Potent non-benzoquinone ansamycin heat shock protein 90 inhibitors from genetic engineering of Streptomyces hygroscopicus.

Inhibition of the protein chaperone Hsp90 is a promising new approach to cancer therapy. We describe the preparation of potent non-benzoquinone ansamycins. One of these analogues, generated by feeding 3-amino-5-chlorobenzoic acid to a genetically engineered strain of Streptomyces hygroscopicus, shows high accumulation and long residence time in tumor tissue, is well-tolerated upon intravenous dosing, and is highly efficacious in the COLO205 mouse tumor xenograft model.

Cytotoxic hypothemycin analogues from Hypomyces subiculosus.

Three new hypothemycin analogues were isolated from the fungal strains Hypomyces subiculosus DSM 11931 and DSM 11932. The structures of these compounds were elucidated by spectroscopic methods, chemical conversion, and X-ray crystallographic analysis. One of the analogues, 4-O-demethylhypothemycin, exhibited potent and selective cytotoxic activity against cell lines with a BRAF mutation.

Characterization of product capture resin during microbial cultivations.

Various bioactive small molecules produced by microbial cultivation are degraded in the culture broth or may repress the formation of additional product. The inclusion of hydrophobic adsorber resin beads to capture these products in situ and remove them from the culture broth can reduce or prevent this degradation and repression. These product capture beads are often subjected to a dynamic and stressful microenvironment for a long cultivation time, affecting their physical structure and performance. Impact and collision forces can result in the fracturing of these beads into smaller pieces, which are difficult to recover at the end of a cultivation run. Various contaminating compounds may also bind in a non-specific manner to these beads, reducing the binding capacity of the resin for the product of interest (fouling). This study characterizes resin bead binding capacity (to monitor bead fouling), and resin bead volume distributions (to monitor bead fracture) for an XAD-16 adsorber resin used to capture epothilone produced during myxobacterial cultivations. Resin fouling was found to reduce the product binding capacity of the adsorber resin by 25-50%. Additionally, the degree of resin bead fracture was found to be dependent on the cultivation length and the impeller rotation rate. Microbial cultivations and harvesting processes should be designed in such a way to minimize bead fragmentation and fouling during cultivation to maximize the amount of resin and associated product harvested at the end of a run.

Production of 8-demethylgeldanamycin and 4,5-epoxy-8-demethylgeldanamycin from a recombinant strain of Streptomyces hygroscopicus.

Two new geldanamycin derivatives produced by genetic engineering of Streptomyces hygroscopicus strain K309-27-1 were isolated and characterized. Removal of the 8-methyl group of geldanamycin was achieved by replacing the AT4 domain of the polyketide synthase with a malonyl AT domain. The resulting strain produced 8-demethyl geldanamycin (2) and 4,5-epoxy-8-demethylgeldanamycin (3). The structures of both molecules were elucidated through interpretation of 1D and 2D NMR data as well as comparison with authentic geldanamycin derivatives. Compounds 2 and 3 displayed moderate cytotoxicity against the human breast cancer cell line SK-BR-3.

Improved bioconversion of 15-fluoro-6-deoxyerythronolide B to 15-fluoro-erythromycin A by overexpression of the eryK Gene in Saccharopolyspora erythraea.

The bioconversion of a 6-deoxyerythronolide B analogue to the corresponding erythromycin A analogue (R-EryA) by a Saccharopolyspora erythraea mutant lacking the ketosynthase in the first polyketide synthase module was significantly improved by changing fluxes at a key branch point affecting the erythromycin congener distribution. This was achieved by integrating an additional copy of the eryK gene into the chromosome under control of the eryAIp promoter. Real-time PCR analysis of RNA confirmed higher expression of eryK in the resulting strain, S. erythraea K301-105B, compared to its parent. In shake flasks, K301-105B produced less of the shunt product 15-fluoro-erythromycin B (15F-EryB), suggesting a shift in congener distribution toward the desired product, 15-fluoro-erythromycin A (15F-EryA). In bioreactor studies, K301-105B produced 1.3 g/L of 15F-EryA with 75-80% molar yield on fed precursor, compared with 0.9 g/L 15F-EryA with 50-55% molar yield on fed precursor by the parent strain. At higher precursor feed rates, K301-105B produced 3.5 g/L of 15F-EryA while maintaining 75-80% molar yield on fed precursor.

A sensitive and robust method for quantification of intracellular short-chain coenzyme A esters.

