Lactococcus lactis and Streptococcus thermophilus are considered as ideal chassis of engineered probiotics,while food-grade genetic tools are limited in those strains.Here,a Zn^(2+)-controlled gene expression(ZICE)sys...Lactococcus lactis and Streptococcus thermophilus are considered as ideal chassis of engineered probiotics,while food-grade genetic tools are limited in those strains.Here,a Zn^(2+)-controlled gene expression(ZICE)system was identified in the genome of S.thermophilus CGMCC7.179,including a transcriptional regulator sczAst and a promoter region of cation transporter czcD(PczcDst).Specific binding of the SczAst to the palindromic sequences in PczcDst was demonstrated by EMSA analysis,suggesting the regulation role of SczAst on PczcDst.To evaluate their possibility to control gene expression in vivo,the sczAst-PczcDst was employed to drive the expression of green fluorescence protein(GFP)gene in L.lactis NZ9000 and S.thermophilus CGMCC7.179,respectively.Both of the transformants could express GFP under Zn^(2+)induction,while no fluorescence without Zn^(2+)addition.For optimal conditions,Zn^(2+)was used at a final concentration of 0.8 mM in L.lactis and 0.16 mM in S.thermophilus at OD600 close to 0.4,and omitting yeast extract powder in the medium unexpectedly improved GFP expression level by 2.2-fold.With the help of the ZICE system,engineered L.lactis and S.thermophilus strains were constructed to secret cytokine interleukin-10(IL-10)with immunogenicity,and the IL-10 content in the supernatant of the engineered L.lactis was 59.37%of that under the nisin controlled expression system.This study provided a tightly controlled expression system by the food-grade inducer Zn^(2+),having potential in development of engineered probiotics.展开更多
基金financially supported by the National Natural Science Foundation of China(NSFC,Grant No.32372290)the National Key R&D Program of China(2019YFA0906700).
文摘Lactococcus lactis and Streptococcus thermophilus are considered as ideal chassis of engineered probiotics,while food-grade genetic tools are limited in those strains.Here,a Zn^(2+)-controlled gene expression(ZICE)system was identified in the genome of S.thermophilus CGMCC7.179,including a transcriptional regulator sczAst and a promoter region of cation transporter czcD(PczcDst).Specific binding of the SczAst to the palindromic sequences in PczcDst was demonstrated by EMSA analysis,suggesting the regulation role of SczAst on PczcDst.To evaluate their possibility to control gene expression in vivo,the sczAst-PczcDst was employed to drive the expression of green fluorescence protein(GFP)gene in L.lactis NZ9000 and S.thermophilus CGMCC7.179,respectively.Both of the transformants could express GFP under Zn^(2+)induction,while no fluorescence without Zn^(2+)addition.For optimal conditions,Zn^(2+)was used at a final concentration of 0.8 mM in L.lactis and 0.16 mM in S.thermophilus at OD600 close to 0.4,and omitting yeast extract powder in the medium unexpectedly improved GFP expression level by 2.2-fold.With the help of the ZICE system,engineered L.lactis and S.thermophilus strains were constructed to secret cytokine interleukin-10(IL-10)with immunogenicity,and the IL-10 content in the supernatant of the engineered L.lactis was 59.37%of that under the nisin controlled expression system.This study provided a tightly controlled expression system by the food-grade inducer Zn^(2+),having potential in development of engineered probiotics.
