CN113699089A - Engineering strain for heterologous expression of histone deacetylase inhibitor FK228 and construction and application thereof - Google Patents
Engineering strain for heterologous expression of histone deacetylase inhibitor FK228 and construction and application thereof Download PDFInfo
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- 108010091666 romidepsin Proteins 0.000 title claims abstract description 50
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Abstract
The invention discloses an engineering strain for heterologous expression of a histone deacetylase inhibitor FK228, which is named as an engineering strain E264: R-tdp-dep and is obtained by integrating a combined biosynthesis gene cluster R-tdp-dep on a genome thereof by using Burkholderia thailandis E264 delta opraBC delta tdpDE5kb as an initial strain through a transposition method. The invention also discloses application of the engineering strain in preparation of a histone deacetylase inhibitor FK 228. The experiment proves that: the engineering strain E264 of the invention has the advantages that the amount of FK228 produced by R-tdp-dep reaches 94mg/L, which is nearly 5 times of that of the prior FK228 industrial production strain, the yield of FK228 is greatly improved, the fermentation cost is reduced, the fermentation production period is shortened, a foundation is laid for large-scale preparation and development of histone deacetylase inhibitor medicines, and the economic value and the social benefit are obvious.
Description
Technical Field
The invention relates to an engineering strain for producing a histone deacetylase inhibitor and construction and application thereof, in particular to an engineering strain for heterologously expressing a histone deacetylase inhibitor FK228 and construction and application thereof in producing FK228 by fermentation, and belongs to the technical field of biosynthesis.
Background
Histone deacetylase inhibitors (HDACi) can prevent Histone Deacetylases (HDACs) from removing acetyl on N-terminal lysine residues of histones, so that chromatin maintains a more open transcriptional activity state, thereby regulating expression of silent tumor suppressor genes. FK228, also known as Romidepsin or Depsipeptide, was originally isolated from Chromobacterium violaceum in Japanese soil samples. FK228 has a very good antitumor activity as a highly potent and selective histone deacetylase inhibitor. The FK228 antineoplastic pharmaceutical formulation (injectable) isotaxax, developed by the company Gloucester Pharmaceuticals, usa, was approved by the FDA in the united states in 2009 for the treatment of peripheral and cutaneous T-cell lymphomas.
The biosynthetic gene cluster dep of FK228 is composed mainly of three non-ribosomal polypeptide synthase genes (depA, depD and depE), two polyketide synthase genes (depB and depC), one disulfide oxidoreductase gene (depH) and one thioesterase gene (tdpH). The current wild strain, namely, the strain Chromobacterium violacea No.968 which produces FK228, has relatively limited yield (19mg/L), and the chemical synthesis thereof has proved to be very difficult, so that a method for efficiently producing the histone deacetylase inhibitor FK228 and an engineered strain for producing the histone deacetylase inhibitor FK228 with high yield are lacked.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an engineering strain for heterologous expression of histone deacetylase inhibitor FK228 and application thereof in construction and fermentation production of FK 228.
The invention relates to an engineering strain for heterologous expression of a histone deacetylase inhibitor FK228, which is characterized in that: the engineering strain is named as engineering strain E264, R-tdp-dep, and the genotype of the engineering strain is as follows: burkholderia thailandrensis E264 delta opraBC delta tdpDE5kb, apramycin resistance, tdpR, tdpA, tdpB, tdpC1, tdp-depDE DE, tdpF, tdpG, tdpH, tdpI and tdpJ are obtained by using Burkholderia thailandrensis E264 delta opraBC delta tdpDE5kb as a starting strain and integrating a combined biosynthesis gene cluster R-tdp-dep on the genome thereof by a transposition method.
