Abstract
BackgroundLactobacillus casei is widely used in the dairy and pharmaceutical industries and a promising candidate for use as cell factories. Recently, genome sequencing and functional genomics provide the possibility for reducing L. casei genome. However, it was still limited by the inefficient and laborious genome deletion methods.ResultsHere, we proposed a genome minimization strategy based on LCABL_13040-50-60 recombineering and Cre-lox site-specific recombination system in L. casei. The LCABL_13040-50-60 recombineering system was used to introduce two lox sites (lox66 and lox71) into 5′ and 3′ ends of the targeted region. Subsequently, the targeted region was excised by Cre recombinase. The robustness of the strategy was demonstrated by single-deletion of a nonessential ~ 39.3 kb or an important ~ 12.8 kb region and simultaneous deletion of two non-continuous genome regions (5.2 and 6.6 kb) with 100% efficiency. Furthermore, a cyclical application of this strategy generated a double-deletion mutant of which 1.68% of the chromosome was sequentially excised. Moreover, biological features (including growth rate, electroporation efficiency, cell morphology or heterologous protein productivity) of these mutants were characterized.ConclusionsTo our knowledge, this strategy is the first instance of sequential deletion of large-scale genome regions in L. casei. We expected this efficient and inexpensive tool can help for rapid genome streamlining and generation restructured L. casei strains used as cell factories.
Highlights
Lactobacillus casei is widely used in the dairy and pharmaceutical industries and a promising candidate for use as cell factories
Scheme for deletion of a large‐scale genome region in L. casei To develop a large genome region deletion strategy in L. casei, the Cre-lox site-specific recombination system was used in this study (Fig. 1)
Quantitative growth curves analysis indicated that the 12.8 kb deletion mutant BLD2 showed a decreased growth rate in MRS medium (Additional file 1: Figure S3C), suggesting that the deletion altered the strain’s phenotype. These results clearly demonstrated that this method could be used to delete a large-scale genome region containing an important gene in L. casei BL23
Summary
Lactobacillus casei is widely used in the dairy and pharmaceutical industries and a promising candidate for use as cell factories. Genome sequencing and functional genomics provide the possibility for reducing L. casei genome. Fast growing numbers of the whole genome sequences and functional genomics of lactic acid bacterial strains have provided an abundant of information for further understanding LAB, including their gene organization, their biological properties, and their ecological roles in animal or human health as well as their environmental interactions [2,3,4,5]. Of all the LAB stains, Lactobacillus casei plays key roles in dairy fermentations and pharmaceutical industries and is the ideal cell factories for production of high-value metabolites [7,8,9,10,11].
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