Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA).

Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways.

FtsK functions in the processing of a Holliday junction intermediate during bacterial chromosome segregation.

In bacteria with circular chromosomes, homologous recombination can generate chromosome dimers that cannot be segregated to daughter cells at cell division. Xer site-specific recombination at dif, a 28-bp site located in the replication terminus region of the chromosome, converts dimers to monomers through the sequential action of the XerC and XerD recombinases. Chromosome dimer resolution requires that dif is positioned correctly in the chromosome, and the activity of FtsK, a septum-located protein that coordinates cell division with chromosome segregation. Here, we show that cycles of XerC-mediated strand exchanges form and resolve Holliday junction intermediates back to substrate irrespective of whether conditions support a complete recombination reaction. The C-terminal domain of FtsK is sufficient to activate the exchange of the second pair of strands by XerD, allowing both intra- and intermolecular recombination reactions to go to completion. Proper positioning of dif in the chromosome and of FtsK at the septum is required to sense the multimeric state of newly replicated chromosomes and restrict complete Xer reactions to dimeric chromosomes.

X-ray structure of aminopeptidase A from Escherichia coli and a model for the nucleoprotein complex in Xer site-specific recombination.

The structure of aminopeptidase A (PepA), which functions as a DNA-binding protein in Xer site-specific recombination and in transcriptional control of the carAB operon in Escherichia coli, has been determined at 2.5 A resolution. In Xer recombination at cer, PepA and the arginine repressor (ArgR) serve as accessory proteins, ensuring that recombination is exclusively intramolecular. In contrast, PepA homologues from other species have no known DNA-binding activity and are not implicated in transcriptional regulation or control of site-specific recombination. PepA comprises two domains, which have similar folds to the two domains of bovine lens leucine aminopeptidase (LAP). However, the N-terminal domain of PepA, which probably plays a significant role in DNA binding, is rotated by 19 degrees compared with its position in LAP. PepA is a homohexamer of 32 symmetry. A groove that runs from one trimer face across the 2-fold molecular axis to the other trimer face is proposed to be the DNA-binding site. Molecular modelling supports a structure of the Xer complex in which PepA, ArgR and a second PepA molecule are sandwiched along their 3-fold molecular axes, and the accessory sequences of the two recombination sites wrap around the accessory proteins as a right-handed superhelix such that three negative supercoils are trapped.

Topology of Xer recombination on catenanes produced by lambda integrase.

Xer site-specific recombination at the psi site from plasmid pSC101 displays topological selectivity, such that recombination normally occurs only between directly repeated sites on the same circular DNA molecule. This intramolecular selectivity is important for the biological role of psi, and is imposed by accessory proteins PepA and ArcA acting at accessory DNA sequences adjacent to the core recombination site. Here we show that the selectivity for intramolecular recombination at psi can be bypassed in multiply interlinked catenanes. Xer site-specific recombination occurred relatively efficiently between antiparallel psi sites located on separate rings of right-handed torus catenanes containing six or more nodes. This recombination introduced one additional node into the catenanes. Antiparallel sites on four-noded right-handed catenanes, the normal product of Xer recombination at psi, were not recombined efficiently. Furthermore, parallel psi sites on right-handed torus catenanes were not substrates for Xer recombination. These findings support a model in which psi sites are plectonemically interwrapped, trapping a precise number of supercoils that are converted to four catenation nodes by Xer strand exchange.

The ArcA/ArcB two-component regulatory system of Escherichia coli is essential for Xer site-specific recombination at psi.

Two recombinases, XerC and XerD, act at the recombination sites psi and cer in plasmids pSC101 and Co1E1 respectively. Recombination at these sites maintains the plasmids in a monomeric state and helps to promote stable plasmid inheritance. The accessory protein PepA acts at both psi and cer to ensure that only intramolecular recombination takes place. An additional accessory protein, ArgR, is required for recombination at cer but not at psi. Here, we demonstrate that the ArcA/ArcB two-component regulatory system of Escherichia coli, which mediates adaptation to anaerobic growth conditions, is required for efficient recombination in vivo at psi. Phosphorylated ArcA binds to psi in vitro and increases the efficiency of recombination at this site. Binding of ArcA to psi may contribute to the formation of a higher order synaptic complex between a pair of psi sites, thus helping to ensure that recombination is intramolecular.

Direct interaction of aminopeptidase A with recombination site DNA in Xer site-specific recombination.

