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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Highly Efficient CYP167A1 (EpoK) dependent Epothilone B Formation and Production of 7-Ketone Epothilone D as a New Epothilone Derivative.

Since their discovery in the soil bacterium Sorangium cellulosum, epothilones have emerged as a valuable substance class with promising anti-tumor activity. Because of their benefits in the treatment of cancer and neurodegenerative diseases, epothilones are targets for drug design and pharmaceutical research. The final step of their biosynthesis - a cytochrome P450 mediated epoxidation of epothilone C/D to A/B by CYP167A1 (EpoK) - needs significant improvement, in particular regarding the efficiency of its redox partners. Therefore, we have investigated the ability of various hetero- and homologous redox partners to transfer electrons to EpoK. Hereby, a new hybrid system was established with conversion rates eleven times higher and Vmax of more than seven orders of magnitudes higher as compared with the previously described spinach redox chain. This hybrid system is the most efficient redox chain for EpoK described to date. Furthermore, P450s from So ce56 were identified which are able to convert epothilone D to 14-OH, 21-OH, 26-OH epothilone D and 7-ketone epothilone D. The latter one represents a novel epothilone derivative and is a suitable candidate for pharmacological tests. The results revealed myxobacterial P450s from S. cellulosum So ce56 as promising candidates for protein engineering for biotechnological production of epothilone derivatives.

Biocatalysis at Work: Applications in the Development of Sagopilone.

For the antitumour agent sagopilone, an epothilone analogue, a large-scale synthesis was developed to synthesise the active pharmaceutical ingredient for clinical trials, exploring enzymatic and microbial methods to produce chiral building blocks on a multi-kilogram scale. The three building blocks were identified as key intermediates in the synthesis and needed to be produced with high optical purity in yields higher than those previously published. The improved syntheses of two of these building blocks are detailed herein. For building block A, the chemical research synthesis was abandoned, and a novel chemical route was developed leading to building block A via an enzymatic hydrolysis process. For building blocks C, replacement of a chemical catalytic procedure by a microbial process meant that the development of a new starting material could be avoided, thereby accelerating the development process. For the clinical development process, a human metabolite of sagopilone was required as a reference. To accelerate the synthesis of the metabolite, no chemical synthesis was investigated; rather, we relied solely on oxidoreductases. The human metabolite of sagopilone was synthesised on a multi-gram scale in a single-step process using genetically engineered E. coli expressing human cytochrome P450 enzyme 2C19. The integration of enzymatic and microbial processes provided tools that enable the synthesis of highly functionalised intermediates and metabolites.

Modular construction of a functional artificial epothilone polyketide pathway.

Natural products of microbial origin continue to be an important source of pharmaceuticals and agrochemicals exhibiting potent activities and often novel modes of action. Due to their inherent structural complexity chemical synthesis is often hardly possible, leaving fermentation as the only viable production route. In addition, the pharmaceutical properties of natural products often need to be optimized for application by sophisticated medicinal chemistry and/or biosynthetic engineering. The latter requires a detailed understanding of the biosynthetic process and genetic tools to modify the producing organism that are often unavailable. Consequently, heterologous expression of complex natural product pathways has been in the focus of development over recent years. However, piecing together existing DNA cloned from natural sources and achieving efficient expression in heterologous circuits represent several limitations that can be addressed by synthetic biology. In this work we have redesigned and reassembled the 56 kb epothilone biosynthetic gene cluster from Sorangium cellulosum for expression in the high GC host Myxococcus xanthus. The codon composition was adapted to a modified codon table for M. xanthus, and unique restriction sites were simultaneously introduced and others eliminated from the sequence in order to permit pathway assembly and future interchangeability of modular building blocks from the epothilone megasynthetase. The functionality of the artificial pathway was demonstrated by successful heterologous epothilone production in M. xanthus at significant yields that have to be improved in upcoming work. Our study sets the stage for future engineering of epothilone biosynthesis and production optimization using a highly flexible assembly strategy.

Co-cultivation of Sorangium cellulosum strains affects cellular growth and biosynthesis of secondary metabolite epothilones.

Sorangium cellulosum, a cellulolytic myxobacterium, is capable of producing a variety of bioactive secondary metabolites. Epothilones are anti-eukaryotic secondary metabolites produced by some S. cellulosum strains. In this study, we analyzed interactions between 12 strains of S. cellulosum consisting of epothilone-producers and non-epothilone producers isolated from two distinct soil habitats. Co-cultivation on filter papers showed that different Sorangium strains inhibited one another's growth, whereas epothilone production by the producing strains changed markedly for most (73%) pairwise mixtures. Using a quantitative polymerase chain reaction, we demonstrated that the expression of epothilone biosynthetic genes in the epothilone producers typically changed significantly when these bacteria were mixed with non-producing strains. The results indicated that intraspecies interactions between different S. cellulosum strains not only inhibited the growth of partners, but also could change epothilone production.

Tandem assembly of the epothilone biosynthetic gene cluster by in vitro site-specific recombination.

We describe a site-specific recombination-based tandem assembly (SSRTA) method for reconstruction of biological parts in synthetic biology. The system was catalyzed by Streptomyces phage φBT1 integrase, which belongs to the large serine recombinase subfamily. This one-step approach was efficient and accurate, and able to join multiple DNA molecules in vitro in a defined order. Thus, it could have applications in constructing metabolic pathways and genetic networks.

Myxobacteria--'microbial factories' for the production of bioactive secondary metabolites.

In this article, we briefly review the potential of myxobacteria as 'natural product factories' by highlighting results from the recently sequenced myxobacterial model strain Myxococcus xanthus. We will focus on the production of polyketides, non-ribosomally-made peptides, and their hybrids, and discuss the evaluation of biosynthetic potential using genome-based methods, as well as biosynthetic process engineering.

Trends in microbial synthesis of natural products and biofuels.

Ever since the era of recombinant DNA technology for natural product biosynthesis emerged (292), microorganisms are increasingly becoming common production platforms for many fine chemicals, including natural products and biofuels, that are currently being produced either through chemical methods or using plant and organ cell cultures. The rapid elucidation of biosynthetic pathways made possible through advanced genomic tools has made natural products again the molecules of choice for drug development. Indeed, half of the drugs currently in clinical use are natural products and it is expected that the market size of biotechnology-derived small molecules will exceed billion U.S.$100 in 2010 and billion U.S.$400 in 2030 (3, 293). There are still many challenges facing the use of microorganisms for high-value chemical synthesis. For example, further developments of recent advances are necessary to make a fermentation-based biobutanol industry that can compete effectively with petrochemically derived butanol. As such, we believe that biocatalyst factories such as E. coli and S. cerevisiae will not only continue to be highly attractive alternatives to traditional chemical manufacturing but the application of powerful systems biology approaches will facilitate their expanded role in industrial applications (294-296).

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.