CN112980864B - Recombinant escherichia coli for synthesizing anticancer drug intermediate, and construction method and application thereof - Google Patents
Recombinant escherichia coli for synthesizing anticancer drug intermediate, and construction method and application thereof Download PDFInfo
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Abstract
The invention provides recombinant escherichia coli for synthesizing an anticancer drug intermediate, a construction method and application thereof. The recombinant plasmid contained in the recombinant escherichia coli carries nucleotide sequences for encoding L-tryptophan oxidase, CPA synthetase and two monooxygenases; the L-tryptophan oxidase is VioA and the CPA synthetase is VioB. In the invention, the combination of the L-tryptophan oxidase, the CPA synthetase and the two monooxygenases can be normally expressed in escherichia coli, and CPA and an anticancer drug intermediate can be efficiently synthesized by taking L-tryptophan as a substrate, thus providing a basis for screening and researching anticancer drugs.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to recombinant escherichia coli, a construction method and application thereof, in particular to recombinant escherichia coli for synthesizing an anticancer drug intermediate, and a construction method and application thereof.
Background
Cancer is a serious threat to human health, and its causative factor often results from abnormal activation of kinases in signaling pathways. In addition, common high-incidence chronic diseases such as autoimmune diseases and neurodegenerative diseases are also closely related to abnormal kinase. In 2001, the first kinase inhibitor, gleevec, was marketed in the U.S. Pat. No. and opened a new era of targeted cancer treatment, with significantly better efficacy than chemotherapy. By the end of 2019, the FDA approved 52 kinase inhibitors altogether. Although there are a large number of cancers, there is still a significant unmet clinical need for treatment due to the variety of cancers, the numerous abnormal kinases involved, the variety of types of variation, and post-use drug resistance. In addition, these marketed drugs target only about 25 kinases, accounting for only 5% of 518 kinases in humans, and there are a large number of kinases that have not been patented. Therefore, there is tremendous space for the development of such drugs.
The antitumor drug mainly comes from natural product analogues. Thus, innovative techniques for efficient synthesis of natural product analogs are critical to new drug discovery. Natural products tend to be structurally complex, containing multiple chiral centers. Biosynthesis is more advantageous than chemical synthesis due to its stringent regio-and stereoselectivity. Especially the increasingly mature principles and techniques of synthetic biology, make efficient synthesis of natural product analogs easy to implement.
Bisindole alkaloids are a natural product containing two indole groups, and representative compounds are Staurosporine (Staurosporine) and butterfly mycin (Rebeccamycin) with antitumor activity. Staurosporine is a natural product derived from actinomycetes and has broad-spectrum strong kinase inhibitory activity. It is not completely counted that staurosporine can effectively inhibit more than 200 human kinases, and the half inhibition concentration (IC 50) of a plurality of kinases can reach the nM level. Butterfly mycin is also a natural product derived from actinomycetes and has potent topoisomerase and kinase inhibitory activities. Development of effective synthesis technology of bisindole alkaloid analogues is a key factor contributing to wide-range patent medicine of the compounds.
The biosynthesis gene cluster DNA sequence of staurosporine was determined in 2002 and the natural synthetase system was initially identified. Subsequently, the catalytic activity of a number of natural synthetases, such as StaO, staD, staP, staC, etc., was demonstrated in vitro biochemical experiments, formula I shows the biosynthetic pathway of staurosporine, wherein the R group represents either=o or=nh, both reversible.
The biosynthetic gene cluster DNA sequence of butterfly mycins was also determined in 2002, a natural synthetase system was initially identified, and formula II shows the biosynthetic pathway of butterfly mycins, where the R group represents either =o or =nh, both reversible.
Comparing formulas I and II shows that the biosynthetic pathways of staurosporine and butterfly mycin are very similar, wherein StaO and RebO are isozymes, staD and RebD are isozymes, staP and RebP are isozymes, but StaC and RebC are non-isozymes, staC catalyzes the synthesis of K252c, and RebC catalyzes the synthesis of 1, 11-dichloro-ARCYRIAFLAVIN A.
Based on the identified enzyme lines of the biosynthetic pathways for staurosporine and butterfly, researchers achieved biosynthesis of multiple bisindole alkaloid analogs in heterologous hosts Streptomyces albus (Streptomyces albus), e.g., synthesis of K252c using enzyme combinations RebO, rebD, rebP and StaC starting with tryptophan and ARCYRIAFLAVIN A using enzyme combinations RebO, rebD, rebP and RebC starting with tryptophan, however, both combinations were expressed in Streptomyces albus and yields were unknown.
Therefore, the provision of a biosynthetic pathway and production strain for the synthesis of staurosporine and butterfly mycin intermediates is of great importance for the improvement of staurosporine and butterfly mycin production, as well as for the screening and development of anticancer drugs.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide recombinant escherichia coli for synthesizing an anticancer drug intermediate, and a construction method and application thereof. The recombinant escherichia coli can express an enzyme combination for synthesizing the intermediate of the anticancer drug, and the enzyme combination can catalyze the raw material L-tryptophan to synthesize the intermediate of the corresponding drug, so that a wide application prospect can be provided for screening and research and development of the anticancer drug.
To achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a recombinant plasmid carrying nucleotide sequences encoding an L-tryptophan oxidase, a CPA synthase and two monooxygenases;
wherein the L-tryptophan oxidase is VioA and the CPA synthetase is VioB.
