Production and bioprocessing of epothilone B from Aspergillus niger, an endophyte of Latania loddegesii, with a conceivable biosynthetic stability: anticancer, anti-wound healing activities and cell cycle analysis.

Epothilones are one of the common prescribed anticancer drugs for solid tumors, for their exceptional binding affinity with ß-tubulin microtubule, stabilizing their disassembly, causing an ultimate arrest to the cellular growth. Epothilones were initially isolated from Sornagium cellulosum, however, their extremely slow growth rate and low yield of epothilone is the challenge. So, screening for a novel fungal endophyte dwelling medicinal plants, with higher epothilone productivity and feasibility of growth manipulation was the objective. Aspergillus niger EFBL-SR OR342867, an endophyte of Latania loddegesii, has been recognized as the heady epothilone producer (140.2 µg/L). The chemical structural identity of the TLC-purified putative sample of A. niger was resolved from the HPLC, FTIR and LC-ESI-MS/MS analyses, with an identical molecular structure of the authentic epothilone B. The purified A. niger epothilone B showed a resilient activity against MCF-7 (0.022 µM), HepG-2 (0.037 µM), and HCT-116 (0.12 µM), with selectivity indices 21.8, 12.9 and 4, respectively. The purified epothilone B exhibited a potential anti-wound healing activity to HepG-2 and MCF-7 cells by ~ 54.07 and 60.0%, respectively, after 24 h, compared to the untreated cells. The purified epothilone has a significant antiproliferative effect by arresting the cellular growth of MCF-7 at G2/M phase by ~ 2.1 folds, inducing the total apoptosis by ~ 12.2 folds, normalized to the control cells. The epothilone B productivity by A. niger was optimized by the response surface methodology, with ~ 1.4 fold increments (266.9 µg/L), over the control. The epothilone productivity by A. niger was reduced by ~ 2.4 folds by 6 months storage as a slope culture at 4 °C, however, the epothilone productivity was slightly restored with ethylacetate extracts of L. loddegesii, confirming the plant-derived chemical signals that partially triggers the biosynthetic genes of A. niger epothilones. So, this is the first report emphasizing the metabolic potency of A. niger, an endophyte of L. loddegesii, to produce epothilone B, that could be a new platform for industrial production of this drug.

Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitalea.

BACKGROUND: Epothilone B is a natural product that stabilizes microtubules, similar to paclitaxel (Taxol); therefore, epothilone B and several derivatives have shown obvious antitumour activities. Some of these products are in clinical trials, and one (ixabepilone, BMS) is already on the market, having been approved by the FDA in 2007. The terminal step in epothilone B biosynthesis is catalysed by the cytochrome P450 enzyme EpoK (CYP167A1), which catalyses the epoxidation of the C12-C13 double bond (in epothilone C and D) to form epothilone A and B, respectively. Although redox partners from different sources support the catalytic activity of EpoK in vitro, the conversion rates are low, and these redox partners are not applied to produce epothilone B in heterologous hosts. RESULTS: Schlegelella brevitalea DSM 7029 contains electron transport partners that efficiently support the catalytic activity of EpoK. We screened and identified one ferredoxin, Fdx_0135, by overexpressing putative ferredoxin genes in vivo and identified two ferredoxin reductases, FdR_0130 and FdR_7100, by whole-cell biotransformation of epothilone C to effectively support the catalytic activity of EpoK. In addition, we obtained strain H7029-3, with a high epothilone B yield and found that the proportion of epothilone A + B produced by this strain was 90.93%. Moreover, the whole-cell bioconversion strain 7029-10 was obtained; this strain exhibited an epothilone C conversion rate of 100% in 12 h. Further RT-qPCR experiments were performed to analyse the overexpression levels of the target genes. Gene knock-out experiments showed that the selected ferredoxin (Fdx_0135) and its reductases (FdR_0130 and FdR_7100) might participate in critical physiological processes in DSM 7029. CONCLUSION: Gene overexpression and whole-cell biotransformation were effective methods for identifying the electron transport partners of the P450 enzyme EpoK. In addition, we obtained an epothilone B high-yield strain and developed a robust whole-cell biotransformation system. This strain and system hold promise for the industrial production of epothilone B and its derivatives.

