Zeaxanthin epoxidase 3 Knockout Mutants of the Model Diatom Phaeodactylum tricornutum Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin.

Carotenoids are pigments that have a range of functions in human health. The carotenoid diatoxanthin is suggested to have antioxidant, anti-inflammatory and chemo-preventive properties. Diatoxanthin is only produced by a few groups of microalgae, where it functions in photoprotection. Its large-scale production in microalgae is currently not feasible. In fact, rapid conversion into the inactive pigment diadinoxanthin is triggered when cells are removed from a high-intensity light source, which is the case during large-scale harvesting of microalgae biomass. Zeaxanthin epoxidase (ZEP) 2 and/or ZEP3 have been suggested to be responsible for the back-conversion of high-light accumulated diatoxanthin to diadinoxanthin in low-light in diatoms. Using CRISPR/Cas9 gene editing technology, we knocked out the ZEP2 and ZEP3 genes in the marine diatom Phaeodactylum tricornutum to investigate their role in the diadinoxanthin-diatoxanthin cycle and determine if one of the mutant strains could function as a diatoxanthin production line. Light-shift experiments proved that ZEP3 encodes the enzyme converting diatoxanthin to diadinoxanthin in low light. Loss of ZEP3 caused the high-light-accumulated diatoxanthin to be stable for several hours after the cultures had been returned to low light, suggesting that zep3 mutant strains could be suitable as commercial production lines of diatoxanthin.

Metabolic and physiological adaptations of microalgal growth-promoting bacterium Azospirillum brasilense growing under biogas atmosphere: a microarray-based transcriptome analysis.

Using microalgal growth-promoting bacteria (MGPB) to improve the cultured microalga metabolism during biotechnological processes is one of the most promising strategies to enhance their benefits. Nonetheless, the culture condition effect used during the biotechnological process on MGPB growth and metabolism is key to ensure the expected positive bacterium growth and metabolism of microalgae. In this sense, the present research study investigated the effect of the synthetic biogas atmosphere (75% CH4-25% CO2) on metabolic and physiological adaptations of the MGPB Azospirillum brasilense by a microarray-based transcriptome approach. A total of 394 A. brasilense differentially expressed genes (DEGs) were found: 201 DEGs (34 upregulated and 167 downregulated) at 24 h and 193 DEGs (140 upregulated and 53 downregulated) under the same conditions at 72 h. The results showed a series of A. brasilense genes regulating processes that could be essential for its adaptation to the early stressful condition generated by biogas. Evidence of energy production is shown by nitrate/nitrite reduction and activation of the hypothetical first steps of hydrogenotrophic methanogenesis; signal molecule modulation is observed: indole-3-acetic acid (IAA), riboflavin, and vitamin B6, activation of Type VI secretion system responding to IAA exposure, as well as polyhydroxybutyrate (PHB) biosynthesis and accumulation. Moreover, an overexpression of ipdC, ribB, and phaC genes, encoding the key enzymes for the production of the signal molecule IAA, vitamin riboflavin, and PHB production of 2, 1.5 and 11 folds, respectively, was observed at the first 24 h of incubation under biogas atmosphere Overall, the ability of A. brasilense to metabolically adapt to a biogas atmosphere is demonstrated, which allows its implementation for generating biogas with high calorific values and the use of renewable energies through microalga biotechnologies.

The fate of antibiotic resistance genes in wastewater containing microalgae treated by chlorination, ultra-violet, and Fenton reaction.

Antibiotic resistance genes (ARGs) and bacteria (ARBs) in the effluent of wastewater treatment plants (WWTPs) are of utmost importance for the dissemination of ARGs in natural aquatic environments. Therefore, there is an urgent need for effective technologies to eliminate WWTP ARGs/ARBs and mitigate the associated risks posed by the discharged ARG in aquatic environments. To test the effective technology for eliminating ARGs/ARBs, we compared the removal of ARGs and ARBs by three different tertiary treatments, namely ultra-violet (UV) disinfection, chlorination disinfection, and Fenton oxidation. Then, the treated wastewater was co-cultured with Chlorella vulgaris (representative of aquatic biota) to investigate the fate of discharged ARGs into the aquatic environment. The results demonstrated that chlorination (at a chlorine concentration of 15 mg/L) and Fenton (at pH 2.73, with 0.005 mol/L Fe2+ and 0.0025 mol/L H2O2) treatment showed higher efficacy in ARG removal (1.8 - 4.17 logs) than UV treatment (15 min) (1.29 - 3.87 logs). Moreover, chlorine at 15 mg/L and Fenton treatment effectively suppressed ARB regeneration while UV treatment for 15 min could not. Regardless of treatments tested in this study, the input of treated wastewater to the Chlorella system increased the number of ARGs and mobile genetic elements (MGEs), indicating the potential risk of ARG dissemination associated with WWTP discharge. Among the wastewater-Chlorella co-culture systems, chlorination resulted in less of an increase in the number of ARGs and MGEs compared to Fenton and UV treatment. When comparing the wastewater systems to the co-culture systems, it was observed that Chlorella vulgaris reduced the number of ARGs and MGEs in chlorination and UV-treated wastewater; however, Chlorella vulgaris promoted ARG survival in Fenton-treated water, suggesting that aquatic microalgae might act as a barrier to ARG dissemination. Overall, chlorination treatment not only effectively removes ARGs and inhibits ARB regeneration but also shows a lower risk of ARG dissemination. Therefore, chlorination is recommended for practical application in controlling the spread of discharged ARGs from WWTP effluent in natural aquatic environments.

