Presentation Information
[P03-374]Development of semi-de novo ATP and NAD+ production cascades using thermophilic enzymes
○Takuma Suzuki1,2, Gladwin Suryatin Alim1,3, Kentaro miyazaki1, Hiroya Tomita1,4, Kohsuke Honda1,4 (1. International Center for Biotechnology, The University of Osaka (Japan), 2. Research Fellow of Japan Society for the Promotion of Science (Japan), 3. Department of Chemistry, University of Basel (Switzerland), 4. Institute for Open and Transdisciplinary Research Initiatives, The University of Osaka (Japan))
Keywords:
ATP,Enzymatic cascade,NAD+,Thermophilic enzymes
Cell-free bio-manufacturing based on enzyme cascades has gained attention because it enables flexible pathway design and low by-product formation. ATP and NAD+ are essential cofactors for many enzymatic reactions; however, owing to their high cost and instability, in situ production systems for them are required for advanced cascade construction.
Non-oxidative glycolysis (NOG) is a carbon-conserving synthetic pathway that converts fructose-6-phosphate into three molecules of acetyl phosphate (AcP), and was later adapted into an ATP-regenerating system (ArNOG) that produces three ATP from glucose-1-phosphate derived from maltodextrin phosphorolysis (Bogorad et al., Nature, 2012; Wei et al., ChemCatChem, 2019). Based on these achievements, we previously constructed a recombinant Escherichia coli strain co-expressing 12 thermophilic ArNOG enzymes (Suryatin Alim et al., ChemBioChem, 2022). Heat treatment of the recombinant E. coli lysate selectively denatures host-derived mesophilic proteins, thereby enabling one-step partial purification of all 12 thermophilic ArNOG enzymes. Here, we expanded this thermophilic ArNOG platform by incorporating the thermophilic adenine nucleotide biosynthetic and salvage enzymes, establishing semi-de novo ATP and NAD+ production cascades.
In ArNOG, three molecules of AcP are generated from glucose-1-phosphate to drive ATP regeneration. Erythrose-4-phosphate is recycled to fructose-6-phosphate through the carbon rearrangement module (CRM). In the designed ATP production cascade, ribose-5-phosphate from the CRM is converted to phosphoribosyl pyrophosphate (PRPP) using ATP. Adenine is phosphoribosylated to AMP, which is converted to ADP by adenylate kinase, followed by ATP regeneration via ArNOG. Although ATP is consumed during PRPP and ADP formation, the cascade results in net ATP production. In this study, ATP was produced from 1 mM adenine with 100 mol% conversion. Next, by incorporating five thermophilic NAD+ biosynthetic and salvage enzymes together with nicotinic acid and ammonium chloride into this ATP production cascade, we designed a semi-de novo NAD+ production cascade driven by internally produced ATP. Reaction optimization of this extended system is ongoing.
Non-oxidative glycolysis (NOG) is a carbon-conserving synthetic pathway that converts fructose-6-phosphate into three molecules of acetyl phosphate (AcP), and was later adapted into an ATP-regenerating system (ArNOG) that produces three ATP from glucose-1-phosphate derived from maltodextrin phosphorolysis (Bogorad et al., Nature, 2012; Wei et al., ChemCatChem, 2019). Based on these achievements, we previously constructed a recombinant Escherichia coli strain co-expressing 12 thermophilic ArNOG enzymes (Suryatin Alim et al., ChemBioChem, 2022). Heat treatment of the recombinant E. coli lysate selectively denatures host-derived mesophilic proteins, thereby enabling one-step partial purification of all 12 thermophilic ArNOG enzymes. Here, we expanded this thermophilic ArNOG platform by incorporating the thermophilic adenine nucleotide biosynthetic and salvage enzymes, establishing semi-de novo ATP and NAD+ production cascades.
In ArNOG, three molecules of AcP are generated from glucose-1-phosphate to drive ATP regeneration. Erythrose-4-phosphate is recycled to fructose-6-phosphate through the carbon rearrangement module (CRM). In the designed ATP production cascade, ribose-5-phosphate from the CRM is converted to phosphoribosyl pyrophosphate (PRPP) using ATP. Adenine is phosphoribosylated to AMP, which is converted to ADP by adenylate kinase, followed by ATP regeneration via ArNOG. Although ATP is consumed during PRPP and ADP formation, the cascade results in net ATP production. In this study, ATP was produced from 1 mM adenine with 100 mol% conversion. Next, by incorporating five thermophilic NAD+ biosynthetic and salvage enzymes together with nicotinic acid and ammonium chloride into this ATP production cascade, we designed a semi-de novo NAD+ production cascade driven by internally produced ATP. Reaction optimization of this extended system is ongoing.
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