Xer site-specific recombination at ColE1 cer converts plasmid multimers into monomers, thus ensuring the heritable stability of ColE1. Two related recombinase proteins, XerC and XerD, catalyse the strand exchange reaction at a 30 bp recombination core site. In addition, two accessory proteins, PepA and ArgR, are required for recombination at cer. These two accessory proteins are thought to act at 180 bp of accessory sequences adjacent to the cer recombination core to ensure that recombination only occurs between directly repeated sites on the same molecule. Here, we demonstrate that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion. We present a model for this synaptic complex, and propose that strand exchange can only occur after the formation of this complex.

Effect of DNA topology on Holliday junction resolution by Escherichia coli RuvC and bacteriophage T7 endonuclease I.

Holliday junctions are key intermediates in homologous genetic recombination. Their resolution requires specialised nucleases that nick pairs of strands at the junction point, leading to the separation of mature recombinants. Resolution occurs in either of two orientations, according to which DNA strands are cut. We show that DNA topology can determine the efficiency and outcome of a recombination reaction. Using two Holliday junction resolvases, Escherichia coli RuvC protein and T7 endonuclease I, we observed that supercoiled figure-8 DNA molecules containing Holliday junctions were resolved with a specific orientation bias, and that this bias was reversed by the presence of a topological tether (catenation). In contrast, when all topological constraints were removed by restriction digestion, the recombination intermediates were resolved equally in the two orientations. These results show that topological constraints affecting Holliday junction structure influence the orientation of resolution by cellular resolvases.

Topological selectivity in Xer site-specific recombination.

The product topology of Xer-mediated site-specific recombination at plasmid sites has been determined. The product of deletion at pSC101 psi is a right-handed antiparallel 4-noded catenane. The ColE1 cer deletion product has an identical topology, except that only one pair of strands is exchanged. These specific product topologies imply that the productive synaptic complex and the strand exchange mechanism have fixed topologies. Further analysis suggests that synapsis traps exactly three negative supercoils between recombining sites, and that strand exchange introduces a further negative topological node in the deletion reaction. We present a model in which the requirement for a specific synaptic stucture, with two recombination sites interwrapped around the accessory proteins ArgR and PepA, ensures that recombination only occurs efficiently between directly repeated sites on the same DNA molecule.

Xer-mediated site-specific recombination in vitro.

The Xer site-specific recombination system acts at ColE1 cer and pSC101 psi sites to ensure that these plasmids are in a monomeric state prior to cell division. We show that four proteins, ArgR, PepA, XerC and XerD are necessary and sufficient for recombination between directly repeated cer sites on a supercoiled plasmid in vitro. Only PepA, XerC and XerD are required for recombination at psi in vitro. Recombination at cer and psi in vitro requires negative supercoiling and is exclusively intramolecular. Strand exchange at cer produces Holliday junction-containing products in which only the top strands have been exchanged. This reaction requires the catalytic tyrosine residue of Xer C but not that of XerD. Recombination at psi gives catenated circular resolution products. Strand exchange at psi is sequential. XerC catalyses the first (top) strand exchange to make a Holiday junction intermediate and XerD catalyses the second (bottom) strand exchange.

Genomic DNA fingerprinting by restriction fragment end labeling.

A typing method for bacteria was developed and applied to several species, including Escherichia coli and Actinobacillus actinomycetemcomitans. Total genomic DNA was digested with a restriction endonuclease, and fragments were enabled with [alpha-32P]dATP by using the Klenow fragment of DNA polymerase and separated by electrophoresis in 6% polyacrylamide/8 M urea (sequencing gel). Depending on the restriction endonuclease and the bacterium, the method produced approximately 30-50 well-separated fragments in the size range of 100-400 nucleotides. For A. actinomycetemcomitans, all strains had bands in common. Nevertheless, many polymorphisms could be observed, and the 31 strains tested could be classified into 29 distinct types. Furthermore, serotype-specific fragments could be assigned for the three serotypes investigated. The method described is very sensitive, allowing more distinct types to be distinguished than other commonly used typing methods. When the method was applied to 10 other clinically relevant bacterial species, both species-specific bands and strain-specific bands were found. Isolates from different locations of one patient showed indistinguishable patterns. Computer-assisted analysis of the DNA fingerprints allowed the determination of similarity coefficients. It is concluded that genomic fingerprinting by restriction fragment end labeling (RFEL) is a powerful and generally applicable technique to type bacterial species.

DNA binding activities of the Caenorhabditis elegans Tc3 transposase.

Tc3 is a member of the Tc1/mariner family of transposable elements. All these elements have terminal inverted repeats, encode related transposases and insert exclusively into TA dinucleotides. We have studied the DNA binding properties of Tc3 transposase and found that an N-terminal domain of 65 amino acids binds specifically to two regions within the 462 bp Tc3 inverted repeat; one region is located at the end of the inverted repeat, the other is located approximately 180 bp from the end. Methylation interference experiments indicate that this N-terminal DNA binding domain of the Tc3 transposase interacts with nucleotides on one face of the DNA helix over adjacent major and minor grooves.

