solution was diluted 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. 200 mu L of standard substances with each concentration are respectively taken and added into a liquid phase sample injection bottle, and three parallel concentrations are respectively arranged. Peak areas of mycosporine glycine at the corresponding concentrations were determined using the HPLC assay methods described above. A standard curve (as shown in fig. 19) was plotted with the standard concentration on the abscissa and the mycosporine glycine peak area on the ordinate.

And (3) measuring the concentration of the sample, namely taking supernatant after carrying out 1.5 mL,10,000 rpm centrifugation on each of 3 fermentation liquor samples, diluting the supernatant by 50 times, filtering the supernatant by a filter membrane, taking 200 mu L of the filtered supernatant, and transferring the filtered supernatant into a sample injection sample bottle. The peak areas were calculated by three separate injections. The fermentation titer was 634.4 mg/L as determined according to the standard curve mycosporine glycine.

Example 3 construction method and application of Streptomyces engineering bacteria for producing 4-deoxygadusol

1. Construction of recombinant plasmid pSET-mysAB

Artificial sequence synthesis the MAA recombinant plasmid contains a MAA biosynthetic operon, which is controlled by a promoter kasOp and a lambda t ₀ terminator. Two genes mysA and mysB (mysAB for short) are inserted between the two genes, and 1 RBS sequence is contained between 2 gene coding sequences. The MAA biosynthesis operon is provided with enzyme cutting sites XbaI/NdeI respectively before and after. MAA biosynthetic operon was constructed on pUC57 to give pUC57-mysAB plasmid. The complete sequence of the operon is shown as Seq ID No. 1.

Cloning of operon:

And (3) enzyme digestion, namely selecting an integrated vector pSET152 as a plasmid skeleton. And selecting proper enzyme cutting sites according to the vector sequence and the target gene sequence. In this example, xbaI and NdeI restriction enzymes were selected to double cleave pSET152 and pUC57-mysAB, respectively. The specific procedure was the same as in example 1, 1.2.

Fragment recovery the reaction mixtures were separated by gel electrophoresis using 1.0% agarose, cut to linearize the pSET152 vector and mysAB insert, respectively.

Ligation linearized pSET152 vector and mysAB insert were mixed in a 1:3 molar ratio and ligation was performed using T ₄ ligase. The specific procedure was the same as the ligation procedure in 1.2 of example 1.

Verification the specific procedure was the same as that in 1.2 of example 1. The PCR detection band is shown in FIG. 20, lanes 8,9 and 14, from which 2400 bp bands can be amplified, are correct recombinant plasmids. The recombinant plasmid pSET-mysAB is obtained by extracting the DH5 alpha strain of the escherichia coli containing the correct plasmid, and is shown in figure 21.

The primer sequence was identical to the primer sequence of example 1.2.

2. Construction of Streptomyces engineering strain producing 4-deoxygadusol

2.1 Transformation of donor bacteria the recombinant plasmid pSET_ mysABC was transformed into competent E.coli ET12567 to give E.coli ET12567 donor bacteria, and the transformant was verified by bacterial liquid PCR, the specific experimental procedure was the same as the transformation procedure of 1.1.2 of example 1. As shown in FIG. 22, lanes 1-16 all amplified to give 2400 bp bands, indicating correct transformants. The verified monoclonal bacteria were resuspended in 1 mL LB medium for use.

2.2 Pretreatment of donor bacteria the specific experimental procedure was the same as the pretreatment procedure of example 1, 2.2.2.

2.3 Pretreatment of recipient bacteria the specific experimental procedure was the same as the pretreatment procedure of example 1, 2.2.3.

2.4 Bond transfer specific experimental procedure was the same as the pretreatment procedure of example 1, 2.2.4.

2.5 PCR verification of the zygote genotype the interface of the operon upstream and downstream of the attB integration site of the chromosomal DNA of Streptomyces using the zygote cell as template and primers (SL-1-F/R and SL-3-F/R) respectively. The specific experimental procedure was the same as the validation procedure of example 1, 2.2.5. PCR detection bands are shown in FIG. 23, and the amplified bands of 1000 bp indicate that the correct recombinant strain was obtained, and lanes 1-16 are all the correct recombinant Streptomyces engineering strains, designated Streptomyces lividans TKmysAB.

The primer sequence was identical to the primer sequence in example 1, 2.5.

