ORCID Profile
0000-0002-5754-5461
Current Organisation
Leidos Inc San Diego
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Publisher: Springer Science and Business Media LLC
Date: 23-05-2022
DOI: 10.1007/S00253-022-11960-9
Abstract: 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. • 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
Publisher: Frontiers Media SA
Date: 05-06-2020
Publisher: MDPI AG
Date: 05-03-2020
DOI: 10.3390/CELLS9030633
Abstract: 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 erse 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.
Publisher: MDPI AG
Date: 05-02-2021
DOI: 10.3390/PH14020125
Abstract: 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.
Publisher: Cold Spring Harbor Laboratory
Date: 28-05-2022
DOI: 10.1101/2022.05.26.493626
Abstract: Diatoms, dinoflagellates, and coccolithophorids are the dominant groups of marine eukaryotic phytoplankton collectively responsible for the majority of primary production in the ocean 1 . These phytoplankton contain additional intracellular membranes around their chloroplasts derived from ancestral engulfment of red microalgae by unicellular heterotrophic eukaryotes that led to secondary endosymbiosis 2 . This symbiogenesis hypothesis for the origin of modern secondary endosymbiotic phytoplankton is supported by a wealth of palaeontologic, morphologic, and genomic evidence 3–6 . However, the selectable evolutionary advantage of these membranes and the physiological significance for extant phytoplankton are unknown. We report that the proton-pumping enzyme V-type H + -ATPase (VHA), ubiquitously used in eukaryotic intercellular digestion, is localized around the chloroplasts of centric diatoms and that VHA-activity significantly enhances photosynthesis over a wide range of oceanic irradiances. Similar results in pennate diatoms, dinoflagellates, and coccolithophorids, but not green or red microalgae, imply a mechanism resulting from the co-option of phagocytic VHA activity into a carbon concentrating mechanism that is common to secondary endosymbiotic phytoplankton. Furthermore, analogous VHA-dependent mechanisms in extant photosymbiotic marine invertebrates 7–9 provide functional evidence for an adaptive advantage throughout the transition from endosymbiosis to symbiogenesis. Our results suggest that VHA-dependent enhancement of photosynthesis contributes at least 7% of primary production in the ocean, providing an ex le of a symbiosis-derived evolutionary innovation with global environmental implications.
Publisher: Springer New York
Date: 22-11-2016
DOI: 10.1007/978-1-4939-3302-0_4
Abstract: The ability to image large numbers of cells at high resolution enhances flow cytometric analysis of cells and cell populations. In particular, the ability to image intracellular features adds a unique aspect to analyses, and can enable correlation between molecular phenomena resulting in alterations in cellular phenotype. Unicellular microalgae are amenable to high-throughput analysis to capture the ersity of cell types in natural s les, or erse cellular responses in clonal populations, especially using imaging cytometry. Using ex les from our laboratory, we review applications of imaging cytometry, specifically using an Amnis(®) ImageStream(®)X instrument, to characterize photosynthetic microalgae. Some of these ex les highlight advantages of imaging flow cytometry for certain research objectives, but we also include ex les that would not necessarily require imaging and could be performed on a conventional cytometer to demonstrate other concepts in cytometric evaluation of microalgae. We demonstrate the value of these approaches for (1) analysis of populations, (2) documentation of cellular features, and (3) analysis of gene expression.
Publisher: Cold Spring Harbor Laboratory
Date: 03-01-2023
DOI: 10.1101/2023.01.02.522487
Abstract: Diatoms are photosynthetic unicellular microalgae that drive global ecological phenomena in the biosphere and are emerging sustainable feedstock for an increasing number of industrial applications. Diatoms exhibit enormous taxonomic and genetic ersity, which often result in peculiar biochemical and biological traits. Transposable elements (TE) represent a substantial portion of diatom genomes and have been hypothesized to exert a relevant role in enriching genetic ersity and centrally contribute to genome evolution. Here, through long-read whole genome sequencing, we identified a novel Mutator-Like Element (MULE) in the model diatom Phaeodactylum tricornutum, and we report the direct observation of its mobilization within the course of one single laboratory experiment. Under selective conditions, this novel TE inactivated the uridine monophosphate synthase ( UMPS ) gene of P. tricornutum, one of the two only endogenous genetic loci currently targeted for selectable auxotrophy in functional genetics and genome editing applications. We report the first, real-time observation of the mobilization of a transposon in diatoms that possesses novel peculiar features. These include the combined presence of a MULE transposase domain with Zinc finger, SWIM-type domains, and of a diatom-specific E3 ubiquitin ligase of the zinc finger UBR type, which indicate a novel 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. Ultimately, this raises relevant concerns on the targeting of loci such as UMPS as selectable markers for functional genetics and biotechnological applications in diatoms. We identified a novel DNA transposon in the diatom Phaeodactylum tricornutum . This new Mutator-Like Element encodes a transposase and a diatom-specific E3 ubiquitin ligase, which suggest a novel mobilization mechanism. We documented independent insertions in real-time, which spontaneously inactivated the uridine monophosphate synthase ( UMPS ) locus, a common selectable marker. We provide new insights on the role of transposons in diatom genome dynamics and evolution and on the unsuitability of UMPS as selection locus in diatoms.
