Abstract
Epothilones are a kind of macrolides with strong cytotoxicity toward cancer cells and relatively lower side effects compared with taxol. Epothilone B derivate ixabepilone has been used for the clinical treatment of advanced breast cancer. However, the low yield of epothilones and the difficulty in the genetic manipulation of Sorangium cellulosum limited their wider application. Transcription activator-like effectors-Trancriptional factor (TALE-TF)-VP64 and clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-VP64 have been demonstrated as effective systems for the transcriptional improvement. In this study, a promoter for the epothilone biosynthesis cluster was obtained and the function has been verified. The TALE-TF-VP64 and CRISPR/dcas9-VP64 target P3 promoter were electroporated into S. cellulosum strain So ce M4, and the transcriptional levels of epothilone biosynthesis-related genes were significantly upregulated. The yield of epothilone B was improved by 2.89- and 1.53-fold by the introduction of recombinant TALE-TF-VP64-P3 and dCas9-VP64-P3 elements into So ce M4, respectively. The epothilone D yield was also improved by 1.12- and 2.18-fold in recombinant dCas9-So ce M4 and TALE-VP64 strains, respectively. The transcriptional regulation mechanism of TALE-TF-VP64 and the competition mechanism with endogenous transcriptional factor were investigated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP), demonstrating the combination of the P3 promoter and TALE-TF element and the competition between TALE-TF and endogenous transcriptional protein. This is the first report on the transcriptional regulation of the epothilone biosynthetic gene cluster in S. cellulosum using the TALE-TF and dCas9-VP64 systems, and the regulatory mechanism of the TALE-TF system for epothilone biosynthesis in S. cellulosum was also firstly revealed, thus shedding light on the metabolic engineering of S. cellulosum to improve epothilone yields substantially and promoting the application of epothilones in the biomedical industry.
Highlights
Epothilones are 16-member macrolides with strong anticancer activity and produced by Sorangium cellulosum
This study was the first report on the transcriptional regulation of epothilone biosynthetic cluster in S. cellulosum strain through the TALETF technology and clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 systems, and this is the first investigation on the transcriptional mechanism of epothilone biosynthetic gene cluster that employs Transcription activator-like effectors-Trancriptional factor (TALE-TF) elements by using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) in S. cellulosum; this study is of significance for the epothilone yield improvement in S. cellulosum So ce M4 by transcriptional regulation approaches
The 440-bp upstream sequence of the epothilone biosynthetic gene cluster from different S. cellulosum strains was cloned and identified, and the results indicated that the sequences of different promoters showed high similarity, whereas the transcriptional activities of these promoters remarkably vary probably because of the presence of stem-loop structures in the promoter sequences
Summary
Epothilones are 16-member macrolides with strong anticancer activity and produced by Sorangium cellulosum. Epothilone yield remains low because of the difficulty in genetically manipulating S. cellulosum and the cytotoxicity of epothilones during the heterologous expression of epothilone biosynthesis cluster. The transcription enhancement of epothilone biosynthetic cluster is an effective approach for improving epothilone yield. There are still few reports on the epothilone yield improvement in S. cellulosum by transcriptional activation approaches. Improving epothilone yield by genetically engineering S. cellulosum through the transcriptional enhancement of epothilone biosynthetic cluster is urgent. The recombinant TALETF and dCas9-VP64+PLX-sgRNA vectors were electroporated into the competent cells of So ce M4 under 2.0 KV in a 2.0cm cuvette. A G52 plate with 50 μg/ml kanamycin and G52 medium with 100 μg/ml ampicillin were used for the selection of the positive clone containing recombinant TALETF element and recombinant dCas9-VP64+PLX-sgRNA vectors, respectively. Primers f1F and f1R, and P43 F and P43 R (Table S1) were used to demonstrate the successful introduction of PLx-sgRNA and pcDNA-dCas9-VP64 vectors
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