WO2019120132A1 - 一株高效异源表达Disorazole Z的工程菌株和构建该菌株的基因簇及其应用 - Google Patents

一株高效异源表达Disorazole Z的工程菌株和构建该菌株的基因簇及其应用 Download PDF

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WO2019120132A1
WO2019120132A1 PCT/CN2018/120969 CN2018120969W WO2019120132A1 WO 2019120132 A1 WO2019120132 A1 WO 2019120132A1 CN 2018120969 W CN2018120969 W CN 2018120969W WO 2019120132 A1 WO2019120132 A1 WO 2019120132A1
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disorazole
dis427
strain
host cell
gene cluster
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张友明
李瑞娟
高运生
涂强
王宗杰
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山东大学
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Definitions

  • the invention belongs to the technical field of microbial gene resources and biosynthesis, and particularly relates to a Disorazole Z biosynthesis gene cluster and an engineered strain of a highly efficient heterologous expression of Disorazole Z constructed by using the gene cluster and an application thereof.
  • Disorazoles was the first structurally novel macrocyclic dilactone compound isolated from the fermentation broth of the genus Sorangium cellulosum So ce 12 by Jansen et al. To date, 29 Disorazoles derivatives have been found in Sorangium cellulosum So ce 12, namely Disorazole A1 to Disorazole I.
  • Disorazoles compounds can inhibit tubulin polymerization, promote tubulin depolymerization, thereby interfering with cell division and inducing apoptosis, and are highly resistant to a variety of tumor cell lines, including multidrug resistant tumor cell lines.
  • Biological activity is a new class of cell microtubule anti-stabilizers.
  • Disorazole A1 and Disorazole C1 are currently the most studied components, and their semi-inhibitory concentrations IC 50 are at pM to nM levels for a variety of human tumor cell lines, including multidrug resistant tumor cell lines.
  • Recent studies have found that Disorazoles compounds also have an inhibitory effect on the invasive cell infection pathway of group A streptococci. Although the activity is remarkable, the Disorazoles-like compounds derived from Sorangium cellulosum So ce 12 have a very short half-life in vivo and are a bottleneck for their medicines.
  • Disorazole Z is a Disorazoles family of compounds derived from Sorangium cellulosum So ce 427. It also has significant antitumor activity compared to Disorazoles derived from Sorangium cellulosum So ce 12, and has a small cyclic skeleton. The structure is more stable and has a longer half-life in the organism. It has been reported that the coupling of this compound with luteinizing hormone releasing hormone for targeted therapy of triple negative breast cancer has entered a phase II clinical study. Therefore, Disorazole Z is an excellent potential anti-tumor or anti-infective new drug.
  • Disorazole Z is expected to be a commercial drug in the near future as a promising anti-tumor drug or anti-infective drug, how to get a large dose of pure substance is one of the biggest limitations today.
  • the wild strain Sorangium cellulosum So ce 427 grows very slowly and is not easy to culture, it is not suitable for large-scale fermentation.
  • the artificial total synthesis method for preparing Disorazole Z is very difficult, and there have been no reports of successful synthesis. In view of this, how to efficiently produce and purify Disorazole Z is an urgent problem to be solved. Therefore, it is necessary to obtain the biosynthetic pathway gene cluster and transfer the gene cluster to the host bacteria which grow fast and easy to culture for heterologous biosynthesis.
  • the wild strain Sorangium cellulosum So ce 427 which currently produces Disorazole Z, grows very slowly, is not easy to culture, and is therefore not suitable for large-scale fermentation.
  • the problem to be solved by the present invention is to genomically excavate the original producing strain So ce 427 to provide a Disorazole Z organism.
  • Synthetic pathway gene cluster (dis427) and the use of this gene cluster to construct a highly efficient heterologous expression of Disorazole Z for efficient heterologous biosynthesis of Disorazole Z.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleoside having at least 80% identity, eg, at least 85%, at least 90%, at least 95% or at least 99% identity to SEQ ID No. 1.
  • percent identity is the relationship between two or more polynucleotide sequences that are determined by comparison of the sequences. "Identity" can be readily calculated by methods known in the art.
  • the isolated nucleic acid molecule described above comprises the nucleotide sequence of SEQ ID No. 1 or a complement thereof.
  • the isolated nucleic acid molecule described above is a biosynthetic gene cluster of Disorazole Z, characterized in that the gene cluster is named dis427, which comprises a polyketide synthase and a non-ribosome which are necessary for the biosynthesis of Disorazole Z.
  • the four core genes of polypeptide synthetase are disA, disB, disC and disD, a hypothetical protein gene orf4 and a post-modification gene orf6; this gene cluster is derived from Sorangium cellulosum So ce 427, whose nucleotide sequence is SEQ. ID No.1 is shown.
  • the Disorazole Z biosynthetic pathway corresponding to the gene cluster is shown in Figure 1.
  • the invention features a vector comprising the isolated nucleic acid molecule described above.
  • Vector refers to an extrachromosomal element that normally carries a gene that is not part of the metabolism of the cell center, and is often in the form of a circular double stranded DNA molecule. Such elements may be autonomously replicating sequences, genomic integration sequences, phage or nucleotide sequences, linear or circular single or double stranded DNA or RNA from any source, many of which have been joined or recombined.
  • the construct is capable of introducing a promoter fragment of the selected gene product and a DNA sequence into the cell along with a suitable 3' non-translated sequence.
  • the invention relates to host cells, such as bacterial cells, fungal cells, such as yeast cells, mammalian cells, insect cells, and plant cells, comprising the nucleic acid molecules or gene clusters or vectors described above.
  • host cells such as bacterial cells, fungal cells, such as yeast cells, mammalian cells, insect cells, and plant cells, comprising the nucleic acid molecules or gene clusters or vectors described above.
  • the host cell is a bacterium, such as Myxococcus xanthus.
  • the host cell is an engineered strain that efficiently heterologously expresses Disorazole Z, characterized in that the strain is named as engineering strain DK1622::Km-Ptet-dis427, and its genotype is: Myxococcus xanthus DK1622, kanamycin Resistance,tetracycline inducible Ptet promoter, disA, disB, disC, orf4, disD and orf6, is a biosynthesis gene cluster of Disorazole Z integrated into its genome by transposition using Mycobacterium xanthos DK1622 as a starting strain. obtain.
  • the invention features a method of producing a host cell that efficiently expresses heterologous Disorazole Z, the method comprising introducing a nucleic acid molecule or gene cluster or vector described above into a heterologous host cell.
  • the invention relates to a method for constructing the engineered strain DK1622::Km-Ptet-dis427 of the highly efficient heterologous expression of Disorazole Z, the steps being:
  • step (2) The plasmid p15A-cm-amp-ccdB-dis427 constructed in step (2) was digested with restriction endonucleases PacI and PmeI and recombined with the tetR-tetO PCR fragment to construct plasmid p15A-cm-tetR. -tetO-dis427;
  • the expression plasmid p15A-tnpA-kan-tetR-tetO-dis427 constructed in step (4) was electrotransferred into Myxococcus xanthus DK1622, and the expression plasmid was expressed in Myxococcus xanthus DK1622.
  • the transposase was distorazole Z biosynthetic gene cluster dis427 Integration into the genome of Myxococcus xanthus DK1622, an engineered strain capable of heterologous expression of Disorazole Z was obtained, and was named as engineering strain DK1622::Km-Ptet-dis427.
