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Serine integrases are phage- (or mobile element-) encoded enzymes that catalyse site-specific recombination reactions between a short DNA sequence on the phage genome (attP) and a corresponding host genome sequence (attB), thereby integrating the phage DNA into the host genome. Each integrase has its unique pair of attP and attB sites, a feature that allows them to be used as orthogonal tools for genome modification applications. In the presence of a second protein, the Recombination Directionality Factor (RDF), integrase catalyses the reverse excisive reaction, generating new recombination sites, attR and attL. In addition to promoting attR x attL reaction, the RDF inhibits attP x attB recombination. This feature makes the directionality of integrase reactions programmable, allowing them to be useful for building synthetic biology devices. In this report, we describe the degree of orthogonality of both integrative and excisive reactions for three related integrases (ϕC31, ϕBT1, and TG1) and their RDFs. Among these, TG1 integrase is the most active, showing near complete recombination in both attP x attB and attR x attL reactions, and the most directional in the presence of its RDF. Our findings show that there is varying orthogonality among these three integrases – RDF pairs. ϕC31 integrase was the least selective, with all three RDFs activating it for attR x attL recombination. Similarly, ϕC31 RDF was the least effective among the three RDFs in promoting the excisive activities of the integrases, including its cognate ϕC31 integrase. ϕBT1 and TG1 RDFs were noticeably more effective than ϕC31 RDF at inhibiting attP x attB recombination by their respective integrases, making them more suitable for building reversible genetic switches. AlphaFold-Multimer predicts very similar structural interactions between each cognate integrase – RDF pair. The binding surface on the RDF is much more conserved than the binding surface on the integrase, an indication that specificity is determined more by the integrase than the RDF. Overall, the observed weak integrase/RDF orthogonality across the three enzymes emphasizes the need for identifying and characterizing more integrase – RDF pairs. Additionally, the ability of a particular integrase's preferred reaction direction to be controlled to varying degrees by non-cognate RDFs provides a path to tunable, non-binary genetic switches. [ABSTRACT FROM AUTHOR]

Abstract Serine integrases are phage- (or mobile element-) encoded enzymes that catalyse site-specific recombination reactions between a short DNA sequence on the phage genome (attP) and a corresponding host genome sequence (attB), thereby integrating the phage DNA into the host genome. Each integrase has its unique pair of attP and attB sites, a feature that allows them to be used as orthogonal tools for genome modification applications. In the presence of a second protein, the Recombination Directionality Factor (RDF), integrase catalyses the reverse excisive reaction, generating new recombination sites, attR and attL. In addition to promoting attR x attL reaction, the RDF inhibits attP x attB recombination. This feature makes the directionality of integrase reactions programmable, allowing them to be useful for building synthetic biology devices. In this report, we describe the degree of orthogonality of both integrative and excisive reactions for three related integrases (ϕC31, ϕBT1, and TG1) and their RDFs. Among these, TG1 integrase is the most active, showing near complete recombination in both attP x attB and attR x attL reactions, and the most directional in the presence of its RDF. Our findings show that there is varying orthogonality among these three integrases – RDF pairs. ϕC31 integrase was the least selective, with all three RDFs activating it for attR x attL recombination. Similarly, ϕC31 RDF was the least effective among the three RDFs in promoting the excisive activities of the integrases, including its cognate ϕC31 integrase. ϕBT1 and TG1 RDFs were noticeably more effective than ϕC31 RDF at inhibiting attP x attB recombination by their respective integrases, making them more suitable for building reversible genetic switches. AlphaFold-Multimer predicts very similar structural interactions between each cognate integrase – RDF pair. The binding surface on the RDF is much more conserved than the binding surface on the integrase, an indication that specificity is determined more by the integrase than the RDF. Overall, the observed weak integrase/RDF orthogonality across the three enzymes emphasizes the need for identifying and characterizing more integrase – RDF pairs. Additionally, the ability of a particular integrase’s preferred reaction direction to be controlled to varying degrees by non-cognate RDFs provides a path to tunable, non-binary genetic switches.

Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and affordability have always been considered. The aim of this study, on the one hand, is to simplify gene cloning by skipping in vitro assembly reactions and, on the other hand, to reduce costs by eliminating relatively expensive materials. We investigated a cloning system using Escherichia coli harboring two plasmids, pLP-AmpR and pScissors-CmR. The pLP-AmpR contains a landing pad (LP) consisting of two genes (λ int and λ gam) that allow the replacement of the transformed linear DNA using site-specific recombination. After the replacement process, the inducible expressing SpCas9 and specific sgRNA from the pScissors-CmR (CRISPR/Cas9) vector leads to the removal of non-recombinant pLP-AmpR plasmids. The function of LP was explored by directly transforming PCR products. The pScissors-CmR plasmid was evaluated for curing three vectors, including the origins of pBR322, p15A, and pSC101. Replacing LP with a PCR product and fast-eradicating pSC101 origin-containing vectors was successful. Recombinant colonies were confirmed following gene replacement and plasmid curing processes. The results made us optimistic that this strategy may potentially be a simple and inexpensive cloning method. Key points: •The in vivo cloning was performed by replacing the target gene with the landing pad. •Fast eradication of non-recombinant plasmids was possible by adapting key vectors. •This strategy is not dependent on in vitro assembly reactions and expensive materials. [ABSTRACT FROM AUTHOR]

Through their involvement in the integration and excision of a large number of mobile genetic elements, such as phages and integrative and conjugative elements (ICEs), site‐specific recombination systems based on heterobivalent tyrosine recombinases play a major role in genome dynamics and evolution. However, despite hundreds of these systems having been identified in genome databases, very few have been described in detail, with none from phages that infect Bacillota (formerly Firmicutes). In this study, we reanalyzed the recombination module of Lactobacillus delbrueckii subsp. bulgaricus phage mv4, previously considered atypical compared with classical systems. Our results reveal that mv4 integrase is a 369 aa protein with all the structural hallmarks of recombinases from the Tn916 family and that it cooperatively interacts with its recombination sites. Using randomized DNA libraries, NGS sequencing, and other molecular approaches, we show that the 21‐bp core‐attP and attB sites have structural similarities to classical systems only if considering the nucleotide degeneracy, with two 7‐bp inverted regions corresponding to mv4Int core‐binding sites surrounding a 7‐bp strand‐exchange region. We also examined the different compositional constraints in the core‐binding regions, which define the sequence space of permissible recombination sites. [ABSTRACT FROM AUTHOR]

The site‐specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that integrate and excise the vial chromosome into and out of the bacterial chromosome. The reactions require 240 bp of phage DNA and 21 bp of bacterial DNA comprising 16 protein binding sites that are differentially used in each pathway by the phage‐encoded Int and Xis proteins and the host‐encoded integration host factor and factor for inversion stimulation proteins. Structures of higher‐order protein–DNA complexes of the four‐way Holliday junction recombination intermediates provided clarifying insights into the mechanisms, directionality, and regulation of these two pathways, which are tightly linked to the physiology of the bacterial host cell. Here we review our current understanding of the mechanisms responsible for regulating and executing λ site‐specific recombination, with an emphasis on key studies completed over the last decade. [ABSTRACT FROM AUTHOR]

Abstract We introduce DEQSeq, a nanopore sequencing approach that rationalizes the selection of favorable genome editing enzymes from directed molecular evolution experiments. With the ability to capture full-length sequences, editing efficiencies, and specificities from thousands of evolved enzymes simultaneously, DEQSeq streamlines the process of identifying the most valuable variants for further study and application. We apply DEQSeq to evolved libraries of Cas12f-ABEs and designer-recombinases, identifying variants with improved properties for future applications. Our results demonstrate that DEQSeq is a powerful tool for accelerating enzyme discovery and advancing genome editing research.

