Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis

Abstract

Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry 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; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed. Genes for the infrared fluorescent protein and for the human IgG-binding DARPin were cloned in all possible combinations to assess the protein yield. The dual promoter plasmid pNZDual enabled balanced expression of the two model proteins. It was exploited for the development of a single-plasmid inducible CRISPR-Cas9 system (pNZCRISPR) by using a nisin promoter, first to drive Cas9 expression and, secondly, to drive single guide RNA transcription. sgRNAs against htrA and ermR directed Cas9 against genomic or plasmid DNA and caused changes in bacterial growth and survival. Replacing Cas9 by dCas9 enabled CRISPR interference-mediated silencing of the upp gene. The present study introduces a new series of plasmids for advanced genetic modification of lactic acid bacterium L. lactis.

Safety Aspects of Genetically Modified Lactic Acid Bacteria

“…Its application to DNA targeting of four genomic islands in Streptococcus thermophilus resulted in generation of stable mutants that collectively lacked a total of 7% of their genome [45]. Cas9 has also been used for removal of plasmids, integrative conjugative elements, and prophages in L. lactis, where specific genetic loci were targeted using the pNZCRISPR and pLABTarget vectors [46,47].…”

“…CRISPR interference is achieved by specific single-guide-RNA-guided binding of nuclease-inactivated Cas9 to target genes and promoters that leads to obstruction of RNA polymerase [49]. Proof of principle of CRISPR interference activity in L. lactis was demonstrated by silencing the upp gene [47]. For some species, Cas9 is toxic and cannot be used for genetic engineering [10]; therefore, alternative Cas proteins have been developed, such as the Cas9 variant ThermoCas9 [50], Cpf1 and C2c1/2/3 [51], and Cas12a, with this last combined with single-stranded DNA recombineering to improve precision [52][53][54].…”

Impact of CRISPR interference on strain development in biotechnology

“…1). CRISPRi has been developed in a diverse group of bacteria, including E. coli [5] , Corynebacterium glutamicum [7] , Lactococcus lactis [18] , Rhodococcus opacus [19] , Burkholderia [20] , Clostridia [21–25] , Bacillus subtilis [26–29] , Bacillus methanolicus [30] , Streptomyces [31,32], and Mycobacteria [33–36], and applied for gene repression to interrogate their physiology or to identify gene targets for biotech applications (see Sections 3 and 4).…”

Metabolic engineering and synthetic biology employing Lactococcus lactis and Bacillus subtilis cell factories

“…Marker-free method for chromosomal mutations/deletions using ssDNA oligo's [17] Cre-loxP recombination system. Site-specific recombination system that allows multiple gene deletions in L. lactis [56] CRISPR-Cas9/CRISPRi-based genome editing [57] Improved expression host L. lactis NZ9000-4 (9k-4) with minimized genome and enhanced heterologous protein production [58] Improved protein secretion Signal peptides (SP). A library of usp45-derived SP for efficient protein secretion [59] several engineered lactococci have reached clinical studies using similar safe containment strategy.…”