Metabolic response to a heterologous poly-3-hydroxybutyrate (PHB) pathway in Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum is an emerging host for metabolic engineering, but little is known about how introduced pathways are integrated into the existing metabolic framework of the host or influence transgene expression. In this study, we expressed the heterologous poly-3-hydroxybutyrate (PHB) pathway using episomal expression, which draws on the precursor acetyl coenzyme-A (AcCoA). By experimentally perturbing cultivation conditions, we gained insight into the regulation of the endogenous metabolism in transgenic lines under various environmental scenarios, as well as on alterations in AcCoA flux within the host cell. Biosynthesis of PHB led to distinct shifts in the metabolome of the host, and further analysis revealed a condition-dependent relationship between endogenous and transgenic metabolic pathways. Under N limitation, which induced a significant increase in neutral lipid content, both metabolic and transcriptomic data suggest that AcCoA was preferably shunted into the endogenous pathway for lipid biosynthesis over the transgenic PHB pathway. In contrast, supply of organic carbon in the form of glycerol supported both fatty acid and PHB biosynthesis, suggesting cross-talk between cytosolic and plastidial AcCoA precursors. This is the first study to investigate the transcriptomic and metabolomic response of diatom cell lines expressing a heterologous multi-gene pathway under different environmental conditions, providing useful insights for future engineering attempts for pathways based on the precursor AcCoA. KEY POINTS: • PHB expression had minimal effects on transcription of adjacent pathways. • N limitation favoured native lipid rather than transgenic PHB synthesis. • Glycerol addition allowed simultaneous lipid and PHB accumulation.

Biosensors in microalgae: A roadmap for new opportunities in synthetic biology and biotechnology.

Biosensors are powerful tools to investigate, phenotype, improve and prototype microbial strains, both in fundamental research and in industrial contexts. Genetic and biotechnological developments now allow the implementation of synthetic biology approaches to novel different classes of microbial hosts, for example photosynthetic microalgae, which offer unique opportunities. To date, biosensors have not yet been implemented in phototrophic eukaryotic microorganisms, leaving great potential for novel biological and technological advancements untapped. Here, starting from selected biosensor technologies that have successfully been implemented in heterotrophic organisms, we project and define a roadmap on how these could be applied to microalgae research. We highlight novel opportunities for the development of new biosensors, identify critical challenges, and finally provide a perspective on the impact of their eventual implementation to tackle research questions and bioengineering strategies. From studying metabolism at the single-cell level to genome-wide screen approaches, and assisted laboratory evolution experiments, biosensors will greatly impact the pace of progress in understanding and engineering microalgal metabolism. We envision how this could further advance the possibilities for unraveling their ecological role, evolutionary history and accelerate their domestication, to further drive them as resource-efficient production hosts.

Mobilization of a diatom mutator-like element (MULE) transposon inactivates the uridine monophosphate synthase (UMPS) locus in Phaeodactylum tricornutum.

Diatoms are photosynthetic unicellular microalgae that drive global ecological phenomena in the biosphere and are emerging as sustainable feedstock for an increasing number of industrial applications. Diatoms exhibit enormous taxonomic and genetic diversity, which often results in peculiar biochemical and biological traits. Transposable elements (TEs) represent a substantial portion of diatom genomes and have been hypothesized to exert a relevant role in enriching genetic diversity and making a core contribution to genome evolution. Here, through long-read whole-genome sequencing, we identified a mutator-like element (MULE) in the model diatom Phaeodactylum tricornutum, and we report the direct observation of its mobilization within the course of a single laboratory experiment. Under selective conditions, this TE inactivated the uridine monophosphate synthase (UMPS) gene of P. tricornutum, one of the few endogenous genetic loci currently targeted for selectable auxotrophy for functional genetics and genome-editing applications. We report the observation of a recently mobilized transposon in diatoms with unique features. These include the combined presence of a MULE transposase with zinc-finger SWIM-type domains and a diatom-specific E3 ubiquitin ligase of the zinc-finger UBR type, which are suggestive of a mobilization mechanism. Our findings provide new elements for the understanding of the role of TEs in diatom genome evolution and in the enrichment of intraspecific genetic variability.

Analysing intracellular isoprenoid metabolites in diverse prokaryotic and eukaryotic microbes.

