CN116239607B - Maytansine derivative and biosynthesis method and application thereof - Google Patents

Maytansine derivative and biosynthesis method and application thereof Download PDF

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CN116239607B
CN116239607B CN202310007908.4A CN202310007908A CN116239607B CN 116239607 B CN116239607 B CN 116239607B CN 202310007908 A CN202310007908 A CN 202310007908A CN 116239607 B CN116239607 B CN 116239607B
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ndei
carbamoylmaytansinol
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王浩鑫
刘清清
李瑶瑶
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Abstract

The invention relates to a maytansine derivative, a biosynthesis method and application thereof. The invention discovers SAM-dependent methyltransferase genes pba295, pba296, pba304 and nan291 from nocardia EGI80425 and metagenome, and can realize the efficient synthesis of maytansine derivative 3-O-carbamoylmaytansinol by coexpression with carbamoyltransferase asc21b, thereby effectively solving the problem that endogenous N-methyltransferase Asm10 cannot recognize and catalyze N-desmethyl-3-O-carbamoylmaytansinol and cannot prepare 3-O-carbamoylmaytansinol. The 3-O-carbamoylmaytansinol provided by the invention has obvious cytotoxicity to human breast cancer cells (MDA-MB-231), human cervical cancer cells (HeLa) and human colon cancer cells (HCT 116), so that the 3-O-carbamoylmaytansinol can replace AP-3 to become a novel maytansinoid medicine raw material.

Description

Maytansine derivative and biosynthesis method and application thereof
Technical Field
The invention relates to a maytansine derivative, a biosynthesis method and application thereof, and belongs to the technical field of microbial pharmacy.
Background
Ansamitocins (ansamitocins) are a class of maytansinoids produced by microorganisms and have extremely high cytotoxicity. Ansamitocins inhibit the polymerization of tubulin by binding to the beta subunit of tubulin, inhibiting the mitosis of tumor cells leading to cell death. The ansamitocin is produced by industrial fermentation of actinomycetes (Actinosynnema pretiosum) ATCC 31565 or ATCC 31280 and derivative strains thereof, and a main fermentation product of ansamitocin P-3 (AP-3) is firstly reduced to remove C-3 ester groups to obtain a key intermediate maytansinol, and then the key intermediate maytansinol is used for synthesizing maytansinoid antibody coupled drugs (such as T-DM 1). However, the current fermentation yield of AP-3 is not high, and the homologous impurities such as P-2, P-4, PND, AGP and ACGP exist at the same time, so that the separation and purification process is complex, and the cost is high.
The biosynthesis of ansamitocin P-3 takes 3-amino-5-hydroxybenzoic acid (AHBA) as a starting unit, the extension of polyketide chains is catalyzed by type I polyketide synthase, including 3 acetic acid units, 3 propionic acid units and 1 methoxy extension unit, followed by cyclization by amide synthase to form a 19-membered macrolide, and finally by a series of post-modification reactions, including chloro, 20-O-methylation, carbamoyl cyclization, 3-O-acylation, epoxidation and amide N-methylation to form ansamitocin. The acyl donor substrate hybridization of the key post-modifier enzyme 3-O-acyltransferase Asm19 is the main cause of ansamitocin homolog impurity formation, however, due to the catalytic timing of the last three post-modification, knocking out the Asm19 gene does not accumulate maytansinol, but rather N-desmethyl-4, 5-deoxymaytansinol (DDM). By exogenously adding valine, sodium isobutyrate or isobutanol to increase the supply of endogenous isobutyryl, the content of homologous impurities such as AP-2 and AP-4 can be reduced to less than 10%, but cannot be completely eliminated.
The carbamoyltransferase Asc21b from the amycolatopsis (ansacarbamitocins) gene cluster has strict acyl donor specificity, and can be used to replace Asm19 to effectively eliminate the acyl diversity at the C-3 position, but since the modified amide N-methyltransferase Asm10 after the last step is catalyzed, does not effectively recognize carbamoylated substrates, only N-desmethyl-3-O-carbamoylmaytansinol is formed. Therefore, the enzyme gene which can identify N-demethyl-3-O-carbamoylmaytansinol and efficiently catalyze the N methylation of amide is screened, and the enzyme gene is used for genetic modification of an ansamitocin biosynthesis pathway, so that the synthesis of 3-O-carbamoylmaytansinol has important significance for simplifying the components of ansamitocins and reducing the cost of maytansinol bulk drugs.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a maytansine derivative, and a biosynthesis method and application thereof.
The technical scheme of the invention is as follows:
a maytansine derivative 3-O-carbamoylmaytansinol having the chemical structure shown below:
an engineering bacterium for biosynthesis of the 3-O-carbamoylmaytansinol is characterized in that the metabolite of the engineering bacterium contains the 3-O-carbamoylmaytansinol shown in the formula.
According to the invention, preferably, the engineering bacteria express both 3-O-carbamoyltransferase responsible for the modification of the C-3 position and methyltransferase responsible for the N-methylation modification of the amide; the coding gene of the 3-O-carbamyl transferase is asc21b, the nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 6; the coding genes of the methyltransferase are pba, pba, 296, pba, 304 or nan291, the nucleotide sequences are shown in SEQ ID NO. 2-5, and the amino acid sequences are shown in SEQ ID NO. 7-10.