  • the invention in another aspect, relates to a method of producing Disorazole Z, the method comprising culturing a host cell as described above, and recovering Disorazole Z from the culture of said host cell.
  • the invention also relates to the use of the host cell described above, in particular the engineered strain DK1622::Km-Ptet-dis427, which efficiently and heterologously expresses Disorazole Z, in the production of Disorazole Z.
  • the engineered strain DK1622::Km-Ptet-dis427 according to the present invention has not been reported in the literature, and is the first to achieve high expression of the biosynthetic gene cluster (dis427) of Disorazole Z in the heterologous host strain Myxococcus xanthus DK1622.
  • the experiment confirmed that the engineering strain DK1622::Km-Ptet-dis427 provided by the present invention has a 10-fold increase in the yield of Disorazole Z compared with the original producing strain Sorangium cellulosum So ce 427, and shortens the fermentation production cycle, which reduces the fermentation production. Cost, the development of new anti-tumor or anti-infective drugs has important research and application value.
  • Figure 1 Disorazole Z biosynthetic gene cluster (dis427) and its synthetic pathway.
  • Module 1 to Module 6 encodes a polyketide synthetase
  • Module 8 encodes a non-ribosomal polypeptide synthetase.
  • KS is a keto synthetase domain in each module
  • KR is a keto reductase domain
  • DH is a dehydratase domain.
  • ACP is an acyl carrier protein domain
  • MT is a methyltransferase domain
  • HC is a heterocyclic domain
  • A is an adenylation domain
  • AT is an acyltransferase domain.
  • Figure 2 Direct cloning of the Disorazole Z biosynthetic gene cluster (dis427).
  • Figure 3 Construction process of expression plasmid p15A-tnpA-kan-tetR-tetO-dis427.
  • Figure 4 Disorazole Z biosynthetic gene cluster (dis427) directly cloned recombinant plasmid p15A-cm-dis427 (1), constructed plasmid p15A-cm-amp-ccdB-dis427(2), p15A-cm-tetR-tetO- Digestion analysis of dis427 (3) and expression plasmid p15A-tnpA-kan-tetR-tetO-dis427 (4).
  • the plasmid was subjected to double digestion analysis with SphI and EcoRV.
  • the left picture shows the theoretical enzyme cut, and the right picture shows the actual enzyme cut.
  • Figure 5 Engineering strain DK1622::Km-Ptet-dis427 constructed by colony PCR detection.
  • A is the result of detection using primers Colony PCR chk01-F and Colony PCR chk01-R;
  • B is the result of detection using primers Colony PCR chk02-F and Colony PCR chk02-R;
  • C is using primers Colony PCR chk03-F and The results of detection by Colony PCR chk03-R;
  • M is TaKaRa DL1000 DNA Marker;
  • N is the original heterologous host Myxococcus xanthus DK1622 as a negative control;
  • P is the recombinant vector p15A-tnpA-Kan-tetR-tetO-dis427, as a positive control ;
  • Numbers 1-6 represent different monoclonals.
  • Figure 6 High performance liquid chromatography-tandem mass spectrometry for the expression of Disorazole Z by engineering strain DK1622::Km-Ptet-dis427.
  • So ce 427_WT is the crude extract of Disorazole Z original producing strain Sorangium cellulosum So ce 427, which is the positive control group;
  • DK1622_WT is the crude extract of the wild type heterologous host Myxococcus xanthus DK1622 fermentation broth, which is the negative control group;
  • DK1622 ::Km-Ptet-dis427 is a crude fermentation broth of the Disorazole Z biosynthetic gene cluster under the control of a tetracycline-inducible promoter expressed in a heterologous host.
  • Escherichia coli GB05, GB05-dir and GBred-gyrA462 recombinase expression plasmid pSC101-BAD-ETgA-tet and plasmid p15A-cm-tetR-tetO-hyg-ccdB, pR6K-amp -cddB and pR6K-oriT-tnpA-kan were purchased from GeneBridges, Germany; T4 DNA polymerase and restriction endonuclease were purchased from NEB, and DNA polymerase for PCR amplification was purchased from TaKaRa; plasmid extraction reagents and DNA agarose gel recovery kit was purchased from Tiangen; wild type Mycobacterium xanthus DK1622 and Sorangium cellulosum So ce427 were deposited by Shandong University-Helmholtz Institute of Biotechnology; Disorazole Z biosynthesis gene The cluster (dis427) nucle
  • Inoculum Sorangium cellulosum So ce 427 was inoculated into VY/2 solid medium (5g/L Angel Yeast, 1.36g/L calcium chloride dihydrate, 0.5mg/L vitamin B12, 15g/L agar powder, adjusted In a pH of 7.2), it was cultured at 30 ° C to a diffusion growth state.
  • Scrape the edge membrane to M26 liquid medium (8g/L potato starch, 2g/L soy peptone, 2g/L yeast extract, 1g/L magnesium sulfate heptahydrate, 1g/L calcium chloride dihydrate, 1mL
  • the /L trace element solution was shaken at 30 ° C to a sufficient amount of bacteria for preparation of genomic DNA.
  • the Sorangium cellulosum So ce 427 genomic DNA prepared by the above method was subjected to RNase digestion treatment and sent to Huada Gene Company for whole genome sequencing.
  • the obtained genomic DNA sequence information was submitted to antiSMASH (https://antismash.secondarymetabolites.org) for prediction of secondary metabolite biosynthesis gene clusters, and the biosynthetic gene cluster of Disorazole Z was analyzed.
  • the resulting gene cluster domain composition was compared with Disorazole Z chemical structure, and the biosynthetic pathway of Disorazole Z was finally determined, as shown in Figure 1.
  • Example 2 Direct cloning of the Disorazole Z biosynthetic gene cluster (dis427)
  • Disorazole Z biosynthetic gene cluster (dis427) is shown in Figure 2.
  • the specific steps are: restriction endonuclease AvaI digestion of plasmid p15A-cm-tetR-tetO-hyg-ccdB to obtain fragment p15A-cm-tetR-tetO (recovering large fragments by enzyme digestion, the glue runs to the bottom and then cut, glue recovery
  • restriction endonuclease AvaI digestion of plasmid p15A-cm-tetR-tetO-hyg-ccdB to obtain fragment p15A-cm-tetR-tetO (recovering large fragments by enzyme digestion, the glue runs to the bottom and then cut, glue recovery
  • glue recovery refer to the instructions of Tiangen Agarose Gel DNA Recovery Kit.
  • p15A-cm-tetR-tetO was used as a PCR template, and PCR amplification was carried out using primers p15A-Cm BstBI and AflII for dis427-F and p15A-Cm BstBI and AflII for dis427-R, and the obtained PCR product p15A-cm vector for The end of dis427 carries a homology arm of the sequence at both ends of the Disorazole Z biosynthetic gene cluster (dis427).
  • PCR primer sequences are as follows (the uppercase letters in the sequence are homologous arms, and the lowercase letters are primers):
  • PCR procedure pre-denaturation at 95 °C for 3 min; denaturation at 98 °C for 15 s; 58 °C (according to primer Tm setting) annealing for 15 s; 72 °C extension for 2 min (extension time is determined according to the length of amplification, 1 kb / 1 min); cycle 30 times The last 72 ° C, 10 min.
  • the primers used in the experiment were p15A-Cm BstBI and AflII for dis427-F and p15A-Cm BstBI and AflII for dis427-R.
  • the template is the product of linearization of p15A-cm-tetR-tetO-hyg-ccdB with restriction endonuclease AvaI.
