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.

Random mutagenesis of Phaeodactylum tricornutum using ultraviolet, chemical, and X-radiation demonstrates the need for temporal analysis of phenotype stability.

We investigated two non-ionising mutagens in the form of ultraviolet radiation (UV) and ethyl methanosulfonate (EMS) and an ionising mutagen (X-ray) as methods to increase fucoxanthin content in the model diatom Phaeodactylum tricornutum. We implemented an ultra-high throughput method using fluorescence-activated cell sorting (FACS) and live culture spectral deconvolution for isolation and screening of potential pigment mutants, and assessed phenotype stability by measuring pigment content over 6 months using high-performance liquid chromatography (HPLC) to investigate the viability of long-term mutants. Both UV and EMS resulted in significantly higher fucoxanthin within the 6 month period after treatment, likely as a result of phenotype instability. A maximum fucoxanthin content of 135 ± 10% wild-type found in the EMS strain, a 35% increase. We found mutants generated using all methods underwent reversion to the wild-type phenotype within a 6 month time period. X-ray treatments produced a consistently unstable phenotype even at the maximum treatment of 1000 Grays, while a UV mutant and an EMS mutant reverted to wild-type after 4 months and 6 months, respectively, despite showing previously higher fucoxanthin than wild-type. This work provides new insights into key areas of microalgal biotechnology, by (i) demonstrating the use of an ionising mutagen (X-ray) on a biotechnologically relevant microalga, and by (ii) introducing temporal analysis of mutants which has substantial implications for strain creation and utility for industrial applications.

Variability in seed salinity tolerance in an island coastal community.

BACKGROUND AND AIMS: Islands, with their long coastlines and increased vulnerability to sea level rise, offer compelling opportunities to investigate the salinity tolerance of coastal plants. Seeds are generally more vulnerable than other plant stages to increased stressors. The aim of this study was to characterize salinity tolerance during germination across a diverse pool of 21 species from 14 plant families found in coastal communities throughout the Hawaiian Islands in order to increase our general understanding of coastal plant ecology for conservation and restoration. METHODS: Seeds of each species were exposed to unfiltered/untreated seawater (35 ppt total salinity) and two salinity treatments (10 and 20 ppt) in which the seawater was diluted with distilled water, and germination percent and timing were compared to seeds in a distilled water control. Non-germinated seeds were then tested for recovery germination. We quantified and compared germination percent, time and recovery among species and across salinity levels and tested for heterogeneity related to seed size, dormancy class, habit and threatened status. KEY RESULTS: Although salinity tolerance varied considerably among species, salinity exposure generally reduced and delayed germination. The greatest effects were detected at higher salinity levels. Recovery germination overall was higher for seeds that had been exposed to higher salinity. None of the factors we explored emerged as predictors of salinity tolerance except seed mass, which tended to enhance germination at higher salinity. CONCLUSIONS: Species responses to salinity exposure indicate high vulnerability of coastal systems to increased salinity stress, and variability among species could lead to shifts in community assembly and composition under sea level rise. These results can help guide coastal ecosystem conservation and restoration management decisions in the face of climate change.

The V-type ATPase enhances photosynthesis in marine phytoplankton and further links phagocytosis to symbiogenesis.

Diatoms, dinoflagellates, and coccolithophores are dominant groups of marine eukaryotic phytoplankton that are collectively responsible for the majority of primary production in the ocean.1 These phytoplankton contain additional intracellular membranes around their chloroplasts, which are derived from ancestral engulfment of red microalgae by unicellular heterotrophic eukaryotes that led to secondary and tertiary endosymbiosis.2 However, the selectable evolutionary advantage of these membranes and the physiological significance for extant phytoplankton remain poorly understood. Since intracellular digestive vacuoles are ubiquitously acidified by V-type H+-ATPase (VHA),3 proton pumps were proposed to acidify the microenvironment around secondary chloroplasts to promote the dehydration of dissolved inorganic carbon (DIC) into CO2, thus enhancing photosynthesis.4,5 We report that VHA is localized around the chloroplasts of centric diatoms and that VHA significantly contributes to their photosynthesis across a wide range of oceanic irradiances. Similar results in a pennate diatom, dinoflagellate, and coccolithophore, but not green or red microalgae, imply the co-option of phagocytic VHA activity into a carbon-concentrating mechanism (CCM) is common to secondary endosymbiotic phytoplankton. Furthermore, analogous mechanisms in extant photosymbiotic marine invertebrates6,7,8 provide functional evidence for an adaptive advantage throughout the transition from endosymbiosis to symbiogenesis. Based on the contribution of diatoms to ocean biogeochemical cycles, VHA-mediated enhancement of photosynthesis contributes at least 3.5 Gtons of fixed carbon per year (or 7% of primary production in the ocean), providing an example of a symbiosis-derived evolutionary innovation with global environmental implications.