The construction method of the engineering bacteria for biosynthesis of 3-O-carbamoylmaytansinol comprises the following steps:
(1) Performing high-fidelity PCR amplification by taking genome DNA of amycolatopsis albus (Amycolatopsis alba) DSM 44262 as a template to obtain an asc21b gene fragment with NdeI and NheI enzyme cutting sites introduced at two ends respectively, connecting a PCR product with a vector pUC-kasOp-T-NdeI/NheI after double enzyme cutting of NdeI and NheI, transforming the connection product into escherichia coli DH5 alpha competent cells, and obtaining a plasmid pUC-asc21b through verification and extraction;
(2) Performing high-fidelity PCR amplification by taking Nocardia (Nocardiopsis ansamitocini) EGI80425 genome DNA as a template to obtain nan gene fragments with NdeI and NheI enzyme cutting sites introduced at two ends respectively, and performing double enzyme cutting on a PCR product by NdeI and NheI to obtain nan-NdeI/NheI fragments; artificially synthesizing SAM-dependent methyltransferases pba295, pba296 and pba304 genes with NdeI and SpeI cleavage sites introduced at both ends, cloning the genes in a pUC57 vector, and obtaining pba-NdeI/SpeI, pba296-NdeI/SpeI and pba-304-NdeI/SpeI fragments by NdeI and SpeI double cleavage; the nan291-NdeI/NheI, the pba295-NdeI/SpeI, the pba296-NdeI/SpeI and the pba-NdeI/SpeI fragments are respectively connected with vector fragments pUC-kasOp-T-NdeI/NheI, and the connection products are respectively transformed into escherichia coli DH5 alpha competent cells, and plasmids pUC-pba295, pUC-pba296, pUC-pba304 and pUC-nan291 are obtained through verification and extraction;
(3) The plasmid pUC-asc21b is subjected to double enzyme digestion by MfeI and SpeI to obtain asc21b-MfeI/SpeI fragments; plasmids pUC-pba295, pUC-pba296, pUC-pba and pUC-nan291 were digested with XbaI and PstI to obtain fragments pba-XbaI/PstI, pba296-XbaI/PstI, pba304-XbaI/PstI and nan 291-XbaI/PstI; the asc21b-MfeI/SpeI and the vector fragment p15ACIS-EcoRI/PstI were ligated with pba295-XbaI/PstI, pba296-XbaI/PstI, pba304-XbaI/PstI, nan291-XbaI/PstI, respectively, to obtain expression plasmids p15ACIS-asc21b-pba295, p15ACIS-asc21b-pba296, p15ACIS-asc21b-pba304 and p15ACIS-asc21b-nan291;
(4) And (3) respectively electrically transforming the expression plasmids obtained in the step (3) into competent cells of escherichia coli ET12567/pUZ8002, respectively introducing the competent cells into DDM producing bacteria HGF052 through conjugation transfer, purifying the zygotes and verifying by PCR to obtain engineering bacteria HGF052-asc21b-pba295, HGF052-asc21b-pba296, HGF052-asc21b-pba304 and HGF052-asc21b-nan291 for producing 3-O-carbamoylmaytansinol.
According to a preferred embodiment of the present invention, in step (1), the PCR amplification primer sequence is as follows:
NdeI-asc21b-F:5’-gaggcggacatatgctggtgctcggactgaac-3’，
NheI-asc21b-R:5’-cccgagtcagctagctcaggccggagtgagggtgaag-3’；
PCR amplification conditions: pre-denaturation, 10min at 95 ℃; denaturation, 30sec at 95 ℃, annealing, 30sec at 65 ℃, extension, 2min at 72 ℃,35 cycles; finally, the extension is carried out at 72 ℃ for 5min.
Preferably, in step (2), the PCR amplification primer sequence is as follows:
NdeI-nan291-F:5’-gaggcggacatatggtgctggaacacgaacg-3’，
NheI-nan291-R:5’-cgcccgagtcagctagcctggacacgggtgtccccttg-3’；
PCR amplification conditions: pre-denaturation, 10min at 95 ℃; denaturation, 30sec at 95 ℃, annealing, 30sec at 72 ℃, extension, 1min at 72 ℃,35 cycles; finally, the extension is carried out at 72 ℃ for 5min.
In the present invention, the vector fragments pUC-kasOp-T-NdeI/NheI and p15 ACIS-kasOp-T-NdeI/NheI were obtained after NdeI and NheI double-digested plasmids pUC-kasOp-T and p15 ACIS-kasOp-T; the vector fragment p15ACIS-EcoRI/PstI was obtained after double digestion of the plasmid p15ACIS-kasOp X-T with EcoRI and PstI. The plasmid pUC-kasOp-T is an existing plasmid.
The plasmid p15 ACIS-kasOp-T was constructed as follows: the plasmid pSET152 is used as a template, GBCIS-F/R is used as a primer, and the apramycin resistance gene and the integration element fragment are obtained through PCR amplification; then using plasmid p15A-Cm-ccdB as a template and GB15Aori-F/R as a primer, and obtaining a p15A replication region fragment through PCR amplification; ecoRI and PstI double cut pUC-kasOp-T to obtain kasOp-T-EcoRI/PstI fragment; the three fragments are spliced by Gibson, a connection system is used for transforming competent cells of escherichia coli DH5 alpha, and plasmid p15 ACIS-kasOp-T is obtained through verification and extraction;
in the construction process of the plasmid p15 ACIS-kasOp-T, the PCR primer sequence is as follows:
GBCIS-F:5’-acactccgctagggcataagggattttggtcatgag-3’
GBCIS-R:5’-gaaacaattgggaattccgatctttgtagaaaccatc-3’
GB15Aori-F:5’-gtaatgcataactgcagacaacttatatcgtatggggctg-3’
GB15Aori-R:5’-gccctagcggagtgtatactg-3’
PCR amplification conditions: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃, annealing, 30sec at 53 ℃, extension, 4min or 1min at 72 ℃,35 cycles; finally, the extension is carried out at 72 ℃ for 5min.
