Figure - available from: Journal of Applied Phycology
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RT-qPCR results and Western Blot results of selected constitutive cell lines. Normalised relative quantities of gene transcripts are represented as bar plots. Presence of the corresponding enzymes in detectable quantities is indicated below each bar plot by green ticks. a: Normalised relative quantity of phaA. b: Normalised relative quantity of phaB. c: Normalised relative quantity of phaC. Labels on the x-axis represent individual cell lines. The corresponding Western blot images can be found in Figure S6. Error bars represent standard deviations of technical replicates
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Diatoms such as Phaeodactylum tricornutum are emerging as sustainable alternatives to traditional eukaryotic microbial cell factories. In order to facilitate a viable process for production of heterologous metabolites, a rational genetic design specifically tailored to metabolic requirements as well as optimised culture conditions are required. In...
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Citations
... In this study, we phenotyped P. tricornutum strains engineered with a phosphate limitation-induced PHB pathway, as this was the most effective strategy for episomal expression of PHB in our previous work (Windhagauer et al. 2022). We aimed to understand whether different cultivation conditions, including the additional deprivation of nitrogen, and the addition of carbon sources (glycerol, acetate and citrate), impact transgenic PHB synthesis, and the native metabolism of P. tricornutum. ...
... org). Plasmids carrying codon-optimised genes of the PHB pathway from Ralstonia eutropha (phaA, phaB and phaC) under the control of the AP1 promoter and FcpB terminator were constructed and transformed into P. tricornutum as previously described (Windhagauer et al. 2022). Transformed cell lines were kept at 4 °C on half strength artificial seawater (ASW) (Darley and Volcani 1969) agar plates supplemented with 100 µg/mL zeocin for mediumterm storage. ...
... The initial screening for PHB production under different conditions was performed on transgenic cell lines G5, G7, G8 and G10. Cell lines G5, G7 and G10, which displayed the most consistent growth across multiple cultivations (Windhagauer et al. 2022), were grown in triplicates and subjected to in-depth condition screening including endpoint metabolite analysis. For the transcriptomic analysis experiment, the highest expressing cell line G8 was grown in 1-L Erlenmeyer flasks with a working volume of 250 mL. ...
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 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.
Diatoms are photosynthetic unicellular microalgae that drive global ecological phenomena in the biosphere and are emerging sustainable feedstock for an increasing number of industrial applications. Diatoms exhibit enormous taxonomic and genetic diversity, which often result in peculiar biochemical and biological traits. Transposable elements (TE) represent a substantial portion of diatom genomes and have been hypothesized to exert a relevant role in enriching genetic diversity and centrally contribute to genome evolution. Here, through long-read whole genome sequencing, we identified a novel 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 novel 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 of a diatom-specific E3 ubiquitin ligase of the zinc finger UBR type, which indicate a novel 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.
