Method for efficiently preparing maytansinol by using heterologous alanyl enzyme

Abstract

The invention relates to a method for efficiently preparing maytansinol by using heterologous alanyl enzyme. The invention provides a genetically engineered bacterium HGFS10 for efficiently preparing maytansinol by using heterologous alanyl enzyme. The genetically engineered bacterium HGFS10 is used for heterologous expression of D-alanyl enzyme, overexpression of a positive regulatory factor gene asm18, overexpression of a C-4 / 5 epoxidase gene asm11 and overexpression of an amide N-methylase gene asm10. The coding gene of the D-alanyl enzyme is smast C, the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the coding gene is as shown in SEQ ID NO. 2. The in-vitro catalytic efficiency of the D-alanyl enzyme SmAstC on N-demethyl-4, 5-deepoxymaytansinol is 8 times that of a homologous protein AstC of the D-alanyl enzyme SmAstC, and a good tool enzyme is provided for biological manufacturing of maytansinol. D-alanine is added into the culture medium, a direct acyl donor of the enzyme SmAstC is optimized, and the yield of maytansinol reaches 16.18 mg / L and is increased by 15.7% compared with the yield of maytansinol without adding D-alanine into the culture medium.

Description

Technical Field

[0001] The invention relates to a method for efficiently preparing maytansinol by utilizing heterologous alanylase, and belongs to the technical field of microbial pharmaceutical manufacturing. Background Art

[0002] Maytansine is a class of microtubule inhibitors with strong cytotoxic activity, which has strong inhibitory activity against a variety of tumor cells. Due to its narrow therapeutic window and excessive toxic side effects, it is difficult to use it clinically as a monomer drug. With the gradual development of antibody-drug conjugates (ADCs), maytansine derivatives have been widely used as small molecule toxins in ADC drug development, and many drugs are currently on the market or in the clinical trial stage. Among them, Trastuzumab Emtansine (T-DM1) was approved by the US FDA in 2013 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive advanced breast cancer, and its indications were added in 2019. In 2020, T-DM1 was approved for marketing in my country and became the first approved ADC drug. In 2022, another ADC drug, mirvetuximabsoravtansine-gynx (Elahere), which uses maytansine derivatives as "warheads", was approved for marketing by the FDA on an accelerated basis, becoming the first ADC drug for ovarian cancer.

[0003] Therefore, with the rapid development of maytansinoid ADC drugs, there is an urgent need to achieve the efficient preparation of maytansinoid target heads. However, due to the extremely low content of maytansinoids in plants, the preparation of maytansinoid derivatives currently usually adopts microbial fermentation to obtain the structural analog ansamitocin, and then removes the acyl group connected to the C-3 hydroxyl group by chemical method to obtain the intermediate maytansinol. Maytansinol is further chemically modified and coupled to the corresponding antibody with a linker to finally prepare an ADC drug. Ansamitocin can be produced by Actinomyces aurantium, and its biosynthetic pathway has been analyzed in detail at the beginning of this century. Among them, 3-O-acyltransferase Asm19 is responsible for the fatty acyl loading of the C-3 hydroxyl group of ansamitocin.

[0004] However, due to the strict post-biosynthetic modification sequence of ansamitocin, knocking out the asm19 gene in vivo does not allow the strain to accumulate maytansinol in large quantities, but mainly accumulates the intermediate metabolite N-demethyl-4,5-deepoxy maytansinol. Therefore, whether it is the ansamitocin-producing bacterium Actinosynnemapretiosum ssp. auranticum ATCC31565 or its asm19 gene knockout mutant HGF052, maytansinol only exists in trace amounts as a branch product rather than a major metabolite. The main method for preparing maytansinol currently used in production is to remove the 3-O-acyl group by chemical methods, but this method not only has harsh reaction conditions but also produces by-products that are difficult to eliminate, which restricts the efficient preparation of maytansinol, an important intermediate.

[0005] Chinese patent document CN111635418A a kind of maytansinoid derivative and its synthesis method and application disclose the integrated expression vector pSBT11-astC comprising alanylase gene astC, and realize heterologous expression of alanylase gene in precious orange actinomycetes mutant. From the crude extract of precious orange actinomycetes mutant expressing alanylase, a modified maytansinoid derivative after alanylation is separated and purified, but the derivative lacks the key C-4 / 5 epoxidation modification and amide N-methylation modification in the maytansinoid structure. Based on this, Chinese patent document CN114875095A a kind of alanyl maytansinol and its synthesis method and application disclose the method for obtaining a hybrid non-ribosomal polypeptide synthetase by replacing the TE domain of AstC, and realize the preparation of alanyl maytansinol by expressing the enzyme. However, the alanyl maytansinol obtained is difficult to determine due to the problem of product configuration and extremely low yield, and it is difficult to be used as the direct source of ADC drug target head. In addition, in the in vitro stability experiment of alanyl maytansinol, it was found that alanyl maytansinol would partially spontaneously hydrolyze into maytansinol.