Bacillus cereus biovar anthracis (Bcbva) is an untypical pathogen causing a fatal anthrax-like disease in a variety of wildlife species in African rainforest areas. In contrast to Bacillus anthracis and most species of the B. cereus group, all strains of the Bcbva cluster contain a 22 kb insertion in the sigK gene which encodes the essential late sporulation sigma factor σK. This insertion is excised during sporulation in a site-specific recombination process resulting in an intact sigK gene and a circular molecule. The sporulation kinetics of two strains each of Bcbva and B. anthracis were compared by the expression analysis of eight sporulation-associated genes, including sigK, using reverse transcriptase quantitative real-time PCR. In addition, morphological sporulation stages were analyzed and quantified by electron microscopy. Our results indicated that the necessary excision of the insertion in Bcbva neither delayed nor inhibited its sporulation. In two spontaneous mutants of Bcbva, the excision of the sigK insertion and sporulation were impeded due to mutations in the spo0A and spoVG regulator genes, respectively. The spo0A frameshift mutation was overcome by intragenic suppression in a revertant which was able to sporulate normally, despite an M171S amino acid exchange in the global regulator Spo0A. A screening of the NCBI database identified further strains of the B. cereus group which possess unrelated insertions in the sigK gene, and two strains containing almost identical insertions at the same gene position. Some of the sigK insertions encode putative prophages, whereas the Bcbva insertion encoded a type I restriction–modification system. The function of these insertions and if they are possibly essential for sporulation remains to be assessed. [ABSTRACT FROM AUTHOR]

Background: Graves' disease (GD), one of the most common forms of autoimmune thyroid disorders, is characterized by hyperthyroidism caused by antibodies (Abs) against the extracellular A-subunit of the thyrotropin receptor (TSHR). Various approaches have been used to create mouse models of GD, including transfected fibroblasts and immunization with plasmids or adenoviruses expressing human TSHR A-subunit (hTSHR A-subunit). These models, however, require repeated immunization and produce inconsistent results. In this study, we established a novel Cre-loxP system-based mouse model that is able to generate the hTSHR A-subunit, mimicking human GD, and characterized the histological changes in Graves' orbitopathy (GO) progression after a single injection. Materials and Methods: A Cre-loxP system-based mouse model was constructed by inserting the CAG-loxP-STOP-loxP-hTSHR A-subunit cassette into the Rosa26 locus of the mouse genome. Conditional expression of the hTSHR A-subunit was successfully achieved by intramuscular injection of the transactivator of transcription-Cre recombinase (GD mice). Blood tests for anti-TSHR Abs and the total thyroxine (T4) level were performed. Magnetic resonance imaging (MRI) was used to monitor morphological changes in the eyes. A histological examination of the thyroid gland and retrobulbar tissues was performed to observe pathological changes. Results: Twenty-four (8 control and 16 GD) mice were investigated. All GD mice exhibited higher levels of TSHR Abs compared with the control group. Moreover, more than 80% of the mouse models showed elevated T4 levels accompanied by thyroid goiter. MRI analysis revealed an increased volume of retrobulbar tissue, while immunohistochemical staining of orbital tissues exhibited macrophage infiltration and muscle fibrosis in the GD mice, contrasting with the control group. Conclusions: Our novel mouse model for GD, which showed the histological features of GO, was successfully established using the Cre-loxP system. This animal model offers improved insights and contributes to advancing methodological developments for GD and GO. [ABSTRACT FROM AUTHOR]

During the development of traditional Chinese hamster ovary (CHO) cell lines, target genes randomly integrate into the genome upon entering the nucleus, resulting in unpredictable productivity of cell clones. The characterization and screening of high-yielding cell lines is a time-consuming and expensive process. Site-specific integration is recognized as an effective approach for overcoming random integration and improving production stability. We have designed a multifunctional expression cassette, called CDbox, which can be manipulated by the site-specific recombination systems Cre/lox and Dre/rox. The CDbox expression cassette was inserted at the Hipp11(H11) locus hotspot in the CHO-K1 genome using CRISPR/Cas9 technology, and a compliant CHO-CDbox cell platform was screened and obtained. The CHO-CDbox cell platform was transformed into a pool of EGFP-expressing cells using Cre/lox recombinase-mediated cassette exchange (RMCE) in only 2 weeks, and this expression remained stable for at least 75 generations without the need for drug stress. Subsequently, we used the Dre/rox system to directly eliminate the EGFP gene. In addition, two practical applications of the CHO-CDbox cell platform were presented. The first was the quick construction of the Pembrolizumab antibody stable expression strain, while the second was a protocol for the integration of surface-displayed and secreted antibodies on CHO cells. The previous research on site-specific integration of CHO cells has always focused on the single functionality of insertion of target genes. This newly developed CHO cell platform is expected to offer expanded applicability for protein production and gene function studies.

