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The subject of this review covers modern experimental procedures for chromosomal gene replacement in Escherichia coli and related bacteria, which enable the specific substitution of targeted genome sequences with copies of those carrying defined mutations. Two principal methods for gene replacement were established. The first "in-out" method is bas...
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... Cloning large DNA inserts in specialized E. coli vectors, e.g. BAC, is very useful approach in functional genetic studies of higher eukaryotes because usually all regulatory gene elements are present in the same construct and after transformation to the original host the gene expression can be studied in its native context. Functional studies are frequently based on preparation of defined mutants. However, usage of classical in vitro cloning techniques is impractical or even impossible in such a large DNA molecules. For this reason in vivo gene replacement techniques based on homologous recombination in E. coli are exploited. Point mutations, large deletions and insertions can be introduced in vivo into plasmids by recombineering, with many protocols and method improvements published recently (Rivero- Müller et al. 2007; Wang et al. 2009; Sharan et al. 2009). Recombineering can be also used for efficient cloning of DNA fragments found on genome or on large plasmids (e.g. BAC) into new vectors ( Fig. 5). In this approach, a plasmid vector containing origin of replication and selectable marker is firstly copied into linear molecule by PCR with primers possessing short ends with homology to DNA to be subcloned. Then the linear vector is transformed into E. coli containing subcloned DNA and λ Red genes. By the activity of the Red genes recombination between ends of the linear vector and the target host DNA occurs resulting in the formation of a circular plasmid by gap repair mechanism. Desired clones are then selected by antibiotic marker present on vector (Lee at al. , 2001 Sharan et al. 2009). The technique is quite simple and fast, its other important advantages include cloning independent of restriction sites, possibility of flanking of the cloned fragment with functional cassettes or restriction sites and a large cloning capacity (up to 80 kb for low-copy plasmids). Another organism which can be mutagenized by recombineering are bacteriophages. In the case of temperature phages, lysogenic state was used for targeted mutant construction and/or in vivo cloning (Burian et al. 1998, Serra-Moreno et al. 2006). However, mutagenesis can be performed also on lytically replicating phages (Oppenheim et al. 2004). Highly effective allele replacement for bacteriophages of Mycobacterium smegmatis was based on simultaneous co-electroporation of phage DNA template and a targeting substrate into cells that have been induced for recombineering functions (Marinelli et al. 2008). Genome engineering in E. coli and other bacteria has undergone remarkable advances during last ten years due to the development of efficient systems for recombination. The new protocols especially that based on λ Red recombineering replaced older tedious techniques. Many sophisticated protocols were described, which enable to construct in frame deletions, reporter fusions and scar-less modifications of selected chromosomal genes as well as great genome scale deletions and insertions. These methods are well suited for high-throughput functional studies of uncharacterized open reading frames in newly sequenced genomes. At the same time, they have a great biotechnological potential in construction of new strains used for heterologous protein expression, live vaccines and in vivo cloning. Acknowledgments. This publication is the result of project implementation: Development of the centre of excellence for utilization of information on bio-macromolecules in disease prevention and in improvement of quality of life (ITMS 26240120027) supported by the Research & Development Operational Program funded by the ERDF. The work was also supported by the grant of Slovak grant agency VEGA ...
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We have developed a new λ Red recombineering methodology for generating transient selection markers that can be used to transfer mutations between bacterial strains of both Escherichia coli and Salmonella enterica. The method is fast, simple and allows for the construction of strains with several mutations without any unwanted sequence changes (sca...
Citations
... The cointegrate is resolved by a second single crossover "out." Only the cells with insertion can survive under selective conditions (Madyagol et al., 2011). The main concern with this technique is its low recurrence, while the addition and erasure leads to weakening the fitness of the cell and recurrence eventually regenerates the wild type genotype. ...
... The plasmid pET28a and the fimA gene were ligated with the restriction enzymes Xba I and Nco I using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). Red homologous recombination was used in the fimA deletion strain of E. coli W1688 (Datsenko and Wanner, 2000;Madyagol et al., 2011). Briefly, the gene of the kanamycin resistance sequence and homologous regions (about 50 bp) were combined by PCR. ...
