The heterologous production of epothilone D in Myxococcus xanthus was improved by 140-fold from an initial titer of 0.16 mg/L with the incorporation of an adsorber resin, the identification of a suitable carbon source, and the implementation of a fed-batch process. To reduce the degradation of epothilone D in the basal medium, XAD-16 (20 g/L) was added to stabilize the secreted product. This greatly facilitated its recovery and enhanced the yield by three-fold. The potential of using oils as a carbon source for cell growth and product formation was also evaluated. From a screen of various oils, methyl oleate was shown to have the greatest impact. At the optimal concentration of 7 mL/L in a batch process, the maximum cell density was increased from 0.4 g dry cell weight (DCW)/L to 2 g DCW/L. Product yield, however, depended on the presence of trace elements in the production medium. With an exogenous supplement of trace metals to the basal medium, the peak epothilone D titer was enhanced eight-fold. This finding demonstrates the significant role of metal ions in cell metabolism and in epothilone biosynthesis. To further increase the product yield, a continuous fed-batch process was used to promote a higher cell density and to maintain an extended production period. The optimized fed-batch cultures consistently yielded a cell density of 7 g DCW/L and an average production titer of 23 mg/L.

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.

No abstract

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 × 105 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 × 106 capture antibodies will result in detection of secretion of this protein at rates as low as 4 molecules/cell/sec. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 214–226, 1998.

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.