A comparison of two gene regions for assessing community composition of eukaryotic marine microalgae from coastal ecosystems.

Two gene regions commonly used to characterise the diversity of eukaryotic communities using metabarcoding are the 18S ribosomal DNA V4 and V9 gene regions. We assessed the effectiveness of these two regions for characterising diverisity of coastal eukaryotic microalgae communities (EMCs) from tropical and temperate sites. We binned amplicon sequence variants (ASVs) into the high level taxonomic groups: dinoflagellates, pennate diatoms, radial centric diatoms, polar centric diatoms, chlorophytes, haptophytes and 'other microalgae'. When V4 and V9 generated ASV abundances were compared, the V9 region generated a higher number of raw reads, captured more diversity from all high level taxonomic groups and was more closely aligned with the community composition determined using light microscopy. The V4 region did resolve more ASVs to a deeper taxonomic resolution within the dinoflagellates, but did not effectively resolve other major taxonomic divisions. When characterising these communities via metabarcoding, the use of multiple gene regions is recommended, but the V9 gene region can be used in isolation to provide high-level community biodiversity to reflect relative abundances within groups. This approach reduces the cost of sequencing multiple gene regions whilst still providing important baseline ecosystem function information.

DNA and RNA Stability of Marine Microalgae in Cold-Stored Sediments and Its Implications in Metabarcoding Analyses.

The ever-increasing applications of metabarcoding analyses for environmental samples demand a well-designed assessment of the stability of DNA and RNA contained in cells that are deposited or buried in marine sediments. We thus conducted a qPCR quantification of the DNA and RNA in the vegetative cells of three microalgae entrapped in facsimile marine sediments and found that >90% of DNA and up to 99% of RNA for all microalgal species were degraded within 60 days at 4 °C. A further examination of the potential interference of the relic DNA of the vegetative cells with resting cyst detection in sediments was performed via a metabarcoding analysis in artificial marine sediments spiked with the vegetative cells of two Kareniaceae dinoflagellates and the resting cysts of another three dinoflagellates. The results demonstrated a dramatic decrease in the relative abundances of the two Kareniaceae dinoflagellates in 120 days, while those of the three resting cysts increased dramatically. Together, our results suggest that a positive detection of microalgae via metabarcoding analysis in DNA or RNA extracted from marine sediments strongly indicates the presence of intact or viable cysts or spores due to the rapid decay of relic DNA/RNA. This study provides a solid basis for the data interpretation of metabarcoding surveys, particularly in resting cyst detection.

Reconstruction of Long-Chain Polyunsaturated Acid Synthesis Pathways in Marine Red Microalga Porphyridium cruentum Using Lipidomics and Transcriptomics.

The marine red microalga Porphyridium can simultaneously synthesize long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (C20:5, EPA) and arachidonic acid (C20:4, ARA). However, the distribution and synthesis pathways of EPA and ARA in Porphyridium are not clearly understood. In this study, Porphyridium cruentum CCALA 415 was cultured in nitrogen-replete and nitrogen-limited conditions. Fatty acid content determination, transcriptomic, and lipidomic analyses were used to investigate the synthesis of ARA and EPA. The results show that membrane lipids were the main components of lipids, while storage lipids were present in a small proportion in CCALA 415. Nitrogen limitation enhanced the synthesis of storage lipids and ω6 fatty acids while inhibiting the synthesis of membrane lipids and ω3 fatty acids. A total of 217 glycerolipid molecular species were identified, and the most abundant species included monogalactosyldiglyceride (C16:0/C20:5) (MGDG) and phosphatidylcholine (C16:0/C20:4) (PC). ARA was mainly distributed in PC, and EPA was mainly distributed in MGDG. Among all the fatty acid desaturases (FADs), the expressions of Δ5FAD, Δ6FAD, Δ9FAD, and Δ12FAD were up-regulated, whereas those of Δ15FAD and Δ17FAD were down-regulated. Based on these results, only a small proportion of EPA was synthesized through the ω3 pathway, while the majority of EPA was synthesized through the ω6 pathway. ARA synthesized in the ER was likely shuttled into the chloroplast by DAG and was converted into EPA by Δ17FAD.

