| Streptococcus thermophilus is a common yogurt starter and the second most used bacterium in the dairy industry.It has been found to ferment milk together with Lactobacillus delbrueckii subsp.bulgaricus for thousands of years.Furthermore,S.thermophilus is also considered a probiotic,which can reduce the risk of pathology and exert beneficial effects after entering the human intestine.Therefore,S.thermophilus is a good candidate for the production of heterologous proteins and a potential microbial cell factory.Food-grade vectors have advantages in heterologous protein expression.However,the food-grade vectors in this species are still relatively few.Therefore,a novel food-grade expression vector needs to be developed.Lactose is the main carbon source for the growth of S.thermophilus.Lactose transportation,metabolism,and the underlying regulatory mechanism have been extensively studied in S.thermophilus.First,lactose is carried into the cytoplasm via lactose permease.Second,lactose is hydrolyzed into glucose and galactose by intracellular β-galactosidase.Glucose is converted into pyruvate through the Embden-Meyerhoff-Parnas pathway,and pyruvate is mainly converted into lactic acid by lactate dehydrogenase.While galactose is usually only weakly metabolized by S.thermophilus and hence released into the medium via the same lactose permease.Therefore,lactose permease and β-galactosidase are essential for the lactose metabolism and the growth of S.thermophilus.In this study,a novel food-grade expression vector was attempted to constructed based on the lactose metabolism of S.thermophilus,which is mainly reflected in the following aspects:(1)α-Complementation phenomenon of β-galactosidase in S.thermophilus.First,a mutant strain S.thermophilus Δα with deletion of 7-36 amino acid residues at the Nterminal of S.thermophilus β-galactosidase was constructed.S.thermophilus Δα lost the ability of lactose utilization and β-galactosidase activity.Subsequently,a series of vectors expressing α-segment with different lengths of 1-36(Sα1),1-53(Sα2),and 1-88(Sα3)amino acids were constructed and transformed into S.thermophilus Δα.The growth andβ-galactosidase activity of the recombinant strains were determined.The recombinant S.thermophilus Δα expressing Sα2 and Sα3 recovered the growth ability in lactose medium.Their β-galactosidase activity accounted for 24.50% and 11.50% of the wild strains,respectively.To figure out the growth ability of complementation strains in natural environments,recombinant S.thermophilus was inoculated into pasteurized milk.Subsequently,L.bulgaricus A101 was used to co-culture with S.thermophilus.The results showed that recombinant S.thermophilus could utilize milk to grow and produce acid.In conclusion,the α-complementation system of β-galactosidase existed in S.thermophilus.(2)Construction of a food-grade expression vector based on α-complementation phenomenon in S.thermophilus.A food-grade vector p SEα was constructed.The antibiotic resistance gene was removed from p SEα,and the Sα2 coding gene of S.thermophilus was used as a selection marker.In addition,the food-grade vector also contains an α-peptide from Escherichia coli β-galactosidase,which facilitated the construction and screening of recombinant plasmids in E.coli DH5α and thus improved the transformation efficiency of S.thermophilus.Due to the α-complementation phenomenon of β-galactosidase that existed both in S.thermophilus and E.coli,their interaction was explored.These results indicated that α-peptides could only complement their own β-galactosidases.Thus,there was no interaction between the two α-complementary systems of S.thermophilus and E.coli.A vector p SEα-Pldh-gfp expressing green fluorescent protein was constructed to test the expression ability of the food-grade vector.Green fluorescent protein as a reporter protein could be highly expressed in S.thermophilus using this vector,and the fluorescence intensity reached a maximum of 120000±14900 RFU.Therefore,p SEα is an effective and safe vector for S.thermophilus with potential applications.(3)The food-grade expression vector p SEα expressing glutamate decarboxylase.The glutamate decarboxylase gene gad B from Bifidobacterium adolescentis was identified by bioinformatics method.First,glutamate decarboxylase was overexpressed in E.coli to analyze the enzymatic properties,and then cloned into the food-grade expression vector.The vector p ET28b-gad B expressing glutamate decarboxylase from B.adolescentis B3 was constructed and transformed into E.coli BL21 for overexpression.The enzyme was determined to be 53 k Da by SDS-PAGE.The determination of enzymatic properties showed that the optimum reaction temperature was 30 ℃,and the optimum p H was 6.0.However,the temperature and p H stability were poor.Subsequently,a vector p SEα-Pldh-gad B expressing glutamate decarboxylase was constructed.The glutamate decarboxylase activity of S.thermophilus Δα/p SEα-Pldh-gad B reached16.35±0.80 U/m L,which was 5.88 times that of S.thermophilus Δα/p SEα.These results indicated that the vector p SEα could express glutamate decarboxylase in S.thermophilus,laying a foundation for the application of food-grade expression vectors.It could be used as a potential tool for enhancing dairy product performance and developing functional yogurt in the future.(4)Preliminary exploration of lactose/ galactose permease as a screening marker in S.thermophilus.The lactose permease-deficient mutant S.thermophilus Δlac S was constructed.S.thermophilus Δlac S lost the ability to utilize lactose and galactose.Subsequently,complementation vectors expressing intact lactose permease and its Nterminal 1-486 amino acid residues of S.thermophilus,and expressing E.coli lactose permease and galactose transportase were constructed and transformed into S.thermophilus Δlac S.The growth of the complementation strains showed that the strain expressing S.thermophilus intact lactose permease recovered the growth ability in both lactose and galactose medium.The strain expressing S.thermophilus N-terminal lactose permease recovered the growth ability only in lactose medium.The transformation of S.thermophilus with a complementation vector expressing E.coli lactose permease has been unsuccessful.The strain expressing E.coli galactose transportase recovered the growth ability only in galactose medium.These results laid a theoretical foundation for the construction of an expression vector using lactose/ galactose permease as a food-grade selection marker,which could further broaden the research toolbox in S.thermophilus. |