Isoprenoids, also known as terpenes or terpenoids, are a very large and diverse group of natural compounds. These compounds fulfil a myriad of critical roles in biology as well as having a wide range of industrial uses. Isoprenoids are produced via two chemically distinct metabolic pathways, the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. Downstream of these two pathways is the shared prenyl phosphate pathway. Because of their importance in both basic physiology and industrial biotechnology, extraction, identification, and quantification of isoprenoid pathway intermediates is an important protocol. Here we describe methods for extraction and analysis of intracellular metabolites from the MVA, MEP, and prenyl phosphate pathways for five key model microbes: the yeast Saccharomyces cerevisiae, the bacterium Escherichia coli, the diatom Phaeodactylum tricornutum, the green algae Chlamydomonas reinhardtii, and the cyanobacterium Synechocystis sp. PCC 6803. These methods also detect several central carbon intermediates. These protocols will likely work effectively, or be readily adaptable, to a variety of related microorganisms and metabolic pathways.

Methyl Jasmonate and Methyl-ß-Cyclodextrin Individually Boost Triterpenoid Biosynthesis in Chlamydomonas Reinhardtii UVM4.

The commercialisation of valuable plant triterpenoids faces major challenges, including low abundance in natural hosts and costly downstream purification procedures. Endeavours to produce these compounds at industrial scale using microbial systems are gaining attention. Here, we report on a strategy to enrich the biomass of the biotechnologically-relevant Chlamydomonas reinhardtii strain UVM4 with valuable triterpenes, such as squalene and (S)-2,3-epoxysqualene. C. reinhardtii UVM4 was subjected to the elicitor compounds methyl jasmonate (MeJA) and methyl-ß-cyclodextrine (MßCD) to increase triterpene yields. MeJA treatment triggered oxidative stress, arrested growth, and altered the photosynthetic activity of the cells, while increasing squalene, (S)-2,3-epoxysqualene, and cycloartenol contents. Applying MßCD to cultures of C. reinhardtii lead to the sequestration of the two main sterols (ergosterol and 7-dehydroporiferasterol) into the growth medium and the intracellular accumulation of the intermediate cycloartenol, without compromising cell growth. When MßCD was applied in combination with MeJA, it counteracted the negative effects of MeJA on cell growth and physiology, but no synergistic effect on triterpene yield was observed. Together, our findings provide strategies for the triterpene enrichment of microalgal biomass and medium.

Overexpression of Key Sterol Pathway Enzymes in Two Model Marine Diatoms Alters Sterol Profiles in Phaeodactylum tricornutum.

Sterols are a class of triterpenoid molecules with diverse functional roles in eukaryotic cells, including intracellular signaling and regulation of cell membrane fluidity. Diatoms are a dominant eukaryotic phytoplankton group that produce a wide diversity of sterol compounds. The enzymes 3-hydroxy-3-methyl glutaryl CoA reductase (HMGR) and squalene epoxidase (SQE) have been reported to be rate-limiting steps in sterol biosynthesis in other model eukaryotes; however, the extent to which these enzymes regulate triterpenoid production in diatoms is not known. To probe the role of these two metabolic nodes in the regulation of sterol metabolic flux in diatoms, we independently over-expressed two versions of the native HMGR and a conventional, heterologous SQE gene in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. Overexpression of these key enzymes resulted in significant differential accumulation of downstream sterol pathway intermediates in P. tricornutum. HMGR-mVenus overexpression resulted in the accumulation of squalene, cycloartenol, and obtusifoliol, while cycloartenol and obtusifoliol accumulated in response to heterologous NoSQE-mVenus overexpression. In addition, accumulation of the end-point sterol 24-methylenecholesta-5,24(24')-dien-3ß-ol was observed in all P. tricornutum overexpression lines, and campesterol increased three-fold in P. tricornutum lines expressing NoSQE-mVenus. Minor differences in end-point sterol composition were also found in T. pseudonana, but no accumulation of sterol pathway intermediates was observed. Despite the successful manipulation of pathway intermediates and individual sterols in P. tricornutum, total sterol levels did not change significantly in transformed lines, suggesting the existence of tight pathway regulation to maintain total sterol content.

Metabolic Engineering Strategies in Diatoms Reveal Unique Phenotypes and Genetic Configurations With Implications for Algal Genetics and Synthetic Biology.

