De Dobbeleer C, Cloutier M, Fouilland M, Legros R, Jolicoeur M
Canada Research Chair on the Development of Metabolic Engineering Tools, Bio-P2, Department of Chemical Engineering, Ecole Polytechnique de Montreal, Station Centre-Ville, Montreal, Quebec, Canada H3C3A7.
Biotechnol Bioeng. 2006 Dec 20;95(6):1126-37. doi: 10.1002/bit.21077.
A perfusion bioreactor allowing continuous extraction of secondary metabolites was designed and challenged for Eschscholtzia californica plant cell suspensions. Four sedimentation columns mounted inside a 2.5-L bioreactor separated single cells and cell aggregates from the culture medium. Cells were elicited with chitin at day 4 and the liquid medium free of cells and debris was then continuously pumped to the extraction columns containing fluidized XAD-7 resins, and then recirculated back to the cell suspension. A medium upward velocity corresponding to cell sedimentation velocity maintained a stable cell/medium separation front in the columns for sedimented cell volume (SCV) of 90% (70% packed cell volume, PCV). Two perfusion bioreactor cultures of 10 and 14 days were performed. A maximum dilution rate of 20.4/day was reached from day 4 to day 6, and was then reduced to 5/day at day 9 for 55% SCV. Control cultures were performed without and with free extraction resins into the cell suspension. Perfusion cultures showed similar specific growth rates of 0.24 +/- 0.04/day before and after elicitation. However, production level in the perfusion cultures was similar to that from the culture without resins with a maximum of 2.06 micromole/gDW total alkaloids, with 1.54 micromole/gDW in the resins. Cultures with free resins resulted in 30.94 micromole/gDW with 28.4 +/- 8.8 micromole/gDW in the resins. Difference in the cells nutritional state from elicitation was identified as a major cause in the production reduction. However, pathway to chelilutine was favored in the continuous extraction culture.
设计了一种允许连续提取次生代谢产物的灌注生物反应器，并将其用于加州罂粟植物细胞悬浮培养。安装在2.5升生物反应器内的四个沉降柱将单细胞和细胞聚集体与培养基分离。在第4天用几丁质诱导细胞，然后将不含细胞和碎片的液体培养基连续泵入装有流化XAD - 7树脂的提取柱，再循环回到细胞悬浮液中。对应于细胞沉降速度的培养基向上流速在柱中维持了90%沉降细胞体积（SCV）（70%填充细胞体积，PCV）的稳定细胞/培养基分离前沿。进行了为期10天和14天的两种灌注生物反应器培养。从第4天到第6天达到了最大稀释率20.4/天，然后在第9天降至5/天以维持55%的SCV。对照培养分别在不添加和添加游离提取树脂到细胞悬浮液的情况下进行。灌注培养在诱导前后显示出相似的比生长速率为0.24±0.04/天。然而，灌注培养的产量水平与不添加树脂的培养相似，总生物碱最大产量为2.06微摩尔/克干重，树脂中为1.54微摩尔/克干重。添加游离树脂的培养产生了30.94微摩尔/克干重，树脂中为28.4±8.8微摩尔/克干重。诱导后细胞营养状态的差异被确定为产量降低的主要原因。然而，在连续提取培养中，向白屈菜碱的途径更受青睐。
