Plant cell cultures of Lithospermum erythrorhizon were performed to produce plant derived secondary metabolizes such as shikonin.
L. erythrorhizon was cultured in 250 mL shake flasks. Dry cell concentration of 13 g/L and fresh cell concentration of 390 g/L were obtained after 210 hours. Specific growth rate of 0.26 $day^{-1}$ was obtained, and it was about 20 times lower than that of microbial cells. The slower growth rate lowers cellular productivity and makes the operation difficult. Owing to the slow growth rate, specific oxygen uptake rate was 7.25 mg/g·h which is much lower than that of microbial cells (Chap. 2).
Plant cell immobilization effect on secondary metabolite production was studied. Plant cells were immobilized in a dual hollow fiber bioreactor to maintain high cell density and to operate continuously. The dual hollow fiber bioreactor consists of outer silicone membrane for oxygen transport and an inner isotopic polypropylene hollow fiber for liquid nutrient transport. its performance was examined by culturing L. erythorhizon cells. The cells have grown well and the dry biomass density of the cells was 325 g/L of void volume for cell growth, which was highest among the methods for plant cell culture ever reported. Volumetric productivity of phenolics as a rough indication of plant cell secondary metabolizes was 221 mg/L·day at a medium flow rate of 0.3 mL/h, which corresponds to a 58 fold increase over that of shake flask culture. The specific productivity of the cells in the reactor was 0.68 mg/g cell·day, which was 2 times higher than that of batch culture (Chap.3).
Effects of cell immobilization and in sits extraction on shikonin production were studied. In sits extraction and cell immobilization in calcium alginate beads enhanced shikonin production, respectively, and they greatly increased shikonin production when they were employed together. This simultaneous treatment increased cellular shikonin productivity and volumetric productivity 25 and 15 times, respectively, and productivities can be further increased if a sufficient level of oxygen was supplied. The most effective solvent addition timing for shikonin production in immobilized cell culture was 5 days after subculture was started. From the above results in situ extraction, cell immobilization, and dissolved oxygen concentration played important roles on shikonin production and cellular metabolism. In the case of in sits extraction, most of the produced shikonin was dissolved in η-hexadecane layer, so it will greatly reduce the costs for shikonin separation. A bioreactor of immobilized cells and in sits extraction with sufficient oxygen supply seems to be a promising system for shikonin and other plant secondary metabolizes production (Chap. 4).
Transformed L. erythrorhizon was cultured to produce shikonin and its shikonin production was compared to that of intact cells. Shikonin production of transformed L. erythrorhizon increased with the enhanced oxygen supply, and in sits extraction also increased sucrose consumption and shikonin production. And in sits extraction at earlier stage significantly enhanced shikonin production. Plant cell permeabilization with 5% DMSO does not help the situation. Sufficient oxygen supply and in situ extraction at earlier stage are required for the effective production of shikonin by the cultures of transformed cells of L. erythrorhizon (Chap. 5).
Oxygen transfer analyses of a dual hollow fiber unit and calcium alginate bead were performed. The effects of Thiele modules and dissolved oxygen concentration of culture medium on oxygen penetration depth were examined. As Thieve modules increased, oxygen penetration depth decreased rapidly. For the case of cell immobilization Thiele modules should be primarily considered as a design criterion and the oxygen concentration of culture medium should be maintained high during the culture (Chap. 6).
Effects of growth hormone and elicitor treatment on shikonin production were studied. The growth hormone (pCPA) in growth stage inhibits shikonin synthesis in induction stage. Thus pCPA in growth medium was deleted (SH-H) or replaced by IAA(SHA) to reduce the growth hormone effect on shikonin production. The cells grown in SH-H or SHA medium were effective for shikonin production. Final shikonin concentration of the cells grown in SH-H medium was about 3 times higher than that of the cells grown in SH medium. And in the case of SHA medium, induction time required for shikonin production was very short and maximum shikonin concentration was obtained within 6 days of the culture in M-9 medium with in situ extraction.
Elicitor treatment with in situ extraction increased shikonin production about 3 times and shikonin was induced within 24 h. Elicitor effects are known to be the results of host-pathogen interaction, but the precise mechanisms are not known and should be studied more (Chap. 7).
Reactor operations were performed to examine the effects of reactor design characteristics and cell immobilization on shikonin production by using suspension culture in stirred tank with in situ extraction and immobilized cells in packed-bed reactor. The packed-bed reactor with immobilized cells and in situ extraction was more effective for shikonin production (chapter 8).
본 논문은 식물세포인 Lithospermum erythrorhizon의 배양에 의한 shikonin 생산에 관한 연구이다.
제1장에서는 식물세포의 배양 특성과 식물세포로부터 생산되는 화합물, 식물세포의 고정화, 그리고 식물세포 배양을 위한 반응기가 소개되었다.
