Abstract
Adenosine triphosphate (ATP) and S-adenosyl-L-methionine (SAM) are important intermediates that are widely present in living organisms. Large-scale preparation and application of ATP or SAM is limited by expensive raw materials. To lower the production costs for ATP/SAM, in this study we used strategies applying engineered multidomain scaffold proteins to synthesize ATP and SAM. An artificial scaffold protein containing CBM3 domain, IM proteins and CL-labeled proteins was assembled to form complex 1 for catalytic reactions to increase ATP production. The ATP synthesis system produced approximately 25 g/L of ATP with approximately 15 g/L of ADP and 5 g/L of AMP using 12.5 g/L of adenosine and 40 g/L of sodium hexametaphosphate reaction at 35 °C and a pH of 8.5 for 6 h. Based on the above ATP synthesis system, two CL-labeled methionine adenosyltransferases (CL9-MAT4 and CL9-MAT5) were applied to construct scaffold protein complex 2 to achieve SAM synthesis. Approximately 25 μg of MAT4 in a reaction system with 0.3 M MgCl2 catalyzed at 20 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 0.9 g/L of SAM. Approximately 25 μg of MAT5 in a reaction system with 0.7 M MgCl2 catalyzed at 35 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 1.2 g/L of SAM. Here, we showed that low-cost substrates can be efficiently converted into high-value additional ATP and SAM via multi-enzyme catalytic reactions by engineered multidomain scaffold proteins.
Highlights
Adenosine triphosphate (ATP), an important metabolite in living organisms, serves as a metabolic intermediate, coenzyme, and energy donor
We showed that low-cost substrates can be efficiently converted into high-value additional ATP and SAM by engineered multidomain scaffold proteins
The expression strain, E. coli BL21 (DE3), the cloned strain, E. coli DH5α, and plasmids (pET-23a(+), pET-28a(+), and pET23a-IM2-IM7-IM8-IM9) were all maintained in our laboratory. 5’-adenosine triphosphate, 5’-adenosine diphosphate, adenine nucleoside, sodium hexametaphosphate, and L-Met were purchased from Shanghai Maklin Biochemical Technology Co., Ltd. (Shanghai, China)
Summary
Adenosine triphosphate (ATP), an important metabolite in living organisms, serves as a metabolic intermediate, coenzyme, and energy donor. ATP participates in a variety of biochemical reactions, providing a direct source of energy required for life. ATP can treat several diseases, such as heart-related diseases, filamentous keratitis, stroke sequelae, gastroptosis, and tumors [1]. ATP has considerable application value in environmental protection [2] and microbiological testing. Researchers have made continuous efforts to synthesize ATP in vitro. Some researchers have used adenylate kinase and acetate kinase coupling to convert adenosine to ATP [3]
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