Huang Ming-Zhi, Cai Xian-Peng, Chen Shuang-Xi, Chu Ju, Zhuang Ying-Ping, Zhang Si-Liang
State Key Laboratory of Bioreactor Engineering, National Engineering Research Center for Biotechnology, and Bioengineering College, East China University of Science and Technology, Shanghai 200237, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Mar;19(2):200-5.
Metabolic engineering has become a powerful tool for optimization of industrial fermentation processes. Metabolic engineering usually undergoes three steps: construction of a recombinant strain with improved properties, genetic and biochemical analysis of the strain, and identification of target for further improvement. Metabolic fluxes analysis is an important part of the biochemical analysis. Based on the law of mass conservation and assuming pseudo-steady-state for the intermediates in the metabolic pathways, we have quantitatively analyzed the time course of the flux distribution in Bacillus subtilis and used the data to reveal the nature of the so-called "40 hour" phenomenon in fermentation of guanosine, a key raw material for the synthesis of additives for human consumption and animal feeds. The phenomenon refers to the observation that guanosine production, which proceeds at high rate from 12 hour on, declines around 40 hour while consumption of glucose keeps increasing, leading to the lower yield of the nucleoside. Equations based upon the metabolic network of Bacillus subtilis consisted of EMP pathway, HMP pathway, TCA cycle, oxidative phosphorylation pathway and others reactions of the intermediates, was constructed. The equations were solved by using the quantitative data obtained in this study. The air flow and volume, concentration of oxygen and carbon dioxide in the exit-gas were monitored online; the concentration of biomass, glucose and guanosine was analyzed manually; and the concentration of acetate, citric acid, pyruvate, and 17 amino acids were HPLC quantified. The solutions of the equation were proved to be valid, as the experimental data on oxygen consumption agrees with that of predicted form the equation. The results indicated that at 40h of the fermentation process the flux of HMP pathway, which provides the precursor of the nucleoside, decreased while that of EMP pathway and the pathways that generate amino acids and organic acids increased. The shift correlated with the accumulation of NH4+ in the broth. The assimilation of NH4+ is an energy consuming process and could shift the metabolism to the energy generating EMP pathway. Accordingly, measures were taken to prevent the accumulation of NH4+. The interference indeed stopped the metabolism shift and boosted the guanosine production at 30 g/L, 70% higher than the level reported in literature.
代谢工程已成为优化工业发酵过程的有力工具。代谢工程通常包括三个步骤：构建具有改良特性的重组菌株、对该菌株进行遗传和生化分析以及确定进一步改良的目标。代谢通量分析是生化分析的重要组成部分。基于质量守恒定律并假设代谢途径中的中间体处于伪稳态，我们定量分析了枯草芽孢杆菌中通量分布的时间进程，并利用这些数据揭示了鸟苷发酵中所谓“40小时”现象的本质，鸟苷是合成人类食用和动物饲料添加剂的关键原料。该现象指的是从12小时起以高速率进行的鸟苷生产在40小时左右下降，而葡萄糖消耗持续增加，导致核苷产量降低。构建了基于枯草芽孢杆菌代谢网络的方程，该网络由EMP途径、HMP途径、TCA循环、氧化磷酸化途径以及中间体的其他反应组成。利用本研究获得的定量数据对方程进行求解。在线监测出口气体中的气流和体积、氧气和二氧化碳浓度；手动分析生物量、葡萄糖和鸟苷的浓度；并通过HPLC对乙酸盐、柠檬酸、丙酮酸和17种氨基酸的浓度进行定量。方程的解被证明是有效的，因为耗氧的实验数据与方程预测的数据一致。结果表明，在发酵过程的40小时，提供核苷前体的HMP途径的通量下降，而EMP途径以及产生氨基酸和有机酸的途径的通量增加。这种转变与肉汤中NH4+的积累相关。NH4+的同化是一个耗能过程，可能会使代谢转向产生能量的EMP途径。因此，采取措施防止NH4+的积累。这种干预确实阻止了代谢转变，并使鸟苷产量提高到30 g/L，比文献报道的水平高出70%。
