CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria

Abstract

It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products' roles in various environments. However, existing genetic tools support only a small fraction of these efforts. Here, we present the development of chassis-independent recombinase-assisted genome engineering (CRAGE), which enables single-step integration of large, complex BGC constructs directly into the chromosomes of diverse bacteria with high accuracy and efficiency. To demonstrate the efficacy of CRAGE, we expressed three known and six previously identified but experimentally elusive non-ribosomal peptide synthetase (NRPS) and NRPS-polyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens in 25 diverse γ-Proteobacteria species. Successful activation of six BGCs identified 22 products for which diversity and yield were greater when the BGCs were expressed in strains closely related to the native strain than when they were expressed in either native or more distantly related strains. Activation of these BGCs demonstrates the feasibility of exploiting their underlying catalytic activity and plasticity, and provides evidence that systematic approaches based on CRAGE will be useful for discovering and identifying previously uncharacterized metabolites.

The Link Between the Ecology of the Prokaryotic Rare Biosphere and Its Biotechnological Potential

“…To this end, coupling culturomics to DNA mutagenesis research will be ultimately necessary to assign novel functions to the wealth of so-far hypothetical proteins which still dominate genome annotations of even the most well-known and studied prokaryotes. Moreover, recent advances in genome editing technologies and synthetic biology hold much promise in leveraging our capacity to engineer e.g., pollutant-removing (Dvořák et al, 2017), drug-producing (Wang et al, 2019) and stress-tolerant (Jia et al, 2014) bacteria, thus facilitating our ability to harness the metabolism of both culturable and thus far unculturable low abundance prokaryotes in biotechnology.…”

Approaches to genetic tool development for rapid domestication of non-model microorganisms

“…Transposons can also be used to insert heterologous pathways into bacteria [140], which have been applied to non-model microbes such as C. ljungdahlii for acetone production [141] and Acidothiobacillus ferroxidans for isobutyric acid biosynthesis [142]. Transposons have also been combined with the Cre-lox site-specific recombination system to insert a landing pad, followed by site-specific insertion of heterologous pathways [143]. Transposons are especially useful for application in organisms that lack other genetic tools such as homologous recombination for integrating DNA into the chromosome.…”

The SAGE genetic toolkit enables highly efficient, iterative site-specific genome engineering in bacteria

“…Homologous recombinationbased allelic exchange relies upon endogenous DNA-repair machinery -which is typically inefficient 27 , precluding the construction of even moderately-sized strain libraries. Integrase-mediated recombination technologies [28][29][30][31][32][33][34][35][36][37] have the potential overcome many of the limitations faced by the above genetic tools. Phage integrases (or site-specific recombinases) are enzymes that catalyze recombination between two specific sequences of DNA 34,38 .…”

“…Phage integrases (or site-specific recombinases) are enzymes that catalyze recombination between two specific sequences of DNA 34,38 . Two tyrosine recombinases, Flp and Cre, are commonly used in molecular genetics 37,39 , but these enzymes recognize identical sites and perform reversible recombination. The other class of phage integrases, serine integrases, are single subunit enzymes that catalyze unidirectional DNA recombination between two short, distinct att sites (attP and attB), generating new attachment sites (attL and attR) in the process.…”