2.6 Product detection and isolation the specific experimental procedure was the same as the detection and isolation procedure of example 1, 2.2.6. HPLC and HPLC-MS analysis are respectively carried out on the fermentation supernatant and the intracellular extract, and the analysis results are shown in figures 24-26. The engineering strain successfully produces the target metabolite 4-deoxygadusol.

The HPLC-MS analysis method is as follows:

Analytical instrument Agilent Technologies 1260 Infinity,6230 TOF LC/MS.

Chromatographic column YMC-Pack-ODS-A (5 μm, 4.6X1250 mm);

As can be seen from the results of FIG. 24, a new chromatographic peak was generated in the recombinant strain broth, and no signal was detected in the cell. As shown in fig. 25, this peak gives a maximum absorption at 268 nm, which shows the uv absorption of typical mycosporine amino acids in the UVB band. As shown in fig. 26, it was determined to be 4-deoxygadusol based on data further passing through high resolution mass spectrometry. Collecting the purified product of 4-deoxygadusol by semi-preparation or preparative chromatography.

Determination of fermentation titres standard curves were determined by analytical HPLC using 4-deoxygadusol pure products. 4.97mg of the standard 4-deoxygadusol is precisely weighed and prepared, and 0.2% (v/v) formic acid water is used for metering the volume into a 5mL volumetric flask to obtain 1.00 mg/mL of standard stock solution. The solution was diluted 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. 200 mu L of standard substances with each concentration are respectively taken and added into a liquid phase sample injection bottle, and three parallel concentrations are respectively arranged. The peak area of 4-deoxygadusol at the corresponding concentration was determined using the HPLC assay described above. A standard curve (as shown in FIG. 27) was plotted with the standard concentration on the abscissa and the peak area of 4-deoxygadusol on the ordinate.

And (3) measuring the concentration of the sample, namely taking supernatant after carrying out 1.5 mL,10,000 rpm centrifugation on each of 3 fermentation liquor samples, diluting the supernatant by 50 times, filtering the supernatant by a filter membrane, taking 200 mu L of the filtered supernatant, and transferring the filtered supernatant into a sample injection sample bottle. The peak areas were calculated by three separate injections. The fermentation titer of 4-deoxygadusol was determined according to the standard curve to be 234.3 mg/L.

Example 4 construction method and application of Streptomyces engineering bacteria for producing porphyra-334

1. Construction of recombinant plasmid pSET-mysABCD _P334

Artificial sequence synthesis the MAA recombinant plasmid contains a MAA biosynthetic operon, which is controlled by a promoter kasOp and a lambda t ₀ terminator. All four genes (mysABCD _P334 for short) are inserted mysA, mysB, mysC, mysD _p334 between the two, and 3 RBS sequences are contained between 4 gene coding sequences. The MAA biosynthesis operon is provided with enzyme cutting sites XbaI/NdeI respectively before and after. MAA biosynthetic operon was constructed on pUC57 to give pUC57-mysABCD _P334 plasmid. Wherein mysD _p334 differs in sequence from mysD in example 1. The complete sequence of the operon comprising mysABCD _p334 is shown in SEQ ID NO. 4.

Cloning of operon:

And (3) enzyme digestion, namely selecting an integrated vector pSET152 as a plasmid skeleton. And selecting proper enzyme cutting sites according to the vector sequence and the target gene sequence. In this example, xbaI and NdeI restriction enzymes were selected to double cleave pSET152 and pUC57-mysABCD _P334, respectively. The specific procedure was the same as in example 1, 1.2.

Fragment recovery the reaction mixtures were separated by gel electrophoresis using 1.0% agarose, cut to linearize the pSET152 vector and mysABCD _P334 insert, respectively.

Ligation linearized pSET152 vector and mysABCD _P334 insert were mixed in a 1:3 molar ratio and ligation was performed using T4 ligase. The specific procedure was the same as the ligation procedure in 1.1.2 of example 1.

The specific procedure for the transformation was the same as in example 1, 1.2.

Verification the specific procedure was the same as that in 1.1.2 of example 1. The PCR detection band is shown in FIG. 28, and the amplified 4800bp band shows that the correct recombinant plasmid is obtained, and the result shows that lanes 2-4 are correct transformants. And extracting the DH5 alpha strain of the escherichia coli containing the correct plasmid to obtain the recombinant plasmid pSET-mysABCD _P334. The plasmid map is shown in FIG. 29.