CRISPR/dCas9-mediated transcriptional improvement of the biosynthetic gene cluster for the epothilone production in Myxococcus xanthus.

BACKGROUND: The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. RESULTS: In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. CONCLUSIONS: Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.

Effects of transcriptional mode on promoter substitution and tandem engineering for the production of epothilones in Myxococcus xanthus.

Promoter optimization is an economical and effective approach to overexpress heterologous genes and improve the biosynthesis of valuable products. In this study, we swapped the original promoter of the epothilone biosynthetic gene cluster in Myxococcus xanthus with two endogenous strong promoters P pilA and P groEL1 , respectively, which, however, decreased the epothilone production ability. The transcriptional abilities by the two promoters were found to be bloomed in the growth stage but markedly decreased after the growth, whereas the original promoter P epo functioned majorly after the exponential growth stage. Tandem repeat engineering on the original promoter P epo remarkably increased epothilone production. The tandem promoter exerted similar expressional pattern as P epo did in M. xanthus. We demonstrated that differential transcriptional modes markedly affected the efficiency of promoters in controlling the gene expressions for the production of the secondary metabolite epothilones. Our study provides an insight into exploiting powerful promoters to produce valuable secondary metabolites, especially in host with limited known promoters.

A bacterial negative transcription regulator binding on an inverted repeat in the promoter for epothilone biosynthesis.

BACKGROUND: Microbial secondary metabolism is regulated by a complex and mostly-unknown network of global and pathway-specific regulators. A dozen biosynthetic gene clusters for secondary metabolites have been reported in myxobacteria, but a few regulation factors have been identified. RESULTS: We identified a transcription regulator Esi for the biosynthesis of epothilones. Inactivation of esi promoted the epothilone production, while overexpression of the gene suppressed the production. The regulation was determined to be resulted from the transcriptional changes of epothilone genes. Esi was able to bind, probably via the N-terminus of the protein, to an inverted repeat sequence in the promoter of the epothilone biosynthetic gene cluster. The Esi-homologous sequences retrieved from the RefSeq database are all of the Proteobacteria. However, the Esi regulation is not universal in myxobacteria, because the esi gene exists only in a few myxobacterial genomes. CONCLUSIONS: Esi binds to the epothilone promoter and down-regulates the transcriptional level of the whole gene cluster to affect the biosynthesis of epothilone. This is the first transcription regulator identified for epothilone biosynthesis.

Isolation and characterisation of the epothilone gene cluster with flanks from high alkalotolerant strain Sorangium cellulosum (So0157-2).

Epothilones are cytotoxic macrolactones having auspicious anti-tumorous activities, but merely produced by rare Sorangium strains. Here, we have focused on the epothilone gene cluster from special niche bacterial strain, S. cellulosum So0157-2. Therefore, we have isolated a high pH tolerant S. cellulosum strain So0157-2 and characterized the epothilones gene cluster and its flanks by cosmid/fosmid libraries preparation and sequencing. The assembly spanned 94,459 bp and consisted of 56,019 bp core region. Remarkably, the core as well as upstream 420 bp and downstream 315 bp were highly conserved, while further neighboring regions varied extremely. Transposase traces were identified near the core of clusters, supporting that the transposon-mediated transgenesis is a naturally evolved strategy for the cluster's dissemination. A predicted neighboring esterase gene was identified as a potential epothilone-resistance gene preventing self-toxicity. Novel modification or regulatory genes, a multi-position-cyclo releasing gene and their relationship with corresponding analogs were identified in strain So0157-2. These findings open the door to discover additional, naturally evolved epothilone-related genes for significant applications in industrial as well as clinical sector.

