10.6084/m9.figshare.9192491.v1
Zhao Aiguo
Zhao
Aiguo
Ding Ruiwen
Ding
Ruiwen
Zhai Meizhi
Zhai
Meizhi
Multi-enzymatic recycling of ATP and NADPH for the synthesis of 5-aminolevulinic acid using a semipermeable reaction system
Taylor & Francis Group
2019
5-Aminolevulinic acid
synthetic biology
multi-enzymatic system
recycling
2019-07-31 07:20:03
Journal contribution
https://tandf.figshare.com/articles/journal_contribution/Multi-enzymatic_recycling_of_ATP_and_NADPH_for_the_synthesis_of_5-aminolevulinic_acid_using_a_semipermeable_reaction_system/9192491
<p>5-Aminolevulinic acid (ALA) is an important cellular metabolic intermediate that has broad agricultural and medical applications. Previously, attempts have been made to synthesize ALA by multiple enzymes in cell free systems. Here we report the development of a semi-permeable system for ALA production using stable enzymes. Glucose, sodium polyphosphate, ATP, tRNA, glutamate and NADPH were used as substrates for ALA synthesis by a total of nine enzymes: adenylate kinase, polyphosphate kinase, glucose-6-phosphate dehydrogenase, phosphogluconolactonase, 6-phosphogluconate dehydrogenase, glutamyl-tRNA synthetase and glutamate-1-semialdehyde aminotransferase from <i>E. coli</i>, hexokinase from yeast, as well as glutamyl-tRNA reductase and its stimulator protein glutamyl-tRNA reductase binding protein (GBP) from Arabidopsis in a semi-permeable system. After reaction for 48 h, the glutamate conversion reached about 95%. This semi-permeable system facilitated the reuse of enzymes, and was helpful for the separation and purification of the product. The ALA production could be further improved by process optimization and enzyme engineering.</p> <p><b>Abbreviations</b>: PPK: polyphosphate kinase; ADK: adenylate kinase; ALA: 5-Aminolevulinic acid; HK: hexokinase; ZWF: glucose-6-phosphatedehydrogenase; PGL: phosphogluconolactonase; GND: 6-phosphogluconate dehydrogenase; GTS: glutamyl-tRNA synthetase; GTR: glutamyl-tRNA reductase; GBP: GTR binding protein; GSAAT: glutamate-1-semialdehyde aminotransferase.</p> <p>The multi-enzyme reactions for 5-aminolevulinic acid (ALA) synthesis through the C5 pathway.
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