[0006] The reason why a small amount of maytansinol can be produced in precious orange actinomycetes (Actinosynnemapretiosum ssp.auranticum ATCC31565) is also due to the hydrolysis of ansamitocin 3-O-acyl, and the hydrolysis rate of alanyl maytansinol is much higher than ansamitocin, which provides the possibility for the direct preparation of maytansinol. However, due to the low catalytic efficiency of the alanylase used, the extremely low yield of alanyl maytansinol is caused. Therefore, digging and screening efficient alanylase, and then realizing the transformation of the ansamitocin biosynthetic pathway, it is expected to achieve efficient biological manufacturing of maytansinol. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a method for efficiently preparing maytansinol using heterologous alanylase. Specifically, the present invention first screens alanylase extensively, and obtains the SmAstC enzyme that can realize the efficient 3-OD-alanylation of N-demethyl-4,5-deepoxy maytansinol; then, by overexpressing the positive regulatory gene asm18 in HGF052, the construction of the high-yield chassis strain HGFS04 of N-demethyl-4,5-deepoxy maytansinol is realized; the smastC gene and the asm11 and asm10 genes responsible for subsequent modification are overexpressed in HGFS04 to construct the maytansinol-producing genetic engineering bacteria HGFS10, and the significant accumulation of maytansinol is achieved; by optimizing the fermentation medium, the maytansinol yield is further improved. The method in the invention will effectively avoid the problems that the steps in the traditional maytansinol preparation process are complicated and there are by-products that are not easy to eliminate.

[0008] The technical solution of the present invention is as follows:

[0009] In a first aspect, the present invention provides a genetically engineered bacterium HGFS10 for efficiently preparing maytansinol using heterologous alanylase, wherein the genetically engineered bacterium HGFS10 heterologously expresses D-alanylase, overexpresses a positive regulatory factor gene asm18, overexpresses a C-4 / 5 cyclooxygenase gene asm11 and an amide N-methylase gene asm10;

[0010] The D-alanylase is derived from Streptomyces MMG1121, the encoding gene is smastC, the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0011] In the genetically engineered bacterium HGFS10, the D-alanylase SmAstC changes the original metabolic route of the precious orange-bundled actinomycetes and can efficiently catalyze the D-alanylation of the C-3 hydroxyl group of N-demethyl-4,5-deepoxymaytansinol; the positive regulatory factor gene asm18 can increase the substrate level; the C-4 / 5 epoxygenase gene asm11 and the amide N-methylase gene asm10 can improve the synergistic catalytic ability of key enzymes, thereby enabling the genetically engineered bacterium HGFS10 to achieve significant accumulation of maytansinol and increase the yield of maytansinol.

[0012] Preferably, according to the present invention, the gene library number of the positive regulatory factor gene asm18 is AF453501.1, and the database number of the encoded protein Asm18 is AAM54096.1.

[0013] Preferably according to the present invention, the gene library number of the C-4 / 5 cyclooxygenase gene asm11 and the amide N-methyltransferase gene asm10 is AF453501.1, the database number of the encoded protein Asm11 is AAM54089.1, and the database number of the protein Asm10 is AAM54088.1.

[0014] In a second aspect, the present invention provides a method for constructing the above-mentioned genetically engineered bacterium HGFS10, comprising the following steps:

[0015] (1) The genome of the precious orange-bundled Actinomycetes mutant strain HGF052 was used as a template for PCR amplification to obtain the positive regulatory factor gene asm18. The PCR primer sequences were:

[0016] asm18-F:5'-CGACCAAAGGAGGGCGGACATATGCGCTTTCAGCTCTTG-3',

[0017] asm18-R:5'-GAATTCCCAATTGCCTCTAGATCAGCGCGCGTGCTGC-3';

[0018] (2) The positive regulatory factor gene asm18 sequence was inserted into the downstream of the strong promoter kasOp* of the integration vector pSV1 by Gibson splicing to obtain the plasmid vector pSV1-kasOp*-asm18; the plasmid vector pSV1-kasOp*-asm18 was then transformed into Escherichia coli ET12567 / pUZ8002 to obtain the chassis strain HGFS04;

[0019] (3) Artificially synthesizing the D-alanylase gene smastC as shown in SEQ ID NO.1, and then ligating it into the plasmid vector pSET5035, so that the gene smastC is located downstream of the kasop* promoter, to obtain the plasmid vector pSET5035-kasOp*-smastC;

[0020] (4) PCR amplification was performed using the genome of the mutant strain HGF052 of the precious orange-bundled Actinomycetes as a template to obtain the C-4 / 5 cyclooxygenase gene asm11 and the amide N-methyltransferase gene asm10. The PCR primer sequences were:

[0021] asm10-F:5'-ACCAAAGGAGCGCGGACATATGAGCCTCCCAGCGG-3',

[0022] asm10-R:5'-CCCAATTGCCTCTAGACTACTACCCCAGCTCGGCC-3';

[0023] asm11-F:5'-CAAGGAGCGGACATGTGAGCCGGGACGGGGTG-3',

[0024] asm11-R:5'-CAATTGCCTCTAGACTATCGCAGCGCCCCCTCCCCG-3';

