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Competing Interests: Declaration of interests No interests are declared.

Background: With the urgent need to reduce carbon emissions, and the dwindling reserves of easily exploitable fossil fuel, microalgae-based biofuels that can be used for transport systems and CO 2 abatement have attracted great attention worldwide in recent years. One useful characteristic of microalgae is their ability to accumulate high levels of lipid content, in particular under conditions of nitrogen deprivation, with numerous species identified so far. However, a trade-off between levels of lipid accumulation and biomass productivity hinders the commercial applicability of lipids from microalgae. Here, we sequenced the genomes of Vischeria sp. CAUP H4302 and Vischeria stellata SAG 33.83, which can accumulate high content of lipids rich in nutraceutical fatty acids and with excellent biomass yield in nitrogen-limiting culture., Results: A whole-genome duplication (WGD) event was revealed in V. sp. CAUP H4302, which is a rare event in unicellular microalgae. Comparative genomic analyses showed that a battery of genes encoding pivotal enzymes involved in fatty acids and triacylglycerol biosynthesis, storage polysaccharide hydrolysis, and nitrogen and amino acid-related metabolisms are expanded in the genus Vischeria or only in V. sp. CAUP H4302. The most highlighted is the expansion of cyanate lyase genes in the genus Vischeria, which may enhance their detoxification ability against the toxic cyanate by decomposing cyanate to NH 3 and CO 2 , especially under nitrogen-limiting conditions, resulting in better growth performance and sustained accumulation of biomass under the aforementioned stress conditions., Conclusions: This study presents a WGD event in microalgae, providing new insights into the genetic and regulatory mechanism underpinning hyper-accumulation of lipids and offering potentially valuable targets for future improvements in oleaginous microalgae by metabolic engineering., (© 2023. The Author(s).)

Although ADP glucose pyrophosphorylase (AGPase), with two large subunits (ls) and two small subunits (ss), is a promising knockout target for increasing the neutral lipid content, the details regarding the sequence-structure features and their distribution within metabolic system in microalgae is rather limited. Against this backdrop, a comprehensive genome-wide comparative analysis on 14 sequenced microalgal genomes was performed. For the first time the heterotetrameric structure of the enzyme and the interaction of the catalytic unit with the substrate was also studied. Novel findings of the present study includes: (i) at the DNA level, the genes controlling the ss are more conserved than those controlling the ls; the variation in both the gene groups is mainly due to exon number, exon length and exon phase distribution; (ii) at protein level, the ss genes are more conserved relative to those for ls; (III) three putative key consensus sequences 'LGGGAGTRLYPLTKNRAKPAV', 'WFQGTADAV' and 'ASMGIYVFRKD' were ubiquitously conserved in all the AGPases; (iv) molecular dynamics investigations revealed that the modeled AGPase heterotetrameric structure, from oleaginous algae Chlamydomonas reinharditii, was completely stable in real time environment; (v) The binding interfaces of catalytic unit, ssAGPase, from C. reinharditii with α-D-glucose 1-phosphate (αGP) was also analyzed. The results of the present study have provided system-based insights into the structure-function of the genes and encoded proteins, which provided clues for exploitation of variability in these genes that, could be further utilized to design site-specific mutagenic experiments for engineering of microalgal strains towards sustainable development of biofuel., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Due to the complexity of metabolic and regulatory networks in microorganisms, it is difficult to obtain robust phenotypes through artificial rational design and genetic perturbation. Adaptive laboratory evolution (ALE) engineering plays an important role in the construction of stable microbial cell factories by simulating the natural evolution process and rapidly obtaining strains with stable traits through screening. This review summarizes the application of ALE technology in microbial breeding, describes the commonly used methods for ALE, and highlights the important applications of ALE technology in the production of lipids and terpenoids in yeast and microalgae. Overall, ALE technology provides a powerful tool for the construction of microbial cell factories, and it has been widely used in improving the level of target product synthesis, expanding the range of substrate utilization, and enhancing the tolerance of chassis cells. In addition, in order to improve the production of target compounds, ALE also employs environmental or nutritional stress strategies corresponding to the characteristics of different terpenoids, lipids, and strains.

Glycerolipids are the most abundant lipids in microalgae, and glycerol-3-phosphate:acyl-CoA acyltransferase (GPAT) plays an important role in their biosynthesis. However, the biochemical and biological functions of algal GPAT remain poorly characterized. Here, we characterized the endoplasmic reticulum (ER)-associated GPAT of the model unicellular green alga Chlamydomonas reinhardtii (CrGPATer). Enzymatic assays indicated that CrGPATer is an sn-1 acyltransferase using a variety of acyl-CoAs as the acyl donor. Subcellular localization revealed that CrGPATer was associated with ER membranes and lipid droplets. We constructed overexpression (OE) and knockdown (KD) transgenic C. reinhardtii lines to investigate the in vivo function of CrGPATer. Lipidomic analysis indicated that CrGPATer OE enhanced the cellular content of galactolipids, especially monogalactosyldiacylglycerol, under nitrogen deficiency stress. Correspondingly, CrGPATer KD lines contained lower contents of galactolipids than the control. Feeding experiments with labeled phosphatidic acid revealed that the intermediate of the eukaryotic Kennedy pathway could be used for galactolipid biosynthesis in the chloroplasts. These results provided multiple lines of evidence that the eukaryotic Kennedy pathway mediated by CrGPATer may be involved in galactolipid biosynthesis in C. reinhardtii. OE of CrGPATer significantly increased the content of triacylglycerol and the yield of biomass. Moreover, the content and yield of 1, 3-olein-2-palmitin, a high-value lipid that can be used as an alternative for human milk fat in infant formula, were significantly enhanced in the OE transgenic lines. Taken together, this study provided insights into the biochemical and biological functions of CrGPATer and its potential as a genetic engineering target in functional lipid manufacturing., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

We provide physiological and metabolic insights into the complex role of γ-aminobutyric acid (GABA) in fine-tuning algal metabolism to improve productivity. Genetic engineering strategies to improve algal GABA biosynthesis are also discussed. Our aim is to provide an understanding of how GABA can be used for cost-competitive algae-based biofuels and bioproducts., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