In the invention, the recombinant plasmid can express L-tryptophan oxidase, CPA synthetase and monooxygenase in the biosynthesis pathway of violacein (Violacein), staurosporine and butterfly mycin; in addition, the L-tryptophan oxidase is VioA, the CPA synthetase is VioB, and compared with StaO/RebO and StaD/rebD in a natural synthetase system, the VioA and the VioB in the violacein biosynthesis pathway used in the invention have higher synthesis efficiency, and CPA can be synthesized efficiently; in addition, compared with StaC, spcC used in the method has higher synthesis efficiency and can synthesize K252c with high efficiency; meanwhile, the plasmid contains two monooxygenases, and the monooxygenases are matched with each other, so that oxidation of CPA can be completed, and target products K252c and ARCYRIAFLAVIN A are obtained.
As a preferred embodiment of the present invention, the two monooxygenases are a combination of RebP and SpcC or a combination of RebP and RebC.
In the invention, the VioA is derived from purple bacillus (Chromobacterium violaceum), the amino acid sequence length is 418aa, and the GenBank number is AAD51808.1;
In the invention, the VioB is derived from purple bacillus (Chromobacterium violaceum), the length of an amino acid sequence is 998aa, and the GenBank number is AAD51809.1;
in the invention, rebP is derived from the hyacinth bean stomata (Lentzea aerocolonigenes), the amino acid sequence length is 397aa, and the GenBank number is AAN01211.1;
in the invention, spcC is derived from streptomyces tricolor (Streptomyces sanyensis), the amino acid sequence length is 542aa, and the GenBank number is AGL96582.1;
In the invention, rebC is derived from the stomata lablab (Lentzea aerocolonigenes), the amino acid sequence length is 529aa, and the GenBank number is AAN01210.1.
Preferably, the recombinant plasmid carries nucleotide sequences encoding L-tryptophan oxidase VioA, CPA synthase VioB, monooxygenase RebP and monooxygenase SpcC; or alternatively
The recombinant plasmid carries nucleotide sequences encoding L-tryptophan oxidase VioA, CPA synthase VioB, monooxygenase RebP and monooxygenase RebC.
In the invention, the enzyme combinations VioA, vioB, rebP and SpcC and the enzyme combinations VioA, vioB, rebP and RebC are constructed on the same plasmid, and the four enzymes are different in sources, but do not affect each other, can normally execute physiological functions and catalytic activities of the enzymes, have certain coordination effects with each other, and the VioA and the VioB oxidize the raw material L-tryptophan and synthesize CPA, so that the whole biosynthesis path is further carried out downstream due to higher synthesis efficiency of CPA, and RebP and SpcC continue to catalyze CPA to form K252c, rebP and RebC continue to catalyze CPA to synthesize ARCYRIAFLAVIN A; thus, the enzyme combination is constructed on a target plasmid and introduced into a host cell, and if the host cell can spontaneously synthesize L-tryptophan, the resulting recombinant host cell can efficiently synthesize the anticancer drug intermediate K252c or ARCYRIAFLAVIN A.
As a preferred technical scheme of the invention, the recombinant plasmid is an escherichia coli expression plasmid.
Preferably, the E.coli expression plasmid comprises any one of pETDuet series, pACYCDuet series, pRSFDuet series, pCOLADuet or pCDFDuet series expression vectors, preferably pCDFDuet series expression vectors.
In the invention, the plasmid can select the currently known commercialized plasmid, and the purpose of the biosynthetic drug intermediate can be achieved without further modification of the commercialized plasmid; likewise, the plasmid may be selected from modified plasmids, and the enzyme combination may be capable of normally expressing the desired enzyme protein in accordance with the plasmid construction principle.
As a preferred embodiment of the present invention, the nucleotide sequence encoding L-tryptophan oxidase and the nucleotide sequence encoding CPA synthase are linked together, and the nucleotide sequences encoding two monooxygenases are linked together.
Preferably, the nucleotide sequences are linked by a ribosome binding site.
In the invention, the nucleotide sequence for encoding the L-tryptophan oxidase VioA and the nucleotide sequence for encoding the CPA synthetase VioB are connected through a ribosome binding site to obtain a nucleotide sequence shown as SEQ ID NO. 1; the nucleotide sequences encoding the two monooxygenases RebP and SpcC are linked to obtain the nucleotide sequence shown in SEQ ID NO. 2; the nucleotide sequences encoding the two monooxygenases RebP and RebC are linked to give the nucleotide sequence shown in SEQ ID NO. 3.
In a second aspect, the present invention provides a recombinant E.coli comprising at least one copy of the recombinant plasmid according to the first aspect.
As a preferred technical scheme of the invention, the yield of the anticancer drug intermediate of the recombinant escherichia coli is not lower than 10 mu M.
Preferably, the anticancer drug intermediate comprises K252c and/or ARCYRIAFLAVIN A.
As a preferred technical scheme of the invention, the recombinant escherichia coli is commercial escherichia coli containing the recombinant plasmid and/or artificially modified escherichia coli.
In the present invention, the type of the E.coli is not limited, that is, the recombinant plasmid can normally realize the expression function in the host E.coli transformed, and in the present invention, the E.coli BL21 (DE 3) is included as an alternative host, but E.coli DH5 alpha cannot be used, assuming that the pETDuet series expression vector is used.
Preferably, the recombinant E.coli is E.coli BL21 (DE 3) containing the recombinant plasmid.
Coli is a common model organism, and its prokaryotic expression system can efficiently express the enzyme combination of the invention. In the invention, if the non-modified escherichia coli is adopted, the yield of the obtained anticancer drug intermediate is not lower than 10 mu M; if E.coli capable of producing L-tryptophan efficiently is selected, the yield of the obtained intermediate will be further improved.
