阅读说明：本技术 一种提高纤维堆囊菌埃博霉素产量的tale-tf载体及其构建方法 (TALE-TF vector for increasing yield of epothilones of sorangium cellulosum and construction method thereof ) 是由 叶伟 刘桃妹 章卫民 朱牧孜 李赛妮 李浩华 孔亚丽 刘珊 于 2019-11-08 设计创作，主要内容包括：本发明公开了一种提高纤维堆囊菌埃博霉素产量的TALE-TF载体及其构建方法。本发明首次公开了一种新型的靶向纤维堆囊菌埃博霉素生物合成基因启动子的新型TALE-TF元件,将其导入纤维堆囊菌,提高了埃博霉素生物合成基因表达水平及埃博霉素产量。本发明通过代谢工程手段提高了埃博霉素产量并降低埃博霉素的生产成本,能促进埃博霉素作为原料药物在肿瘤治疗方面的应用。(The invention discloses a TALE-TF vector for improving the yield of epothilone of sorangium cellulosum and a construction method thereof. The invention discloses a novel TALE-TF element of a novel target epothilone biosynthesis gene promoter of sorangium cellulosum, which is introduced into sorangium cellulosum, so that the expression level of the epothilone biosynthesis gene and the epothilone yield are improved. The invention improves the yield of the epothilone and reduces the production cost of the epothilone by means of metabolic engineering, and can promote the application of the epothilone as a raw material medicament in the aspect of tumor treatment.)

1. A TALE-TF vector for improving yield of epothilone of sorangium cellulosum is characterized by being constructed by the following method:

determining a TALE-TF target sequence according to a nucleotide sequence of a promoter of an epothilone biosynthesis gene P3 of sorangium cellulosum, constructing a TALE-TF element according to the target sequence, inserting the nucleotide sequence coding the TALE-TF element into a vector PtalenR36, then carrying out enzyme digestion on the vector and a VP64 element containing sites of endonuclease BamHI and SacI on the upstream and the downstream by using endonucleases BamHI and SacI, and then carrying out ligation reaction to insert the VP64 element between the sites BamHI and SacI instead of a FokI element to obtain a PTALE-VP64 vector; and (3) carrying out enzyme digestion on the PTALE-VP64 vector and a P43 promoter containing the AscI and SpeI sites at the upstream and downstream by using endonuclease AscI and SpeI, and then carrying out a connection reaction to enable the P43 promoter to replace a pGPD promoter to be inserted between the AscI and SpeI sites, so as to obtain a PTALE-VP64-P43 recombinant vector, namely the TALE-TF vector for improving the yield of the epothilone of sorangium cellulosum.

2. The TALE-TF vector for increasing the yield of the epothilone of Sorangium cellulosum according to claim 1, wherein the TALE-TF target sequence is shown in Seq ID No. 5.

3. The TALE-TF vector for increasing the yield of the epothilone of the sorangium cellulosum according to claim 1, wherein the VP64 element is obtained by PCR amplification by using VP64F and VP64R as primers and pCDNA-dCas9/VP64 vector as a template; the primer sequence of VP64F is shown in Seq ID No.3, and the primer sequence of VP64R is shown in Seq ID No. 4.

4. The TALE-TF vector for improving the yield of the epothilone of the sorangium cellulosum according to claim 1, wherein the P43 promoter is obtained by PCR amplification by taking P43TALE F and P43TALE R as primers and taking a pBEP43 vector as a template; the P43TALE F primer sequence is shown in Seq ID No.1, and the P43TALE R sequence is shown in Seq ID No. 2.

5. The TALE-TF vector for increasing the yield of sorangium cellulosum epothilone according to claim 1, wherein the sorangium cellulosum is So ce M4.

6. The genetic engineering bacteria of sorangium cellulosum for producing the epothilone are characterized in that the TALE-TF vector is transferred into sorangium cellulosum So ce M4.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a TALE-TF vector for improving the yield of epothilone of sorangium cellulosum and a construction method thereof.

