A study on L-threonine and L-serine uptake in Escherichia coli K-12.

In the current study, we report the identification and characterization of the yifK gene product as a novel amino acid carrier in E. coli K-12 cells. Both phenotypic and biochemical analyses showed that YifK acts as a permease specific to L-threonine and, to a lesser extent, L-serine. An assay of the effect of uncouplers and composition of the reaction medium on the transport activity indicates that YifK utilizes a proton motive force to energize substrate uptake. To identify the remaining threonine carriers, we screened a genomic library prepared from the yifK-mutant strain and found that brnQ acts as a multicopy suppressor of the threonine transport defect caused by yifK disruption. Our results indicate that BrnQ is directly involved in threonine uptake as a low-affinity but high-flux transporter, which forms the main entry point when the threonine concentration in the external environment reaches a toxic level. By abolishing YifK and BrnQ activity, we unmasked and quantified the threonine transport activity of the LIV-I branched chain amino acid transport system and demonstrated that LIV-I contributes significantly to total threonine uptake. However, this contribution is likely smaller than that of YifK. We also observed the serine transport activity of LIV-I, which was much lower compared with that of the dedicated SdaC carrier, indicating that LIV-I plays a minor role in the serine uptake. Overall, these findings allow us to propose a comprehensive model of the threonine/serine uptake subsystem in E. coli cells.

Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.

Despite advances in bacterial genome engineering, delivery of large synthetic constructs remains challenging in practice. In this study, we propose a straightforward and robust approach for the markerless integration of DNA fragments encoding whole metabolic pathways into the genome. This approach relies on the replacement of a counterselection marker with cargo DNA cassettes via λRed recombineering. We employed a counterselection strategy involving a genetic circuit based on the CI repressor of λ phage. Our design ensures elimination of most spontaneous mutants, and thus provides a counterselection stringency close to the maximum possible. We improved the efficiency of integrating long PCR-generated cassettes by exploiting the Ocr antirestriction function of T7 phage, which completely prevents degradation of unmethylated DNA by restriction endonucleases in wild-type bacteria. The employment of highly restrictive counterselection and ocr-assisted λRed recombineering allowed markerless integration of operon-sized cassettes into arbitrary genomic loci of four enterobacterial species with an efficiency of 50-100%. In the case of Escherichia coli, our strategy ensures simple combination of markerless mutations in a single strain via P1 transduction. Overall, the proposed approach can serve as a general tool for synthetic biology and metabolic engineering in a range of bacterial hosts.

Excision of selectable markers from the Escherichia coli genome without counterselection using an optimized λRed recombineering procedure.

The introduction of chromosomal mutations into the E. coli genome using λRed-mediated recombineering includes two consecutive steps-the insertion of an antibiotic resistance gene and the subsequent excision of the marker. The second step usually requires a counterselection method, because the efficiency of recombination is not high enough to find recombinants among non-recombinant cells. Most counterselection methods require the introduction of additional mutations into the genome or the use of expensive chemicals. In this paper, we describe the development of a reliable procedure for the removal of an antibiotic resistance marker from the E. coli genome without the need for counterselection. For this purpose, we used dsDNA cassettes consisting of two regions homologous to the sequences that flank the marker on the chromosome. We optimized the length of the homologous regions, the electroporation conditions, and the duration of recovery for the electroporated cells in order to maximize the recombination efficiency. Using the optimal parameters identified, we obtained a rate of 4-6% recombinants among the transformed cells. This high efficiency allowed us to find marker-less, antibiotic-sensitive recombinants by replica plating without the need for selection.

Development of new versatile plasmid-based systems for λRed-mediated Escherichia coli genome engineering.

Plasmid-based systems are the most appropriate for multistep lambda Red (λRed)-mediated recombineering, such as the assembly of strains for biotechnological applications. Currently, the widely used λRed-expressing plasmids use a temperature-sensitive origin of replication or temperature shift control of λRed expression. In this work, we have constructed a new, conditionally replicating vector that can be efficiently eliminated from the host strain through passaging in medium containing isopropyl-ß-d-thiogalactopyranoside. Using the new vector, we have developed two improved helper plasmids (viz., pDL17 and pDL14) for dsDNA and oligonucleotide-mediated recombineering, respectively. The plasmid pDL14 contains a dominant negative mutSK622A allele that suppresses methyl-directed mismatch repair (MMR). The coexpression of λRed and mutSK622A provides efficient oligonucleotide-mediated recombineering in the presence of active host MMR. The expression of λRed was placed under the control of the tightly regulated PrhaB promoter. Because of their low expression level under uninduced conditions, both plasmids could be maintained without elimination for multiple recombineering steps. The temperature-independent replication of the plasmids and control of λRed expression by l-rhamnose allow for all procedures to be performed at 37 °C. Thus, the new plasmids are robust, convenient, and versatile tools for Escherichia coli genome editing.