In a third aspect, the present invention also provides a method for producing recombinant E.coli according to the second aspect, comprising:
Transferring the recombinant plasmid according to the first aspect into escherichia coli by an electrotransformation or chemical transformation method to obtain the recombinant escherichia coli.
As a preferred technical scheme of the invention, the construction method of the recombinant plasmid comprises the following steps:
the synthesized nucleotide sequences for encoding the L-tryptophan oxidase, the CPA synthetase and at least two monooxygenases are inserted into an escherichia coli expression plasmid in an enzyme digestion and connection mode, so that the recombinant plasmid is obtained.
Preferably, the method of enzyme digestion is double enzyme digestion.
Preferably, the combination of sites for double cleavage comprises a combination of cleavage sites NcoI and BamHI and/or a combination of cleavage sites NdeI and XhoI.
In a fourth aspect, the invention also provides the use of a recombinant plasmid as described in the first aspect or a recombinant E.coli as described in the second aspect for the preparation of an anticancer drug or an anticancer drug intermediate.
Preferably, the anticancer drug comprises staurosporine and/or butterfly mycin.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention constructs a recombinant plasmid which expresses L-tryptophan oxidase, CPA synthetase and two monooxygenases, and the biosynthesis path constructed by the recombinant plasmid can express and synthesize an anticancer drug intermediate by taking L-tryptophan as a raw material; the recombinant plasmid is transferred into escherichia coli, so that an anticancer drug intermediate can be efficiently synthesized in the escherichia coli, and compared with streptomyces, the escherichia coli is the most commonly used host strain in laboratories and industry, and the genetic operation and the fermentation technology are very mature and the operation is simple;
(2) In the invention, the synthesis efficiency of the enzyme combination VioA and VioB is higher, the synthesis activity is far higher than that of StaO/RebO and StaD/RebD in a natural synthesis system, and meanwhile, the synthesis efficiency of SpcC used in the enzyme combination is also far higher than that of StaC, so that the recombinant escherichia coli BL21 (DE 3)/pCDFDuet-1_vioAvioBribbPspcC and BL21 (DE 3)/pCDFDuet-1_vioAvioBribbPrebC constructed by the invention can respectively express enzyme combinations VioA, vioB, rebP and SpcC and enzyme combinations VioA, vioB, rebP and RebC, and can respectively and effectively synthesize K252c and ARCYRIAFLAVIN A; the recombinant escherichia coli is fermented and cultured, so that a large amount of K252c and ARCYRIAFLAVIN A can be obtained quickly, conveniently and at low cost, and the recombinant escherichia coli has strong kinase inhibition activity; therefore, the recombinant escherichia coli has wide application prospects in the fields of preparation of anticancer drug intermediates, screening and research and development of anticancer drugs and the like.
Drawings
FIG. 1 is a LC/MS detection spectrum of the product obtained in example 2, wherein I is the ultraviolet absorbance spectrum at 290nm of the broth extracted sample, and II is the ion spectrum with a mass-to-charge ratio (m/z) of 312 in the broth extracted sample.
FIG. 2 is a full-wavelength scan spectrum corresponding to the UV absorption peak of K252c in the LC/MS detection spectrum of example 2.
FIG. 3 is a LC/MS detection spectrum of the product obtained in example 3, wherein I is the ultraviolet absorbance spectrum at 316nm of the broth extracted sample, and II is the ion spectrum of the broth extracted sample with a mass to charge ratio (m/z) of 326.
FIG. 4 is a full wavelength scan of the peak corresponding to ARCYRIAFLAVIN A UV absorbance peaks in the LC/MS detection spectrum of example 3.
Detailed Description
The following embodiments are further described with reference to the accompanying drawings, but the following examples are merely simple examples of the present invention and do not represent or limit the scope of the invention, which is defined by the claims.
In the following examples, all reagents and consumables were purchased from the reagent manufacturers routine in the art unless specifically indicated; unless otherwise indicated, all methods and techniques used are those conventional in the art.
Example 1
This example was used to prepare recombinant plasmids pCDFDuet-1_vioAvioBribPspcC and pCDFDuet-1_vioAvioBribPrebC.
The recombinant plasmid is constructed according to the following method:
(1) Nucleotide sequences encoding proteins VioA, vioB, rebP, spcC and RebC were designed according to E.coli codon bias;
wherein, the GenBank database numbers of the five enzyme proteins are shown in Table 1:
TABLE 1
(2) Combining nucleotide fragments for coding the VioA and the VioB to obtain a nucleotide sequence shown as SEQ ID NO.1, wherein a ribosome binding site is arranged between the two genes, and NcoI and BamHI enzyme cutting sites are respectively carried at the upstream and downstream;
wherein the nucleotide sequence of the ribosome binding site is:
ACCAACAAGGACCATAGATT(SEQ ID NO.4)；
Combining nucleotide fragments of codes RebP and SpcC to obtain a nucleotide sequence shown as SEQ ID NO.2, wherein a ribosome binding site is arranged between the two genes, and NdeI and XhoI restriction sites are respectively carried on the upstream and downstream;
wherein the nucleotide sequence of the ribosome binding site is:
ATTAAAGAGGAGAAATTAACT(SEQ ID NO.5)；
Similarly, nucleotide fragments of codes RebP and RebC are combined to obtain a nucleotide sequence shown as SEQ ID NO.3, wherein a ribosome binding site (the site sequence is the nucleotide sequence shown as SEQ ID NO. 5) is arranged between the two genes, and NdeI and XhoI enzyme cutting sites are respectively carried at the upstream and downstream;
After designing the nucleotide sequences SEQ ID NO. 1-3, the synthesis of the gene synthesis company is entrusted.