The construction method of the engineering strain for heterologous expression of the histone deacetylase inhibitor FK228 comprises the following steps:
(1) assembling the synthesized three fragments of depD and depE genes PartI, PartII and PartIII with a vector pBR322-amp-ccdB fragment by using an ExoCET assembly technology to construct a plasmid pBR 322-amp-ccdB-depD-depE;
(2) replacing a tdpDE gene in a plasmid p15A-cm-tdp with a km-ccdB fragment by utilizing a Red/ET homologous recombination technology to construct a plasmid p 15A-cm-tdp-delDE-km-ccdB;
(3) digesting the plasmid pBR322-amp-ccdB-depD-depE constructed in the step (1) by using a restriction enzyme PacI to obtain a depDE linear fragment, and digesting the plasmid p15A-cm-tdp-delDE-km-ccdB constructed in the step (2) by using restriction enzymes BstZ171 and BamHI to release the vector p15A-cm-tdp-deltdpDE linear fragment; then treating the two linear fragments in vitro by T4 DNA polymerase, and then electrically transferring the two linear fragments to E.coli GB2005 to obtain a plasmid p 15A-cm-tdp-dep;
(4) inserting km-ccdB into the plasmid p15A-cm-tdp-dep constructed in the step (3) by utilizing a Red/ET homologous recombination technology for knocking out a tetracycline promoter to obtain a plasmid p 15A-cm-dep-deltetR-km-ccdB;
(5) digesting the plasmid p15A-cm-dep-deltetR-km-ccdB by using a restriction enzyme BstZ171 to release a linear fragment of the vector p 15A-cm-dep-deltetR-km-ccdB; the p15A-cm-dep-deltetR-km-ccdB linear fragment and tdpR are treated in vitro by T4 DNA polymerase, and then are electrically transferred into E.coli GB2005 to obtain a plasmid p 15A-cm-R-tdp-dep;
(6) inserting transposable elements into the plasmids p15A-cm-R-tdp-dep constructed in the step (5) respectively to construct expression plasmids p 15A-apra-tnpA-R-tdp-dep;
(7) the expression plasmid p15A-apra-tnpA-R-tdp-dep is electrically transferred into E.coli WM3064, then combined with Burkholderia thailandrosis E264 delta opraBC delta tdpDE5kb for transfer, and finally the combined biosynthesis gene cluster R-tdp-dep is integrated on a host chromosome, so that an engineering strain capable of producing FK228 is obtained, and the engineering strain is named as engineering strain E264:: R-tdp-dep.
The invention discloses application of an engineering strain for heterologously expressing a histone deacetylase inhibitor FK228 in preparation of FK 228.
Wherein:
the method for preparing FK228 by fermenting the engineering strain for heterologously expressing the histone deacetylase inhibitor FK228 comprises the following steps:
first-order seed culture: the preserved seed E264 is that R-tdp-dep picks a ring-scribing plate on a solid LB culture medium by using an inoculating ring and cultures for 12 plus or minus 2 hours in a constant temperature incubator at 37 plus or minus 1 ℃;
secondary seed culture: the cultured first-stage seeds are picked by an inoculating loop, inoculated into a 250mL shaking bottle with the liquid loading capacity of 50mL liquid LB culture medium, and are cultured at 37 +/-1 ℃ and 180 +/-20 r.min-1Culturing for 16 +/-2 h under the condition;
fermentation culture: inoculating the second-stage seeds into a 250mL shake flask containing 65mL fermentation medium according to the inoculation amount of 1% of the volume ratio, and performing fermentation at 28 +/-1 ℃ and 180 +/-20 r.min-1Culturing for 12 +/-2 h under the condition of (1); adding XAD-16 macroporous adsorbent resin suspension with final concentration of 4 wt%, at 28 + -1 deg.C and 180 + -20 r.min-1Continuously culturing for 38 +/-2 h under the condition of (1) to obtain a fermentation liquor containing FK 228;
the method for separating the fermentation liquor containing FK228 comprises the following steps:
conventionally centrifuging fermentation liquor containing FK228 to collect resin and thallus, and freeze-drying; soaking the product in dichloromethane for 4 + -1 h, filtering and separating the leaching solution, repeating for 3 + -1 times, mixing the leaching solutions, concentrating under low pressure, and drying to obtain crude extract;
primary separation and purification by using a liquid phase: ultrapure water is used as a mobile phase A, chromatographic pure acetonitrile is used as a mobile phase B, and gradient elution conditions are 0-5 min 30% B, 5-60 min 30-80% B and 8 mL/min; collecting fractions to obtain a FK228 crude product;
and (3) HPLC separation and purification: and (3) taking ultrapure water as a mobile phase A, taking chromatographic pure acetonitrile as a mobile phase B, performing gradient elution under the conditions of 0-5 min 5% B, 5-60 min 5-45% B and 3mL/min, collecting fractions, and performing low-pressure concentration and drying to obtain the FK228 pure product.
R-tdp-dep is not reported in the existing literature, and is a heterozygous gene cluster R-tdp-dep constructed by the applicant for the first time by utilizing the biosynthetic gene cluster tdp of Thailandepsins through a combined biosynthesis strategy, and the efficient FK228 expression is realized in Burkholderia thailandisis E264 delta OPrABC delta tdPE 5 kb. The experiment proves that: the engineering strain E264 provided by the invention has the advantages that the yield of FK228 produced by R-tdp-dep reaches 94mg/L, is nearly 5 times of that of the conventional FK228 industrial production strain, greatly improves the yield of FK228, reduces the fermentation production cost, shortens the fermentation production period, and lays a foundation for large-scale preparation and development of histone deacetylase inhibitor medicines. Has important research and application values for developing antitumor drugs.
Drawings
FIG. 1: the process for constructing plasmid pBR 322-amp-ccdB-depD-depE.
FIG. 2: cleavage analysis of plasmid pBR322-amp-ccdB-depD-depE recombinant.
The pBR322-amp-ccdB-depD-depE recombinant was subjected to restriction analysis using the restriction enzyme PstI. The right panel is the theoretical cleavage map, and the left panel is the actual cleavage map. The correct recombinants are marked by grey arrows.