It is urgent to develop techniques that can simultaneously detect multiple microalgae, due to the diversity of harmful algal blooms (HABs)-forming algal species. The target algae species in this study are Heterosigma akashiwo, Prorocentrum donghaiense and Karenia mikimotoi. These algae are the dominant species that cause HABs in the East China Sea, and the multiple detection technique focusing on these three algae is not common. Therefore, this study established a multiplex polymerase chain reaction(mPCR) to diagnose the three algae, which is simple and low cost. First, the corresponding specific primers were designed based on the D1-D2 region of the large subunit (LSU) ribosomal DNA sequence. Then, mPCR was established and the reaction conditions were optimized. And the specificity, sensitivity, and stability of mPCR were evaluated. The result of specificity test showed that the established mPCR had good specificity for the target microalgae and did not cross-react with eighteen non-target microalgae. The sensitivity of experiment was 3.3 × 10 -1  ng μL -1 , and the established mPCR was not affected by the interfering microalgae. Moreover, the practicability evaluation of mPCR by using the simulated natural water samples showed that the detection limit of target microalgae was 100 cells mL -1 , which could meet the demand for early warning of HABs. In summary, the established mPCR is characterized by strong specificity, good stability, and multiple analysis to detect H. akashiwo, P. donghaiense, and K. mikimotoi., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Rare earth element tailings (REEs) wastewater, which has the characteristics of high ammonia nitrogen (NH 4 + -N) and low COD. It can cause eutrophication and biotoxicity in water which is produced in high volumes, requiring treatment before final disposal. Microalgae-Bacteria symbiotic (MBS) system can be applied in REEs wastewater, but its low extent of nitrogen removal and instability limit its application. By adding biodegradable carrier as both carbon source and carrier, the system can be stabilized and the efficiency can be improved. In this work, the extent of NH 4 + -N removal reached 100% within 24 h in a MBS system after adding loofah under optimal conditions, and the removal rate reached 127.6 mg NH 4 + -N·L -1 ·d -1 . In addition, the carbon release from loofah in 3 d reached 408.7 mg/L, which could be used as a carbon source to support denitrification. During 90 d of operation of the MBS system loaded with loofah, the effluent NH 4 + -N was less than 15 mg/L. At phylum level, Proteobacteria were dominant which accounted for 78.2%. Functional gene analysis showed that enhancement of microalgae assimilation was the main factor affecting NH 4 + -N removal. This work expands our understanding of the enhanced role of carbon-based carriers in the denitrification of REEs wastewater., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Microalgal domestication is an expanding research field that aims to multiply and accelerate the potential of microalgae for various biotechnological purposes. We investigated the stability of improved lipid traits and genetic changes of a domesticated strain of the haptophyte Tisochrysis lutea, TisoS2M2, previously obtained by a mutation-selection improvement program. After 7 years of maintenance, TisoS2M2 still displayed improved lipid traits compared with the native strain, demonstrating that a mutation-selection improvement program is suitable for obtaining a domesticated strain with stable, improved phenotype over time. We identified specific genetic variations between the native and domesticated strains and focused on the dynamics of transposable elements (TEs). DNA transposons mainly caused specific TE indels of the domesticated strain TisoS2M2, and some specific TE indels may have impacted genes associated to the neutral lipid pathway. We revealed transposition events for TEs in T. lutea and discussed on the potential role of the improvement program on their activity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Berthelier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

CRISPR-Cas has revolutionized genetic modification with its comparative simplicity and accuracy, and it can be used even at the genomic level. Microalgae are excellent feedstocks for biofuels and nutraceuticals because they contain high levels of fatty acids, carotenoids, and other metabolites; however, genome engineering for microalgae is not yet as developed as for other model organisms. Microalgal engineering at the genetic and metabolic levels is relatively well established, and a few genomic resources are available. Their genomic information was used for a "safe harbor" site for stable transgene expression in microalgae. This review proposes further genome engineering schemes including the construction of sgRNA libraries, pan-genomic and epigenomic resources, and mini-genomes, which can together be developed into synthetic biology for carbon-based engineering in microalgae. Acetyl-CoA is at the center of carbon metabolic pathways and is further reviewed for the production of molecules including terpenoids in microalgae., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Phenotypic plasticity, the change in the phenotype of a given genotype in response to its environment of development, is a ubiquitous feature of life, enabling organisms to cope with variation in their environment. Theoretical studies predict that, under stationary environmental variation, the level of plasticity should evolve to match the predictability of selection at the timing of development. However, the extent to which patterns of evolution of plasticity for more integrated traits are mirrored by their underlying molecular mechanisms remains unclear, especially in response to well-characterized selective pressures exerted by environmental predictability. Here, we used experimental evolution with the microalgae Dunaliella salina under controlled environmental fluctuations, to test whether the evolution of phenotypic plasticity in responses to environmental predictability (as measured by the squared autocorrelation ρ2) occurred across biological levels, going from DNA methylation to gene expression to cell morphology. Transcriptomic analysis indicates clear effects of salinity and ρ2 × salinity interaction on gene expression, thus identifying sets of genes involved in plasticity and its evolution. These transcriptomic effects were independent of DNA methylation changes in cis. However, we did find ρ2-specific responses of DNA methylation to salinity change, albeit weaker than for gene expression. Overall, we found consistent evolution of reduced plasticity in less predictable environments for DNA methylation, gene expression, and cell morphology. Our results provide the first clear empirical signature of plasticity evolution at multiple levels in response to environmental predictability, and highlight the importance of experimental evolution to address predictions from evolutionary theory, as well as investigate the molecular basis of plasticity evolution., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Leung et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Astaxanthin is a valuable ketocarotenoid with various pharmaceutical and nutraceutical applications. Green microalgae harbor natural capacities for pigment accumulation due to their 2- C -methyl-d-erythritol 4-phosphate (MEP) pathway. Recently, a redesigned ß-carotene ketolase (BKT) was found to enable ketocarotenoid accumulation in the model microalga Chlamydomonas reinhardtii , and transformants exhibited reduced photoinhibition under high-light. Here, a systematic screening by synthetic transgene design of carotenoid pathway enzymes and overexpression from the nuclear genome identified phytoene synthase (PSY/crtB) as a bottleneck for carotenoid accumulation in C. reinhardtii . Increased ß-carotene hydroxylase (CHYB) activity was found to be essential for engineered astaxanthin accumulation. A combined BKT, crtB, and CHYB expression strategy resulted in a volumetric astaxanthin production of 9.5 ± 0.3 mg L -1 (4.5 ± 0.1 mg g -1 CDW) in mixotrophic and 23.5 mg L -1 (1.09 mg L -1 h -1 ) in high cell density conditions, a 4-fold increase compared to previous reports in C. reinhardtii . This work presents a systematic investigation of bottlenecks in astaxanthin accumulation in C. reinhardtii and the phototrophic green cell factory design for competitive use in industrial biotechnology.

