Development of Zn^(2+)-controlled expression system for lactic acid bacteria and its application in engineered probiotics

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.

Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli(ETEC) K88-infected piglet

Engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties.In this study,we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32(WB800-KR32)using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2(Nrf2)-Kelch-like ECH-associated protein 1(Keap1)pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli(ETEC)K88 in weaned piglets.Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet.The feed of the control group(CON)was infused with normal sterilized saline;meanwhile,the ETEC,ETEC+WB800,and ETEC+WB800-KR32 groups were orally administered normal sterilized saline,5×10^(10)CFU(CFU:colony forming units)WB800,and 5×10^(10)CFU WB800-KR32,respectively,on Days 1-14 and all infused with ETEC K881×10^(10)CFU on Days 15-17.The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance,improved the mucosal activity of antioxidant enzyme(catalase(CAT),superoxide dismutase(SOD),and glutathione peroxidase(GPx))and decreased the content of malondialdehyde(MDA).More importantly,WB800-KR32 downregulated genes involved in antioxidant defense(GPx and SOD1).Interestingly,WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum.WB800-KR32 markedly changed the richness estimators(Ace and Chao)of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces.The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway,providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.