Fig. 1: Construction of the PAPS regeneration system.

a Natural PAPS synthesis and decomposition pathways in E. coli. b The design of the PAPS regeneration pathway. c Enzyme activity comparison between different PPK2s using AMP as a substrate. PPK2A^p.a. PPK2B^p.a, PPK2C^p.a, PPK2C^p.a-N (ppk2C^p.a(A1-G753)), and PPK2C^p.a-C (ppk2C^{p.a(C748-G1491)}) are from P. aeruginosa, PPK2^r.p is from R. pomeroyi, PPK2^s.e is from S. epidermidis, and PPK2^d.r is from M. ruber. The catalytic reaction time was 60 min. d Comparison of the affinity of PPK2^s.e to AMP and ADP. e PAP 3’-dephosphatase enzyme activity assays from different species. cgCysQ, C. glutamicum. ecCysQ, E. coli str. K-12 substr. MG1655. paCysQ P. aeruginosa PAO1. scCysQ, S. cerevisiae S288C. kpCysQ, K. phaffii GS115. Significance (P value) was evaluated by two-sided t-test, *, **, *** denote P value < 0.05, <0.01, <0.001, respectively. f Conformation that PAP 3′-dephosphatase from K. phaffii exhibits no PAPS degradation activity. ATP, adenosine 5'-triphosphate. ADP, adenosine 5′-diphosphate. AMP, adenosine 5′-monophosphate. APS, adenosine-5′-phosphosulfate. PAPS, 3′-phosphoadenosine-5'-phosphosulfate. PAP, 3′-phosphoadenosine-5′-phosphate, polyP₆, hexametaphosphate. PPK2, polyphosphate kinase family 2. ASAK, bifunctional ATP sulfurylase-APS kinase. All the data are expressed as the mean ± S.D. from three (n = 3) biologically independent replicates. Source data are provided as a Source Data file.