[0025] (5) The C-4 / 5 cyclooxygenase gene asm11 and the amide N-methyltransferase gene asm10 were respectively connected to the plasmid vector pSET5035, so that the gene asm11 and the gene asm10 were located downstream of the kasop* promoter, to obtain the plasmid vectors pSET5035-kasOp*-asm11 and pSET5035-kasOp*-asm10; then the plasmid vectors pSET5035-kasOp*-smastC, pSET5035-kasOp*-asm11 and pSET5035-kasOp*-asm10 were digested and assembled to obtain the integration plasmid vector pSET5035-kasOp*-smastC-kasOp*-asm11-kasOp*-asm10;

[0026] (6) The plasmid vector pSET5035-kasOp*-smastC-kasOp*-asm11-kasOp*-asm10 was transferred into the chassis strain HGFS04 by conjugation transfer to obtain the genetically engineered bacterium HGFS10 that efficiently produces maytansinol.

[0027] In a third aspect, the present invention provides a method for efficiently preparing maytansinol using the above-mentioned genetically engineered bacteria HGFS10, comprising the following steps:

[0028] 1) streaking the genetically engineered bacteria HGFS10 on a YMG agar medium for activation, culturing at 28-30° C. for 3-5 days until white aerial hyphae grow, then inoculating the bacteria on a YMGA agar medium, and fermenting and culturing the bacteria at 28-30° C. for 10-15 days to obtain a solid culture;

[0029] 2) Cut the solid culture into 1 cm 3 The small cubes are soaked and extracted with ethyl acetate / methanol with a volume ratio of 4:1 at 20-30°C for 18-24 hours, and the extraction is repeated 2-4 times, and the leached product is evaporated to dryness under reduced pressure to obtain a fermentation crude extract;

[0030] 3) The fermentation crude extract is dissolved in methanol, and separated by medium pressure liquid chromatography (MPLC) column chromatography to obtain a maytansinol methanol extract; the maytansinol methanol extract is then subjected to thin layer chromatography (TLC), the components containing maytansinol are combined and concentrated under reduced pressure, and after preparation by semi-preparative HPLC, the eluates with the same components are combined and evaporated to obtain maytansinol.

[0031] Preferably according to the present invention, in step 1), the YMG agar medium comprises: 0.4% yeast extract, 0.4% glucose, 1% malt extract, 2% agar powder and 96.2% distilled water, the pH value is 7.2-7.4, and it is sterilized at 121°C for 30 min.

[0032] Preferably according to the present invention, in step 1), the YMGA agar medium comprises: 0.4% yeast extract, 0.4% glucose, 1% malt extract, 0.0445% D-alanine, 2% agar powder and 96.2% distilled water, with a pH value of 7.2-7.4, and is sterilized at 121°C for 30 min.

[0033] Preferably according to the present invention, in step 3), the medium pressure liquid chromatography (MPLC) column chromatography is eluted with water, 35%, 40%, 50%, 60%, 70%, 80%, and 100% methanol in sequence, and collected at 200 mL / tube to obtain the maytansine methanol extract.

[0034] Preferably according to the present invention, in step 3), the developing solvent condition of the thin layer chromatography (TLC) is ethyl acetate:methanol at a ratio of 15:1; and the elution gradient of the semi-preparative HPLC is 45% acetonitrile.

[0035] In a fourth aspect, the present invention provides a maytansinol prepared by the above method, the chemical structure of which is shown in the following formula:

[0036]

[0037] Where the present invention is not detailed, the prior art may be used. Where the "%" is not specifically stated in the present invention, it refers to the mass percentage.

[0038] The beneficial effects of the present invention are:

[0039] 1. The present invention provides a method for obtaining maytansinol by biological fermentation, which avoids the process of first obtaining ansamitocin by biological fermentation and then removing the C-3 acyl group by chemical method in the traditional method. It avoids the disadvantages of redundant synthesis steps, harsh conditions and the generation of by-products that are difficult to separate.

[0040] 2. Through in-depth exploration and screening, the present invention identified the D-alanylase SmAstC that can achieve the unstable 3-OD-alanylation modification of N-demethyl-4,5-deepoxymaytansinol. The in vitro catalytic efficiency of this enzyme for N-demethyl-4,5-deepoxymaytansinol is 8 times that of its homologous protein AstC, providing a good tool enzyme for the biomanufacturing of maytansinol.

[0041] 3. The present invention constructs an expression vector comprising D-alanylase SmAstC, C-4 / 5 epoxidase asm11 and amide N-methylase asm10, and realizes heterologous expression of the expression vector in the chassis strain HGFS04, thereby constructing a genetically engineered bacterium HGFS10 for efficiently preparing maytansinol, providing a candidate strain for preparing maytansinol by direct biofermentation.

