Marker‐Free System Using Ribosomal Promoters Enhanced Xylose/Glucose Isomerase Production in Streptomyces rubiginosus

Xylose/glucose isomerases are important industrial enzymes that are most widely used in food industries; however, their previously reported expression levels do not meet the requirements for industrial application. Here, an antibiotic resistance marker (ARM)‐free system driven by ribosomal RNA (rRNA) promoters is developed to obtain high‐level xylose/glucose isomerase (XI/GI) expression in Streptomyces rubiginosus ( S. rubiginosus ). The rRNA promoter rrn D yields the highest glucose isomerase production titer of XIs/GIs, which is eight times higher than that of ermEp * and 2.6 times higher than that of kasOp *. The integrated ARM gene is removed by further introduction of the Cre plasmid with a temperature‐sensitive replicon. The production titer of XIs/GIs is further improved by replacing the xylR gene with an additional expression glucose isomerase cassette at the xylR locus. Ultimately, the glucose isomerase activity reaches up to 79.7 ± 7.5 U mL −1 at 96 h. The results support the robustness and stability of XI/GI production with this ARM‐free system using optimal ribosomal promoters in S. rubiginosus , demonstrating strong potential in large‐scale industrial applications. Besides, the results imply that rRNA promoters are strong promoters that can be used for protein engineering or metabolic engineering.