Ruchir P. Desai

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.

Preparation of Erythromycin Analogs Having Functional Groups at C-15

Chemobiosynthesis has been used to prepare analogs of erythromycins having unique functional groups at the 15-position. Using diketide thioester feeding to genetically engineered Streptomyces coelicolor, analogs of 6-deoxyerythronolide B were prepared having 15-fluoro, 15-chloro, and 15-azido groups. Bioconversion using a genetically engineered mutant of Saccharopolyspora erythraea was used to produce 15-fluoroerythromycin A and 15-azidoerythromycin A. These new erythromycin analogs provide antibacterial macrolides with unique physicochemical properties and functional groups that allow for selective derivatization.

Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues is improved by removal of the initial catalytic sites of the polyketide synthase

Precursor-directed biosynthesis has been shown to be a powerful tool for the production of polyketide analogues that would be difficult or cost prohibitive to produce from medicinal chemistry efforts alone. It has been most extensively demonstrated using a KS1 null mutation (KS10) to block the first round of condensation in the biosynthesis of the erythromycin polyketide synthase (DEBS) for the production of analogues of its aglycone, 6-deoxyerythronolide B (6-dEB). Here we show that removing the DEBS loading domain and first module (mod1Δ), rather than using the KS10 system, can lead to an increase in the utilization of some chemical precursors and production of 6-dEB analogues (R-6dEB) in both Streptomyces coelicolor and Saccharopolyspora erythraea. While the difference in utilization of the precursor was diketide specific, in strains fed (2R*, 3S*)-5-fluoro-3-hydroxy-2-methylpentanoate N-propionylcysteamine thioester, twofold increases in both utilization of the diketide and 15-fluoro-6dEB (15F-6dEB) production were observed in S. coelicolor, and S. erythraea exhibited a tenfold increase in production of 15-fluoro-erythromycin when utilizing the mod1Δ rather than the KS10 system.

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.

Approaches to stabilization of inter-domain recombination in polyketide synthase gene expression plasmids

Abstract. Regions of extremely high sequence identity are recurrent in modular polyketide synthase (PKS) genes. Such sequences are potentially detrimental to the stability of PKS expression plasmids used in the combinatorial biosynthesis of polyketide metabolites. We present two different solutions for circumventing intra-plasmid recombination within the megalomicin PKS genes in Streptomyces coelicolor. In one example, a synthetic gene was used in which the codon usage was reengineered without affecting the primary amino acid sequence. The other approach utilized a heterologous subunit complementation strategy to replace one of the problematic regions. Both methods resulted in PKS complexes capable of 6-deoxyerythronolide B analogue biosynthesis in S. coelicolor CH999, permitting reproducible scale-up to at least 5-l stirred-tank fermentation and a comparison of diketide precursor incorporation efficiencies between the erythromycin and megalomicin PKSs.