Development of a Recombineering System for the Acetogen Eubacterium limosum with Cas9 Counterselection for Markerless Genome Engineering

Eubacterium limosum is
a Clostridial
acetogen that efficiently utilizes a wide range of single-carbon substrates
and contributes to metabolism of health-associated compounds in the
human gut microbiota. These traits have led to interest in developing
it as a platform for sustainable CO2-based biofuel production
to combat carbon emissions, and for exploring the importance of the
microbiota in human health. However, synthetic biology and metabolic
engineering in E. limosum have been
hindered by the inability to rapidly make precise genomic modifications.
Here, we screened a diverse library of recombinase proteins to develop
a highly efficient oligonucleotide-based recombineering system based
on the viral recombinase RecT. Following optimization, the system
is capable of catalyzing ssDNA recombination at an efficiency of up
to 2%. Addition of a Cas9 counterselection system eliminated unrecombined
cells, with up to 100% of viable cells encoding the desired mutation,
enabling creation of genomic point mutations in a scarless and markerless
manner. We deployed this system to create a clean knockout of the
extracellular polymeric substance (EPS) gene cluster, generating a
strain incapable of biofilm formation. This approach is rapid and
simple, not requiring laborious homology arm cloning, and can readily
be retargeted to almost any genomic locus. This work overcomes a major
bottleneck in E. limosum genetic engineering
by enabling precise genomic modifications, and provides both a roadmap
and associated recombinase plasmid library for developing similar
systems in other Clostridia of interest.