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

Adenosine triphosphate (ATP), as a universal energy currency, takes a central role in many biochemical reactions with potential for the synthesis of numerous high-value products. However, the high cost of ATP limits industrial ATP-dependent enzyme-catalyzed reactions. Here, we investigated the effect of cell-surface display of phosphotransferase on ATP regeneration in recombinant Escherichia coli. By N-terminal fusion of the super-folder green fluorescent protein (sfGFP), we successfully displayed the phosphotransferase of Pseudomonas brassicacearum (PAP-Pb) on the surface of E. coli cells. The catalytic activity of sfGFP-PAP-Pb intact cells was 2.12 and 1.47 times higher than that of PAP-Pb intact cells, when the substrate was AMP and ADP, respectively. The conversion of ATP from AMP or ADP were up to 97.5% and 80.1% respectively when catalyzed by the surface-displayed enzyme at 37 °C for only 20 min. The whole-cell catalyst was very stable, and the enzyme activity of the whole cell was maintained above 40% after 40 rounds of recovery. Under this condition, 49.01 mg/mL (96.66 mM) ATP was accumulated for multi-rounds reaction. This ATP regeneration system has the characteristics of low cost, long lifetime, flexible compatibility, and great robustness.

Aerobic glycolysis in cancer cells supplies ATP while preventing excess metabolic thermogenesis

A general feature of cancer metabolism is ATP regeneration via substrate-level phosphorylation even under normoxic conditions (aerobic glycolysis). However, it is unclear why cancer cells prefer the inefficient aerobic glycolysis over the highly efficient process of oxidative phosphorylation for ATP regeneration. Here, we show that a beneficial aspect of aerobic glycolysis is that it reduces metabolic heat generation during ATP regeneration. 13C-metabolic flux analysis of 12 cultured cancer cell lines and in silico metabolic simulation revealed that metabolic heat production during ATP regeneration via aerobic glycolysis was considerably lesser than that produced via oxidative phosphorylation. The dependency on aerobic glycolysis was partly alleviated upon culturing under low temperatures. In conclusion, thermogenesis is required for maintaining thermal homeostasis and can govern aerobic glycolysis in cancer cells.

Recombinant Production of Arginyl Dipeptides by l-Amino Acid Ligase RizA Coupled with ATP Regeneration

Arginyl dipeptides like Arg-Ser, Arg-Ala, and Arg-Gly are salt-taste enhancers and can potentially be used to reduce the salt content of food. The l-amino acid ligase RizA from B. subtilis selectively synthesizes arginyl dipeptides. However, industrial application is prevented by the high cost of the cofactor adenosine triphosphate (ATP). Thus, a coupled reaction system was created consisting of RizA and acetate kinase (AckA) from E. coli providing ATP regeneration from acetyl phosphate. Both enzymes were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were varied and analyzed by RP-HPLC with fluorescence detection. Under optimal conditions the system produced up to 5.9 g/L Arg-Ser corresponding to an ATP efficiency of 23 g Arg-Ser per gram ATP. Using similar conditions with alanine or glycine as second amino acid, 2.6 g/L Arg-Ala or 2.4 g/L Arg Gly were produced. The RizA/AckA system selectively produced substantial amounts of arginyl dipeptides while minimizing the usage of the expensive ATP.

Synthesis of L-asparagine Catalyzed by a Novel Asparagine Synthase Coupled With an ATP Regeneration System

Compared with low-yield extraction from plants and environmentally unfriendly chemical synthesis, biocatalysis by asparagine synthetase (AS) for preparation of L-asparagine (L-Asn) has become a potential synthetic method. However, low enzyme activity of AS and high cost of ATP in this reaction restricts the large-scale preparation of L-Asn by biocatalysis. In this study, gene mining strategy was used to search for novel AS with high enzyme activity by expressing them in Escherichia coli BL21 (DE3) or Bacillus subtilis WB600. The obtained LsaAS-A was determined for its enzymatic properties and used for subsequent preparation of L-Asn. In order to reduce the use of ATP, a class III polyphosphate kinase 2 from Deinococcus ficus (DfiPPK2-Ⅲ) was cloned and expressed in E. coli BL21 (DE3), Rosetta (DE3) or RosettagamiB (DE3) for ATP regeneration. A coupling reaction system including whole cells expressing LsaAS-A and DfiPPK2-Ⅲ was constructed to prepare L-Asn from L-aspartic acid (L-Asp). Batch catalytic experiments showed that sodium hexametaphosphate (>60 mmol L−1) and L-Asp (>100 mmol L−1) could inhibit the synthesis of L-Asn. Under fed-batch mode, L-Asn yield reached 90.15% with twice feeding of sodium hexametaphosphate. A final concentration of 218.26 mmol L−1 L-Asn with a yield of 64.19% was obtained when L-Asp and sodium hexametaphosphate were fed simultaneously.

ATP regeneration by a single polyphosphate kinase powers multigram-scale aldehyde synthesis in vitro

Towards scalable ATP recycling: a newly identified PPK2-III biocatalyst unlocked fully in vitro multigram-scale aldehyde synthesis employing a carboxylic acid reductase.

Overview of Inositol and Inositol Phosphates on Chemoprevention of Colitis-Induced Carcinogenesis

Chronic inflammation is one of the most common and well-recognized risk factors for human cancer, including colon cancer. Inflammatory bowel disease (IBD) is defined as a longstanding idiopathic chronic active inflammatory process in the colon, including ulcerative colitis and Crohn’s disease. Importantly, patients with IBD have a significantly increased risk for the development of colorectal carcinoma. Dietary inositol and its phosphates, as well as phospholipid derivatives, are well known to benefit human health in diverse pathologies including cancer prevention. Inositol phosphates including InsP3, InsP6, and other pyrophosphates, play important roles in cellular metabolic and signal transduction pathways involved in the control of cell proliferation, differentiation, RNA export, DNA repair, energy transduction, ATP regeneration, and numerous others. In the review, we highlight the biologic function and health effects of inositol and its phosphates including the nature and sources of these molecules, potential nutritional deficiencies, their biologic metabolism and function, and finally, their role in the prevention of colitis-induced carcinogenesis.