The complete gene synthesis of SAM-dependent methyltransferase genes pba295, pba296, pba304 according to the present invention was carried out with reference to the prior art, and the vector pUC57 was an existing plasmid vector.
The DDM-producing strain HGF052 of the present invention is an asm19 knockout mutant strain of actinomycetes treponini ATCC 31565, which is derived from the literature (Journal of American Chemical Society,2002, 124 (23), 6544-6545).
The preparation method of the maytansine derivative 3-O-carbamoylmaytansinol comprises the following steps:
1) Inoculating the engineering bacteria on YMG solid culture medium, and performing expansion culture for 5-10 days at 25-35 ℃;
2) Cutting YMG solid culture of engineering bacteria into blocks, soaking and extracting for 3 times by using ethyl acetate as an extracting solution, merging the extracting solutions, concentrating the extracting solution under reduced pressure to 200-400mL, extracting for 3 times by using ethyl acetate with equal volume, merging the extracted ethyl acetate phases, and concentrating under reduced pressure to dryness to obtain an ethyl acetate extract;
3) Extracting ethyl acetate extract with equal volume petroleum ether and methanol to obtain methanol phase and petroleum ether phase, concentrating methanol phase under reduced pressure, separating by sephadex column chromatography to obtain crude product, and preparing the crude product by HPLC to obtain 3-O-carbamoylmaytansinol shown in the above formula.
According to a preferred embodiment of the present invention, in step 1), the expansion culture is 8L expansion culture, and the culture condition is 30℃for 8 days.
According to a preferred embodiment of the present invention, in step 1), the YMG solid medium is formulated as follows: yeast extract 0.4%, malt extract 1%, glucose 0.4%,10N NaOH pH adjusted to 7.2-7.4,1.5-2% agar, and autoclaved at 115℃for 30min.
The application of the maytansinoid derivative 3-O-carbamoylmaytansinol in preparing antitumor drugs.
Pharmacological test researches show that the 3-O-carbamoylmaytansinol provided by the invention has cytotoxic activity on human breast cancer cells (MDA-MB-231), human cervical cancer cells (HeLa) and human colon cancer cells (HCT 116), has IC50 values of 5.85, 0.32 and 1.24 mu M respectively, and can be used for preparing antitumor drugs. Preferably, the tumor is breast cancer, cervical cancer or colon cancer.
The present invention is not limited to the details of the prior art.
The invention has the beneficial effects that:
1. the in vitro anti-tumor activity experiment shows that the 3-O-carbamoylmaytansinol provided by the invention has obvious cytotoxicity on human breast cancer cells (MDA-MB-231), human cervical cancer cells (HeLa) and human colon cancer cells (HCT 116), and the IC50 values are 5.85, 0.32 and 1.24 mu M respectively, so that the 3-O-carbamoylmaytansinol can be used for preparing anti-tumor drugs.
2. The maytansine derivative 3-O-carbamoylmaytansinol provided by the invention can be reduced into maytansinol by the prior art and is used for preparing maytansinol antibody coupling drugs, so that the maytansinol can be used as a new maytansinol drug raw material instead of AP-3.
3. The invention discovers SAM-dependent methyltransferase genes pba295, pba296, pba304 and nan291 which are derived from metagenome and nocardiopsis EGI80425, and can realize the efficient synthesis of maytansine derivative 3-O-carbamoylmaytansinol by coexpression with carbamoyltransferase asc21b, thereby effectively solving the problem that endogenous N-methyltransferase Asm10 cannot recognize and catalyze N-desmethyl-3-O-carbamoylmaytansinol and cannot prepare 3-O-carbamoylmaytansinol.
Drawings
FIG. 1 is a schematic representation of the genetically engineered metabolic pathway of the synthesis of 3-O-carbamoylmaytansinol according to the present invention.
FIG. 2 is a schematic flow chart for constructing a plasmid for co-expression of a carbamoyltransferase gene and a SAM-dependent methyltransferase gene according to the present invention.
FIG. 3 is a diagram showing agarose gel electrophoresis of PCR verification of 3-O-carbamoylmaytansinol producing engineering bacterium according to the present invention.
In the figure, the left panels are the strains HGF052-asc21b and HGF052-asc21 b-asc 10, and the right panels are the strains HGF052-asc21b-pba295, HGF052-asc21b-pba296, HGF052-asc21b-pba304, HGF052-asc21b-nan291.
FIG. 4 is a HPLC detection chart of the metabolite of the 3-O-carbamoylmaytansinol producing engineering bacterium of the present invention.
Detailed Description
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
Human breast cancer cells (MDA-MB-231), human cervical cancer cells (HeLa) and human colon cancer cells (HCT 116), a sea cell bank is available in the national academy of sciences. Doxorubicin hydrochloride (Dox), MCE (MedChemExpress), is commercially available.
The genetically engineered metabolic pathway for the synthesis of 3-O-carbamoylmaytansinol according to the present invention is shown in FIG. 1, and the specific procedures are described in the examples.
