An α2,3‐sialyltransferase from Photobacterium phosphoreum with broad substrate scope: Controlling hydrolytic activity by directed evolution

Defined sialoglycoconjugates are important molecular probes to study the role of sialylated glycans in biological systems. We show that the  α 2,3-sialyltransferase from  Photobacterium phosphoreum  JT-ISH-467 (2,3SiaT pph ) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution and C5 stereoinversion. To reduce the enzyme's hydrolytic activity that erodes the product yield, an extensive structure-guided mutagenesis study identified 3 variants that show up to 5 times higher catalytic efficiency for sialyltransfer, up to 10 times lower efficiency for substrate hydrolysis and drastically reduced product hydrolysis. Variant 2,3SiaT pph (A151D) displayed overall best performance in the synthesis of the GM3 trisaccharide ( α 2,3-Neu5Ac-Lac) from lactose in a one-pot, two-enzyme cascade. Our study demonstrates that several complementary solutions can be found to suppress the common problem of undesired hydrolysis activity of microbial GT80 sialyltransferases. The new enzymes are powerful catalysts for the synthesis of a wide variety of complex natural and new-to-nature sialoconjugates for biological studies .