FIG. 3: construction of plasmid p 15A-apra-tnpA-R-tdp-dep.
FIG. 4: and (3) carrying out enzyme digestion analysis on the plasmid p15A-cm-tdp-delDE-km-ccdB recon.
The p15A-cm-tdp-delDE-km-ccdB recombinants were subjected to double-restriction analysis with the restriction enzymes AscI and NcoI. The left panel is the actual cleavage map and the right panel is the theoretical cleavage map. In the theoretical cleavage map, E represents p15A-cm-tdp-delDE-km-ccdB, and C represents a control plasmid p 15A-cm-tdp. The correct recombinants are marked by grey arrows.
FIG. 5: cleavage analysis of the plasmid p15A-cm-tdp-dep recombinants.
The p15A-cm-tdp-dep recombinants were subjected to restriction analysis with the restriction enzyme PstI. The left panel is the actual cleavage map and the right panel is the theoretical cleavage map. In the theoretical cleavage map, E represents p15A-cm-tdp-dep, and C represents a control plasmid p 15A-cm-tdp-delDE-km-ccdB. The correct recombinants are marked by grey arrows.
FIG. 6: and (3) carrying out enzyme digestion analysis on a plasmid p15A-cm-dep-deltetR-km-ccdB recon.
The p15A-cm-dep-deltetR-km-ccdB recombinants were subjected to double-restriction analysis using the restriction enzymes AscI and SacI. The left panel is the actual cleavage map and the right panel is the theoretical cleavage map. In the theoretical cleavage map, E represents p15A-cm-dep-deltetR-km-ccdB, and C represents the control plasmid p 15A-cm-tdp-dep. The correct recombinants are marked by grey arrows.
FIG. 7: restriction analysis of the plasmid p15A-cm-R-tdp-dep recombinants.
The p15A-cm-R-tdp-dep recombinants were subjected to double-restriction analysis with the restriction enzymes PstI and NcoI. The left panel is the actual cleavage map and the right panel is the theoretical cleavage map. In the theoretical cleavage map, E represents p15A-cm-R-tdp-dep, and C represents the control plasmid p 15A-cm-dep-deltetR-km-ccdB. The correct recombinants are marked by grey arrows.
FIG. 8: and (3) carrying out enzyme digestion analysis on the plasmid p15A-apra-tnpA-R-tdp-dep recon.
The p15A-apra-tnpA-R-tdp-dep recombinants were subjected to double-restriction analysis with the restriction enzymes PstI and NcoI. The left panel is the actual cleavage map and the right panel is the theoretical cleavage map. In the theoretical enzymatic cleavage map, E represents p15A-apra-tnpA-R-tdp-dep, and C represents the control plasmid p 15A-cm-R-tdp-dep. The correct recombinants are marked by grey arrows.
FIG. 9: quantitative analysis of R-tdp-dep FK228 production of the engineering strain E264.
Detailed Description
The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
General description:
the strain E.coli GB2005, the recombinase-expressing strain E.coliGB08-red, the recombinase-expressing strain E.coliGBdir-gyrA462 and the recombinase-expressing strain E.coliGBred-gyrA462 which are referred to in the following examples are all purchased from GeneBridges, Germany; coliwm3064 and Burkholderia thailandris E264 Δ oprac Δ tdpDE5kb were from university of shandong-helmholtz biotechnology institute.
The recombinase expression plasmid pSC101-BAD-ETgA-tet, plasmid p15A-cm-tetR-tetO-hyg-ccdB and pR6K-oriT-tnpA-apra were all purchased from GeneBridges, Germany.
LB culture medium: 10 g.L of Tryptone-1，Yeast extract 5g·L-1，NaCl 10g·L-1In the solid medium, 12 g/L of agar is added-1(ii) a Fermentation medium: glucose 10 g.L-1Tryptone 1.67 g.L-1，K2HPO4·3H2O 9.17g·L-1， KH2PO4 2g·L-1Sodium citrate 0.5 g.L-1，MgSO4·7H2O 0.1g·L-1，pH 7.0。
The sequences of the genes of the biosynthesis gene cluster of Thailandepsins tdp and FK228 dep are reported in NCBI.
Gene sequencing in plasmid construction was accomplished by Huada Gene. The plasmids are all conventional plasmids which are sold on the market, and the method for electrotransformation into recipient bacteria is a conventional method.
In the following examples, other reagents and consumables are all made in China. Unless otherwise specified, the experimental methods and reagents in the examples are conventional in the art and commercially available, and are commercially available.
Example 1: construction of plasmid pBR322-amp-ccdB-depD-depE
The procedure for constructing plasmid pBR322-amp-ccdB-depD-depE is shown in FIG. 1.
