Screening lactic acid bacteria with high production of AI-2 and cloning and expression of the Pfs gene

Abstract:

[Objective] The study in order to screen the strain with high yield of autoinducer-2 (AI-2) of lactic acid bacteria strains which isolated from koumiss of Ximeng region in Inner Mongolia, and optimize the condition of recombinant protein to synthesize signal molecule AI-2 in vitro. [Methods] Using biological luminescence method to compare the contents of signal molecule AI-2 which produced by different lactic acid bacteria, with high production of signal molecule AI-2 lactic acid bacteria genomic DNA as a template, expanded its S-adenosine homocysteine nucleoside enzyme (Pfs) gene to build the prokaryotic expression vector. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce expression of recombinant proteins. The culture medium, induction temperature, the density of bacteria, IPTG concentration and inducing time were optimized to get the high expression of Pfs protein, and finally synthesized signal molecule AI-2 in vitro. [Results] Ten strains of lactic acid bacteria could produce AI-2, and the relative luminescence intensity of AI-2 secreted by Enterococcus faecium 8-3 was obviously higher than other strains. The optimal inducing condition of the recombinant protein was as follows: when the OD600 was 0.5–0.7, using SOC medium as the induction medium, the induction were initiated with 0.1 mmol/L IPTG at 37 °C for 12 h; The optimal inducing condition was used to induce the target protein and obtained the purified Pfs protein with the concentration of 4.08 g/L, and successfully synthesized AI-2 in vitro. [Conclusion] The strains of ten lactic acid bacteria isolated from Koumiss could produce AI-2 and the signal molecule AI-2 of Enterococcus faecium 8-3 could be synthesized by Pfs gene.