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Glycosaminoglycans (GAGs) are extensively utilized in clinical, cosmetic, and healthcare field, as well as in the treatment of thrombosis, osteoarthritis, rheumatism, and cancer. The biological production of GAGs is a strategy that has garnered significant attention due to its numerous advantages over traditional preparation methods. In this review, we embark on a journey to decode the intricate molecular symphony that orchestrates the biosynthesis of glycosaminoglycans. By unraveling the complex interplay of related enzymes and thorough excavation of the intricate metabolic cascades involved, GAGs chain aggregation and transportation, which efficiently and controllably modulate GAGs sulfation patterns involved in biosynthetic pathway, we endeavor to offer a thorough comprehension of how these remarkable GAGs are intricately assembled and pushes the boundaries of our understanding in GAGs biosynthesis. • The enzymes involved in GAGs synthesis pathways are summarized. • Microbial production systems and its synthesis mechanism are discussed. • The sulfation model involved in GAGs biosynthesis pathway was analyzed. [ABSTRACT FROM AUTHOR]

Industrial biotechnology employs cells for producing valuable products and serving as biocatalysts sustainably, addressing resource, energy, and environmental issues. Bacillus subtilis is a preferred host for creating microbial chassis cells and producing industrial enzymes and functional nutritional products. In this study, a dual-module T7 integration expression system in B. subtilis was established. The first module, driven by the T7 RNA polymerase, was integrated into the genome via the CRISPR/Cas9 system. Another module responsible for expression control was systematically integrated into 28 discrete chromosomal loci and the impact of different genomic positions on gene expression was explored, resulting in a high-intensity integrated expression system. Furthermore, by modifying the LacI repressor factor for biological regulation, we achieved a strong expression intensity without the inducer addition. This system was successfully used to express phospholipase D and hyaluronic acid lyase, resulting in extracellular enzyme activities of 339.12 U/mL and 2.60 × 10 4 U/mL, respectively. Additionally, by exclusively targeting the HA gene cluster for expression, a production yield of 6.86 g/L was achieved on a 5 L fermentation scale. The system eliminates the use of antibiotics and inducers, offering a controllable, efficient, and promising gene expression regulation tool in B. subtilis , enhancing its potential for biomanufacturing applications.