high-throughput-metabolic-profiling-for-model-refinements

High-Throughput Metabolic Profiling for Model Refinements of Microalgae

Metabolic models are reconstructed based on an organism&#39;s available genome annotation and provide predictive tools to study metabolic processes at a ...

New York University Abu Dhabi

Zhejiang University

As a major primary producer in marine environments, diatoms have been considered as promising feedstocks for their applications in functional foods, bioactive pharmaceuticals, and cosmetics. This review focuses on the biotechnology potential of diatoms for value-added products like carotenoids. The impact of abiotic environmental stresses, such as intensity and quality of incident light, nutrient deficiency and silicon depletion, on diatoms has been examined to determine key factors that affect the growth performance and the accumulation of valuable compounds. Previous studies suggested that adaptive evolution could be an efficient method to improve the diatom productivity of valuable compounds. Light emitting diode (LED)-based photobioreactors were introduced and proposed as a promising new technology for producing quality products from diatoms. Currently available molecular biology tools were also summarized and discussed in relation to their application in the production of carotenoids and other valuable products. Taken together, systems biology and synthetic biology approaches have the potential to address the challenges faced while working toward the industrial application of diatoms. 

Developing diatoms for value-added products: challenges and opportunities.

Marine diatoms have recently gained much attention as they are expected to be a promising resource for sustainable production of bioactive compounds such as carotenoids and biofuels as a future clean energy solution. To develop photosynthetic cell factories, it is important to improve diatoms for value-added products. In this study, we utilized UVC radiation to induce mutations in the marine diatom Phaeodactylum tricornutum and screened strains with enhanced accumulation of neutral lipids and carotenoids. Adaptive laboratory evolution (ALE) was also used in parallel to develop altered phenotypic and biological functions in P. tricornutum and it was reported for the first time that ALE was successfully applied on diatoms for the enhancement of growth performance and productivity of value-added carotenoids to date. Liquid chromatography-mass spectrometry (LC-MS) was utilized to study the composition of major pigments in the wild type P. tricornutum, UV mutants and ALE strains. UVC radiated strains exhibited higher accumulation of fucoxanthin as well as neutral lipids compared to their wild type counterpart. In addition to UV mutagenesis, P. tricornutum strains developed by ALE also yielded enhanced biomass production and fucoxanthin accumulation under combined red and blue light. In short, both UV mutagenesis and ALE appeared as an effective approach to developing desired phenotypes in the marine diatoms via electromagnetic radiation-induced oxidative stress.

Photo-Oxidative Stress-Driven Mutagenesis and Adaptive Evolution on the Marine Diatom Phaeodactylum tricornutum for Enhanced Carotenoid Accumulation.

Advances in Biotechnology for Sustainable Development.

Through iterative cycles of selection, amplification, and mutagenesis, in vitro selection provides the ability to isolate molecules of desired properties and function from large pools (libraries) of random molecules with as many as 10(16) distinct species. This review, in recognition of a quarter of century of scientific discoveries made through in vitro selection, starts with a brief overview of the method and its history. It further covers recent developments in in vitro selection with a focus on tools that enhance the capabilities of in vitro selection and its expansion from being purely a nucleic acids selection to that of polypeptides and proteins. In addition, we cover how next generation sequencing and modern biological computational tools are being used to complement in vitro selection experiments. On the very least, sequencing and computational tools can translate the large volume of information associated with in vitro selection experiments to manageable, analyzable, and exploitable information. Finally, in vivo selection is briefly compared and contrasted to in vitro selection to highlight the unique capabilities of each method.

The in vitro selection world.

With the advent of modern biotechnology, microorganisms from diverse lineages have been used to produce bio-based feedstocks and bioactive compounds. Many of these compounds are currently commodities of interest, in a variety of markets and their utility warrants investigation into improving their production through strain development. In this review, we address the issue of strain improvement in a group of organisms with strong potential to be productive "cell factories": the photosynthetic microalgae. Microalgae are a diverse group of phytoplankton, involving polyphyletic lineage such as green algae and diatoms that are commonly used in the industry. The photosynthetic microalgae have been under intense investigation recently for their ability to produce commercial compounds using only light, CO₂, and basic nutrients. However, their strain improvement is still a relatively recent area of work that is under development. Importantly, it is only through appropriate engineering methods that we may see the full biotechnological potential of microalgae come to fruition. Thus, in this review, we address past and present endeavors towards the aim of creating productive algal cell factories and describe possible advantageous future directions for the field.