The urge for food security and sustainability has advanced the field of microalgal biotechnology. Microalgae are microorganisms able to grow using (sun)light, fertilizers, sugars, CO 2 , and seawater. They have high potential as a feedstock for food, feed, energy, and chemicals. Microalgae grow faster and have higher areal productivity than plant crops, without competing for agricultural land and with 100% efficiency uptake of fertilizers. In comparison with bacterial, fungal, and yeast single-cell protein production, based on hydrogen or sugar, microalgae show higher land-use efficiency. New insights are provided regarding the potential of microalgae replacing soy protein, fish oil, and palm oil and being used as cell factories in modern industrial biotechnology to produce designer feed, recombinant proteins, biopharmaceuticals, and vaccines., Competing Interests: Declaration of interests The authors have no interests to declare., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Interactions between microalgae and bacteria can directly influence the global biogeochemical cycles but the majority of such interactions remain unknown. 16S rRNA gene-based co-occurrence networks have potential to help identify microalgal-bacterial interactions. Here, we used data from 10 Earth microbiome projects to identify potential microalgal-bacterial associations in aquatic ecosystems. A high degree of clustering was observed in microalgal-bacterial modules, indicating densely connected neighbourhoods. Proteobacteria and Bacteroidetes predominantly co-occurred with microalgae and represented hubs of most modules. Our results also indicated that species-specificity may be a global characteristic of microalgal associated microbiomes. Several previously known associations were recovered from our network modules, validating that biologically meaningful results can be inferred using this approach. A range of previously unknown associations were recognised such as co-occurrences of Bacillariophyta with uncultured Planctomycetes OM190 and Deltaproteobacteria order NB1-j. Planctomycetes and Verrucomicrobia were identified as key associates of microalgae due to their frequent co-occurrences with several microalgal taxa. Despite no clear taxonomic pattern, bacterial associates appeared functionally similar across different environments. To summarise, we demonstrated the potential of 16S rRNA gene-based co-occurrence networks as a hypothesis-generating framework to guide more focused research on microalgal-bacterial associations., (© 2023. The Author(s).)

The comprehensive effect of exogenous pollutants on the dispersal and abundance of antibiotic-resistance genes (ARGs) in the phycosphere, bacterial community and algae-bacteria interaction remains poorly understood. We investigated community structure and abundance of ARGs in free-living (FL) and particle-attached (PA) bacteria in the phycosphere under nanoparticles (silver nanoparticles (AgNPs) and hematite nanoparticles (HemNPs)) and antibiotics (tetracycline and sulfadiazine) stress using high-throughput sequencing and real-time quantitative PCR. Meanwhile, the intrinsic connection of algae-bacteria interaction was explored by transcriptome and metabolome. The results showed that the relative abundance of sulfonamide and tetracycline ARGs in PA and FL bacteria increased 103-129 % and 112-134 %, respectively, under combined stress of nanoparticles and antibiotics. Antibiotics have a greater effect on ARGs than nanoparticles at environmentally relevant concentrations. Proteobacteria, Firmicutes, and Bacteroidetes, as the primary potential hosts of ARGs, were the dominant phyla. Lifestyle, i.e., PA and FL, significantly determined the abundance of ARGs and bacterial communities. Moreover, algae can provide bacteria with nutrients (carbohydrates and amino acids), and can also produce antibacterial substances (fatty acids). This algal-bacterial interaction may indirectly affect the distribution and abundance of ARGs. These findings provide new insights into the distribution and dispersal of ARGs in microalgae-bacteria symbiotic systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Lutein is a valuable metabolite widely used in the food, pharmaceutical, cosmetic, and aquaculture industries. Marigold flowers are the most common source of commercial lutein, but cultivation area, weather conditions, and high manpower costs are among the disadvantages of lutein production from marigold flowers. Microalgae are an excellent alternative to plant sources of lutein as they do not have the limitations of plant extraction. Auxenochlorella protothecoides is a promising candidate for commercial production of lutein. In the present research, a genome-scale metabolic model was applied to introduce some strategies to improve lutein production in A. protothecoides. The effective reactions to improve lutein production were determined based on analysis of multiple optimal solutions. The enzymatic regulators of candidate reactions were identified using the BRENDA database. The effect of 13 activators was investigated experimentally. Our results showed that sodium citrate has the greatest effect on lutein production, so it was introduced as the most effective compound for increasing lutein production by A. protothecoides., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Even though sunlight energy far outweighs the energy required by human activities, its utilization is a key goal in the field of renewable energies. Microalgae have emerged as a promising new and sustainable feedstock for meeting rising food and feed demand. Because traditional methods of microalgal improvement are likely to have reached their limits, genetic engineering is expected to allow for further increases in the photosynthesis and productivity of microalgae. Understanding the mechanisms that control photosynthesis will enable researchers to identify targets for genetic engineering and, in the end, increase biomass yield, offsetting the costs of cultivation systems and downstream biomass processing. This review describes the molecular events that happen during photosynthesis and microalgal productivity through genetic engineering and discusses future strategies and the limitations of genetic engineering in microalgal productivity. We highlight the major achievements in manipulating the fundamental mechanisms of microalgal photosynthesis and biomass production, as well as promising approaches for making significant contributions to upcoming microalgal-based biotechnology.

Microalgae are considered a suitable production platform for high-value lipids and oleochemicals. Several species including Nannochloropsis oceanica produce large amounts of essential [Formula: see text]-3 polyunsaturated fatty acids (PUFAs) which are integral components of food and feed and have been associated with health-promoting effects. N. oceanica can further accumulate high contents of non-polar lipids with chemical properties that render them a potential replacement for plant oils such as palm oil. However, biomass and lipid productivities obtained with microalgae need to be improved to reach commercial feasibility. Genetic engineering can improve biomass and lipid productivities, for instance by increasing carbon flux to lipids. Here, we report the overexpression of glycerol-3-phosphate acyltransferase (GPAT) in N. oceanica during favorable growth conditions as a strategy to increase non-polar lipid content. Transformants overproducing either an endogenous (NoGPAT) or a heterologous (Acutodesmus obliquus GPAT) GPAT enzyme targeted to the endoplasmic reticulum had up to 42% and 51% increased non-polar lipid contents, respectively, compared to the wild type. Biomass productivities of transformant strains were not substantially impaired, resulting in lipid productivities that were increased by up to 37% and 42% for NoGPAT and AoGPAT transformants, respectively. When exposed to nutrient stress, transformants and wild type had similar lipid contents, suggesting that GPAT enzyme exerts strong flux control on lipid synthesis in N. oceanica under favorable growth conditions. NoGPAT transformants further accumulated PUFAs in non-polar lipids, reaching a total of 6.8% PUFAs per biomass, an increase of 24% relative to the wild type. Overall, our results indicate that GPAT is an interesting target for engineering of lipid metabolism in microalgae, in order to improve non-polar lipid and PUFAs accumulation in microalgae., (© 2023. The Author(s).)

