Heterologous Production and Yield Improvement of Epothilones in Burkholderiales Strain DSM 7029

Abstract

The cloning of microbial natural product biosynthetic gene clusters and their heterologous expression in a suitable host have proven to be a feasible approach to improve the yield of valuable natural products and to begin mining cryptic natural products in microorganisms. Myxobacteria are a prolific source of novel bioactive natural products with only limited choices of heterologous hosts that have been exploited. Here, we describe the use of Burkholderiales strain DSM 7029 as a potential heterologous host for the functional expression of myxobacterial secondary metabolites. Using a newly established electroporation procedure, the 56 kb epothilone biosynthetic gene cluster from the myxobacterium Sorangium cellulosum was introduced into the chromosome of strain DSM 7029 by transposition. Production of epothilones A, B, C, and D was detected despite their yields being low. Optimization of the medium, introduction of the exogenous methylmalonyl-CoA biosynthetic pathway, and overexpression of rare tRNA genes resulted in an approximately 75-fold increase in the total yields of epothilones to 307 μg L. These results show that strain DSM 7029 has the potential to produce epothilones with reasonable titers and might be a broadly applicable host for the heterologous expression of other myxobacterial polyketide synthases and nonribosomal peptide synthetases, expediting the process of genome mining.

An Easy and Efficient Strategy for the Enhancement of Epothilone Production Mediated by TALE-TF and CRISPR/dcas9 Systems in Sorangium cellulosum

“…The epothilones are potent anticancer drugs that bind to tubulin and inhibit the disassembly of microtubules (Bian et al, 2017). The epothilone B derivative ixabepilone has been approved for use in the treatment of breast and ovarian cancer patients, showing remarkable curative effect compared with taxol (Roché et al, 2007;Thomas et al, 2007;Sparano et al, 2010).…”

“…However, only the essential elements for promoter were found in the P3 promoter, and only the P3 promoter showed strong transcriptional activity. The complete genome of the epothilone-producing strain S. cellulosum So0157-2 was sequenced (Schneiker et al, 2007;Han et al, 2013;Bian et al, 2017), the Fis family regulatory protein was identified (Schneiker et al, 2007;Zhu et al, 2012), and the binding activity of this regulatory protein epothilone biosynthesis promoter was confirmed by EMSA assay in vitro. The binding activity of Etf1 with epothilone biosynthesis promoter was demonstrated by the co-expression of Etf1 and the promoter ligated with reporter gene EGFP.…”

“…However, the highest yield of epothilone B in recombinant M. xanthus strain was <5 mg/L because of the cytotoxicity of epothilones. The disruption of the epoK gene in the M. xanthus strain can improve the epothilone D yield to 20 mg/L (Julien and Shah, 2002;Bian et al, 2017;Peng et al, 2018). The CRISPR/dCas9 system was introduced into the M. xanthus strain, thus improving epothilone B yield 2-fold to 15 mg/L.…”

Discovery of novel bioactive natural products driven by genome mining

“…To simultaneously assess large numbers of closely related bacterial strains and their pan-metabolomes, molecular networking should be improved to recognize the MS/MS fragmentation patterns with the availability of biosynthetic gene gene clusters (14,36). The exploitation of secondary metabolic gene clusters requires the continuous development of transgenic cluster techniques among different organisms, including the optimization of super-hosts and the improvement of the methods for constructing/expressing multiple genes in heterologous systems (37,38).…”

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“…epothilone C 34) has been significantly improved using a novel electroporation procedure. 23 30 As iron complexes are generally poor catalysts for C-H amination, it is believed that the enzyme's protein framework provides function that the iron cofactor cannot achieve alone.…”