The biofilm (BF) provides favorable growth conditions to cells, which has been exploited in the field of industrial biotechnology. Based on our previous research works on type I fimbriae for the biosynthesis of L-threonine (LT) in Escherichia coli, in this study, a fimA-overexpressing strain was engineered, which improved BF formation under industrial fermentation conditions. The morphological observation and characterization of BF formation were conducted to verify the function of the subunit FimA. However, it was not suitable for repeated-batch immobilized fermentation as the LT titer was not elevated significantly. The underlying molecular mechanisms of BF formation and the LT carbon flux were explored by transcriptomic analysis. The results showed that fimA regulated E. coli BF formation but affected LT carbon distribution. This study will stimulate thoughts about how the fimbriae gene regulated biofilms and amino acid excretion and will bring some consideration and provide a reference for the development of BF-based biomanufacturing processes in E. coli.
... Genes in E. coli W3110 were deleted using a Red/ ET recombination system [37]. The deletion cassettes were prepared by PCR amplification, and pKD46 was transformed into E. coli W3110. ...
Background
In recent years, there has been a growing demand for microbial production of trans -4-hydroxy-L-proline ( t 4Hyp), which is a value-added amino acid and has been widely used in the fields of medicine, food, and cosmetics. In this study, a multivariate modular metabolic engineering approach was used to remove the bottleneck in the synthesis pathway of t 4Hyp.
Results
Escherichia coli t 4Hyp synthesis was performed using two modules: a α-ketoglutarate (α-KG) synthesis module (K module) and L-proline synthesis with hydroxylation module (H module). First, α-KG attrition was reduced, and then, L-proline consumption was inhibited. Subsequently, to improve the contribution to proline synthesis with hydroxylation, optimization of gene overexpression, promotor, copy number, and the fusion system was performed. Finally, optimization of the H and K modules was performed in combination to balance metabolic flow. Using the final module H1K4 in a shaking flask culture, 8.80 g/L t 4Hyp was produced, which was threefold higher than that produced by the W0 strain.
Conclusions
These strategies demonstrate that a microbial cell factory can be systematically optimized by modular engineering for efficient production of t 4Hyp.
... Considerable research efforts have been devoted to developing techniques for synthetic integrations (21)(22)(23). The two tools most widely used in prokaryotes for targeted integration of constructs are homologous recombination by expression of the RecET or -Red proteins (24)(25)(26) and site-specific recombination (27). ...
Chromosomal integration of recombinant genes is desirable compared with expression from plasmids due to increased stability, reduced cell-to-cell variability, and elimination of the need for antibiotics for plasmid maintenance. Here, we present a new approach for tuning pathway gene expression levels via random integration and high-throughput screening. We demonstrate multiplexed gene integration and expression-level optimization for isobutanol production in Escherichia coli . The integrated strains could, with far lower expression levels than plasmid-based expression, produce high titers (10.0 ± 0.9 g/liter isobutanol in 48 hours) and yields (69% of the theoretical maximum). Close examination of pathway expression in the top-performing, as well as other isolates, reveals the complexity of cellular metabolism and regulation, underscoring the need for precise optimization while integrating pathway genes into the chromosome. We expect this method for pathway integration and optimization can be readily extended to a wide range of pathways and chassis to create robust and efficient production strains.
... The copyright holder for this preprint (which this version posted July 29, 2020. . https://doi.org/10.1101/2020.07.29.226290 doi: bioRxiv preprint Significant research efforts have been devoted to developing techniques for synthetic integrations [22][23][24] . The two tools most widely used in prokaryotes for targeted integration of constructs are homologous recombination by expression of the RecET or λ-Red proteins [25][26][27] and site-specific recombination 28 . ...