[0042] 4. The present invention also provides a method for efficiently preparing maytansinol using the genetically engineered bacterium HGFS10. By adding D-alanine to the culture medium and optimizing the direct acyl donor of the enzyme SmAstC, the maytansinol yield was further increased to 16.18 mg / L, which is 15.7% higher than the yield without adding D-alanine to the culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram (A) and mass spectrometry detection results (B) of 3-OD-alanylation of N-demethyl-4,5-deepoxymaytansol catalyzed by D-alanylase SmAstC in the present invention.

[0044] Figure 2 Schematic diagram for comparison of the enzyme activities of different D-alanylases.

[0045] Figure 3 Schematic diagram of the metabolic pathway for preparing maytansinol in the present invention.

[0046] Figure 4 This is a HPLC detection diagram of the metabolites of the genetically engineered bacteria HGFS10 and the chassis strain HGFS4 in the present invention.

[0047] Figure 5 This is a schematic diagram showing the effect of adding different concentrations of D-alanine to the culture medium on the yield of maytansine in the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. The experimental methods and reagents without specific conditions in the examples are all based on conventional conditions in the art.

[0049] The integrative vector pSV1 and plasmid vector pSET5035-kasOp* involved in the examples are both existing vectors.

[0050] Example 1, screening of D-alanylase SmAstC

[0051] 1. Taking ansatriene D-alanylase AstC (AHH25592.1) as the starting enzyme, five homologous proteins with sequence similarity >80% were found from the NCBI protein database through BLAST search and sequence alignment. They may be D-alanylases that catalyze the D-alanylation of the C-3 hydroxyl group of N-demethyl-4,5-deepoxymaytansol. Their names and numbers are SmAstC (WP_053663711.1), SyAstC (KUN02983.1), MycC (AFG19416.1), AnsC (AHW80292.1) and SlAstC (WP_069773361.1). According to sequence information, respectively by synthetic above-mentioned D-alanylase gene, and be connected on the NdeI / XhoI restriction site of pET-28a carrier, obtain the protein expression plasmid of six D-alanylases.Wherein, the plasmid containing smastC gene is pET28a-smastC, and the pET28a-astC containing astC gene constructs expression plasmid as control.Expression plasmid is transformed into BL21 (DE3) competent cell respectively by the mode of chemical transformation, obtains recombinant Escherichia coli, then obtains recombinant D-alanylase by mass fermentation and protein purification.The information of described astC gene is disclosed in Chinese patent literature CN111635418A.

[0052] The specific steps are as follows:

[0053] (1) picking up the recombinant E. coli monoclone growing on the kanamycin-resistant plate and placing it in a resistant LB liquid medium, and culturing it with shaking at 37° C. overnight to obtain a seed solution;

[0054] (2) The seed solution was inoculated into LB liquid medium at a volume ratio of 1:100, and cultured at 37°C with shaking until the OD value was 0.6-0.8. After cooling to 18°C, IPTG with a final concentration of 0.1 mM was added for induction overnight;

[0055] (3) The recombinant E. coli cells were collected by centrifugation and resuspended in a resuspension buffer. After mechanical disruption, the cells were centrifuged at 12,000 rpm for 1 hour, and the supernatant was collected. The supernatant protein was then purified by nickel column. The purified protein was dialyzed overnight to obtain the recombinant D-alanylase and its concentration was determined.

[0056] 2. The six recombinant D-alanylases were assayed for enzyme activity using the same enzyme activity system. The specific enzyme activity system is as follows: 20 mM Tris-HCl (pH 8.0), 10 μM D-alanylase, 0.5 μM SFP protein, 1 mM ATP, 10 mM MgCl2, 1 mM CoA, 1 mM D-alanine, 10 μM N-demethyl-4,5-deepoxymaytansol, and the remaining volume was filled with ddH2O.

[0057] The enzyme activity system was incubated at 30°C for 3 hours and then terminated with an equal volume of methanol. After centrifugation, HPLC and high-resolution mass spectrometry were performed. Figure 1 As shown, D-alanylase can catalyze the D-alanylation of C-3 position of N-demethyl-4,5-deepoxy maytansinol to generate N-demethyl-3-OD-alanyl-4,5-deepoxy maytansinol. The relative conversion rates of the six homologous proteins to the substrate N-demethyl-4,5-deepoxy maytansinol were calculated by substrate conversion rate. The results are shown in Figure 2 shown.

[0058] Depend on Figure 2 It can be seen that the D-alanylase SmAstC from Streptomyces MMG1121 is a protein with the best catalytic activity for the substrate N-demethyl-4,5-deepoxymaytansinol, and its in vitro activity is 8 times that of AstC. The nucleotide sequence of the D-alanylase SmAstC is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0059] HPLC detection conditions are as follows: THERMO Ultimate 3000HPLC, YMC-Pack Pro C18 (4.6×250mm, 5μm), detection wavelength 254nm. The mobile phases are acetonitrile and water with 0.5‰ formic acid added, respectively, the injection volume is 10μL, and the flow rate is 1mL / min. The gradient elution conditions are: 0-5min, 20%-35% acetonitrile; 19-23min, 50%-60% acetonitrile; 24-27min, 100% acetonitrile; 28-30min 20% acetonitrile.