(3) The nucleotide sequence shown in SEQ ID No.1 is subjected to double digestion by NcoI and BamHI, and then inserted into a commercial vector pCDFDuet-1 subjected to the same digestion treatment, so as to obtain an intermediate plasmid pCDFDuet-1_vioAvioB;
Then, after double digestion of SEQ ID No.2 and SEQ ID No.3 with NdeI and XhoI, respectively, the intermediate plasmid pCDFDuet-1_vioAvioB treated by the same digestion was inserted to obtain expression plasmids pCDFDuet-1_vioAvioBribPspcC and pCDFDuet-1_vioAvioBribPrebC.
Example 2
Recombinant E.coli was constructed using recombinant plasmid pCDFDuet-1_vioAvioBrebPspcC in this example, and the resultant recombinant E.coli was subjected to test tube fermentation and product collection.
The method comprises the following specific steps:
(1) The plasmid pCDFDuet-1_vioAvioBrebPspcC obtained in example 1 was transferred into E.coli BL21 (DE 3), and the single clone was picked up and inoculated into a test tube containing 2mL of LB medium, and cultured at 37℃with shaking at 200 rpm;
(2) When the bacterial liquid OD 600 grows to 0.6-0.8 (multiple test tubes are simultaneously cultured), 0.4mM IPTG is added to induce the expression of the enzyme protein, the temperature is reduced to 20 ℃, and the shaking culture is carried out for 24 hours;
(3) After fermentation, heating the metal bath at 85 ℃ for 15min;
(4) Cooling to room temperature of 25 ℃, adding ethyl acetate with volume of 1 time, fully and uniformly mixing, extracting, centrifuging at 37 ℃ for 30min at 200rpm, collecting an organic phase supernatant, and repeating for one time;
(5) The collected organic phase supernatants were combined, evaporated to dryness at 40℃and dissolved in 50. Mu.L of DMSO for LC/MS detection.
The synthetic route of K252c in the escherichia coli is shown as a formula III:
The LC/MS detection result is shown in figure 1, the ultraviolet absorption peak of K252c appears in the fermentation liquid extraction sample (I diagram) at 3.527min, the mass/charge ratio (M/z) of the ion corresponding to the peak, namely [ M+1], is 312 (II diagram), and the full-wavelength scanning result corresponding to the peak is shown in figure 2;
The strain BL21 (DE 3)/pCDFDuet-1_vioAvioBrebPspcC successfully synthesizes K252c, the retention time, the mass/charge ratio (M/z) of [ M+1] ions and the full-wavelength scan are consistent with the standard;
The concentration in the broth was 13. Mu.M according to the concentration standard curve of K252 c.
Example 3
The difference from example 2 is that in this example, the plasmid pCDFDuet-1_vioAvioBribPspcC is replaced with the plasmid pCDFDuet-1_vioAvioBribPrebC, and the rest of the procedure is the same as in example 3;
the synthetic route of ARCYRIAFLAVIN A in the escherichia coli is shown as a formula IV:
The LC/MS detection result is shown in FIG. 3, the ultraviolet absorption peak of ARCYRIAFLAVIN A appears in the fermentation broth extract sample (I graph) at 3.867min, the mass/charge ratio (M/z) of the ion corresponding to the peak, namely [ M+1], is 326 (II graph), and the full-wavelength scanning result corresponding to the peak is shown in FIG. 4;
Strain BL21 (DE 3)/pCDFDuet-1_vioAvioBrebPrebC successfully synthesized ARCYRIAFLAVIN A, its retention time, [ M+1] ion mass/charge ratio (M/z), and the full wavelength scans were also consistent with standards.
The concentration in the broth was 96. Mu.M according to the ARCYRIAFLAVIN A concentration standard curve.
In contrast, the present invention also constructs plasmid pCDFDuet-1_staOstaDstaPstaC and plasmid pCDFDuet-1_staOstaDstaPspcC to synthesize K252c; construction of plasmid pCDFDuet-1_rebOrebDrebPrebC to synthesize ARCYRIAFLAVIN A;
The recombinant E.coli post-fermentation was obtained by the same method, the amount of the obtained product was significantly lower than in examples 2 and 3, and the specific data are shown in Table 2:
TABLE 2
As can be seen from Table 2 above, the concentration of the E.coli product constructed in the present invention is significantly higher than that of other enzyme combinations; and as can be seen from the comparison of pCDFDuet-1_vioaAvioBribPspcC and pCDFDuet-1_staOstaDstaPstac, the enzyme combination yield of the present invention is superior to that of the enzyme combination in the natural path; likewise, pCDFDuet-1_vioaAvioBrebPrebC and pCDFDuet-1_rebOrebDrebPrebC also demonstrated this conclusion.
