Recombineering-Based, High-Throughput Microbial Engineering Methods

High-efficiency recombineering has made E. coli a powerful chassis for synthetic biology over the past two decades as it enables both easy genomic modification and advanced techniques such as multiplex editing (MAGE), directed evolution of targeted genomic loci (DIvERGE), and excision and manipulation of large chromosomal segments (REXER). Recombineering technology, however, has been limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, highly efficient SSAPs must first be found. We developed a high-throughput method for SSAP discovery that we call Serial Enrichment for Efficient Recombineering (SEER). With SEER we screened libraries of SSAPs through E. coli and identified a highly active variant, CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5 to 10-fold in E. coli. Expanding the SEER screen allowed us to enable efficient recombineering in six well-studied bacteria: Agrobacterium tumefaciens, Caulobacter crescentus, Corynebacterium glutamicum, Lactococcus lactis, Mycobacterium smegmatis, Pseudomonas aeruginosa, and Staphylococcus aureus. The deployment of SEER in new species will pave the way toward new genetic engineering tools in these organisms. Recombineering-powered genome engineering techniques include easy gene knockouts or gene fusions, evolution or library generation on chromosomal loci, high-throughput allelic profiling, and genome-scale reverse genetics. We demonstrate some of these methods in newly recombineering-enabled hosts in a series of small vignettes. The methods that we demonstrate should improve microbiologists' ability to conduct high-throughput genetic experimentation in varied bacterial species to arrive at important biological insights.