S. cerevisiae as a Process Organism
To produce recombinant proteins in high yields and of optimum quality, S.cerevisiae has been subjected to a series of genetic manipulations. The success of the design of the disintegration vector, designed as whole 2-µm vectors in a cir° background (Figure 1), dispels the common view that yeast episomal plasmids are too unstable for industrial use, as no plasmid loss occurs during the production process. The stability of this system was demonstrated in semicontinuous operation over a time scale of months.
The process is robust and only two physiological consequences of the genetic changes resulting from many accumulative genetic changes have been observed.
The first phenomenon is a reduction in the critical growth, µcrit, which is the highest rate at which growth is fully aerobic without production of ethanol or acetate. Values above µcrit will result in the build-up of unwanted by-products. Although a lower µcrit value theoretically results in a reduction in bioreactor productivity, this is of little economic significance, since at large scale, factors such as mass and heat transfer limit the maximum growth rate. The change in µcrit can be resolved by lowering the parameter used in the control algorithm that determines the effective growth rate in the process.
The second phenomenon is a tendency of the organism to produce acetate under conditions where there is a slight excess in nutrient supply. Ethanol production is readily detected by a rise in Respiratory Quotient (RQ) determined by exit gas analysis. Hence, the control algorithm is designed to adjust the feed rate automatically. Acetic acid cannot be detected by a change in RQ but can be identified by changes in conductivity. Novozymes Delta used this principle to develop a subroutine in the automatic control procedure to adjust the feed rate appropriately.
If acetate is present, there is a second substrate available for oxidation in addition to the sucrose in the feed. The result is that metabolism is not strictly limited by the feed rate. This can be detected by a limitation check. Under conditions of carbon limitation by feed supply, a reduction in feed rate causes an almost immediate reduction in catabolism and respiratory rate.
The first indication of this is a rise in DOT or fall in stirrer speed if it is controlling DOT followed by a decline in oxygen uptake and CO2 evolution determined by exit gas analysis. The procedure here is to make a periodic reduction, once every few hours, in growth rate by about 20% for a period of 20 minutes. If the expected changes indicating reduced respiratory rate are observed, the culture was in limitation before the reduction and the previous feed rate can be restored.
If the respiration rate remains unchanged, this indicates the presence of acetate, whose increased consumption compensates for the reduction in feed rate. Since the feed rate cut has already caused an increase in acetate consumption, no further action is usually required. A subsequent limitation check a few hours later will show that the process is now under control. If necessary, repeated cuts can be imposed.