Design and biocatalytic applications of genetically fused multifunctional enzymes.

Gram-scale biocatalytic preparation of the non-natural cofactor nicotinamide cytosine dinucleotide

Microbial synthesis of glycosaminoglycans and their oligosaccharides.

Metabolic engineering of Escherichia coli for the production of an antifouling agent zosteric acid.

The compound 3'-phosphoadenosine-5'-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of ATP (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5'-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided "ADP expulsion" strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme's flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1 ) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor, and achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. Then, the PAPS catalytic system was combined with the chondroitin 4-sulfotransferase (C4ST) using a one-pot method. Finally, chondroitin sulfate was transformed from chondroitin at a conversion rate of 98.75%. This strategy has great potential for scale biosynthesis of PAPS and chondroitin sulfate. This article is protected by copyright. All rights reserved.

Yunlu Zhong

Papers

Rational design of a highly efficient catalytic system for the production of PAPS from ATP and its application in the synthesis of chondroitin sulfate