Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.

Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli, usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expression of CcdB is toxic to E. coli in the absence of the CcdA antidote so counterselection is initiated by the removal of CcdA expression. CcdB counterselection is robust and does not require titrations or experiment-to-experiment optimization. Because counterselection strategies necessarily differ according to the copy number of the target, we describe two variations. For multi-copy targets, we use two E. coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature shift to remove the expression plasmid. To reduce unwanted intramolecular recombination, we also combined CcdB counterselection with Redα omission. These options improve the use of counterselection in recombineering with BACs, plasmids and the E. coli chromosome.

The exponentially increasing volumes of DNA sequence data highlight the need for new DNA cloning methods to explore the new information. Here, we describe ‘ExoCET’ (Exonuclease Combined with RecET recombination) to directly clone any chosen region from bacterial and mammalian genomes with nucleotide precision into operational plasmids. ExoCET combines in vitro exonuclease and annealing with the remarkable capacity of full length RecET homologous recombination (HR) to retrieve specified regions from genomic DNA preparations. Using T4 polymerase (T4pol) as the in vitro exonuclease for ExoCET, we directly cloned large regions (>50 kb) from bacterial and mammalian genomes, including DNA isolated from blood. Employing RecET HR or Cas9 cleavage in vitro, the directly cloned region can be chosen with nucleotide precision to position, for example, a gene into an expression vector without the need for further subcloning. In addition to its utility for bioprospecting in bacterial genomes, ExoCET presents straightforward access to mammalian genomes for various applications such as region-specific DNA sequencing that retains haplotype phasing, the rapid construction of optimal, haplotypic, isogenic targeting constructs or a new way to genotype that presents advantages over Southern blotting or polymerase chain reaction. The direct cloning capacities of ExoCET present new freedoms in recombinant DNA technology.

Many studies have demonstrated that intravenously administered bacteria can target and proliferate in solid tumors and then quickly be released from other organs. Here, we employed the tumor-targeting property of Escherichia coli Nissle 1917 to inhibit mouse B16 melanoma and 4T1 breast tumors through the expression of azurin protein. For this purpose, recombinant azurinexpressing E. coli Nissle 1917 was developed. The levels of in vitro and in vivo azurin secretion in the engineered bacterium were determined by immunochemistry. Our results demonstrated that B16 melanoma and orthotopic 4T1 breast tumor growth were remarkably restrained and pulmonary metastasis was prevented in immunocompetent mice. It is worth noting that this therapeutic effect partially resulted from the antitumor activity of neutrophils and lymphocytes due to inflammatory responses caused by bacterial infections. No toxicity was observed in the animal during the experiments. This study indicates that E. coli Nissle 1917 could be a potential carrier to deliver antitumor drugs effectively for cancer therapy.

The reconstruction of a natural product biosynthetic pathway from bacteria in a vector and subsequent heterologous expression in a technically amenable microbial system represents an efficient alternative to empirical traditional methods for functional discovery, yield improvement, and genetic engineering to produce "unnatural" derivatives. However, the traditional cloning procedure based on genomic library construction and screening are complicated due to the large size (>10 kb) of most biosynthetic pathways. Here, we describe the direct cloning of a partial syringolin biosynthetic gene cluster (sylCDE, 19 kb) from a digested genomic DNA mixture of Pseudomonas syringae into a plasmid in which sylCDE is under the control of an inducible promoter by one step linear-plus-linear homologous recombination (LLHR) in Escherichia coli. After expression in E. coli GB05-MtaA, two new syringolin derivatives were discovered. The complete syringolin gene cluster was assembled by addition of sylAB and exchange of a synthetic bidirectional promoter against the native promoter to drive sylB and sylC expression by using Red/ET recombineering. The varying production distribution of syringolin derivatives showed the different efficiencies of native and synthetic promoters in E. coli. The successful reconstitution and expression of the syringolin biosynthetic pathway shows that Red/ET recombineering is an efficient tool to clone and engineer secondary metabolite biosynthetic pathways.

Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed ‘recombineering’, in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5’-3’ exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced recombineering efficiency. Results from bioinformatic analysis and recombineering assays suggest that Plu2934 may be functionally equivalent to Redγ, which inhibits the major endogenous E. coli nuclease, RecBCD. The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.

Spinosyn and its analogs, produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. They are macrolides with a 21-carbon, 12-membered tetracyclic lactones that are attached to two deoxysugars, tri-O-methylrhamnose and forosamine. Labeling studies, analysis of the biosynthetically blocked mutants, and the genetic identification of the spinosyn gene cluster have provided detailed information concerning the mechanism of spinosyn biosynthesis and have enabled combinatorial biosynthesis of a large group of new spinosyns. The following developments have recently impacted the field of spinosyn biology: (1) A second-generation spinosyn called spinetoram (XDE-175) was launched in late 2007; it is a semisynthesized spinosyn derivative produced through the modification of 3'-O-methyl group of rhamnose and the double bond between C5 and C6 of spinosyn J and L. This molecule was shown to have improved insecticidal activity, enhanced duration of control, and an expanded pest spectrum. (2) A new class of spinosyns, the butenyl-spinosyns, was discovered from Saccharopolyspora pogona. The butenyl-spinosyns are similar to spinosyns, but differ in the length of the side chain at C-21. In addition to structural similarities with the spinosyns, the butenyl-spinosyns exhibit a high level of similarity in insecticidal activity to spinetoram. (3) Spinosyn analogs, 21-cyclobutyl-spinosyn A and 21-cyclobutyl-spinosyn D were generated by metabolic engineering of the spinosyn biosynthetic gene cluster. They showed better insecticidal activities against cotton aphid and tobacco budworm than that of spinosyn A and D. Future progress toward the development of more potent spinosad analogs, as well as enhancements in production yields will likely result from these recent advances in the genetics and biochemistry of spinosyns.

Summary The limited efficiency of the available tools for genetic manipulation of Pseudomonas limits fundamental research and utilization of this genus. We explored the properties of a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (RecTE Psy ) from Pseudomonas syringae pv. syringae B728a. Compared with RecTE Psy , the BAS operon was functional at a higher temperature indicating potential to be a generic system for Pseudomonas . Owing to the lack of RecBCD inhibitor in the BAS operon, we added Redγ or Pluγ and found increased recombineering efficiencies in P . aeruginosa and Pseudomonas fluorescens but not in Pseudomonas putida and P . syringae . Overexpression of single-stranded DNA-binding protein enhanced recombineering in several contexts including RecET recombination in E . coli . The utility of these systems was demonstrated by engineering P. aeruginosa genomes to create an attenuated rhamnolipid producer. Our work enhances the potential for functional genomics in Pseudomonas .