This chapter will discuss the diversity of algae and show that the diversity is much greater than just obligately oxygenic photosynthetic algae and that it includes many mixotrophic and heterotrophic organisms that are more similar to the major groups of microorganisms. The photosynthetic groups are seen as part of the plant kingdom, whereas the non-photosynthetic groups are not related to plants at all. The organisation of algal groups has become complex and confusing - The chapter will address the problems within this area of eukaryotic taxonomy. The metabolic diversity of algae and the ability to genetically engineer algae are key components in developing the biotechnology of algae. As more researchers become interested in exploiting algae for a number of industrial products, it is important to understand the relationships between different groups of algae and the relationships of algae with the rest of the living world., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Microalga-microbiome interactions are central to both health and disease of aquatic environments. Despite impressive advances in deciphering how microorganisms participate in and impact aquatic ecosystems, the evolution and ecological involvement of microalgae and the microbiome in polluted waters are typically studied independently. Here, the phycosphere (i.e., the consortia of microalgae and the related microbiome) is regarded as an independent and integrated life form, and we summarize the survival strategies exhibited by this symbiont when exposed to anthropogenic pollution. We highlight the cellular strategies and discuss the modulation at the transcriptional and population levels, which reciprocally alters community structure or genome composition for medium-term acclimation or long-term adaptation. We propose a 'PollutantBiome' concept to help the understanding of microalga-microbiome interactions and development of beneficial microbial synthetic communities for pollutant remediation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

Carbon dioxide is the major greenhouse gas and regards as the critical issue of global warming and climate changes. The inconspicuous microalgae are responsible for 40% of carbon fixation among all photosynthetic plants along with a higher photosynthetic efficiency and convert the carbon into lipids, protein, pigments, and bioactive compounds. Genetic approach and metabolic engineering are applied to accelerate the growth rate and biomass of microalgae, hence achieve the mission of carbon neutrality. Meanwhile, CRISPR/Cas9 is efficiently to enhance the productivity of high-value compounds in microalgae for it is easier operation, more affordable and is able to regulate multiple genes simultaneously. The genetic engineering strategies provide the multidisciplinary concept to evolute and increase the CO 2 fixation rate through Calvin-Benson-Bassham cycle. Therefore, the technologies, bioinformatics tools, systematic engineering approaches for carbon neutrality and circular economy are summarized and leading one step closer to the decarbonization society in this review., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors are grateful for the financial support received from the Ministry of Science and Technology to I-Son Ng (MOST 111-2221-E-006-012-MY3 and MOST 110-2221-E-006-030-MY3) and to Tse-Min Lee (MOST 110-2311-B-110 -003, MOST 111-2311-B-110-002, MOST 111-2923-B-110 -001-MY2, and MOST 110-2622-E-110-016) in Taiwan., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Microalgae are organisms that have the ability to perform photosynthesis, capturing CO 2 from the atmosphere to produce different metabolites such as vitamins, sugars, lipids, among others, many of them with different biotechnological applications. Recently, these microorganisms have been widely studied due to their possible use to obtain clean energy. It has been postulated that the growth of microalgae and the production of high-energy metabolites depend on the correct function of cellular organelles such as mitochondria and chloroplasts. Thus, the development of different genetic tools to improve the function of these organelles is of high scientific and technological interest. In this paper we review the recent advances in microalgae engineering and the role of cellular organelles in order to increase cell productivity and biomass., Competing Interests: Diego F. Gomez-Casati is an Academic Editor for Peer J., (© 2022 Gomez-Casati et al.)

Microbial communities in the world ocean are affected strongly by oceanic circulation, creating characteristic marine biomes. The high connectivity of most of the ocean makes it difficult to disentangle selective retention of colonizing genotypes (with traits suited to biome specific conditions) from evolutionary selection, which would act on founder genotypes over time. The Arctic Ocean is exceptional with limited exchange with other oceans and ice covered since the last ice age. To test whether Arctic microalgal lineages evolved apart from algae in the global ocean, we sequenced four lineages of microalgae isolated from Arctic waters and sea ice. Here we show convergent evolution and highlight geographically limited HGT as an ecological adaptive force in the form of PFAM complements and horizontal acquisition of key adaptive genes. Notably, ice-binding proteins were acquired and horizontally transferred among Arctic strains. A comparison with Tara Oceans metagenomes and metatranscriptomes confirmed mostly Arctic distributions of these IBPs. The phylogeny of Arctic-specific genes indicated that these events were independent of bacterial-sourced HGTs in Antarctic Southern Ocean microalgae., (© 2022 Dorrell et al.)

Development of integrated multi-trophic aquaculture (IMTA) systems constitutes a step change in the sustainable production of freshwater fish to meet emerging needs for high-protein foods globally. Recently, there has been a paradigm shift away from harvesting peat as a fuel towards the development of wettable peatland innovation (termed 'paludiculture'), such as aquaculture. Such eco-innovations support carbon sequestration and align with a balanced environmental approach to protecting biodiversity. This novel peatland-based IMTA process in the Irish midlands relies upon natural microalgae for waste treatment, recirculation and water quality where there is no use of pesticides or antibiotics. This novel IMTA system is powered with a wind turbine and the process has 'organic status'; moreover, it does not discharge aquaculture effluent to receiving water. However, there is a significant lack of understanding as to diversity of microalgae in this 'paludiculture'-based IMTA processes. This constitutes the first case study to use conventional microscopy combined with next-generation sequencing and bioinformatics to profile microalgae occurring in this novel IMTA system from pooled samples over a 12 month period in 2020. Conventional microscopy combined with classic identification revealed twenty genera of algae; with Chlorophyta and Charophyta being the most common present. However, algal DNA isolation, 16 s sequencing and bioinformatics revealed a combined total of 982 species from 341 genera across nine phyla from the same IMTA system, which emphasized a significant underestimation in the number and diversity of beneficial or potentially harmful algae in the IMTA-microbiome. These new methods also yield rich data that can be used by digital technologies to transform future monitoring and performance of the IMTA system for sustainability. The findings of this study align with many sustainability development goals of the United Nations including no poverty, zero hunger, good health and well-being, responsible consumption and production, climate change, and life below water., Competing Interests: Declaration of competing interest The authors declare that there are no competing interests of conflicts of interest with respect to the publication of this article., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

While algae demonstrate potential as a sustainable fuel source, low productivities limit the economic realization of algal biofuels. High-throughput strain engineering, omics-informed genome-scale modeling, and microbiome engineering are key technologies for enabling algal biofuels. High-throughput strain engineering efforts generate improved traits, including high biomass productivity and lipid content, in diverse algal species. Genome-scale models, constructed with the aid of omics data, provide insight into metabolic limitations and guide rational algal strain engineering efforts. As outdoor cultivation systems introduce exogenous organisms, microbiome engineering seeks to eliminate harmful organisms and introduce beneficial species. Optimizing algal biomass production and lipid content using these technologies may overcome the productivity barrier for the commercialization of algal biofuels., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The biotechnological potential of microalgae has been the target of a range of research aimed at using its potential to produce macromolecules with high added value. Particular focus has been given to biofuels' production, such as biohydrogen, biodiesel, and bioethanol from lipids and carbohydrates extracted from microalgal biomass. Bioprospecting and accurate identification of microalgae from the environment are important in the search for strains with better performance. Methodologies that combine morphology and molecular techniques allow more precise knowledge of species. Thereby, this work aimed to identify the new strain LGMM0013 collected at Iraí Reservoir, located in Paraná state, Brazil, and to evaluate the production of biomass, carbohydrates, and lipids from this new microalgal strain. Based on morphology and phylogenetic tree from internal transcribed spacer (ITS), strain LGMM0013 was identified as Desmodesmus abundans. D. abundans accumulated 1500 mg L-1 of dried biomass after 22 days of cultivation in autotrophic conditions, 50% higher than Tetradesmus obliquus (LGMM0001) (Scenedesmaceae-Chlorophyceae), usually grown in photobioreactors located at NPDEAS at the Federal University of Paraná (UFPR) to produce biomass. Analysis of the D. abundans biomass from showed an accumulation of 673.39 mg L-1 of carbohydrates, 130% higher than T. obliquus (LGMM0001). Lipid production was 259.7 mg L-1, equivalent to that of T. obliquus. Nitrogen deprivation increased the production of biomass and carbohydrates in D. abundans LGMM0013, indicating this new strain greater biomass production capacity.