  • the prepared Sorangium cellulosum So ce 427 genomic DNA was digested with restriction endonucleases BstBI and AflII to release the target gene fragment to be cloned.
  • the enzyme digestion system is shown in the following table:
  • the digestion reaction solution was treated at 37 ° C for 4 h, and 10 ⁇ l was taken for agarose gel electrophoresis.
  • the remaining reaction solution was extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and then sodium acetate-ethanol was used. precipitation.
  • the digested genomic DNA was finally dissolved in an appropriate amount of sterile deionized water, and the concentration was measured by Nanodrop 2000, approximately 2 ⁇ g/ ⁇ l, and placed at 4 ° C until use.
  • the cloning vector fragment and the digested genomic DNA were first treated with T4 DNA polymerase, and then electroporated to transform the recombinant enzyme-expressing E. coli to further complete the final recombination reaction in vivo.
  • 35 ⁇ l of 10% L-arabinose was added to the culture, placed on an Eppendorf thermomixer at 37 ° C, and incubated at 950 rpm for 40 min.
  • the cells were collected by centrifugation at 9400 g for 30 sec. The supernatant was discarded and the pellet was suspended in 1 ml of H 2 O.
  • the centrifugation was repeated, resuspended, centrifuged again, the supernatant was discarded, and the cells were suspended with 20 ⁇ l of H 2 O.
  • the DNA treated with T4 polymerase and desalted was added, and the mixture of cells and DNA was transferred to a 1 mm electric shock cup, and subjected to electric shock with an Eppendorf electroporator 2510, a voltage of 1350 V, a capacitance of 10 Mf, and a resistance of 600 ⁇ .
  • 1 ml LB was added to the electric shock cup, the cells were washed and transferred to a 1.5 ml tube in a well, placed on an Eppendorf thermomixer at 37 ° C, and incubated at 950 rpm for 1 h. Finally, all the bacterial liquid was applied to an LB plate to which 15 ⁇ g/ml of chloramphenicol was added, and cultured at 37 ° C overnight.
  • Example 3 Construction of Disorazole Z biosynthetic gene cluster (dis427) expression plasmid p15A-tnpA-kan-tetR-tetO-dis427
  • the present invention constructs a plasmid which is modified by the promoter of the dis427 gene cluster to strictly regulate its expression.
  • the DNA fragment containing amp-ccdB is amplified by PCR using the primers Amp-ccdB PCR-F and Amp-ccdB PCR-R, and the PCR reaction system and amplification conditions are referred to in Example 2.1.
  • the gel was recovered, it was eluted with sterile deionized water, and the concentration was determined to be about 200 ng/ ⁇ l by Nanodrop 2000.
  • the DNA fragment and the recombinant expression vector were co-transformed with arabinose-induced Escherichia coli GBred-gyr A462 at a low temperature, and resuscitated at 37 ° C. After 1 h, it was applied to an LB plate to which 15 ⁇ g/ml chloramphenicol and 100 ⁇ g/ml ampicillin antibody were added, and cultured overnight at 37 ° C to grow a single colony.
  • PCR primer sequences are as follows (the uppercase letters in the sequence are homologous arms, the lower case letters are primers, and the underlined letters are the restriction enzyme sites of restriction enzymes PacI and PmeI):
  • Amp-ccdB PCR-F CCGCATATGATCAATTCAAGGCCGAATAAG TTAATTAAGTTTAAA C tttgttcaaaaaaaagcc
  • Amp-ccdB PCR-R CGTCCTGCTCTACGTGATTCCCGCTGCTCAT TTAATTAAGTTTAAA C tttgtttatttttctaaatac
  • sequencing primer sequences are as follows:
  • the plasmid p15A-cm-amp-ccdB-disZ427 was digested with restriction endonucleases PacI and PmeI, and the digested product was precipitated by sodium acetate-ethanol and dissolved in an appropriate amount of sterile deionized water to obtain a linear fragment.
  • the DNA fragment containing the tetracycline-inducible promoter was amplified by PCR using the primers tetR-tetO PCR-F and tetR-tetO PCR-R to obtain tetR-tetO PCR for dis427, and the PCR reaction system and amplification conditions were as described in Example 2.1.
  • a single colony was picked to prepare plasmid DNA, which was digested with restriction endonucleases SphI and EcoRV, and the correct recombinant plasmid p15A-cm-tetR-tetO-dis427 was screened (see Figure 4 for enzymatic electrophoresis analysis).
  • the correctly cleaved plasmid was sequenced using the sequencing primers Promoter substitution seq-03 and Promoter substitution seq-04.
  • PCR primer sequences are as follows (the uppercase letters in the sequence are homologous arms, and the lowercase letters are primers):
  • tetR-tetO PCR-F CCGCATATGATCAATTC
  • tetR-tetO PCR-R CGTCCTGCTCTACGTGATTCCCGCTGCTCAtagatcctttctcctctttagatc
  • sequencing primer sequences are as follows:
  • the specific steps are: restriction endonuclease AseI digesting plasmid pR6K-oriT-tnpA-kan to obtain fragment oriT-tnpA-kan (recovering large fragments by enzyme digestion, the glue runs to the bottom and then cutting the glue, and the specific method of rubber recovery refers to Tiangen Company) Agarose Gel DNA Recovery Kit Instructions).
  • the fragment oriT-tnpA-kan was flanked by homologous arms at both ends of the chloramphenicol gene in plasmid p15A-cm-tetR-tetO-dis427.
  • the chloramphenicol gene on plasmid p15A-cm-tetR-tetO-dis427 was replaced with oriT-tnpA-kan, thereby obtaining recombinant plasmid p15A-tnpA-kan-tetR-tetO-dis427.
  • the bacterial solution after the resuscitation was applied to an LB plate to which 15 ⁇ g/ml kanamycin was added, and cultured at 37 ° C overnight.
  • Example 4 Construction of engineering strain DK1622::Km-Ptet-dis427 expressing Disorazole Z according to the present invention
  • the plasmid p15A-tnpA-kan-tetR-tetO-dis427 was electrothermally transformed into Mycobacterium aureus Myxococcus xanthus DK1622 after normal temperature desalting treatment.
  • the electroporation step was: inoculation of Myxococcus xanthus DK1622 in CTT liquid medium (Casitone 10 g/L, MgSO 4 ).
  • the cells were finally resuspended in 50 ⁇ L of sterile deionized water for use as electroporation competent cells.
  • 3 ⁇ g of the plasmid DNA after desalting was added to the prepared competent cells for mixing, and the mixture was transferred to a 1 mm electric cup and subjected to electroporation at a voltage of 1250 V.
  • the bacteria were suspended in 1 mL of CTT. In a liquid medium, it was placed in a shaker at 30 ° C for 4-6 hours.
  • CTT liquid medium melted and cooled to 42 ° C to form a soft agar suspension, and pour 50 ⁇ g/mL kanamycin.
  • CTT plate (containing 1.5% Agar), after the soft agar was solidified, the plate was placed in a 30 ° C incubator for 5-7 days to grow a single colony.
  • Colony PCR identification and identification were performed using 3 pairs of primers (Colony PCR chk01-F and Colony PCR chk01-R, Colony PCR chk02-F and Colony PCR chk02-R, Colony PCR chk03-F and Colony PCR chk03-R). The results are shown in Figure 5.
