This is one element that I love about Kosan.... any success, and they'll have an incredible nucleus of experienced process-science sorts. There will always be a need for those with a leg up in the fermentation sciences......
J Bacteriol. 2003 Nov;185(21):6325-30.
Characterization of the Integrase Gene and Attachment Site for the Myxococcus xanthus Bacteriophage Mx9.
Julien B.
Kosan Biosciences, Inc., Hayward, California 94545.
Bacteriophage Mx9 is a temperate phage that infects Myxococcus xanthus. It lysogenizes the bacteria by integrating into the bacterial chromosome by site-specific recombination at one of two sites, attB1 or attB2. Integration at attB1 results in deletion of DNA between the two attB sites. The attB2 site lies within the 5' region of the M. xanthus tRNA(Gly) gene. Mx9 integration requires a single protein, Int. Analysis of integration revealed that the phage attachment site (attP) is contained in the int gene and that upon integration, the 3' end of the int gene is altered. Plasmids containing fusions of the pilA or mgl promoter to lacZ integrated at either Mx9 attB site have higher levels of transcription than the same fusions integrated at the Mx8 attB site.
background........
Appl Microbiol Biotechnol. 2003 Jun;61(5-6):451-5. Epub 2003 Mar 06.
Nutrient regulation of epothilone biosynthesis in heterologous and native production strains.
Regentin R, Frykman S, Lau J, Tsuruta H, Licari P.
Department of Process Sciences, Kosan Biosciences, 3832 Bay Center Place, Hayward, CA 94545, USA. regentin@kosan.com
Fermentation media with different initial concentrations of ammonium and phosphate salts were used to study the inhibitory effects of those ions on growth and production of epothilone in Sorangium cellulosum and Myxococcus xanthus. The native epothilone producer, S. cellulosum was more sensitive to ammonium and phosphate than the heterologous producer, M. xanthus. An ammonium concentration of 12 mM reduced epothilone titers by 90% in S. cellulosum but by only 40% in M. xanthus. When 5 mM phosphate was added to the medium, production in both strains was 60% lower. Higher phosphate concentrations had little additional effect on M. xanthus titers, but epothilone production with 17 mM extra-cellular phosphate in S. cellulosum was 95% lower than in the control condition. The effect of iron supplementation to the fermentation medium was also investigated. Both strains showed best production with 20 microM iron added to the medium.
Antimicrob Agents Chemother. 2002 Sep;46(9):2772-8.
Heterologous expression of epothilone biosynthetic genes in Myxococcus xanthus.
Julien B, Shah S.
Kosan Biosciences, Inc., Hayward, California 94545, USA. julien@kosan.com
Epothilones are potential anticancer drugs that stabilize microtubules in a manner similar to paclitaxel (Taxol). Epothilones are produced from the myxobacterium Sorangium cellulosum, which has a 16-h doubling time and produces only milligram-per-liter amounts of epothilone A and epothilone B. Furthermore, genetic manipulation of S. cellulosum is difficult. To produce epothilones in a more genetically amenable and rapidly growing host, we chose the closely related and best-characterized myxobacteria Myxococcus xanthus. We inserted 65.4 kb of S. cellulosum DNA that encompassed the entire epothilone gene cluster into the chromosome of M. xanthus by a series of homologous recombination events. The resulting strain produced epothilones A and B. Construction of a strain that contained a mutation in epoK, the P450 epoxidase, resulted in production of epothilones C and D.
Biotechnol Prog. 2002 Sep-Oct;18(5):913-20.
Control of secondary metabolite congener distributions via modulation of the dissolved oxygen tension.
Frykman SA, Tsuruta H, Starks CM, Regentin R, Carney JR, Licari PJ.
Department of Process Science, Kosan Biosciences, Inc, 3832 Bay Center Place, Hayward, California 94545, USA. frykman@kosan.com
Many secondary metabolites, including various polyketides, require complex enzymatic pathways for modification into their final biologically active forms. Limitation of the dissolved oxygen supplied during cultivation of various microbial strains can decrease the activity of cytochrome P-450 monooxygenases required for the processing of pathway intermediates into their final forms, resulting in the accumulation of these intermediates as the primary products. Here, a generalized oxygen-limited cultivation strategy is specifically demonstrated with a myxobacterial strain engineered to heterologously express the epothilone polyketide synthase (PKS) gene cluster under either an excess (the dissolved oxygen tension is maintained at 50% of saturation) or a depleted (no residual dissolved oxygen detected) level of oxygenation during cultivation. Cultivation of this myxobacterial strain with excess oxygenation resulted in the production of epothilones A and B as the primary products, while cultivation of this same strain under depleted oxygenation resulted in the production of epothilones C and D as the primary products. Additionally, the peak cell density in the oxygen-depleted cultivations was 60% higher than that observed in oxygen-excess cultivations. Finally, an active EpoK epoxidase was found to catalyze the production of a novel epothilone (Epo506) with an unexpected structure during the cultivation of another myxobacterial strain expressing a genetically modified epothilone PKS under excess oxygenation. The structure of Epo506 was determined by high-resolution mass spectrometry and one- and two-dimensional NMR.
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