In conclusion, the recombinant escherichia coli containing the enzyme combinations VioA, vioB, rebP and SpcC and the enzyme combinations VioA, vioB, rebP and RebC constructed in the invention can efficiently synthesize the intermediates of staurosporine and butterfly mycin, and provides wide application prospects for screening and research and development of anticancer drugs.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
Sequence listing
<110> The Baizhu pharmaceutical Co., ltd
<120> Recombinant E.coli for synthesizing anticancer drug intermediate, construction method and application thereof
<130> 20210309
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4274
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<213> Synthesis of the product
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atgaagcaca gcagcgatat ctgcattgtt ggtgcgggta ttagcggtct gacctgcgcg 60
agccacctgc tggatagccc ggcgtgccgt ggtctgagcc tgcgtatctt cgacatgcag 120
caagaggcgg gtggccgtat tcgtagcaaa atgctggacg gcaaggcgag catcgaactg 180
ggtgcgggcc gttacagccc gcaactgcac ccgcactttc agagcgcgat gcaacactac 240
agccagaaaa gcgaggtgta tccgttcacc cagctgaagt ttaaaagcca cgttcagcaa 300
aagctgaaac gtgcgatgaa cgaactgagc ccgcgtctga aagagcacgg caaggagagc 360
ttcctgcaat ttgtgagccg ttaccagggt cacgatagcg cggttggcat gattcgtagc 420
atgggttacg atgcgctgtt cctgccggac atcagcgcgg agatggcgta tgacatcgtg 480
ggcaagcacc cggaaattca aagcgttacc gacaacgatg cgaaccagtg gtttgcggcg 540
gagaccggtt ttgcgggtct gatccaaggt attaaggcga aagttaaagc ggcgggtgcg 600
cgttttagcc tgggttaccg tctgctgagc gttcgtaccg acggtgatgg ctatctgctg 660
cagctggcgg gtgacgatgg ctggaaactg gaacaccgta cccgtcacct gattctggcg 720
attccgccga gcgcgatggc gggtctgaac gttgattttc cggaggcgtg gagcggtgcg 780
cgttacggca gcctgccgct gttcaaaggt tttctgacct acggcgaacc gtggtggctg 840
gattataagc tggacgatca agtgctgatc gttgacaacc cgctgcgtaa gatctacttc 900
aagggcgata agtacctgtt cttttatacc gacagcgaga tggcgaacta ctggcgtggt 960
tgcgtggcgg agggtgaaga cggctatctg gagcaaattc gtacccatct ggcgagcgcg 1020
ctgggtattg ttcgtgaacg tattccgcag ccgctggcgc acgtgcacaa gtactgggcg 1080
cacggtgttg agttctgccg tgacagcgat attgaccatc cgagcgcgct gagccaccgt 1140
gatagcggca tcattgcgtg cagcgacgcg tataccgagc actgcggttg gatggaaggt 1200
ggcctgctga gcgcgcgtga agcgagccgt ctgctgctgc agcgtattgc ggcgtaaacc 1260
aacaaggacc atagattatg agcatcctgg atttcccgcg tattcacttt cgtggttggg 1320
cgcgtgtgaa cgcgccgacc gcgaaccgtg atccgcacgg ccacattgac atggcgagca 1380
acaccgttgc gatggcgggt gaaccgtttg acctggcgcg tcacccgacc gaatttcatc 1440
gtcacctgcg tagcctgggt ccgcgttttg gtctggatgg tcgtgcggac ccggagggtc 1500
cgtttagcct ggcggaaggc tacaacgcgg cgggtaacaa ccacttcagc tgggagagcg 1560
cgaccgtgag ccacgttcag tgggacggtg gcgaagcgga tcgtggtgac ggcctggtgg 1620
gcgcgcgtct ggcgctgtgg ggtcactaca acgattatct gcgtaccacc tttaaccgtg 1680
cgcgttgggt tgatagcgac ccgacccgtc gtgatgcggc gcagatctat gcgggtcaat 1740
tcaccattag cccggcgggt gcgggtccgg gtaccccgtg gctgtttacc gcggatattg 1800
acgatagcca tggtgcgcgt tggacccgtg gtggccacat tgcggagcgt ggtggccact 1860
tcctggacga ggaatttggc ctggcgcgtc tgttccagtt tagcgtgccg aaggatcacc 1920
cgcacttcct gtttcatccg ggtccgtttg acagcgaggc gtggcgtcgt ctgcaactgg 1980
cgctggaaga cgatgacgtg ctgggcctga ccgttcagta cgcgctgttc aacatgagca 2040
ccccgccgca accgaacagc ccggtgtttc acgatatggt tggcgtggtt ggtctgtggc 2100
gtcgtggcga actggcgagc tacccggcgg gtcgtctgct gcgtccgcgt cagccgggcc 2160
tgggtgacct gaccctgcgt gtgagcggtg gccgtgttgc gctgaacctg gcgtgcgcga 2220
tcccgttcag cacccgtgcg gcgcaaccga gcgcgccgga tcgtctgacc ccggacctgg 2280
gtgcgaaact gccgctgggt gacctgctgc tgcgtgatga ggatggtgcg ctgctggcgc 2340
gtgtgccgca ggcgctgtac caagattatt ggaccaacca cggcattgtt gacctgccgc 2400
tgctgcgtga accgcgtggt agcctgaccc tgagcagcga gctggcggaa tggcgtgagc 2460
aggattgggt tacccaaagc gacgcgagca acctgtatct ggaggcgccg gatcgtcgtc 2520
atggccgttt ctttccggag agcatcgcgc tgcgtagcta tttccgtggt gaagcgcgtg 2580
cgcgtccgga catcccgcac cgtattgaag gcatgggtct ggtgggcgtt gagagccgtc 2640
aggatggtga tgcggcggag tggcgtctga ccggtctgcg tccgggtccg gcgcgtattg 2700
