A RAGE Based Strategy for the Genome Engineering of the Human Respiratory Pathogen Mycoplasma pneumoniae

Abstract

Genome engineering of microorganisms has become a standard in microbial biotechnologies. Several efficient tools are available for the genetic manipulation of model bacteria such as Escherichia coli and Bacillus subtilis, or the yeast Saccharomyces cerevisiae. Difficulties arise when transferring these tools to nonmodel organisms. Synthetic biology strategies relying on genome transplantation (GT) aim at using yeast cells for engineering bacterial genomes cloned as artificial chromosomes. However, these strategies remain unsuccessful for many bacteria, including Mycoplasma pneumoniae (MPN), a human pathogen infecting the respiratory tract that has been extensively studied as a model for systems biology of simple unicellular organisms. Here, we have designed a novel strategy for genome engineering based on the recombinase-assisted genomic engineering (RAGE) technology for editing the MPN genome. Using this strategy, we have introduced a 15 kbp fragment at a specific locus of the MPN genome and replaced 38 kbp from its genome by engineered versions modified either in yeast or in E. coli. A strain harboring a synthetic version of this fragment cleared of 13 nonessential genes could also be built and propagated in vitro. These strains were depleted of known virulence factors aiming at creating an avirulent chassis for SynBio applications. Such a chassis and technology are a step forward to build vaccines or deliver therapeutic compounds in the lungs to prevent or cure respiratory diseases in humans.

A genetic toolkit and gene switches to limit Mycoplasma growth for a synthetic vaccine chassis

“…Recent advances in Mycoplasma research have led to designing different chassis with potential as live attenuated vectors, whether for vaccination or drug delivery systems 16,41,87,88 . For the aim of engineering live biotherapeutics for livestock or humans, it will be essential to ensure the biosafety of the products, during bioproduction as well.…”

“…For example, while inducible gene expression systems have been widely exploited in model bacteria, like E. coli or Bacillus subtilis, that is not the case for Mycoplasma, despite their huge potential as cell chassis. With new advances in genome engineering techniques [40][41][42][43][44] , it is more important than ever to increase the gene regulation toolkit for these microorganisms.…”

Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans

“…Different strategies could also be evaluated to optimise the delivery of Cre recombinase, such as self-excising Cre expression cassettes. 22 The use of a split pyrG marker between recombination sites could be evaluated to minimise the background by random integration and abortive colonies, or to further evaluate AMA1-based donor vectors. 46…”

“…Different strategies could also be evaluated to optimise the delivery of Cre recombinase, such a selfexcising Cre expression cassettes. 22 The use of a split pyrG marker between recombination sites could be evaluated to minimise the background by random integration and abortive colonies, or to further evaluate AMA1-based donor vectors. 45 To sum up, Cre/lox-mediated integration of BGCs has the potential to speed up the process of constructing strains to produce heterologous metabolites in A. nidulans.…”

“…RMCE has been used for the integration of large constructs in a wide range of cell factories and model organisms. [19][20][21][22][23] However, to this date there is no system for site-specific recombinases-mediated chromosomal integration in filamentous fungi.…”