The method comprises the following specific steps: the genes depD and depE were synthesized in three segments based on the DNA sequence of the FK228 biosynthetic gene cluster in NCBI. Three DNA sequences are respectively named as PartI, PartII and PartIII, and the three fragments are respectively provided with homologous arms and can be assembled with a vector. And (3) amplifying a pBR322-amp-ccdB fragment by using pBR322-amp-ccdB-neo-rpsL as a template and using primers BR 322-ampcdB-DE-1 and BR 322-ampcdB-DE-2 to obtain a PCR product pBR322-amp-ccdB PCR. Two ends of the pBR322-amp-ccdB PCR are respectively provided with homologous arms of PartI and PartIII, and the pBR322-amp-ccdB PCR is subjected to gel cutting recovery. PartI, PartII, PartIII and pBR322-amp-ccdB PCR were incubated with T4 DNA polymerase in vitro, then transferred electrically to E. coliGBdir-gyrA462 induced by 10% L-arabinose, after 1h of cell recovery, spread evenly on LB plates containing 50. mu.g/ml ampicillin, placed in an incubator at 37 ℃ and cultured overnight. Single colonies were picked, digested with the restriction enzyme PstI (see FIG. 2 for restriction analysis), and the correct recombinant plasmid pBR322-amp-ccdB-depD-depE was selected. The recombinant plasmid with the correct restriction enzyme analysis was sequenced with the primers ccdB-1-seq and pBR322-1-seq, respectively, to ensure that the sequences in the homologous arm regions were unchanged.
T4 DNA polymerase in vitro incubation step: 200ng of pBR322-amp-ccdB PCR, PartI, PartII and PartIII were taken, respectively, and 0.2U T4 DNA polymerase (New England Biolabs, cat.no. M0203) was added to 20. mu.L of the reaction mixture and incubated at 25 ℃ for 1 hour, 75 ℃ for 20min, 50 ℃ for 2 hours and 4 ℃.
The electrotransformation steps are as follows: coli GBdir-gyrA462 was cultured in LB medium (low salt, 1% Triptone, 0.5% yeast extract, 0.1% NaCl) at 37 ℃ Overnight (OD)6003-4). 40 μ l of overnight culture (OD)6003-4) were transferred to 1.3ml LB, incubated at 950rpm for 2h (OD) on Eppendorf thermomixer at 30 ℃6000.35 to 0.4). To the cultureMu.l of 10% L-arabinose was added thereto, and the mixture was incubated at 37 ℃ and 950rpm for 40min on an Eppendorf thermomixer. Cells were harvested by centrifugation at 9,400g for 30 sec. Discard the supernatant and precipitate with 1ml H2And O is suspended. Repeated centrifugation, resuspension, recentrifugation, discarding the supernatant, 20. mu. l H2O suspending the cells. A DNA mixture incubated with T4 NDA polymerase was added, and the mixture of cells and DNA was transferred to a 1mm cuvette and shocked with Eppendorf electroporator 2510 at 1350V, a capacitance of 10 μm f, and a resistance of 600. omega. Add 1ml LB to the cuvette, wash the cells and transfer them to a 1.5ml perforated tube, incubate them at 37 ℃ and 950rpm for 1h on an Eppendorf thermomixer. Finally, all the bacterial solutions were spread on LB plates to which 50. mu.g/ml ampicillin had been added, and cultured overnight at 37 ℃.
BR322-ampccdB-DE-1:
BR322-ampccdB-DE-2:
The specific method for PCR amplification of fragment pBR322-amp-ccdB by using primers BR 322-ampcdB-DE-1 and BR 322-ampcdB-DE-2 is as follows:
PCR amplification System:
PCR procedure: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 15 s; annealing at 60 deg.C (set according to the Tm of the primer) for 30 s; extension at 72 ℃ for 4min (extension time determined by the amplified length, 1kb/1 min); circulating for 30 times; finally 72 ℃ for 10 min.
The sequences of the primers used to sequence the regions of homology arms were as follows:
ccdB-1-seq:TGGTGCATATCGGGGATGAAAGC
pBR322-1-seq:GTATCCGGTAAGCGGCAGGGTC
example 2: construction of plasmid p15A-apra-tnpA-R-tdp-dep
The procedure for constructing the plasmid p15A-apra-tnpA-R-tdp-dep is shown in FIG. 3.
(1) Construction of plasmid p15A-cm-tdp-delDE-km-ccdB
Using pR6K-km-ccdB as a template, using kmccdB-1 and kmccdB-2 as primers to amplify a km-ccdB fragment, respectively carrying homologous arms at two ends of a tdpDE gene, carrying out gel recovery on the obtained fragment km-ccdB PCR, then electrically transferring the purified km-ccdB PCR fragment to E.coli GBred-gyrA462 carrying plasmid p15A-cm-tdp and induced by 10% L-arabinose, after the cells are recovered for 1h, uniformly coating the fragment on an LB plate added with 30 mu g/ml kanamycin, placing the plate in a constant temperature incubator at 37 ℃ and carrying out overnight culture. Single colonies were picked, double digested with restriction enzymes PstI and AscI (see FIG. 4 for restriction enzyme electrophoresis analysis), and the correct recombinant plasmid p15A-cm-tdp-delDE-km-ccdB was selected. The recombinant plasmid with the correct restriction enzyme analysis was sequenced with primers neo-5out-seq and neo-3out-seq, respectively, to ensure that the sequences in the homologous arm regions were not altered.
kmccdB-1:
kmccdB-2: 
(lower case letters are homology arms and bold upper case letters are BstZ171 and BamHI cleavage sites).
