Jia Luqiang, Li Teng, Wu Yixuan, Wu Chunsen, Li Huaxiang, Huang Agen
School of Food Science and Technology, Yangzhou University, Yangzhou, 225127, China.
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
Appl Microbiol Biotechnol. 2021 Feb;105(3):1041-1050. doi: 10.1007/s00253-021-11100-9. Epub 2021 Jan 14.
In human lysozyme (hLYZ) production by Pichia pastoris, the glycerol fed-batch phase was generally implemented under the environment of "oxygen sufficient-glycerol limited" to achieve high cell-density cultivation during the cell growth phase. However, the structural and functional components in P. pastoris cells were irreversible damaged with more and more reactive oxygen species (ROS) accumulation when cells were exposed to the oxygen sufficient environments for long time, leading to a failure of hLYZ expression. In this study, a novel periodic glycerol and dissolved oxygen concentration (DO) control strategy was proposed to solve these problems. This strategy periodically switched the cultivation environments from "oxygen sufficient-glycerol limited" to "oxygen limited-glycerol sufficient" for 5 cycles. When using this strategy: (1) the highest dry cell weight (DCW) of 143.02 g-DCW/L and the lowest distribution of glycerol towards to cell maintenance (0.0400 1/h) were achieved during the glycerol feeding phase by maintaining ROS levels below 48.39 Fluorescence intensity/g-DCW; (2) the adaption time of P. pastoris cells to methanol induction environments was shortened for about 50%; (3) P. pastoris cell metabolic activities reflected by the activities of alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and methanol consumption rate, etc., in the successive induction phase were largely enhanced; (4) hLYZ activity reached the highest level of 2.45 × 10 IU/mL, which was about 2-fold than that obtained with the strategy of "oxygen sufficient-glycerol limited," when the same methanol induction strategy was adopted. KEY POINTS: • A novel periodic glycerol feeding strategy proposed/used for P. pastoris cell growth. • Higher cell density was obtained by controlling ROS at low level via this strategy. • The highest hLYZ activity was achieved when initiating induction at higher cell density.
在毕赤酵母生产人溶菌酶（hLYZ）的过程中，甘油补料分批培养阶段通常在“氧气充足-甘油受限”的环境下进行，以便在细胞生长阶段实现高细胞密度培养。然而，当细胞长时间暴露于氧气充足的环境时，毕赤酵母细胞中的结构和功能成分会因活性氧（ROS）积累越来越多而受到不可逆的损害，导致hLYZ表达失败。在本研究中，提出了一种新的周期性甘油和溶解氧浓度（DO）控制策略来解决这些问题。该策略将培养环境从“氧气充足-甘油受限”周期性地切换到“氧气受限-甘油充足”，共进行5个循环。使用该策略时：（1）在甘油补料阶段，通过将ROS水平维持在48.39荧光强度/g-DCW以下，实现了最高干细胞重量（DCW）为143.02 g-DCW/L，以及最低的甘油用于细胞维持的分配率（0.0400 1/h）；（2）毕赤酵母细胞对甲醇诱导环境的适应时间缩短了约50%；（3）在连续诱导阶段，由醇氧化酶、甲醛脱氢酶、甲酸脱氢酶活性以及甲醇消耗率等反映的毕赤酵母细胞代谢活性大大增强；（4）当采用相同的甲醇诱导策略时，hLYZ活性达到最高水平2.45×10 IU/mL，约为采用“氧气充足-甘油受限”策略时的2倍。要点：• 提出/使用了一种新的周期性甘油补料策略用于毕赤酵母细胞生长。• 通过该策略将ROS控制在低水平从而获得更高的细胞密度。• 在更高细胞密度下开始诱导时实现了最高的hLYZ活性。
