A Slovenian research organization has developed a new tool for recombinant protein expression and gene regulation in Lactococcus lactis - a food-grade lactic acid bacterium that is used in the dairy industry and as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis. Its advantages are generally recognized as safe (GRAS) status and absence of endotoxins. L. lactis was recently recognized as a probiotic and has been genetically engineered as a vector for the delivery of antigens and therapeutic proteins to the mucosal surfaces.
Tools for recombinant protein expression have been relatively well developed. L. lactis is therefore comparable to other well established bacterial expression systems, such as Escherichia coli and Bacillus subtilis.
Advanced techniques for genetic engineering are required to develop L. lactis further as a microbial cell factory. Simultaneous expression of two or more proteins is beneficial for various applications, including the expression of multi-subunit proteins, the use of L. lactis as a mucosal delivery vehicle or as a multistep biocatalyst.
Here, plasmids for co-expression of two recombinant proteins in L. lactis have been developed and their effectiveness assessed by the expression of model proteins. Plasmids were further upgraded and a single plasmid CRISPR-Cas9 system has been developed. Duplication of the nisin promoter enabled the balanced, inducible expression of two model proteins in L. lactis, thus constituting a new tool for recombinant protein expression in this organism. A similar strategy resulted in a single plasmid CRISPR-Cas9 system that can be used, among other possible applications, for plasmid curing or CRISPRi-mediated gene regulation in L. lactis.
Plasmids will be applied in the future research in L. lactis for concomitant expression of therapeutic and reporter proteins, as well as for plasmid curing and gene silencing.
Slovenian research group expertise is related to probiotic and other lactic acid bacteria and best described by the following three focal points and references:
(i) genetically engineered lactic acid bacteria as vehicles for the delivery of therapeutic molecules to different mucosal surfaces – treatment of inflammatory bowel disease, binding the Shiga toxin, targeting cancer cells and delivering viral antigen for vaccination;
(ii) methods that facilitate the study of lactic acid bacteria - assessment of novel anchors for nonGMO surface display, in vivo imaging of lactic acid bacteria in mice, and first CRISPR-based tools for genomic engineering of Lactococcus lactis;
(iii) advanced delivery systems for probiotics – lactobacilli incorporated in viable nanofibers enabling the formation of dosage form for probiotics.
The Slovenian research team is seeking collaboration with companies and academia related to its expertise in producing proteins for development and research.
This represents a broad range of R&D topics. The Slovenian research team is seeking both technical as well as research cooperation’s and is also interested to participate in consortia created in the framework of forthcoming FP8 calls.
Partners sought are companies and academia related to the field of biotechnology, more specifically molecular biology, protein production, enzymology and medical imaging. Partners are sought for:
• Technical cooperation: due to broad range application field of this kind of plasmid systems, partners interested in genome editing, gene regulation, production of next-generation antimicrobials, DNA imaging and other applications are invited for cooperation.
• Research cooperation: future research in L. lactis for concomitant expression of therapeutic and reporter proteins, as well as for plasmid curing and gene silencing.