The specific method for PCR amplification of fragment kan-ccdB by primers kmcccdB-1 and kmcccdB-2 is as follows:
PCR amplification System:
PCR procedure: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 15 s; annealing at 58 deg.C (set according to the Tm value of the primer) for 30 s; extension at 72 ℃ for 4min (extension time determined by the amplified length, 1kb/1 min); circulating for 30 times; finally 72 ℃ for 10 min.
The sequences of the primers used to sequence the regions of homology arms were as follows:
neo-5out-seq:AATCCATCTTGTTCAATCAT
neo-3out-seq:TGGCGGCGAATGGGCTGACC
(2) construction of plasmid p15A-cm-tdp-dep
The plasmid pBR322-amp-ccdB-depD-depE was digested with the restriction enzyme PacI to release the depD-depE fragment. The plasmid p15A-cm-tdp-delDE-km-ccdB is subjected to double digestion by restriction enzymes BstZ171 and BamHI to release a linear fragment of the vector p15A-cm-tdp-deltdpDE, and a digestion product is recovered by alcohol precipitation. Then, the recovered p15A-cm-tdp-deltdpDE linear fragment and depD-depE fragment were treated in vitro with T4 DNA polymerase, and then were electrically transferred to E.coli GB2005, after the cells were recovered for 1 hour, they were uniformly spread on LB plates of 30. mu.g/ml chloramphenicol, and placed in a 37 ℃ incubator for overnight culture. Single colonies were picked and double digested with restriction enzymes PstI and PvuII (see FIG. 5 for restriction analysis), and the correct recombinant plasmid p15A-cm-tdp-dep was selected.
T4 DNA polymerase treatment step: to a 0.2ml PCR tube were added 200ng of p15A-cm-tdp-deltdpDE linear fragment, 200ng of depD-depE fragment, 2ul of 10 XNEB buffer 2.1 and 0.13ul of 3U ul of-1T4 DNA polymerase (T4pol), 20ul was made up with water. The following cycles were then performed in the PCR instrument: 25 ℃ for 20min, 75 ℃ for 20min, 50 ℃ for 30min, and finally 4 ℃ for heat preservation. The reaction product was desalted with a Millipore membrane filter (Merck-Millipore, cat. No. VSWP01300) for 30min at room temperature before electrotransformation.
(3) Construction of plasmid p15A-cm-dep-deltetR-km-ccdB
Firstly, pR6K-km-ccdB is taken as a template, tdp-km-1 and tdp-km-2 are taken as templates to amplify a fragment km-ccdB, homology arms at two ends of a tetracycline promoter sequence are arranged on the fragment, and the amplified fragment is recovered and purified by glue. The purified fragment, km-ccdB, was electroporated into E.coli GBred-gyrA462 containing plasmid p15A-cm-tdp-dep and induced with 10% L-arabinose, after 1h of cell recovery, it was spread evenly onto LB plates containing 30. mu.g/ml kanamycin, placed in an incubator at 37 ℃ and cultured overnight. Single colonies were picked, double digested with restriction enzymes BstZ171 and PstI (see FIG. 6 for restriction electrophoresis), and the correct recombinant plasmid p15A-cm-dep-deltetR-km-ccdB was selected. The recombinant plasmid with the correct restriction enzyme analysis was sequenced with primers neo-5out-seq and neo-3out-seq, respectively, to ensure that the sequences in the homologous arm regions were not altered.
tdp-km-1:
tdp-km-2:
The specific method for amplifying the fragment km-ccdB by using the primers tdp-km-1 and tdp-km-2PCR is as follows:
PCR amplification System:
PCR procedure: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 15 s; annealing at 58 deg.C (set according to the Tm value of the primer) for 30 s; extension at 72 ℃ for 4min (extension time determined by the amplified length, 1kb/1 min); circulating for 30 times; finally 72 ℃ for 10 min.
The sequences of the primers used to sequence the regions of homology arms were as follows:
neo-5out-seq:AATCCATCTTGTTCAATCAT
neo-3out-seq:TGGCGGCGAATGGGCTGACC
(4) construction of plasmid p15A-cm-R-tdp-dep
The Burkholderia thailandrensis E264 genome is taken as a template, tdpR-1 and tdpR-2 are taken as primers, a target fragment tdpR is amplified, both ends of the target fragment tdpR are provided with homologous arms of sequences at both ends of a tetracycline promoter sequence, and the amplified fragment is recovered and purified by glue.