Serine integrases are emerging as one of the most powerful biological tools for biotechnology. Over the past decade, many research papers have been published on the use of serine integrases in synthetic biology. In this review, we aim to systematically summarize the various studies ranging from structure and the catalytic mechanism to genetic design and interdisciplinary applications. First, we introduce the classification, structure, and catalytic model of serine integrases. Second, we present a timeline with milestones that describes the representative achievements. Then, we summarize the applications of serine integrases in genome engineering, genetic design, and DNA assembly. Finally, we discuss the potential of serine integrases for advancing interdisciplinary research. We anticipate that serine integrases will be further expanded as a versatile genetic toolbox for synthetic biology applications. [ABSTRACT FROM AUTHOR]

Single‐molecule techniques have been demonstrated as powerful tools to investigate individual protein‐DNA interactions that are difficult to access by conventional biochemical techniques. These methods are popularly used to obtain valuable and detailed molecular mechanisms of enzyme functions. Currently, we have used single‐molecule tethered particle motion to investigate protein‐DNA interactions, including homologous recombination, site‐specific recombination, DNA package, and the nucleosome remodeling process, and useful information was obtained. Here, we will describe the experimental designs and present the information obtained. [ABSTRACT FROM AUTHOR]

Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories. For some uncommon microbial hosts, such as Mycolicibacterium and Mycobacterium species, however, it is a challenge. Here, we present a multiplexed integrase-assisted site-specific recombination (miSSR) method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories. First, a single-step multi-copy integration method was established in M. neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy, the efficiencies of which were ∼100% for no more than three-copy integration events and decreased sharply to ∼20% for five-copy integration events. Second, the R4, Bxb1 and ΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events. Third, a reconstructed mycolicibacterial Xer recombinases (Xer-cise) system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation. As a proof of concept, the biosynthetic pathway of ergothioneine (EGT) in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system. With six copies of the biosynthetic gene clusters (BGCs) of EGT and pentose phosphate isomerase (PRT), the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L, which was 3.77 times of that in the wild strain. The improvements indicated that the miSSR system was an effective, flexible, and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.

Site-specific recombinases (integrases) can mediate the horizontal transfer of genomic islands. The ability to integrate large DNA sequences into target sites is very important for genetic engineering in prokaryotic and eukaryotic cells. Here, we characterized an unprecedented catalogue of 530 tyrosine-type integrases by examining genes potentially encoding tyrosine integrases in bacterial genomic islands. The phylogeny of putative tyrosine integrases revealed that these integrases form an evolutionary clade that is distinct from those already known and are affiliated with novel integrase groups. We systematically searched for candidate integrase genes, and their integration activities were validated in a bacterial model. We verified the integration functions of six representative novel integrases by using a two-plasmid integration system consisting of a donor plasmid carrying the integrase gene and attP site and a recipient plasmid harboring an attB site in recA-deficient Escherichia coli. Further quantitative reverse transcription-PCR (qRT-PCR) assays validated that the six selected integrases can be expressed with their native promoters in E. coli. The attP region reductions showed that the extent of attP sites of integrases is approximately 200 bp for integration capacity. In addition, mutational analysis showed that the conserved tyrosine at the C terminus is essential for catalysis, confirming that these candidate proteins belong to the tyrosine-type recombinase superfamily, i.e., tyrosine integrases. This study revealed that the novel integrases frombacterial genomic islands have site-specific recombination functions, which is of physiological significance for their genomic islands in bacterial chromosomes. More importantly, our discovery expands the toolbox for genetic engineering, especially for efficient integration activity. [ABSTRACT FROM AUTHOR]

Bacillus cereus biovar anthracis (Bcbva) is an untypical pathogen causing a fatal anthrax-like disease in a variety of wildlife species in African rainforest areas. In contrast to Bacillus anthracis and most species of the B. cereus group, all strains of the Bcbva cluster contain a 22 kb insertion in the sigK gene which encodes the essential late sporulation sigma factor σK. This insertion is excised during sporulation in a site-specific recombination process resulting in an intact sigK gene and a circular molecule. The sporulation kinetics of two strains each of Bcbva and B. anthracis were compared by the expression analysis of eight sporulation-associated genes, including sigK, using reverse transcriptase quantitative real-time PCR. In addition, morphological sporulation stages were analyzed and quantified by electron microscopy. Our results indicated that the necessary excision of the insertion in Bcbva neither delayed nor inhibited its sporulation. In two spontaneous mutants of Bcbva, the excision of the sigK insertion and sporulation were impeded due to mutations in the spo0A and spoVG regulator genes, respectively. The spo0A frameshift mutation was overcome by intragenic suppression in a revertant which was able to sporulate normally, despite an M171S amino acid exchange in the global regulator Spo0A. A screening of the NCBI database identified further strains of the B. cereus group which possess unrelated insertions in the sigK gene, and two strains containing almost identical insertions at the same gene position. Some of the sigK insertions encode putative prophages, whereas the Bcbva insertion encoded a type I restriction–modification system. The function of these insertions and if they are possibly essential for sporulation remains to be assessed.

Genetic tools for targeted modification of the mycobacterial genome contribute to the understanding of the physiology and virulence mechanisms of mycobacteria. Human and animal pathogens, such as the Mycobacterium tuberculosis complex, which causes tuberculosis, and M. leprae, which causes leprosy, are of particular importance. Genetic research opens up novel opportunities to identify and validate new targets for antibacterial drugs and to develop improved vaccines. Although mycobacteria are difficult to work with due to their slow growth rate and a limited possibility to transfer genetic information, significant progress has been made in developing genetic engineering methods for mycobacteria. The review considers the main approaches to changing the mycobacterial genome in a targeted manner, including homologous and site-specific recombination and use of the CRISPR/Cas system. [ABSTRACT FROM AUTHOR]

Site-specific recombination systems are widely used as bioengineering tools. However, the traditional site-specific recombination system requires a consensus sequence for the specific site. Such sequence-level constraints limit effective recombination between sites. Therefore, in order to develop an efficient site-specific recombination system, we investigated the attC site of the bacterial integration subsystem and built a predictive model to infer the important features that contribute to recombination. Here, we design an attC site prediction algorithm based on a combination optimisation strategy. Based on the structural features of attC sites, the prediction algorithm realises the high-precision prediction of the recombination frequencies between sites and the screening of the top 20 important features that play a role in recombination, which are effective for improving the design method of attC sites. The algorithm has better portability and higher prediction accuracy compared with the existing advanced algorithms, among which the Pearson correlation coefficient is 0.87, explained variance score is 0.73, root mean square error is 0.006 and mean absolute error is 0.041. This can not only provide ideas for the research of efficient recombination systems but also provide a theoretical basis for developing genetic engineering further. [ABSTRACT FROM AUTHOR]

Sinorhizobium meliloti is a free-living soil Gram-negative bacterium that participates in nitrogen-fixation symbiosis with several legumes. S. meliloti has the potential to be utilized for the production of high-value nutritional compounds, such as vitamin B 12 . Advances in gene editing tools play a vital role in the development of S. meliloti strains with enhanced characteristics for biotechnological applications. Several novel genetic engineering strategies have emerged in recent years to investigate genetic modifications in S. meliloti . This review provides a comprehensive overview of the mechanism and application of the extensively used Tn5-mediated genetic engineering strategies. Strategies based on homologous recombination and site-specific recombination were also discussed. Subsequently, the development and application of the genetic engineering strategies utilizing various CRISPR/Cas systems in S. meliloti are summarized. This review may stimulate research interest among scientists, foster studies in the application areas of S. meliloti , and serve as a reference for the utilization of genome editing tools for other Rhizobium species.

