Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany.
Microb Cell Fact. 2019 Jan 17;18(1):8. doi: 10.1186/s12934-019-1057-5.
In vivo protein formation is a crucial part of cellular life. The process needs to adapt to growth conditions and is exploited for the production of technical and pharmaceutical proteins in microbes such as Escherichia coli. Accordingly, the elucidation of basic regulatory mechanisms controlling the in vivo translation machinery is of primary interest, not only to improve heterologous protein production but also to elucidate fundamental regulation regimens of cellular growth.
The current modeling analysis elucidates the impact of diffusion for the stochastic supply of crucial substrates such as the elongation factor EFTu, and tRNA species, all regarded as key elements for ensuring optimum transcriptional elongation. Together with the consideration of cellular ribosome numbers, their impact on the proper functioning of the translation machinery was investigated under different in vivo and in vitro conditions and utilizing the formation of non-native GFP and native EFTu as target proteins. The results show that translational elongation was diffusion limited. However, this effect was much more pronounced for the translation of non-native proteins than for the formation of codon-optimized native proteins.
Cellular ATP requirements constrain the options of improving protein production. In the case of non-native protein sequences, an optimized tRNA supply may be the most economical solution, as cells necessarily have to invest in ATP-costly ribosome synthesis to boost translation and increase growth rates.
体内蛋白质的合成是细胞生命的关键部分。该过程需要适应生长条件，并被用于生产大肠杆菌等微生物中的技术和药物蛋白。因此，阐明控制体内翻译机制的基本调控机制是首要任务，不仅要提高异源蛋白的生产效率，还要阐明细胞生长的基本调控规律。
目前的建模分析阐明了扩散对关键底物（如延伸因子 EFTu 和 tRNA 种类）的随机供应的影响，这些都是确保转录延伸最佳的关键因素。同时考虑到细胞核糖体的数量，在不同的体内和体外条件下，利用非天然 GFP 和天然 EFTu 的形成作为靶蛋白，研究了它们对翻译机制正常运转的影响。结果表明，翻译延伸受到扩散限制。然而，对于非天然蛋白质的翻译，这种影响比形成密码子优化的天然蛋白质更为显著。
细胞的 ATP 需求限制了提高蛋白质产量的选择。对于非天然蛋白质序列，优化的 tRNA 供应可能是最经济的解决方案，因为细胞必须投资于 ATP 成本高昂的核糖体合成，以提高翻译效率并增加生长速率。