The plasmid p15A-cm-dep-deltetR-km-ccdB is subjected to enzyme digestion by using a restriction enzyme BstZ171 to release a vector p15A-cm-dep-deltetR-km-ccdB linear fragment, and an enzyme digestion product is recovered by alcohol precipitation. Then, the recovered p15A-cm-dep-deltetR-km-ccdB linear fragment and tdpR fragment were treated with T4 DNA polymerase in vitro, then transferred electrically to E.coli GB2005, and after the cells were recovered for 1 hour, they were uniformly spread on LB plates of 30. mu.g/ml chloramphenicol, and placed in an incubator at 37 ℃ for overnight culture. Single colonies were picked and double digested with restriction enzymes PstI and NcoI (see FIG. 7 for restriction electrophoresis), and the correct recombinant plasmid p15A-cm-R-tdp-dep was selected. The recombinant plasmids with the correct restriction enzyme analysis were sequenced with primers R1-3in-seq, R1-3out-seq, R2-seq, R3-seq and R4-seq, respectively, to ensure that no changes in the PCR product sequence occurred.
tdpR-1:cttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattTCAGCTGAATTGTGACGACT
tdpR-2: ccgacttaaccttgcttttcagatgcgcatcgcttcggggctcgaccacTCGTGCAACTCTTTATCAACG (lower case homology arms).
The specific method for PCR amplification of the tdpR fragment by using the primers tdpR-1 and tdpR-2 is as follows:
PCR amplification System:
PCR procedure: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 15 s; annealing at 60 deg.C (set according to the Tm of the primer) for 30 s; extension at 72 ℃ for 4min (extension time determined by the amplified length, 1kb/1 min); circulating for 30 times; finally 72 ℃ for 10 min.
The primer sequences used for sequencing the PCR products were as follows:
R1-3in-seq:GCGCTGGAGAATCTCTTGCC
R1-3out-seq:GGCAAGAGATTCTCCAGCGC
R2-seq:CGGAATGCATGGGTGGCCTC
R3-seq:AATGCGGCGCGGATGTCATG
R4-seq:GTTTTAAATGAATCGATATG
(5) construction of plasmid p15A-apra-tnpA-R-tdp-dep
Firstly, pR6K-oriT-tnpA-apra is taken as a template, oriT-TnpA-apra-1 and oriT-TnpA-apra-2 are taken as templates to amplify a fragment oriT-tnpA-apra, wherein the fragment oriT-tnpA-apra is provided with homologous arms at two ends of a chloramphenicol gene sequence on a p15A-cm-R-tdp-dep plasmid, and the amplified fragment is recovered and purified by glue. The purified fragment oriT-tnpA-apra was electroporated into E.coli GB08-red containing plasmid p15A-cm-R-tdp-dep and induced with 10% L-arabinose, and after 1h of cell recovery, it was spread evenly on LB plates of 20. mu.g/ml apramycin and placed in a 37 ℃ incubator for overnight culture. Single colonies were picked and double digested with restriction enzymes PstI and NcoI (see FIG. 8 for restriction analysis), and the correct recombinant plasmid p15A-apra-tnpA-R-tdp-dep was selected. The recombinant plasmid with correct restriction enzyme analysis is sequenced by using primers R1-3out-seq and apra-3out-seq respectively, and the sequences of the primer sequences used for sequencing the homologous arm regions are as follows:
oriT-TnpA-apra-1:ACTAGTGCTTGGATTCTCAC
oriT-TnpA-apra-2:
tccgttgacgaagtcgcgcaaactacgtatatcgaaacacagtcgtcacaattcagctgaCATGCTACGTATGCCGGCAC (lower case letters are homology arms).
The specific method for PCR amplification of fragment oriT-tnpA-apra by primers oriT-TnpA-apra-1 and oriT-TnpA-apra-2 is as follows:
PCR amplification System:
PCR procedure: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 15 s; annealing at 60 deg.C (set according to the Tm of the primer) for 30 s; extension at 72 ℃ for 4min (extension time determined by the amplified length, 1kb/1 min); circulating for 30 times; finally 72 ℃ for 10 min.
The sequences of the primers used to sequence the regions of homology arms were as follows:
R1-3out-seq:GGCAAGAGATTCTCCAGCGC
apra-3out-seq:GAATGACCACTGCTGTGAG
example 3: the construction of the engineering strain E264 of the invention R-tdp-dep
The expression plasmid p15A-apra-tnpA-R-tdp-dep is electrically transferred into E.coli WM3064, then combined with Burkholderia thailandris E264 delta oprABC delta tdpDE5kb for transfer, and finally the combined biosynthesis gene cluster R-tdp-dep is integrated into a host chromosome, so that the engineering strain E264:: R-tdp-dep capable of producing FK228 is obtained.
