Jorge Galazzo

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.

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°) 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.

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.

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.

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° 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.