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. We developed an antimicrobial cocktail that can be added to solid media to inhibit bacterial and fungal growth without impairing germination, allowing us to bypass the surface sterilization step. Adding a combination of terbinafine (1 μM) and timentin (200 mg l−1) to Murashige and Skoog agar delayed the onset of observable microbial growth and did not affect germination of non-sterile seeds from 10 different wild-type and mutant Arabidopsis thaliana accessions. We named this antimicrobial solid medium “MSTT agar”. Seedlings sown in non-sterile conditions could be maintained on MSTT agar for up to a week without observable contamination. This medium was compatible with rapid screening methods for hygromycin B, phosphinothricin (BASTA) and nourseothricin resistance genes, meaning that positive transformants can be identified from non-sterile seeds in as little as 4 days after stratification, and transferred to soil before the onset of visible microbial contamination. By using MSTT agar we were able to select genetic transformants on solid media without seed surface sterilization, eliminating a tedious and time-consuming step.</p

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. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-β-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco (Nicotiana benthamiana) chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, in vitro kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies

Systems-level engineering and characterization 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. autoethanogenum is currently being deployed on a commercial scale for ethanol production. Expanding the product spectrum of acetogens will enhance the economics of gas fermentation. To achieve efficient heterologous product synthesis, limitations in redox and energy metabolism must be overcome. Here, we engineered and characterised at a systems-level, a recombinant poly-3-hydroxybutyrate (PHB)-producing strain of C. autoethanogenum. Cells were grown in CO-limited steady-state chemostats on two gas mixtures, one resembling syngas (20% H) and the other steel mill off-gas (2% H). Results were characterized using metabolomics and transcriptomics, and then integrated using a genome-scale metabolic model reconstruction. PHB-producing cells had an increased expression of the Rnf complex, suggesting energy limitations for heterologous production. Subsequent optimization of the bioprocess led to a 12-fold increase in the cellular PHB content. The data suggest that the cellular redox state, rather than the acetyl-CoA pool, was limiting PHB production. Integration of the data into the genome-scale metabolic model showed that ATP availability limits PHB production. Altogether, the data presented here advances the fundamental understanding of heterologous product synthesis in gas-fermenting acetogens

Unraveling acetogen gas fermentation using quantitative systems biology

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Clathrin-mediated post-fusion membrane retrieval influences the exocytic mode of endothelial Weibel-Palade bodies.

Weibel-Palade bodies (WPBs), the storage organelles of endothelial cells, are essential to normal haemostatic and inflammatory responses. Their major constituent protein is von Willebrand factor (VWF) which, following stimulation with secretagogues, is released into the blood vessel lumen as large platelet-catching strings. This exocytosis changes the protein composition of the cell surface and also results in a net increase in the amount of plasma membrane. Compensatory endocytosis is thought to limit changes in cell size and retrieve fusion machinery and other misplaced integral membrane proteins following exocytosis; however, little is known about the extent, timing, mechanism and precise function of compensatory endocytosis in endothelial cells. Using biochemical assays, live-cell imaging and correlative spinning-disk microscopy and transmission electron microscopy assays we provide the first in-depth high-resolution characterisation of this process. We provide a model of compensatory endocytosis based on rapid clathrin- and dynamin-mediated retrieval. Inhibition of this process results in a change of exocytic mode: WPBs then fuse with previously fused WPBs rather than the plasma membrane, leading, in turn, to the formation of structurally impaired tangled VWF strings.This article has an associated First Person interview with the first authors of the paper

Investigations of cytochromes P450 using the DNA family shuffling method.