A bonding transfer step: firstly, electrically transferring a plasmid p15A-apra-tnpA-R-tdp-dep into E.coli WM3064, and selecting a single clone for enzyme digestion identification and screening of a correct clone; e.coli WM3064 containing plasmid p15A-apra-tnpA-R-tdp-dep was streaked on LB plate with 20. mu.g/ml apramycin and 1mM DAP, and cultured in an inverted state at 37 ℃ overnight; burkholderia thailandrensis E264 delta opraBC delta tdpDE5kb was streaked on an LB plate, and was cultured in an inverted manner at 37 ℃ overnight; picking overnight cultured Burkholderia thailandrensis E264. delta. opraBC. delta. tdpDE5kb on LB plate containing 1mM DAP, streaking overnight cultured E.coli WM3064 containing plasmid p 15A-apra-tnpA-R-tdp-dep; after 6h of culture, the lawn was washed out with LB, 100ul was aspirated, and streaked onto LB plates containing 20. mu.g/ml apramycin. The single clone was picked and subjected to colony PCR to identify the correct clone.
Example 4: the invention discloses a fermentation method of an engineering strain for efficiently producing FK228
First-order seed culture: selecting a ring-scribing plate from the R-tdp-dep by using an inoculating ring, and culturing for 12h in a constant-temperature incubator at 37 ℃; second stageSeed culture: the cultured first-stage seeds are picked up by an inoculating loop, inoculated into a 250mL shaking flask containing 50mL liquid LB culture medium, and subjected to 180 r.min at 37 DEG C-1Culturing for 16h under the condition; fermentation culture: inoculating the second-stage seeds into a 250mL shake flask containing 65mL fermentation medium according to the inoculation amount of 1% of the volume ratio, and performing fermentation at 28 ℃ for 180 r.min-1Culturing for 12h under the condition of (1); adding XAD-16 macroporous adsorbent resin suspension with final concentration of 4 wt%, and culturing at 28 deg.C for 38 hr under 180r min-1 to obtain FK 228-containing fermentation broth.
Example 5: the invention relates to a method for separating and preparing FK228
Centrifugally collecting resin and thalli from the obtained fermentation liquor containing FK228 by a conventional method, and freeze-drying; soaking the product in dichloromethane for 4 hr, vacuum filtering to separate the leaching solution, repeating for 3 times, mixing the leaching solutions, concentrating under low pressure, and drying to obtain crude extract; and (3) performing primary separation and purification by using a liquid phase: gradient elution conditions (0-5 min 30% B, 5-60 min 30-80% B,8mL/min) with ultrapure water as mobile phase A and chromatographic pure acetonitrile as mobile phase B; collecting fractions to obtain a FK228 crude product; HPLC separation purification (shimadzu): taking ultrapure water as a mobile phase A and chromatographic pure acetonitrile as a mobile phase B, collecting fractions under gradient elution conditions (0-5 min 5% B, 5-60 min 5-45% B,3mL/min), concentrating and drying at low pressure to obtain the FK228 pure product.
Claims (4)
1. An engineering strain for heterogeneously expressing a histone deacetylase inhibitor FK228 is characterized in that: the engineering strain is named as engineering strain E264, R-tdp-dep, and the genotype of the engineering strain is as follows: burkholderia thailandrensis E264 delta opraBC delta tdpDE5kb, apramycin resistance, tdpR, tdpA, tdpB, tdpC1, tdp-depDE DE, tdpF, tdpG, tdpH, tdpI and tdpJ are obtained by using Burkholderia thailandrensis E264 delta opraBC delta tdpDE5kb as a starting strain and integrating a combined biosynthesis gene cluster R-tdp-dep on the genome thereof by a transposition method.