Example 1 construction of plasmid pUC-asc21b
PCR amplification is carried out by taking the white amycolatopsis DSM 44262 genome DNA containing the carbamoyltransferase gene asc21b as a template to obtain a 3-O-carbamoyltransferase asc21b sequence (SEQ ID NO. 1), and primers are designed by bioinformatics software to respectively introduce NdeI and NheI enzyme cutting sites at two ends of the sequence, wherein the sequences of the primers are as follows:
NdeI-asc21b-F:5’-gaggcggaCATATGctggtgctcggactgaac-3’；
NheI-asc21b-R:5’-cccgagtcaGCTAGCtcaggccggagtgagggtgaag-3’。
the PCR amplification system is as follows: 50-100ng of DNA template, 0.4 mu M of primer, 0.2mM of dNTP mix, 25 mu L of 2 XGC buffer, phanta Max Super-Fidelity DNA Polymerase U, and adding pure water to fill up to 50 mu L;
PCR amplification conditions: pre-denaturation, 10min at 95 ℃; denaturation, 30sec at 95 ℃, annealing, 30sec at 62 ℃, extension, 2min at 72 ℃,35 cycles; finally, the extension is carried out at 72 ℃ for 5min.
The PCR product is recovered and purified by a gel recovery kit, and the asc21b-NdeI/NheI fragment is obtained by double enzyme digestion; plasmid pUC-kasOp-T was digested with NdeI and NheI and dephosphorylated to obtain vector fragment pUC-kasOp-T-NdeI/NheI; through ligase connection, the connection system is chemically transformed into escherichia coli DH5 alpha competent cells, the competent cells are coated on LB solid medium containing 100 mug/mL ampicillin, the culture is carried out at 37 ℃ overnight, positive transformants are picked up, and plasmid pUC-asc21b is extracted from the positive transformants by using a plasmid extraction kit.
Example 2 construction of plasmids pUC-pba295, pUC-pba296, pUC-pba304 and pUC-nan291
PCR amplification is carried out by taking Nocardia mimetic Nocardiopsisansamitocini EGI80425 genome DNA as a template to obtain methyltransferase nan291 gene (SEQ ID NO. 5), ndeI and NheI restriction enzyme sites are respectively introduced at two ends of the sequence, and the primer sequences are as follows:
NdeI-nan291-F:5’-gaggcggaCATATGgtgctggaacacgaacg-3’；
NheI-nan291-R:5’-cgcccgagtcaGCTAGCctggacacgggtgtccccttg-3’。
the PCR amplification system comprises: 50-100ng of DNA template, 0.4 mu M of primer, 0.2mM of dNTP mix, 25 mu L of 2 XGC buffer, phanta Max Super-Fidelity DNA Polymerase U, and adding pure water to fill up to 50 mu L;
PCR amplification conditions: pre-denaturation, 10min at 95 ℃; denaturation, 30sec at 95 ℃, annealing, 30sec at 62 ℃, extension, 1min at 72 ℃,35 cycles; finally, the extension is carried out at 72 ℃ for 5min.
The PCR product was recovered and purified by a gel recovery kit, the PCR product and the vector plasmid pUC-kasOp-T were digested with NdeI and NheI, respectively, the digested products were ligated by ligase, the ligation system was chemically transformed into E.coli DH 5. Alpha. Competent cells, and the competent cells were plated on LB solid medium containing 100. Mu.g/mL ampicillin, cultured overnight at 37℃to select clones and positive transformants were selected by plasmid size, and plasmid pUC-nan291 was extracted from the positive transformants using a plasmid extraction kit.
Artificially synthesizing SAM-dependent methyltransgeration gene pba295 (SEQ ID NO. 2), pba296 (SEQ ID NO. 3), pba304 (SEQ ID NO. 4), adding NdeI and SpeI enzyme cutting sites at two ends during synthesis, cloning to pUC57 vector to obtain plasmids pUC57-pba295, pUC57-pba296 and pUC57-pba304; the above plasmid was digested with NdeI and SpeI to obtain pba295-NdeI/SpeI, pba296-NdeI/SpeI, and pba304-NdeI/SpeI fragments; the above fragment was ligated with the vector fragment pUC-kasOp-T-NdeI/NheI via ligase, and the ligation system was chemically transformed into E.coli DH 5. Alpha. Competent cells, plated on LB solid plates containing 100. Mu.g/mL ampicillin, cultured overnight at 37℃to pick up positive transformants, and plasmids pUC-pba295, pUC-pba296 and pUC-pba were extracted from the positive transformants using a plasmid extraction kit.
EXAMPLE 3 construction of expression plasmids p15ACIS-asc21b-pba295, p15ACIS-asc21b-pba296, p15ACIS-asc21b-pba304 and p15ACIS-asc21b-nan291
As shown in FIG. 2, plasmid pUC-asc21b was digested with MfeI and SpeI to obtain asc21b-MfeI/SpeI fragment; plasmids pUC-pba295, pUC-pba296, pUC-pba and pUC-nan291 were digested with XbaI and PstI to obtain fragments pba295-XbaI/PstI, pba296-XbaI/PstI, pba304-XbaI/PstI and nan 291-XbaI/PstI; plasmid p15 ACIS-kasOp-T was digested with EcoRI and PstI to obtain the p15ACIS-EcoRI/PstI vector fragment.
The asc21b-MfeI/SpeI and the vector fragment p15ACIS-EcoRI/PstI were ligated with pba-XbaI/PstI, pba296-XbaI/PstI, pba304-XbaI/PstI and nan-XbaI/PstI fragments, respectively, by means of a ligase, the ligation system was chemically transformed into E.coli DH 5. Alpha. Competent cells, and the cells were spread on LB solid medium containing 30. Mu.g/mL of apramycin, and cultured overnight at 37℃to pick up positive transformants, and plasmids p15ACIS-asc21b-pba295, p15ACIS-asc21b-pba296, p15ACIS-asc21b-pba and p15ACIS-asc21b-nan were extracted from the positive transformants using a plasmid extraction kit.