A procedure for the analysis of short-chain intracellular coenzyme A (CoA) esters and adenine nucleotide pools in microbial cells is described. The simultaneous isolation of bacterial cells from media, quenching of their metabolism, and extraction of metabolites was accomplished by centrifugation of cells through a layer of silicone oil into a denser solution of trichloroacetic acid. The acid was neutralized by extraction into Freon containing tri-n-octylamine to provide a salt-free solution of cell metabolites. After high-performance liquid chromatography separation, CoA, CoA esters, and adenine-containing nucleotides were derivatized by postcolumn reaction with bromoacetaldehyde to form the fluorescent 1,N6-ethenoadenine adducts which were analyzed by a fluorescence detector at picomolar levels.

Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia coli.

A robust high cell-density fed-batch bioprocess was developed for the heterologous production of 6-deoxyerythronolide B (6-dEB), the macrocyclic core of the antibiotic erythromycin, with a recombinant Escherichia coli strain expressing the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. Initial evaluation of the E. coli strain in a 5-l bioreactor with the addition of exogenous propionate for polyketide biosynthesis resulted in a maximum cell density of 30 g l(-1) (OD600 approximately 60) and the production of 700 mg l(-1) of 6-dEB. Retention of the two plasmids harboring the heterologous genes was maintained between 90 and 100% even in the absence of antibiotic selection. However, the accumulation of excess ammonia in the culture medium was found to significantly decrease the productivity of the cells. Through optimization of the medium composition and fermentation conditions, the maximum cell density was increased by two-fold, and a final titer of 1.1 g l(-1) of 6-dEB was achieved. This represents an 11-fold improvement compared to the highest reported titer of 100 mg l(-1) with E. coli as the production host.

Combining classical, genetic, and process strategies for improved precursor-directed production of 6-deoxyerythronolide B analogues.

A process for the production of erythromycin aglycone analogues has been developed by combining classical strain mutagenesis techniques with modern recombinant DNA methods and traditional process improvement strategies. A Streptomyces coelicolor strain expressing the heterologous 6-deoxyerythronolide B (6-dEB) synthase (DEBS) for the production of erythromycin aglycones was subjected to random mutagenesis and selection. Several strains exhibiting 2-fold higher productivities and reaching >3 g/L total macrolide aglycones were developed. These mutagenized strains were cured of the plasmid carrying the DEBS genes and a KS1 degrees mutant DEBS operon was introduced for the production of novel analogues when supplemented with a synthetic diketide precursor. The strains expressing the mutant DEBS were screened for improved 15-methyl-6-dEB production, and the best clone, strain B9, was found to be 50% more productive as compared to the parent host strain used for 15-methyl-6-dEB production. Strain B9 was evaluated in 5-L fermenters to confirm productivity in a scalable process. Although peak titers of 0.85 g/L 15-methyl-6-dEB by strain B9 confirmed improved productivity, it was hypothesized that the low solubility of 15-methyl-6-dEB limited productivity. The solubility of 15-methyl-6-dEB in water was determined to be 0.25-0.40 g/L, although higher titers are possible in fermentation medium. The incorporation of the hydrophobic resin XAD-16HP resulted in both the in situ adsorption of the product and the slow release of the diketide precursor. The resin-containing fermentation achieved 1.3 g/L 15-methyl-6-dEB, 50% higher than the resin-free process. By combining classical mutagenesis, recombinant DNA techniques, and process development, 15-methyl-6-dEB productivity was increased by over 100% in a scalable fermentation process.

Precursor-directed biosynthesis of novel triketide lactones.

Precursor-directed biosynthesis was used to produce different triketide lactones (R-TKLs) in a fermentation process. Plasmids expressing engineered versions of the first subunit of 6-deoxyerythronolide B synthase (DEBS1) fused to the terminal DEBS thioesterase (TE) were introduced into three different Streptomyces strains. The DEBS1 protein fused to TE had either an inactivated ketosynthase domain (KS1 degrees ) or a partial DEBS1 lacking module 1 but containing module 2 (M2+TE). Different synthetic precursors were examined for their effect on R-TKL production. An overproducing strain of S. coelicolor expressing the M2+TE protein was found to be best for production of R-TKLs. Racemic precursors were as effective as enantiomerically pure precursors in the fermentation process. The R group on the precursor significantly affected titer (propyl >> chloromethyl > vinyl). The R-TKLs were unstable in fermentation broth at pH 6-8. A two-phase fermentation with a pH shift was implemented to stabilize the products. The fermentation pH initially was controlled at optimal values for cell growth (pH 6.5) and then shifted to 5.5 during production. This doubled peak titers and stabilized the product. Finally, the concentration of synthetic precursor in the fermentation was optimized to improve production. A maximum titer of 500 mg/L 5-chloromethyl-TKL was obtained using 3.5 g/L precursor.