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Abstract: [Background] Glucosamine (GlcN) and its derivative N-acetylglucosamine (GlcNAc) are important precursors for the synthesis of glycosaminoglycans, which have a wide range of applications in the fields of medicine, cosmetics, and healthcare products. The traditional production methods of GlcN and GlcNAc have many drawbacks, such as environmental pollution, raw material limit, and unsuitability for people with seafood allergies. Therefore, the microbial fermentation for producing GlcN and GlcNAc has attracted increasing attention. [Objective] To explore the molecular modification for producing GlcNAc by microbial fermentation and optimize the fermentation conditions for increasing the production. [Methods] Firstly, the glmS from Escherichia coli MG1655 and gna1 from Saccharomyces cerevisiae were co-expressed in E. coli MG1655 by the pTrcHisA vector. CRISPR/Cas9 was then employed to knock out the genes responsible for GlcN and GlcNAc transport and metabolism to improve the production of GlcNAc. Finally, the fermentation conditions were optimized to further increase the production of GlcNAc. [Results] The RY-5 strain was constructed by the molecular modification. The production of GlcNAc by RY-5 reached 2.36 g/L after shake flask fermentation for 20 h, which was 29 times that by RY-1 strain. After optimization of the fermentation conditions, the production of GlcNAc reached 7.74 g/L, which increased by 2.3 times compared with that before optimization. [Conclusion] This study successfully developed a recombinant E. coli strain producing GlcN and GlcNAc, which launched a foundation for the industrial production of GlcN and GlcNAc by microbial fermentation.
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