Wherein the plasmid p15 ACIS-kasOp-T is constructed as follows: the plasmid pSET152 is used as a template, GBCIS-F/R is used as a primer, and the apramycin resistance gene and the integration element fragment are obtained through PCR amplification; then using plasmid p15A-Cm-ccdB as a template and GB15Aori-F/R as a primer, and obtaining a p15A replication region fragment through PCR amplification; ecoRI and PstI double cut pUC-kasOp-T to obtain kasOp-T-EcoRI/PstI fragment; the three fragments are spliced by Gibson, the connection system is used for transforming competent cells of escherichia coli DH5 alpha, and the plasmid p15 ACIS-kasOp-T is obtained through verification and extraction. pSET152 and p15A-Cm-ccdB are existing plasmids.
In the above construction of plasmid p15 ACIS-kasOp-T, the PCR primer sequences were as follows:
GBCIS-F:5’-acactccgctagggcataagggattttggtcatgag-3’
GBCIS-R:5’-gaaacaattgggaattccgatctttgtagaaaccatc-3’
GB15Aori-F:5’-gtaatgcataactgcagacaacttatatcgtatggggctg-3’
GB15Aori-R:5’-gccctagcggagtgtatactg-3’
PCR amplification conditions: pre-denaturation, 5min at 95 ℃; denaturation, 30sec at 95 ℃, annealing, 30sec at 53 ℃, extension, 4min or 1min at 72 ℃,35 cycles; finally, the extension is carried out at 72 ℃ for 5min.
Example 4 construction of engineering bacteria HGF052-asc21b-pba295, HGF052-asc21b-pba296, HGF052-asc21b-pba304 and HGF052-asc21b-nan291
The expression plasmids p15ACIS-asc21b-pba295, p15ACIS-asc21b-pba296, p15ACIS-asc21b-pba304 and p15ACIS-asc21b-nan291 are respectively and electrically transformed into competent cells of escherichia coli ET12567/pUZ8002, and then the expression plasmids are respectively joined and transferred into actinomycetes rarius HGF052 to obtain engineering bacteria HGF052-asc21b-pba295, HGF052-asc21b-pba296, HGF052-asc21b-pba304 and HGF052-asc21b-nan291. Then respectively coating the engineering bacteria on the substrateContaining 10mM MgCl 2 The preparation method comprises the steps of (1) inversely culturing at 30 ℃ for 12 hours, covering a joint plate with 1mg of nalidixic acid and 1mg of apramycin, picking the joint when inversely culturing at 30 ℃ for about 5 days, transferring to YMG solid culture medium containing 25 mug/mL of nalidixic acid and 30 mug/mL of apramycin for purification culture, picking the pure joint after 5 days in liquid YMG culture medium containing 30 mug/mL of apramycin, culturing at 30 ℃ for about 3 days at 220rpm, and extracting genome DNA of the joint. The primers cam-verF/cam-verR were designed and used to perform PCR verification of the genomic DNA of the zygote, which was performed by agarose gel electrophoresis as shown in FIG. 3.
As can be seen from FIG. 3 (right), the expected sizes of PCR products of engineering bacteria HGF052-asc21b-pba295, HGF052-asc21b-pba296, HGF052-asc21b-pba304 and HGF052-asc21b-nan291 are 3818bp, 3821bp, 3845bp and 3806bp, respectively. The PCR product size accords with the expected zygote, namely engineering bacteria HGF052-asc21b-pba295, HGF052-asc21b-pba296, HGF052-asc21b-pba304 and HGF052-asc21b-nan291 for producing 3-O-carbamoylmaytansinol.
The formula of the YMG solid culture medium is as follows: yeast extract 0.4%, malt extract 1%, glucose 0.4%,10N NaOH pH adjusted to 7.2-7.4,1.5-2% agar, and steam sterilizing at 115deg.C for 30min.
The extraction of the DNA of the pure zygote was performed according to the prior art, and the PCR validation primer sequences were as follows:
cam-verF:5’-gtagcggcgtagcgagacac-3’；
cam-verR:5’-agccagttacctcggttcaaag-3’。
PCR conditions: pre-denaturation, 10min at 95 ℃; denaturation, 30sec at 95 ℃, annealing, 30sec at 63 ℃, extension, 2min at 72 ℃,30 cycles; finally, the extension is carried out at 72 ℃ for 5min.
EXAMPLE 5 fermentation culture for producing 3-O-carbamoylmaytansinol engineering bacterium
The engineering bacteria HGF052-asc21b-pba295, HGF052-asc21b-pba296, HGF052-asc21b-pba and HGF052-asc21b-nan291 prepared in example 4 are respectively inoculated in YMG solid medium, fermented and cultured for 8d at 30 ℃, the fermented culture is diced and soaked and extracted overnight with extraction liquid ethyl acetate-methanol-glacial acetic acid (80:15:5, v/v/v), the extraction liquid is concentrated under reduced pressure at 30 ℃ to obtain crude extract, the crude extract is dissolved by methanol, supernatant is taken for HPLC detection, and HPLC detection results are shown in figure 4.
The HPLC detection elution conditions are as follows: THERMO FISHER Ultimate, YMC-Pack Pro C18 (4.6X250 mm,5 μm), detection wavelength 254nm. The mobile phases are acetonitrile and water added with 1%o formic acid respectively, the sample injection volume is 20 mu L, and the flow rate is 1mL/min. The gradient elution conditions were: 0-5min,20% -35% acetonitrile; 20min, rising to 55% acetonitrile; 23min, raising to 100% acetonitrile, and maintaining for 4min; the reaction time was reduced to 20 acetonitrile for 28min and maintained for 2min.