Algal Cell Factories: Approaches, Applications, and Potentials.

To investigate the phenomic and genomic traits that allow green algae to survive in deserts, we characterized a ubiquitous species, , which we isolated from multiple locations in the United Arab Emirates (UAE). Metabolomic analyses of  indicated that the alga accumulates a broad range of carbon sources, including several desiccation tolerance-promoting sugars and unusually large stores of palmitate. Growth assays revealed capacities to grow in salinities from zero to 60 g/L and to grow heterotrophically on >40 distinct carbon sources. Assembly and annotation of genomic reads yielded a 52.5 Mbp genome with 8153 functionally annotated genes. Comparison with other sequenced green algae revealed unique protein families involved in osmotic stress tolerance and saccharide metabolism that support phenomic studies. Our results reveal the robust and flexible biology utilized by a green alga to successfully inhabit a desert coastline.

The genome and phenome of the green alga  UTEX 3007 reveal adaptive traits for desert acclimatization.

Diatoms, considered as one of the most diverse and largest groups of algae, can provide the means to reach a sustainable production of petrochemical substitutes and bioactive compounds. However, a prerequisite to achieving this goal is to increase the solar-to-biomass conversion efficiency of photosynthesis, which generally remains less than 5% for most photosynthetic organisms. We have developed and implemented a rapid and effective approach, herein referred to as intracellular spectral recompositioning (ISR) of light, which, through absorption of excess blue light and its intracellular emission in the green spectral band, can improve light utilization. We demonstrate that ISR can be used chemogenically, by using lipophilic fluorophores, or biogenically, through the expression of an enhanced green fluorescent protein (eGFP) in the model diatom . Engineered  cells expressing eGFP achieved 28% higher efficiency in photosynthesis than the parental strain, along with an increased effective quantum yield and reduced nonphotochemical quenching (NPQ) induction levels under high-light conditions. Further, pond simulator experiments demonstrated that eGFP transformants could outperform their wild-type parental strain by 50% in biomass production rate under simulated outdoor sunlight conditions. Transcriptome analysis identified up-regulation of major photosynthesis genes in the engineered strain in comparison with the wild type, along with down-regulation of NPQ genes involved in light stress response. Our findings provide a proof of concept for a strategy of developing more efficient photosynthetic cell factories to produce algae-based biofuels and bioactive products.

Intracellular spectral recompositioning of light enhances algal photosynthetic efficiency.

Diatoms are a major group of unicellular algae that are rich in lipids and carotenoids. However, sustained research efforts are needed to improve the strain performance for high product yields towards commercialization. In this study, we generated a number of mutants of the model diatom , a cosmopolitan species that has also been found in Nordic region, using the chemical mutagens ethyl methanesulfonate (EMS) and -methyl-'-nitro--nitrosoguanidine (NTG). We found that both chlorophyll  and neutral lipids had a significant correlation with carotenoid content and these correlations were better during exponential growth than in the stationary growth phase. Then, we studied  common metabolic pathways and analyzed correlated enzymatic reactions between fucoxanthin synthesis and pigmentation or lipid metabolism through a genome-scale metabolic model. The integration of the computational results with liquid chromatography-mass spectrometry data revealed key compounds underlying the correlative metabolic pathways. Approximately 1000 strains were screened using fluorescence-based high-throughput method and five mutants selected had 33% or higher total carotenoids than the wild type, in which four strains remained stable in the long term and the top mutant exhibited an increase of 69.3% in fucoxanthin content compared to the wild type. The platform described in this study may be applied to the screening of other high performing diatom strains for industrial applications.

Chemical Mutagenesis and Fluorescence-Based High-Throughput Screening for Enhanced Accumulation of Carotenoids in a Model Marine Diatom Phaeodactylum tricornutum.