  • Colony PCR chk03-R CGACGAGCAGGGTGGCGTATCC
  • PCR procedure pre-denaturation at 94 °C for 1 min; denaturation at 98 °C for 10 s; annealing at 55 °C (according to the Tm value of the primer) for 15 s; elongation at 68 °C for 1 min (extension time determined according to the length of amplification, 1 kb / 1 min); 30 cycles After extending 68 ° C, 10 min; last 4 ° C insulation.
  • Example 5 Application of the engineering strain DK1622::Km-Ptet-dis427 of the invention in preparing Disorazole Z
  • the engineered strain DK1622::Km-Ptet-dis427 was inoculated into a CTT liquid medium containing kanamycin (50 ⁇ g/mL), and cultured overnight at 30 ° C on a shaker. The overnight culture was inoculated into shake flasks containing 50 ml of fresh CTT liquid medium at a 1% inoculum size. After incubation for 2 days at 30 ° C, 200 rpm, anhydrous tetracycline was added at a final concentration of 0.5 ⁇ g/ml. After continuing the culture for 1 d, 2% of XAD-16 macroporous adsorption resin was added, and then the culture was continued for 1 d until the end of the fermentation.
  • the cells and macroporous adsorption resin were collected by centrifugation at 8000 rpm for 10 min, and then extracted with methanol.
  • the methanol extract was filtered through a filter paper, and the filtrate was evaporated to dryness under reduced pressure at 40 ° C to obtain a crude extract, and the obtained crude extract was dissolved in 1 ml of chromatography methanol.
  • HPLC Model UltiMate TM 3000 RSLC The chromatographic conditions were: AcclaimTM RSLC 120C18, 5 ⁇ m, 4.6 ⁇ 250 mm; solvent A was ultrapure water (0.1% formic acid) and B acetonitrile (0.1% formic acid); solvent gradient was 0-5 min, 5% B, 5-25 min, 5%-95% B, 25-30 min, 95% B; column flow rate was 0.75 ml/min.
  • the high resolution mass spectrometer was modeled as Bruker microOTOF-Q II, ESI-Q-TOF MS (electrospray quadrupole time-of-flight mass spectrometer).
  • the mass spectrometry conditions were: Auto MS 2 , Mass range (50-1500), and precursor ion 2.
  • the collected liquid data were analyzed by Data Analysis software.
  • the crude extract of Soorium cellulosum So ce 427 was used as the positive control, and the crude extract of wild type heterologous host Myxococcus xanthus DK1622 was used as the negative control.
  • the peaks of [M+H] + were extracted for comparison and analysis.
  • the results showed that the Disorazole Z biosynthetic gene cluster (dis427) was successfully expressed in Myxococcus xanthus DK1622, and the results are shown in Fig. 6.
  • Example 6 Comparison of the amount of Disorazole Z produced by the engineered strain DK1622::Km-Ptet-dis427 and the original producing strain Sorangium cellulosum So ce 427
  • the comparison between the amount of the engineering strain DK1622::Km-Ptet-dis427 constructed by the present invention and the Disorazole Z produced by the wild strain Sorangium cellulosum So ce 427 is mainly based on the peak area comparison method, as follows: First, the ion current is extracted from Disorazole Z (EIC). The [M+H] + peak (EIC 747.3121 ⁇ 0.05+All MS) was integrated to obtain the peak area; then the ratio of the peak area was made, and the ratio was close to 2:1.
  • Disorazole Z-expressing engineering strain DK1622::Km-Ptet-dis427 constructed according to the present invention has a 10-fold increase in Disorazole Z production compared to the wild strain Sorangium cellulosum So ce 427.

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Abstract

一种Disorazole Z的生物合成基因簇dis427，其核苷酸序列如SEQ ID No.1所示。利用所述dis427基因簇构建的异源表达Disorazole Z的工程菌株DK1622：：Km-Ptet-dis427。该菌株是利用黄色粘球菌Myxococcus xanthus DK1622为出发菌株，通过转座的方法在其基因组上整合了Disorazole Z的生物合成基因簇dis427获得。工程菌株DK1622：：Km-Ptet-dis427在制备Disorazole Z中的应用。Disorazole Z生物合成途径及其在异源宿主中的表达方法为开发新的抗肿瘤或抗感染药物、降低发酵生产成本具有研究和应用价值。

Description

