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Plants and cyanobacteria are promising heterologous hosts for metabolic engineering, and particularly suited for expression of cytochrome P450 (P450s), enzymes that catalyse key steps in biosynthetic pathways leading to valuable natural products such as alkaloids, terpenoids and phenylpropanoids. P450s are often difficult to express and require a membrane-bound NADPH-dependent reductase, complicating their use in metabolic engineering and bio-production. We previously demonstrated targeting of heterologous P450s to thylakoid membranes both in N. benthamiana chloroplasts and cyanobacteria, and functional substitution of their native reductases with the photosynthetic apparatus via the endogenous soluble electron carrier ferredoxin. However, because ferredoxin acts as a sorting hub for photosynthetic reducing power, there is fierce competition for reducing equivalents, which limits photosynthesis-driven P450 output. This study compares the ability of four electron carriers to increase photosynthesis-driven P450 activity. These carriers, three plant ferredoxins and a flavodoxin-like engineered protein derived from cytochrome P450 reductase, show only modest differences in their electron transfer to our model P450, CYP79A1 in vitro. However, only the flavodoxin-like carrier supplies appreciable reducing power in the presence of competition for reduced ferredoxin, because it possesses a redox potential that renders delivery of reducing equivalents to endogenous processes inefficient. We further investigate the efficacy of these electron carrier proteins in vivo by expressing them transiently in N. benthamiana fused to CYP79A1. All but one of the fusion enzymes show improved sequestration of photosynthetic reducing power. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison - nearly 25-fold on a per protein basis. Thus, this study demonstrates that synthetic electron transfer pathways with optimal redox potentials can alleviate the problem of endogenous competition for reduced ferredoxin and sets out a new metabolic engineering strategy useful for producing valuable natural products. • Photosynthesis can supply reducing power for cytochrome P450 catalysis directly. • Competing host metabolism limits the amount of reducing power channeled towards heterologous P450s. • Four soluble electron carriers were tested as partners for a plant P450 expressed in N. benthamiana thylakoids. • All carriers could reduce the P450 in vitro , but had different electron transfer efficiencies with photosystem I and P450. • A flavodoxin-like carrier greatly enhanced in vivo flux of reducing power to the P450 when carrier and P450 were fused. [ABSTRACT FROM AUTHOR]

Several commercial assay kits exist with limited explanation of the kit components and reagent constituents, which greatly increases potential incompatibility issues resulting in the loss of samples, time, and data. Herein we explore such issues via the redox ion [Fe(CN) 6 ]3/4- in two commercial l -lactate and pyruvate assay kits. We clearly demonstrate significant interference from redox compounds with the l -lactate and pyruvate assays; a significance in signal inhibition/mechanism restriction, and false/mechanism exhaustion, respectively. Potential mechanisms are explored to explain interference. The need for transparency is crucial for consistency of assay kit performance from lab to lab. There is a need for suppliers to list the components of kits and/or list the potential for interference from specific agents to ensure that results obtained from these kits are reliable and reproducible. [Display omitted] • Discusses the growing field of bioelectronics and implementation of electrochemistry to detect and quantify biomarkers. • Demonstrates how components of assay kits interfere with electron facilitating compounds resulting in inaccurate data. • Discusses potential electron transfer kinetics between assay kits and underlying reactions responsible for inaccurate data. • Discusses the need for transparency regarding interference; a wider problem in the scientific community. [ABSTRACT FROM AUTHOR]