Engineering a novel adenine-sulfotransferase for efficient synthesis of PAPS and chondroitin sulfate in microbial cells

Sulfonated compounds are widely utilized in feed additives, daily commodities, industrial manufacturing, and healthcare applications. Their production relies on the sulfonate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS). This study identified a novel adenine-sulfotransferase with sulfotransferase activity toward adenosine monophosphate for adenosine phosphosulfate formation. The identified enzyme was rationally engineered, yielding the mutant Bta APSST H8M/L117D , which exhibited a 0.93-fold increase in sulfotransfer efficiency. The mutant Bta APSST H8M/L117D was subsequently combined with additional enzymes to reconstruct what we term the RPA pathway, enabling the synthesis of PAPS at titers of 7.6 g/l and 5.03 g/l in Escherichia coli and Bacillus subtilis , respectively, using adenine and ribose as substrates. The RPA pathway was further integrated into the chondroitin producing strain E. coli GZ17, to construct E. coli CSA-02, which produced 1.89 g/l chondroitin sulfate A (CSA) with a sulfation rate of 76%. These results offer a promising way to enhance the biosynthesis of sulfonated compounds in microbial cell factories.