Development of an interspecies genome recombination method
using cell fusion and CRISPR/Cas9 techniques
Tomoya MAEDA
RIKEN Center for Biosystems Dynamics Research
Report: A Corynebacterium glutamicum strain carrying a single-copy plasmid sequence from
Escherichia coli was constructed. This strain was then fused with an Escherichia coli strain
using the heterologous protoplast fusion technique. The obtained fusant strain was confirmed
to carry both E. coli and C. glutamicum genomes; however, the introduced C. glutamicum
genome was unstable in the fusant strain.
Research aims
Acquisition of heterologous genes and genome segments i.e. horizontal genetransfer (HGT) accelerates evolution in the natural world. However, construction ofrecombinant strains carrying functional large insertions from foreign genome segments isstill challenging. In this study, I aimed to develop a methodology for constructing
recombinant strains carrying large insertion sequences derived from a heterologous genome.
Methods
Heterologous protoplast fusion
A protoplast is a cell whose cell wall has been removed, and in which thecytoplasmic membrane is the outermost layer. Protoplasts can be obtained by treating cellswith lytic enzymes such as lysozyme to remove the cell wall. When two or more protoplast
cells come into contact, they adhere to one another spontaneously to form hybrid protoplasts
with heteroplasmic cytoplasm and two fused parent nuclei [1]. E. coli protoplast cells weregenerated by treatment with 30 mg/ml lysozyme, followed by incubation in GP medium
containing 500 μg/ml ampicillin. However, C. glutamicum protoplast cells could not begenerated using the same method as E. coli due to the presence of a thick mycolic acid layer.
Therefore, instead of protoplast formation, C. glutamicum L-form cells were obtained.
Briefly, 109 C. glutamicum cells were spread onto medium containing BHI/MSM buffer (20mM MgCl2, 0.5 M Sucrose, 20 mM maleic acid, pH 7), 400 μg/ml D-cycloserine, and 2%
agar and incubated for 5 days at 30°C. Heterologous cell fusion was then performed asfollows. E. coli protoplast cells and C. glutamicum L-form cells were suspended into 40%
PEG6000 buffer (40% PEG6000, 20 mM MgCl2, 0.5 M Sucrose, 20 mM maleic acid, 10mM CaCl2, 5% DMSO, pH7) and incubated at 25°C for 5 min. The fused cells were further

subjected to protoplast regeneration by suspending in GP medium containing 1% BSA andincubating at 30°C for 2 days.
Site-directed recombination of a large heterologous genome segment using protoplast fusion
and the CRISPR/Cas9 system
To insert the homologous arms of the recombination sites into the genome of an E.
coli strain, approximately 1 kbp of NCgl1683 and NCgl1768 flanking regions derived fromthe C. glutamicum ATCC13032 strain was inserted into the DH5α strain. The newly
constructed strain was named ECH2. Two gRNA genes targeting the NCgl1683 andNCgl1783 genes were inserted into the ECH2 genome and the resulting newly constructedstrain was named ECHG2. A plasmid pRedCas9DG was constructed to express Cas9 andλRED and the ECH2 strain was transformed using this plasmid. Next, the cglIR geneencoding a type II restriction endonuclease was disrupted by inserting the chloramphenicol
resistance gene, cat, into the ATCC13032 strain genome. The newly constructed strain wasnamed as the cglIR::cat strain. Heterologous genomic recombination between E. coli and C.
glutamicum was then attempted by the expression of gRNAs, Cas9, and λRed recombinaseduring protoplast fusion.
Results
Protoplast cells of ECHG2/pRedCas9DG and L-form cells of cglIR::cat were fusedusing the protoplast fusion technique. However, recombination of the ~100 kbp genome
segment derived from the C. glutamicum genome into the E. coli genome was not observed.
On the contrary, markerless gene deletions and heterologous gene insertion using thispRedCasDG plasmid were successful. This suggests a short duration of the co-existence of
two heterologous genomes during heterologous protoplast fusion. To replicate the C.
glutamicum genome as a large single-copy plasimid in E. coli cells, an E. coli single-copy
plasmid derived from miniF was inserted into the cglIR::cat genome. To observe theco-existence of two heterologous genomes, the mCherry and EGFP genes were inserted into
the E. coli and C. glutamicum genomes respectively. The newly constructed cells were thensubjected to a heterologous protoplast fusion experiment. The obtained single colonies wereisolated and examined by fluorescent microscopy and PCR to determine whether the fusantclones carried two heterologous genomes (Fig. 1). As shown in Fig. 1, the fusant cells
expressed both mCherry and EGFP and carried both E. coli and C. glutamicum genes(randomly selected genes mreB and leuA, respectively). However, after subculturing threetimes, the fusant cells were confirmed to have lost the C. glutamicum genome. These results
suggest that the miniF-derived replication system can maintain the heterologous genome forseveral generations, even though maintenance of the heterologous genome is unstable.

Fig. 1. Heterologous protoplast fusion of E. coli and C. glutamicum. (A) Fluorescencemicroscopy of the E. coli and C. glutamicum fusant cells. (B) PCR product derived from thefusant cells.
Conclusion
In this study, I showed that the C. glutamicum genome can be introduced into E.
coli cells using the heterologous protoplast fusion technique. The results suggested that theminiF-derived replication system can maintain the heterologous genome for severalgenerations in E. coli, even though the maintenance of the heterologous genome is unstable.
Therefore, developments in the methodology to maintain the heterologous genome stably
and for site-directed recombination between two heterologous genomes are needed infuture studies.
References
1) Hopwood, D.A. (1981) Genetic studies with bacterial protoplasts. Annu Rev Microbiol.
35: 237–272.