Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures

Abstract

BACKGROUND: A temperature limited fed-batch (TLFB) technique is described and used for Pichia pastoris Mut+ strain cultures and compared with the traditional methanol limited fed-batch (MLFB) technique. A recombinant fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced and secreted by this strain. RESULTS: A protein concentration of about 1 g L-1 was produced in the MLFB process. However, this product was considerably degraded by protease(s). By applying the TLFB process, the yield was increased to 2 g L-1 full-length product and no proteolytic degradation was observed. Flow cytometry analysis showed that the percentage of dead cells increased rapidly during the initial methanol feed phase in the MLFB process and reached a maximum of about 12% after about 40-70 hours of methanol feeding. In the TLFB process, cell death rate was low and constant and reached 4% dead cells at the end of cultivation (about 150 hours methanol feeding time). The lower cell death rate in the TLFB correlated with a lower protease activity in the culture supernatant. The specific alcohol oxidase (AOX) activity in the TLFB process was 3.5 times higher than in the MLFB process. CONCLUSION: Three mechanisms that may contribute to the much higher accumulation of product in the TLFB process are: 1) reduced proteolysis due to lower temperature, 2) reduced proteolysis due to lower cell death and protease release to the medium, 3) increased synthesis rate due to higher AOX activity.

Figure 1

A: Characteristics of a substrate limited fed-batch technique with constant temperature. B: Characteristics of a temperature limited fed-batch technique in which the substrate concentration is controlled at a non-limiting concentration while DOT is controlled at a set-point by means of the temperature controller. Parameters in A and B correspond to growth of P. pastoris on glycerol which promotes cell accumulation only. After a certain cell density is reached, the production phase can be started by a shift to methanol feed. F: feed rate; X: biomass concentration; My (μ): specific growth rate; T: temperature; DOT: dissolved oxygen tension; S: substrate concentration.

Figure 2

Arrhenius plot of the growth rate of P. pastoris SMD1168 grown on methanol and producing CBM-CALB. Dots represent experimental data.

Figure 3

Characteristics of a temperature limited fed-batch culture of P. pastoris. F: methanol feed rate; M: methanol concentration in the culture medium; DOT: dissolved oxygen tension; X: (open circles), biomass concentration; and T: temperature

Figure 4

Comparison of biomass concentrations (X) and consumption of oxygen per methanol (Yo/m) in two similar TLFB processes (filled symbols, solid lines) and in two similar MLFB processes at 30°C (open symbols, dotted lines). The methanol phase is divided in temperature limitation and methanol limitation only in the TLFB process. Both types of processes were controlled at DOT = 25% with similar oxygen transfer rate (stirrer speed and aeration rate in the same bioreactor).

Figure 5

Cell death in three TLFB processes (filled symbols) and in two MLFB processes at 30°C (open symbols). Solid line designated M represents the methanol concentration in culture broth in one of the TLFB processes. In the MLFB process, the methanol concentration is close to zero (not visible in the figure).

Figure 6

Comparison of the specific AOX activity in TLFB (open circles) and MLFB (closed circles) processes. The temperature (T) and methanol concentration (M) profiles of the TLFB process are also shown. During the methanol limitation phases, the methanol signal is close to zero and not visible in the figure.

Figure 7

Lipase activity produced in two MLFB (open symbols) and three TLFB processes (filled symbols).

Figure 8

A: Western blot analysis of samples from the MLFB run at pH 5.0 and 30°C. B: Western blot analysis of samples from the TLFB. C: Western blot analysis of samples from the TLFB with an early induction. Antibodies directed against the CALB moiety were used. CBM-CALB appears as diffuse bands in the range 39–44 kDa due to glycosylation [16].

Figure 9

Comparison of XET stability in cell-free supernatants withdrawn from a TLFB and an MLFB process. Incubation temperature was 22°C in both cases.