Publisher: Elsevier BV
Date: 05-2012
Publisher: Cold Spring Harbor Laboratory
Date: 09-12-2020
DOI: 10.1101/2020.12.09.418384
Abstract: Acidification of the ocean due to high atmospheric CO 2 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 CO 2 . Specifically, we have discovered, through EGFP-tagging, a plastid membrane localized putative Na + (K + )/H + antiporter that is significantly upregulated at 800 ppm CO 2 , 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 CO 2 conditions. In future acidified oceanic conditions, dramatic oscillations of -fold change between nighttime (high) and daytime (low) in transcript abundances of the antiporter gene were associated with increased resilience of T. pseudonana . By analyzing metatranscriptomic data from the Tara Oceans project, we demonstrate that phylogenetically erse 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 CO 2 conditions in marine environments.
Publisher: American Chemical Society (ACS)
Date: 07-02-2020
Publisher: Frontiers Media SA
Date: 17-03-2020
Publisher: Elsevier BV
Date: 02-2017
DOI: 10.1016/J.PROTIS.2016.10.005
Abstract: Evidence suggests that diatom photorespiratory metabolism is distinct from other photosynthetic eukaryotes in that there may be at least two routes for the metabolism of the photorespiratory metabolite glycolate. One occurs primarily in the mitochondria and is similar to the C2 photorespiratory pathway, and the other processes glycolate through the peroxisomal glyoxylate cycle. Genomic analysis has identified the presence of key genes required for glycolate oxidation, the glyoxylate cycle, and malate metabolism, however, predictions of intracellular localization can be ambiguous and require verification. This knowledge gap leads to uncertainties surrounding how these in idual pathways operate, either together or independently, to process photorespiratory intermediates under different environmental conditions. Here, we combine in silico sequence analysis, in vivo protein localization techniques and gene expression patterns to investigate key enzymes potentially involved in photorespiratory metabolism in the model diatom Thalassiosira pseudonana. We demonstrate the peroxisomal localization of isocitrate lyase and the mitochondrial localization of malic enzyme and a glycolate oxidase. Based on these analyses, we propose an updated model for photorespiratory metabolism in T. pseudonana, as well as a mechanism by which C2 photorespiratory metabolism and its associated pathways may operate during silicon starvation and growth arrest.
Publisher: Oxford University Press (OUP)
Date: 04-09-2023
DOI: 10.1093/AOB/MCAD129
Publisher: Wiley
Date: 04-02-2016
DOI: 10.1111/NPH.13843
Abstract: 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.
Publisher: Elsevier BV
Date: 04-2017
Publisher: Elsevier BV
Date: 06-2013
DOI: 10.1016/J.CBPA.2013.02.027
Abstract: Microalgae are among the most erse 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 erse algal classes and to synthetic systems.
Publisher: Springer Science and Business Media LLC
Date: 09-07-2022
DOI: 10.1007/S10811-022-02795-Y
Abstract: 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 in idual 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 CO 2 on biomass and PHB accumulation. While ambient CO 2 cultivation resulted in a maximum PHB yield of 2.3% cell dry weight (CDW) on day 11, under elevated CO 2 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 CO 2 . 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.