[0060] Example 2: Construction of chassis strain HGFS04

[0061] The specific construction steps of chassis strain HGFS04 are as follows:

[0062] (1) The genome of the precious orange-bundled Actinomycetes mutant strain HGF052 was used as a template for PCR amplification to obtain the positive regulatory factor gene asm18. The PCR primer sequences were:

[0063] asm18-F:5'-CGACCAAAGGAGGGCGGACATATGCGCTTTCAGCTCTTG-3',

[0064] asm18-R:5'-GAATTCCCAATTGCCTCTAGATCAGCGCGCGTGCTGC-3';

[0065] The PCR system was as follows: 10 ng of template plasmid, 2 μl of each 10 μM primer, 25 μl of 2× PrimeSTAR Max Premix, 2 μl of DMSO, and made up to 50 μl with ddH2O;

[0066] PCR amplification conditions: pre-denaturation, 95℃5min; denaturation, 95℃10sec; annealing, 60℃30sec; extension, 72℃2min, 30 cycles; termination extension, 72℃10min; insulation at 16℃;

[0067] (2) Recovering the PCR product, assembling the recovered positive regulatory factor gene asm18 gene fragment with the integrative vector pSV1 cut with NdeI / XbaI by Gibson splicing, and inserting it into the downstream of the strong promoter kasOp* of the integrative vector pSV1 to obtain the plasmid vector pSV1-kasOp*-asm18;

[0068] The Gibson splicing reaction system is as follows: 50 ng of restriction digested vector, 30 ng of PCR product fragment, 5 μl of 2×HB infusion Master Mix, and ddH2O to make up to 10 μl; react at 50°C for 50 min;

[0069] (3) The plasmid vector pSV1-asm18 was transformed into DH5α competent cells, spread on LB plates containing ampicillin and cultured at 37°C overnight. A clone was picked from the resistant plate and inoculated on a LB liquid medium containing ampicillin. A small amount of plasmid was extracted and sequenced for verification. The correctly sequenced plasmid vector pSV1-asm18 was transformed into Escherichia coli ET12567 / pUZ8002 competent cells by conjugation transfer, and then transferred into the precious orange-colored Actinomycetes mutant strain HGF052 by conjugation transfer. After culturing at 37°C for 16 hours, the plate was covered with 1 mg of nalidixic acid and 1 mg of thiostrepton. After inverted culture at 30°C for 5 to 7 days, the orange-yellow conjugate was picked and plated on a resistant YMG plate containing thiostrepton for resistance verification, and the genome was extracted for PCR verification to obtain the chassis strain HGFS04.

[0070] The precious aurantiacus mutant strain HGF052 comes from the document J.Am.Chem.Soc.2002, (124), 6544-6545.

[0071] Example 3: Construction of Genetically Engineered Bacteria HGFS10 for Efficient Preparation of Maytansol

[0072] A method for constructing a genetically engineered bacterium HGFS10 comprises the following steps:

[0073] (1) The plasmid vector pET28a-smastC was obtained according to the method described in Example 1, and then PCR amplification was performed using the plasmid vector pET28a-smastC as a template to obtain the D-alanylase gene smastC gene fragment. The PCR primer sequences were:

[0074] smastC-F:5'-CCAAAGGAGGGCGGACATATGCTGGTGAAGGCGC-3',

[0075] smastC-R:5'-CCAATTGCCTCTAGACTATCAGTCCCCGAGGTGA-3';

[0076] (2) PCR amplification was performed using the genome of the mutant strain HGF052 of the precious orange-bundled Actinomycetes as a template to obtain the C-4 / 5 cyclooxygenase gene asm11 and the amide N-methyltransferase gene asm10. The PCR primer sequences were:

[0077] asm10-F:5'-ACCAAAGGAGCGCGGACATATGAGCCTCCCAGCGG-3',

[0078] asm10-R:5'-CCCAATTGCCTCTAGACTACTACCCCAGCTCGGCC-3';

[0079] asm11-F:5'-CAAGGAGCGGACATGTGAGCCGGGACGGGGTG-3',

[0080] asm11-R:5'-CAATTGCCTCTAGACTATCGCAGCGCCCCCTCCCG-3'.

[0081] The PCR system and PCR amplification conditions were the same as in Example 2;

[0082] (2) Recovering the PCR products, and assembling the D-alanylase gene smastC fragment, the C-4 / 5 cyclooxygenase gene asm11, and the amide N-methyltransferase gene asm10 fragment with the pSET5035 vector digested with NdeI / XbaI by Gibson splicing, so that the genes asm11 and asm10 are located downstream of the kasop* promoter, to obtain plasmid vectors pSET5035-kasOp*-smastC, pSET5035-kasOp*-asm11, and pSET5035-kasOp*-asm10;

[0083] The Gibson splicing reaction system is the same as in Example 2;

[0084] (3) The plasmid vectors pSET5035-kasOp*-smastC, pSET5035-kasOp*-asm11 and pSET5035-kasOp*-asm10 were transformed into DH5α competent cells, spread on resistant LB plates containing apramycin and cultured at 37°C overnight. The clones were picked from the resistant plates and inoculated into resistant LB liquid medium containing apramycin. The plasmids were extracted in small amounts and sequenced for verification.