제2장에서는 L. erythrorhizon의 shake flask에서의 성장 kinetics가 연구되었다. 210시간 배양후 건조 세포 농도는 13 g/L였고 습윤세포 농도는 390 g/L였다. 비성장속도는 0.26/day 이고, 이는 다른 미생물인 박테리아에 비해 약 20배정도 느린 것이다. 비산소 소비속도는 7.25 mg/g.h 로서 역시 다른 호기성 미생물에 비하여 월등히 낮았다.
제3장에서는 식물세포의 고정화 효과가 연구 되었다. L. erythrorhizon을 이중실관 생물 반응기에 고정화하고 연속조업을 하면서 세포고정화 효과를 조사하였다. 이중실관 생물 반응기에서 건조세포 농도는 325 g/L였고, 이는 다른 식물세포 고정화 방법에 비하여 월등히 높은 것이다. 식물세포의 2차대사물로서 phenolics의 생산을 shake flask와 비교하여 보았는데 이때 productivity가 221 mg/L.day로서 shake flask의 그것에 비하여 58배 향상되었다. 또한 비생산성을 비교하여 본 결과 0.68 mg/g cell.day로 shake flask에 비하여 약 2배 증가하였다.
제4장에서는 세포의 고정화와 in situ 추출이 shikonin 생산에 미치는 영향을 조사하였다. 세포의 calcium alginate bead에의 고정화와 n-hexadecane 을 사용한 in situ 추출은 shikonin 생산성을 향상시켰는데 이들 둘을 같이 사용한 경우 shikonin의 비생산성과 부피 생산성이 controlled 배양에 비해 각각 25, 15배 증가하였다. 고정화 세포의 경우 용매의 첨가시기는 배양 후 5일째가 가장 좋았다. 세포의 shikonin 생산이나 sucrose 소비는 용존산소 농도에 많은 영향을 받으므로 적절한 산소를 공급해 줄 수 있는 반응기 시스템이 필요하다. 생산된 shikonin은 거의 모두 녹아 분리, 정제비용을 절감하는 효과를 기대할 수 있다.
제5장에서는 유전자가 변이된 transformed L. erythrorhizon을 배양하여 shikonin 생산을 하여 wild type의 그것과 비교하였다. 변이세포는 산소전달이 증가함에 따라 shikonin 생산이 증가하였고 in situ 추출은 shikonin 의 생산뿐만 아니라 sucrose 의 소비도 높였다. 식물세포를 5% DMSO로 처리하여 세포벽을 헐어서 shikonin의 세포로부터의 분비를 촉진시키려했으나 효과가 없었다. 배양 초기의 in situ 추출과 충분한 산소 공급이 shikonin 생산에 효과적이었다.
제6장에서는 이중실관 생물 반응기와 calcium alginate에 고정화된 세포의 산소전달을 simulation을 통하여 해석하였다. Thiele modulus와 배지의 용존산소 농도가 산소 투과깊이에 어떻게 영향을 미치는가가 연구되었다. Thiele modulus가 증가함에 따라 산소의 투과깊이는 급격히 감소하였고, 배지의 용존산소 농도의 증가는 산소 투과깊이를 증가시켰다.
제7장에서는 shikonin 생산에 성장호르몬과 elicitor의 효과가 연구되었다. SH-H나 SHA 배지에서 성장한 세포는 shikonin 생산에 매우 효과적이어서 SH 배지에서 성장한 것에 비하여 약 3배 정도 생산이 향상되었으며 SHA 배지에서 성장한 경우 shikonin induction 시간이 짧아 져서 shikonin의 생산성이 향상되었다. Elicitor를 처리한 세포의 경우 shikonin 생산량이 다른 것에 비하여 3배 정도 향상되었고 용매첨가 시기에 따라 전체 shikonin이 하룻만에 생산되어 생산성 향상에 크게 기여하였다
제8장에서는 반응기 종류에 따른 shikonin의 생산을 비교하였다. 교반 반응기에서는 suspension culture를 in situ 추출과 함께 행하였고 충전층 반응기에서는 calcium alginate에 고정화된 세포를 in situ 추출과 함께 배양하여 shikonin을 생산하였다. 그 결과 고정화 세포를 이용한 충전충 반응기의 생산성이 교반 반응기에 비해 높았다.
이상의 연구 결과에서 세포고정화, in situ 추출, 성장호르몬 조절, 그리고 elicitor의 첨가는 shikonin 생산성을 향상시켰으며 이들의 동시 사용에 의한 synergistic effect가 기대된다. 반응기 조업을 고려해 볼 때는 고정화 세포를 in situ 추출을 할 수 있는 airlift 나 bubble column 반응기가 매우 적절한 것으로 사료된다.
