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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-3β-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.

Diatoms are a broadly distributed and evolutionarily diversified group of microalgae that produce a diverse range of sterol compounds. Sterols are triterpenoids that play essential roles in membrane-related processes in eukaryotic cells. Some sterol compounds possess bioactivities that promote human health and are currently used as nutraceuticals. The relationship between sterol diversity in diatoms and their acclimation to different environments is not well understood. In this study, we investigated the occurrence of different sterol types across twelve diatom species, as well as the effect of temperature reduction and changes in salinity on the sterol contents of three model diatom species. In the diatoms Thalassiosira pseudonana, Phaeodactylum tricornutum and Chaetoceros muelleri, we found that changes in the relative contents of minor sterols accompanied shifts in temperature and salinity. This may be indicative of acquired adaptive traits in diatom metabolism.

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-β-cyclodextrine (MβCD) 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 MβCD 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 MβCD 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.

Rhizogenic Agrobacterium strains comprise biotrophic pathogens that cause hairy root disease (HRD) on hydroponically grown Solanaceae and Cucurbitaceae crops, besides being widely explored agents for the creation of hairy root cultures for the sustainable production of plant-specialized metabolites. Hairy root formation is mediated through the expression of genes encoded on the T-DNA of the root-inducing (Ri) plasmid, of which several, including root oncogenic locus B (rolB), play a major role in hairy root development. Despite decades of research, the exact molecular function of the proteins encoded by the rol genes remains enigmatic. Here, by means of TurboID-mediated proximity labeling in tomato (Solanum lycopersicum) hairy roots, we identified the repressor proteins TOPLESS (TPL) and Novel Interactor of JAZ (NINJA) as direct interactors of RolB. Although these interactions allow RolB to act as a transcriptional repressor, our data hint at another in planta function of the RolB oncoprotein. Hence, by a series of plant bioassays, transcriptomic and DNA-binding site enrichment analyses, we conclude that RolB can mitigate the TPL functioning so that it leads to a specific and partial reprogramming of phytohormone signaling, immunity, growth, and developmental processes. Our data support a model in which RolB manipulates host transcription, at least in part, through interaction with TPL, to facilitate hairy root development. Thereby, we provide important mechanistic insights into this renowned oncoprotein in HRD.

En este artículo se determinaron las mejores condiciones para la remoción del colorante Azul Ácido 9 (AA9) a través de un diseño factorial completo 23 y su posterior optimización mediante un diseño de superficie Box-Behnken utilizando tallos de flores (TF) como material adsorbente. Las variables evaluadas fueron dosis de adsorbente (D), concentración de colorante (C) y tiempo de contacto (t). El contenido del colorante se cuantificó por espectrometría UV-Vis. El modelo estadístico presentó un adecuado coeficiente de ajuste (R2 = 99,18 %), permitiendo alcanzar una remoción del 98,5% con una dosificación de 7,8 gL-1, concentración de colorante de 11,7 mgL-1 y tiempo de contacto de 104 min. Estos resultados sugieren que los residuos de flores constituyen un material adsorbente alternativo y potencial para el tratamiento de colorantes ácidos disueltos.

Diatoms are a large group of eukaryotic microalgae that arose through secondary endosymbiosis and are renowned for their wide ecological distribution. Diatoms have genetically diversified their physiology, metabolism and natural products, while adapting to dynamic environments. Among these metabolic products are an expanded repertoire of phytosterols, a class of essential terpenoids that are involved in the regulation of membrane dynamics, signalling, and membrane-bound protein functions in higher plants, algae, fungi, and vertebrates. Phytosterols are considered a marker of eukaryotic life and have been used to identify and date evolutionary events. They are also useful natural products due to their wide range of biological applications. The principal therapeutic and nutraceutical properties of phytosterols include cholesterol-lowering, anti-inflammatory and anti-diabetic activities. The global phytosterol market by 2013 was US$ 300 million and it is growing at about 7-9% per annum. In order to meet this demand, diatom microalgae are proposed as an alternative source of natural products.The function, distribution and biosynthesis of sterols is well characterised and conserved in model animal, plant and fungal organisms. However, the biological role and metabolism of the high diversity of sterols produced by diatoms is not well understood. To establish diatoms as a suitable platform for phytosterols production, in this PhD project we provide insight into key aspects of sterol compounds from diatoms: i) The response of sterol levels to changes in environmental conditions, ii) The reconstruction of the sterol biosynthesis pathways of multiple diatom species, and iii) Genetic investigations and engineering of diatoms to alter sterol product profiles.In Chapter 1, I provide an updated review of the phytosterol repertoire in diatoms, including the biology and regulation of sterol biosynthesis according to the latest primary studies, and new genetic approaches by which the productive metabolisms of these organisms can be further optimised.In the first data chapter, Chapter 2, I investigated the occurrence of different sterol types in twelve different diatom species, as well as the effect of temperature reduction and changes in salinity on the sterol contents of three model diatoms. In Chapter 3, I experimentally examined the sterol biosynthesis pathways of three divergent diatom species, using empirical biochemical profiling and comparative ‘omics. This Chapter experimentally explored hypotheses with regard to what extent the sterol biosynthesis pathways of three diatom species are conserved, and where each of these has diverged to produce different phytosterols. This study introduces in-depth multi-species analyses in order to compare and contrast the biosynthesis pathways of distantly related species. The results expand our understanding of sterol biosynthesis in diatoms, including a new model for cholesterol synthesis in diatoms.Finally, in Chapter 4, I implemented and performed genetic engineering technologies to test the extent to which natural sterol levels can be rationally manipulated in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. Three different genetic targets were chosen, including i) The overexpression of a rate-limiting enzyme in sterol biosynthesis, HMGR, and ii) the expression of a N-terminal truncated HMGR and introduction of a heterologous squalene epoxidase enzyme from the microalgae Nannochloropsis oceanica.

Jasmonic acid (JA) is the precursor of the bioactive molecule jasmonoyl-isoleucine (JA-Ile), a plant hormone that regulates fitness and development. Although JA biosynthesis, signaling, and responses have been intensively studied, the catabolism of JA remains incompletely understood. Here, we used the recently developed technique of limited proteolysis-coupled mass spectrometry (LiP-MS) to investigate metabolite–protein interactions in plants, aiming to discover enzymes involved in JA metabolism. We identified several previously reported JA-binding proteins, thus validating the robustness of the method, along with recognized enzymes of the JA pathway and a series of novel potential JA-binding proteins. We performed functional characterization of a set of identified JA-interacting UDP-glucuronosyltransferase (UGT) enzymes through omics, biochemical, enzymatic, and structural analyses. Our results demonstrate that two tomato UGTs effectively glucosylate JA to form JA-glucosyl esters, potentially playing a role in the regulation of bioactive JA homeostasis. With this, our findings uncovered a missing step in the metabolism of JA.

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-3β-ol was observed in all P. tricornutum overexpression lines, and campesterol increased 3-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. Competing Interest Statement The authors have declared no competing interest.