ABSTRACT In bacteria, the mechanisms used to repair DNA lesions during genome replication include homologous recombination between sister chromosomes. This can lead to the formation of chromosome dimers if an odd number of crossover events occurs. The dimers must be resolved before cell separation to ensure genomic stability and cell viability. Dimer resolution is achieved by the broadly conserved dif/Xer system, which catalyzes one additional crossover event immediately prior to cell separation. While dif/Xer systems have been characterized or predicted in the vast majority of proteobacteria, no homologs to dif or xer have been identified in the order Legionellales. Here, we report the discovery of a distinct single-recombinase dif/Xer system in the intracellular pathogen Legionella pneumophila. The dif site was uncovered by our analysis of Legionella mobile element-1 (LME-1), which harbors a dif site mimic and integrates into the L. pneumophila genome via site-specific recombination. We demonstrate that lpg1867 (here named xerL) encodes a tyrosine recombinase that is necessary and sufficient for catalyzing recombination at the dif site and that deletion of dif or xerL causes filamentation along with extracellular and intracellular growth defects. We show that the dif/XerL system is present throughout Legionellales and that Coxiella burnetii XerL and its cognate dif site can functionally substitute for the native system in L. pneumophila. Finally, we describe an unexpected link between C. burnetii dif/Xer and the maintenance of its virulence plasmids. IMPORTANCE The maintenance of circular chromosomes depends on the ability to resolve aberrant chromosome dimers after they form. In most proteobacteria, broadly conserved Xer recombinases catalyze single crossovers at short, species-specific dif sites located near the replication terminus. Chromosomal dimerization leads to the formation of two copies of dif within the same molecule, leading to rapid site-specific recombination and conversion back into chromosome monomers. The apparent absence of chromosome dimer resolution mechanisms in Legionellales has been a mystery to date. By studying a phage-like mobile genetic element, LME-1, we have identified a previously unknown single-recombinase dif/Xer system that is not only widespread across Legionellales but whose activity is linked to virulence in two important human pathogens.

Escherichia coli K-12 is one of the most well-studied species of bacteria. This species, however, is much more difficult to modify by homologous recombination (HR) than other model microorganisms. Research on HR in E. coli has led to a better understanding of the molecular mechanisms of HR, resulting in technical improvements and rapid progress in genome research, and allowing whole-genome mutagenesis and large-scale genome modifications. Developments using λ Red (exo, bet, and gam) and CRISPR-Cas have made E. coli as amenable to genome modification as other model microorganisms, such as Saccharomyces cerevisiae and Bacillus subtilis. This review describes the history of recombination research in E. coli, as well as improvements in techniques for genome modification by HR. This review also describes the results of large-scale genome modification of E. coli using these technologies, including DNA synthesis and assembly. In addition, this article reviews recent advances in genome modification, considers future directions, and describes problems associated with the creation of cells by design. [ABSTRACT FROM AUTHOR]

In the worldwide health threat posed by antibiotic-resistant bacterial pathogens, mobile genetic elements (MGEs) play a critical role in favoring the dissemination of resistance genes. Among them, the genomic island Glsul2 and the ISCR-related element CR2-sul2 unit are believed to participate in this dissemination. However, the mobility of the two elements has not yet been demonstrated. Here, we found that the Glsul2 and CR2-sul2 units can excise from the host chromosomal attachment site (attB) in Shigella flexneri. Through establishing a two-plasmid mobilization system composed of a donor plasmid bearing the Glsul2 and a trap plasmid harboring the attB in recA-deficient Escherichia coli, we reveal that the integrase of Glsul2 can perform the excision and integration of Glsul2 and CR2-sul2 unit by site-specific recombination between att core sites. Furthermore, we demonstrate that the integrase and the att sites are required for mobility through knockout experiments. Our findings provide the first experimental characterization of the mobility of Glsul2 and CR2-sul2 units mediated by integrase. They also suggest a potential and unappreciated role of the Glsul2 integrase family in the dissemination of CR2-sul2 units carrying various resistance determinants in between. [ABSTRACT FROM AUTHOR]

Understanding the genetic basis of reproductive isolation and adaptive traits in natural populations is one of the fundamental goals in evolutionary biology. Genome editing technologies based on CRISPR-Cas systems and site-specific recombinases have enabled us to modify a targeted genomic region as desired and thus to conduct functional analyses of target loci, genes and mutations even in non-conventional model organisms. Here, we review the technical properties of genome editing techniques by classifying them into the following applications: targeted gene knock-out for investigating causative gene functions, targeted gene knock-in of marker genes for visualizing expression patterns and protein functions, precise gene replacement for identifying causative alleles and mutations, and targeted chromosomal rearrangement for investigating the functional roles of chromosomal structural variations. We describe examples of their application to demonstrate functional analysis of naturally occurring genetic variations and discuss how these technologies can be applied to speciation and adaptation research. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'. [ABSTRACT FROM AUTHOR]

Vibrio cholerae, the causative agent of the diarrheal disease cholera, is a microbe capable of inhabiting two different ecosystems: chitinous surfaces in brackish, estuarine waters and the epithelial lining of the human gastrointestinal tract. V. cholerae defends against competitive microorganisms with a contact-dependent, contractile killing machine called the type VI secretion system (T6SS) in each of these niches. The T6SS resembles an inverted T4 bacteriophage tail and is used to deliver toxic effector proteins into neighboring cells. Pandemic strains of V. cholerae encode a unique set of T6SS effector proteins, which may play a role in pathogenesis or pandemic spread. In our recent study (Santoriello et al. (2020), Nat Commun, doi: 10.1038/s41467-020-20012-7), using genomic and molecular biology tools, we demonstrated that the T6SS island Auxiliary Cluster 3 (Aux3) is unique to pandemic strains of V. cholerae. We went on to show that Aux3 is related to a phage-like element circulating in environmental V. cholerae strains and that two genetic domestication events formed the pandemic Aux3 cluster during the evolution of the pandemic clone. Our findings support two main conclusions: (1) Aux3 evolution from phage-like element to T6SS cluster offers a snapshot of phage domestication in early T6SS evolution and (2) chromosomal maintenance of Aux3 was advantageous to the common ancestor of V. cholerae pandemic strains.

Genome integration enables host organisms to stably carry heterologous DNA messages, introducing new genotypes and phenotypes for expanded applications. While several genome integration approaches have been reported, a scalable tool for DNA message storage within site-specific genome landing pads is still lacking. Here, we introduce an iterative genome integration method utilizing orthogonal serine integrases, enabling the stable storage of multiple heterologous genes in the chromosome of Escherichia coli MG1655. By leveraging serine integrases TP901-1, Bxb1, and PhiC31, along with engineered integration vectors, we demonstrate high-efficiency, marker-free integration of DNA fragments up to 13 kb in length. To further simplify the procedure, we then develop a streamlined integration method and showcase the system's versatility by constructing an engineered E. coli strain capable of storing and expressing multiple genes from diverse species. Additionally, we illustrate the potential utility of these engineered strains for synthetic biology applications, including in vivo and in vitro protein expression. Our work extends the application scope of serine integrases for scalable gene integration cascades, with implications for genome manipulation and gene storage applications in synthetic biology.

