Regulation of tagatose catabolic gene cluster and development of a tagatose-inducible gene expression system in the probiotic Escherichia coli Nissle 1917

Escherichia coli Nissle 1917 (EcN), a well-known Gram-negative probiotic bacterium, has been widely used to treat various intestinal disorders and has gained recognition as a promising platform for diverse biotechnological applications, validated by numerous institutions and researchers over the years However, despite its importance, developing suitable expression systems in EcN has been challenging due to the difficulty in meeting key criteria such as non-toxicity, biocompatibility, and tunable expression. Recently, we identified a gene cluster in EcN responsible for tagatose utilization, challenging the conventional belief that E. coli cannot metabolize tagatose. D-tagatose, a rare, low-calorie sugar naturally found in small amounts in dairy products and fruits, is recognized as safe by the FDA. Due to its poor digestibility in humans, only certain enteric microorganisms are capable of metabolizing it. In this study, we investigated the regulatory elements within this gene cluster using high-throughput differential RNA sequencing (dRNA-seq) and developed a tagatose-inducible expression plasmid, creating a tunable gene expression system for engineering EcN. We evaluated the performance of this novel system and successfully applied it to overproduce a pharmaceutical protein and an industrial enzyme under both aerobic and anaerobic conditions. Sample from the exponential phase of modified R (MR) medium containing D-tagatose were collected for total RNA extraction. The isolated RNA was then used for differential RNA sequencing (dRNA-seq) library preparation, following the established protocol.