Optimized transgene expression in the red alga Porphyridium purpureum and efficient recombinant protein secretion into the culture medium.

Microalgae represent a promising but yet underexplored production platform for biotechnology. The vast majority of studies on recombinant protein expression in algae have been conducted in a single species, the green alga Chlamydomonas reinhardtii. However, due to epigenetic silencing, transgene expression in Chlamydomonas is often inefficient. Here we have investigated parameters that govern efficient transgene expression in the red microalga Porphyridium purpureum. Porphyridium is unique in that the introduced transformation vectors are episomally maintained as autonomously replicating plasmids in the nucleus. We show that full codon optimization to the preferred codon usage in the Porphyridium genome confers superior transgene expression, not only at the level of protein accumulation, but also at the level of mRNA accumulation, indicating that high translation rates increase mRNA stability. Our optimized expression constructs resulted in YFP accumulation to unprecedented levels of up to 5% of the total soluble protein. We also designed expression cassettes that target foreign proteins to the secretory pathway and lead to efficient protein secretion into the culture medium, thus simplifying recombinant protein harvest and purification. Our study paves the way to the exploration of red microalgae as expression hosts in molecular farming for recombinant proteins and metabolites.

Characterization of halotolerant microalga isolated from waterlogged habitats: Deciphering the biochemical profiling and unraveling the molecular identity.

The primary objective of this study was to comprehensively explore the biochemical profile of the novel halotolerant microalgae strain, biogas laboratory scenedesmus (BGLRS), previously isolated from waterlogged regions in the southwest zone of Punjab, India. To achieve this, three distinct drying methods viz. freeze-drying, oven-drying, and shade-drying were employed and biochemical composition and antioxidant analyses on the microalgal biomass were conducted. Utilizing advanced analytical techniques, including high-performance liquid chromatography (HPLC), inductively coupled plasma-atomic emission spectroscopy (ICP-AES), and gas chromatography-mass spectroscopy (GC-MS) on freeze-dried biomass, its carbohydrate profile, micronutrient composition, and presence of bioactive compounds with potential therapeutic and nutraceutical significance were sought to unravel. Among the drying methods evaluated, freeze-drying exhibited the most promising experimental results, prompting its selection for further investigation. Notably, ICP-AES unveiled elevated concentrations of essential elements such as calcium, iron, magnesium, and phosphorus in BGLRS, with negligible traces of heavy metals, underscoring its safety for human consumption. GC-MS analysis further divulged the existence of numerous biologically active compounds, indicating potential applications in medical and nutraceutical fields. Through molecular identification using sequencing of the internal transcribed spacer (ITS) region, a close taxonomic resemblance between BGLRS and Scenedesmus sp. MKB was established, solidifying its unique position within the microalgal taxonomy. The deposition of ITS sequences into the NCBI GenBank, obtaining accession number MN796425, attests to the rigor and transparency of this research. Overall, these findings strongly suggest that microalgae BGLRS possesses high-quality biochemical attributes of significant therapeutic and nutraceutical importance.

The effect of PK gene overexpression on content and antioxidant properties of carotenoids in marine microalga Dunaliella parva.

Dunaliella parva can extensively accumulate carotenoids, which is a promising raw material for carotenoids production. Carotenoids have important medicinal value. D. parva is an ideal organism for studying the mechanism of carotenoid synthesis. Our previous study identified a transcription factor DpAP2 which could regulate carotenoid synthesis in D. parva. In addition, DpAP2 could interact with three proteins with different activities (DNA binding transcription factor activity, protein kinase activity, and alpha-D-phosphohexomutase). To investigate the function of PK gene encoding interacting protein of DpAP2 with protein kinase activity in D. parva, PK gene was cloned into vector pBI221-GFP-UbiΩ-CAT and transformed into D. parva in this study. The results showed that overexpression of PK gene enhanced the contents of carotenoids, total sugars, proteins, and antioxidant activities of carotenoid extract such as superoxide radical scavenging activity, reducing power, hydroxyl radical scavenging activity in transgenic D. parva with overexpression of PK gene. This study explored the function of PK gene, and improved the medicinal value of D. parva.

Synthetic biology in microalgae towards fucoxanthin production for pharmacy and nutraceuticals.

