Construction of EcN-engineered bacteria for targeted utilization of alginate oligosaccharides and its probiotic evaluation

• 1、School of life sciences and health engineering,Jiangnan University,Wuxi 214122

Abstract：Escherichia coli Nissle 1917 (EcN) is a widely studied and applied probiotics. In order to further enhance the colonization ability and probiotic performance of EcN, CRISPR/Cas9 gene editing technology was utilized to construct an engineered probiotics that can selectively utilize alginate oligosaccharides (AOS). This studyinvestigated the integration sites and promoters, and constructed an engineering strain of EcN-MED with a complete AOS metabolic pathway by introducing two key enzymes, the exolytic alginate lyase Oal17A and the 4-deoxy-L-erythro-hexoseulose uronic acid reductase A1-R. After being cultured for 36 hwith AOS as the sole carbon source, the OD600 of the engineered strain EcN-MED increased by 4 times compared to the wild-type.No significant changes obseved in the tolerance of simulated gastrointestinal digestive fluidand intestinal adhesion ability.The probiotic effect of EcN-MED combined with AOS was investigated using co-cultivation method.The engineered bacteria can significantly promote the growth of aprobioticsof Lactobacillus rhamnosus GG and inhibit the proliferation of a pathogenic bacteria of Enterobacter hormaechei, demonstrating good probiotic performances.

Keywords： Escherichia coli Nissle 1917 alginate oligosaccharides engineered probiotics co-culture