Currently, the generation of isoprenoid factories in microalgae relies on two strategies: 1) enhanced production of endogenous isoprenoids; or 2) production of heterologous terpenes by metabolic engineering. Nevertheless, low titers and productivity are still a feature of isoprenoid biotechnology and need to be addressed. In this context, the mechanisms underlying isoprenoid biosynthesis in microalgae and its relationship with central carbon metabolism are reviewed. Developments in microalgal biotechnology are discussed, and a new approach of integrated "design-build-test-learn" cycle is advocated to the trends, challenges and prospects involved in isoprenoid engineering. The emerging and promising strategies and tools are discussed for microalgal engineering in the future. This review encourages a systematic engineering perspective aimed at potentiating progress in isoprenoid engineering of photosynthetic microalgae., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Microalgae are increasingly used to generate a wide range of commercial products, and there is growing evidence that microalgae-based products can be produced sustainably. However, industrial production of microalgal biomass is not as developed as other biomanufacturing platform technologies. In addition, results of bench-scale research often fail to translate to large-scale or mass production systems. This disconnect may result from trait drift and evolution occurring, through time, in response to unique drivers in each environment, such as cultivation regimes, weather, and pests. Moreover, outdoor and indoor cultivation of microalgae has the potential to impose negative selection pressures, which makes the maintenance of desired traits a challenge. In this context, this review sheds the light on our current understanding of trait drift and evolution in microalgae. We delineate the basics of phenotype plasticity and evolution, with a focus on how microalgae respond under various conditions. In addition, we review techniques that exploit phenotypic plasticity and evolution for strain improvement in view of industrial commercial applications, highlighting associated advantages and shortcomings. Finally, we suggest future research directions and recommendations to overcome unwanted trait drift and evolution in microalgae cultivation., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

CO 2 sequestration carried by biological methodologies shows enhanced potential and has the advantage that biomass produced from the captured CO 2 can be used for different applications. Bio-mitigation of carbons through a micro-algal system addresses a promising and feasible option. This review mostly focused on the role of algae, particular mechanisms, bioreactors in algae cultivation, and genetically modified algae in CO 2 fixation and energy generation systems. A combination of CO 2 bio-mitigation and biofuel production might deliver an extremely promising alternative to current CO 2 mitigation systems. Bio mitigation in which the excess carbon is captured and bio fixation which the carbon is captured by algae or autotrophs and used for producing biofuel. This review revealed that steps for biofuel production from algae include factors affecting, harvesting techniques, oil extraction and transesterification. This review helps environmentalists and researchers to assess the effect of algae-based biorefinery on the green environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Photosynthetic algae are the main primary producers in polar regions, form the basis of polar food webs, and are responsible for a significant portion of global carbon fixation. Many cold-water algae are psychrophiles that thrive in the cold but cannot grow at moderate temperatures (≥20°C). Polar regions are at risk of rapid warming caused by climate change, and the sensitivity of psychrophilic algae to rising temperatures makes them, and the ecosystems they inhabit, particularly vulnerable. Recent research on the Antarctic psychrophile Chlamydomonas priscuii, an emerging algal model, has revealed unique adaptations to life in the permanent cold. Additionally, genome sequencing of C. priscuii and its relative Chlamydomonas sp. ICE-L has given rise to a plethora of computational tools that can help elucidate the genetic basis of psychrophily. This minireview summarizes new advances in characterizing the heat stress responses in psychrophilic algae and examines their extraordinary sensitivity to temperature increases. Further research in this field will help determine the impact of climate change on psychrophiles from threatened polar environments., (© 2022 Scandinavian Plant Physiology Society.)

Background: Adaptive laboratory evolution (ALE) is a powerful method for strain optimization towards abiotic stress factors and for identifying adaptation mechanisms. In this study, the green microalga Picochlorum sp. BPE23 was cultured under supra-optimal temperature to force genetic adaptation. The robustness and adaptive capacity of Picochlorum strains turned them into an emerging model for evolutionary studies on abiotic stressors such as temperature, salinity, and light., Results: Mutant strains showed an expanded maximal growth temperature of 44.6 °C, whereas the maximal growth temperature of the wild-type strain was 42 °C. Moreover, at the optimal growth temperature of 38 °C, the biomass yield on light was 22.3% higher, and the maximal growth rate was 70.5% higher than the wild type. Genome sequencing and transcriptome analysis were performed to elucidate the mechanisms behind the improved phenotype. A de novo assembled phased reference genome allowed the identification of 21 genic mutations involved in various processes. Moreover, approximately half of the genome contigs were found to be duplicated or even triplicated in all mutants, suggesting a causal role in adaptation., Conclusions: The developed tools and mutant strains provide a strong framework from whereupon Picochlorum sp. BPE23 can be further developed. Moreover, the extensive strain characterization provides evidence of how microalgae evolve to supra-optimal temperature and to photobioreactor growth conditions. With this study, microalgal evolutionary mechanisms were identified by combining ALE with genome sequencing., (© 2022. The Author(s).)

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 diversity within, and the 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 in virus evolution. We assessed the diversity 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 optimized to detect highly divergent RNA viruses, we identified 10 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 substantial diversity of RNA viruses likely exists undetected within the unicellular microalgae. It also highlights the necessity for RNA viral characterization and for investigation of the effects of viral infections on microalgal physiology, biology, and growth, considering their environmental and industrial roles. IMPORTANCE Our knowledge of the diversity of RNA viruses infecting microbial algae-the microalgae-is minimal. However, describing the RNA viruses infecting these organisms is of primary importance at both the ecological and economic scales 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. Accordingly, we identified 10 new divergent RNA virus species belonging to RNA virus families as diverse as the double-stranded Totiviridae , Endornaviridae , and Partitiviridae and the single-stranded Marnaviridae . By expanding the known diversity 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.

