재조합 효모의 유가식 배양을 통한 외래 단백질 생산의 최적화

나정걸, 김현한*, 장용근*, 정봉현**

(주)에스티알바이오텍 중앙연구소, 한국과학기술원 화학공학과*, 생명공학연구원**

Optimization of Foreign Protein Production in Fed-batch Culture of Recombinant Yeast

Jeong-Geol Na, Hyun Han Kim*, Yong Keun Chang*, Bong Hyun Chung**

STR Biotech. Co., Ltd., Department of chemical engineering, KAIST*, Korea research institute of bioscience and bioengineering**

Introduction

In the recombinant plasmid systems, cell mass maximization doesn't necessarily guarantee the maximization of protein production. Moreover, in GAL system, the manipulated variables are glucose feeding rate and galactose feeding rate. Optimization of bioreactors with two control variables is considered very difficult and there have been few attempts.

The dynamics of fed-batch fermentation in recombinant yeast will be analyzed in terms of galactose uptake rate, induction time of galactose, and the effect of glucose and ethanol. The adaptive optimization strategy developed will be applied to this expression system.

Theory

In fed-batch culture of recombinant yeast cells on glucose and galactose, it is assumed that the formations of by-products except ethanol, substrate consumption for cell maintenance, and inhibition of growth by protein are negligible. Then, mass balance is represented by

[eqn: d(XV) over dt ~&=~ mu XV # {d(G_1 V)} over dt ~&=~ G_F1 F_1 - sigma_1 XV # {d(G_2 V)} over dt ~&=~ G_F2 F_2 - sigma_2 XV # {d(EV)} over dt ~&=~ (pi_E - eta_E ) XV # {d(PV)} over dt ~&=~ (pi_P - eta_P ) XV # dV over dt ~&=~ F_1 + F_2]	(1)

where, X, G1, G2, E, and P are the concentrations of cells, glucose, galactose, ethanol, and hPTH, respectively, F1 is feed flow rate of glucose, F2 is feed flow rate of galactose, V is culture volume, GF1 is glucose concentration in the glucose feed, GF2 is galactose concentration in the galactose feed, □ are specific growth rates on glucose, galactose and ethanol, □1 is specific glucose uptake rate, □2 is specific galactose uptake rate, □E is specific ethanol production rate, □E is specific ethanol consumption rate, □P is specific hPTH production rate, □P is specific hPTH degradation rate.

A model based on self-organizing genetic-fuzzy system was developed and used as the adaptive model. The concentrations of cells, glucose, galactose and ethanol were measured every one hour. When the difference between the model estimates of those concentrations and the measurement values exceeded a certain thresh-hold value, adaptation was carried out by using genetic algorithm. With the updated model parameters, a new optimal feed rate profile for the remaining culture time was calculated.

Experimentals

The strain used for protein production was a hPTH-producing recombinant yeast Saccharomyces cerevisiae L3262 disrupted in the YAP3 coding gene. One hundred mililiters of seed culture was transferred to a 5 L jar fermentor containing 900 mL of start-up medium. When glucose was exhausted and the dissolved oxygen concentration began to increase rapidly, a concentrated feed medium started to be fed to the fermentor. The culture temperature and pH were maintained at 30 ℃ and 5.5, respectively.

Cell growth was monitored by measuring the optical density at 600 nm. Glucose and galactose concentration was determined by using a chemical analyzer. Ethanol concentration was measured by using a gas chromatograph.

Result and discussion

A series of fed-batch culture experiments were conducted with the model strain, a hPTH producing recombinant S. cerevisiae to characterize the process dynamics as well as the relationships among cell growth, galactose consumption, and hPTH production. Profiles for the fed-batch fermentation are shown in Fig. 1~3. Examination of the fed-batch dynamics reveals some interesting features of this fermentation.

Figure 1 shows that, at about 20 hr after inoculation, ethanol is depleted from the medium, cell growth rate decreases, and hPTH is degraded. Galactose was not consumed until 13 hr after inoculation and was consumed at an almost constant rate (about 1.25 g/L/hr). The consumption pattern of galactose is in agreement with earlier studies. The reasons of long lag time observed could be considered as follows; (1) time for enzyme synthesis related to galactose metabolic pathway, (2) repression effect of ethanol. In order to examine the dynamics of galactose consumption, another fermentation with early induction was conducted.

Profile of the hPTH fermentation with early induction is shown in Fig. 2. Galactose consumption was repressed until 13 hr in like manner. The difference of galactose consumption rate owing to induction time was negligible. Experiemental results indicate that the dynamics of galactose consumption and hPTH production is not influenced by the induction time.

To improve the production of intact hPTH by promoting cell growth, the fed-batch fermentation was conducted with combined feeding of glucose and galactose. The maximum concentration of intact hPTH (~150 mg/L) was produced at 30 hr after inoculation as shown in Fig. 3. It is thought that this improvement of performance resulted from sustained growth until 30 hr even if relative low productivity. Thus, it is necessary to maintain high growth rate for overproduction of intact hPTH. Since gene expression is repressed while glucose is present in the medium, productivity is lower than that of galactose-only feeding. Experimental results suggested that the productivity could be maximized by fed-batch cultivation under conditions of active cell growth and galactose consumption, with a dual feeding of glucose and galactose. This controlled growth can be achieved in a fed-batch operation which has distinctive feature that cell growth and protein production is achieved by glucose feeding and galactose feeding, respectively.

Before attempting to formulate control algorithms for such fed-batch operations, constant feed rate of glucose fed-batch fermentation was conducted to examine the characteristics of operation when glucose, galactose, and ethanol coexist in the medium. Figure 4 shows the concentration profile and off-gas analysis. The intact hPTH concentration reached 184 mg/L at 24 hr of culture (1.5 times with galactose-only feeding). Improvement of fermentation performance results from the active cell growth.

An on-line optimization algorithm was applied for the optimization of hPTH production. As shown in Figure 5, the model followed the actual process fairly well. The cell mass concentration reached 32 g/L at about 20 hr. The performance index was 381.81 mg and the maximum intact hPTH concentration was 178 mg/L at 20 hr, while the predicted value was about 197 mg/L. It was found to be very successful in maintaining cell growth and galactose consumption at high levels, thus resulting in significant improvements in the productivity (up to 2.1 times) and intact hPTH cocentration (up to 1.5 times) compared with the case of an intermittent glucose and galactose, or galactose feeding.

Fig 1. intermittent feeding of galactose               Fig.2 early induction by galactose

Fig. 3 Intermittent feeding of glucose and galactose    Fig. 4 Dual feeding of glucose and galactose

Fig. 5 On-line optimization of hPTH production