[0085] (4) The plasmid vector pSET5035-kasOp*-smastC was digested with PstI / XbaI to obtain the kasOp*-smastC fragment, the plasmid vector pSET5035-kasOp*-asm11 was digested with PstI / XbaI to obtain the kasOp*-asm11 fragment, the plasmid vector pSET5035-kasOp*-asm11 was digested with SpeI / EcoRI to obtain the kasOp*-asm10 fragment, the pSET5035 vector was digested with PstI / EcoRI to obtain the digested vector, and the kasOp*-smastC fragment was obtained. The tC fragment, kasOp*-asm11 fragment and kasOp*-asm10 fragment were connected to the plasmid vector pSET5035 by T4 ligase to obtain the integration plasmid vector pSET5035-kasOp*-smastC-kasOp*-asm11-kasOp*-asm10; then the integration plasmid vector pSET5035-kasOp*-smastC-kasOp*-asm11-kasOp*-asm10 was transformed into DH5α competent cells, spread on apramycin-containing resistant LB plate and cultured at 37°C overnight;

[0086] The integration site of the integration plasmid vector pSET5035-kasOp*-smastC-kasOp*-asm11-kasOp*-asm10 is the ΦC31 integration site, and the promoter is the artificial strong promoter kasOp*; the genes smastC, asm11 and asm10 in the plasmid are all overexpressed;

[0087] (5) The clones were picked from the resistant plate and inoculated into resistant LB liquid medium containing apramycin, and a small amount of plasmids were extracted and verified by enzyme digestion. The correctly sequenced integration plasmid vector pSET5035-kasOp*-smastC-kasOp*-asm11-kasOp*-asm10 was transformed into Escherichia coli ET12567 / pUZ8002 competent cells by conjugation transfer, and then transferred into the chassis strain HGFS04 by conjugation transfer. After culturing at 37°C for 16 hours, 1 mg of nalidixic acid and 1 mg of apramycin were covered on the plate, and then inverted and cultured at 30°C for 5 to 7 days. The orange-yellow conjugates were picked and plated on a resistant YMG plate containing apramycin for resistance verification, and the genome was extracted for PCR verification to obtain the genetically engineered bacterium HGFS10 for efficient production of maytansinol.

[0088] Example 4, Fermentation Preparation of Maytansol

[0089] 1. Biotransformation pathways of maytansine in vivo Figure 3 As shown. Figure 3 It can be seen that the 3-OD-alanylation of N-demethyl-4,5-deepoxymaytansinol catalyzed by D-alanylase SmAstC is the key step of this biotransformation, and in vitro results show that the direct acyl donor of D-alanylase SmAstC is D-alanine rather than L-alanine. Therefore, exogenous feeding of alanine will help increase the conversion rate of this step, thereby further increasing the yield of maytansinol.

[0090] The gradient concentration alanine addition experiment is as follows:

[0091] Prepare 1M D-alanine stock solution, take different volumes of stock solution and mix with 1ml sterilized water and apply to YMG agar medium to obtain YMG agar medium containing 0mM, 1mM, 2mM, 3mM, 5mM, 8mM, and 10mM D-alanine. Pick the mycelium on the seed plate and streak it on the above medium, and culture it in a 30℃ incubator for 13 days. Extract a small amount of fermentation products and perform HPLC detection. Plot the yield of maytansinol according to different culture medium conditions Figure 5 .

[0092] like Figure 5As shown in the figure, the results of the gradient concentration alanine addition experiment show that 1-10 mM D-alanine can effectively increase the yield of maytansinol. According to the optimal conditions, the optimized culture medium containing 5 mM D-alanine was used to ferment the maytansinol production strain HGFS10 to increase the yield of maytansinol.

[0093] 2. A method for efficiently preparing maytansinol using genetically engineered bacteria HGFS10, comprising the following steps:

[0094] 1) The genetically engineered bacteria HGFS10 was streaked on YMG agar medium for activation and cultured at 30°C for 4 days until white aerial hyphae grew, and then the hyphae were evenly streaked on YMGA agar medium with an inoculation loop, and fermented and cultured at 30°C for 13 days to obtain a solid culture;

[0095] 2) Cut the solid culture into 1 cm 3 The small cubes were soaked and extracted with ethyl acetate / methanol with a volume ratio of 4:1 at 25°C for 20 hours, and the extraction was repeated 3 times. The leached product was evaporated to dryness under reduced pressure to obtain a fermentation crude extract;

[0096] 3) After the fermentation crude extract is dissolved in methanol, it is separated by medium pressure liquid chromatography (MPLC) column chromatography, and eluted with water, 35%, 40%, 50%, 60%, 70%, 80%, and 100% methanol in sequence, and collected at 200 mL / tube; then it is detected by thin layer chromatography (TLC), and the developing solvent condition is ethyl acetate: methanol at a ratio of 15:1. According to the test results, the components containing maytansinol are combined and concentrated under reduced pressure, and prepared by semi-preparative HPLC with an elution gradient of 45% acetonitrile. The obtained product is evaporated to dryness to obtain maytansinol.

