Pretreated sugarcane bagasse matches performance of synthetic media for lipid production with Yarrowia lipolytica.

Engineered strains of Yarrowia lipolytica with modified lipid profiles and other desirable properties for microbial oil production are widely reported but are almost exclusively characterized in synthetic laboratory-grade media. Ensuring translatable performance between synthetic media and industrially scalable lignocellulosic feedstocks is a critical challenge. Yarrowia lipolytica growth and lipid production were characterized in media derived from two-step acid-catalyzed glycerol pretreatment of sugarcane bagasse.

The methylerythritol phosphate pathway as an oxidative stress sense and response system.

The methylerythritol phosphate (MEP) pathway is responsible for biosynthesis of the precursors of isoprenoid compounds in eubacteria and plastids. It is a metabolic alternative to the well-known mevalonate pathway for isoprenoid production found in archaea and eukaryotes. Recently, a role for the MEP pathway in oxidative stress detection, signalling, and response has been identified.

Development of Lyophilized Eukaryotic Cell-Free Protein Expression System Based on .

Eukaryotic cell-free protein expression systems enable rapid production of recombinant multidomain proteins in their functional form. A cell-free system based on the rapidly growing protozoan (LTE) has been extensively used for protein engineering and analysis of protein interaction networks. However, like other eukaryotic cell-free systems, LTE deteriorates at ambient temperatures and requires deep freezing for transport and storage.

Biosensor-guided rapid screening for improved recombinant protein secretion in Pichia pastoris.

Pichia pastoris (Komagataella phaffii) is widely used for industrial production of heterologous proteins due to high secretory capabilities but selection of highly productive engineered strains remains a limiting step. Despite availability of a comprehensive molecular toolbox for construct design and gene integration, there is high clonal variability among transformants due to frequent multi-copy and off-target random integration. Therefore, functional screening of several hundreds of transformant clones is essential to identify the best protein production strains.

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.

Systems-level engineering and characterisation of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB).

Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO + H as its sole carbon and energy sources. Fermentation of C.

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.

Maintenance of ATP Homeostasis Triggers Metabolic Shifts in Gas-Fermenting Acetogens.

Acetogens are promising cell factories for producing fuels and chemicals from waste feedstocks via gas fermentation, but quantitative characterization of carbon, energy, and redox metabolism is required to guide their rational metabolic engineering. Here, we explore acetogen gas fermentation using physiological, metabolomics, and transcriptomics data for Clostridium autoethanogenum steady-state chemostat cultures grown on syngas at various gas-liquid mass transfer rates. We observe that C.

Arginine deiminase pathway provides ATP and boosts growth of the gas-fermenting acetogen Clostridium autoethanogenum.

Acetogens are attractive organisms for the production of chemicals and fuels from inexpensive and non-food feedstocks such as syngas (CO, CO and H). Expanding their product spectrum beyond native compounds is dictated by energetics, particularly ATP availability. Acetogens have evolved sophisticated strategies to conserve energy from reduction potential differences between major redox couples, however, this coupling is sensitive to small changes in thermodynamic equilibria.

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.

2,2-Diphenyl-1-picrylhydrazyl as a screening tool for recombinant monoterpene biosynthesis.

Background: Monoterpenes are a class of natural C10 compounds with a range of potential applications including use as fuel additives, fragrances, and chemical feedstocks. Biosynthesis of monoterpenes in heterologous systems is yet to reach commercially-viable levels, and therefore is the subject of strain engineering and fermentation optimization studies. Detection of monoterpenes typically relies on gas chromatography/mass spectrometry; this represents a significant analytical bottleneck which limits the potential to analyse combinatorial sets of conditions.

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.