The YMG solid culture medium is prepared by the following steps: yeast extract 0.4%, malt extract 1%, glucose 0.4%,10N NaOH pH adjusted to 7.2-7.4,1.5-2% agar, and steam sterilizing at 115deg.C for 30min.
EXAMPLE 6 preparation of 3-O-carbamoylmaytansinol
A method for preparing a maytansine derivative 3-O-carbamoylmaytansinol, which comprises the following steps:
1) Inoculating engineering bacteria HGF052-asc21b-pba296 to YMG solid agar culture medium, and culturing for 8d at 30 ℃ in an inverted way;
2) Cutting YMG solid culture medium, soaking with ethyl acetate overnight for 3 times, mixing the leaching solutions, concentrating the leaching solution at 30deg.C under reduced pressure to 200-400mL, extracting with ethyl acetate of equal volume for 3 times, mixing the extracted ethyl acetate phases, concentrating under reduced pressure to dryness to obtain ethyl acetate extract A;
3) Ethyl acetate extract a was extracted with an equal volume of petroleum ether and methanol, and then the methanol phase was recovered, and after evaporation of the methanol phase under reduced pressure, the crude product was isolated by Sephadex column chromatography (Sephadex LH-20, 100g, methanol, 10 s/drop, 3 mL/tube) and prepared by HPLC (48% acetonitrile, 4mL/min, UV 254 nm) to give compound 1.
Example 7 structural identification of 3-O-carbamoylmaytansinol
The compound 1 prepared in example 6 was subjected to spectroscopic data analysis by 1D and 2D NMR, UV, IR and HRMS, and the analysis results are shown in table 1.
TABLE 1 Compound 1 1 H and 13 C NMR(DSMO-d 6 ) Data
Compound 1:UV-vis(CH3CN)λmax:204,233,252,280nm；IR:3365,2935,1701,1651,1388,1342,1309,1080,1048。
as can be seen from Table 1, the molecular formula of Compound 1 was C as measured by high resolution Mass Spectrometry (HR-ESIMS) 29 H 38 C l N 3 O 9 (m/z 608.2366,calculated 608.2369 for[M+H]And (c) determining that the compound 1 is 3-O-carbamoylmaytansinol, wherein the specific structural formula is shown in the following figure:
example 8 in vitro anti-tumor Activity assay of 3-O-carbamoylmaytansinol
The CCK-8 kit was used to determine the cell growth inhibition.
The specific method comprises the following steps:
(1) Human breast cancer cells (MDA-MB-231), human cervical cancer cells (HeLa) and human colon cancer cells (HCT 116) were prepared as single cell suspensions with a culture solution containing 10% fetal bovine serum, and each well was inoculated with 90. Mu.L, 5X 10 4 Wall-attached cells per mL to 96-well plates, 5% CO 2 Pre-culturing at 37 ℃ for 24 hours;
(2) Adding 10 mu L of 3-O-carbamoylmaytansinol sample solution into each well, setting 10 gradients of 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 5, 10 and 20 mu M respectively, setting 3 compound wells for each concentration, and culturing for 48h;
(3) Aspiration of old medium and drug solution, addition of dilution tenfold per well100. Mu.L of CCK-8 solution, 37℃and 5% CO 2 Continuing to culture for 1-4 days (light-shielding operation);
(4) Measuring absorbance at 450nm by using an enzyme-labeled instrument, and recording an original data result;
(5) The raw data is standardized by Excel software, the cell proliferation inhibition rate is calculated through the OD value of each hole, and the inhibition rate is counted. IC50 values were calculated by GraphPad Prism 8 and the results are shown in table 2.
Table 2, results of cytotoxicity test of 3-O-carbamoylmaytansinol against 3 tumor cells (IC 50 ，μM)
* Is a positive control drug
Cell growth inhibition = (1-mean OD value of dosing group/OD value of control group wells) ×100%.
As can be seen from Table 2, the 3-O-carbamoylmaytansinol provided by the present invention shows cytotoxic activity against human breast cancer cells (MDA-MB-231), human cervical cancer cells (HeLa) and human colon cancer cells (HCT 116), and the IC50 values thereof are 5.85, 0.32 and 1.24. Mu.M, respectively.
Conclusion of the test: through pharmacological tests, the 3-O-carbamoylmaytansinol provided by the invention has shown cytotoxic activity on human breast cancer cells (MDA-MB-231), human cervical cancer cells (HeLa) and human colon cancer cells (HCT 116). Therefore, the 3-O-carbamoylmaytansinol provided by the invention can be used for preparing antitumor drugs, can be prepared into antitumor drug compositions with other drugs, and can be coupled with different antibodies and linkers to prepare antibody conjugates.
Construction of comparative example 1, HGF052-asc21b-asm10 Strain and metabolite detection
PCR amplification is carried out by taking the DNA of the genome of the actinomycetes with precious beam filament HGF052 as a template to obtain a methyltransferase gene asm10 fragment (the registration number of a gene bank is AF 453501.1), ndeI and NheI restriction sites are respectively introduced at two ends of the sequence, and the primer sequences are as follows:
NdeI-asm10-F:5’-accaaaggaggcggaCATATGagcctcccagcggactcac-3’；
NheI-asm10-R:5’-cgcccgagtcaGCTAGCctaccccagctcggcctcga-3’。
the PCR product obtained was recovered by purification with a gel recovery kit, the PCR product and the vector plasmid p15 ACIS-kasOp-T were digested with NdeI and NheI, respectively, the asm10-NdeI/NheI obtained by digestion was ligated with the vector fragment p15 ACIS-kasOp-T-NdeI/NheI via a ligase, the ligation system was chemically transformed into E.coli DH 5. Alpha. Competent cells, plated on LB solid medium containing 30. Mu.g/mL apramycin, cultured overnight at 37℃to select positive transformants by plasmid size selection, and plasmid p15ACIS-asm10 was obtained from the positive transformants by extraction with a plasmid extraction kit.
