Closed-Loop System Driven by ADP Phosphorylation from Pyrophosphate Affords Equimolar Transformation of ATP to 3′-Phosphoadenosine-5′-phosphosulfate

Abstract

3′-Phosphoadenosine-5′-phosphosulfate (PAPS) is a universal sulfate group donor for all biological sulfation reactions in living organisms. Ambitions to biomanufacture sulfate-containing compounds such as heparin and chondroitin sulfate also promote the study on PAPS in vitro synthesis. However, the established enzymatic synthesis of PAPS faces hurdles of the natural low theoretical transformation rate of 50% (two ATP to one PAPS) and high cost. Here, we developed a PAPS synthesis route with 100% theoretical transformation rate which affords equimolar transformation of ATP to PAPS. By fusing the identified adenosine 5′-triphosphate sulfurylase and adenosine 5′-phosphosulfate kinase from different species, we created an artificial active bifunctional enzyme to directly convert ATP to PAPS. To maximize the conversion from ATP to PAPS, a polyphosphate (polyP)-dependent ATP regeneration system was designed and engineered by screening polyP kinases which consumes the low-cost polyP as the phosphate donor. In addition, we found that PPi could be used as the phosphate donor for phosphorylating ADP to ATP by polyP kinases. After demonstration of the wide distribution of PPi kinase activity in polyP kinases, a closed-loop ATP regeneration route was thereupon created to convert one ATP to one PAPS in theory. Using PPi as the phosphate donor, the conversion rate of ATP to PAPS reached 92.3%. The efficient enzymatic route that is constructed here for PAPS synthesis with low cost would boost the biosynthesis of sulfated compounds and peptides.

Enzymatic Production of Chondroitin Oligosaccharides and Its Sulfate Derivatives

“…According to our recent study on PAPS biosynthesis ( Xu et al, 2021 ), a new green sulfation system that directly from ATP and sulfate for CS biosynthesis was established ( Figure 4A ). C4ST and ASAK S5 were purified from recombinant P. pastoris ( Jin et al, 2020 ) and E. coli ( Xu et al, 2021 ) respectively according to our previous research ( Supplementary Figures S12A,S12B ).…”

“…According to our recent study on PAPS biosynthesis ( Xu et al, 2021 ), a new green sulfation system that directly from ATP and sulfate for CS biosynthesis was established ( Figure 4A ). C4ST and ASAK S5 were purified from recombinant P. pastoris ( Jin et al, 2020 ) and E. coli ( Xu et al, 2021 ) respectively according to our previous research ( Supplementary Figures S12A,S12B ). In consideration of the critical effect of temperature on enzyme reaction ( Arcus & Mulholland, 2020 ), the temperature of the entire catalytic system with C4ST and ASAK S5 was explored.…”

“…The protein concentration and the purity of the C4ST were 0.2 g/L and 86.9%, respectively. The ASAK S5 (a bifunctional PAPS synthase that converts ATP to PAPS) was prepared according to the previous study (Xu et al, 2021). In vitro sulfation of chondroitin was performed in the reaction solvent containing Tris-HCl (50 mM, pH 7.5), ATP (10 mM), MgSO 4 (20 mM), ASAK S5 (0.5 g/L), C4ST (1.0 g/L) and chondroitin (2.0 g/L) for 48 h. To promote efficient chondroitin sulfation, the temperature of the reaction was investigated at of 25 °C, 30 °C, 35 °C, 37 °C, and 40 °C.…”

“…To this end, the depolymerization process of chondroitin with an engineered chondroitinase ABC I (csABC I) was quantitatively analyzed and optimized for preparing specific o-CHs disaccharides, tetrasaccharides, hexasaccharides, octasaccharides and decasaccharides with high conversion rates. On the basis of the construction of an artificially bifunctional enzyme for converting ATP (adenosine 5′-triphosphate) to PAPS ( Xu et al, 2021 ), a new low-cost sulfation system which directly initiates from ATP and sulfate was constructed and optimized for the enzymatic production of chondroitin sulfate A (CSA) and o-CHs sulfate derivatives.…”

Synthesis of bioengineered heparin by recombinant yeast Pichia pastoris

“…1). To this end, P. pastoris that has already been engineered for the biosynthesis of chondroitin sulfate A 38,43 was chosen as the host for the active expression of all the enzymes NDST, C5 epi, 2-OST, 6-OST and 3-OST.…”