Increasing marine microplastics (MPs) pollution potentially threatens the stability of phytoplankton community structures in marine environments. MPs toxicities to microalgae are largely determined by particle size, while the size-dependent mechanisms are still not fully understood. In this study, two sizes (0.1 µm and 1 µm) of polystyrene (PS) MPs were used as experimental targets to systemically compare their different effecting mechanisms on the marine model diatom Thalassiosira pseudonana with respect to oxidative stress and photosynthesis. The results indicated the toxicity of 1 µm sized MPs was higher than 0.1 µm sized MPs regarding to population growth. In condition of similar microalgal population inhibition rates, we found more enhanced cellular oxidative stress and cell death happened in the 1 µm MPs treatments, which could be linked to higher zeta potential of 1 µm MPs and more severe cell surface damage; microalgal surface light shading and cellular pigments decline were more obvious in the 0.1 µm MPs treatment, which could be linked to high aggregation abilities of 0.1 µm MPs. Gene expressions supported the morphological and physiological findings on the transcriptional level. Environmental related MPs concentrations (5 μg L -1 ) also aroused gene expression changes of T. pseudonana while more changing genes were found under 0.1 µm MPs than 1 µm MPs. These results provide novel insights into the size-dependent mechanisms of MPs toxicity on marine microalgae, as well as their potential influence on the marine environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Oils are among the most important agricultural commodities and have wide applications in food/nutrition, biofuels, and oleochemicals. The oleaginous microalga Nannochloropsis oceanica can produce large amounts of oils and the high-value ω-3 eicosapentaenoic acid, which represents a promising resource for oil production targeting biodiesel, nutraceutical, and aquaculture industries. In recent years, with the availability of omics databases and the development of genetic tools, N. oceanica has been extensively investigated as a model photosynthetic organism for studying lipid metabolism and as a green cellular factory to produce lipids for industrial applications. This review summarizes the current knowledge on the lipid composition and biosynthetic pathways of N. oceanica and reviews the recent advances in metabolic engineering of lipid production in this microalga.

Oleaginous microalgae represent potential feedstocks for the sustainable production of lipids thanks to their ability to accumulate triacylglycerols (TAGs). TAG accumulation in several algal species is strongly induced under specific conditions such as nutrient deprivation and high light which, however, also negatively impact growth. Genetic modification of lipogenic pathways can potentially enhance TAG accumulation without negatively affecting growth, avoiding the trade-off between biomass and lipid productivity. In this study, the phospholipid: diacylglycerol acyltransferase (PDAT), an enzyme involved in membrane lipid recycling, was overexpressed in the seawater alga Nannochloropsis gaditana. PDAT overexpression induced increased TAG content in actively growing algae cultures while no effects were observed in conditions naturally stimulating strong lipid accumulation such as high light and nitrogen starvation. The increase of TAG content was confirmed also in a strain cultivated in industrially relevant conditions even though PDAT overexpression, if too strong, the gene overexpression becomes detrimental for growth in the longer term. Results overall suggest that genetic modulation of the PDAT gene represents a promising strategy to increase microalgae lipid content by minimizing negative effects on biomass productivity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

This study investigated the effects of microalgae growth on antibiotic removal and the attenuation of antibiotic resistance genes (ARGs)/ARGs host bacteria in algal-bacterial granular sludge (ABGS) system. In the presence of tetracycline (TC) and sulfadiazine (SDZ) mixture (2-4 mg/L), microalgae could grow on bacterial granular sludge (BGS) to form ABGS, with a chlorophyll-a content of 7.68-8.13 mg/g-VSS being achieved. The removal efficiencies of TC and SDZ by ABGS were as high as 79.0 % and 94.0 %, which were 4.3-5.0 % higher than those by BGS. Metagenomic analysis indicated that the relative abundances of TC/SDZ- related ARGs and mobile genetic elements (MGEs) in BGS were 56.1 % and 22.1 % higher than those in ABGS. A total of 26 ARGs were detected from the granules, and they were identified to associate with 46 host bacteria. 13 out of 26 ARGs and 13 out of 46 hosts were shared ARGs and hosts, respectively. The total relative abundance of host bacteria in BGS was 30.8 % higher than that in ABGS. Scenedesmus and Chlorella were the dominant microalgae that may reduce the diversity of ARGs hosts. Overall, ABGS is a promising biotechnology for antibiotic-containing wastewater treatment., (Copyright © 2022. Published by Elsevier B.V.)

Phenotypic plasticity, the ability of a given genotype to produce alternative phenotypes in response to its environment of development, is an important mechanism for coping with variable environments. While the mechanisms underlying phenotypic plasticity are diverse, their relative contributions need to be investigated quantitatively to better understand the evolvability of plasticity across biological levels. This requires relating plastic responses of the epigenome, transcriptome, and organismal phenotype, and investigating how they vary with the genotype. Here we carried out this approach for responses to osmotic stress in Dunaliella salina, a green microalga that is a model organism for salinity tolerance. We compared two strains that show markedly different demographic responses to osmotic stress, and showed that these phenotypic responses involve strain- and environment-specific variation in gene expression levels, but a relative low-albeit significant-effect of strain × environment interaction. We also found an important genotype effect on the genome-wide methylation pattern, but little contribution from environmental conditions to the latter. However, we did detect a significant marginal effect of epigenetic variation on gene expression, beyond the influence of genetic differences on epigenetic state, and we showed that hypomethylated regions are correlated with higher gene expression. Our results indicate that epigenetic mechanisms are either not involved in the rapid plastic response to environmental change in this species, or involve only few changes in trans that are sufficient to trigger concerted changes in the expression of many genes, and phenotypic responses by multiple traits., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

The performance of photo-sequencing batch reactor (PSBR) in removing multiple antibiotics and nutrients from aquaculture wastewater as well as the antibiotic resistance genes (ARGs) proliferation were firstly investigated during the long-term experiments. The operational conditions (i.e. light intensity, light time, aeration and solid retention time) were optimised to realise the simultaneous removal of antibiotics and nutrients. It was found that, compared with traditional SBR, PSBR has similar nutrient removal rate and a 30% higher antibiotics removal rate due to the corporation of microalgae ( Chlorella ) and bacteria, and the absolute abundance of ARGs decreased by 78% in PSBR. Further investigation showed that PSBR had certain advantages in removing quinolones and the corresponding removal rate could reach up to 90%. In terms of the mechanisms, the possible metabolic pathway of antibiotic was analysed and the intermediate metabolites were different from that of the reported studies. The microbial communities were also affected by microalgae and the relative abundance of certain bacteria (such as members of the families Rhodocyclaceae and Burkholderiaceae ), which were positively correlated with some ARGs, decreased in PSBR. This study provides an alternative and effective method to aquaculture wastewater treatment, which present high nutrients and antibiotics removal efficiencies and low ARGs transmission.