A new approach for improving epothilone B yield in Sorangium cellulosum by the introduction of vgb epoF genes.

Epothilone B has drawn great attention due to its much stronger anticancer activity and weaker side effects compared with taxol. The relative low yield of epothilone B limited its application. In this study, we report the successful introduction of the vgb gene and the epoF gene into Sorangium cellulosum So ce M4 by electroporation for the first time, which was demonstrated by Southern blot analysis. Results of qRT-PCR, SDS-PAGE and western blot analysis confirmed the transcription and expression of the vgb and epoF genes. LC-MS results showed that the epothilones B, A yields were improved and epothilones D, C yields were decreased. The yields of epothilone B were improved by 57.9 ± 0.3, 62.7 ± 0.8 and 122.4 ± 0.7 % through the introduction of vgb gene, epoF gene and both genes into strain So ce M4, respectively. Our study provides a new approach for improving epothilone B yield in S. cellulosum.

Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition.

Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to heterologous hosts. This possibility is particularly relevant to the expression of large secondary metabolite gene clusters in various heterologous hosts. Recently, we showed that the engineering of large gene clusters like type I polyketide/nonribosomal peptide pathways for heterologous expression is no longer a bottleneck. Here, we apply recombineering to engineer either the epothilone (epo) or myxochromide S (mchS) gene cluster for transpositional delivery and expression in heterologous hosts. The 58-kb epo gene cluster was fully reconstituted from two clones by stitching. Then, the epo promoter was exchanged for a promoter active in the heterologous host, followed by engineering into the MycoMar transposon. A similar process was applied to the mchS gene cluster. The engineered gene clusters were transferred and expressed in the heterologous hosts Myxococcus xanthus and Pseudomonas putida. We achieved the largest transposition yet reported for any system and suggest that delivery by transposon will become the method of choice for delivery of large transgenes, particularly not only for metabolic engineering but also for general transgenesis in prokaryotes and eukaryotes.

Promoter Screening Facilitates Heterologous Production of Complex Secondary Metabolites in Burkholderiales Strains.

Burkholderiales are an emerging source of bioactive secondary metabolites and have the potential to be a robust chassis for metabolites from Gram-negative bacteria. However, only a few constitutive promoters can be utilized in Burkholderiales. Herein, we described the screening of strong constitutive promoters from Burkholderiales strain DSM 7029, and 37 promoters identified from transcriptome sequencing were cloned and characterized using a firefly luciferase reporter and were further verified by qPCR analysis. These promoters were then used to drive a complex 56-kb epothilone BGC from myxobacterium and a 23-kb rhizomide BGC from Paraburkholderia rhizoxinica, and the successful production of epothilone and rhizomide was observed in DSM 7029, with improved yields compared to the production achieved by previously used promoters. Additionally, these promoters are also functional in other Burkholderiales species. Thus, these promoters are highly useful for optimizing yields of important metabolites in Burkholderiales and for mining cryptic biosynthetic pathways in DSM 7029.

Reassembly of the Biosynthetic Gene Cluster Enables High Epothilone Yield in Engineered Schlegelella brevitalea.

Epothilones, as a new class of microtubule-stabilizing anticancer drugs, exhibit strong bioactivity against taxane-resistant cells and show clinical activity for the treatment of advanced breast cancer. Additionally, they also show great potential for a central nervous system injury and Alzheimer's disease. However, due to the long fermentation period of the original producer and challenges of genetic engineering of nonribosomal peptide/polyketide (NRP/PK) megasynthase genes, the application of epothilones is severely limited. Here, we addressed these problems by reassembling a novel 56-kb epothilone biosynthetic gene cluster, optimizing the promoter of each gene based on RNA-seq profiling, and completing precursor synthetic pathways in engineered Schlegella brevitalea. Furthermore, we debottlenecked the cell autolysis by optimizing culture conditions. Finally, the yield of epothilones in shake flasks was improved to 82 mg/L in six-day fermentation. Overall, we not only constructed epothilone overproducers for further drug development but also provided a rational strategy for high-level NRP/PK compound production.

