High efficient production of adenine in Bacillus subtilis with novel metabolic engineering strategies.

Adenine, serves as a crucial precursor for nucleic acid drugs and food additives, currently suffered from limited availability since its high production costs associated with expensive precursors and enzyme preparations. Here, we developed an effective strategy for high-yield adenine production using Bacillus subtilis as the host. Beginning with an adenosine-producing strain, we first knocked-out the apt and adeC genes, which were involved in product degradation pathways, and deleted the purR gene to alleviate the feedback inhibition on purine operon through CRISPR-Cas9 technology. Then, we overexpressed the nucleoside hydrolase RihC, PRPP amidotransferase PurF, and ribokinase RbsK to enhance the de novo adenine biosynthesis and enable the recycling of D-ribose. The production of adenine reached 18 g/L after the fed-batch fermentation, and the titer continued to reach 78 g/L when adenosine was added as the substrate subsequently, revealing the significant catalytic role of RihC produced during the fermentation. This work innovatively integrated de novo biosynthesis and enzymatic conversion strategies to achieve high yield adenine production in Bacillus subtilis, demonstrating a promising scalable process for industrial adenine manufacturing.