Protein production in Escherichia coli is guided by the trade-off between intracellular substrate availability and energy cost

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.