High level production of diacetylchitobiose deacetylase by refactoring genetic elements and cellular metabolism

• High levels of Dac secretion were realized by signal peptide engineering. • The efficient expression of Dac were realized by RBS sequence optimization. • The stable and efficient expression of Dac was achieved through genomic integration. • B. subtilis C6 was constructed by CRISPR/Cpf1 system to facilitate gene editing. • The extracellular Dac activity(6357.38 U/mL) was the highest level reported so far. Diacetylchitobiose deacetylase (Dac) from Pyrococcus horikoshii can realize the one-step production of glucosamine (GlcN). The efficient expression and secretion of Dac play a central role in the green production of GlcN. In this study, Bacillus subtilis WB600 was used as the expression host. Firstly, we screened 12 signal peptides, among which signal peptide NprB had the strongest ability of guiding Dac secretion. Further optimization of the functional region showed that the extracellular Dac activity of NprB mutant was increased to 3682.2 U/mL. Next, the extracellular Dac activity was increased to 4807.6 U/mL by RBS sequence optimization. Then we got a new recombinant B. subtilis C6 for plasmid-free expression of Dac by integrating comK gene and silencing bpr , nprB , aprE , mpr and nprE genes. Finally, the extracellular Dac activity of genome-integrating strain reached 6357.38 U/mL, which was the highest level reported so far.