Enzymatic Synthesis Of 3′-phosphoadenosine-5′-phosphosulfate In Vitro

3′-phosphoadenosine-5′-phosphosulfate（PAPS）is the active form of sulfate acid group,and the only known active sulfate acid group donor.PAPS is expensive and unstable,which is one of the important reasons limiting the efficient synthesis of the sulfonated products,for example,the enzymatic synthesis of important drugs heparin and chondroitin sulfate in vitro.At present,the synthesis of PAPS by Aryl sulfotransferase and ATP sulfurylase and APS kinase are the main methods,but the former has many disadvantages,such as expensive substrate and toxic substances during the reaction process.In this study,through the construction and optimization of PAPS synthesis pathway in vitro by expressing ATP sulfurylase and APS kinase,a high efficiency,low cost and green synthesis method of PAPS was achieved.The main conclusions of this study are as follows:（1）The best sources of ATP sulfurylase and APS kinase were selected.In the Escherichia coli expression system,ATP sulfurylase from Saccharomyces cerevisiae and Kluyveromyces cerevisiae and APS kinase from Penicillium chrysogenum,Escherichia coli,Saccharomyces cerevisiae,Mycobacterium tuberculosis were expressed efficiently and purified effectively.We have used effective methods for optimizating the expression of target genes,such as the N-terminal codon optimization,co-expression with thioredoxin Trx B.The results showed that the combination ATP sulfurylase from S.cerevisiae and APS kinase from Escherichia coli was the best for PAPS synthesis,and the convention rate of PAPS is 31.8%。（2）Bifunctional enzyme was artificially constructed to improve the efficiency of PAPS synthesis.In the process of PAPS synthesis,the substrate ATP,intermediate product APS and target product PAPS were all high-energy compounds with lively and unstable properties.For the efficiency PAPS synthesis,a linker used for fusing of ATP sulfurylase and APS kinase narrowed the distance between substrates and enzymes.Through the analysis of the activity of the fusion proteins with different linkers,the results showed that the activity of the fusion protein was the highest when the linker was（Gly4Ser）₆,and the optimal reaction temperature was 35℃,pH was 7.5 and the optimal substrate ATP concentration was 3.0 g·L^-1.Under the most optimal conditions,the conversion rate of PAPS synthesis is 38.4%.（3）The ATP regeneration system with by-product self-supplying is constructed for improving the conversion rate of PAPS.In order to achieve efficient recycling of by-product ADP,ATP regeneration systems coupled with pyruvate kinase and polyphosphate kinase were constructed,and it was verified that the conversion rate of PAPS was increased to 78.0%and69.0%,respectively.In addition,the ATP regeneration system coupled with polyphosphate kinase from C.glutamicum can simultaneously use the by-products ADP and pyrophosphate to achive the self-supplement of ATP re-synthesis,which has a great economic advantage.Realized the effective purification of PAPS,and successfully applied to the enzymatic synthesis of chondroitin sulfate A,which realized the green production of chondroitin sulfate A and greatly reduced the production cost.