The integration of mobile genetic elements into their host chromosome influences the immediate fate of cellular organisms and gradually shapes their evolution. Site-specific recombinases catalyzing this integration have been extensively characterized both in bacteria and eukarya. More recently, a number of reports provided the in-depth characterization of archaeal tyrosine recombinases and highlighted new particular features not observed in the other two domains. In addition to being active in extreme environments, archaeal integrases catalyze reactions beyond site-specific recombination. Some of these integrases can catalyze low-sequence specificity recombination reactions with the same outcome as homologous recombination events generating deep rearrangements of their host genome. A large proportion of archaeal integrases are termed suicidal due to the presence of a specific recombination target within their own gene. The paradoxical maintenance of integrases that disrupt their gene upon integration implies novel mechanisms for their evolution. In this review, we assess the diversity of the archaeal tyrosine recombinases using a phylogenomic analysis based on an exhaustive similarity network. We outline the biochemical, ecological and evolutionary properties of these enzymes in the context of the families we identified and emphasize similarities and differences between archaeal recombinases and their bacterial and eukaryal counterparts. [ABSTRACT FROM AUTHOR]

Escherichia coli K-12 is one of the most well-studied species of bacteria. This species, however, is much more difficult to modify by homologous recombination (HR) than other model microorganisms. Research on HR in E. coli has led to a better understanding of the molecular mechanisms of HR, resulting in technical improvements and rapid progress in genome research, and allowing whole-genome mutagenesis and large-scale genome modifications. Developments using λ Red (exo, bet, and gam) and CRISPR-Cas have made E. coli as amenable to genome modification as other model microorganisms, such as Saccharomyces cerevisiae and Bacillus subtilis. This review describes the history of recombination research in E. coli, as well as improvements in techniques for genome modification by HR. This review also describes the results of large-scale genome modification of E. coli using these technologies, including DNA synthesis and assembly. In addition, this article reviews recent advances in genome modification, considers future directions, and describes problems associated with the creation of cells by design.

The genus Habenivirus which includes Ralstonia virus ϕRSM encodes a site-specific integrase of a small serine recombinase belonging to the resolvase/invertase family. Here we describe the integrative/excisive recombination reactions mediated by ϕRSM integrase using in vitro assays. The products of attP/attB recombination, i.e. attL and attR, were exactly identical to those found in the prophage ϕRSM in R. solanacearum strains. The minimum size of attB required for integration was determined to be 37 bp, containing a 13 bp core and flanking sequences of 4 bp on the left and 20 bp on the right. ϕRSM integrative recombination proceeds efficiently in vitro in the absence of additional proteins or high-energy cofactors. Excision of a functional phage genome from a prophage fragment was demonstrated in vitro, demonstrating two-way activity of ϕRSM1 integrase. This is the first example of a small serine recombinase from the resolvase/invertase group that functions in integrative and excisive recombination for filamentous phages. This serine integrase could be used as a tool for several genome engineering applications. [ABSTRACT FROM AUTHOR]

Abstract Efficient methods for multigene transformation are important for developing novel crop varieties. Methods based on random integrations of multiple genes have been successfully used for metabolic engineering in plants. However, efficiency of co‐integration and co‐expression of the genes could present a bottleneck. Recombinase‐mediated integration into the engineered target sites is arguably a more efficient method of targeted integration that leads to the generation of stable transgenic lines at a high rate. This method has the potential to streamline multigene transformation for metabolic engineering and trait stacking in plants. Therefore, empirical testing of transgene(s) stability from the multigene site‐specific integration locus is needed. Here, the recombinase technology based on Cre‐lox recombination was evaluated for developing multigenic lines harboring constitutively‐expressed and inducible genes. Targeted integration of a five genes cassette in the rice genome generated a precise full‐length integration of the cassette at a high rate, and the resulting multigenic lines expressed each gene reliably as defined by their promoter activity. The stable constitutive or inducible expression was faithfully transmitted to the progeny, indicating inheritance‐stability of the multigene locus. Co‐localization of two distinctly inducible genes by heat or cold with the strongly constitutive genes did not appear to interfere with each other's expression pattern. In summary, high rate of co‐integration and co‐expression of the multigene cassette installed by the recombinase technology in rice shows that this approach is appropriate for multigene transformation and introduction of co‐segregating traits. Significance Statement Recombinase‐mediated site‐specific integration approach was found to be highly efficacious in multigene transformation of rice showing proper regulation of each gene driven by constitutive or inducible promoter. This approach holds promise for streamlining gene stacking in crops and expressing complex multigenic traits.

Abstract Vector control programs based on population reduction by matings with mass-released sterile insects require the release of only male mosquitoes, as the release of females, even if sterile, would increase the number of biting and potentially disease-transmitting individuals. While small-scale releases demonstrated the applicability of sterile males releases to control the yellow fever mosquito Aedes aegypti, large-scale programs for mosquitoes are currently prevented by the lack of efficient sexing systems in any of the vector species. Different approaches of sexing are pursued, including classical genetic and mechanical methods of sex separation. Another strategy is the development of transgenic sexing systems. Such systems already exist in other insect pests. Genome modification tools could be used to apply similar strategies to mosquitoes. Three major tools to modify mosquito genomes are currently used: transposable elements, site-specific recombination systems, and genome editing via TALEN or CRISPR/Cas. All three can serve the purpose of developing sexing systems and vector control strains in mosquitoes in two ways: first, via their use in basic research. A better understanding of mosquito biology, including the sex-determining pathways and the involved genes can greatly facilitate the development of sexing strains. Moreover, basic research can help to identify other regulatory elements and genes potentially useful for the construction of transgenic sexing systems. Second, these genome modification tools can be used to apply the gained knowledge to build and test mosquito sexing strains for vector control.

