Nutrient regulation of epothilone biosynthesis in heterologous and native production strains.

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.

Modulation of epothilone analog production through media design.

Recently, the epothilone polyketide synthase (PKS) was successfully introduced into a heterologous production host for the large-scale production of epothilone D. We have found that at least three other epothilones can also be produced as the major fermentation product of this recombinant strain by supplementation of specific substrates to the production media. Addition of acetate or propionate to the media results in modulation of the epothilone D:C ratio, whereas addition of L-serine with either acetate or propionate yields epothilone H(1) or H(2) as the major product. This strategy permits production of at least four novel epothilones by culturing a single host with a genetically modified epothilone PKS in various media.

Precursor-directed production of erythromycin analogs by Saccharopolyspora erythraea.

Diketide N-acetylcysteamine (diketide NAC) thioester precursors were fed to 6-Deoxyerythronolide B synthase (DEBS) ketosynthase-1 inactivated (KS1 degree) Saccharopolyspora erythraea strains to produce 13-substituted erythromycin analogs. This direct feeding process potentially represents a simplified production process over the current analog production system. Titers of these analogs were observed to increase linearly with the diketide concentration up to a precursor-specific saturation level. However, the rate of product formation was lower and the rate of diketide consumption higher with S. erythraea than was previously observed with a recombinant strain of Streptomyces coelicolor. Several strategies were pursued to address the issue of these high diketide consumption rates: (1) elucidation of the locale of diketide degradation, (2) addition of beta-oxidation inhibitors to the cultures, and (3) addition of a sacrificial diketide enantiomer to occupy putative degradative enzymes. Additionally, repeated addition of diketide to an S. erythraea KS1 degrees culture indicated that the titer of these erythromycin analogs is also currently limited by a shorter production period than observed during erythromycin synthesis by the parent strain. These results indicate potential avenues for expanding the use of this precursor-directed system from the generation of limited quantities of erythromycin analogs to a large-scale production system for these compounds.

Cell cycle-dependent protein secretion by Saccharomyces cerevisiae.

Synchronized Saccharomyces cerevisiae cell populations were used to examine secretion rates of a heterologous protein as a function of cell cycle position. The synchronization procedure had a profound effect on the type and quality of data obtained. When cell synchrony was induced by cell cycle-arresting drugs, a significant physiological perturbation of cells was observed that obscured representative secretion data. In contrast, synchronization with centrifugal elutriation resulted in synchronized first-generation daughter cells with undetectable perturbation of the physiological state. The synchronized cells did not secrete significant amounts of protein until they reached cell division, suggesting that the secretion process in these cells is strongly cell cycle dependent. However, the maximum secretion rate of the synchronized culture (7-14 molecules/cell/second) was significantly lower than that of an asynchronous culture (29-51 molecules/cell/second). This result indicates that young daughter cells isolated in the synchronization process exhibit different protein secretion behavior than older mother cells that are absent in the synchronized cell population but present in the asynchronous culture.

Quantitating secretion rates of individual cells: design of secretion assays.

To observe events occurring in the microenvironment surrounding individual cells, a mathematical framework has been developed describing the behavior of a compound following its secretion by a single cell. This description is based on the diffusional and binding processes taking place in the vicinity of the cell surface. It allows prediction of the rate of capture and accumulation of a secreted compound around a single cell. This concept provides the basis for the design of two experimental assays for measuring single-cell secretion rates: (1) Cells are immobilized in hydrogel microbeads which contain capture sites for the secreted compound; and (2) artificial receptors are bound directly to the cell surface which are capable of binding molecules secreted by individual cells. This general methodology is developed in the specific case of the model organism Saccharomyces cerevisiae secreting a heterologous protein, but can be applied to any cell/secreted protein combination. Binding studies have shown that approximately 2 x 10(5) of these artificial receptors can be attached to the surface of a single yeast cell. At this surface density of a putative artificial receptor, it is predicted that single-cell secretion rates of 47 molecules/cell/sec of a 150 kDa protein can be detected. Simulations indicate that a microbead loaded with 5 x 10(6) capture antibodies will result in detection of secretion of this protein at rates as low as 4 molecules/cell/sec.

Effects of ibuprofen on postoperative bowel motility and propulsion.

Eighteen mongrel dogs were randomized into two groups, and all underwent right segmental colectomy. One group received ibuprofen, 12 mg/kg, preoperatively and postoperatively q6h for 24 hours; the second group served as a saline control. Propulsion was measured radiographically (q8h X 24h, then q12h) by following intraluminal radiopaque markers. Motility was measured with a multilumen monometric catheter on postoperative days 1, 2, and 3 for a 3-hour period. Propulsion, as defined by movement of more than 80 percent of the markers to the rectum, averaged 46 hours in the saline animals vs. 40.8 hours in the ibuprofen animals for an average decrease of 13 percent (P = .42). Postoperative motility showed a significant increase in the ibuprofen-treated animals on the first postoperative day (26.8 percent, P = .043), most marked at the level of the ileum (48 percent, P = .005), with insignificant differences on the second and third days. Studies comparing ileum, proximal, and distal colon suggest that ileum and proximal colon may be the major site of ibuprofen effect (10.7 percent, P = .23 and 11.96 percent, P = .41 respectively). The data suggest that ibuprofen may have a beneficial effect on postoperative bowel motility but the clinical significance of this still needs to be determined.