一株高效异源表达Disorazole Z的工程菌株和构建该菌株的基因簇及其应用 技术领域
本发明属于微生物基因资源和生物合成技术领域，具体涉及一种Disorazole Z生物合成基因簇和利用该基因簇构建的一株高效异源表达Disorazole Z的工程菌株及其应用。
背景技术
Disorazoles是最早由Jansen等人于1994年从纤维堆囊菌Sorangium cellulosum So ce 12的发酵液中分离得到的结构新颖的大环双内酯类化合物。至今，在Sorangium cellulosum So ce 12中已经发现29个Disorazoles衍生物，分别为Disorazole A1至Disorazole I。
研究表明，Disorazoles类化合物能够抑制微管蛋白聚合，促进微管蛋白解聚，从而干扰细胞分裂，诱导细胞凋亡，对于多种肿瘤细胞系，包括多药耐药肿瘤细胞系均具有很强的生物活性，是一类新型的细胞微管抗稳定剂。Disorazole A1和Disorazole C1是目前研究较多的组分，对于多种人类肿瘤细胞系，包括多药耐药肿瘤细胞系，其半抑制浓度IC 50在pM至nM水平。近期研究发现，Disorazoles类化合物还对A群链球菌的入侵细胞感染途径具有抑制作用。虽然活性显著，但是来源于Sorangium cellulosum So ce 12的Disorazoles类化合物在生物体内的半衰期非常短，是其成药的瓶颈。
Disorazole Z是来源于纤维堆囊菌Sorangium cellulosum So ce 427的Disorazoles家族化合物，与来源于Sorangium cellulosum So ce 12的Disorazoles类化合物相比也具有显著的抗肿瘤活性，同时具有较小的环状骨架，结构更为稳定，在生物体内具有更长的半衰期。已有报道将该化合物与促黄体激素释放激素偶联用于三阴性乳腺癌的靶向治疗已进入二期临床研究。因此，Disorazole Z是一种优良的潜在抗肿瘤或者抗感染新药物。
Figure PCTCN2018120969-appb-000001
尽管Disorazole Z作为一种极具开发前景的抗肿瘤药物或者抗感染药物有望在不远的将来作为商品药物推广，但是如何得到大剂量的纯品物质是当今最大限制之一。一方面，由于野生菌株Sorangium cellulosum So ce 427生长非常缓慢、不易培养因而不适合大规模发酵，另一方面，人工全合成方法制备Disorazole Z非常困难，至今尚未有成功合成的报道。鉴于此，如何高效地生产并提纯Disorazole Z是目前亟待解决的问题。因此，获取其生物合成途径基因簇并将该基因簇转移至生长快速且易于培养的宿主菌中进行异源生物合成显得十分必要，对于开发新的抗肿瘤或抗感染药物、降低发酵生产成本具有重要的应用价值。经检索，Disorazole Z的生物合成基因簇(dis427)以及利用该基因簇在异源宿主菌黄色粘球菌Myxococcus xanthus DK1622中实现高效表达Disorazole Z的文献或专利还未见报道。
发明内容
针对目前产Disorazole Z的野生菌株Sorangium cellulosum So ce 427生长非常缓慢、不易培养因而不适合大规模发酵的不足，本发明要解决的问题是基因组挖掘原始产生菌So ce 427来提供一种Disorazole Z生物合成途径基因簇(dis427)以及利用该基因簇构建一株高效异源表达Disorazole Z的工程菌株用于Disorazole Z的高效异源生物合成。
一方面，本发明涉及一种分离的核酸分子，其包含与SEQ ID No.1具有至少80％同一性，例如，至少85％、至少90％、至少95％或至少99％同一性的核苷酸序列或其互补物。本文中使用的百分比同一性是两条或更多条多核苷酸序列之间的关系，该关系通过对序列进行比较确定。“同一性”能够通过本领域已知的方法容易地计算。
在一个实施方案中，上文所述的分离的核酸分子包含SEQ ID No.1的核苷酸序列或其互补物。
具体地，上文所述的分离的核酸分子是Disorazole Z的生物合成基 因簇，其特征在于：该基因簇命名为dis427，其包含Disorazole Z生物合成所必需的编码聚酮合成酶及非核糖体多肽合成酶的四个核心基因disA，disB，disC和disD，一个假设蛋白基因orf4和一个后修饰基因orf6；该基因簇来源于纤维堆囊菌Sorangium cellulosum So ce 427，其核苷酸序列如SEQ ID No.1所示。所述基因簇对应的Disorazole Z生物合成途径如图1所示。
另一方面，本发明涉及包含上文所述分离的核酸分子的载体。“载体”是指通常携带有不属于细胞中心代谢的部分的基因的染色体外元件，并且常常是环状双链DNA分子的形式。此类元件可为得自任何来源的自主复制序列、基因组整合序列、噬菌体或核苷酸序列、线性或环状的单链或双链DNA或RNA，其中许多核苷酸序列已经被接合或重组到特定构建体中，该构建体能够将所选基因产物的启动子片段和DNA序列连同合适的3′非翻译序列一起导入细胞。
另一方面，本发明涉及包含上文所述核酸分子或基因簇或载体的宿主细胞，例如细菌细胞、真菌细胞，例如酵母细胞、哺乳动物细胞、昆虫细胞和植物细胞。
在一个实施方案中，所述宿主细胞是细菌，例如黄色粘球菌(Myxococcus xanthus)。
在一个实施方案中，所述宿主细胞是高效异源表达Disorazole Z的工程菌株，其特征在于：该菌株命名为工程菌株DK1622：：Km-Ptet-dis427，其基因型为：Myxococcus xanthus DK1622，kanamycin resistance，tetracycline inducible Ptet promoter，disA，disB，disC，orf4，disD and orf6，是利用黄色粘球菌Myxococcus xanthus DK1622为出发菌株，通过转座的方法在其基因组上整合了Disorazole Z的生物合成基因簇dis427获得。另一方面，本发明涉及生产高效异源表达Disorazole Z的宿主细胞的方法，该方法包括将上文所述的核酸分子或基因簇或载体导入异源宿主细胞。
在一个实施方案中，本发明涉及所述高效异源表达Disorazole Z的工程菌株DK1622：：Km-Ptet-dis427的构建方法，步骤是：
(1)利用Red/ET DNA重组技术将Disorazole Z的生物合成基因簇dis427直接克隆至p15A-cm-tetR-tetO-hyg-ccdB载体上，构建得到质粒p15A-cm-dis427；
(2)在步骤(1)构建的质粒p15A-cm-dis427上插入反向筛选标记amp-ccdB，构建得到质粒p15A-cm-amp-ccdB-dis427；
(3)步骤(2)构建的质粒p15A-cm-amp-ccdB-dis427通过限制性内切酶PacI和PmeI酶切后与tetR-tetO PCR片段进行线线重组，构建得到质粒p15A-cm-tetR-tetO-dis427；
(4)在步骤(3)构建的质粒p15A-cm-tetR-tetO-dis427上插入转座元件，构建得到表达质粒p15A-tnpA-kan-tetR-tetO-dis427；
(5)将步骤(4)构建的表达质粒p15A-tnpA-kan-tetR-tetO-dis427电转至Myxococcus xanthus DK1622中，表达质粒在Myxococcus xanthus DK1622中表达转座酶将Disorazole Z的生物合成基因簇dis427整合到Myxococcus xanthus DK1622的基因组上，得到能高效异源表达Disorazole Z的工程菌株，命名为工程菌株DK1622：：Km-Ptet-dis427。
另一方面，本发明涉及生产Disorazole Z的方法，该方法包括培养上文所述的宿主细胞，并且从所述宿主细胞的培养物中回收Disorazole Z。
另一方面，本发明还涉及上文所述的宿主细胞，特别是高效异源表达Disorazole Z的工程菌株DK1622：：Km-Ptet-dis427在生产Disorazole Z中的应用。
本发明所涉及的工程菌株DK1622：：Km-Ptet-dis427在文献中未见报道，是首次对Disorazole Z的生物合成基因簇(dis427)在异源宿主菌Myxococcus xanthus DK1622中实现高效表达。实验证实：本发明提供的工程菌株DK1622：：Km-Ptet-dis427与原始产生菌Sorangium cellulosum So ce 427相比，Disorazole Z的产量提高了1倍，而且缩短了发酵生产周期，这对于降低发酵生产成本，开发新的抗肿瘤或抗感染药物具有重要的研究和应用价值。
附图说明
图1：Disorazole Z生物合成基因簇(dis427)及其合成途径。