Chromosomal integration of recombinant genes is desirable compared to expression from plasmids due to increased stability, reduced cell-to-cell variability, and the elimination of antibiotics for plasmid maintenance. Here, we present a new approach for tuning pathway gene expression levels via random integrations followed by high-throughput screening. We demonstrate multiplexed pathway gene integration and optimization of expression levels for isobutanol production in Escherichia coli. The integrated strains could, with significantly lower expression levels than observed from multicopy plasmid-based expression, produce high titers (up to 10.0 ± 0.9 g/L isobutanol in 48 h) and yields (up to 69 % of the theoretical maximum). Close examination of pathway gene expression in the top-performing, as well as other isolates, reveals the complexity of cellular metabolism and regulation, underscoring the need for precise optimization while integrating pathway genes into the chromosome. This new method for multiplexed pathway gene integration and expression optimization could be readily extended and applied to a wide range of pathways and chassis to create robust and efficient production strains.
... Although the efficiency of elimination of the antibiotic resistance gene is high (more than 80% cells lost the antibiotic resistance gene in genome), more than 80-100 nt scar, which included an FRT site, would be left behind the disrupted genes. After more than five times of chromosomal recombination experiments, the scars as mentioned above present in the host chromosome can influence expression of surrounding genes and caused the failure to achieve continuous knockout or integration experiments (Madyagol et al., 2011). To avoid this shortage, two rounds of recombination were designed and carried out Abbreviations: Ap R , ampicillin resistance gene; Cm R , chloramphenicol resistance gene; E. coli, Escherichia coli; IPTG, isopropyl-β-D-thiogalactoside; Km R , kanamycin resistance gene; TyrRS, tyrosyl-tRNA synthetase; UAA, unnatural amino acid; UCM, UAA conditional selectable marker. ...
Genetic manipulations including chromosome engineering are essential techniques used to restructure cell metabolism. Lambda/Red (λ/Red)-mediated recombination is the most commonly applied approach for chromosomal modulation in Escherichia coli. However, the efficiency of this method is significantly hampered by the laborious removal of the selectable markers. To overcome the problem, the integration helper plasmid was constructed, pSBC1a-CtR, which contains Red recombinase, Cre recombinase, and exogenous orthogonal aminoacyl-transfer RNA (tRNA) synthetase/tRNA pairs, allows an unnatural amino acid (UAA) to be genetically encoded at the defined site of the antibiotic resistance gene-encoded protein. When UAAs are not in the culture medium, there was no expression in the antibiotic resistance gene-encoded protein. Accordingly, the next procedure of antibiotic gene excising is not needed. To verify this method, poxB gene was knocked out successfully. Furthermore, sequential deletion of three target genes (galR, ptsG, and pgi) was able to generate neurosporene-producing strain marked by high growth rate. Thus, the site-specific incorporation UAA mutagenesis system were used to control and expand the use of conditional selectable marker, and the technique is used to facilitate a rapid continuous genome editing in Escherichia coli.
... The final engineered strain was named E. coli W1688-fimH * with Kanamycin resistance for screening. On the other hand, fimH from E. coli W1688 was deleted by Red homologous recombination, resulting in an E. coli W1688-fimH (Madyagol et al., 2011). Briefly, a PCR-generated Kanamycin resistance marker was used as knock-in DNA fragment. ...
Biofilms provide cells favorable growth conditions, which have been exploited in industrial biotechnological processes. However, industrial application of the biofilm has not yet been reported in Escherichia coli, one of the most important platform strains, though the biofilm has been extensively studied for pathogenic reasons. Here, we engineered E. coli by overexpressing the fimH gene, which successfully enhanced its biofilm formation under industrial aerobic cultivation conditions. Subsequently, a biofilm-based immobilized fermentation strategy was developed. L-threonine production was increased from 10.5 to 14.1 g/L during batch fermentations and further to 17.5 g/L during continuous (repeated-batch) fermentations with enhanced productivities. Molecular basis for the enhanced biofilm formation and L-threonine biosynthesis was also studied by transcriptome analysis. This study goes beyond the conventional research focusing on pathogenic aspects of E. coli biofilm and represents a successful application case of engineered E. coli biofilm to industrial processes.