Diatoms are photosynthetic microeukaryotes that dominate phytoplankton populations and have increasing applicability in biotechnology. Uncovering their complex biology and elevating strains to commercial standards depends heavily on robust genetic engineering tools. However, engineering microalgal genomes predominantly relies on random integration of transgenes into nuclear DNA, often resulting in detrimental "position-effects" such as transgene silencing, integration into transcriptionally-inactive regions, and endogenous sequence disruption. With the recent development of extrachromosomal transgene expression via independent episomes, it is timely to investigate both strategies at the phenotypic and genomic level. Here, we engineered the model diatom Phaeodactylum tricornutum to produce the high-value heterologous monoterpenoid geraniol, which, besides applications as fragrance and insect repellent, is a key intermediate of high-value pharmaceuticals. Using high-throughput phenotyping we confirmed the suitability of episomes for synthetic biology applications and identified superior geraniol-yielding strains following random integration. We used third generation long-read sequencing technology to generate a complete analysis of all transgene integration events including their genomic locations and arrangements associated with high-performing strains at a genome-wide scale with subchromosomal detail, never before reported in any microalga. This revealed very large, highly concatenated insertion islands, offering profound implications on diatom functional genetics and next generation genome editing technologies, and is key for developing more precise genome engineering approaches in diatoms, including possible genomic safe harbour locations to support high transgene expression for targeted integration approaches. Furthermore, we have demonstrated that exogenous DNA is not integrated inadvertently into the nuclear genome of extrachromosomal-expression clones, an important characterisation of this novel engineering approach that paves the road to synthetic biology applications.

Emerging Technologies in Algal Biotechnology: Toward the Establishment of a Sustainable, Algae-Based Bioeconomy.

Mankind has recognized the value of land plants as renewable sources of food, medicine, and materials for millennia. Throughout human history, agricultural methods were continuously modified and improved to meet the changing needs of civilization. Today, our rapidly growing population requires further innovation to address the practical limitations and serious environmental concerns associated with current industrial and agricultural practices. Microalgae are a diverse group of unicellular photosynthetic organisms that are emerging as next-generation resources with the potential to address urgent industrial and agricultural demands. The extensive biological diversity of algae can be leveraged to produce a wealth of valuable bioproducts, either naturally or via genetic manipulation. Microalgae additionally possess a set of intrinsic advantages, such as low production costs, no requirement for arable land, and the capacity to grow rapidly in both large-scale outdoor systems and scalable, fully contained photobioreactors. Here, we review technical advancements, novel fields of application, and products in the field of algal biotechnology to illustrate how algae could present high-tech, low-cost, and environmentally friendly solutions to many current and future needs of our society. We discuss how emerging technologies such as synthetic biology, high-throughput phenomics, and the application of internet of things (IoT) automation to algal manufacturing technology can advance the understanding of algal biology and, ultimately, drive the establishment of an algal-based bioeconomy.

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae.

Microalgae exhibit great potential for recombinant therapeutic protein production, due to lower production costs, immunity to human pathogens, and advanced genetic toolkits. However, a fundamental aspect to consider for recombinant biopharmaceutical production is the presence of correct post-translational modifications. Multiple recent studies focusing on glycosylation in microalgae have revealed unique species-specific patterns absent in humans. Glycosylation is particularly important for protein function and is directly responsible for recombinant biopharmaceutical immunogenicity. Therefore, it is necessary to fully characterise this key feature in microalgae before these organisms can be established as industrially relevant microbial biofactories. Here, we review the work done to date on production of recombinant biopharmaceuticals in microalgae, experimental and computational evidence for N- and O-glycosylation in diverse microalgal groups, established approaches for glyco-engineering, and perspectives for their application in microalgal systems. The insights from this review may be applied to future glyco-engineering attempts to humanize recombinant therapeutic proteins and to potentially obtain cheaper, fully functional biopharmaceuticals from microalgae.

Extrachromosomal Genetic Engineering of the Marine Diatom Phaeodactylum tricornutum Enables the Heterologous Production of Monoterpenoids.

