Vibrio natriegens constitutes one of the fastest-growing nonpathogenic bacteria and a potential novel workhorse for many biotechnological applications. Here, we report the development of a Vibrio-based cell-free protein synthesis system (CFPS). Specifically, up to 0.4 g L-1 eGFP could be successfully synthesized in small-scale batch reactions using cell-free extract obtained from fast-growing V. natriegens cultures. Versatile CFPS system characterization attained by combining the analyses of key metabolites for translation and ribosomes revealed limitations regarding rRNA stability and critical substrate consumption (e.g., amino acids). Alternatively, rRNA showed increased stability by inducing Mg2+homeostasis in the reaction. Although the enormous translation capacity of the CFPS system based on the available ribosome concentration could not yet be fully exploited, its potential was successfully demonstrated by activating an endogenous transcription unit with V. natriegensRNA polymerase (RNAP) for protein expression. This allowed the use of in vitro screening for promoter strength, a critical factor for efficient gene expression in vitro and in vivo. Three different promoters were tested and output signals corresponded well with the expected affinity for V. natriegens RNAP. This established CFPS toolbox may provide a foundation to establish V. natriegens as a valuable platform in biotechnology as well as synthetic biology.

Cell-free protein synthesis is a versatile protein production system. Performance of the protein synthesis depends on highly active cytoplasmic extracts. Extracts from E. coli are believed to work best; they are routinely obtained from exponential growing cells, aiming to capture the most active translation system. Here, we report an active cell-free protein synthesis system derived from cells harvested at non-growth, stressed conditions. We found a downshift of ribosomes and proteins. However, a characterization revealed that the stoichiometry of ribosomes and key translation factors was conserved, pointing to a fully intact translation system. This was emphasized by synthesis rates, which were comparable to those of systems obtained from fast-growing cells. Our approach is less laborious than traditional extract preparation methods and multiplies the yield of extract per cultivation. This simplified growth protocol has the potential to attract new entrants to cell-free protein synthesis and to broaden the pool of applications. In this respect, a translation system originating from heat stressed, non-growing E. coli enabled an extension of endogenous transcription units. This was demonstrated by the sigma factor depending activation of parallel transcription. Our cell-free expression platform adds to the existing versatility of cell-free translation systems and presents a tool for cell-free biology.

Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell-free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i) the cell-free extract preparation and (ii) the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein S1, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems.

Cell-free (in vitro) protein synthesis (CFPS) systems provide a versatile tool that can be used to investigate different aspects of the transcription-translation machinery by reducing cells to the basic functions of protein formation. Recent improvements in reaction stability and lysate preparation offer the potential to expand the scope of in vitro biosynthesis from a research tool to a multifunctional and versatile platform for protein production and synthetic biology. To date, even the best-performing CFPS systems are drastically slower than in vivo references. Major limitations are imposed by ribosomal activities that progress in an order of magnitude slower on the mRNA template. Owing to the complex nature of the ribosomal machinery, conventional "trial and error" experiments only provide little insight into how the desired performance could be improved. By applying a DNA-sequence-oriented mechanistic model, we analyzed the major differences between cell-free in vitro and in vivo protein synthesis. We successfully identified major limiting elements of in vitro translation, namely the supply of ternary complexes consisting of EFTu and tRNA. Additionally, we showed that diluted in vitro systems suffer from reduced ribosome numbers. On the basis of our model, we propose a new experimental design predicting 90% increased translation rates, which were well achieved in experiments. Furthermore, we identified a shifting control in the translation rate, which is characterized by availability of the ternary complex under in vitro conditions and the initiation of translation in a living cell. Accordingly, the model can successfully be applied to sensitivity analyses and experimental design.

Ribosomes are a crucial component of the physiological state of a cell. Therefore, we aimed to monitor ribosome dynamics using a fast and easy fluorescence readout. Using fluorescent-labeled ribosomal proteins, the dynamics of ribosomes during batch cultivation and during nutritional shift conditions was investigated. The fluorescence readout was compared to the cellular rRNA content determined by capillary gel electrophoresis with laser-induced fluorescence detection during exponentially accelerating and decelerating growth. We found a linear correlation between the observed fluorescence and the extracted rRNA content throughout cultivation, demonstrating the applicability of this method. Moreover, the results show that ribosome dynamics, as a result of slowing growth, are accompanied by the passive effect of dilution of preexisting ribosomes, de novo ribosome synthesis and ribosome degradation. In light of the challenging task of deciphering ribosome regulatory mechanisms, our approach of using fluorescence to follow ribosome dynamics will allow more comprehensive studies of biological systems.