Construction And Application Of Genome Editing Tools Based On Prophage Recombinases In Lactic Acid Bacteria

Lactic acid bacteria have been used in food fermentation industry for a long time Some lactic acid bacteria strains could produce high value-added metabolites,such as organic acid,vitamin and polysaccharide,that endow fermented products with flavor,texture and shelf life.Recently,researchers have reported that some lactic acid strains are beneficial to human health.Lactobacillus casei,as a lactic acid bacteira,is an important industrial microorganism for dairy fermented products.Nowadays,the development of whole genome sequencing technology and functional genomics have provided the theoretical basis for the functional analysis of hypothetical proteins,the development of metabolic engineering and synthetic biology in Lactobacillus casei However,genome editing for L.casei are mainly using inefficient and cumbersome methods based on the homologous double crossover,which hinder the development of the above researches.Moreover,L.casei could act as cell factories for the production of antigens,bioactive molecules and some enzymes.However,to employ L.casei as a cell factory,many problems should be solved,for example,it is still unable to efficiently design and reduce the genome to construct optimal cell factories.The sufficient quantity of recombinant protein cannot be expressed from chromosome to overcome the low genetic stability of expression of recombinant protein on plasmid.It is inefficient to screen the prophage deleted strains,so as to prevent the fermentation abortion caused by the prophage induced from the chromosomes by the various stresses during fermentation process.In this thesis,we used L.casei as the cell host to focus on the construction of genome editing tools to solve the problems encountered in each of the above application areas.Firstly,a prophage-derived recombinase operon LCABL 13040-50-60 was identified by bioinformatic analysis,and its recombination function was confirmed by in vivo experiments.Based on this prophage-derived recombinase operon LCABL₁3040-50-60,a two-plasmids genome editing tool was developed and used for gene deletion?s?,insertion?s?and precision point mutations,while a single-plasmid genome editing tools was also constructed and used for metabolic engineering in L.casei.Secondly,by combination of the LCABL 13040-50-60 recombineering system with the Cre/loxP site-specific recombination system,both a simplified large-scale genome deletion system and a targeted and repetitive chromosomal integration tool were also established,which provided two effective systems respectively for the construction of ideal cell factories by designing and reducing genome and the hyper-production of chromosomal recombinant proteins.Moreover,the CRISPR/Cas9 system was demonstrated to be functional in L.casei,and used for screening a series of prophage deleted strains to study the potential of prophage deleted strains as cell factories in industrial fermentation.Furthermore,a counterselectable mutagenesis system PheS*/pG+host9 was developed for seamless gene deletions in Lc.lactis.The detailed contents and results are listed as follows:1.Identification and functional analysis of potential prophage-derived recombinases LCABL 13040-50-60 for genome editing in Lactobacillus caseiGenome editing is necessary for the discovery of novel functional genes and the modification of metabolic pathways.However,genome editing tools for L.casei is inefficient and cumbersome.In this study,a ? Red-like recombinase operon LCABL 13040-50-60 was identified from a prophage PLE3 in Lactobacillus casei BL23 genome by bioinformatic analysis,and its recombination function was confirmed by replacement of a 167 bp galK fragment with chloramphenicol-resistant gene in the L.casei BL23 genome.Further functional analysis showed that LCABL₁3040 and LCABL 13060 were analogs to the host nuclease inhibitor?Red??and 5'-3' exonuclease?Red?/RecE?.respectively.After optimization of recombineering conditions,including induction,homology length,recovery time and double-strand DNA?dsDNA?substrates quantity,the recombineering efficiency reached?2.2×10-7.Subsequently,combining Cre/loxP technology,the optimal LCABL₁3040-50-60 proteins could catalyze markerless deletion of a 167 bp galK fragment and insertion of the gfp gene as well as precision point mutation of rpoB gene in the L.casei BL23 genome.Moreover,with the assistance of Red?,the LCABL 13040-50-60 proteins also showed recombinase activity in six other L.casei strains,L.paracasei OY and L.plantarum WCFS1.All the results demonstrated that the prophage-associated recombinases LCABL 13040-50-60 have great potential to be used for genome editing in LAB,providing an efficient method for the functional analysis of hypothetical proteins,the development of metabolic engineering and synthetic biology in LABs.2.A single-plasmid genome editing system based on prophage recombinases LCABL 13040-50-60 for metabolic engineering of Lactobacillus caseiAlthough the two-plasmid genome editing system established in this thesis is efficient,it is still time-consuming for the sequentially deletion of multiple genes in metabolic engineering.In this part,we developed a single-plasmid and easy-to-use genome engineering strategy for metabolic engineering of L.casei for acetoin production.Plasmid pMSP456-Cre,that contains prophage recombinase operon LCABL₁3040-50-60 driven by the nisin-controlled inducible expression?NICE?system and the site-specific recombinase gene cre under the control of the promoter of the lactose operon from L.casei,was constructed.Using this plasmid,integration of a hicD3 gene linear donor cassette?up-lox66-cat-lox71-down?was catalyzed by the LCABL 13040-50-60 recombinase and the cat gene was excised by the Crx/loxP system with an efficiency of 60%.To demonstrate this system for sequential and iterative knocking out genes in L.casei.another three genes?pflB,Idh and pdhC?related to acetoin production were deleted with the efficiencies of 60%,40%and 60%,respectively.The yielding quadruple mutant could produce a?1 8-fold higher amount of acetoin than the wild-type