Efficient genome engineering is critical to understand and use microbial functions. Despite recent development of tools such as CRISPR-Cas gene editing, efficient integration of exogenous DNA with well-characterized functions remains limited to model bacteria. Here, we describe serine recombinase–assisted genome engineering, or SAGE, an easy-to-use, highly efficient, and extensible technology that enables selection marker–free, site-specific genome integration of up to 10 DNA constructs, often with efficiency on par with or superior to replicating plasmids. SAGE uses no replicating plasmids and thus lacks the host range limitations of other genome engineering technologies. We demonstrate the value of SAGE by characterizing genome integration efficiency in five bacteria that span multiple taxonomy groups and biotechnology applications and by identifying more than 95 heterologous promoters in each host with consistent transcription across environmental and genetic contexts. We anticipate that SAGE will rapidly expand the number of industrial and environmental bacteria compatible with high-throughput genetics and synthetic biology.

Exposure to the mycotoxin aflatoxin B1 (AFB1) strongly correlates with hepatocellular carcinoma (HCC). P450 enzymes convert AFB1 into a highly reactive epoxide that forms unstable 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) DNA adducts, which convert to stable mutagenic AFB1 formamidopyrimidine (FAPY) DNA adducts. In CYP1A2-expressing budding yeast, AFB1 is a weak mutagen but a potent recombinagen. However, few genes have been identified that confer AFB1 resistance. Here, we profiled the yeast genome for AFB1 resistance. We introduced the human CYP1A2 into ∼90% of the diploid deletion library, and pooled samples from CYP1A2-expressing libraries and the original library were exposed to 50 mM AFB1 for 20 hs. By using next generation sequencing (NGS) to count molecular barcodes, we initially identified 86 genes from the CYP1A2-expressing libraries, of which 79 were confirmed to confer AFB1 resistance. While functionally diverse genes, including those that function in proteolysis, actin reorganization, and tRNA modification, were identified, those that function in postreplication DNA repair and encode proteins that bind to DNA damage were over-represented, compared to the yeast genome, at large. DNA metabolism genes also included those functioning in checkpoint recovery and replication fork maintenance, emphasizing the potency of the mycotoxin to trigger replication stress. Among genes involved in postreplication repair, we observed that CSM2, a member of the CSM2(SHU) complex, functioned in AFB1-associated sister chromatid recombination while suppressing AFB1-associated mutations. These studies thus broaden the number of AFB1 resistance genes and have elucidated a mechanism of error-free bypass of AFB1-associated DNA adducts.

Sustainable enhancements to crop productivity and increased resilience to adverse conditions are critical for modern agriculture, and application of plant growth promoting rhizobacteria (PGPR) is a promising method to achieve these goals. However, many desirable PGPR traits are highly regulated in their native microbe, limited to certain plant rhizospheres, or insufficiently active for agricultural purposes. Synthetic biology can address these limitations, but its application is limited by availability of appropriate tools for sophisticated, high-throughput genome engineering that function in environments where selection for DNA maintenance is impractical. Here we present an orthogonal, Serine-integrase Assisted Genome Engineering (SAGE) system, which enables iterative, site-specific integration of up to 10 different DNA constructs at efficiency on par or better than replicating plasmids. SAGE does not require use of replicating plasmids to deliver recombination machinery, and employs a secondary serineintegrase to excise and recycle selection markers. Furthermore, unlike the widely utilized pBBR1 origin, DNA transformed using SAGE is stable without selection. We highlight SAGE's utility by constructing a 287-member constitutive promoter library with a ~40,000-fold dynamic range in P. fluorescens SBW25. We show that SAGE functions robustly in diverse ⍺and "-proteobacteria, thus providing evidence that it will be broadly useful for engineering industrial or environmental bacteria.

Metabolite exchange between plant roots and their associated rhizosphere microbiomes underpins plant growth promotion by microbes. Sorghum bicolor is a cereal crop that feeds animals and humans and is used for bioethanol production. Its root tips exude large amounts of a lipophilic benzoquinone called sorgoleone. Sorgoleone is an allelochemical that suppresses the growth of competing plant seedlings and is mineralized by microbes in soil. As an avenue to understand how sorghum and its root microbiome may be connected through root exudates, we identified the molecular determinants of microbial sorgoleone degradation and the distribution of this trait among microbes. We isolated and studied from sorghum-associated soils, three bacterial strains classified as Acinetobacter, Burkholderia, and Pseudomonas species that grow with sorgoleone as a sole carbon and energy source. The genomes of these strains were sequenced and subjected to transcriptomic and gene fitness analyses to identify candidate sorgoleone degradation genes. Follow up mutational analysis showed that sorgoleone catabolism is dependent on four contiguous genes that are conserved among the species we sequenced. Phylogenetic analysis of the sorgoleone degradation gene cluster showed that sorgoleone catabolism is enriched in sorghum-associated Streptomyces strains. The discovery of bacteria that grow on a compound like sorgoleone that is plant specific and not widely distributed in the environment, provides an opportunity to study how a plant exudate can enforce the development of a rhizosphere specific microbiome for the mutual benefit of plant and microbe.

Exposure to the mycotoxin aflatoxin B1 (AFB 1 ) strongly correlates with hepatocellular carcinoma. P450 enzymes convert AFB 1 into a highly reactive epoxide that forms unstable 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB 1 -N 7 -Gua) DNA adducts, which convert to stable mutagenic AFB 1 formamidopyrimidine (FAPY) DNA adducts. In CYP1A2-expressing budding yeast, AFB 1 is a weak mutagen but a potent recombinagen. However, few genes have been identified that confer AFB 1 resistance. Here, we profiled the yeast genome for AFB 1 resistance. We introduced the human CYP1A2 into ~90% of the diploid deletion library, and pooled samples from CYP1A2-expressing libraries and the original library were exposed to 50 M AFB 1 for 20 hs. By using next generation sequencing to count molecular barcodes, we identified 85 AFB 1 resistant genes from the CYP1A2-expressing libraries. While functionally diverse genes, including those that function in proteolysis, actin reorganization, and tRNA modification, were identified, those that function in post-replication DNA repair and encode proteins that bind to DNA damage were over-represented, compared to the yeast genome, at large. DNA metabolism genes included those functioning in DNA damage tolerance, checkpoint recovery and replication fork maintenance, emphasizing the potency of the mycotoxin to trigger replication stress. Among genes involved in error-free DNA damage tolerance, we observed that CSM2, a member of the CSM2(SHU) complex, functioned in AFB 1 -associated sister chromatid recombination while suppressing AFB 1 -associated mutations. These studies thus broaden the number of AFB 1 resistant genes and have elucidated a mechanism of error-free bypass of AFB 1associated DNA adducts.