We've spent most of our time focusing on epothilone D and 17-AAG, but Kosan's pipeline is showing signs of deepening, with a number of unrelated preclinical programs ongoing, along with development of manufacturing capabilities for them.
kosan.com
This is for the motilide program . . .
>>Biotechnol Prog. 2004 Feb 6;20(1):38-43.
Combining Classical, Genetic, and Process Strategies for Improved Precursor-Directed Production of 6-Deoxyerythronolide B Analogues.
Desai RP, Leaf T, Hu Z, Hutchinson CR, Hong A, Byng G, Galazzo J, Licari P.
Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, California 94545, and MDS Pharma Services, Fermentation Laboratories, 158 Li-Teh Road, Peitou, Taipei, Taiwan 112.
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.<<
While this one describes improved production techniques for a certain class of polyketides that were described in one article as antiparasytics . . .
>>Biotechnol Prog. 2004 Feb 6;20(1):122-127.
Precursor-Directed Biosynthesis of Novel Triketide Lactones.
Regentin R, Kennedy J, Wu N, Carney JR, Licari P, Galazzo J, Desai R.
Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, California 94545.
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.<<
Cheers, Tuck |
