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

• Published in: Biotechnology and bioengineering Vol. 118; no. 11; pp. 4503 - 4515
• Main Authors: Liu, Kaifang, Chen, Xiulai, Zhong, Yunlu, Gao, Cong, Hu, Guipeng, Liu, Jia, Guo, Liang, Song, Wei, Liu, Liming
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.11.2021
• Subjects: 3′‐phosphoadenosine‐5′‐phosphosulfate; Adenosine diphosphate; Adenosine kinase; Adenosine triphosphate; Adenosine Triphosphate - metabolism; ATP; Biomedical materials; Bioreactors; Biosynthesis; Catalytic converters; catalytic efficiency; Chondroitin sulfate; Chondroitin Sulfates - biosynthesis; Chondroitin Sulfates - genetics; Conformation; E coli; Enzymes; Escherichia coli - genetics; Escherichia coli - metabolism; Expulsion; Fungal Proteins - genetics; Fungal Proteins - metabolism; Fungi; In vivo methods and tests; Kinases; Molecular dynamics; pathway design; Penicillium chrysogenum - enzymology; Penicillium chrysogenum - genetics; Phosphoadenosine Phosphosulfate - biosynthesis; Phosphoadenosine Phosphosulfate - genetics; Phosphotransferases (Alcohol Group Acceptor) - genetics; Phosphotransferases (Alcohol Group Acceptor) - metabolism; protein engineering; Sulfates; Sulfotransferase; pathway design; protein engineering; 3′-phosphoadenosine-5′-phosphosulfate; chondroitin sulfate; catalytic efficiency
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.27919

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 adenosine triphosphate (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 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. An efficient and controllable catalytic system was designed and constructed for the conversion of ATP into PAPS. Based on analysis of the catalytic system, an “ADP expulsion” strategy was developed to change the enzyme's flexibility and lid structure to a more open conformation and improve the catalytic efficiency by 73.27 times. 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%.