Background

Epothilones are macrolide compounds with strong antitumor activity generated by sorangium cellulosum, and the reported antitumor activity of epothilone B is 10-100 times that of taxol, and epothilones show strong antitumor activity to p-glycoprotein expressed multidrug resistant tumor cell lines, so that tumor cells are not easy to generate drug resistance to the compounds. The research on Sorangium cellulosum mainly focuses on improving the yield of the epothilone by means of optimizing fermentation conditions, ultraviolet mutagenesis and the like. At present, the yield of the epothilone A can reach 18-22mg/L and the yield of the epothilone B reaches 8-9mg/L through physical and chemical methods, and a certain distance is reserved from large-scale production. The epothilone gene clusters of Sorangium cellulosum SMP44 and So ce90 have been identified, but because the genetic manipulation of Sorangium cellulosum is difficult, the epothilone gene clusters are cloned into myxococcus aurantiacus, Escherichia coli and Streptomyces coelicolor for expression, but the yield of the heterologous expression of epothilone is lower due to the cytotoxicity of epothilone. The highest yield of epothilone B was only 2.5 mg/L. Sorangium cellulosum is still the most ideal host for expression of epothilones. Therefore, if the sorangium cellulosum can be modified by genetic manipulation means to prepare the high-yield epothilone B engineering strain, the development of a novel efficient anti-cancer drug can be promoted.

TALEs are a class of highly specific DNA binding proteins from the plant pathogenic bacterium xanthomonas (Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F.A TAL effector toolboxfor genome engineering. nature Protocols,2012,7: 171-. The TALE element mainly comprises a TALEN element and a TALE-TF element, and the TALE-TF element is widely applied to the activation of target genes in hosts such as mammalian cells, yeasts and the like due to the high specificity of the combined DNA and the characteristic that a transcription activation element activates the expression of the target genes, and achieves better effect. However, the TALE-TF technology is not successfully applied to Sorangium cellulosum at present.

Disclosure of Invention

The inventor finds that the application of the TALE-TF element in activating the expression of the epothilone biosynthesis gene can improve the epothilone biosynthesis efficiency of sorangium cellulosum, thereby improving the yield of epothilone.

The inventor previously isolated a sorangium cellulosum Soce M4(GDIM 1.777) capable of producing epothilone from Guangdong Xinyi soil, and amplified the sorangium cellulosum Soce M4 epothilone biosynthesis gene promoter P3. The inventors found that the biosynthesis efficiency of epothilone of Sorangium cellulosum can be improved by targeting the P3 promoter sequence with a TALE-TF element.

Therefore, the first purpose of the invention is to provide a TALE-TF vector for improving the yield of the epothilone of sorangium cellulosum and a construction method thereof.

The TALE-TF vector for improving the yield of the epothilone of the sorangium cellulosum is characterized by being constructed by the following method:

determining a TALE-TF target sequence according to a nucleotide sequence of a promoter of an epothilone biosynthesis gene P3 of sorangium cellulosum, constructing a TALE-TF element according to the target sequence, inserting the nucleotide sequence coding the TALE-TF element into a vector PtalenR36, then carrying out enzyme digestion on the vector and a VP64 element containing sites of endonuclease BamHI and SacI on the upstream and the downstream by using endonucleases BamHI and SacI, and then carrying out ligation reaction to insert the VP64 element between the sites BamHI and SacI instead of a FokI element to obtain a PTALE-VP64 vector; and (3) carrying out enzyme digestion on the PTALE-VP64 vector and a P43 promoter containing the AscI and SpeI sites at the upstream and downstream by using endonuclease AscI and SpeI, and then carrying out a connection reaction to enable the P43 promoter to replace a pGPD promoter to be inserted between the AscI and SpeI sites, so as to obtain a PTALE-VP64-P43 recombinant vector, namely the TALE-TF vector for improving the yield of the epothilone of sorangium cellulosum.

Preferably, the TALE-TF target sequence is shown as Seq ID No. 5;

preferably, the VP64 element is obtained by PCR amplification by using VP64F and VP64R as primers and pCDNA-dCas9/VP64 vector as a template; the primer sequence of VP64F is shown as Seq ID No.3, and the primer sequence of VP64R is shown as Seq ID No. 4;

preferably, the P43 promoter is obtained by PCR amplification by taking P43TALE F and P43TALE R as primers and a pBEP43 vector as a template; the P43TALE F primer sequence is shown as Seq ID No.1, and the P43TALE R sequence is shown as Seq ID No. 2;

preferably, the sorangium cellulosum is sorangium cellulosum So ce M4.

