Redesign of Lactococcus Lactis genome for Increasing pDNA and Recombinant Protein Production Using a CRISPR/Cas9 Strategy

The degradation of recombinant plasmid DNA by endonucleases is one of the major bottlenecks that is keeping Lactococcus lactis from reaching its full potential as cell factories for pDNA and recombinant protein production with pharmaceutical applications.

Escherichia coli has been the gold standard, since it is able to achieve high pDNA and protein yields, but L. lactis is a promising alternative, with its food-grade status. The purification process itself is already more cost effective for L. lactis, due to the absence of co-purifying lipopolysaccharides, a good secretion system and the presence of only one major extracellular protease. If endonuclease genes could be removed, the pDNA and, consequently, protein yields, should increase and turn L. lactis in a laboratory and industrial work horse.

There are not easy and effective systems for editing genes in L. lactis, so we decided for the CRISPR/Cas9 strategy, for a scarless and targeted removal. The present work consists in the adaptation of the Lactobacillus casei pLCNICK one-step system to L. lactis.

The nth gene was chosen as the first knockout target, since it codes for an endonuclease III that could be responsible for creating gaps in the plasmid vectors, contributing for its quick degradation. The pLCNICK is currently being engineered in order to Cas9 and the homologous recombination system be able to recognize and knockout the nth gene.

Several L. lactis strains were transformed with the designed plasmid and the nth knockout confirmed by PCR amplification and sequencing. After plasmid curing, the strains were transformed with a GFP reporter plasmid and the effect of the nth gene knockout in the plasmid yield and quality analyzed by agarose gel electrophoresis, spectrophotometry and chromatography of the purified pDNA, fluorimetry, fluorescence microscopy and flow cytometry using whole cells. Real-time quantitative PCR was used to quantify the plasmid copy number.