High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration

Abstract

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.

Maximizing Heterologous Expression of Engineered Type I Polyketide Synthases: Investigating Codon Optimization Strategies

“…To ensure greater accuracy and consistency of our results in C. glutamicum and P. putida , we utilized serine recombinase-assisted genome engineering (SAGE) to integrate our codon variants into the genome of these hosts. , The strains AG5577 and AG6212 are derived from P. putida and C. glutamicum , respectively, and contain a total of 9 unique attB sites in their genome (Adam Guss, personal communication). These genomic “landing pads” enable highly efficient and precise gene integration via the SAGE system (Figure a).…”

“…Integrations of pBH026 or pAN001 vectors and backbone excisions were performed using the SAGE system. 24 Excision of the backbones of pBH026 or pAN001 using pGW30 or pALC412 leads to the removal of the repressor lacI, the selection marker, and origin of replication. Serine recombinase-assisted integrations into the hosts AG5577 and AG6212 were confirmed by colony PCR (cPCR) using primers that flank the specific attB site in the host genome and primers that anneal to the 5′ and 3′ end of the target gene.…”

“…Given that our systematic approach amounted to 11 codon variants of a relatively large gene (>7 kb), we sought to avoid relying exclusively on plasmid-based expression systems. Besides well-known issues such as plasmid stability, copy number variations and growth defects, intercompatibility between our different organisms might have become challenging as well. ,− The SAGE system has proven to be an indispensable tool for genetic engineering when reproducibility and throughput are limiting factors. Stable integrations of large genes into the chosen host genomes can be multiplexed, are highly efficient and require minimal knowledge about the targeted host.…”

“…Heterologous genes were integrated into the host genome or expressed from plasmid DNA. Integrations of pBH026 or pAN001 vectors and backbone excisions were performed using the SAGE system . Excision of the backbones of pBH026 or pAN001 using pGW30 or pALC412 leads to the removal of the repressor lacI , the selection marker, and origin of replication.…”

Automated Platform for the Plasmid Construction Process

“…This vector thus enables episomal replication in S. cerevisiae for DNA assembly, episomal replication in E. coli for cloning and potentially rapid expression profiling, and reversible chromosomal integration into a compatible host organism with an integrated Bxb1 attB site. As Elmore et al demonstrated, there is a wide variety of organisms that can potentially be made SAGE -compatible with the integration of a Bxb1 attB site, which means that cross-species comparison of our assembled PKSs is feasible . We additionally incorporated two promoters into the design for each possible PKS partP lacUV5 and P T7 with a linked N-terminal 6x-His Tagto enable two different applications, namely, cross-species isopropyl-β- d -thiogalactopyranoside (IPTG)-inducible expression with P lacUV5 and high inducible expression in E. coli with P T7 for protein purification and cell-free assay purposes.…”