• The cellular efficiency of signal relative to coding end joining in V(D)J recombination is not known. • ddPCR permits independent analysis of signal (SJ) and coding joints (CJ) on inversion substrates. • SJ and CJ formation are temporally uncoupled such that one joint can often remain incomplete when the other has formed. • Despite their temporal gap, the overall ratio of SJ/CJ is close to 1 in a human pre-B cell line. In vertebrate antigen receptor gene rearrangement, V(D)J recombination events can occur by deletion or by inversion. For deletional events, the signal joint is deleted from the genome. Nearly half of the immunoglobulin light chain genes undergo V(D)J recombination in an inversional manner, and both signal and coding joint formation must occur to retain chromosomal integrity. But given the undetermined amount of pre-B and pre-T cell death that occurs during V(D)J recombination, the efficiency with which both joints are completed is not known, nor is the relative efficiency (balance) of signal versus coding joint formation. Signal joint formation only requires Ku and XRCC4:DNA ligase 4 of the nonhomologous DNA end joining repair pathway. Coding joint formation requires these proteins as well, but in addition requires Artemis and DNA-dependent protein kinase to open the hairpin DNA coding ends, which the RAG complex generated; and further processing is required because the hairpin opening generates incompatible 3′ overhangs. Mutations in some of the end processing enzymes affect one, but only minimally the other joint. We have devised a precise cellular assay that does not have any cellular, enzymatic or biochemical selective bias to assess signal and coding joint formation independently, and it can detect intermediates for which one joint has formed but not the other. We find that intermediates with only one completed joint are more abundant than molecules with both joints completed. This indicates that either joint can form independent of the other and joint formation can be a relatively slow process. [ABSTRACT FROM AUTHOR]

XerCD‐dif site‐specific recombination is a well characterized system, found in most bacteria and archaea. Its role is resolution of chromosomal dimers that arise from homologous recombination. Xer‐mediated recombination is also used by several plasmids for multimer resolution to enhance stability and by some phage for integration into the chromosome. In the past decade, it has been hypothesized that an alternate and novel function exists for this system in the dissemination of genetic elements, notably antibiotic resistance genes, in Acinetobacter species. Currently the mechanism underlying this apparent genetic mobility is unknown. Multidrug resistant Acinetobacter baumannii is an increasingly problematic pathogen that can cause recurring infections. Sequencing of numerous plasmids from clinical isolates of A. baumannii revealed the presence of possible mobile modules: genes were found flanked by pairs of Xer recombination sites, called plasmid‐dif (pdif) sites. These modules have been identified in multiple otherwise unrelated plasmids and in different genetic contexts suggesting they are mobile elements. In most cases, the pairs of sites flanking a gene (or genes) are in inverted repeat, but there can be multiple modules per plasmid providing pairs of recombination sites that can be used for inversion or fusion/deletion reactions; as many as 16 pdif sites have been seen in a single plasmid. Similar modules including genes for surviving environmental toxins have also been found in strains of Acinetobacter Iwoffi isolated from permafrost cores; this suggests that these mobile modules are an ancient adaptation and not a novel response to antibiotic pressure. These modules bear all the hallmarks of mobile genetic elements, yet, their movement has never been directly observed to date. This review gives an overview of the current state of this novel research field. [ABSTRACT FROM AUTHOR]

Site-specific recombinases are widely used tools for analysis of genetics, development, and cell biology, and many schemes have been devised to alter gene expression by recombinase-mediated DNA rearrangements. Because the FRT and lox target sites for the commonly used FLP and Cre recombinases are asymmetrical, and must pair in the same direction to recombine, construct design must take into account orientation of the target sites. Both direct and inverted configurations have been used. However, the outcome of recombination between target sites on sister chromatids is frequently overlooked. This is especially consequential with inverted target sites, where exchange between oppositely oriented target sites on sisters will produce dicentric and acentric chromosomes. By using constructs that have inverted target sites in Drosophila melanogaster and in mice, we show here that dicentric chromosomes are produced in the presence of recombinase, and that the frequency of this event is quite high. The negative effects on cell viability and behavior can be significant, and should be considered when using such constructs. [ABSTRACT FROM AUTHOR]

Efficient methods for multigene transformation are important for developing novel crop varieties. Methods based on random integrations of multiple genes have been successfully used for metabolic engineering in plants. However, efficiency of co‐integration and co‐expression of the genes could present a bottleneck. Recombinase‐mediated integration into the engineered target sites is arguably a more efficient method of targeted integration that leads to the generation of stable transgenic lines at a high rate. This method has the potential to streamline multigene transformation for metabolic engineering and trait stacking in plants. Therefore, empirical testing of transgene(s) stability from the multigene site‐specific integration locus is needed. Here, the recombinase technology based on Cre‐lox recombination was evaluated for developing multigenic lines harboring constitutively‐expressed and inducible genes. Targeted integration of a five genes cassette in the rice genome generated a precise full‐length integration of the cassette at a high rate, and the resulting multigenic lines expressed each gene reliably as defined by their promoter activity. The stable constitutive or inducible expression was faithfully transmitted to the progeny, indicating inheritance‐stability of the multigene locus. Co‐localization of two distinctly inducible genes by heat or cold with the strongly constitutive genes did not appear to interfere with each other's expression pattern. In summary, high rate of co‐integration and co‐expression of the multigene cassette installed by the recombinase technology in rice shows that this approach is appropriate for multigene transformation and introduction of co‐segregating traits. Significance Statement: Recombinase‐mediated site‐specific integration approach was found to be highly efficacious in multigene transformation of rice showing proper regulation of each gene driven by constitutive or inducible promoter. This approach holds promise for streamlining gene stacking in crops and expressing complex multigenic traits. [ABSTRACT FROM AUTHOR]

• Engineered KO of DNA-PKcs expression impacts V(D)J coding joint formation much more than signal joint formation. • Here, chemical inhibition of DNA-PKcs reduces Artemis endonuclease activity in a purified system. • Cellular V(D)J effects of a DNA-PKcs chemical inhibitor show a reduction in coding joint formation relative to signal joint formation. • The recombination signal DNA end resection observed in DNA-PKcs genetic mutants is not seen with chemical inhibition. Spontaneous DNA-PKcs deficiencies in animals result in a severe combined immunodeficiency (SCID) phenotype because DNA-PKcs is required to activate Artemis for V(D)J recombination coding end hairpin opening. The impact on signal joint formation in these spontaneous mutant mammals is variable. Genetically engineered DNA-PKcs null mice and cells from them show a >1,000-fold reduction in coding joint formation and minimal reduction in signal joint formation during V(D)J recombination. Does chemical inhibition of DNA-PKcs mimic this phenotype? M3814 (also known as Nedisertib) is a potent DNA-PKcs inhibitor. We find here that M3814 causes a quantitative reduction in coding joint formation relative to signal joint formation. The sequences of signal and coding junctions were within normal limits, though rare coding joints showed novel features. The signal junctions generally did not show evidence of resection into the signal ends that is often seen in cells that have genetic defects in DNA-PKcs. Comparison of the chemical inhibition findings here with the known results for spontaneous and engineered DNA-PKcs mutant mammals is informative for considering pharmacologic small molecule inhibition of DNA-PKcs in various types of neoplasia. [ABSTRACT FROM AUTHOR]

