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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.

Acidification of the ocean due to high atmospheric CO2 levels may increase the resilience of diatoms causing dramatic shifts in abiotic and biotic cycles with lasting implications on marine ecosystems. Here, we report a potential bioindicator of a shift in the resilience of a coastal and centric model diatom Thalassiosira pseudonana under elevated CO2. Specifically, we have discovered, through EGFP-tagging, a plastid membrane localized putative Na+(K+)/H+ antiporter that is significantly upregulated at >800 ppm CO2, with a potentially important role in maintaining pH homeostasis. Notably, transcript abundance of this antiporter gene was relatively low and constant over the diel cycle under contemporary CO2 conditions. In future acidified oceanic conditions, dramatic oscillation with >10-fold change between nighttime (high) and daytime (low) transcript abundances of the antiporter was associated with increased resilience of T. pseudonana. By analyzing metatranscriptomic data from the Tara Oceans project, we demonstrate that phylogenetically diverse diatoms express homologs of this antiporter across the globe. We propose that the differential between night- and daytime transcript levels of the antiporter could serve as a bioindicator of a shift in the resilience of diatoms in response to high CO2 conditions in marine environments.

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. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: We identified a DNA transposon in the diatom Phaeodactylum tricornutum. This mutator‐like element encodes a transposase and a diatom‐specific E3 ubiquitin ligase, suggestive of a mobilization mechanism. We documented independent insertions that spontaneously inactivated the uridine monophosphate synthase (UMPS) locus, a common selectable marker. We provide insights on the role of transposons in diatom genome dynamics and evolution, and on the potential challenges of choosing UMPS as a selection locus in diatoms. [ABSTRACT FROM AUTHOR]

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 this study we investigated the effect of constitutive and inducible expression of the heterologous poly-3-hydroxybutyrate (PHB) pathway in P. tricornutum using non-integrative episomes in 3 different configurations. Constitutive expression led to downregulation of at least one individual gene out of three (phaA, phaB and phaC) and was outperformed by inducible expression. To further asses and optimise the dynamics of PHB accumulation driven by the inducible alkaline phosphatase 1 promoter, we upscaled the production to lab-scale bioreactors and tested the effect of supplemented CO2 on biomass and PHB accumulation. While ambient CO2 cultivation resulted in a maximum PHB yield of 2.3% cell dry weight (CDW) on day 11, under elevated CO2 concentrations PHB yield peaked at 1.7% CDW on day 8, coincident with PHB titres at 27.9 mg L−1 that were approximately threefold higher than ambient CO2. With other more valuable bio-products in mind, these results highlight the importance of the genetic design as well as substrate availability to supply additional reduction equivalents to boost biomass accumulation and relieve potential enzymatic bottlenecks for improved product accumulation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The algal endosymbiont Durusdinium trenchii enhances the resilience of coral reefs under thermal stress. D. trenchii can live freely or in endosymbiosis, and the analysis of genetic markers suggests that this species has undergone whole-genome duplication (WGD). However, the evolutionary mechanisms that underpin the thermotolerance of this species are largely unknown. Here, we present genome assemblies for two D. trenchii isolates, confirm WGD in these taxa, and examine how selection has shaped the duplicated genome regions using gene expression data. We assess how the free-living versus endosymbiotic lifestyles have contributed to the retention and divergence of duplicated genes, and how these processes have enhanced the thermotolerance of D. trenchii. Our combined results suggest that lifestyle is the driver of post-WGD evolution in D. trenchii, with the free-living phase being the most important, followed by endosymbiosis. Adaptations to both lifestyles likely enabled D. trenchii to provide enhanced thermal stress protection to the host coral. [ABSTRACT FROM AUTHOR]