PubMed · 6690992
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A H Bretag. What is the solution?. https://doi.org/10.1038/307116a0
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Cultures of Saccharomyces cerevisiae strain GL7 (a sterol auxotroph) were incubated with nonradioactive and tritium-labeled cycloartenol, and the sterol composition of the cells was examined by chemical (GLC, TLC, HPLC, MS, 1H-NMR, and 13C-NMR) and radiotracer techniques. Several novel sterols were isolated from the cells including 14 alpha-methyl ergosta-9(11),24(28)-dien-3 beta-ol, 24 beta-methyl-9 beta,19-cyclopropyl ergost-8(14)-en-3 beta-ol, and 9 beta,19-cyclopropyl ergosta-7(8),24(28)-dien-3 beta-ol. GL7 converted [2-3H]cycloartenol to [2-3H]ergosterol in low yield (1% incorporation), whereas [2-3H]lanosterol was converted to [2-3H]ergosterol in high yield (41% incorporation). The degree of sterol absorption and transformation by GL7 was dependent on the type and amount of sterol(s) in the growth medium. The results demonstrate for the first time that yeast may transform 9 beta,19-cyclopropyl sterols to 9(11)-sterols and delta 5-sterols and that 14-demethylation of sterols may proceed in GL7 to double bond formation either in the 8(14)-position or in the 14(15)-position.
In order to develop a cost-effective recovery process for an intracellular product, crossflow microfiltration was studied for the harvest of a recombinant yeast under severe time constraint. It was required to process yeast broth in a short period of time to minimize the risk for product degradation. Preliminary microfiltration studies employing flat sheet membranes showed high throughout with initial fluxes on the order of water fluxes (> 1000 LMH, regime I, < 2 min), followed by a rapid decay towards a low pseudo-steady state flux (20 LMH, regime II, > 2 min). Exploitation of these high fluxes and control of their eventual decline were crucial in establishing a rapid crossflow filtration process. The effect of several parameters, such as initial cell concentration, shear rate, transmembrane pressure, membrane pore size and medium composition on filtration performance were investigated to better understand the flux decline mechanisms. We found that the major contributor to flux decay was reversible fouling by the cake formation on the membrane surface. Within the operating boundaries of our microfiltration system, large-pore membrane (0.65 micron) was much more desirable for harvesting our yeast (10 microns size) without cell leakage than smaller pore ones (0.22 micron and 0.45 micron). Among adjustable operating parameters, feed flow rate (i.e., shear rate) exerted significant impact on average flux, whereas manipulation of transmembrane pressure afforded little improvement. Although initial cell concentration affected adversely the permeation rates, growth medium components, especially soy-peptone, was deemed pivotal in determining the characteristics of cell cake, thus controlling yeast microfiltration.
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