Publisher: Cold Spring Harbor Laboratory
Date: 11-04-2022
DOI: 10.1101/2022.04.10.487810
Abstract: Coral reefs are fundamentally sustained by symbioses involving dinoflagellate algae in the Family Symbiodiniaceae. The coral symbiont Durusdinium trenchii is notable for enhancing the resilience of coral holobionts under thermal stress. Believed to have experienced whole-genome duplication (WGD), D. trenchii offers a valuable model system to understand how selection acts on the genome of a facultative symbiont after WGD. We present genome assemblies for two isolates of D. trenchii and confirm WGD in these taxa, providing the first ex le of this phenomenon in a single-celled eukaryotic symbiont. We assess how the facultative lifestyle has contributed to the retention and ergence of duplicated genes, and how these results intersect with the observed thermotolerance of corals hosting D. trenchii symbionts. Our findings reveal that the free-living lifestyle is the main driver of post-WGD evolution, however, they also implicate symbiosis in this process, with both lifestyles increasing algal fitness. Our results demonstrate that WGD, driven by selection in the free-living phase, has converted D. trenchii into a coral symbiont that serendipitously provides increased thermal stress protection to the host coral.
Publisher: Cold Spring Harbor Laboratory
Date: 15-05-2022
DOI: 10.1101/2022.05.14.491972
Abstract: Unicellular microalgae are of immense ecological importance with growing commercial potential in industries such as renewable energy, food and pharmacology. Viral infections can have a profound impact on the growth and evolution of their hosts. However, very little is known of the ersity within, and effect of, unicellular microalgal RNA viruses. In addition, identifying RNA viruses in these organisms that could have originated more than a billion years ago constitutes a robust data set to dissect molecular events and address fundamental questions on virus evolution. We assessed the ersity of RNA viruses in eight microalgal cultures including representatives from the diatom, eustigmatophyte, dinoflagellate, red algae and euglenid groups. Using metatranscriptomic sequencing combined with bioinformatic approaches optimised to detect highly ergent RNA viruses, we identified ten RNA virus sequences, with nine constituting new viral species. Most of the newly identified RNA viruses belonged to the double-stranded Totiviridae, Endornaviridae and Partitiviridae , greatly expanding the reported host range for these families. Two new species belonging to the single-stranded RNA viral clade Marnaviridae , commonly associated with microalgal hosts, were also identified. This study highlights that a great ersity of RNA viruses likely exists undetected within the unicellular microalgae. It also highlights the necessity for RNA viral characterisation and to investigate the effects of viral infections on microalgal physiology, biology and growth, considering their environmental and industrial roles. In comparison to animals or plants, our knowledge of the ersity of RNA viruses infecting microbial algae – the microalgae – is minimal. Yet describing the RNA viruses infecting these organisms is of primary importance at both the ecological and economical levels because of the fundamental roles these organisms play in aquatic environments and their growing value across a range of industrial fields. Using metatranscriptomic sequencing we aimed to reveal the RNA viruses present in cultures of eight microalgae species belonging to the diatom, dinoflagellate, eustigmatophyte, rhodophyte and euglena major clades of algae. This work identified ten new ergent RNA virus species, belonging to RNA virus families as erse as the double-stranded Totiviridae, Endornaviridae, Partitiviridae and the single-stranded Marnaviridae . By expanding the known ersity of RNA viruses infecting unicellular eukaryotes, this study contributes to a better understanding of the early evolution of the virosphere and will inform the use of microalgae in industrial applications.
Publisher: MDPI AG
Date: 23-12-2021
DOI: 10.3390/MD20010019
Abstract: 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 s les 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.
Publisher: The Oceanography Society
Date: 09-2011
Publisher: Informa UK Limited
Date: 03-2012
DOI: 10.4155/BFS.11.157
Publisher: Elsevier BV
Date: 06-2018
Publisher: Wiley
Date: 20-06-2023
DOI: 10.1111/TPJ.16271
Abstract: 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 ersity, 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 ersity 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.
Publisher: MDPI AG
Date: 21-12-2020
DOI: 10.3390/PH13120481
Abstract: Sterols are a class of triterpenoid molecules with erse 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 ersity 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 c esterol 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 in idual 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.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C5MB00804B
Abstract: In both eukaryotes and prokaryotes, fatty acid synthases are responsible for the biosynthesis of fatty acids in an iterative process, extending the fatty acid by two carbon units every cycle.
Location: United States of America
No related grants have been discovered for Raffaela Abbriano.