MicroRNAs (miRNAs) can efficiently regulate gene expression at intracellular and extracellular levels. Plant-derived miRNAs are highly enriched in animal haemolymph and regulate mammalian gene expression. However, evidence for food-derived miRNAs in Mollusca species is lacking. In this study, we fed the microalga Nannochloropsis oculata to the pearl oyster Pinctada fucata martensii and detected dietary miRNAs in exosomes isolated from the haemolymph by RNA-seq. In total, 273 endogenous miRNAs were identified in all biological replicates. We identified 23 microalgae-derived miRNAs in the exosomes of pearl oyster haemolymph. Most microalgae-derived miRNAs showed high expression levels in both exosomes and microalgae and exhibited apparent variation among individuals. These food-derived miRNAs were predicted to participate in endocytosis, apoptosis, signal transduction, energy metabolism, and biomineralization by targeting multiple genes. These findings demonstrated the cross-kingdom transport of miRNAs from microalgae to bivalves and provide insights into novel nutrient transmission through the food chain., (Copyright © 2022. Published by Elsevier Inc.)

The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99-100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A cheap cultivation of microalgae greatly reduces the biodiesel production cost. Subsequently in this study, citric acid and effluents from sugar and tannery industries were used as the nutritional supplements for the improvement of biomass and TAG production in Phaeodactylum tricornutum using two-step cultivation. When compared to control (media without supplementation), a considerable increase in biomass and chlorophyll a was obtained with citric acid (CA) and sugar industry effluent (SIE) supplemented media. In the two-step cultivation method, biomass raised from CA (100 mg·L -1 ) and SIE (1.5 mL·L -1 ) supplementations in the first step, viz. biomass production (BP) step was allowed for lipid accumulation in the second step, viz. lipid production (LP) step, and thus yielded enhanced lipids of 11.5 ± 0.7 mg·L -1 ·day -1 and 13.5 ± 1.9 mg·L -1 ·day -1 respectively, with improved TAG synthesis. Further, differential expression analysis of TAG biosynthetic genes of P. tricornutum under single-step and two-step cultivation modes were performed, and the gene expression patterns were studied., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Incongruent phylogenies have been widely observed between nuclear and plastid or mitochondrial genomes in terrestrial plants and animals. However, few studies have examined these patterns in microalgae or the discordance between the two organelles. Here we investigated the nuclear-mitochondrial-plastid phylogenomic incongruence in Emiliania-Gephyrocapsa, a group of cosmopolitan calcifying phytoplankton with enormous populations and recent speciations. We assembled mitochondrial and plastid genomes of 27 strains from across global oceans and temperature regimes, and analyzed the phylogenomic histories of the three compartments using concatenation and coalescence methods. Six major clades with varying morphology and distribution are well recognized in the nuclear phylogeny, but such relationships are absent in the mitochondrial and plastid phylogenies, which also differ substantially from each other. The rampant phylogenomic discordance is due to a combination of organellar capture (introgression), organellar genome recombination, and incomplete lineage sorting of ancient polymorphic organellar genomes. Hybridization can lead to replacements of whole organellar genomes without introgression of nuclear genes and the two organelles are not inherited as a single cytoplasmic unit. This study illustrates the convoluted evolution and inheritance of organellar genomes in isogamous haplodiplontic microalgae and provides a window into the phylogenomic complexity of marine unicellular eukaryotes., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Microalgae including diatoms are of interest for environmentally friendly manufacturing such as production of biofuels, chemicals, and materials. The highly oil-accumulating marine diatom Fistulifera solaris has been studied as a promising host organism to be employed for these applications. Recently reported large-scale genetic engineering based on episomal vectors for diatoms could be useful to further enhance the potential of F. solaris, whereas we need to understand more the mode-of-action of diatom centromeres to rationally design the episomal vectors for stable extrachromosomal maintenance. Our previous genome analysis with pyrosequencing (short read sequencing) had generated the fragmented scaffolds which were not useful to predict centromeres on each chromosome. Here, we report the almost complete chromosomal structure of the genome of F. solaris using a long-read nanopore sequencing platform MinION. From just one single run using a MinION flow-cell, the chromosome-scale assembly with telomere-to-telomere resolution was achieved for 41 out of 44 chromosomes. Putative centromere regions were predicted from the 16 chromosomes, and we discovered putative consensus motifs in the predicted centromeres. Similar motif search had been performed in model diatoms, but no consensus motif was found. Therefore, this is the first study to successfully estimate consensus motifs in diatom centromeres. The chromosome-scale assembly also suggests the potential existence of multi-copy mini-chromosomes and tandemly repeated lipogenesis genes related to the oleaginous phenotype of F. solaris. Findings of this study are useful to understand and further engineer the oleaginous phenotype of F. solaris., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Genetic compensation has been proposed to explain phenotypic differences between gene knockouts and knockdowns in several metazoan and plant model systems. With the rapid development of reverse genetic tools such as CRISPR/Cas9 and RNAi in microalgae, it is increasingly important to assess whether genetic compensation affects the phenotype of engineered algal mutants. While exploring triacylglycerol (TAG) biosynthesis pathways in the model alga Chlamydomonas reinhardtii, it was discovered that knockout of certain genes catalyzing rate-limiting steps of TAG biosynthesis, type-2 diacylglycerol acyltransferase genes (DGTTs), triggered genetic compensation under abiotic stress conditions. Genetic compensation of a DGTT1 null mutation by a related PDAT gene was observed regardless of the strain background or mutagenesis approach, for example, CRISPR/Cas 9 or insertional mutagenesis. However, no compensation was found in the PDAT knockout mutant. The effect of PDAT knockout was evaluated in a Δvtc1 mutant, in which PDAT was upregulated under stress, resulting in a 90% increase in TAG content. Knockout of PDAT in the Δvtc1 background induced a 12.8-fold upregulation of DGTT1 and a 272.3% increase in TAG content in Δvtc1/pdat1 cells, while remaining viable. These data suggest that genetic compensation contributes to the genetic robustness of microalgal TAG biosynthetic pathways, maintaining lipid and redox homeostasis in the knockout mutants under abiotic stress. This work demonstrates examples of genetic compensation in microalgae, implies the physiological relevance of genetic compensation in TAG biosynthesis under stress, and provides guidance for future genetic engineering and mutant characterization efforts., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The carotenoids, including lycopene, lutein, astaxanthin, and zeaxanthin belong to the isoprenoids, whose basic structure is made up of eight isoprene units, resulting in a C40 backbone, though some of them are only trace components in Euglena . They are essential to all photosynthetic organisms due to their superior photoprotective and antioxidant properties. Their dietary functions decrease the risk of breast, cervical, vaginal, and colorectal cancers and cardiovascular and eye diseases. Antioxidant functions of carotenoids are based on mechanisms such as quenching free radicals, mitigating damage from reactive oxidant species, and hindering lipid peroxidation. With the development of carotenoid studies, their distribution, functions, and composition have been identified in microalgae and higher plants. Although bleached or achlorophyllous mutants of Euglena were among the earliest carotenoid-related microalgae under investigation, current knowledge on the composition and biosynthesis of these compounds in Euglena is still elusive. This review aims to overview what is known about carotenoid metabolism in Euglena , focusing on the carotenoid distribution and structure, biosynthesis pathway, and accumulation in Euglena strains and mutants under environmental stresses and different culture conditions. Moreover, we also summarize the potential applications in therapy preventing carcinogenesis, cosmetic industries, food industries, and animal feed.