... These two plasmids were used to transform the expression strain E. coli BL21(DE3), resulting in strains BL21(DE3)/pET24a-pta and BL21(DE3)/pET24a-pta1. Genetic manipulations were performed in mutant alleles by using a two-step scarless gene replacement technique employing λ Red recombination [21,22]. For scarless gene replacement, plasmid pMD-SK was created by cloning two separate PCR products, kan and sacB, into the plasmid vector pMD18-T. ...
Escherichia coli FB-04(pta1), a recombinant l-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (l-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key l-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher l-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, l-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g⁻¹ during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.
... Antibiotic-resistant marker genes such as neo, bla, cat, and tetA [11][12][13][14][15] are used to select the recombinants, whereas SceI, sacB, rpsL, tolC, galK, and thyA [16][17][18] have been used to select for positive-marker, gene-free clones; PCR-generated mutations in these negative-selection markers lead to the loss of their critical function, resulting in the selection of false-positive clones [16][17][18][19]. A dual selection system using a single gene for both positive and negative selection is considered as a more promising strategy in selecting desired recombinants because of its double functionality and elimination of inactive-marker genes caused by non-specific mutations [15,20]. TetA exports tetracycline from bacterial cells, conferring resistance to the antibiotic tetracycline [21][22][23], and pumps cadmium [24] and nickel [25] cations into the cell, resulting in cell death. ...
The ability to precisely and seamlessly modify a target genome is needed for metabolic engineering and synthetic biology techniques aimed at creating potent biosystems. Herein, we report on a promising method in Escherichia coli that relies on the insertion of an optimized tetA dual selection cassette followed by replacement of the same cassette with short, single-stranded DNA (oligos) or long, double-stranded DNA and the isolation of recombinant strains by negative selection using NiCl2. This method could be rapidly and successfully used for genome engineering, including deletions, insertions, replacements, and point mutations, without inactivation of the methyl-directed mismatch repair (MMR) system and plasmid cloning. The method we describe here facilitates positive genome-edited recombinants with selection efficiencies ranging from 57 to 92%. Using our method, we increased lycopene production (3.4-fold) by replacing the ribosome binding site (RBS) of the rate-limiting gene (dxs) in the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway with a strong RBS. Thus, this method could be used to achieve scarless, proficient, and targeted genome editing for engineering E. coli strains. © 2017 Ryu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
... Therefore, the ability to precisely clone large chromosomal fragments and insert them directly into specific loci in multiple copies is a recurrent need. To achieve this goal, different techniques have been developed, as reviewed, for example, in (Karas et al., 2015;Madyagol et al., 2011). ...
Despite the abundance of genetic manipulation approaches, particularly for Escherichia coli, new techniques and increased flexibility in the application of existing techniques are required to address novel aims. The most widely used approaches for chromosome editing are based on bacteriophage site-specific and λRed/RecET-mediated homologous recombination. In the present study, these techniques were combined to develop a novel approach for in vivo cloning and targeted long-length chromosomal insertion. This approach permits direct λRed-mediated cloning of DNA fragment with lengths of 10kb or greater from the E. coli chromosome into the plasmid vector pGL2, which carries the ori of pSC101, the ϕ80-attP site of ϕ80 phage, and an excisable Cm(R) marker bracketed by λ-attL/attR sites. In pGL2-based recombinant plasmids, the origin of replication can be eliminated in vitro via hydrolysis by SceI endonuclease and recircularization by DNA ligase. The resulting ori-less circular recombinant DNA can be used for targeted insertion of the cloned sequence into the chromosome at a selected site via ϕ80 phage-specific integrase-mediated recombination using the Dual-In/Out approach (Minaeva et al., 2008). At the final stage of chromosomal editing, the Cm(R)-marker can be excised from the chromosome due to expression of the λint/xis genes. Notably, the desired fragment can be inserted as multiple copies in the chromosome by combining insertions at different sites in one strain using the P1 general transduction technique (Moore, 2011). The developed approach is useful for the construction of plasmidless, markerless recombinant strains for fundamental and industrial purposes.