Xenobiotic-metabolising cytochromes P450 (P450s) are capable of metabolising an extraordinarily diverse range of substrates. These enzymes are arranged into evolutionarily-related homologous sub-families, a property that makes them amenable to recombination using DNA family shuffling (a method for the creation of libraries of mosaic nucleotide sequences by recombining fragments of homologous genes). This thesis presents an exploration of the utility of this method in the directed evolution of xenobiotic-metabolising P450s. In the first part of this thesis, DNA family shuffling is used to investigate the determinants of P450 expression in Escherichia coli as specific to P450 2F1, a form found in the human respiratory tract that had not previously been expressed heterologously. Heterologous expression of P450s is critical to obtaining sufficient quantities of enzyme for detailed structural and functional characterisation. Common strategies for achieving bacterial expression have proven unsuccessful for P450 2F1, despite such strategies achieving expression of P450 2F3 (a goat homologue with 84 % sequence identity to P450 2F1). Initial experiments analysing protein expression revealed that P450 2F1 is successfully translated in E. coli but does not fold correctly. To achieve heterologous expression of P450 2F1, the coding sequences for P450s 2F1 and 2F3 were recombined using DNA family shuffling. Shuffled mutants were analysed for expression of folded P450 enzyme in E. coli, and expressed mutants were sequenced. Two specific regions between residues 65-92 and 266-301 were found where P450 2F1 sequence did not occur in mutants that expressed in E. coli. Three rounds of directed evolution with the dual selection criteria of increased incorporation of P450 2F1 sequence and detectable P450 expression resulted in a mutant with 95.8 % amino acid identity to P450 2F1. This mutant (JH_2F_F3_1_007) expressed 120 ± 40 pmol of folded enzyme per 1 mL culture (mean ± 1 standard deviation) and was demonstrated to be catalytically active. Structural comparisons were made between P450 2F1, shuffled mutant JH_2F_F3_1_007, and P450 2F3, alongside P450s from the CYP2 family that had been expressed in E. coli previously and for which structures were available. A limited number of specific amino acid residues that may be responsible for misfolding of P450 2F1 were identified, and the most probable candidates being Met65, Ser117, Gln266, Lys301 and His308 on the grounds that they represented mismatches to otherwise strongly conserved residues in successfully-expressed P450 2F forms. The roles of these amino acid residues in protein folding were not obvious when modelled on predicted structures for P450 2F1 and the JH_2F_F3_1_007 mutant, and chimeras constructed to specifically test the importance of residues 65-92 and 266-313 did not achieve significant expression. Incremental elimination of remaining non-CYP2F1 sequence from the JH_2F_F3_1_007 mutant may further clarify the determinants of P450 2F1 folding in E. coli. In the second part of this thesis, DNA family shuffling was used to create libraries of novel P450s, in order to examine the effect of differences in the nucleotide fragmentation step on progeny P450 characteristics. This study focused on one particular aspect of the DNA family shuffling method, the fragmentation of nucleotide sequences. A library of shuffled enzymes was made from P450s 3A4, 3A5, 3A7 and 3A9 using identical methodology to a shuffled library already available, except that different combinations of restriction endonucleases were used during the fragmentation of coding sequences prior to shuffling. A library of similar size to the comparator library was produced (107 vs. 110 mutants). Comparing shuffled mutants from the two libraries demonstrated that an increased frequency of fragmentation during DNA family shuffling resulted in an increase in recombination during the reassembly reaction. However, the increase in recombination came at the cost of sequence diversity with a bias observed toward recombination between two of the four parents, P450s 3A5 and 3A7. Increased recombination also resulted in a decreased frequency of mutants that expressed as properly folded haemoprotein, and a decrease in catalytically active mutants as determined using a set of probe substrates. Finally the limitations of using DNA family shuffling to recombine more distantly-related P450s were tested. Two libraries were made using different fragmentation approaches in attempts to shuffle P450 coding sequences sharing less than 70 % nucleotide sequence identity (human CYP3A4, CYP3A27 from trout, and CYP3A37 from turkey). Recombination was observed in one library but in both libraries a high frequency of reassembled parental P450 sequences was observed. It was concluded that the frequency of shuffled mutants was insufficient for DNA family shuffling to be considered a useful method for generating libraries of novel enzymes by recombination of diverse sequences. In summary, the work in this thesis demonstrates the utility of DNA family shuffling as a problem solving tool for answering fundamental questions regarding P450 folding in recombinant systems, and provides insights into practical aspects of using this method for creating libraries of novel P450 enzymes

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. P450s can catalyse dehalogenation and denitration of a range of environmentally persistent pollutants including halogenated hydrocarbons and nitroamine explosives. P450-mediated reductive dehalogenations were first discovered in the context of human pharmacology but have since been observed in a variety of organisms. Additionally, P450-mediated reductive denitration of synthetic explosives has been discovered in bacteria that inhabit contaminated soils. This review will examine the distribution of P450-mediated reductive dehalogenations and denitrations in nature and discuss synthetic biology approaches to developing P450-based reagents for bioremediation.</p

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. To date, synthetic scaffolding has focused primarily on soluble enzyme systems, but many metabolic pathways for high-value secondary metabolites depend on membrane-bound enzymes. The compositional diversity of biological membranes and general challenges associated with modifying membrane proteins complicate scaffolding with membrane-requiring enzymes. Several recent studies have introduced new approaches to protein scaffolding at membrane surfaces, with notable success in improving product yields from specific metabolic pathways.</p

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

Abstract 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. Screening methods are commonly based on deep-well plate cultures with analysis by immunoblotting or enzyme activity assays of post-induction samples, and each heterologous protein produced may require development of bespoke assays with multiple sample processing steps. In this work, we developed a generic system based on a P. pastoris strain that uses a protein-based biosensor to identify highly productive protein secretion clones from a heterogeneous set of transformants. The biosensor uses a split green fluorescent protein where the large GFP fragment (GFP1-10) is fused to a sequence-specific protease from Tobacco Etch Virus (TEV) and is targeted to the endoplasmic reticulum. Recombinant proteins targeted for secretion are tagged with the small fragment of the split GFP (GFP11). Recombinant protein production can be measured by monitoring GFP fluorescence, which is dependent on interaction between the large and small GFP fragments. The reconstituted GFP is cleaved from the target protein by TEV protease, allowing for secretion of the untagged protein of interest and intracellular retention of the mature GFP. We demonstrate this technology with four recombinant proteins (phytase, laccase, β-casein and β-lactoglobulin) and show that the biosensor directly reports protein production levels that correlate with traditional assays. Our results confirm that the split GFP biosensor can be used for facile, generic, and rapid screening of P. pastoris clones to identify those with the highest production levels

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. Although these enzymes have not evolved in Nature to perform the reactions required for modern chemical industries, many P450s show relaxed substrate specificity and exhibit some degree of activity towards non-natural substrates of relevance to applications such as drug development. Directed evolution and other protein engineering methods can be used to improve upon this low level of activity and convert these promiscuous generalist enzymes into specialists capable of mediating reactions of interest with exquisite regio- and stereo-selectivity. Although there are some notable successes in exploiting P450s from natural sources in metabolic engineering, and P450s have been proven repeatedly to be excellent material for engineering, there are few examples to date of practical application of engineered P450s. The purpose of the present review is to illustrate the progress that has been made in altering properties of P450s such as substrate range, cofactor preference and stability, and outline some of the remaining challenges that must be overcome for industrial application of these powerful biocatalysts