and converted 59.8%of glucose to acetoin in aerobic.Therefore,these results proved this simple genome engineering strategy have potential in metabolic engineering of L.casei for production of high value-added metabolites.3.Coupling the LCABL 13040-50-60 to Cre/loxP system enables simplified large-scale genome deletion in Lactobacillus caseiIn recent years,designing and streamlining of the genome to construct optimal cell factories is receiving more and more attention.However,the genome editing tools constructed in the first two parts are inefficient for large-scale genome deletions,limiting the rapid and efficient genome reduction of L.casei.Here,we proposed a genome minimization strategy based on LCABL 13040-50-60 recombineering and Cre/loxP 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 kb 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.To 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 for production of high value-added metabolites.4.Targeted and repetitive chromosomal integration enables high yield ofheterologous gene expression in Lactobacillus caseiThe problem of low genetic stability of recombinant proteins expressed by plasmids can be solved by expressing recombinant proteins by chromosomes in microbial cell factories.However,the yields of the recombinant protein expressed by the L.casei engineered strains constructed using the existing chromosomal integration method was not enough for the actual production.Here,we developed a two-step integration strategy in L.casei by combination of the LCABL 13040-50-60 recombineering system with the Cre/loxP site-specific recombination system,with an efficiency of?3.7×103 CFU/?g DNA.A gfp gene was successfully integrated into six selected chromosomal sites,and the relative fluorescence intensities?RFU?of the resulting integrants varied up to?3.7-fold depending on the integrated site,among which the LCABL₀7270 site gfp integration showed the highest RFU.However,integrants with gfp gene?s?integrated into the LCABL 07270 site showed varying RFUs,from 993±89 to 7,289±564,corresponding to 1 to 13.68±1.08 copies of gfp gene integration.Moreover,the integrant with 13.68± 1.08 copies of the gfp gene had a stable RFU after 63 generations compared with a plasmid-engineered strain.To investigate the feasibility of this system for bioactive molecules with high expression levels,the fimbrial adhesin gene,faeG,from Escherichia coli was tested and successfully integrated into the LCABL₀7270 site with 5.51±0.25 copies,and the integrated faeG achieved stable expression.All results demonstrate that this two-step integration system could achieve a high yield of heterologous gene expression by repetitive integration at a targeted chromosomal location,providing an effective method for L.casei cell factories to express chromosomal heterogenous proteins.5.CRISPR/Cas9 system for screening prophage deleted strains in LactobacilluscaseiScreening prophage deleted strains of L.casei as cell factories for industrial fermentation can avoid fermentation abortion caused by prophage excision from the chromosomes.However,it is inefficient to screen prophage deleted strains with current methods.Here,we explored the impacts of the prophages?PLE1,PLE2 and PLE3?on the host survival at the hostile environments in Lactobacilus casei BL23 by simply selection of prophage deletion variants using type II CRISPR/Cas9 system from Streptococcus pyogenes.Firstly,introduction of a gene galK or plasmid pNZ8148 targeting plasmid into L.casei BL23 or L.casei BL23/pNZ8148 led to a strong reduction in the number of transformants obtained compared to that of a non-targeting plasmid,demonstrating the feasibility and lethality of the CRISPR/Cas9 system in L.casei.Subsequently,three single prophage deletion mutants?BLP1,BLP2 and BLP3?were respectively selected by introduction of prophage PLE1.PLE2 and PLE3-targeting plasmids to break the chromosome of wild-type strain L.casei BL23.Finally,a triple prophage deletion strain?BLP123?was also screened by a sequential manner.The survival rates of the prophage deletion derivates BLP123 and the wild-type strain under various environments were compared and found that the survival rates of BLP123 was almost similar to that of the wild-type strains,demonstrationg that BLP123 has the potential to be used as a cell factory.6.Development of a counterselectable seamless mutagenesis system in lactic acid bacteriaNowadays,molecular tools for genetic modifying LAB strains are mainly vector-based double-crossover strategies,which are laborious and inefficient.To address this problem,several counterselectable markers have been developed,while few of them could be used in the wild-type host cells without pretreatment.The gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome.When mutant pheS gene?pheS*?under the control of the Lc.lactis NZ9000 L-lactate dehydrogenase promoter?Pldh?was expressed from a plasmid,the resulted PheS*with an A312G substitution rendered cells sensitive to the phenylalanine analog p-chloro-phenylalanine?p-Cl-Phe?.This result suggested pheS*was suitable to be used as a counterselectable marker in Lc.lactis.However,the expression level of pheS*from a chromosomal copy was too low to confer p-Cl-Phe sensitivity.Therefore,a strategy of cascading promoters was attempted for strengthening the expression level of pheS*.Expectedly,a cassette 5Pldh-pheS*with five tandem repetitive promoters Pldh resulted in a sensitivity to 15 mM p-Cl-Phe.Subsequently,a counterselectable seamless mutagenesis system PheS*/pG+host9 based on a temperature-sensitive plasmid pG+host9 harboring a 5Pldh-pheS*cassette was developed in Lc.lactis.We also demonstrated the possibility of applying pheS*to be a counterselectable marker in Lactobacillus casei BL23.As reported in E.coli,pheS*as a counterselectable marker has been demonstrated to be functional in targeted gene?s?deletion in Lc.lactis as well as in L.casei.Moreover,the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG+host9 has potential to be applied to other LAB strains and provides an effective way for the development of metabolic engineering and synthetic biology in lactic acid bacteria.