| Probiotics are "living microorganisms" that are beneficial to the health of host.After reasonable genetical modification,probiotics can have better physiological effects and more efficient biosynthesis ability in the field of biotherapy and industrial production.Polysaccharides are biopolymers with specific characteristics and physiological functions.They can not only be biosynthesized as metabolites in probiotics but also used as biomaterial to effectively solve the bottleneck in the application of probiotics.Therefore,there is a mutually beneficial relationship between the biosynthesis of polysaccharides and the application of probiotics.Streptococcus thermophilus is a traditional probiotic with the characteristics of favorable biological safety and the potential for polysaccharides production,but it has the defects of low polysaccharides production efficiency,weak biological activity,poor stability,and single function.Therefore,it is of great significance to develop new S.thermophilus strains for achieving efficient polysaccharides production and excellent physiological function.Hyaluronic acid is a glycosaminoglycan that exists in the bacterial capsule.It is not only allowed to be used as food material and dietary supplement,but also as biomaterial to maintain the activity and stability of probiotics.However,the production and application of food-grade hyaluronic acid need further study.Based on the above research background,this thesis research successfully constructed the engineered probiotic strains coated with hyaluronic acid shell by using synthetic biology technologies to modify the metabolic pathways in S.thermophilus.At the same time,the engineered S.thermophilus showed good hyaluronic acid biosynthesis ability,enhanced biological activity,increased stability,and better probiotic effects.The main research contents and results are as follows.(1)Levansucrase was identified as a negative selection marker,and 10%sucrose concentration was determined as the screening condition in S.thermophilus LMD-9.A traceless operation cassette for gene manipulation was successfully constructed based on the negative selection marker levansucrase.In order to avoid false-positive strains in the screening process,200 bp was defined as the optimal length of direct repeat(DR)in the operation cassette.The optimized traceless genetic operation system could complete different types of genome modification(knockout,mutation,and insertion),and it would not leave marks to realize safe and efficient genome editing in S.thermophilus.(2)Based on the analysis of endogenous CRISPR/Cas gene clusters and the verification of the self-targeting in S.thermophilus LMD-9,the CRISPR1/Cas system was determined as the modified object to establish a tool for regulating gene expression.The endogenous Cas protein Sthl was successfully modified into dCas by mutating the key amino acids using the traceless genetic modification system.The expression of dCas was further optimized by promoter substitution.The construction of CRISPR/dCas system was completed after introducing the corresponding sgRNA.The green fluorescent protein reporting system showed that the CRISPR/dCas system could effectively inhibit the expression of target genes,and the design of sgRNAs would affect the efficiency of interference.(3)The established traceless genetic modification and CRISPR/dCas systems were used to reform the metabolic pathway of hyaluronic acid in S.thermophilus LMD-9.By optimizing the expression of hyaluronic acid synthase,strengthening the biosynthetic pathway of precursors,and weakening the competition of by-products(exopolysaccharides and lactic acid),the biosynthesis ability of the original strain with 3.40 mg/L hyaluronic acid had been continuously enhanced.The yield of hyaluronic acid in engineered strain L1 reached 77.83 mg/L,and the molecular weight was 496.67 kDa.(4)The biosynthesis of endogenous hyaluronic acid achieved self-encapsulation of the microorganism by characterizing the morphology of the engineered strain L1.The evaluation of the resistance to gastrointestinal fluid in vitro confirmed that the hyaluronic acid coating could effectively protect the bacteria and maintain the complete cell morphology.Through the optimization of fermentation conditions(2%sucrose as carbon source,100 r/min,37℃ for 30 h),the biosynthesis ability of hyaluronic acid in engineered S.thermophilus L1 was further enhanced.The fermented milk of engineered strain L1 contained about 5.1×108 CFU/mL engineered bacteria and 200 mg/L hyaluronan with a molecular weight of 1133.33 kD,and the fermented milk showed good storage stability at low temperature.Moreover,the S.thermophilus L1 coated with endogenous hyaluronic acid in fermented milk showed enhanced gastrointestinal fluid resistance than that in M17 medium.(5)Hemolysis tests,body weight monitoring,and histological observation confirmed that the fermented milk prepared by self-encapsulated S.thermophillus L1 had excellent biological safety.The fermented milk of engineered strain L1 made a good combination of S.thermophilus and hyaluronic acid.On one hand,compared with the wild-type strain,the engineered strain L1 exhibited greater survival and longer colonization time in the gut due to the protection of hyaluronic acid shell;On the other hand,hyaluronic acid in fermented milk could be absorbed,and the engineered probiotic strain could also produce hyaluronic acid after intestinal colonization.The fermented milk of engineered strain L1 showed positive effects on strengthening the intestinal barrier and regulating intestinal microorganisms related to the biosynthesis of short-chain fatty acids through staining analysis of intestinal tissue sections,16S rDNA sequencing,and metabolite determination of mouse feces.To sum up,the engineered S.thermophilus coated with endogenous hyaluronic acid promotes the further combination of the application of S.thermophilus and the biosynthesis of hyaluronic acid,which not only provides an ideal reference for the effective and multifunctional oral administration of probiotics,but also provides an excellent cell factory for the fermentation preparation of food-grade hyaluronic acid and other biological products. |
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