[0097] The YMG agar culture medium comprises the following components: 0.4% yeast extract, 0.4% glucose, 1% malt extract, 2% agar powder and 96.2% distilled water, the pH value is 7.2-7.4, and the medium is sterilized at 121°C for 30 minutes.

[0098] The YMGA agar medium comprises the following components: 0.4% yeast extract, 0.4% glucose, 1% malt extract, 0.0445% D-alanine, 2% agar powder and 96.2% distilled water, the pH value is 7.2-7.4, and the medium is sterilized at 121°C for 30 minutes.

[0099] Example 5. Detection and identification of products of genetically engineered bacteria HGFS10 and chassis strain HGFS4

[0100] Electrospray ionization high resolution mass spectrometry (ESI-HRMS) measured the quasi-molecular ion peak of the compound obtained in Example 4 to be m / z 565.2304 [M+H] +, which is consistent with the expected molecular weight of maytansinol. In addition, the one-dimensional and two-dimensional NMR results are consistent with maytansinol.

[0101] Table 1. Nuclear magnetic resonance data of maytansinol

[0102]

[0103]

[0104] The structure of the compound obtained in Example 4 is shown in the following formula, which indicates that the method of the present invention successfully prepared maytansinol.

[0105]

[0106] 2. According to the method described in Example 4, the fermentation products of HGFS04 and HGFS10 strains were dissolved in methanol and tested by HPLC with an injection volume of 10 μl. The results are as follows: Figure 4 shown.

[0107] Depend on Figure 4 It can be seen that the main metabolite N-demethyl-4,5-deepoxymaytansinol (1) in the chassis strain HGFS04 was significantly reduced in HGFS10, while the trace component maytansinol (2) in HGFS04 was significantly accumulated in HGFS10 and became the main metabolite of this strain, indicating that D-alanylase SmAstC has a high catalytic efficiency for N-demethyl-4,5-deepoxymaytansinol and can effectively increase the production of maytansinol.

[0108] A standard solution of 5 mg / ml of maytansinol was prepared with 5 mg of maytansinol standard and gradient concentration dilution was performed to obtain a series of standard solutions with a concentration of 1.25 to 500 μg / ml. The standard solution was subjected to HPLC detection with an injection volume of 10 μl, and a standard curve of maytansinol concentration and peak area was drawn. The concentration of the extract of the maytansinol producing strain was determined according to the obtained standard curve, and it was finally determined that the maytansinol yield per liter of the genetically engineered bacteria HGFS10 fermentation product in Example 4 was 16.18 mg / L.

[0109] The HPLC detection conditions were the same as those in Example 1.

[0110] Comparative Example 1

[0111] A method for efficiently preparing maytansinol using genetically engineered bacteria HGFS10, the specific method is the same as that of Example 4, except that YMG agar medium is used for culture in step 1).

[0112] In this comparative example, the maytansinol yield per liter of the genetically engineered bacteria HGFS10 fermentation product is 13.98 mg / L. By comparing Example 5 with Comparative Example 1, it can be seen that the use of YMGA agar medium containing 5 mM D-alanine for fermentation of the maytansinol production strain HGFS10 can effectively increase the maytansinol yield, which is 15.7% higher than the yield without adding D-alanine to the culture medium.

[0113] In summary, the present invention utilizes heterologous D-alanylase SmAstC in Actinomycetes aurantifolia to change the original metabolic pathway and achieve significant accumulation of maytansinol. The present invention can realize direct biomanufacturing of maytansinol, avoid the redundant steps of chemical deacylation in the existing maytansinol synthesis pathway, and directly obtain maytansinol by microbial fermentation, providing a new method for the efficient preparation of key intermediates of antibody-drug conjugates.

[0114] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A genetically engineered bacterium HGFS10 for efficiently preparing maytansinol using heterologous alanylase, characterized in that: The genetically engineered bacterium HGFS10 heterologously expresses D-alanylase, overexpresses the positive regulatory factor gene asm18, overexpresses the C-4 / 5 cyclooxygenase gene asm11 and the amide N-methyltransferase gene asm10; The D-alanylase is derived from Streptomyces MMG1121, the encoding gene is smastC, the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. The genetically engineered bacterium HGFS10 according to claim 1, characterized in that The gene library number of the positive regulatory factor gene asm18 is AF453501.1, and the database number of the encoded protein Asm18 is AAM54096.

1.

3. The genetically engineered bacterium HGFS10 according to claim 1, characterized in that The gene library number of the C-4 / 5 cyclooxygenase gene asm11 and the amide N-methyltransferase gene asm10 is AF453501.1, the database number of the encoded protein Asm11 is AAM54089.1, and the database number of the protein Asm10 is AAM54088.

1.

