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.

Metabolic evolution and (13) C flux analysis of a succinate dehydrogenase deficient strain of Yarrowia lipolytica.

Bio-based succinic acid production can redirect industrial chemistry processes from using limited hydrocarbons to renewable carbohydrates. A fermentation process that does not require pH-titrating agents will be advantageous to the industry. Previously, a Yarrowia lipolytica strain that was defective for succinate dehydrogenase was constructed and was found to accumulate up to 17.5 g L(-1) of succinic acid when grown on glycerol without buffering. Here, a derivative mutant was isolated that produced 40.5 g L(-1) of succinic acid in 36 h with a yield of 0.32 g g(-1) glycerol. A combination approach of induced mutagenesis and metabolic evolution allowed isolation of another derivative that could utilize glucose efficiently and accumulated 50.2 g L(-1) succinic acid in 54 h with a yield of 0.43 g g(-1) . The parent strain of these isolated mutants was used for [1,6-(13) C2 ]glucose assimilation analysis. At least 35% glucose was estimated to be utilized through the pentose phosphate pathway, while ≥84% succinic acid was formed through the oxidative branch of the tricarboxylic acid cycle. Biotechnol. Bioeng. 2016;113: 2425-2432. © 2016 Wiley Periodicals, Inc.

Repetitive genomic sequences as a substrate for homologous integration in the Rhizopus oryzae genome.

The vast number of repetitive genomic elements was identified in the genome of Rhizopus oryzae. Such genomic repeats can be used as homologous regions for integration of plasmids. Here, we evaluated the use of two different repeats: the short (575 bp) rptZ, widely distributed (about 34 copies per genome) and the long (2053 bp) rptH, less prevalent (about 15 copies). The plasmid carrying rptZ integrated, but did so through a 2256-bp region of homology to the pyrG locus, a unique genomic sequence. Thus, the length of rptZ was below the minimal requirements for homologous strand exchange in this fungus. In contrast, rptH was used efficiently for homologous integration. The plasmid bearing this repeat integrated in multicopy fashion, with up to 25 copies arranged in tandem. The latter vector, pPyrG-H, could be a valuable tool for integration at homologous sequences, for such purposes as high-level expression of proteins.

Cell surface display of Yarrowia lipolytica lipase Lip2p using the cell wall protein YlPir1p, its characterization, and application as a whole-cell biocatalyst.

The Yarrowia lipolytica lipase Lip2p was displayed on the yeast cell surface via N-terminal fusion variant using cell wall protein YlPir1p. The hydrolytic activity of the lipase displayed on Y. lipolytica cells reached 11,900 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. The calculated number of recombinant enzyme displayed on the cell surface corresponds to approximately 6 × 10(5) molecules per cell, which is close to the theoretical maximum (2 × 10(6) molecules/cell). Furthermore, the leaking enzyme was presented as three N-glycosylated proteins, one of which corresponds to the whole hybrid protein. Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, the surface-displayed lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. Cell-bound lipase retained 74 % of its original activity at 60 °C for 5 min of incubation, and 83 % of original activity after incubation at 50 °C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1 and 71.0 % methyl esters after 33- and 45-h reactions, respectively.

Is it possible to produce succinic acid at a low pH?

Bio-based succinate is still a matter of special emphasis in biotechnology and adjacent research areas. The vast majority of natural and engineered producers are bacterial strains that accumulate succinate under anaerobic conditions. Recently, we succeeded in obtaining an aerobic yeast strain capable of producing succinic acid at low pH. Herein, we discuss some difficulties and advantages of microbial pathways producing "succinic acid" rather than "succinate." It was concluded that the peculiar properties of the constructed yeast strain could be clarified in view of a distorted energy balance. There is evidence that in an acidic environment, the majority of the cellular energy available as ATP will be spent for proton and anion efflux. The decreased ATP:ADP ratio could essentially reduce the growth rate or even completely inhibit growth. In the same way, the preference of this elaborated strain for certain carbon sources could be explained in terms of energy balance. Nevertheless, the opportunity to exclude alkali and mineral acid waste from microbial succinate production seems environmentally friendly and cost-effective.

Production of succinic acid at low pH by a recombinant strain of the aerobic yeast Yarrowia lipolytica.

Biotechnological production of weak organic acids such as succinic acid is most economically advantageous when carried out at low pH. Among naturally occurring microorganisms, several bacterial strains are known to produce considerable amounts of succinic acid under anaerobic conditions but they are inefficient in performing the low-pH fermentation due to their physiological properties. We have proposed therefore a new strategy for construction of an aerobic eukaryotic producer on the basis of the yeast Yarrowia lipolytica with a deletion in the gene coding one of succinate dehydrogenase subunits. Firstly, an original in vitro mutagenesis-based approach was proposed to construct strains with Ts mutations in the Y. lipolytica SDH1 gene. These mutants were used to optimize the composition of the media for selection of transformants with the deletion in the Y. lipolytica SDH2 gene. Surprisingly, the defects of each succinate dehydrogenase subunit prevented the growth on glucose but the mutant strains grew on glycerol and produced succinate in the presence of the buffering agent CaCO(3). Subsequent selection of the strain with deleted SDH2 gene for increased viability allowed us to obtain a strain capable of accumulating succinate at the level of more than 45 g L(-1) in shaking flasks with buffering and more than 17 g L(-1) without buffering. The possible effect of the mutations on the utilization of different substrates and perspectives of constructing an industrial producer is discussed.