The low solubility and aggregation properties of HIV-1 integrase (IN) are major obstacles for biochemical and structural studies. The lens epithelium–derived growth factor (LEDGF) is a cellular factor that binds IN and tethers preintegration complexes to chromatin before integration. The LEDGF also stimulates HIV-1 IN DNA strand transfer activity and improves its solubility in vitro. We show that these properties are conferred by a short peptide spanning residues 178 to 197 of the LEDGF that encompasses its AT-hook DNA-binding elements. The peptide stimulates HIV-1 IN activity both in trans and in cis. Fusion of the peptide to either the N- or C-terminus of IN results in maximal stimulation of concerted integration activity and greatly improves the solubility of the protein and nucleoprotein complexes of IN with viral DNA ends (intasomes). High-resolution structures of HIV-1 intasomes are required to understand the mechanism of IN strand transfer inhibitors (INSTIs), which are front-line drugs for the treatment of HIV-1, and how the virus can develop resistance to INSTIs. We have previously determined the structure of the HIV-1 strand transfer complex intasome. The improved biophysical properties of intasomes assembled with LEDGF peptide fusion IN have enabled us to determine the structure of the cleaved synaptic complex intasome, which is the direct target of INSTIs. Image 1 • Structural studies of the HIV-1 intasomes have been frustrated by aggregation. • Structures of intasomes are needed to understand inhibitor mechanism and resistance. • Fusion of a peptide to HIV-1 integrase (IN) stimulates catalytic activity. • The peptide fusion integrase assembles intasomes with lesser aggregation. • We have used the fusion IN to determine the structure of the HIV-1 cleaved synaptic complex intasome. [ABSTRACT FROM AUTHOR]

Cre recombinase exchanges DNA strands at the LoxP recognition site via transphosphorylation reactions that involve pentacoordinate transition states. We demonstrate that meta-vanadate ion (VO(3)(-)) and appropriate DNA substrates assemble a transition-state analog-like complex in the Cre active site. Meta-vanadate inhibits recombination of LoxP-derived oligonucleotide substrates that contain a gap at either or both scissile phosphates, but does not inhibit reactions with intact LoxP. The 3(')-hydroxyl group of the gapped substrate is required for inhibition, suggesting that vanadate is ligated by three oxo ligands. Assembly of the inhibited complex is slow (t(1/2)=19min at 4mM NaVO(3)) and requires Cre, substrates, and meta-vanadate. Holliday junction intermediates accumulated at lower meta-vanadate concentrations, suggesting that the second strand exchange is inhibited more readily than the first. The apparent K(D) for meta-vanadate is 1.5-2mM and binding shows positive cooperativity. This methodology may have general application for mechanistic studies of recombinase/topoisomerase-mediated strand exchange reactions.

Abstract Background Direct manipulation of the genome is a widespread technique for genetic studies and synthetic biology applications. The tyrosine and serine site-specific recombination systems of bacteriophages HK022 and ΦC31 are widely used for stable directional exchange and relocation of DNA sequences, making them valuable tools in these contexts. We have developed site-specific recombination tools that allow the direct selection of recombination events by embedding the attB site from each system within the β-lactamase resistance coding sequence (bla). Results The HK and ΦC31 tools were developed by placing the attB sites from each system into the signal peptide cleavage site coding sequence of bla. All possible open reading frames (ORFs) were inserted and tested for recombination efficiency and bla activity. Efficient recombination was observed for all tested ORFs (3 for HK, 6 for ΦC31) as shown through a cointegrate formation assay. The bla gene with the embedded attB site was functional for eight of the nine constructs tested. Conclusions The HK/ΦC31 att-bla system offers a simple way to directly select recombination events, thus enhancing the use of site-specific recombination systems for carrying out precise, large-scale DNA manipulation, and adding useful tools to the genetics toolbox. We further show the power and flexibility of bla to be used as a reporter for recombination.

The topological properties of DNA molecules, supercoiling, knotting, and catenation, are intimately connected with essential biological processes, such as gene expression, replication, recombination, and chromosome segregation. Non-trivial DNA topologies present challenges to the molecular machines that process and maintain genomic information, for example, by creating unwanted DNA entanglements. At the same time, topological distortion can facilitate DNA-sequence recognition through localized duplex unwinding and longer-range loop-mediated interactions between the DNA sequences. Topoisomerases are a special class of essential enzymes that homeostatically manage DNA topology through the passage of DNA strands. The activities of these enzymes are generally investigated using circular DNA as a model system, in which case it is possible to directly assay the formation and relaxation of DNA supercoils and the formation/resolution of knots and catenanes. Some topoisomerases use ATP as an energy cofactor, whereas others act in an ATP-independent manner. The free energy of ATP hydrolysis can be used to drive negative and positive supercoiling or to specifically relax DNA topologies to levels below those that are expected at thermodynamic equilibrium. The latter activity, which is known as topology simplification, is thus far exclusively associated with type-II topoisomerases and it can be understood through insight into the detailed non-equilibrium behavior of type-II enzymes. We use a non-equilibrium topological-network approach, which stands in contrast to the equilibrium models that are conventionally used in the DNA-topology field, to gain insights into the rates that govern individual transitions between topological states. We anticipate that our quantitative approach will stimulate experimental work and the theoretical/computational modeling of topoisomerases and similar enzyme systems.

The circular chromosomes of bacteria can be concatenated into dimers by homologous recombination. Dimers are solved by the addition of a cross-over at a specific chromosomal site, dif, by 2 related tyrosine recombinases, XerC and XerD. Each enzyme catalyzes the exchange of a specific pair of strands. Some plasmids exploit the Xer machinery for concatemer resolution. Other mobile elements exploit it to integrate into the genome of their host. Chromosome dimer resolution is initiated by XerD. The reaction is under the control of a cell-division protein, FtsK, which activates XerD by a direct contact. Most mobile elements exploit FtsK-independent Xer recombination reactions initiated by XerC. The only notable exception is the toxin-linked cryptic satellite phage of Vibrio cholerae, TLCΦ, which integrates into and excises from the dif site of the primary chromosome of its host by a reaction initiated by XerD. However, the reaction remains independent of FtsK. Here, we show that TLCΦ carries a Xer recombination activation factor, XafT. We demonstrate in vitro that XafT activates XerD catalysis. Correspondingly, we found that XafT specifically interacts with XerD. We further show that integrative mobile elements exploiting Xer (IMEXs) encoding a XafT-like protein are widespread in gamma- and betaproteobacteria, including human, animal, and plant pathogens. [ABSTRACT FROM AUTHOR]

