Exploring the fitness benefits of genome reduction in Escherichia coli by a selection-driven approach.

Artificial simplification of bacterial genomes is thought to have the potential to yield cells with reduced complexity, enhanced genetic stability, and improved cellular economy. Of these goals, economical gains, supposedly due to the elimination of superfluous genetic material, and manifested in elevated growth parameters in selected niches, have not yet been convincingly achieved. This failure might stem from limitations of the targeted genome reduction approach that assumes full knowledge of gene functions and interactions, and allows only a limited number of reduction trajectories to interrogate. To explore the potential fitness benefits of genome reduction, we generated successive random deletions in E. coli by a novel, selection-driven, iterative streamlining process. The approach allows the exploration of multiple streamlining trajectories, and growth periods inherent in the procedure ensure selection of the fittest variants of the population. By generating single- and multiple-deletion strains and reconstructing the deletions in the parental genetic background, we showed that favourable deletions can be obtained and accumulated by the procedure. The most reduced multiple-deletion strain, obtained in five deletion cycles (2.5% genome reduction), outcompeted the wild-type, and showed elevated biomass yield. The spectrum of advantageous deletions, however, affecting only a few genomic regions, appears to be limited.

Enhancing the Translational Capacity of E. coli by Resolving the Codon Bias.

Escherichia coli is a well-established and popular host for heterologous expression of proteins. The preference in the choice of synonymous codons (codon bias), however, might differ for the host and the original source of the recombinant protein, constituting a potential bottleneck in production. Codon choice affects the efficiency of translation by a complex and poorly understood mechanism. The availability of certain tRNA species is one of the factors that may curtail the capacity of translation. Here we provide a tRNA-overexpressing strategy that allows the resolution of the codon bias, and boosts the translational capacity of the popular host BL21(DE3) when rare codons are encountered. In the BL21(DE3)-derived strain, called SixPack, copies of the genes corresponding to the six least abundant tRNA species have been assembled in a synthetic fragment and inserted into a rRNA operon. This arrangement, while not interfering with the growth properties of the new strain, allows dynamic control of the transcription of the extra tRNA genes, providing significantly elevated levels of the rare tRNAs in the exponential growth phase. Results from expression assays of a panel of recombinant proteins of diverse origin and codon composition showed that the performance of SixPack surpassed that of the parental BL21(DE3) or a related strain equipped with a rare tRNA-expressing plasmid.

Engineered ribosomal RNA operon copy-number variants of E. coli reveal the evolutionary trade-offs shaping rRNA operon number.

Ribosomal RNA (rrn) operons, characteristically present in several copies in bacterial genomes (7 in E. coli), play a central role in cellular physiology. We investigated the factors determining the optimal number of rrn operons in E. coli by constructing isogenic variants with 5-10 operons. We found that the total RNA and protein content, as well as the size of the cells reflected the number of rrn operons. While growth parameters showed only minor differences, competition experiments revealed a clear pattern: 7-8 copies were optimal under conditions of fluctuating, occasionally rich nutrient influx and lower numbers were favored in stable, nutrient-limited environments. We found that the advantages of quick adjustment to nutrient availability, rapid growth and economic regulation of ribosome number all contribute to the selection of the optimal rrn operon number. Our results suggest that the wt rrn operon number of E. coli reflects the natural, 'feast and famine' life-style of the bacterium, however, different copy numbers might be beneficial under different environmental conditions. Understanding the impact of the copy number of rrn operons on the fitness of the cell is an important step towards the creation of functional and robust genomes, the ultimate goal of synthetic biology.