The plasmid pUC-asc21b is subjected to double enzyme digestion by MfeI and SpeI to obtain asc21b-MfeI/SpeI fragments; the plasmid p15ACIS-asm10 is subjected to double digestion by XbaI and PstI to obtain an asm10-XbaI/PstI fragment; the vector plasmid p15 ACIS-kasOp-T was digested with EcoRI and PstI to obtain the p15ACIS-EcoRI/PstI vector fragment.
The asc21b-MfeI/SpeI, asm10-XbaI/PstI and p15ACIS-EcoRI/PstI carrier fragments are connected by ligase, the connection system is chemically transformed into E.coli DH5 alpha competent cells, the competent cells are coated on LB solid medium containing 30 mu g/mL of apramycin, the culture is carried out at 37 ℃ overnight, positive transformants are picked up, and plasmid p15ACIS-asc21b-asm10 is obtained from the positive transformants by using a plasmid extraction kit
The expression plasmid p15ACIS-asc21 b-asc 10 is electrically transformed into competent cells of escherichia coli ET12567/pUZ8002, and is introduced into actinomycetes titude HGF052 through conjugation transfer to obtain engineering bacteria HGF052-asc21 b-asc 10. PCR verification of the genomic DNA of the zygote was performed using primers cam-verF/cam-verR, and agarose gel electrophoresis was verified by PCR as shown in FIG. 3.
As can be seen from FIG. 3 (left), the expected size of the PCR product of engineering bacterium HGF052-asc21b-asm10 is 3815bp.
The method for extracting the mutant strain genome DNA is carried out according to the prior art, and the PCR verification primer sequence is as follows:
cam-verF:5’-gtagcggcgtagcgagacac-3’；
cam-verR:5’-agccagttacctcggttcaaag-3’。
PCR conditions: pre-denaturation, 10min at 95 ℃; denaturation, 30sec at 95 ℃, annealing, 30sec at 62 ℃, extension, 2min at 72 ℃,30 cycles; finally, the extension is carried out at 72 ℃ for 5min.
Engineering bacteria HGF052-asc21 b-asc 10 are inoculated in YMG solid culture medium, fermentation culture is carried out for 8d at 30 ℃, the fermentation culture is diced and soaked and extracted overnight with extracting solution ethyl acetate-methanol-glacial acetic acid (80:15:5, v/v/v), the extracting solution is concentrated under reduced pressure at 30 ℃ to obtain crude extract, the crude extract is dissolved by methanol, supernatant is taken for HPLC detection, and the HPLC detection result is shown in figure 4.
As is clear from FIG. 4, the engineering bacterium HGF052-asc21b obtained by introducing asc21b into the DDM-producing bacterium HGF052 accumulated N-desmethyl-3-O-carbamoylmaytansinol (DCAM) in a large amount, and the target compound 3-O-Carbamoylmaytansinol (CAM) was not detected, which indicates that the endogenous N-methyltransferase Asm10 originally present could not recognize and catalyze the N-methyl synthesis of DCAM into the target compound CAM efficiently. On the basis, the expression of asm10 is enhanced by extra copy, and the obtained engineering bacterium HGF052-asc21b-asm10 can not obviously improve the condition. The engineering bacteria HGF052-asc21b-pba304, HGF052-asc21b-pba296 and HGF052-asc21b-nan291 accumulate a large amount of the main product CAM, which means that methyltransferases expressed by genes pba304, pba296 and nan291 can efficiently recognize and catalyze the N-methylation of DCAM. Engineering bacteria HGF052-asc21b-pba295 can also synthesize CAM in large quantity, but precursor DCAM still has large accumulation, which indicates that pba295 coded enzyme has insufficient catalytic efficiency and is a suboptimal choice for producing CAM.

Claims (8)

1. A maytansine derivative 3-O-carbamoylmaytansinol characterized in that its chemical structure is shown below:
2. an engineered bacterium for producing 3-O-carbamoylmaytansinol according to claim 1, characterized in that the engineered bacterium expresses both a 3-O-carbamoyltransferase responsible for modification at the C-3 position and a methyltransferase responsible for N-methylation modification of amides; the coding gene of the 3-O-carbamyl transferase is asc21b, the nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 6; the coding genes of the methyltransferase are pba295, pba296, pba304 or nan291, the nucleotide sequences are shown in SEQ ID NO. 2-5, and the amino acid sequences are shown in SEQ ID NO. 7-10;
the metabolite of the engineering bacteria contains the 3-O-carbamoylmaytansinol shown in the claim 1.
