ABSTRACT
Pharmaceuticals have historically been produced by either chemical synthesis or whole cell fermentation. The former is applied to synthetic small molecules while the latter to natural products. As a result of recent advances in rapid discovery of enzymes through genome mining and metagenomics, and their tunability in functions and stability through directed evolution, biocatalysis is emerging to be a transformational technology for drug discovery and production. Enzymes can catalyze reactions otherwise challenging by chemical approaches. Furthermore, enzymatic catalysis is a powerful tool for green chemistry development. This manuscript gives a brief overview of current status in integrating chemical and biological transformations for the synthesis of small molecular therapeutics.
Subject(s)
Biocatalysis , Drug Design , Enzymes , Pharmaceutical Preparations/chemical synthesis , Protein Engineering/methods , Anti-Bacterial Agents/biosynthesis , Anticholesteremic Agents/chemical synthesis , Antidepressive Agents/chemical synthesis , Antineoplastic Agents/chemical synthesis , Biopharmaceutics , Biotechnology , HumansABSTRACT
Directed evolution of 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase for microbial synthesis of shikimate pathway products provides an alternate strategy to circumvent the competition for phosphoenolpyruvate between 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase and the phosphoenolpyruvate:carbohydrate phosphotransferase system in Escherichia coli. E. coli KDPGal aldolase was evolved using a combination of error-prone polymerase chain reaction, DNA shuffling, and multiple-site-directed mutagenesis to afford KDPGal aldolase variant NR8.276-2, which exhibits a 60-fold improvement in the ratio kcat/KM relative to that of wild-type E. coli KDPGal aldolase in catalyzing the addition of pyruvate to d-erythrose 4-phosphate to form DAHP. On the basis of its nucleotide sequence, NR8.276-2 contains seven amino acid changes from the wild-type E. coli KDPGal aldolase. Amplified expression of NR8.276-2 in the DAHP synthase and shikimate dehydrogenase-deficient E. coli strain NR7 under fed-batch fermentor-controlled cultivation conditions resulted in synthesis of 13 g/L 3-dehydroshikimic acid in 6.5% molar yield from glucose. Increased coexpression of the irreversible downstream enzyme 3-dehydroquinate synthase increased production of 3-dehydroshikimic acid to 19 g/L in 9.7% molar yield from glucose. Coamplification with transketolase, which increases d-erythrose 4-phosphate availability, afforded 16 g/L 3-dehydroshikimic acid in 8.5% molar yield.
Subject(s)
3-Deoxy-7-Phosphoheptulonate Synthase , Aldehyde-Lyases , Directed Molecular Evolution , Escherichia coli/enzymology , Recombinant Fusion Proteins , 3-Deoxy-7-Phosphoheptulonate Synthase/chemistry , 3-Deoxy-7-Phosphoheptulonate Synthase/genetics , 3-Deoxy-7-Phosphoheptulonate Synthase/metabolism , Aldehyde-Lyases/chemistry , Aldehyde-Lyases/genetics , Aldehyde-Lyases/metabolism , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Glucose/metabolism , Models, Molecular , Molecular Structure , Mutagenesis, Site-Directed , Phosphoenolpyruvate/metabolism , Plasmids , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Shikimic Acid/analogs & derivatives , Shikimic Acid/metabolismABSTRACT
The competition between the Escherichia coli carbohydrate phosphotransferase system and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase for phosphoenolpyruvate limits the concentration and yield of natural products microbially synthesized via the shikimate pathway. To circumvent this competition for phosphoenolpyruvate, a shikimate pathway variant has been created. 2-Keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases encoded by Escherichia coli dgoA and Klebsiella pneumoniae dgoA are subjected to directed evolution. The evolved KDPGal aldolase isozymes exhibit 4-8-fold higher specific activities relative to that for native KDPGal aldolase with respect to catalyzing the condensation of pyruvate and d-erythrose 4-phosphate to produce DAHP. To probe the ability of the created shikimate pathway variant to support microbial growth and metabolism, growth rates and synthesis of 3-dehydroshikimate are examined for E. coli constructs that lack phosphoenolpruvate-based DAHP synthase activity and rely on evolved KDPGal aldolase for biosynthesis of shikimate pathway intermediates and products.