To convert waste CO from flue gases of power plants into value-added products, bio-mitigation technologies show promise. In this study, we cultivated a fast-growing species of green microalgae, Chlorella vulgaris, in different sizes of photobioreactors (PBRs) and developed a strategy using small doses of sugars for enhancing CO sequestration under light-emitting diode illumination. Glucose supplementation at low levels resulted in an increase of photoautotrophic growth-driven biomass generation as well as CO capture by 10% and its enhancement corresponded to an increase of supplied photon flux. The utilization of urea instead of nitrate as the sole nitrogen source increased photoautotrophic growth by 14%, but change of nitrogen source didn't compromise glucose-induced enhancement of photoautotrophic growth. The optimized biomass productivity achieved was 30.4% higher than the initial productivity of purely photoautotrophic culture. The major pigments in the obtained algal biomass were found comparable to its photoautotrophic counterpart and a high neutral lipids productivity of 516.6 mg/(L·day) was achieved after optimization. A techno-economic model was also developed, indicating that LED-based PBRs represent a feasible strategy for converting CO into value-added algal biomass.

Sugar-stimulated CO sequestration by the green microalga Chlorella vulgaris.

The data presented in this article are related to the research article entitled "Sugar-stimulated CO sequestration by the green microalga " (Fu et al., 2019) [1]. The data describe a rational design and scale-up of LED-based photobioreactors for producing value-added algal biomass while removing waste CO from flu gases from power plants. The dataset were created from growth rate experiments for biomass production including direct biomass productivity data, PBR size and setup parameters, medium composition as well as indirect energy cost and overhead in Iceland. A complete economic analysis is formed through a cost breakdown as well as PBR scalability predictions.

Dataset on economic analysis of mass production of algae in LED-based photobioreactors.

The activities of microalgae support nutrient cycling that helps to sustain aquatic and terrestrial ecosystems. Most microalgal species, especially those from the subtropics, are genomically uncharacterized. Here we report the isolation and genomic characterization of 22 microalgal species from subtropical coastal regions belonging to multiple clades and three from temperate areas. Halotolerant strains including Halamphora, Dunaliella, Nannochloris, and Chloroidium comprised the majority of these isolates. The subtropical-based microalgae contained arrays of methyltransferase, pyridine nucleotide-disulfide oxidoreductase, abhydrolase, cystathionine synthase, and small-molecule transporter domains present at high relative abundance. We found that genes for sulfate transport, sulfotransferase, and glutathione S-transferase activities were especially abundant in subtropical, coastal microalgal species and halophytic species in general. Our metabolomics analyses indicate lineage- and habitat-specific sets of biomolecules implicated in niche-specific biological processes. This work effectively expands the collection of available microalgal genomes by ∼50%, and the generated resources provide perspectives for studying halophyte adaptive traits.

Potential for Heightened Sulfur-Metabolic Capacity in Coastal Subtropical Microalgae.

Microalgae have been investigated for the photosynthetic production of natural products with industrial and biomedical applications. Their rapid growth offers an advantage over higher plants, while their complex metabolic capacities allow for the production of various molecules. Despite their potentials, molecular techniques are underdeveloped in microalgae compared to higher plants, fungi, and bacteria. However, recent advances in genome sequencing, strain development, and genome editing technologies, are providing thrust to enhance research on microalgal species that have branched out from several focal model organisms to encompass a great diversity of species. In this review, we highlight the recent, significant advances in microalgal research, with a focus on the development of new resources that can enhance work on model and non-model species.

Advances in microalgal research and engineering development.

Diatoms, which can accumulate large amounts of carotenoids, are a major group of microalgae and the dominant primary producer in marine environments. Phaeodactylum tricornutum, a model diatom species, acquires little silicon for its growth although silicon is known to contribute to gene regulation and play an important role in diatom intracellular metabolism. In this study, we explored the effects of artificial high-silicate medium (i.e. 3.0 mM sodium metasilicate) and LED illumination conditions on the growth rate and pigment accumulation in P. tricornutum, which is the only known species so far that can grow without silicate. It's well known that light-emitting diodes (LEDs) as novel illuminants are emerging to be superior monochromatic light sources for algal cultivation with defined and efficient red and blue lights.

Combined artificial high-silicate medium and LED illumination promote carotenoid accumulation in the marine diatom Phaeodactylum tricornutum.

Surface colonization allows diatoms, a dominant group of phytoplankton in oceans, to adapt to harsh marine environments while mediating biofoulings to human-made underwater facilities. The regulatory pathways underlying diatom surface colonization, which involves morphotype switching in some species, remain mostly unknown. Here, we describe the identification of 61 signaling genes, including G-protein-coupled receptors (GPCRs) and protein kinases, which are differentially regulated during surface colonization in the model diatom species, Phaeodactylum tricornutum. We show that the transformation of P. tricornutum with constructs expressing individual GPCR genes induces cells to adopt the surface colonization morphology. P. tricornutum cells transformed to express GPCR1A display 30% more resistance to UV light exposure than their non-biofouling wild-type counterparts, consistent with increased silicification of cell walls associated with the oval biofouling morphotype. Our results provide a mechanistic definition of morphological shifts during surface colonization and identify candidate target proteins for the screening of eco-friendly, anti-biofouling molecules.

