Chen W, Graham C, Ciccarelli R B
Apollon, Inc, Malvern, PA 19355, USA.
J Ind Microbiol Biotechnol. 1997 Jan;18(1):43-8. doi: 10.1038/sj.jim.2900355.
A fermentation process in Escherichia coli for production of supercoiled plasmid DNA for use as a DNA vaccine was developed using an automated feed-back control nutrient feeding strategy based on dissolved oxygen (DO) and pH. The process was further automated through a computer-aided data processing system to regulate the cell growth rate by controlling interactively both the nutrient feed rate and agitation speed based on DO. The process increased the total yield of the plasmid DNA by approximately 10-fold as compared to a manual fed-batch culture. The final cell yield from the automated process reached 60 g L-1 of dry cell weight (OD600 = 120) within 24 h. A plasmid DNA yield of 100 mg L-1 (1.7 mg g-1 cell weight) was achieved by using an alkaline cell lysis method. Plasmid yield was confirmed using High Performance Liquid Chromatography (HPLC) analysis. Because cells had been grown under carbon-limiting conditions in the automated process, acetic acid production was minimal (below 0.01 g L-1) throughout the fed-batch stage. In contrast, in the manual process, an acid accumulation rate as high as 0.36 g L-1 was observed, presumably due to the high nutrient feed rates used to maintain a maximum growth rate. The manual fed-batch process produced a low cell density averaging 10-12 g L-1 (OD600 = 25-30) and plasmid yields of 5-8 mg L-1 (approximately 0.7 mg g-1 cells). The improved plasmid DNA yields in the DO- and pH-based feed-back controlled process were assumed to be a result of a combination of increased cell density, reduced growth rate (mu) from 0.69 h-1 to 0.13 h-1 and the carbon/nitrogen limitation in the fed-batch stage. The DO- and pH-based feed-back control, fed-batch process has proven itself to be advantageous in regulating cell growth rate to achieve both high cell density and plasmid yield without having to use pure oxygen. The process was reproducible in triplicate fermentations at both 7-L and 80-L scales.
利用基于溶解氧（DO）和pH值的自动反馈控制营养物进料策略，开发了一种在大肠杆菌中生产用作DNA疫苗的超螺旋质粒DNA的发酵工艺。该工艺通过计算机辅助数据处理系统进一步实现自动化，通过基于溶解氧交互式控制营养物进料速率和搅拌速度来调节细胞生长速率。与手动补料分批培养相比，该工艺使质粒DNA的总产量提高了约10倍。自动工艺的最终细胞产量在24小时内达到60 g L-1干细胞重量（OD600 = 120）。通过碱性细胞裂解方法，质粒DNA产量达到100 mg L-1（1.7 mg g-1细胞重量）。使用高效液相色谱（HPLC）分析确认了质粒产量。由于在自动工艺中细胞是在碳限制条件下生长的，因此在整个补料分批阶段乙酸产量极低（低于0.01 g L-1）。相比之下，在手动工艺中，观察到酸积累速率高达0.36 g L-1，这可能是由于为维持最大生长速率而使用的高营养物进料速率所致。手动补料分批工艺产生的细胞密度较低，平均为10-12 g L-1（OD600 = 25-30），质粒产量为5-8 mg L-1（约0.7 mg g-1细胞）。基于溶解氧和pH值的反馈控制工艺中质粒DNA产量的提高被认为是细胞密度增加、生长速率（μ）从0.69 h-1降低到0.13 h-1以及补料分批阶段碳/氮限制共同作用的结果。基于溶解氧和pH值的反馈控制补料分批工艺已证明自身在调节细胞生长速率以实现高细胞密度和质粒产量方面具有优势，而无需使用纯氧。该工艺在7-L和80-L规模的三次重复发酵中均可重复。