The mechanism of transposition of Tc3 in C. elegans.

The Tc3 transposon of C. elegans belongs to a family of inverted repeat DNA transposons, found in many different phyla. We studied the mechanism of Tc3 transposition by expression of Tc3 transposase from a heat-shock promoter in transgenic nematodes. Transposition is accompanied by the appearance of linear extrachromosomal Tc3 DNA. Analysis of the ends of this presumed transposition intermediate shows that the transposon is excised incompletely: the 5' ends of the transposon lack two nucleotides. The 3' ends coincide with the last nucleotide of the integrated element and carry 3' hydroxyls. The nucleotides that are not coexcised with the transposon remain at the donor site and result in a characteristic footprint. A model is derived for the mechanism of Tc3 jumping that probably applies to the entire family of Tc1/mariner transposable elements.

Xer-mediated site-specific recombination at cer generates Holliday junctions in vivo.

Normal segregation of the Escherichia coli chromosome and stable inheritance of multicopy plasmids such as ColE1 requires the Xer site-specific recombination system. Two putative lambda integrase family recombinases, XerC and XerD, participate in the recombination reactions. We have constructed an E. coli strain in which the expression of xerC can be tightly regulated, thereby allowing the analysis of controlled recombination reactions in vivo. Xer-mediated recombination in this strain generates Holliday junction-containing DNA molecules in which a specific pair of strands has been exchanged in addition to complete recombinant products. This suggests that Xer site-specific recombination utilizes a strand exchange mechanism similar or identical to that of other members of the lambda integrase family of recombination systems. The controlled in vivo recombination reaction at cer requires recombinase and two accessory proteins, ArgR and PepA. Generation of Holliday junctions and recombinant products is equally efficient in RuvC- and RuvC+ cells, and in cells containing a multicopy RuvC+ plasmid. Controlled XerC expression is also used to analyse the efficiency of recombination between variant cer sites containing sequence alterations and heterologies within their central regions.

Mobilization of quiet, endogenous Tc3 transposons of Caenorhabditis elegans by forced expression of Tc3 transposase.

The commonly studied Caenorhabditis elegans strain Bristol N2 contains approximately 15 copies per genome of the transposon Tc3. However, Tc3 is not active in Bristol N2. Tc3 contains one major open reading frame (Tc3A). We have fused this open reading frame to an inducible promoter and expressed it in a transgenic Bristol N2 line. Tc3A expression resulted in frequent excision and transposition of endogenous Tc3 elements. This shows that the Bristol N2 genome contains Tc3 transposons that are cis proficient for transposition, but are immobile because Tc3A is absent. We demonstrate that recombinant Tc3A binds specifically to the terminal nucleotides of the Tc3 inverted repeat, indicating that Tc3A is the Tc3 transposase. Activation of Tc3 transposition in vivo was accompanied by the appearance of extrachromosomal, linear copies of Tc3. These may be intermediates in Tc3 transposition.

Recombination at ColE1 cer requires the Escherichia coli xerC gene product, a member of the lambda integrase family of site-specific recombinases.

Site-specific recombination at the plasmid ColE1 cer site requires the Escherichia coli chromosomal gene xerC. The xerC gene has been localized to the 85-min region of the E. coli chromosome, between cya and uvrD. The nucleotide sequences of the xerC gene and flanking regions have been determined. The xerC gene encodes a protein with a calculated molecular mass of 33.8 kDa. This protein has substantial sequence similarity to the lambda integrase family of site-specific recombinases and is probably the cer recombinase. The xerC gene is expressed as part of a multicistronic unit that includes the dapF gene and two other open reading frames.

xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase.

The heritable stability of ColE1 is dependent on a site-specific recombination system which acts to resolve plasmid multimers into monomers. This plasmid stabilizing recombination system requires the presence in cis of the ColE1 cer region, plus at least two trans-acting factors encoded by the xerA and xerB genes of Escherichia coli. The xerB gene has been cloned and sequenced and found to encode a polypeptide with a calculated mol. wt of 55.3 kd. The predicted amino acid sequence of this protein exhibits striking similarity to that of bovine lens leucine aminopeptidase (53 kd). The biological significance of this similarity is corroborated by genetic and biochemical evidence which suggests that xerB is identical to the E.coli and S.typhimurium pepA genes that encode aminopeptidase A.