Direct Genome Transfer from Acholeplasma laidlawii to Yeast

Cloning bacterial genomes in yeast or other hosts is a crucial step in workflows for creating highly engineered strains. The genome of Acholeplasma laidlawii PG-8A can be cloned in yeast if a toxic gene is removed, but this deletion has not been tested in live bacteria. Here, we demonstrate cloning the 1.46 Mb genome of A. laidlawii strain DN-E by integrating a yeast vector into the toxic gene and transferring the genome directly to yeast by using cell fusion. Genomic integration was possible only when A. laidlawii was complemented with a functional recA gene. Following cell fusion, 13 out of 20 screened yeast colonies contained the A. laidlawii genome, as determined by multiplex PCR. This is the first demonstration of cloning entire A. laidlawii genomes by simply mixing donor and recipient cells in fusion buffer, bringing us one step closer to creating fully synthetic Acholeplasma strains.