Geraniol is a commercially relevant plant-derived monoterpenoid that is a main component of rose essential oil and used as insect repellent. Geraniol is also a key intermediate compound in the biosynthesis of the monoterpenoid indole alkaloids (MIAs), a group of over 2000 compounds that include high-value pharmaceuticals. As plants naturally produce extremely small amounts of these molecules and their chemical synthesis is complex, industrially sourcing these compounds is costly and inefficient. Hence, microbial hosts suitable to produce MIA precursors through synthetic biology and metabolic engineering are currently being sought. Here, we evaluated the suitability of a eukaryotic microalga, the marine diatom Phaeodactylum tricornutum, for the heterologous production of monoterpenoids. Profiling of endogenous metabolism revealed that P. tricornutum, unlike other microbes employed for industrial production of terpenoids, accumulates free pools of the precursor geranyl diphosphate. To evaluate the potential for larger synthetic biology applications, we engineered P. tricornutum through extrachromosomal, episome-based expression, for the heterologous biosynthesis of the MIA intermediate geraniol. By profiling the production of geraniol resulting from various genetic and cultivation arrangements, P. tricornutum reached the maximum geraniol titer of 0.309 mg/L in phototrophic conditions. This work provides (i) a detailed analysis of P. tricornutum endogenous terpenoid metabolism, (ii) a successful demonstration of extrachromosomal expression for metabolic pathway engineering with potential gene-stacking applications, and (iii) a convincing proof-of-concept of the suitability of P. tricornutum as a novel production platform for heterologous monoterpenoids, with potential for complex pathway engineering aimed at the heterologous production of MIAs.

Phytosterol biosynthesis and production by diatoms (Bacillariophyceae).

Diatoms are abundant unicellular marine photosynthetic algae that have genetically diversified their physiology and metabolism while adapting to numerous environments. The metabolic repertoire of diatoms presents opportunities to characterise the biosynthesis and production of new and potentially valuable microalgal compounds, including sterols. Sterols of plant origin, known as phytosterols, have been studied for health benefits including demonstrated cholesterol-lowering properties. In this review we summarise sterol diversity, the unique metabolic features of sterol biosynthesis in diatoms, and prospects for the extraction of diatom phytosterols in comparison to existing sources. We also review biotechnological efforts to manipulate diatom biosynthesis, including culture conditions and avenues for the rational engineering of metabolism and cellular regulation.

A widespread alternative squalene epoxidase participates in eukaryote steroid biosynthesis.

Steroids are essential triterpenoid molecules that are present in all eukaryotes and modulate the fluidity and flexibility of cell membranes. Steroids also serve as signalling molecules that are crucial for growth, development and differentiation of multicellular organisms1-3. The steroid biosynthetic pathway is highly conserved and is key in eukaryote evolution4-7. The flavoprotein squalene epoxidase (SQE) catalyses the first oxygenation reaction in this pathway and is rate limiting. However, despite its conservation in animals, plants and fungi, several phylogenetically widely distributed eukaryote genomes lack an SQE-encoding gene7,8. Here, we discovered and characterized an alternative SQE (AltSQE) belonging to the fatty acid hydroxylase superfamily. AltSQE was identified through screening of a gene library of the diatom Phaeodactylum tricornutum in a SQE-deficient yeast. In accordance with its divergent protein structure and need for cofactors, we found that AltSQE is insensitive to the conventional SQE inhibitor terbinafine. AltSQE is present in many eukaryotic lineages but is mutually exclusive with SQE and shows a patchy distribution within monophyletic clades. Our discovery provides an alternative element for the conserved steroid biosynthesis pathway, raises questions about eukaryote metabolic evolution and opens routes to develop selective SQE inhibitors to control hazardous organisms.

Terpenoid Metabolic Engineering in Photosynthetic Microorganisms.

Terpenoids are a group of natural products that have a variety of roles, both essential and non-essential, in metabolism and in biotic and abiotic interactions, as well as commercial applications such as pharmaceuticals, food additives, and chemical feedstocks. Economic viability for commercial applications is commonly not achievable by using natural source organisms or chemical synthesis. Engineered bio-production in suitable heterologous hosts is often required to achieve commercial viability. However, our poor understanding of regulatory mechanisms and other biochemical processes makes obtaining efficient conversion yields from feedstocks challenging. Moreover, production from carbon dioxide via photosynthesis would significantly increase the environmental and potentially the economic credentials of these processes by disintermediating biomass feedstocks. In this paper, we briefly review terpenoid metabolism, outline some recent advances in terpenoid metabolic engineering, and discuss why photosynthetic unicellular organisms-such as algae and cyanobacteria-might be preferred production platforms for the expression of some of the more challenging terpenoid pathways.

The transcription factor bZIP14 regulates the TCA cycle in the diatom Phaeodactylum tricornutum.