Industrial biotechnology is reliant on native pathway engineering or foreign pathway introduction for efficient biosynthesis of target products. Chromosomal integration, with intrinsic genetic stability, is an indispensable step for reliable expression of homologous or heterologous genes and pathways in large-scale and long-term fermentation. With advances in synthetic biology and CRISPR-based genome editing approaches, a wide variety of novel enabling technologies have been developed for single-step, markerless, multi-locus genomic integration of large biochemical pathways, which significantly facilitate microbial overproduction of chemicals, pharmaceuticals and other value-added biomolecules. Notably, the newly discovered homology-mediated end joining strategy could be widely applicable for high-efficiency genomic integration in a number of homologous recombination-deficient microbes. In this review, we explore the fundamental principles and characteristics of genomic integration, and highlight the development and applications of targeted integration approaches in the three representative industrial microbial systems, including Escherichia coli , actinomycetes and yeasts. [ABSTRACT FROM AUTHOR]

The bacteria of the genus Streptomyces are the most valuable source of natural products of industrial and medical importance. A recent explosion of Streptomyces genome sequence data has revealed the enormous genetic potential of new biologically active compounds, although many of them are silent under laboratory conditions. Efficient and stable manipulation of the genome is necessary to induce their production. Comprehensive studies in the past have led to a large and versatile collection of molecular biology tools for gene manipulation of Streptomyces, including various replicative plasmids. However, biotechnological applications of these bacteria require stable genome alterations/mutations. To accomplish such stable genome editing, two major strategies for streptomycetes have been developed: (1) integration into the chromosome through Att/Int site-specific integration systems based on Streptomyces actinophages (ΦC31, ΦBT1, VWB, TG1, SV1, R4, ΦJoe, μ1/6) or pSAM2 integrative plasmid; (2) integration by homologous recombination using suicidal non-replicating vectors. The present review is an attempt to provide a comprehensive summary of both approaches for stable genomic engineering and to outline recent advances in these strategies, such as CRISPR/Cas9, which have successfully manipulated Streptomyces strains to improve their biotechnological properties and increase production of natural or new gene-manipulated biologically active compounds. [ABSTRACT FROM AUTHOR]

Key message: A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination. Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools. [ABSTRACT FROM AUTHOR]

Abstract Chromosomal integration of genes and pathways is of particular importance for large-scale and long-term fermentation in industrial biotechnology. However, stable, multi-copy integration of long DNA segments (e.g., large gene clusters) remains challenging. Here, we describe a plug-and-play toolkit that allows for high-efficiency, single-step, multi-locus integration of natural product (NP) biosynthetic gene clusters (BGCs) in actinomycetes, based on the innovative concept of "multiple integrases-multiple attB sites". This toolkit consists of 27 synthetic modular plasmids, which contain single- or multi-integration modules (from two to four) derived from five orthogonal site-specific recombination (SSR) systems. The multi-integration modules can be readily ligated into plasmids containing large BGCs by Gibson assembly, which can be simultaneously inserted into multiple native attB sites in a single step. We demonstrated the applicability of this toolkit by performing stabilized amplification of acetyl-CoA carboxylase genes to facilitate actinorhodin biosynthesis in Streptomyces coelicolor. Furthermore, using this toolkit, we achieved a 185.6% increase in 5-oxomilbemycin titers (from 2.23 to 6.37 g/L) in Streptomyces hygroscopicus via the multi-locus integration of the entire 5-oxomilbemycin BGC (72 kb) (up to four copies). Compared with previously reported methods, the advanced multiplex site-specific genome engineering (aMSGE) method does not require the introduction of any modifications into host genomes before the amplification of target genes or BGCs, which will drastically simplify and accelerate efforts to improve NP production. Considering that SSR systems are widely distributed in a variety of industrial microbes, this novel technique also promises to be a valuable tool for the enhanced biosynthesis of other high-value bioproducts. Highlights • An innovative methodology was established for multi-locus genomic integration of genes/pathways in microbial systems. • A plug-and-play, multi-copy integration toolkit derived from five orthogonal SSR systems was developed in actinomycetes. • Multi-copy overexpression of presurcor biosynthetic genes accA2BE facilitated actinorhodin biosynthesis by 4.6-fold. • Stable amplification of 5-oxomilbemycin BGC achieved a 185.6% increase in 5-oxomilbemycin titers (from 2.23 to 6.37 g/L). [ABSTRACT FROM AUTHOR]

Highlights • A full-length clone system to produce infectious VSV was constructed by site-specific recombination. • The virulence of rescued VSV was evaluated in pigs. • Site-specific recombination represents a useful and rapid methodology for cloning vesiculoviruses. Abstract This study reports the use of a site-specific recombination cloning technique for rapid development of a full-length cDNA clone that can produce infectious vesicular stomatitis New Jersey virus (VSNJV). The full-length genome of the epidemic VSNJV NJ0612NME6 strain was amplified in four overlapping cDNA fragments which were linked together and cloned into a vector plasmid by site-specific recombination. Furthermore, to derive infectious virus, three supporting plasmid vectors containing either the nucleoprotein (N), phosphoprotein (P) or polymerase (L) genes were constructed using the same cloning methodology. Recovery of recombinant VSNJV was achieved after transfecting all four vectors on into BSR-T7/5 cells, a BHK-derived cell line stably expressing T7 RNA polymerase (PMID: 9847328). In vitro characterization of recombinant and parental viruses revealed similar growth kinetics and plaque morphologies. Furthermore, experimental infection of pigs with the recombinant virus resulted in severe vesicular stomatitis with clinical signs similar to those previously reported for the parental field strain. These results validate the use of site-directed specific recombination cloning as a useful alternative method for rapid construction of stable full-length cDNA clones from vesicular stomatitis field strains. The approach reported herein contributes to the improvement of previously published methodologies for the development of full-length cDNA clones of this relevant virus. [ABSTRACT FROM AUTHOR]

Integrons play an important role in multidrug resistance. The integron platform codes for integrase (intI) that is required for gene cassette integration through site-specific recombination. The recombination crossover occurs between the G and TT nucleotides in non-palindromic attI and palindromic attC sites. The aim of this study was to establish an efficient in vitro assay for integrase purification and activity detection. To this end, the intI gene was cloned into the pET-22b plasmid. Then, the resulting recombinant plasmid was transformed into Escherichia coli Origami™ strain. The recombinant protein expression was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot assays. The recombinant intI protein was purified by nickel–nitrilotriacetic acid (Ni–NTA) affinity chromatography, and its activity was measured by a newly introduced assay. Briefly, specific primers for each side of attI and attC were used, thereby, a polymerase chain reaction would be performed, if a fused plasmid containing both attI and attC sites was created upon recombination. SDS-PAGE and western blotting confirmed the presence of a 38-kDa recombinant protein. Optimum conditions were established for the measurement of the integrase activity and a new model assay was conducted to analyse the recombination activity in vitro. Although the electrophoretic mobility shift assay is an efficient and reliable method, the newly introduced assay provided new or enhanced capability to determine the integrase activity, suggesting that there is no need for expensive and advanced equipment.