Development of Zn²⁺-controlled expression system for lactic acid bacteria and its application in engineered...

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 Zn²⁺-controlled gene expression (ZICE) system was identified in the genome of S. thermophilus CGMCC7.179, including a transcriptional regulator sczA and a promoter region of cation transporter czcD (P). Specific binding of the SczA_st to the palindromic sequences in P was demonstrated by EMSA analysis, suggesting the regulation role of SczA_st on P. To evaluate their possibility to control gene expression in vivo, the sczA-P 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 Zn²⁺ induction, while no fluorescence without Zn²⁺ addition. For optimal conditions, Zn²⁺ 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 Zn²⁺, having potential in development of engineered probiotics.