其中：模块1至模块6编码聚酮合成酶，模块8编码非核糖体多肽合成酶，各模块中KS为酮基合成酶结构域，KR为酮基还原酶结构域，DH为脱水酶结构域，ACP为酰基载体蛋白结构域，MT为甲基转移酶结构域，HC为杂环化结构域，A为腺苷酰化结构域，AT为酰基 转移酶结构域。
图2：Disorazole Z生物合成基因簇(dis427)的直接克隆过程。
图3：表达质粒p15A-tnpA-kan-tetR-tetO-dis427的构建过程。
图4：Disorazole Z生物合成基因簇(dis427)直接克隆重组子质粒p15A-cm-dis427(1)，构建的质粒p15A-cm-amp-ccdB-dis427(2)、p15A-cm-tetR-tetO-dis427(3)及表达质粒p15A-tnpA-kan-tetR-tetO-dis427(4)的酶切分析。
用SphI和EcoRV对质粒进行双酶切分析。左图为理论酶切图，右图为实际酶切图。
图5：菌落PCR检测构建的工程菌株DK1622：：Km-Ptet-dis427。
A为利用引物Colony PCR chk01-F和Colony PCR chk01-R进行检测的结果；B为利用引物Colony PCR chk02-F和Colony PCR chk02-R进行检测的结果；C为利用引物Colony PCR chk03-F和Colony PCR chk03-R进行检测的结果；M为TaKaRa DL1000 DNA Marker；N为原始异源宿主Myxococcus xanthus DK1622，作为阴性对照；P为重组载体p15A-tnpA-Kan-tetR-tetO-dis427，作为阳性对照；数字1-6代表不同的单克隆。
图6：工程菌株DK1622：：Km-Ptet-dis427表达Disorazole Z的高效液相色谱-质谱检测。
其中，So ce 427_WT为Disorazole Z原始产生菌Sorangium cellulosum So ce 427发酵液粗提物，为阳性对照组；DK1622_WT为野生型异源宿主菌Myxococcus xanthus DK1622发酵液粗提物，为阴性对照组；DK1622：：Km-Ptet-dis427为四环素诱导启动子调控下的Disorazole Z生物合成基因簇在异源宿主中进行表达的发酵液粗提物。
具体实施方式
以下结合附图及具体实例详细描述本发明，以便更好地理解本发明，但所述内容并不限制本发明的保护内容。
一般性说明：如下实施例所涉及的大肠杆菌GB05、GB05-dir和GBred-gyrA462，重组酶表达质粒pSC101-BAD-ETgA-tet以及质粒p15A-cm-tetR-tetO-hyg-ccdB、pR6K-amp-cddB和pR6K-oriT-tnpA-kan均购于德国GeneBridges公司；T4 DNA聚合酶和限制性内切酶购于 NEB公司，用于PCR扩增的DNA聚合酶购于TaKaRa公司；质粒提取试剂和DNA琼脂糖凝胶回收试剂盒购于天根公司；野生型黄色粘球菌Myxococcus xanthus DK1622和纤维堆囊菌Sorangium cellulosum So ce427为山东大学-亥姆霍兹生物技术研究所保藏；Disorazole Z生物合成基因簇(dis427)核苷酸序列见序列表SEQ ID No.1；基因测序由华大基因公司完成；寡核苷酸合成由上海生工生物公司完成；其他涉及的试剂和耗材均为国产，实施例中的实验方法及试剂如无特殊说明，均为本领域常规方法与市售试剂。
实施例1：Disorazole Z生物合成基因簇(dis427)的挖掘
将纤维堆囊菌Sorangium cellulosum So ce 427接种至VY/2固体培养基(5g/L安琪酵母、1.36g/L二水合氯化钙、0.5mg/L维生素B12、15g/L琼脂粉，调节pH值为7.2)中，置于30℃培养至扩散生长状态。刮取边缘菌膜转接到M26液体培养基(8g/L马铃薯淀粉、2g/L大豆蛋白胨、2g/L酵母提取物、1g/L七水合硫酸镁、1g/L二水合氯化钙、1mL/L微量元素溶液，调节pH值为7.2)中，置于30℃摇床培养至足够的菌体量以用于制备基因组DNA。
离心收集菌体后，将其重悬于10mM Tris-HCl缓冲液中(pH值为8.0)。向菌悬液中加入终浓度为1mg/ml的蛋白酶K及终浓度为1％的SDS，置于50℃水浴处理至少2h。向处理后的裂解液中加入等体积的DNA提取液(苯酚∶氯仿∶异戊醇＝25∶24∶1)，充分混匀后离心得到上清液。向上清液中加入1/10体积的3M醋酸钠(pH值为8.0)，混匀后再加入3倍体积的无水乙醇，充分混匀后可见絮状基因组DNA沉淀。将絮状沉淀挑取至75％乙醇中，离心后弃上清得到基因组DNA，自然晾干后溶解于10mM Tris-HCl缓冲液中(pH值为8.0)置于4℃备用。
上述方法制备的Sorangium cellulosum So ce 427基因组DNA经过RNA酶消化处理之后送至华大基因公司进行全基因组测序。将获得的基因组DNA序列信息提交至antiSMASH(https：//antismash.secondarymetabolites.org)进行次生代谢产物生物合成基因簇预测，分析得到Disorazole Z的生物合成基因簇。将得到的基因簇结构域构成与Disorazole Z化学结构进行比较分析，最终确定了 Disorazole Z的生物合成途径，如图1所示。
实施例2：Disorazole Z生物合成基因簇(dis427)的直接克隆
Disorazole Z生物合成基因簇(dis427)直接克隆过程见图2。
2.1 Disorazole Z生物合成基因簇(dis427)直接克隆载体的制备
具体步骤为：限制性内切酶AvaI酶切质粒p15A-cm-tetR-tetO-hyg-ccdB得到片段p15A-cm-tetR-tetO(酶切回收大片段，胶跑到底部再切胶，胶回收具体做法参照天根公司琼脂糖凝胶DNA回收试剂盒说明书)。然后以p15A-cm-tetR-tetO作为PCR模板，用引物p15A-Cm BstBI and AflII for dis427-F和p15A-Cm BstBI and AflII for dis427-R进行PCR扩增，得到的PCR产物p15A-cm vector for dis427末端带有Disorazole Z生物合成基因簇(dis427)两端序列的同源臂。
PCR引物序列如下(序列中大写字母为同源臂，小写字母为引物)：
p15A-cm BstBI and AflII for dis427-F：AAGCCGTCACGGGCGCTCTGGTCTCCCTTAGTAGCAGGACACGGGCCAGGGCTCGGCCTGACAGATTTCCCGCGTTTACCagttacggatcttaaggatctc
p15A-cm BstBI and AflII for dis427-R：CGATTGCTCGGGGGCGCCGGAGACCGCCGGCAGGGGCTTCGATTTCCGCGGGTATCTGGCGCGCATGGCCGCCACGGAGActtattcggccttgaattgatc
用引物p15A-Cm BstBI and AflII for dis427-F和p15A-Cm BstBI and AflII for dis427-R PCR扩增片段p15A-cm vector for dis427的具体做法如下：
PCR扩增体系：
Figure PCTCN2018120969-appb-000002
PCR程序：95℃预变性3min；98℃变性15s；58℃(根据引 物Tm值设定)退火15s；72℃延伸2min(延伸时间根据所扩增的长度确定，1kb/1min)；循环30次；最后72℃，10min。实验过程中所用的引物是p15A-Cm BstBI and AflII for dis427-F和p15A-Cm BstBI and AflII for dis427-R。模板是p15A-cm-tetR-tetO-hyg-ccdB用限制性内切酶AvaI线性化的产物。
2.2 基因组DNA的限制性内切酶处理
将制备的Sorangium cellulosum So ce 427基因组DNA用限制性内切酶BstBI和AflII进行酶切处理以释放出待克隆的目的基因片段，酶切体系如下表所示：
Figure PCTCN2018120969-appb-000003
将酶切反应液置于37℃处理4h，取10μl进行琼脂糖凝胶电泳检测，剩余的反应液利用苯酚∶氯仿∶异戊醇(25∶24∶1)抽提，然后用醋酸钠-乙醇沉淀。酶切后的基因组DNA最终溶解于适量无菌去离子水，利用Nanodrop 2000测浓度，大约2μg/μl，置于4℃备用。
2.3 Disorazole Z生物合成基因簇(dis427)克隆子的获得
克隆载体片段和酶切后的基因组DNA首先利用T4 DNA聚合酶进行处理，然后电击转化表达重组酶的大肠杆菌来进一步在体内完成最终的重组反应。
