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Engineered cardoon calli as novel platform for production of renewable feedstock

ABSTRACT: In this study, we investigated transcriptomic changes in two cardoon (Cynara cardunculus L. var. altilis) calli cell cultures lines engineered for production of valuable fatty acids, therefore suitable for large-scale production of biofuel precursors, as alternative to triacylglycerols (TAGs) engineered plants. Namely to gain insight in the molecular effects of genetic transformation, we analyzed the transcriptome of SAD-OE line (overexpressing stearic acid desaturase), and FAD-KD (a fatty acid desaturase RNA interference line, FAD2.2-RNAi) accumulating oleic and linoleic acids, respectively. Differentially expressed genes (DEGs) involved in fatty acid metabolism and streamed pathways such as glycolysis, pyruvate, glycerolipid, and glycerophospholipid metabolism, were enriched in the KEGG analysis in both lines. Above all DEGs, we also monitored by qRT-PCR key regulatory and biosynthetic genes involved in TAG formation and assembly to understand transcriptional behavior during ten days of cellular growth in liquid culture. In addition, we explored how genetic transformation affected sugar metabolism. Consistently, with transcriptional induction of TAG biosynthetic genes, fructan depletion supported their possible usage for TAG formation. Further, confocal microscopy confirmed a high accumulation of oil bodies in both transgenic lines as compared to WT calli. In conclusion, in this study we set up an efficient and stable platform for massive production of vegetable oils in in cardoon cells which, unlike plants, do not encounter growth and metabolic defects.

ORGANISM(S): Cynara cardunculus

PROVIDER: GSE284690 | GEO | 2025/10/14

REPOSITORIES: GEO

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Project description:The oleaginous green microalga Neochloris oleoabundans accumulates both starch and lipids to high levels under stress conditions such as nitrogen starvation. In order to steer the metabolism towards starch or lipids only, it is important to understand the mechanisms behind it. In this study physiological changes and gene expression upon nitrogen starvation were analysed in controlled flat-panel photobioreactors over both short- and long-term time-scales. Starch accumulation is transient and occurs rapidly within 24 hrs upon starvation, while lipid accumulation lasts longer and reaches a maximum after 4 days. The major fraction of accumulated lipids is composed of de novo synthesized neutral lipids - triacylglycerides (TAG) - and is characterized by a decreased composition of the polyunsaturated fatty acids (PUFAs) C18:3 and C16:3 and an increased composition of the mono-unsaturated and saturated fatty acids C18:1/C16:1 and C18:0/C16:0, respectively. RNA-sequencing revealed that genes related to starch biosynthesis and degradation show different temporal expression dynamics compared to those of lipid biosynthesis. An immediate rapid increase in starch synthesis gene expression is followed by an increase in starch degradation and decrease in starch synthesis gene expression. In contrast, increased gene expression for fatty acid and TAG synthesis is initiated later and occurs more gradually. Expression of several fatty acid desaturase (FAD) genes was decreased upon starvation, which corresponds to the observed changes to higher levels of mono-unsaturated and saturated fatty acids. Moreover, several homologs of transcription regulators that were implicated in controlling starch and lipid metabolism in other microalgae showed differential gene expression and might be key regulators of starch and lipid metabolism in N. oleoabundans as well. Promising candidates for future metabolic engineering are a DYRKP homolog that in Chlamydomonas acts as a negative regulator of carbon storage and photosynthetic efficiency under N-starvation, and two bZIP-type regulators that have been implicated to control several steps in microalgal TAG synthesis. Our data for the first time show the temporal dynamics of storage compound accumulation and transcriptional changes on a short- and long-term time-scale during nitrogen starvation in N. oleoabundans, and increases insight into the genetic foundation for starch and lipid metabolism in this microalga. This information and the identified target genes can now be used for metabolic engineering strategies towards tailored N. oleoabundans strains for industrial applications with increased lipid production and altered fatty acid composition.

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Engineered cardoon calli as novel platform for production of renewable feedstock

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