Genome-Scale Model Reveals Metabolic Basis of Biomass Partitioning in a Model Diatom

Abstract

Diatoms are eukaryotic microalgae that contain genes from various sources, including bacteria and the secondary endosymbiotic host. Due to this unique combination of genes, diatoms are taxonomically and functionally distinct from other algae and vascular plants and confer novel metabolic capabilities. Based on the genome annotation, we performed a genome-scale metabolic network reconstruction for the marine diatom Phaeodactylum tricornutum. Due to their endosymbiotic origin, diatoms possess a complex chloroplast structure which complicates the prediction of subcellular protein localization. Based on previous work we implemented a pipeline that exploits a series of bioinformatics tools to predict protein localization. The manually curated reconstructed metabolic network iLB1027_lipid accounts for 1,027 genes associated with 4,456 reactions and 2,172 metabolites distributed across six compartments. To constrain the genome-scale model, we determined the organism specific biomass composition in terms of lipids, carbohydrates, and proteins using Fourier transform infrared spectrometry. Our simulations indicate the presence of a yet unknown glutamine-ornithine shunt that could be used to transfer reducing equivalents generated by photosynthesis to the mitochondria. The model reflects the known biochemical composition of P. tricornutum in defined culture conditions and enables metabolic engineering strategies to improve the use of P. tricornutum for biotechnological applications.

Transcriptional Orchestration of the Global Cellular Response of a Model Pennate Diatom to Diel Light Cycling under Iron Limitation

“…However, little is known regarding the specific role of the OUC across diverse physiological states. Recent work by Levering et al [120] identified the existence of a chloroplast ornithine cycle, the function of which was hypothesized to exchange NAD(P)H and ATP between the chloroplast and mitochondria. To gain insight into the biological role of these pathways and potential interactions between them, the diel and Fe-specific expression of genes and key metabolites involved in the OUC cycle were analyzed.…”

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

“…In 10 rescued NR knockouts, YFP expression was also induced when the cells were switched from NH 4 + to NO 3 2 media (Supplemental Figure 3). FTIR spectroscopy, previously calibrated using biochemical measurements (Levering et al, 2016;Mayers et al, 2013;Stehfest Figure 3A). During a 7-d time course experiment, three NR-KO lines (#9, 14, and 15) and the wild type were initially grown in NH 4 + media and then transferred into NO 3 2 -amended media and sampled periodically.…”

“…Indeed, expression profiles of genes encoding ACOAT, unCPS, GLDH, and ICDH (Supplemental Figures 5D to 5F and 11B) are more similar to the upstream NR cohort genes and support their substantive links to N-assimilation and C-metabolism. Plastidic ACOAT, a component of the recently proposed in silico ornithine-glutamine shuttle, transfers reducing equivalents generated by photosynthesis to the mitochondria (Levering et al, 2016). During N-stress, its secondary product, chloroplast-produced 2OG, may supplement the 2OG produced by ICDH in the mitochondria.…”

“…Prior to scanning, samples were dried with a lyophilizer, maintained in a desiccator, crushed to a powder, and placed on the sample holder. Percentages of protein, carbohydrate, and lipid per biomass, derived from peak values in scans, were compared by paired t tests for significant differences over the 7-d time course (Levering et al, 2016). Each NR-KO line (1 and 14) was individually compared with the wild type.…”

A Phaeodactylum tricornutum literature database for interactive annotation of content

“…It streamlines the manual annotation and verification of metabolic pathways by providing a literature resource that is focused on biochemistry and physiology [19,20]. We created a bibliome of P. tricornutum-specific literature to facilitate a model that describes its metabolism [21]. The result is a comprehensive literature database enriched in biochemical, physiological, and molecular biology studies on P. tricornutum literature.…”