Preparation and separation of epothilones with anticancer activity.

Epothilone is a kind of macrolide compound, which has anticancer activity and strong inhibitory effect on breast cancer and rectal cancer cells. Herein, the chemical and biological synthesis of epothilones and how to improve fermentation yield and separation methods were summarized and discussed.

Epothilones as lead structures for new anticancer drugs--pharmacology, fermentation, and structure-activity-relationships.

Epothilones (Epo's) A and B are naturally occurring microtubule-stabilizers, which inhibit the growth of human cancer cells in vitro at low nM or sub-nM concentrations. In contrast to taxol (paclitaxel, Taxol) epothilones are also active against different types of multidrug-resistant cancer cell lines in vitro and against multidrug-resistant tumors in vivo. Their attractive preclinical profile has made epothilones important lead structures in the search for improved cytotoxic anticancer drugs and Epo B (EPO906, patupilone) is currently undergoing Phase III clinical trials. Numerous synthetic and semisynthetic analogs have been prepared since the absolute stereochemistry of epothilones was first disclosed in mid-1996 and their in vitro biological activity has been determined. Apart from generating a wealth of SAR information, these efforts have led to the identification of at least six compounds (in addition to Epo B), which are currently at various stages of clinical evaluation in humans. The most advanced of these compounds, Epo B lactam BMS-247550 (ixabepilone), has recently obtained FDA approval for the treatment of metastatic and advanced breast cancer. This chapter will first provide a summary of the basic features of the biological profile of Epo B in vitro and in vivo. This will be followed by a review of the processes that have been developed for the fermentative production of Epo B. The main part of the chapter will focus on the most relevant aspects of the epothilone SAR with regard to effects on tubulin polymerization, in vitro antiproliferative activity, and in vivo antitumor activity. Particular emphasis will be placed on work conducted in the authors' own laboratories, but data from other groups will also be included. In a final section, the current status of those epothilone analogs undergoing clinical development will be briefly discussed.

Selective production of epothilone B by heterologous expression of propionyl-CoA synthetase in Sorangium cellulosum.

The metabolic engineering of epothilones, as secondary metabolites, was investigated using Sorangium cellulosum to achieve the selective production of epothilone B, a potent anticancer agent. Thus, the propionyl-CoA synthetase gene (prpE) from Ralstonia solanacearum was heterologously expressed in S. cellulosum to increase the production of epothilone B. Propionyl-CoA synthetase converts propionate into propionyl-CoA, a potent precursor of epothilone B. The recombinant S. cellulosum containing the prpE gene exhibited a significant increase in the resolution of epothilones B/A, with an epothilone B to A ratio of 127 to 1, which was 100 times higher than that of the wild-type cells, demonstrating its potential use for the selective production of epothilone B.

Isolation of Sorangium cellulosum carrying epothilone gene clusters.

Epothilone and its analogs are a potent new class of anticancer compounds produced by myxobacteria. Thus, in an effort to identify new myxobacterial strains producing epothilone and its analogs, cellulose-degrading myxobacteria were isolated from Korean soils, and 13 strains carrying epothilone biosynthetic gene homologs were screened using a polymerase chain reaction. A migration assay revealed that Sorangium cellulosum KYC3013, 3016, 3017, and 3018 all produced microtubule-stabilizing compounds, and an LCMS/ MS analysis showed that S. cellulosum KYC3013 synthesized epothilone A.

Yield improvement of epothilones in Burkholderia strain DSM7029 via transporter engineering.