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.

Unassembled cell wall proteins form aggregates in the extracellular space of Chlamydomonas reinhardtii strain UVM4.

The green microalga Chlamydomonas reinhardtii is emerging as a promising cell biofactory for secreted recombinant protein (RP) production. In recent years, the generation of the broadly used cell wall-deficient mutant strain UVM4 has allowed for a drastic increase in secreted RP yields. However, purification of secreted RPs from the extracellular space of C. reinhardtii strain UVM4 is challenging. Previous studies suggest that secreted RPs are trapped in a matrix of cell wall protein aggregates populating the secretome of strain UVM4, making it difficult to isolate and purify the RPs. To better understand the nature and behaviour of these extracellular protein aggregates, we analysed and compared the extracellular proteome of the strain UVM4 to its cell-walled ancestor, C. reinhardtii strain 137c. When grown under the same conditions, strain UVM4 produced a unique extracellular proteomic profile, including a higher abundance of secreted cell wall glycoproteins. Further characterization of high molecular weight extracellular protein aggregates in strain UVM4 revealed that they are largely comprised of pherophorins, a specific class of cell wall glycoproteins. Our results offer important new insights into the extracellular space of strain UVM4, including strain-specific secreted cell wall proteins and the composition of the aggregates possibly related to impaired RP purification. The discovery of pherophorins as a major component of extracellular protein aggregates will inform future strategies to remove or prevent aggregate formation, enhance purification of secreted RPs, and improve yields of recombinant biopharmaceuticals in this emerging cell biofactory. KEY POINTS: • Extracellular protein aggregates hinder purification of recombinant proteins in C. reinhardtii • Unassembled cell wall pherophorins are major components of extracellular protein aggregates • Known aggregate composition informs future strategies for recombinant protein purification.

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.

Comparative Study Highlights the Potential of Spectral Deconvolution for Fucoxanthin Screening in Live Phaeodactylum tricornutum Cultures.

Microalgal biotechnology shows considerable promise as a sustainable contributor to a broad range of industrial avenues. The field is however limited by processing methods that have commonly hindered the progress of high throughput screening, and consequently development of improved microalgal strains. We tested various microplate reader and flow cytometer methods for monitoring the commercially relevant pigment fucoxanthin in the marine diatom Phaeodactylum tricornutum. Based on accuracy and flexibility, we chose one described previously to adapt to live culture samples using a microplate reader and achieved a high correlation to HPLC (R2 = 0.849), effectively removing the need for solvent extraction. This was achieved by using new absorbance spectra inputs, reducing the detectable pigment library and changing pathlength values for the spectral deconvolution method in microplate reader format. Adaptation to 384-well microplates and removal of the need to equalize cultures by density further increased the screening rate. This work is of primary interest to projects requiring detection of biological pigments, and could theoretically be extended to other organisms and pigments of interest, improving the viability of microalgae biotechnology as a contributor to sustainable industry.

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.

Transcript level coordination of carbon pathways during silicon starvation-induced lipid accumulation in the diatom Thalassiosira pseudonana.

Diatoms are one of the most productive and successful photosynthetic taxa on Earth and possess attributes such as rapid growth rates and production of lipids, making them candidate sources of renewable fuels. Despite their significance, few details of the mechanisms used to regulate growth and carbon metabolism are currently known, hindering metabolic engineering approaches to enhance productivity. To characterize the transcript level component of metabolic regulation, genome-wide changes in transcript abundance were documented in the model diatom Thalassiosira pseudonana on a time-course of silicon starvation. Growth, cell cycle progression, chloroplast replication, fatty acid composition, pigmentation, and photosynthetic parameters were characterized alongside lipid accumulation. Extensive coordination of large suites of genes was observed, highlighting the existence of clusters of coregulated genes as a key feature of global gene regulation in T. pseudonana. The identity of key enzymes for carbon metabolic pathway inputs (photosynthesis) and outputs (growth and storage) reveals these clusters are organized to synchronize these processes. Coordinated transcript level responses to silicon starvation are probably driven by signals linked to cell cycle progression and shifts in photophysiology. A mechanistic understanding of how this is accomplished will aid efforts to engineer metabolism for development of algal-derived biofuels.

Metabolic and cellular organization in evolutionarily diverse microalgae as related to biofuels production.

Microalgae are among the most diverse organisms on the planet, and as a result of symbioses and evolutionary selection, the configuration of core metabolic networks is highly varied across distinct algal classes. The differences in photosynthesis, carbon fixation and processing, carbon storage, and the compartmentation of cellular and metabolic processes are substantial and likely to transcend into the efficiency of various steps involved in biofuel molecule production. By highlighting these differences, we hope to provide a framework for comparative analyses to determine the efficiency of the different arrangements or processes. This sets the stage for optimization on the based on information derived from evolutionary selection to diverse algal classes and to synthetic systems.