The primer sequence was identical to the primer sequence of example 1.2.

2. Construction of Streptomyces engineering strain for producing porphyra-334

2.1 Transformation of donor bacteria recombinant plasmid pSET-mysABCD _P334 was transformed into competent E.coli ET12567 to give E.coli ET12567 donor bacteria, and the transformant was verified by bacterial liquid PCR, and the specific experimental procedure was the same as the transformation procedure of 1.1.2 of example 1. As shown in FIG. 30, the agarose gel electrophoresis analysis results show that the correct transformant was obtained by amplifying the band of 4800 bp, and the results show that lanes 8 and 11 were the correct transformants. The verified monoclonal bacteria were resuspended in 1mL LB medium for use.

2.5 PCR verification of the zygote genotype the interface of the operon upstream and downstream of the attB integration site of the chromosomal DNA of Streptomyces using the zygote cell as template and primers (SL-1-F/R and SL-3-F/R) respectively. The specific experimental procedure was the same as the validation procedure of example 1, 2.2.5. The PCR detection band is shown in FIG. 31, and the amplified band of 1000 bp shows that the correct recombinant strain is obtained, and lanes 1-8 are all the correct recombinant Streptomyces engineering strain, designated Streptomyces lividans TKmysABCD _P334.

The primer sequence was identical to the primer sequence in example 1, 2.5.

2.6 Product detection and isolation the specific experimental procedure was the same as the detection and isolation procedure of example 1, 2.2.6. The fermentation supernatants were subjected to HPLC and HPLC-MS analysis. The analysis results are shown in fig. 32 to 33. The engineering strain successfully produces the target metabolite porphyra-334.

The HPLC-MS analysis method is as follows:

Analytical instrument Agilent Technologies 1260 Infinity,6230 TOF LC/MS.

Chromatographic column YMC-Pack-ODS-A (5 μm, 4.6X1250 mm);

As shown in FIG. 32, a new chromatographic peak appears in the recombinant strain broth. As shown in fig. 33, this chromatographic peak gives a maximum absorption at 334 nm, showing the typical uv absorption of typical retinoid amino acids in the UVA band. Further, as shown in fig. 34, it was determined to be porphyra-334 by high-resolution mass spectrometry data. Collecting the purified product porphyra-334 by semi-preparation or preparative chromatography.

Determination of fermentation titres standard curves were determined by analytical HPLC using porphyra-334 pure. Accurately weighing and preparing porphyra-334 standard substance 5.05 mg, and fixing the volume to a5 mL volumetric flask by using 0.2% (v/v) formic acid water to obtain 1.01 mg/mL standard substance stock solution. The solution was diluted 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. 200 mu L of standard substances with each concentration are respectively taken and added into a liquid phase sample injection bottle, and three parallel concentrations are respectively arranged. Peak areas of porphyra-334 at the corresponding concentrations were determined using the HPLC assay methods described above. Standard curves (as shown in fig. 35) were plotted with the standard concentration on the abscissa and the peak area porphyra-334 on the ordinate.

And (3) measuring the concentration of the sample, namely taking supernatant after carrying out 1.5 mL,10,000 rpm centrifugation on each of 3 fermentation liquor samples, diluting the supernatant by 50 times, filtering the supernatant by a filter membrane, taking 200 mu L of the filtered supernatant, and transferring the filtered supernatant into a sample injection sample bottle. The peak areas were calculated by three separate injections. The fermentation titer of porphyra-334 was found to be 394.3 mg/L according to the standard curve.

The foregoing embodiments are merely illustrative of the technical solutions of the present invention and are not intended to be limiting, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof, without departing from the spirit and scope of the technical solutions as claimed in the present invention.