GPCR Genes as Activators of Surface Colonization Pathways in a Model Marine Diatom.

Being integral primary producers in diverse ecosystems, microalgal genomes could be mined for ecological insights, but representative genome sequences are lacking for many phyla. We cultured and sequenced 107 microalgae species from 11 different phyla indigenous to varied geographies and climates. This collection was used to resolve genomic differences between saltwater and freshwater microalgae. Freshwater species showed domain-centric ontology enrichment for nuclear and nuclear membrane functions, while saltwater species were enriched in organellar and cellular membrane functions. Further, marine species contained significantly more viral families in their genomes (p = 8e-4). Sequences from Chlorovirus, Coccolithovirus, Pandoravirus, Marseillevirus, Tupanvirus, and other viruses were found integrated into the genomes of algal from marine environments. These viral-origin sequences were found to be expressed and code for a wide variety of functions. Together, this study comprehensively defines the expanse of protein-coding and viral elements in microalgal genomes and posits a unified adaptive strategy for algal halotolerance.

Large-scale genome sequencing reveals the driving forces of viruses in microalgal evolution.

Diatoms are a major group of microalgae that initiate biofouling by surface colonization of human-made underwater structures; however, the involved regulatory pathways remain uncharacterized. Here, we describe a protocol for identifying and validating regulatory genes involved in the morphology shift of the model diatom species  during surface colonization. We also provide a workflow for characterizing biofouling transformants. By using this protocol, gene targets such as  signaling genes could be identified and manipulated to turn off diatom biofouling. For complete information on the generation and use of this protocol, please refer to Fu et al. (2020).

Article	Year
Developing diatoms for value-added products: challenges and opportunities. New biotechnology\| PubMed ID: 25882605	2015
Photo-Oxidative Stress-Driven Mutagenesis and Adaptive Evolution on the Marine Diatom Phaeodactylum tricornutum for Enhanced Carotenoid Accumulation. Marine drugs\| PubMed ID: 26426027	2015
Advances in Biotechnology for Sustainable Development. BioMed research international\| PubMed ID: 27110569	2016
The in vitro selection world. Methods (San Diego, Calif.)\| PubMed ID: 27312879	2016
Algal Cell Factories: Approaches, Applications, and Potentials. Marine drugs\| PubMed ID: 27983586	2016
The genome and phenome of the green alga UTEX 3007 reveal adaptive traits for desert acclimatization. eLife\| PubMed ID: 28623667	2017
Intracellular spectral recompositioning of light enhances algal photosynthetic efficiency. Science advances\| PubMed ID: 28879232	2017
Chemical Mutagenesis and Fluorescence-Based High-Throughput Screening for Enhanced Accumulation of Carotenoids in a Model Marine Diatom Phaeodactylum tricornutum. Marine drugs\| PubMed ID: 30081564	2018
Sugar-stimulated CO sequestration by the green microalga Chlorella vulgaris. The Science of the total environment\| PubMed ID: 30445327	2019
Dataset on economic analysis of mass production of algae in LED-based photobioreactors. Data in brief\| PubMed ID: 30581917	2019
Potential for Heightened Sulfur-Metabolic Capacity in Coastal Subtropical Microalgae. iScience\| PubMed ID: 30684492	2019
Advances in microalgal research and engineering development. Current opinion in biotechnology\| PubMed ID: 31252302	2019
Combined artificial high-silicate medium and LED illumination promote carotenoid accumulation in the marine diatom Phaeodactylum tricornutum. Microbial cell factories\| PubMed ID: 31791335	2019
GPCR Genes as Activators of Surface Colonization Pathways in a Model Marine Diatom. iScience\| PubMed ID: 32798972	2020
Large-scale genome sequencing reveals the driving forces of viruses in microalgal evolution. Cell host & microbe\| PubMed ID: 33434515	2021
Protocol to generate and characterize biofouling transformants of a model marine diatom. STAR protocols\| PubMed ID: 34401782	2021

Weiqi Fu

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