Exploiting photosynthesis-driven P450 activity to produce indican in tobacco chloroplasts.

Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules.

Curvature thylakoid 1 proteins modulate prolamellar body morphology and promote organized thylakoid biogenesis in .

The term "de-etiolation" refers to the light-dependent differentiation of etioplasts to chloroplasts in angiosperms. The underlying process involves reorganization of prolamellar bodies (PLBs) and prothylakoids into thylakoids, with concurrent changes in protein, lipid, and pigment composition, which together lead to the assembly of active photosynthetic complexes. Despite the highly conserved structure of PLBs among land plants, the processes that mediate PLB maintenance and their disassembly during de-etiolation are poorly understood.

Reductive Cytochrome P450 Reactions and Their Potential Role in Bioremediation.

Cytochrome P450 enzymes, or P450s, are haem monooxygenases renowned for their ability to insert one atom from molecular oxygen into an exceptionally broad range of substrates while reducing the other atom to water. However, some substrates including many organohalide and nitro compounds present little or no opportunity for oxidation. Under hypoxic conditions P450s can perform reductive reactions, contributing electrons to drive reductive elimination reactions.

Antimicrobial solid media for screening non-sterile Arabidopsis thaliana seeds.

Stable genetic transformation of plants is a low-efficiency process, and identification of positive transformants usually relies on screening for expression of a co-transformed marker gene. Often this involves germinating seeds on solid media containing a selection reagent. Germination on solid media requires surface sterilization of seeds and careful aseptic technique to prevent microbial contamination, but surface sterilization techniques are time consuming and can cause seed mortality if not performed carefully.

Synthetic Protein Scaffolding at Biological Membranes.

Protein scaffolding is a natural phenomenon whereby proteins colocalize into macromolecular complexes via specific protein-protein interactions. In the case of metabolic enzymes, protein scaffolding drives metabolic flux through specific pathways by colocalizing enzyme active sites. Synthetic protein scaffolding is increasingly used as a mechanism to improve product specificity and yields in metabolic engineering projects.

Membrane-Bound Protein Scaffolding in Diverse Hosts Using Thylakoid Protein CURT1A.

Protein scaffolding is a useful strategy for controlling the spatial arrangement of cellular components via protein-protein interactions. Protein scaffolding has primarily been used to colocalize soluble proteins in the cytoplasm, but many proteins require membrane association for proper function. Scaffolding at select membrane domains would provide an additional level of control over the distribution of proteins within a cell and could aid in exploiting numerous metabolic pathways that contain membrane-associated enzymes.

Non-photosynthetic plastids as hosts for metabolic engineering.

Using plants as hosts for production of complex, high-value compounds and therapeutic proteins has gained increasing momentum over the past decade. Recent advances in metabolic engineering techniques using synthetic biology have set the stage for production yields to become economically attractive, but more refined design strategies are required to increase product yields without compromising development and growth of the host system. The ability of plant cells to differentiate into various tissues in combination with a high level of cellular compartmentalization represents so far the most unexploited plant-specific resource.

Prospects for Applying Synthetic Biology to Toxicology: Future Opportunities and Current Limitations for the Repurposing of Cytochrome P450 Systems.

The 30 years since the inception of Chemical Research in Toxicology, game-changing advances in chemical and molecular biology, the fundamental disciplines underpinning molecular toxicology, have been made. While these have led to important advances in the study of mechanisms by which chemicals damage cells and systems, there has been less focus on applying these advances to prediction, detection, and mitigation of toxicity. Over the last ∼15 years, synthetic biology, the repurposing of biological "parts" in systems engineered for useful ends, has been explored in other areas of the biomedical and life sciences, for such applications as detecting metabolites, drug discovery and delivery, investigating disease mechanisms, improving medical treatment, and producing useful chemicals.

Systems analysis of methylerythritol-phosphate pathway flux in E. coli: insights into the role of oxidative stress and the validity of lycopene as an isoprenoid reporter metabolite.

Background: High-throughput screening methods assume that the output measured is representative of changes in metabolic flux toward the desired product and is not affected by secondary phenotypes. However, metabolic engineering can result in unintended phenotypes that may go unnoticed in initial screening. The red pigment lycopene, a carotenoid with antioxidant properties, has been used as a reporter of isoprenoid pathway flux in metabolic engineering for over a decade.

Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity.

Cytochrome P450 enzymes are renowned for their ability to insert oxygen into an enormous variety of compounds with a high degree of chemo- and regio-selectivity under mild conditions. This property has been exploited in Nature for an enormous variety of physiological functions, and representatives of this ancient enzyme family have been identified in all kingdoms of life. The catalytic versatility of P450s makes them well suited for repurposing for the synthesis of fine chemicals such as drugs.

Restriction enzyme-mediated DNA family shuffling.

DNA shuffling is an established recombinatorial method that was originally developed to increase the speed of directed evolution experiments beyond what could be accomplished using error-prone PCR alone. To achieve this, mutated copies of a protein-coding sequence are fragmented with DNase I and the fragments are then reassembled in a PCR without primers. The fragments anneal where there is sufficient sequence identity, resulting in full-length variants of the original gene that have inherited mutations from multiple templates.

DNA shuffling of cytochrome P450 enzymes.

DNA family shuffling is an efficient method for creating libraries of novel enzymes, in which a high proportion of mutants exhibit correct folding and possess catalytic properties distinct from the starting material. The evolutionary arrangement of cytochromes P450 into subfamilies of enzymes with highly similar nucleotide sequences but distinct catalytic properties renders them excellent starting material for DNA family shuffling experiments. This chapter provides a general method for creating libraries of shuffled P450s from two or more related sequences and incorporates several recent improvements to previously published methods.

Directed evolution reveals requisite sequence elements in the functional expression of P450 2F1 in Escherichia coli.

Cytochrome P450 2F1 (P450 2F1) is expressed exclusively in the human respiratory tract and is implicated in 3-methylindole (3MI)-induced pneumotoxicity via dehydrogenation of 3MI to a reactive electrophilic intermediate, 3-methyleneindolenine (3-MEI). Studies of P450 2F1 to date have been limited by the failure to express this enzyme in Escherichia coli. By contrast, P450 2F3, a caprine homologue that shares 84% sequence identity with P450 2F1 (86 amino acid differences), has been expressed in E.