体外T4 DNA聚合酶处理的反应体系如下表所示：
Figure PCTCN2018120969-appb-000004
体外T4 DNA聚合酶处理的反应条件如下表所示：
Figure PCTCN2018120969-appb-000005
电转化步骤为：将含有温敏复制子的重组酶表达质粒pSC101-BAD-ETgA-tet的菌株GB05-dir在加有4μg/ml四环素的LB培养基(低盐，1％Triptone，0.5％酵母提取物，0.1％NaCl)中30℃培养过夜(OD 600＝3～4)。将40μl过夜培养物(OD 600＝3～4)转接到加有4μg/ml四环素的1.3ml LB中，置于Eppendorf thermomixer上30℃，950rpm培养2h(OD 600＝0.35～0.4)。向培养物中加35μl 10％L-阿拉伯糖，置于Eppendorf thermomixer上37℃，950rpm培养40min。9400g离心30sec收集细胞。弃上清，沉淀用1ml H 2O悬浮。重复离心、重悬、再离心、弃上清，用20μl H 2O悬浮细胞。加入T4聚合酶处理并脱盐的DNA，将细胞和DNA的混合液转入1mm电击杯中，用Eppendorf electroporator 2510进行电击，电压1350V，电容10Mf，电阻600Ω。加1ml LB至电击杯中，洗涤细胞并将其转移至扎孔的1.5ml管中，置于Eppendorf thermomixer上37℃，950rpm培养1h。最后将所有菌液涂布到加有15μg/ml氯霉素的LB平板上，37℃过夜培养。
挑取单菌落在加有10μg/ml氯霉素的LB培养基中置于37℃培养过夜，利用碱裂解和异丙醇沉淀法提取质粒DNA，经限制性内切酶SphI和EcoRV消化后进行电泳检测，筛选得到正确的重组质粒p15A-cm-dis427(酶切电泳分析见图4)。
实施例3：Disorazole Z生物合成基因簇(dis427)表达质粒p15A-tnpA-kan-tetR-tetO-dis427的构建
3.1 质粒p15A-cm-tetR-tetO-disZ427的构建
质粒p15A-cm-tetR-tetO-disZ427的构建过程见图3。
已有报道Disorazoles类化合物生物合成基因簇的组成型表达可能 影响异源宿主生长及正常代谢过程，因此本发明构建了一种对dis427基因簇进行启动子改造以严谨调控其表达的质粒。
具体步骤为：首先用引物Amp-ccdB PCR-F和Amp-ccdB PCR-R通过PCR扩增含有amp-ccdB的DNA片段，PCR反应体系及扩增条件参照实施例2.1。胶回收之后用无菌去离子水洗脱，利用Nanodrop 2000测浓度，大约200ng/μl，将该DNA片段与重组表达载体在低温条件下共同转化阿拉伯糖诱导的大肠杆菌GBred-gyrA462，37℃复苏1h后涂布至加有15μg/ml氯霉素和100μg/ml氨苄霉素双抗的LB平板，37℃过夜培养至长出单菌落。
然后挑取单菌落制备质粒DNA，用限制性内切酶SphI和EcoRV酶切，筛选得到正确的重组质粒p15A-cm-amp-ccdB-dis427(酶切电泳分析见图4)，并对酶切正确的质粒用引物Promoter substitution seq-01和Promoter substitution seq-02进行测序。
PCR引物序列如下(序列中大写字母为同源臂，小写字母为引物，下划线字母为限制性内切酶PacI和PmeI的酶切位点)：
Amp-ccdB PCR-F：CCGCATATGATCAATTCAAGGCCGAATAAG TTAATTAAGTTTAAA Ctttgttcaaaaaaaagcc
Amp-ccdB PCR-R：CGTCCTGCTCTACGTGATTCCCGCTGCTCAT TTAATTAAGTTTAAA Ctttgtttatttttctaaatac
测序引物序列如下：
Promoter substitution seq-01：CAACGGTGGTATATCCAGTG
Promoter substitution seq-02：CGAAATCAGGGGAATAATAGG
3.2 质粒p15A-cm-tetR-tetO-dis427的构建
质粒p15A-cm-tetR-tetO-dis427的构建过程见图3。
用限制性内切酶PacI和PmeI对质粒p15A-cm-amp-ccdB-disZ427进行双酶切，酶切反应产物经醋酸钠-乙醇沉淀后溶解于适量无菌去离子水中得到线性片段。用引物tetR-tetO PCR-F和tetR-tetO PCR-R通过PCR扩增含有四环素诱导启动子的DNA片段得到tetR-tetO PCR for dis427，PCR反应体系及扩增条件参照实施例2.1。参照实施例2.3中的T4 DNA聚合酶作用条件将酶切后的线性DNA片段和PCR扩增的 启动子片段tetR-tetO PCR for dis427进行体外连接，脱盐处理后电击转化大肠杆菌GB05，涂布至加有15μg/ml氯霉素的LB平板，37℃过夜培养至长出单菌落。
挑取单菌落制备质粒DNA，用限制性内切酶SphI和EcoRV酶切，筛选正确的重组质粒p15A-cm-tetR-tetO-dis427(酶切电泳分析见图4)。并对酶切正确的质粒用测序引物Promoter substitution seq-03和Promoter substitution seq-04进行测序。
PCR引物序列如下(序列中大写字母为同源臂，小写字母为引物)：
tetR-tetO PCR-F：CCGCATATGATCAATTC
tetR-tetO PCR-R：CGTCCTGCTCTACGTGATTCCCGCTGCTCAtagatcctttctcctctttagatc
测序引物序列如下：
Promoter substitution seq-03：GTGAGTATGGTGCCTATCTA
Promoter substitution seq-04：GAAGGGGAAAGCTGGCAAGA
3.3 表达质粒p15A-tnpA-kan-tetR-tetO-dis427的构建
表达质粒p15A-tnpA-kan-tetR-tetO-dis427的构建过程见图5。
具体步骤为：限制性内切酶AseI酶切质粒pR6K-oriT-tnpA-kan得到片段oriT-tnpA-kan(酶切回收大片段，胶跑到底部再切胶，胶回收具体做法参照天根公司琼脂糖凝胶DNA回收试剂盒说明书)。片段oriT-tnpA-kan两端带有质粒p15A-cm-tetR-tetO-dis427中氯霉素基因两端的同源臂。然后将200ng DNA片段oriT-tnpA-kan和200ng质粒p15A-cm-tetR-tetO-dis427共电转化到35μl 10％L-阿拉伯糖诱导表达了Redα/β/γ重组酶的菌株GBred-gyrA462中进行线环重组。在重组酶的作用下，质粒p15A-cm-tetR-tetO-dis427上的氯霉素基因被oriT-tnpA-kan替换，从而得到重组质粒p15A-tnpA-kan-tetR-tetO-dis427。复苏之后的菌液涂布到加有15μg/ml卡那霉素的LB平板上，37℃培养过夜。然后挑取单菌落制备质粒DNA，用限制性内切酶SphI和EcoRV酶切，筛选正确的重组质粒p15A-tnpA-kan-tetR-tetO-dis427(酶切电泳分析见图4)。
实施例4：本发明所述表达Disorazole Z的工程菌株DK1622：：Km-Ptet-dis427的构建
将质粒p15A-tnpA-kan-tetR-tetO-dis427常温脱盐处理之后电转化至黄色粘球菌Myxococcus xanthus DK1622，电转化步骤为：将Myxococcus xanthus DK1622接种于CTT液体培养基(Casitone 10g/L，MgSO 4-7H 2O 1.97g/L，1mol/L Tris HCI(pH＝7.6)10mL，0.1mol/L KPO 4 buffer(pH＝7.6)10mL，pH＝7.6)中，置于30℃摇床培养过夜，取100μL过夜培养物转接到新的1.7mL CTT液体培养基中继续培养约24h至OD 600为0.6，低温9400g离心1min收集菌体，将菌体重悬于1mL预冷的无菌去离子水中，重复一次，菌体最终重悬于50μL无菌去离子水中以用作电转感受态细胞。取3μg除盐处理之后的质粒DNA加入到制备的感受态细胞中混匀，将混匀液转入1mm电转杯中并置于1250V电压下进行电击转化，电转化之后将菌体重悬于1mL CTT液体培养基中，置于30℃摇床复苏培养4-6h。向复苏培养液中加入1mL CTT液体培养基和1mL融化并冷却至42℃的CTT固体培养基(含1.5％Agar)混匀以形成软琼脂菌悬液，倾倒含50μg/mL卡那霉素的CTT平板(含1.5％Agar)，待软琼脂凝固之后将平板倒置于30℃培养箱中培养5-7d至长出单菌落。