Claims

1. A recombinant engineering strain of streptomyces producing a mycosporine-like metabolite, characterized in that the genome of the recombinant engineering strain of streptomyces is integrated with a gene expression cassette, the gene expression cassette is recombined to attB sites of chromosomes in a chassis strain of streptomyces, 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 _p334 biosynthetic gene elements in the retinoid metabolite synthesis pathway;

when the operon comprises MysA and MysB biosynthesis gene elements in the synthesis pathway of the mycosporine-like metabolite, the nucleic acid sequence of the operon is shown as SEQ ID NO.1, and the corresponding metabolite of the recombinant engineering strain of Streptomyces is 4-deoxygadusol;

when the operon comprises MysA, mysB and MysC biosynthesis gene elements in the synthesis path of the mycosporine-like metabolite, the nucleic acid sequence of the operon is shown as SEQ ID NO.2, and the corresponding metabolite of the recombinant engineering strain of Streptomyces is mycosporine-glycine;

When the operon comprises MysA, mysB, mysC and MysD biosynthetic gene elements of the retinoid metabolite synthesis pathway, the nucleic acid sequence of the operon is shown as SEQ ID NO.3, and the corresponding metabolite of the recombinant engineering strain of Streptomyces is shinorine;

When the operon comprises MysA, mysB, mysC and mysD _p334 biosynthesis gene elements in the synthesis pathway of the mycosporine-like metabolite, the nucleic acid sequence of the operon is shown as SEQ ID NO.4, and the corresponding metabolic product of the recombinant engineering strain of Streptomyces is porphyra-334;

The Streptomyces chassis strain is Streptomyces lividans LIVIDANS TK;

the nucleic acid sequence of the promoter kasOp and the kasOp promoter is shown in SEQ ID NO. 11;

The nucleic acid sequence of mysA is shown in GenBank: BBY28658, the nucleic acid sequence of mysB is shown in GenBank: BBY28659, the nucleic acid sequence of mysC is shown in GenBank: BBY28660, the nucleic acid sequence of mysD is shown in GenBank: BBY28661, and the nucleic acid sequence of mysDP334 is shown in GenBank: BAY79928.

2. The method for constructing a recombinant engineering strain of Streptomyces producing a retinoid metabolite according to claim 1, comprising the steps of constructing recombinant plasmids pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD _p334, integrating the recombinant plasmids pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD _p334 into the attB site of a chromosome in a Streptomyces chassis strain by means of combination transfer, achieving the integrated expression of gene expression cassettes and the production of different target metabolites, comprising the steps of:

(1) Constructing recombinant plasmids pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD _p334, respectively converting competent E.coli ET12567, and selecting transformants as donor strains for joint transfer;

(2) The recipient bacteria are chosen to be Streptomyces lividans Streptomyces lividansTK as conjunctive transfer recipient bacteria, and spores of the recipient bacteria are collected to be prepared into spore suspension for conjunctive transfer;

3. The method for constructing a recombinant engineering strain of Streptomyces producing a retinoid metabolite according to claim 2, wherein the recombinant plasmid pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD _p334 is a recombinant plasmid for constructing a retinoid metabolite with an integrative vector pSET152, specifically comprising:

S5) plasmid verification, namely respectively extracting plasmids of the transformants, and obtaining recombinant plasmids pSET-mysAB, pSET-mysABC, pSET-mysABCD or pSET-mysABCD _p334 after verifying sequence correctness.

4. The method according to claim 3, wherein the sequence of insert mysAB in step S2) is shown in SEQ ID NO.1, the sequence of insert mysABC is shown in SEQ ID NO.2, the sequence of insert mysABCD is shown in SEQ ID NO.3, the sequence of insert mysABCD _p334 is shown in SEQ ID NO.4, the pSET-mysAB in step S5) comprises and expresses two upstream gene elements mysA and mysB, the pSET-mysABC comprises and expresses two upstream gene elements mysA and mysB and one downstream gene element mysC, the pSET-mysABCD or pSET-mysABCD _p334 comprises 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 engineering strain of Streptomyces lividans producing a metabolite of a kind of mycosporine according to claim 2, wherein the engineering strain of Streptomyces lividans in step (4) is named S.lividans TK K B, S.lividans TK K B, S.lividans TK B C and S.lividans TK K B C D _p334, and the four engineering strains are capable of producing 4-deoxygadusol, mycosporine-glycine, shinorine or porphyra-334, respectively, according to the differences of the four recombinant plasmids in step (1).

6. The recombinant engineering strain of Streptomyces according to claim 1 or the recombinant engineering strain of Streptomyces constructed by the method according to any one of claims 2 to 5 for use in the preparation of a mycosporine-like metabolite, comprising the steps of inoculating the recombinant engineering strain of Streptomyces into a fermentation medium for culturing, enriching and purifying to obtain the mycosporine-like metabolite, wherein the mycosporine-like metabolite comprises 4-deoxygadusol, mycosporine-glycine, shinorine or porphyra-334.