Diatoms are amongst the most important marine microalgae in terms of biomass, but little is known concerning the molecular mechanisms that regulate their versatile metabolism. Here, the pennate diatom Phaeodactylum tricornutum was studied at the metabolite and transcriptome level during nitrogen starvation and following imposition of three other stresses that impede growth. The coordinated upregulation of the tricarboxylic acid (TCA) cycle during the nitrogen stress response was the most striking observation. Through co-expression analysis and DNA binding assays, the transcription factor bZIP14 was identified as a regulator of the TCA cycle, also beyond the nitrogen starvation response, namely in diurnal regulation. Accordingly, metabolic and transcriptional shifts were observed upon overexpression of bZIP14 in transformed P. tricornutum cells. Our data indicate that the TCA cycle is a tightly regulated and important hub for carbon reallocation in the diatom cell during nutrient starvation and that bZIP14 is a conserved regulator of this cycle.

Flux balance analysis of primary metabolism in the diatom Phaeodactylum tricornutum.

Diatoms (Bacillarophyceae) are photosynthetic unicellular microalgae that have risen to ecological prominence in oceans over the past 30 million years. They are of interest as potential feedstocks for sustainable biofuels. Maximizing production of these feedstocks will require genetic modifications and an understanding of algal metabolism. These processes may benefit from genome-scale models, which predict intracellular fluxes and theoretical yields, as well as the viability of knockout and knock-in transformants. Here we present a genome-scale metabolic model of a fully sequenced and transformable diatom: Phaeodactylum tricornutum. The metabolic network was constructed using the P. tricornutum genome, biochemical literature, and online bioinformatic databases. Intracellular fluxes in P. tricornutum were calculated for autotrophic, mixotrophic and heterotrophic growth conditions, as well as knockout conditions that explore the in silico role of glycolytic enzymes in the mitochondrion. The flux distribution for lower glycolysis in the mitochondrion depended on which transporters for TCA cycle metabolites were included in the model. The growth rate predictions were validated against experimental data obtained using chemostats. Two published studies on this organism were used to validate model predictions for cyclic electron flow under autotrophic conditions, and fluxes through the phosphoketolase, glycine and serine synthesis pathways under mixotrophic conditions. Several gaps in annotation were also identified. The model also explored unusual features of diatom metabolism, such as the presence of lower glycolysis pathways in the mitochondrion, as well as differences between P. tricornutum and other photosynthetic organisms.

Profiling of the Early Nitrogen Stress Response in the Diatom Phaeodactylum tricornutum Reveals a Novel Family of RING-Domain Transcription Factors.

Diatoms often inhabit highly variable habitats where they are confronted with a wide variety of stresses, frequently including starvation of nutrients such as nitrogen. In this study, the transcriptome of the model diatom Phaeodactylum tricornutum was profiled during the onset of nitrogen starvation by RNA sequencing, and overrepresented motifs were determined in promoters of genes that were early and strongly up-regulated during the nitrogen stress response. One of these motifs could be bound by a nitrogen starvation-inducible RING-domain protein termed RING-GAF-Gln-containing protein (RGQ1), which was shown to act as a transcription factor and belongs to a previously uncharacterized family that is conserved in heterokont algae.

Tracking the sterol biosynthesis pathway of the diatom Phaeodactylum tricornutum.

Diatoms are unicellular photosynthetic microalgae that play a major role in global primary production and aquatic biogeochemical cycling. Endosymbiotic events and recurrent gene transfers uniquely shaped the genome of diatoms, which contains features from several domains of life. The biosynthesis pathways of sterols, essential compounds in all eukaryotic cells, and many of the enzymes involved are evolutionarily conserved in eukaryotes. Although well characterized in most eukaryotes, the pathway leading to sterol biosynthesis in diatoms has remained hitherto unidentified. Through the DiatomCyc database we reconstructed the mevalonate and sterol biosynthetic pathways of the model diatom Phaeodactylum tricornutum in silico. We experimentally verified the predicted pathways using enzyme inhibitor, gene silencing and heterologous gene expression approaches. Our analysis revealed a peculiar, chimeric organization of the diatom sterol biosynthesis pathway, which possesses features of both plant and fungal pathways. Strikingly, it lacks a conventional squalene epoxidase and utilizes an extended oxidosqualene cyclase and a multifunctional isopentenyl diphosphate isomerase/squalene synthase enzyme. The reconstruction of the P. tricornutum sterol pathway underscores the metabolic plasticity of diatoms and offers important insights for the engineering of diatoms for sustainable production of biofuels and high-value chemicals.

CathaCyc, a metabolic pathway database built from Catharanthus roseus RNA-Seq data.

