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Microbiology and Biotechnology Letters

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Biocatalysis and Bioprocess Engineering  |  Enzyme, Protein Engineering, and Metabolic Engineering

Microbiol. Biotechnol. Lett. 2019; 47(4): 536-545

https://doi.org/10.4014/mbl.1905.05008

Received: May 20, 2019; Accepted: June 18, 2019

Application of Solanum lycopersicum Glucose-6-phosphate Dehydrogenase to NADPH-generating System for Cytochrome P450 Reactions

Chan Mi Park , Heon Jeong , Sang Hoon Ma , Hyun Min Kim , Young Hee Joung and Chul-Ho Yun *

Chonnam National University

Cytochrome P450 (P450 or CYP) is involved in the metabolism of endogenous and exogenous compounds in most organisms. P450s have great potential as biocatalysts in the pharmaceutical and fine chemical industries because they catalyze diverse oxidative reactions using a wide range of substrates. The high-cost nicotinamide cofactor, NADPH, is essential for P450 reactions. Glucose-6-phosphate dehydrogenase (G6PDH) has been commonly used in NADPH-generating systems (NGSs) to provide NADPH for P450 reactions. Currently, only two G6PDHs from Leuconostoc mesenteroides and Saccharomyces cerevisiae can be obtained commercially. To supply high-cost G6PDH cost-effectively, we cloned the cytosolic G6PDH gene of Solanum lycopersicum (tomato) with 6xHis tag, expressed it in Escherichia coli, and purified the recombinant G6PDH (His-G6PDH) using affinity chromatography. In addition, enzymatic properties of His-G6PDH were investigated, and the His-G6PDH-coupled NGS was optimized for P450 reactions. His-G6PDH supported CYP102A1-catalyzed hydroxylation of omeprazole and testosterone by NADPH generation. This result suggests that tomato His-G6PDH could be a cost-effective enzyme source for NGSs for P450-catalyzed reactions as well as other NADPH-requiring reactions.

Keywords: Cytochrome P450, tomato glucose-6-phosphate dehydrogenase, heterologous expression, NADPH-generating system

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