Dept. of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, 2628 BC Delft, The Netherlands.
Biotechnol Prog. 2012 Mar-Apr;28(2):337-48. doi: 10.1002/btpr.1503. Epub 2011 Dec 30.
Although penicillin-G (PenG) production by the fungus Penicillium chrysogenum is a well-studied process, little is known about the mechanisms of transport of the precursor phenylacetic acid (PAA) and the product PenG over the cell membrane. To obtain more insight in the nature of these mechanisms, in vivo stimulus response experiments were performed with PAA and PenG in chemostat cultures of P. chrysogenum at time scales of seconds to minutes. The results indicated that PAA is able to enter the cell by passive diffusion of the undissociated acid at a high rate, but is at the same time actively excreted, possibly by an ATP-binding cassette transporter. This results in a futile cycle, dissipating a significant amount of metabolic energy, which was confirmed by increased rates of substrate and oxygen consumption, and carbon dioxide production. To estimate the kinetic properties of passive import and active export of PAA over the cell membrane, a dynamic mathematical model was constructed. With this model, a good description of the dynamic data could be obtained. Also, PenG was found to be rapidly taken up by the cells upon extracellular addition, indicating that PenG transport is reversible. The measured concentration gradient of PenG over the cell membrane corresponded well with facilitated transport. Also, for PenG transport, a dynamic model was constructed and validated with experimental data. The outcome of the model simulations was in agreement with the presence of a facilitated transport system for PenG.
尽管青霉素 G（PenG）的生产过程在真菌产黄青霉中已经得到了深入研究，但对于前体苯乙酸（PAA）和产物 PenG 穿过细胞膜的运输机制知之甚少。为了更深入地了解这些机制的本质，我们在产黄青霉的恒化器培养物中进行了体内刺激响应实验，实验时间尺度从秒到分钟不等。结果表明，PAA 可以通过未离解酸的被动扩散以高速度进入细胞，但同时也被主动排出，可能是通过 ATP 结合盒转运体。这导致了无效循环，消耗了大量的代谢能量，这可以通过增加底物和氧气消耗以及二氧化碳产生来证实。为了估计 PAA 穿过细胞膜的被动导入和主动导出的动力学特性，构建了一个动态数学模型。通过该模型，可以很好地描述动态数据。此外，还发现 PenG 在细胞外添加后会迅速被细胞摄取，表明 PenG 运输是可逆的。测量的 PenG 跨细胞膜的浓度梯度与促进运输很好地吻合。此外，还为 PenG 运输构建并验证了一个动态模型。模型模拟的结果与存在促进运输系统的结果一致。
