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.

On the Importance of the Thiazole Nitrogen in Epothilones: Semisynthesis and Microtubule-Binding Affinity of Deaza-Epothilone C

Deaza-epothilone C, which incorporates a thiophene moiety in place of the thiazole heterocycle in the natural epothilone side chain, has been prepared by semisynthesis from epothilone A, in order to assess the contribution of the thiazole nitrogen to microtubule binding. The synthesis was based on the esterification of a known epothilone A-derived carboxylic acid fragment and a fully synthetic alcohol building block incorporating the modified side chain segment and subsequent ring-closure by ring-closing olefin metathesis. The latter proceeded with unfavorable selectivity and in low yield. Distinct differences in chemical behavior were unveiled between the thiophene-derived advanced intermediates and what has been reported for the corresponding thiazole-based congeners. Compared to natural epothilone C, the free energy of binding of deaza-epothilone C to microtubules was reduced by ca. 1 kcal/mol or less, thus indicating a distinct but non-decisive role of the thiazole nitrogen in the interaction of epothilones with the tubulin/microtubule system. In contrast to natural epothilone C, deaza-epothilone C was devoid of antiproliferative activity in vitro up to a concentration of 10 μM, presumably due to an insufficient stability in the cell culture medium.

Metagenomics and Faecal Metabolomics Integrative Analysis towards the Impaired Glucose Regulation and Type 2 Diabetes in Uyghur-Related Omics

Objective. Gut microbiota and their metabolites play an important role in the development of type 2 diabetes mellitus (T2DM). This research was designed to study the relationship between gut microbiota and faecal metabolites of Uyghur newly onset T2DM and impaired glucose regulation (IGR) patients. Materials and Methods. A total of 60 different glycemic Uyghur subjects were enrolled and divided into T2DM, IGR, and normal glucose tolerance (NGT) groups. Metagenomics and LC-MS-based untargeted faecal metabolomics were employed. Correlations between bacterial composition and faecal metabolomics were evaluated. Results. We discovered that the composition and diversity of gut microbiota in newly onset T2DM and IGR were different from those in NGT. The α-diversity was higher in NGT than in T2DM and IGR; β-diversity analysis revealed apparent differences in the bacterial community structures between patients with T2DM, IGR, and NGT. LC-MS faecal metabolomics analysis discovered different metabolomics features in the three groups. Alchornoic acid, PE (14 : 0/20 : 3), PI, L-tyrosine, LysoPC (15 : 0), protorifamycin I, pimelic acid, epothilone A, 7-dehydro-desmosterol, L-lysine, LysoPC (14 : 1), and teasterone are the most significant differential enriched metabolites. Most of the differential enriched metabolites were involved in metabolic processes, including carbohydrate metabolism, starch and sucrose metabolism, phenylpropanoid biosynthesis, and biosynthesis of amino acids. Procrustes analysis and correlation analysis identified correlations between gut microbiota and faecal metabolites. Matricin was positively correlated with Bacteroides and negatively correlated with Actinobacteria; protorifamycin I was negatively correlated with Actinobacteria; epothilone A was negatively correlated with Actinobacteria and positively correlated with Firmicutes; PA was positively correlated with Bacteroides and negatively correlated with Firmicutes; and cristacarpin was positively correlated with Actinobacteria; however, this correlation relationship does not imply causality. Conclusions. This study used joint metagenomics and metabolomics analyses to elucidate the relationship between gut microbiota and faecal metabolites in different glycemic groups, and the result suggested that metabolic disorders and gut microbiota dysbiosis occurred in Uyghur T2DM and IGR. The results provide a theoretical basis for studying the pathological mechanism for further research.

A Scalable Approach for the Synthesis of Epothilone Thiazole Fragment (C12–C21 unit) Via Wacker Oxidation

A new scalable synthesis of the common thiazole fragment (C12–C21 unit) of epothilone family of molecules (epothilone A–D) has been developed via palladium-catalyzed Wacker oxidation as a key step using (S)-2,2,3,3,9,9,10,10-octamethyl-5-vinyl-4,8-dioxa-3,9-disilaundecane, which has been prepared from commercially available chiral synthon (R)-ethyl-4-cyano-3-hydroxybutanoate. Then further chemical modifications using Horner–Wadsworth–Emmons reaction and Wittig reaction result in the common thiazole fragment (C12–C21 unit) in good yields.