Evaluation of Engineering Potential in Undomesticated Microbes with VECTOR

Abstract

Genetic engineering research has predominantly focused on well-characterized organisms like Escherichia coli and Bacillus subtilis, with methods that often fail to translate to other microorganisms. This limitation presents a significant challenge, particularly given the increasing isolation of large microbial collections through high-throughput culturomics. In response, we developed a scalable, high-throughput pipeline to evaluate the engineerability of diverse microbial community members we named VECTOR (Versatile Engineering and Characterization of Transferable Origins and Resistance). We utilized a library of vectors with the Bacterial Expression Vector Archive (BEVA) architecture that included combinations of three antibiotic resistance genes, and three broad host range origins of replication (pBBR1, RK2 and RSF1010) or the restricted host range R6K with an integrative mariner transposon. We tagged each vector with green fluorescent protein and a unique nucleotide barcode. The resulting plasmids were delivered en masse to libraries of undomesticated microbes from plant microbiomes in workflows designed to evaluate their ability to be engineered. Utilizing OD600 and relative fluorescence measurements, we were able to monitor genetic cargo transfer in real time, indicating successfully engineered strains. Next-generation sequencing of plasmid molecular barcodes allowed us to identify specific vector architectures that worked well in particular bacterial strains from a large community. Modifications to the procedure facilitated isolation of engineered microbes. Our results underscore the potential of this approach to rapidly develop toolkits for the efficient engineering of a wide range of cultivatable microorganisms. ImportanceUndomesticated, cultured microbial strains contain a largely untapped reservoir of genetic potential for synthetic biology, and are increasingly being utilized in synthetic communities for microbial ecology research or biotechnology.. However, these strains often have unique physiological or ecological characteristics that make them difficult to engineer using traditional methods. Current approaches are often restricted by inefficient plasmid delivery and integration, which stifles progress in unlocking the promise of undomesticated strains. Our research addresses this challenge by developing VECTOR (Versatile Engineering and Characterization of Transferable Origins and Resistance), a scalable, high-throughput pipeline that utilizes modular vectors and efficient engineering workflows to identify host range and improve plasmid uptake. By optimizing plasmid architectures and pooling them for simultaneous screening across a range of bacterial strains, VECTOR enhances engineering efficiency and opens new avenues for advancements in biotechnology.