Cost-Effective Production of ATP and S-Adenosylmethionine Using Engineered Multidomain Scaffold Proteins

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.

Whole-Cell Display of Phosphotransferase in Escherichia coli for High-Efficiency Extracellular ATP Production

“…As shown in Figure 5 , the optimal pH was 8.5 for the synthesis reaction of ATP respectively whether using ADP or AMP as the substrate ( Figure 5 a). The optimal concentration of Mg 2+ was 30 mM ( Figure 5 b) using ADP or AMP as the substrate, that was different to the data reported [ 1 , 12 , 13 , 17 , 34 ]. The results showed a relatively broad pH and concentration ranges of Mg 2+ for the reaction.…”

“…ATP, ADP, and AMP were monitored with HPLC (Welch), equipped with a Welch Ultimate LP-C18 column (4.6 × 250 mm, 5 μm, Welch Materials, Inc, Shanghai, China) after 0.22 μm filtration. The mobile phase was methanol with 50 mM ammonium formate buffer (pH 4.5) (95% v/v ) at a flow rate of 0.5 mL/min [ 34 ], and the effluent was detected at a wavelength of 260 nm. The detection times were as follows: ATP (7.453 s), ADP (8.489 s), and AMP (14.009 s).…”

Biomimetic S‐Adenosylmethionine Regeneration Starting from Multiple Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes**

“…2.5.1.6), nature's SAM-synthesising enzyme. [42][43][44] Alternatively, chlorinases (SalL, E.C. 2.5.1.94) are ChemBioChem employed to synthesise SAM from l-Met and 5'-chloro-5'deoxyadenosine (CldA) in their reverse reaction.…”

“…Biomimetic SAM supply starts from l ‐methionine ( l ‐Met) and adenosine 5'‐triphosphate (ATP) using methionine adenosyl‐transferase (MAT, E.C. 2.5.1.6), nature's SAM‐synthesising enzyme [42–44] . Alternatively, chlorinases (SalL, E.C.…”

Development of a synthetic transcription factor-based S-adenosylmethionine biosensor in Saccharomyces cerevisiae

“…Recently, SAM used alone or in combination with other drugs is emerging as a potentially effective strategy for cancer treatment and chemoprophylaxis. In addition, SAM as a dietary supplement aroused great interest and has enormous commercial value (Silveri et al 2003; Yan et al 2021). Although recent efforts have been made to enhance the yield of SAM, it is still not enough to meet the needs of industrial production (Y. W. Chen et al 2018; Kanai et al 2017;Qin et al 2020).…”