挑取单菌落接种到加有50μg/mL卡那霉素的1.5mL CTT液体培养基中置于30℃摇床培养过夜以用于菌落PCR鉴定。分别用3对引物(Colony PCR chk01-F和Colony PCR chk01-R、Colony PCR chk02-F和Colony PCR chk02-R、Colony PCR chk03-F和Colony PCR chk03-R)对其进行菌落PCR鉴定，鉴定结果见图5。
上述菌落PCR引物的序列为：
Colony PCR chk01-F：CAGAAGAACTCGTCAAGAAG
Colony PCR chk01-R：GAACAAGATGGATTGCACGC
Colony PCR chk02-F：GGATCGTGAGTACCTGGAGAAG
Colony PCR chk02-R：GAGCGTCCGGGAGGTCGTGGGC
Colony PCR chk03-F：GCAGAAGTACGTGGGCCTCAGC
Colony PCR chk03-R：CGACGAGCAGGGTGGCGTATCC
菌落PCR扩增体系：
Figure PCTCN2018120969-appb-000006
PCR程序：94℃预变性1min；98℃变性10s；55℃(根据引物Tm值设定)退火15s；68℃延伸1min(延伸时间根据所扩增的长度确定，1kb/1min)；循环30次；后延伸68℃，10min；最后4℃保温。
实施例5：本发明所述工程菌株DK1622：：Km-Ptet-dis427在制备Disorazole Z中的应用
将工程菌株DK1622：：Km-Ptet-dis427接种至含有卡那霉素(50μg/mL)的CTT液体培养基中，30℃摇床培养过夜。按1％的接种量，将过夜培养物接种到含有50ml新鲜CTT液体培养基的摇瓶中。30℃，200rpm培养2d之后加入终浓度为0.5μg/ml的无水四环素。继续培养1d之后加入2％的XAD-16大孔吸附树脂，然后继续培养1d至发酵结束。8000rpm离心10min收集细胞和大孔吸附树脂，然后用甲醇提取。甲醇提取液用滤纸过滤，将滤液在40℃下减压旋转蒸干得到粗提物，并将得到的粗提物溶解于1ml色谱甲醇中。
利用0.22μm滤膜过滤之后取5μl用于HPLC-MS分析。高效液相色谱仪型号为UltiMate TM 3000 RSLC。色谱条件为：AcclaimTM RSLC 120C18，5μm，4.6×250mm；溶剂A为超纯水(0.1％甲酸)和B乙腈(0.1％甲酸)；溶剂梯度为，0-5min，5％B，5-25min，5％-95％B，25-30min，95％B；柱流速是0.75ml/min。高分辨质谱仪的型号为Bruker microOTOF-Q II，ESI-Q-TOF MS(电喷雾四级杆飞行时间质谱仪)。质谱条件为：Auto MS 2，Mass range(50-1500)，precursor ion 2。
采用Data Analysis软件对采集到的液质数据进行分析，以Disorazole Z原始产生菌Sorangium cellulosum So ce 427的粗提物为阳 性对照，以野生型异源宿主菌Myxococcus xanthus DK1622的粗提物为阴性对照，提取[M+H] +的峰进行比较和分析，结果显示，Disorazole Z生物合成基因簇(dis427)在Myxococcus xanthus DK1622中能够成功表达，结果见图6。
实施例6：构建的工程菌株DK1622：：Km-Ptet-dis427与原始产生菌Sorangium cellulosum So ce 427产Disorazole Z的量的比较
本发明构建的工程菌株DK1622：：Km-Ptet-dis427与野生菌株Sorangium cellulosum So ce 427产Disorazole Z的量的比较主要是采用峰面积比较法，具体如下：首先，对Disorazole Z提取离子流(EIC)的[M+H] +峰(EIC 747.3121±0.05+All MS)进行积分，得到峰面积；然后对峰面积进行比值，比值接近2∶1。实验证明，本发明构建的表达Disorazole Z的工程菌株DK1622：：Km-Ptet-dis427与野生菌株Sorangium cellulosum So ce 427相比，Disorazole Z的产量提高了1倍。
Figure PCTCN2018120969-appb-000007
Figure PCTCN2018120969-appb-000008
Figure PCTCN2018120969-appb-000009
Figure PCTCN2018120969-appb-000010
Figure PCTCN2018120969-appb-000011
Figure PCTCN2018120969-appb-000012
Figure PCTCN2018120969-appb-000013
Figure PCTCN2018120969-appb-000014
Figure PCTCN2018120969-appb-000015
Figure PCTCN2018120969-appb-000016
Figure PCTCN2018120969-appb-000017
Figure PCTCN2018120969-appb-000018
Figure PCTCN2018120969-appb-000019
Figure PCTCN2018120969-appb-000020
Figure PCTCN2018120969-appb-000021
Figure PCTCN2018120969-appb-000022
Figure PCTCN2018120969-appb-000023
Figure PCTCN2018120969-appb-000024
Figure PCTCN2018120969-appb-000025
Figure PCTCN2018120969-appb-000026
Figure PCTCN2018120969-appb-000027
Figure PCTCN2018120969-appb-000028
Figure PCTCN2018120969-appb-000029
Figure PCTCN2018120969-appb-000030
Figure PCTCN2018120969-appb-000031
Figure PCTCN2018120969-appb-000032
Figure PCTCN2018120969-appb-000033
Figure PCTCN2018120969-appb-000034
Figure PCTCN2018120969-appb-000035

Claims (10)

  1. 一种分离的核酸分子，其包含与SEQ ID No.1具有至少80％同一性，例如，至少85％、至少90％、至少95％或至少99％同一性的核苷酸序列或其互补物。
  2. 权利要求1的分离的核酸分子，其包含SEQ ID No.1的核苷酸序列或其互补物。
  3. 权利要求1的分离的核酸分子，其如SEQ ID No.1的核苷酸序列所示。
  4. 包含权利要求1-3的任一项所述的核酸分子的载体。
  5. 重组宿主细胞，其包含权利要求1-3的任一项所述的核酸分子或权利要求4所述的载体。
  6. 权利要求5的宿主细胞，其中所述宿主细胞是细菌，例如黄色粘球菌(Myxococcus xanthus)。
  7. 权利要求6的宿主细胞，其中所述宿主细胞是Myxococcus xanthus DK1622：：Km-Ptet-dis427，其基因型为：Myxococcus xanthus DK1622，kanamycin resistance，tetracycline inducible Ptet promoter，disA，disB，disC，orf4，disD and orf6，是利用黄色粘球菌Myxococcus xanthus DK1622为出发菌株，通过转座的方法在其基因组上整合了Disorazole Z的生物合成基因簇dis427获得。
  8. 生产高效异源表达Disorazole Z的宿主细胞的方法，该方法包括将权利要求1-3的任一项所述的核酸分子或权利要求4所述的载体导入异源宿主细胞。
  9. 生产Disorazole Z的方法，该方法包括培养权利要求5-7的任一项的宿主细胞，并且从所述宿主细胞的培养物中回收Disorazole Z。
  10. 权利要求5-7的任一项的宿主细胞在生产Disorazole Z中的应用。
PCT/CN2018/120969 2017-12-18 2018-12-13 一株高效异源表达Disorazole Z的工程菌株和构建该菌株的基因簇及其应用 WO2019120132A1 (zh)

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