tgctggatga cggtgcggaa gcgatcccgc tgcgtgttct gccggatgac tgggcgctgg 2760
atgacgcgac cgtggaggaa gttgattacg cgttcctgta tcgtcacgtg atggcgtact 2820
atgagctggt ttacccgttc atgagcgata aggtgtttag cctggcggac cgttgcaaat 2880
gcgaaaccta tgcgcgtctg atgtggcaga tgtgcgaccc gcaaaaccgt aacaagagct 2940
actatatgcc gagcacccgt gagctgagcg cgccgaaggc gcgtctgttt ctgaaatacc 3000
tggcgcacgt tgaaggccag gcgcgtctgc aggctccgcc gccggcgggc ccggcgcgca 3060
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ttatgctgca gtacctgtat gcggcgtaca gcatcccgaa ctatgcgcaa ggccagcaac 3180
gtgttcgtga tggtgcgtgg accgcggagc agctgcaact ggcgtgcggc agcggtgatc 3240
gtcgtcgtga tggtggcatc cgtgcggcgc tgctggaaat cgcgcacgag gaaatgattc 3300
actacctggt ggttaacaac ctgctgatgg cgctgggtga accgttctat gcgggcgtgc 3360
cgctgatggg tgaagcggcg cgtcaggcgt ttggtctgga taccgaattc gcgctggagc 3420
cgtttagcga aagcaccctg gcgcgtttcg ttcgtctgga gtggccgcac tttatcccgg 3480
cgccgggcaa gagcattgcg gactgctacg cggcgatccg tcaagcgttc ctggatctgc 3540
cggacctgtt tggtggcgag gcgggtaaac gtggtggcga acaccacctg ttcctgaacg 3600
agctgaccaa ccgtgcgcac ccgggttacc agctggaagt gtttgatcgt gacagcgcgc 3660
tgttcggcat tgcgtttgtt accgatcaag gtgaaggtgg cgcgctggac agcccgcact 3720
atgagcacag ccacttccag cgtctgcgtg aaatgagcgc gcgtatcatg gcgcaaagcg 3780
cgccgtttga accggcgctg ccggcgctgc gtaacccggt gctggatgaa agcccgggtt 3840
gccaacgtgt tgcggatggt cgtgcgcgtg cgctgatggc gctgtaccag ggtgtgtatg 3900
agctgatgtt cgcgatgatg gcgcaacact ttgcggttaa accgctgggc agcctgcgtc 3960
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gttgcgagcg tctgctggaa caggcgagca tgctggagcc gggttggctg ccggatgcgc 4200
agatggaact gctggacttc taccgtcgtc aaatgctgga cctggcgtgc ggtaaactga 4260
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<212> DNA
<213> Synthesis of the product
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atgaaaccgt tcgatctgaa agcatttacc ggtgcagatc tggcagatcc gtatccggtt 60
tatcgtgaat atctgaccgg tgatccggtt catcataatg gtgaagcatg gtatgtgttt 120
ggttatgatg gtgttgcaca tgttctgacc agccgtgatt atggtcgtcg tggtccgggt 180
ggtcgtgcaa ccccgattcc gcctagccat gataccctga gccgtattgt tgaaaattgg 240
ctggtttttc tggatccgcc tcgtcatacc gcactgcgta gcctgctggc aaaagaattt 300
agtccggcag ttgttaccgg tctgcgtgaa cgtgttcgta aaattgccgg tgaactgctg 360
gcaggcctgg gtgatgcggg tgaaattgat ctggttgaag attttgcagc accgctgccg 420
attctggtta ttagcgagct gctgggtgtt ccggcacgtc tgcgtagctg gtttcgtcgt 480
tgtgcagttg atctgcaaga ggcaagcacc gcacgtgcca cccgtaatcc gggtgcactg 540
gcacgcgcag atggtgcagc aagcgaactg gttgaatttt ttggtggtga actgggcacc 600
cgcaaaccgg atgatgaaga tctggtggca ctgctggtta atgcacagcg tcgtggtgaa 660
gccctgaccg atgaagaaat tgttagcacc tgtgttcatc tgctgaccgc aggccatgaa 720
accaccacca atctgattag caaaagcgtt ctggccctgc tggccaatcc ggcagcagca 780
gctgaacctc tggctggtct ggatgttacc cctcaggttg ttgaagaact gaatcgtttt 840
gatacaccgg ttcagatggt tacccgttgg gcacatcagg atacagcact gggtggtaaa 900
ccgattcgtc gcggtgataa agttgttctg gttctgggta gcgcaaatcg tgatcctgca 960
gcatttgcag aacctgatcg tctggatctg cgtcgtgata gccgtcgtca ttgtggtttt 1020
ggtctgggta ttcattattg tctgggtgca gccctggcac gtaccgaagc agaaattggt 1080
ctgagcgttc tgtttaccaa ttttccgggt ctgcgtctgg gtggcgaacc ggtgcgttat 1140
gccgatgatc tggtatttca tggtccagcc cgtctgccga tgctgacccg ttagattaaa 1200
gaggagaaat taactatgcg caaaggccgc aacgcgatgc gtcatagtgg cgcgaggact 1260
gacgtcctta tcgctggagg agggccagta gggatggcgc tggcgctgga tctggcgtat 1320
cgcggcattg attgcatggt ggcggatgcg ggcgatggga ctgtacggca cccaaaggtc 1380
tcgaccattg gcccgcgcag catggaactg tttcgccgct ggggcctggc tggcgcgatt 1440
cgcgatgcgg gctggccggc ggatcatccg ctggatattg cgtgggtgac ccgcgtgggc 1500
ggccatgaag tgtatcgcta tcgccgcggc accgcgggcg atcgcccggc gtttgcgcat 1560
acaccagagc cggatcagat atgtccagca cactggctca atcccgtctt aactcgtgct 1620
gtaggagtcc acccggatgg cccgctgcgc ctgaacacca ccgtggaacg cgtggaacag 1680
agcgaagaac atgtagacgc tgtccttact gaccacgctg ctggtacttc tgggacggtc 1740
agggccagat acttagtagc ctgtgacgga gctgcctctc caatcaggcg tgcctgtggg 1800