3. The construction method of engineering bacteria for biosynthesis of 3-O-carbamoylmaytansinol according to claim 2, characterized in that the steps are as follows:
(1) Performing high-fidelity PCR amplification by taking genome DNA of amycolatopsis albus (Amycolatopsis alba) DSM 44262 as a template to obtain an asc21b gene fragment with NdeI and NheI enzyme cutting sites introduced at two ends respectively, connecting a PCR product with a vector pUC-kasOp-T-NdeI/NheI after double enzyme cutting of NdeI and NheI, transforming the connection product into escherichia coli DH5 alpha competent cells, and obtaining a plasmid pUC-asc21b through verification and extraction;
(2) High-fidelity PCR amplification is carried out by taking the genomic DNA of nocardia (Nocardiopsis ansamitocini) EGI80425 as a template, ndeI and NheI restriction enzyme sites nan gene fragments are respectively introduced at two ends of the PCR product, and the PCR product is subjected to NdeI and NheI double restriction enzyme digestion, so as to obtain nan291-NdeI/NheI fragments; artificially synthesizing SAM-dependent methyltransferases pba295, pba296 and pba304 genes with NdeI and SpeI cleavage sites introduced at both ends, cloning the genes in a pUC57 vector, and obtaining pba-NdeI/SpeI, pba296-NdeI/SpeI and pba-304-NdeI/SpeI fragments by NdeI and SpeI double cleavage; the nan291-NdeI/NheI, the pba295-NdeI/SpeI, the pba296-NdeI/SpeI and the pba-NdeI/SpeI fragments are respectively connected with vector fragments pUC-kasOp-T-NdeI/NheI, and the connection products are respectively transformed into escherichia coli DH5 alpha competent cells, and plasmids pUC-pba295, pUC-pba296, pUC-pba304 and pUC-nan291 are obtained through verification and extraction;
(3) The plasmid pUC-asc21b is subjected to double enzyme digestion by MfeI and SpeI to obtain asc21b-MfeI/SpeI fragments; plasmids pUC-pba295, pUC-pba296, pUC-pba and pUC-nan291 were digested with XbaI and PstI to obtain fragments pba-XbaI/PstI, pba296-XbaI/PstI, pba304-XbaI/PstI and nan 291-XbaI/PstI; the asc21b-MfeI/SpeI and the vector fragment p15ACIS-EcoRI/PstI were ligated with pba295-XbaI/PstI, pba296-XbaI/PstI, pba304-XbaI/PstI, nan291-XbaI/PstI, respectively, to obtain expression plasmids p15ACIS-asc21b-pba295, p15ACIS-asc21b-pba296, p15ACIS-asc21b-pba304 and p15ACIS-asc21b-nan291;
(4) And (3) respectively electrically transforming the expression plasmids obtained in the step (3) into competent cells of escherichia coli ET12567/pUZ8002, respectively introducing the competent cells into DDM producing bacteria HGF052 through conjugation transfer, purifying the zygotes and verifying by PCR to obtain engineering bacteria HGF052-asc21b-pba295, HGF052-asc21b-pba296, HGF052-asc21b-pba304 and HGF052-asc21b-nan291 for producing 3-O-carbamoylmaytansinol.
4. The method of claim 3, wherein in step (1), the PCR amplification primer sequences are as follows:
NdeI-asc21b-F:5’-gaggcggacatatgctggtgctcggactgaac-3’，
NheI-asc21b-R:5’-cccgagtcagctagctcaggccggagtgagggtgaag-3’；
PCR amplification conditions: pre-denaturation, 10min at 95 ℃; denaturation, 30sec at 95 ℃, annealing, 30sec at 65 ℃, extension, 2min at 72 ℃,35 cycles; finally, the extension is carried out at 72 ℃ for 5min.
5. The method of claim 3, wherein in step (2), the PCR amplification primer sequences are as follows:
NdeI-nan291-F:5’-gaggcggacatatggtgctggaacacgaacg-3’，
NheI-nan291-R:5’-cgcccgagtcagctagcctggacacgggtgtccccttg-3’；
PCR amplification conditions: pre-denaturation, 10min at 95 ℃; denaturation, 30sec at 95 ℃, annealing, 30sec at 72 ℃, extension, 1min at 72 ℃,35 cycles; finally, the extension is carried out at 72 ℃ for 5min.
6. A process for the preparation of the maytansinol derivative 3-O-carbamoylmaytansinol according to claim 1, characterized in that it comprises the steps of:
1) Inoculating the engineering bacteria of claim 2 on YMG solid culture medium, and performing expansion culture for 5-10 days at 25-35 ℃;
2) Cutting YMG solid culture of engineering bacteria into blocks, soaking and extracting for 3 times by using ethyl acetate as an extracting solution, merging the extracting solutions, concentrating the extracting solution under reduced pressure to 200-400mL, extracting for 3 times by using ethyl acetate with equal volume, merging the extracted ethyl acetate phases, and concentrating under reduced pressure to dryness to obtain an ethyl acetate extract;
3) Extracting ethyl acetate extract with equal volume petroleum ether and methanol to obtain methanol phase and petroleum ether phase, concentrating methanol phase under reduced pressure, separating by sephadex column chromatography to obtain crude product, and preparing the crude product by HPLC to obtain 3-O-carbamoylmaytansinol shown in the above formula.
7. The method according to claim 6, wherein in step 1), the expansion culture is 8L expansion culture, and the culture condition is 30℃for 8 days; the formula of the YMG solid culture medium is as follows: yeast extract 0.4%, malt extract 1%, glucose 0.4%,10N NaOH pH adjusted to 7.2-7.4,1.5-2% agar, and autoclaved at 115℃for 30min.
8. Use of the maytansinoid derivative 3-O-carbamoylmaytansinol according to claim 1 for the preparation of an antitumor drug, wherein the tumor is breast cancer, cervical cancer or colon cancer.
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US4263294A (en) * 1978-11-20 1981-04-21 Takeda Chemical Industries, Ltd. Maytansinoids, pharmaceutical compositions thereof and method of use thereof
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