The invention also provides an epothilone-producing genetic engineering bacterium, which is characterized in that the TALE-TF vector is transferred into Sorangium cellulosum Soce M4.

The inventor subjects the group to separate sorangium cellulosum So ce M4 from soil in Credit City of Guangdong province at the earlier stage, and found that the bacterium can produce epothilone, but the yield is relatively low. TALE-TF is a transcription regulation element capable of improving the expression level of a target gene. The invention constructs a TALE-TF element of a target epothilone biosynthesis gene promoter P3 and introduces the TALE-TF element into Sorangium cellulosum So ce M4 through electric conversion. The expression level of the epothilone biosynthesis genes was analyzed by fluorescent quantitative PCR, and the yields of epothilone in wild and recombinant bacteria were analyzed by HPLC and LC-MS. The invention provides a novel method for improving the biosynthesis efficiency of sorangium cellulosum epothilone by transcription regulation so as to improve the yield of the epothilone, thereby greatly improving the yield of the epothilone and reducing the production cost of the epothilone by metabolic engineering means in the later period, and promoting the application of the epothilone as a raw material medicament in the aspect of tumor treatment.

The nucleotide sequence of the promoter of the epothilone biosynthesis gene P3 of sorangium cellulosum So ce M4 is disclosed in the patent application numbers: 201811486406.X, the invention name is: a novel epothilone biosynthesis gene P3 promoter, and a preparation method and application thereof.

The sorangium cellulosum So ce M4 is preserved in Guangdong province microorganism culture collection center (GDMCC), and the preservation number is as follows: GIM1.777, which was marketed before the filing date of this patent.

Drawings

FIG. 1 is a sequencing validation diagram for constructing a TALE-TF element;

FIG. 2 is a vector targeting the TALE-TF element of the promoter of the epothilone biosynthetic gene: PTALEN R36;

FIG. 3 is a PCR validation of the targeted recombinant vector construction: wherein, the diagram A is PtalenR36 vector which is obtained by inserting the VP64 element after enzyme digestion into BamHI and SacI double-enzyme digestion and contains TALE-TF element of the target P3 promoter; lanes 1-10 are different colony PCR product identification; FIG. B shows the P43 promoter after digestion inserted into the PTALE-VP64 vector after digestion; lanes 1-4 are different colony PCR product identification; lane 5 is a positive control;

FIG. 4 is a drawing showing the confirmation of introduction of the recombinant vector PTALE-VP64-P43 into Sorangium cellulosum So ce M4;

FIG. 5 is a fluorescent quantitative PCR analysis of the expression levels of epoA, epoC and epoK genes for epothilone biosynthesis; wherein dCas9-VP64 represents Sorangium cellulosum So ce M4 transferred into pCDNA-dCas9/VP64 vector, and RP3-TALE-VP64 represents PTALE-VP64-P43 recombinant vector Sorangium cellulosum So ce M4;

FIG. 6 is a graph of LC-MS analysis of yields of wild Sorangium cellulosum So ce M4 and recombinant Sorangium cellulosum So ce M4 epothilone B: a is a yield comparison analysis chart, wherein So ce M4 represents wild Sorangium cellulosum So ce M4, and TALE-So ce M4 represents recombinant Sorangium cellulosum So ce M4; and the B picture and the C picture are respectively LC-MS analysis of wild Sorangium cellulosum So ce M4 and recombinant Sorangium cellulosum So ce M4 epothilone B.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

The G52 liquid medium used had the following composition: 8g/L of potato starch, 2g/L of glucose, 2g/L of soybean protein zymolyte, 2g/L of yeast extract, 8mg/L of EDTA iron sodium, 1g/L of MgSO4.7H2O, 1g/L of CaCl2, 0.5g/L of Tris and water as a solvent, and adjusting the pH to 7.4 by hydrochloric acid.