atagaggcgc cgccgcgcca tcgcccgcag gtgtttcgca acattctgtt tcgcgcgccg 1860
ggcctgcgcg aacgcctggg cgaacgcgcg gcgctggtgt atttcctcat gctgagcccg 1920
accctgcgct ttccgctgcg cagcctggat ggcagcgatc tgtataacct ggtggtgggc 1980
gcggatgaag tggcgcgcgt gggcgcggaa gaactgattc gccaggcggt ggcgctggat 2040
gtgccggtgg aactgctggg cgatggcgaa tggcacttga cgcaccgagt agccgacagg 2100
tatcgtgccg gtcgggtcct attagccggt gacgctgcac acaccctgtc tccaagtggt 2160
gggttcggcc tgaacaccgg catagccgac gcagccgacc ttggttggaa gttagcggcc 2220
gcgcttgatg gatgggcggg cccgcgcctg ctggatacct atgaagcgga acgccgcccg 2280
attgcggtgg aaagcctgga tgaagcgaac cgcaacctgc agcgcaccct gggccgcgaa 2340
ctgccgccgg atatactcgt cgacggacct gagggtgaca gagcaagagc tgagacagcc 2400
gagcgcctgc gcaacagcgg cgcgcagcgc gaatttgatg cgccggaaat tcatttcggt 2460
ttgcgctatc gcagcccgct gattgtggag gacccaggtg ttcctgtccg gcacggtaag 2520
ccaactgctg actggcggcc aggatcagag ccgggttacc gagcagccca cgcgtggtgg 2580
gaagatagca ccagcaccct ggatctgttt ggccgcggct tcgtcctctt agtattaact 2640
ggtcaagcag agacgggtgg tgtggaacgt gccttcgcag agcgcggcat tccgctgaca 2700
gtcagagggg gagggggcga agaagtggcg aaactgtatg agagagcctt cgttctagtg 2760
cgcccggatg gccatgtggc gtggcgagga gacacactac cagaagatcc tgctgcctta 2820
gctgacactg ttagaggagc ttaa 2844
<210> 3
<211> 2805
<212> DNA
<213> Synthesis of the product
<400> 3
atgaaaccgt tcgatctgaa agcatttacc ggtgcagatc tggcagatcc gtatccggtt 60
tatcgtgaat atctgaccgg tgatccggtt catcataatg gtgaagcatg gtatgtgttt 120
ggttatgatg gtgttgcaca tgttctgacc agccgtgatt atggtcgtcg tggtccgggt 180
ggtcgtgcaa ccccgattcc gcctagccat gataccctga gccgtattgt tgaaaattgg 240
ctggtttttc tggatccgcc tcgtcatacc gcactgcgta gcctgctggc aaaagaattt 300
agtccggcag ttgttaccgg tctgcgtgaa cgtgttcgta aaattgccgg tgaactgctg 360
gcaggcctgg gtgatgcggg tgaaattgat ctggttgaag attttgcagc accgctgccg 420
attctggtta ttagcgagct gctgggtgtt ccggcacgtc tgcgtagctg gtttcgtcgt 480
tgtgcagttg atctgcaaga ggcaagcacc gcacgtgcca cccgtaatcc gggtgcactg 540
gcacgcgcag atggtgcagc aagcgaactg gttgaatttt ttggtggtga actgggcacc 600
cgcaaaccgg atgatgaaga tctggtggca ctgctggtta atgcacagcg tcgtggtgaa 660
gccctgaccg atgaagaaat tgttagcacc tgtgttcatc tgctgaccgc aggccatgaa 720
accaccacca atctgattag caaaagcgtt ctggccctgc tggccaatcc ggcagcagca 780
gctgaacctc tggctggtct ggatgttacc cctcaggttg ttgaagaact gaatcgtttt 840
gatacaccgg ttcagatggt tacccgttgg gcacatcagg atacagcact gggtggtaaa 900
ccgattcgtc gcggtgataa agttgttctg gttctgggta gcgcaaatcg tgatcctgca 960
gcatttgcag aacctgatcg tctggatctg cgtcgtgata gccgtcgtca ttgtggtttt 1020
ggtctgggta ttcattattg tctgggtgca gccctggcac gtaccgaagc agaaattggt 1080
ctgagcgttc tgtttaccaa ttttccgggt ctgcgtctgg gtggcgaacc ggtgcgttat 1140
gccgatgatc tggtatttca tggtccagcc cgtctgccga tgctgacccg ttagattaaa 1200
gaggagaaat taactatgaa cgcgccgatc gagaccgacg tgctgattct gggtggcggt 1260
ccggttggta tggcgctggc gctggacctg gcgcaccgtc aagtgggcca cctggtggtt 1320
gagcaaaccg atggtaccat cacccacccg cgtgtgggta ccattggtcc gcgtagcatg 1380
gaactgtttc gtcgttgggg cgttgcgaaa cagatccgta ccgcgggttg gccgggtgat 1440
cacccgctgg atgcggcgtg ggtgacccgt gttggcggtc acgaggttta tcgtattccg 1500
ctgggtaccg cggacacccg tgcgaccccg gagcacaccc cggaaccgga tgcgatttgc 1560
ccgcagcact ggctggcgcc gctgctggcg gaggcggtgg gtgaacgtct gcgtacccgt 1620
agccgtctgg acagcttcga acaacgtgac gatcacgttc gtgcgaccat taccgatctg 1680
cgtaccggtg cgacccgtgc ggtgcatgcg cgttacctgg tggcgtgcga tggtgcgagc 1740
agcccgaccc gtaaggcgct gggtattgat gcgccgccgc gtcaccgtac ccaagtgttt 1800
cgtaacattc tgtttcgtgc gccggaactg cgtagcctgc tgggtgaacg tgcggcgctg 1860
ttctttttcc tgatgctgag cagcagcctg cgttttccgc tgcgtgcgct ggatggtcgt 1920
ggtctgtatc gtctgaccgt gggtgttgac gatgcgagca aaagcaccat ggatagcttt 1980
gagctggtgc gtcgtgcggt tgcgttcgac accgaaatcg aggttctgag cgatagcgaa 2040
tggcacctga cccatcgtgt ggcggacagc tttagcgcgg gtcgtgtttt cctgaccggt 2100
gatgcggcgc acaccctgag cccgagcggc ggtttcggta tgaacaccgg tattggtagc 2160
gcggcggacc tgggttggaa gctggcggcg accctgcgtg gttgggcggg tccgggtctg 2220
ctggcgacct acgaggaaga gcgtcgtccg gtggcgatca ccagcctgga agaggcgaac 2280