2. The method for constructing the engineering strain for heterologously expressing the histone deacetylase inhibitor FK228 as claimed in claim 1 comprises the following steps:
(1) assembling the synthesized three fragments of depD and depE genes PartI, PartII and PartIII with a vector pBR322-amp-ccdB fragment by using an ExoCET assembly technology to construct a plasmid pBR 322-amp-ccdB-depD-depE;
(2) replacing a tdpDE gene in a plasmid p15A-cm-tdp with a km-ccdB fragment by utilizing a Red/ET homologous recombination technology to construct a plasmid p 15A-cm-tdp-delDE-km-ccdB;
(3) digesting the plasmid pBR322-amp-ccdB-depD-depE constructed in the step (1) by using a restriction enzyme PacI to obtain a depDE linear fragment, and digesting the plasmid p15A-cm-tdp-delDE-km-ccdB constructed in the step (2) by using restriction enzymes BstZ171 and BamHI to release the vector p15A-cm-tdp-deltdpDE linear fragment; then treating the two linear fragments in vitro by T4 DNA polymerase, and then electrically transferring the two linear fragments to E.coli GB2005 to obtain a plasmid p 15A-cm-tdp-dep;
(4) inserting km-ccdB into the plasmid p15A-cm-tdp-dep constructed in the step (3) by utilizing a Red/ET homologous recombination technology for knocking out a tetracycline promoter to obtain a plasmid p 15A-cm-dep-deltetR-km-ccdB;
(5) digesting the plasmid p15A-cm-dep-deltetR-km-ccdB by using a restriction enzyme BstZ171 to release a linear fragment of the vector p 15A-cm-dep-deltetR-km-ccdB; the p15A-cm-dep-deltetR-km-ccdB linear fragment and tdpR are treated in vitro by T4 DNA polymerase, and then are electrically transferred into E.coli GB2005 to obtain a plasmid p 15A-cm-R-tdp-dep;
(6) inserting transposable elements into the plasmids p15A-cm-R-tdp-dep constructed in the step (5) respectively to construct expression plasmids p 15A-apra-tnpA-R-tdp-dep;
(7) the expression plasmid p15A-apra-tnpA-R-tdp-dep is electrically transferred into E.coli WM3064, then combined with Burkholderia thailandrosis E264 delta opraBC delta tdpDE5kb for transfer, and finally the combined biosynthesis gene cluster R-tdp-dep is integrated on a host chromosome, so that an engineering strain capable of producing FK228 is obtained, and the engineering strain is named as engineering strain E264:: R-tdp-dep.
3. The use of the engineered strain heterologously expressing the histone deacetylase inhibitor FK228 as defined in claim 1 in the preparation of FK 228.
4. Use according to claim 1, characterized in that:
the method for preparing FK228 by fermenting the engineering strain for heterologously expressing the histone deacetylase inhibitor FK228 comprises the following steps:
first-order seed culture: the preserved seed E264 is that R-tdp-dep picks a ring-scribing plate on a solid LB culture medium by using an inoculating ring and cultures for 12 plus or minus 2 hours in a constant temperature incubator at 37 plus or minus 1 ℃;
secondary seed culture: the cultured first-stage seeds are picked by an inoculating loop, inoculated into a 250mL shaking bottle with the liquid loading capacity of 50mL liquid LB culture medium, and are cultured at 37 +/-1 ℃ and 180 +/-20 r.min-1Culturing for 16 +/-2 h under the condition;
fermentation culture: inoculating the second-stage seeds into a 250mL shake flask containing 65mL fermentation medium according to the inoculation amount of 1% of the volume ratio, and performing fermentation at 28 +/-1 ℃ and 180 +/-20 r.min-1Culturing for 12 +/-2 h under the condition of (1); adding XAD-16 macroporous adsorbent resin suspension with final concentration of 4 wt%, at 28 + -1 deg.C and 180 + -20 r.min-1Continuously culturing for 38 +/-2 h under the condition of (1) to obtain a fermentation liquor containing FK 228;
the method for separating the fermentation liquor containing FK228 comprises the following steps:
conventionally centrifuging fermentation liquor containing FK228 to collect resin and thallus, and freeze-drying; soaking the product in dichloromethane for 4 + -1 h, filtering and separating the leaching solution, repeating for 3 + -1 times, mixing the leaching solutions, concentrating under low pressure, and drying to obtain crude extract;
primary separation and purification by using a liquid phase: ultrapure water is used as a mobile phase A, chromatographic pure acetonitrile is used as a mobile phase B, and gradient elution conditions are 0-5 min 30% B, 5-60 min 30-80% B and 8 mL/min; collecting fractions to obtain a FK228 crude product;
and (3) HPLC separation and purification: and (3) taking ultrapure water as a mobile phase A, taking chromatographic pure acetonitrile as a mobile phase B, performing gradient elution under the conditions of 0-5 min 5% B, 5-60 min 5-45% B and 3mL/min, collecting fractions, and performing low-pressure concentration and drying to obtain the FK228 pure product.
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| CN114410560A (en) * | 2022-01-28 | 2022-04-29 | 山东大学 | Engineering strain for high yield of FK228 and construction and application thereof |
| CN115947774A (en) * | 2022-09-15 | 2023-04-11 | 山东大学 | Compound spiroruchostatin E and preparation method and application thereof |
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| WO2018149282A1 (en) * | 2017-02-20 | 2018-08-23 | 复旦大学 | Method for heterologous expression of epothilone |
| CN109486848A (en) * | 2018-11-23 | 2019-03-19 | 山东大学 | A kind of construction method containing the more operon artificial gene cluster plasmids of pleocidin and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114410560A (en) * | 2022-01-28 | 2022-04-29 | 山东大学 | Engineering strain for high yield of FK228 and construction and application thereof |
| CN114410560B (en) * | 2022-01-28 | 2023-09-15 | 山东大学 | Engineering strain for high-yield FK228 and construction and application thereof |
| CN115947774A (en) * | 2022-09-15 | 2023-04-11 | 山东大学 | Compound spiroruchostatin E and preparation method and application thereof |
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