The medicinal plant Madagascar periwinkle (Catharanthus roseus) synthesizes numerous terpenoid indole alkaloids (TIAs), such as the anticancer drugs vinblastine and vincristine. The TIA pathway operates in a complex metabolic network that steers plant growth and survival. Pathway databases and metabolic networks reconstructed from 'omics' sequence data can help to discover missing enzymes, study metabolic pathway evolution and, ultimately, engineer metabolic pathways. To date, such databases have mainly been built for model plant species with sequenced genomes. Although genome sequence data are not available for most medicinal plant species, next-generation sequencing is now extensively employed to create comprehensive medicinal plant transcriptome sequence resources. Here we report on the construction of CathaCyc, a detailed metabolic pathway database, from C. roseus RNA-Seq data sets. CathaCyc (version 1.0) contains 390 pathways with 1,347 assigned enzymes and spans primary and secondary metabolism. Curation of the pathways linked with the synthesis of TIAs and triterpenoids, their primary metabolic precursors, and their elicitors, the jasmonate hormones, demonstrated that RNA-Seq resources are suitable for the construction of pathway databases. CathaCyc is accessible online (http://www.cathacyc.org) and offers a range of tools for the visualization and analysis of metabolic networks and 'omics' data. Overlay with expression data from publicly available RNA-Seq resources demonstrated that two well-characterized C. roseus terpenoid pathways, those of TIAs and triterpenoids, are subject to distinct regulation by both developmental and environmental cues. We anticipate that databases such as CathaCyc will become key to the study and exploitation of the metabolism of medicinal plants.

The metabolic blueprint of Phaeodactylum tricornutum reveals a eukaryotic Entner-Doudoroff glycolytic pathway.

Diatoms are one of the most successful groups of unicellular eukaryotic algae. Successive endosymbiotic events contributed to their flexible metabolism, making them competitive in variable aquatic habitats. Although the recently sequenced genomes of the model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana have provided the first insights into their metabolic organization, the current knowledge on diatom biochemistry remains fragmentary. By means of a genome-wide approach, we developed DiatomCyc, a detailed pathway/genome database of P. tricornutum. DiatomCyc contains 286 pathways with 1719 metabolic reactions and 1613 assigned enzymes, spanning both the central and parts of the secondary metabolism of P. tricornutum. Central metabolic pathways, such as those of carbohydrates, amino acids and fatty acids, were covered. Furthermore, our understanding of the carbohydrate model in P. tricornutum was extended. In particular we highlight the discovery of a functional Entner-Doudoroff pathway, an ancient alternative for the glycolytic Embden-Meyerhof-Parnas pathway, and a putative phosphoketolase pathway, both uncommon in eukaryotes. DiatomCyc is accessible online (http://www.diatomcyc.org), and offers a range of software tools for the visualization and analysis of metabolic networks and 'omics' data. We anticipate that DiatomCyc will be key to gaining further understanding of diatom metabolism and, ultimately, will feed metabolic engineering strategies for the industrial valorization of diatoms.

Transcriptional profiling in the lumbar spinal cord of a mouse model of amyotrophic lateral sclerosis: a role for wild-type superoxide dismutase 1 in sporadic disease?

Mice bearing mutations of copper-zinc-superoxide dismutase recapitulate spinal cord motor neuron degeneration and disease progression occurring in human amyotrophic lateral sclerosis. We have investigated the relationship between disease progression and altered gene expression by comparing the transcriptional profiles in lumbar spinal cord, fronto-parietal cortex and hippocampus of mutant G93A-SOD1, wild-type SOD1 transgenic and non-transgenic mice. Gene expression was evaluated at 55 and 110 days of age, representing pre-symptomatic and advanced disease stages of G93A mice, respectively. Whereas no significant variations were detectable in cortical and hippocampal areas, several mutation-related changes were detected in the lumbar spinal cord at the symptomatic stage, consistent with a condition of neuronal distress. Also, at both ages, we found a number of transgene-related changes, i.e. variations occurring in both transgenic groups independently of the G93A mutation, with wild-type SOD1- and G93A-SOD1-overexpressing mice displaying global transcriptional similarity at 110 days of age. Some of the changes in common between the two transgenic groups involve genes implicated in oxidative stress, inflammation, spinocerebellar degeneration and other neurodegenerative disorders. The finding that gene expressional alterations potentially associated to cellular distress are shared by wild-type and mutant human SOD1-overexpressing mice raises the possibility that mutated (in familial ALS) or otherwise dysregulated (in sporadic ALS) SOD1 expression is a common pathogenetic substrate of the disease.