gttaacctgc gtcgtaccat ggaccgtgag ctgccgccgg gtctgcatga tgatggtccg 2340
cgtggtgaac gtattcgtgc ggcggtggcg gagaaactgg aacgtagcgg tgcgcgtcgt 2400
gagtttgatg cgccgggcat ccacttcggt cacacctatc gtagcagcat tgtgtgcggc 2460
gaaccggaga ccgaagttgc gaccggcggt tggcgtccga gcgcgcgtcc gggtgcgcgt 2520
gcgccgcacg cgtggctgac cccgaccacc agcaccctgg acctgtttgg ccgtggtttc 2580
gtgctgctga gctttggcac caccgacggt gtggaggcgg ttacccgtgc gtttgcggat 2640
cgtcatgtgc cgctggaaac cgttacctgc catgcgccgg agatccatgc gctgtatgaa 2700
cgtgcgcatg tgctggtgcg tccggatggt catgttgcgt ggcgtggtga tcacctgccg 2760
gcggaactgg gcggtctggt ggataaagtt cgtggtgcgg cgtaa 2805
<210> 4
<211> 20
<212> DNA
<213> Synthesis of the product
<400> 4
accaacaagg accatagatt 20
<210> 5
<211> 21
<212> DNA
<213> Synthesis of the product
<400> 5
attaaagagg agaaattaac t 21
Claims (17)
1. A recombinant plasmid carrying nucleotide sequences encoding an L-tryptophan oxidase, a CPA synthase and two monooxygenases;
Wherein the L-tryptophan oxidase is VioA and the CPA synthetase is VioB; the two monooxygenases are a combination of RebP and SpcC.
2. The recombinant plasmid of claim 1, wherein the recombinant plasmid comprises an e.coli expression plasmid.
3. The recombinant plasmid according to claim 2, wherein the escherichia coli expression plasmid comprises any one of petdouet series, pACYCDuet series, pRSFDuet series, pCOLADuet or pcdfduret series expression vectors.
4. The recombinant plasmid according to claim 3, wherein the E.coli expression plasmid is a pCDFDuet series expression vector.
5. The recombinant plasmid of claim 1, wherein the nucleotide sequence encoding an L-tryptophan oxidase is linked to the nucleotide sequence encoding a CPA synthase and the nucleotide sequence encoding a monooxygenase is linked.
6. The recombinant plasmid of claim 5, wherein the nucleotide sequences are linked by a ribosome binding site.
7. A recombinant escherichia coli comprising the recombinant plasmid of any one of claims 1-6.
8. The recombinant escherichia coli according to claim 7, wherein the yield of the anticancer drug intermediate of the recombinant escherichia coli is not lower than 10 μm.
9. The recombinant escherichia coli of claim 8, wherein the anticancer drug intermediate comprises K252c.
10. The recombinant escherichia coli according to claim 7, wherein the recombinant escherichia coli is a commercial escherichia coli and/or an artificially engineered escherichia coli comprising the recombinant plasmid.
11. The recombinant escherichia coli according to claim 10, wherein the recombinant escherichia coli is escherichia coli BL21 (DE 3) containing the recombinant plasmid.
12. A method for preparing the recombinant escherichia coli as set forth in any one of claims 7 to 11, wherein the preparation method comprises:
Transferring the recombinant plasmid according to any one of claims 1-6 into escherichia coli by an electrotransformation or chemical transformation method to obtain the recombinant escherichia coli.
13. The method of claim 12, wherein the method of constructing the recombinant plasmid comprises:
The synthesized nucleotide sequences for encoding the L-tryptophan oxidase, the CPA synthetase and the two monooxygenases are inserted into an escherichia coli expression plasmid in an enzyme digestion and connection mode, so that the recombinant plasmid is obtained.
14. The method of claim 13, wherein the cleavage is performed in a double cleavage mode.
15. The method of claim 14, wherein the combination of sites for double cleavage comprises a combination of cleavage sites NcoI and BamHI and/or a combination of cleavage sites NdeI and XhoI.
16. Use of the recombinant plasmid according to any one of claims 1 to 6 or the recombinant escherichia coli according to any one of claims 7 to 11 for preparing an anticancer drug or an anticancer drug intermediate.
17. The use according to claim 16, wherein the anticancer drug comprises staurosporine.
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