Transporter engineering has been shown to be a positive approach for enhancing natural product titers in microbial cell factories by expelling target compounds out of feasible hosts. In this work, two multidrug efflux pumps, Orf14 and Orf3, were modulated in the epothilone production strain Burkholderia DSM7029::Tn5-km-epo (named G32) via Red/ET engineering to increase heterologous polyketide epothilone yields. Compared with the prior G32 strain, the total production of several epothilones in the G32::orf14-orf3 mutant was meaningfully doubled according to high-performance liquid chromatography-mass spectrometer analysis. Typically for epothilone B, in simple and clear liquid medium CYMG, the overall productivity in the engineered high-yield producer G32::orf14-orf3 was improved for almost 3-fold, from 2.7 to about 8.1 µg/l. Additionally, the ratio of extracellular to intracellular accumulation of epothilone B was raised from 9.3:1 to 13.7:1 in response to expression of two putative transport genes orf14 and orf3. Hence, we strongly recommend that the Orf14 and Orf3 transporters export epothilone, thus promotes the forward reaction of biosynthesis on epothilone manufacture inside the cells. Our results afford a practical stage for yield improvement of other heterologous natural products in broad chassis cells.

Repeated batch production of epothilone B by immobilized Sorangium cellulosum.

Production of extracellular epothilone B, one of the potent anticancer agents, by free and immobilized Sorangium cellulosum was studied using the repeated batch culture process. The concentration of alginate used in immobilization was directly related to the mass transfer rate of nutrients, mechanical stability, and the epothilone B production yield. With the optimized 3% (w/v) calcium alginate carrier, a prolonged repeated batch culture was investigated for the 5 repeated batches for 24 days. The maximum productivity of epothilone B obtained from the alginate-immobilized cells was 5.03 mg/l/day, which is 3 times higher than that of free cells (1.68 mg/l/day).

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

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-1. 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.

The epothilones and related analogues-a review of their syntheses and anti-cancer activities.

The macrocylic polyketide class of compounds known as the epothilones has generated substantial interest over the last few years in the areas of chemistry, biology, and medicine due to their interesting structure and, more importantly, their activity against numerous cancer cell lines, including drug-resistant, especially Taxol-resistant, cancer cell lines. To date, numerous total syntheses have been published, hundreds of epothilone analogues have been synthesized, and detailed structure activity relationship studies have been conducted. The purpose of this review is to give a brief summary of the latest advances made concerning the epothilones. Recent total or partial syntheses will be presented along with the syntheses of new epothilone analogues and their corresponding biological data. In addition, we will look at the current state of research into an economically viable method for the biosynthesis of the epothilones and related analogues.

Quantifying intermediates in template-directed natural product biosynthesis.

High-performance mass spectrometry is providing new experimental windows into the enzymology of natural product biosynthesis. The first quantitative assessments of covalently attached biosynthetic intermediates promise to shine new light on template-directed biosynthesis.

Mass spectrometric interrogation of thioester-bound intermediates in the initial stages of epothilone biosynthesis.

Direct detection of thioester intermediate mixtures bound to EpoC, a 195 kDa polyketide synthase, has been achieved using limited proteolysis and Fourier-transform mass spectrometry (FTMS). Incubation with various N-acetylcysteamine thioester (S-NAC) substrate mimics produced mass shifts on the EpoC ACP domain consistent with their condensation with an enzyme-bound carbanion produced by the decarboxylation of methylmalonyl-S-EpoC. Reconstitution of EpoA-ACP, EpoB, and EpoC gave a +165.0 Da mass shift consistent with the formation of the methylthiazolyl-methacrylyl product by incorporation of acetyl-CoA, cysteine, and methylmalonyl-CoA. Thioester-templated reaction intermediates and products are typically characterized by quantifying radioactive substrates, either enzyme bound or chemically hydrolyzed. In contrast, the MS-based methodology described here provides semiquantifiable ratios of free enzyme, intermediate, and product occupancy and reveals that certain substrates result in a >50% formation of nonproductive intermediates.