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Cytochrome P450 Enzymes for the Synthesis of Novel and Known Drugs and Drug Metabolites
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Sanjana Haque, Yuqing Gong, Sunitha Kodidela, Mohammad A. Rahman, Sabina Ranjit, Santosh Kumar
Plants produce more than 200,000 metabolites to interact with and adapt to their environment. Plant specific metabolites, such as terpenoids, phenylpropanoids, and nitrogen-containing compounds are the major source of new drugs and bioactive compounds. Plant CYP enzymes have the potential to be used for industrial hydroxylation reactions. However, plant CYP enzymes typically have low turnover rates (ten to a few hundred per minute range), poor solubility, less suitable expression systems, and requirement of redox proteins (Hamberger and Bak, 2013; Renault et al., 2014). Therefore, gene-fusion approaches have been created for plant CYP enzymes to maximize enzymatic activity. Recently, the plant P450 isoflavone synthase 1 (IFS) from Glycine max (soybean) was used to create a redox-self-sufficient CYP by fusing with the bacterial reductase Rhf-RED. The IFSn-Rhf-RED fusion enzyme can be expressed in E. coli, and transform the flavonoid substrate naringenin to genistein, which is an angiogenesis inhibitor and a phytoestrogen. They also created an active CYP fusion enzyme for CYP73A5, a cinnamate-4-hydroxylase (C4H) from Arabidopsis thaliana. The enzyme fusion C4H-Rhf-RED was expressed in E. coli and can be used for the hydroxylation of cinnamic acid to p-coumaric acid. The fusion system expresses both plant and bacterial CYP enzymes, and has the potential to be used in industrial biocatalysis (Schuckel et al., 2012).
Impact of copper treatment on phenylpropanoid biosynthesis in adventitious root culture of Althaea officinalis L.
Published in Preparative Biochemistry & Biotechnology, 2022
Yun Ji Park, Nam Su Kim, Ramaraj Sathasivam, Yong Suk Chung, Sang Un Park
The real-time PCR reactions contained, at a total volume of 20 μL, 5 μL of diluted cDNA (1:20), 1 μL of each forward and reverse primer (10 pmol/μL), 10 μL of 2X Real-Time PCR Master Mix (Including SFC green® I) (BIOFACT, Korea), and 3 μL of distilled water. Gene expression profiling was performed using a CFX96 real-time system (BIO-RAD Laboratories, USA), under the following thermal cycling conditions: 15 min at 95 °C, 40 cycles of denaturation at 95 °C for 20 s, alignment step at 56 °C for 40 s, and elongation step at 72 °C for 20 s. Glyceraldehyde 3-phosphate dehydrogenase gene from A. officinalis (AoGAPDH) was used as a housekeeping gene to determine relative transcript abundance. Moreover, seventeen phenylpropanoid biosynthetic genes, including phenylalanine ammonia-lyase (AoPALs), cinnamate 4-hydroxylase (AoC4H), 4-coumarate-CoA ligase, (Ao4CL), chalcone synthase (AoCHSs), chalcone isomerase (AoCHIs), flavanone 3-hydroxylase (AoF3H), flavonol synthase (AoFLS), anthocyanidin reductase (AoANR), dihydroflavonol 4-reductase (AoDFR), anthocyanidin synthase (AoANS), 4-coumarate 3-hydroxylase (AoC3Hs), Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (AoHCT), and Hydroxycinnamoyl CoA quinate transferase (AoHQT) were determined from A. officinalis[21]. The primers used in this study were designed using the GenScript Real-time PCR (TaqMan) Primer Design (www.genscript.com) and are listed in Table 1. For each condition, qRT-PCR experiments were conducted in triplicate. The specificity of the PCR reaction was confirmed by melting curve analysis of each amplified product. Standard curves were drawn to confirm the efficiency (E) for the qPCR assay and the coefficient (R2) of the serial dilutions. The relative gene expression levels were measured using the 2 –ΔΔCt method.[22]