The water-insoluble part of Parachlorella kessleri HY1 biomass was subjected to the extraction of cell-wall polysaccharides using polar aprotic solvents (DMSO, LiCl/DMSO) and aqueous alkaline solutions (0.1, 1 and 4 mol·l -1 of NaOH). Proteins predominated in all the crude extracts and in the insoluble residues were partially removed by treatment with proteolytic enzymes (pepsin and pronase), and in some cases with the HCl/H 2 O 2 reagent, yielding purified polysaccharide-enriched fractions. These treatments led to the solubilisation of some products in water. The composition and structure of isolated polysaccharides were characterised based on monosaccharide composition, glycosidic linkage and spectroscopic analyses. The DMSO extract contained mainly proteins, and polysaccharides were not detected. The water-soluble parts isolated from the LiCl/DMSO extract contained α-l-rhamnan, α-d-glucan and β-d-glucogalactan; the water-insoluble part contained (1 → 4)-β-d-xylan, first isolated from the biomass of green microalgae. The alkali extracts contained polysaccharides of similar structure, and also water-insoluble (1 → 4)-β-d-mannan. The insoluble part after all extractions contained α-chitin as the main polysaccharide, which was confirmed by spectroscopic methods. All these polysaccharides can play a certain role in the cell wall structure of this microalga., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Random mutagenesis is an effective strategy for enhancing cellular traits. In this study, we used the mutagen ethyl methanesulfonate to create fast-growing Nannochloropsis oculata mutants. When cultivated in a photobioreactor with a diel cycle, two mutants exhibited 2.2-fold higher carbohydrate productivity and 3.5-4.0-fold higher pigment productivity than the wild type, while one of them also showed 2.5-fold higher lipid productivity. A comprehensive physiological, metabolomic, and lipidomic study showed that the mutants had high levels of glucose-, galactose-, and xylose-based carbohydrates. Their high growth rate was attributed to increased chlorophyll a content, improved nitrogen assimilation, storage, and recycling, and low monogalactosyldiacyl glycerol/digalactosyldiacyl glycerol ratio, which was responsible for higher biomass productivity. The investigation revealed upregulation of lipid precursors, shedding light on high lipid accumulation. The derived algae strains are capable of increasing the biosynthesis of value-added storage molecules without impairing growth, rendering them promising candidates for commercial development in future biorefineries.

Antibiotic resistance gene (ARG) pollution is critical environmental problem, and horizontal gene transfer acts as a driving evolutionary force. In theory, due to the phylogenetic distance between eukaryotes and prokaryotes, eukaryotic microalgae can be a natural barrier that plays a negative role in ARG transfer among the symbiotic bacteria to decrease ARG abundance in sludge during wastewater treatment. However, this hypothesis is far from proven and needs to be tested experimentally, so this study investigated the influence of eukaryote microalgae (Scenedesmus) on the ARG profile of symbiotic bacteria based on aerobic granular reactor. The results indicated that Scenedesmus symbiosis could affect ARG diversity of bacteria, and the detected numbers of ARG in aerobic granular sludge (AG) group and algae-bacteria granular consortia (AAG) group were 45-53 and 44-47, respectively. In terms of relative abundance, after target microalgae symbiosis, the total abundance of ARGs significantly decreased from 1.17 × 10°, 2.69 × 10° and 1.36 × 10 -1 to 6.53 × 10 -1 , 9.64 × 10 -1 and 1.04 × 10 -1 in the systems with the addition of streptomycin, azithromycin and vancomycin, respectively (P < 0.05), yet there was no significant difference between AG and AAG under the stress of ampicillin, sulfamethazine and tetracycline (P > 0.05). Redundancy analysis showed that the eukaryotic microalgae were significant factor explaining the change in ARG relative abundance (P < 0.05), which contributed 15.3% of ARG variation. Furthermore, the results show that, except for the tetracycline treatment system, the total relative abundances of MGEs in the AAG under the stress of the other five antibiotics were 3.54 × 10 -2 -7.13 × 10 -1 , which were all significantly lower than those in the AG (8.38 × 10 -2 -1.59 × 10°). There was a more significant positive correlation relationship between ARGs and mobile genetic elements (MGEs) than that between ARGs and dominated bacteria., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Microalgae have become a promising novel and sustainable feedstock for meeting the rising demand for food and feed. However, microalgae-based products are currently hindered by high production costs. One major reason for this is that commonly cultivated wildtype strains do not possess the robustness and productivity required for successful industrial production. Several strain improvement technologies have been developed towards creating more stress tolerant and productive strains. While classical methods of forward genetics have been extensively used to determine gene function of randomly generated mutants, reverse genetics has been explored to generate specific mutations and target phenotypes. Site-directed mutagenesis can be accomplished by employing different gene editing tools, which enable the generation of tailor-made genotypes. Nevertheless, strategies promoting the selection of randomly generated mutants avoid the introduction of foreign genetic material. In this paper, we review different microalgal strain improvement approaches and their applications, with a primary focus on random mutagenesis. Current challenges hampering strain improvement, selection, and commercialization will be discussed. The combination of these approaches with high-throughput technologies, such as fluorescence-activated cell sorting, as tools to select the most promising mutants, will also be discussed.

Triacylglycerols (TAGs) are the main storage lipids in photosynthetic organisms under stress. In the oleaginous alga Nannochloropsis oceanica, while multiple acyl CoA:diacylglycerol (DAG) acyltransferases (NoDGATs) are involved in TAG production, the role of the unique phospholipid:DAG acyltransferase (NoPDAT) remains unknown. Here, we performed a functional complementation assay in TAG-deficient yeast (Saccharomyces cerevisiae) and an in vitro assay to probe the acyltransferase activity of NoPDAT. Subcellular localization, overexpression, and knockdown (KD) experiments were also conducted to elucidate the role of NoPDAT in N. oceanica. NoPDAT, residing at the outermost plastid membrane, does not phylogenetically fall into the clades of algae or plants and uses phosphatidylethanolamine (PE) and phosphatidylglycerol with 16:0, 16:1, and 18:1 at position sn-2 as acyl-donors in vivo. NoPDAT KD, not triggering any compensatory mechanism via DGATs, led to an ∼30% decrease of TAG content, accompanied by a vast accumulation of PEs rich in 16:0, 16:1, and 18:1 fatty acids (referred to as "LU-PE") that was positively associated with CO2 availability. We conclude that the NoPDAT pathway is parallel to and independent of the NoDGAT pathway for oil production. LU-PE can serve as an alternative carbon sink for photosynthetically assimilated carbon in N. oceanica when PDAT-mediated TAG biosynthesis is compromised or under stress in the presence of high CO2 levels., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)