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

Abstract

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_st and a promoter region of cation transporter czcD (P_czcDst). Specific binding of the SczA_st to the palindromic sequences in P_czcDst was demonstrated by EMSA analysis, suggesting the regulation role of SczA_st on P_czcDst. To evaluate their possibility to control gene expression in vivo, the sczA_st-P_czcDst 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.

Keywords: Engineered probiotic; Food-grade expression system; Lactococcus lactis; Streptococcus thermophilus.