4. The method for constructing the genetically engineered bacterium HGFS10 according to claim 1, characterized in that: The steps include: (1) The genome of the precious orange-bundled Actinomycetes mutant strain HGF052 was used as a template for PCR amplification to obtain the positive regulatory factor gene asm18. The PCR primer sequences were: asm18-F:5'-CGACCAAAGGAGGGCGGACATATGCGCTTTCAGCTCTTG-3', asm18-R:5'-GAATTCCCAATTGCCTCTAGATCAGCGCGCGTGCTGC-3'; (2) The positive regulatory factor gene asm18 sequence was inserted into the downstream of the strong promoter kasOp* of the integration vector pSV1 by Gibson splicing to obtain the plasmid vector pSV1-kasOp*-asm18; the plasmid vector pSV1-kasOp*-asm18 was then transformed into Escherichia coli ET12567 / pUZ8002 to obtain the chassis strain HGFS04; (3) Artificially synthesizing the D-alanylase gene smastC as shown in SEQ ID NO.1, and then ligating it into the plasmid vector pSET5035, so that the gene smastC is located downstream of the kasop* promoter, to obtain the plasmid vector pSET5035-kasOp*-smastC; (4) PCR amplification was performed using the genome of the mutant strain HGF052 of the precious orange-bundled Actinomycetes as a template to obtain the C-4 / 5 cyclooxygenase gene asm11 and the amide N-methyltransferase gene asm10. The PCR primer sequences were: asm10-F:5'-ACCAAAGGAGCGCGGACATATGAGCCTCCCAGCGG-3', asm10-R:5'-CCCAATTGCCTCTAGACTACTACCCCAGCTCGGCC-3'; asm11-F:5'-CAAGGAGCGGACATGTGAGCCGGGACGGGGTG-3', asm11-R:5'-CAATTGCCTCTAGACTATCGCAGCGCCCCCTCCCCG-3'; (5) The C-4 / 5 cyclooxygenase gene asm11 and the amide N-methyltransferase gene asm10 were respectively connected to the plasmid vector pSET5035, so that the gene asm11 and the gene asm10 were located downstream of the kasop* promoter, to obtain the plasmid vectors pSET5035-kasOp*-asm11 and pSET5035-kasOp*-asm10; Then the plasmid vectors pSET5035-kasOp*-smastC, pSET5035-kasOp*-asm11 and pSET5035-kasOp*-asm10 were digested and assembled to obtain the integration plasmid vector pSET5035-kasOp*-smastC-kasOp*-asm11-kasOp*-asm10; (6) The plasmid vector pSET5035-kasOp*-smastC-kasOp*-asm11-kasOp*-asm10 was transferred into the chassis strain HGFS04 by conjugation transfer to obtain the genetically engineered bacterium HGFS10 that can efficiently produce maytansinol.

5. A method for efficiently preparing maytansinol using the genetically engineered bacterium HGFS10 according to any one of claims 1 to 3, characterized in that: The steps include: 1) streaking the genetically engineered bacteria HGFS10 on a YMG agar medium for activation, culturing at 28-30° C. for 3-5 days until white aerial hyphae grow, then inoculating the bacteria on a YMGA agar medium, and fermenting and culturing the bacteria at 28-30° C. for 10-15 days to obtain a solid culture; 2) Cut the solid culture into 1 cm 3 The small cubes are soaked and extracted with ethyl acetate / methanol with a volume ratio of 4:1 at 20-30°C for 18-24 hours, and the extraction is repeated 2-4 times, and the leached product is evaporated to dryness under reduced pressure to obtain a fermentation crude extract; 3) dissolving the crude fermentation extract in methanol, and separating by medium pressure liquid chromatography (MPLC) column chromatography to obtain a maytansinol methanol extract; Then, the maytansinol methanol extract was subjected to thin layer chromatography (TLC), and the components containing maytansinol were combined and concentrated under reduced pressure. After preparation using semi-preparative HPLC, the eluates with the same components were combined and evaporated to dryness to obtain maytansinol.

6. The method according to claim 5, characterized in that In step 1), the components of the YMG agar medium are: 0.4% yeast extract, 0.4% glucose, 1% malt extract, 2% agar powder and 96.2% distilled water, pH value is 7.2-7.4, high temperature sterilization at 121℃ for 30min.

7. The method according to claim 5, characterized in that In step 1), the components of the YMGA agar medium are: 0.4% yeast extract, 0.4% glucose, 1% malt extract, 0.0445% D-alanine, 2% agar powder and 96.2% distilled water, pH value is 7.2-7.4, high temperature sterilization at 121℃ for 30min.

8. The method according to claim 5, characterized in that In step 3), the medium pressure liquid chromatography (MPLC) column chromatography is eluted with water, 35%, 40%, 50%, 60%, 70%, 80%, and 100% methanol in sequence, and collected at 200 mL / tube to obtain the maytansine methanol extract.

9. The method according to claim 5, characterized in that In step 3), the developing solvent of the thin layer chromatography (TLC) is ethyl acetate:methanol at a ratio of 15:1; and the elution gradient of the semi-preparative HPLC is 45% acetonitrile.

10. The present invention provides a maytansinol prepared by the method of claims 5 to 9, the chemical structure of which is shown in the following formula:

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