Synthetic biology has emerged as a powerful tool for engineering biological systems to produce valuable compounds, including pharmaceuticals and nutraceuticals. Microalgae, in particular, offer a promising platform for the production of bioactive compounds due to their high productivity, low land and water requirements, and ability to perform photosynthesis. Fucoxanthin, a carotenoid pigment found predominantly in brown seaweeds and certain microalgae, has gained significant attention in recent years due to its numerous health benefits, such as antioxidation, antitumor effect and precaution osteoporosis. This review provides an overview of the principles and applications of synthetic biology in the microbial engineering of microalgae for enhanced fucoxanthin production. Firstly, the fucoxanthin bioavailability and metabolism in vivo was introduced for the beneficial roles, followed by the biological functions of anti-oxidant activity, anti-inflammatory activity, antiapoptotic role antidiabetic and antilipemic effects. Secondly, the cultivation condition and strategy were summarized for fucoxanthin improvement with low production costs. Thirdly, the genetic engineering of microalgae, including gene overexpression, knockdown and knockout strategies were discussed for further improving the fucoxanthin production. Then, synthetic biology tools of CRISPR-Cas9 genome editing, transcription activator-like effector nucleases as well as modular assembly and chassis engineering were proposed to precise modification of microalgal genomes to improve fucoxanthin production. Finally, challenges and future perspectives were discussed to realize the industrial production and development of functional foods of fucoxanthin from microalgae.

Exploring the dynamics of algae-associated microbiome during the scale-up process of Tetraselmis sp. microalgae: A metagenomics approach.

Microalgae have become a key source of valuable compounds, promoting commercial scale applications. However, biological contamination is one of the most critical problems associated with large scale algal production, especially in open systems such as raceway ponds. The current research is the first to assess the effectiveness of open raceway ponds in maintaining a pure culture of Tetraselmis sp., starting from 20 L culture up to 10,000 L culture. Microbial profiling of each successive stage revealed lower abundance of eukaryotic organisms, whereas bacterial abundance increased notably resulting in a significant decrease in Tetraselmis sp. abundance. Furthermore, several bacteria with algae growth-promoting properties were found throughout the various culture stages including Balneola, Roseovarius, and Marinobacter. However, some algae-suppressive bacteria were evidenced at later stages such as Ulvibacter, Aestuariicoccus, and Defluviimonas. Overall, due to the increasing bacterial concentration, considerations limiting bacterial contamination need to be taken.

Toxicity of tigecycline on the freshwater microalga Scenedesmus obliquus: Photosynthetic and transcriptional responses.

Tigecycline (TGC) is a new tetracycline antibiotic medication against multidrug-resistant bacteria. However, the toxicity of TGC to microalgae remains largely unknown. In this study, the toxicity of TGC on Scenedesmus obliquus was examined, focusing on changes in algal growth, photosynthetic activity, and transcriptome. According to an acute toxicity test, the IC10 and IC50 values were 0.72 mg/L and 4.15 mg/L, respectively. Analyses of photosynthetic efficiency and related parameters, such as light absorption, energy capture, and electron transport, identified a 35% perturbation in the IC50 group, while the IC10 group remained largely unaffected. Transcriptomic analysis showed that in the IC10 and IC50 treatment groups, there were 874 differentially expressed genes (DEGs) (220 upregulated and 654 downregulated) and 4289 DEGs (2660 upregulated and 1629 downregulated), respectively. Gene Ontology enrichment analysis showed that TGC treatment markedly affected photosynthesis, electron transport, and chloroplast functions. In the IC50 group, a clear upregulation of genes related to photosynthesis and chloroplast functions was observed, which could be an adaptive stress response. In the IC10 group, significant downregulation of DEGs involved in ribosomal pathways and peptide biosynthesis processes was observed. Kyoto Encyclopedia of Gene and Genomes enrichment analysis showed that treatment with TGC also disrupted energy production, protein synthesis, and metabolic processes in S. obliquus. Significant downregulation of key proteins related to Photosystem II was observed under the IC10 TGC treatment. Conversely, IC50 TGC treatment resulted in substantial upregulation across a broad array of photosystem-related proteins from both Photosystems II and I. IC10 and IC50 TGC treatments differentially influenced proteins involved in the photosynthetic electron transport process. This study emphasizes the potential risks of TGC pollution to microalgae, which contributes to a better understanding of the effects of antibiotic contamination in aquatic ecosystems.

Pioneering DNA assembling techniques and their applications in eukaryotic microalgae.

Assembling DNA fragments is a fundamental manipulation of cloning microalgal genes and carrying out microalgal synthetic biological studies. From the earliest DNA recombination to current trait and metabolic pathway engineering, we are always accompanied by homology-based DNA assembling. The improvement and modification of pioneering DNA assembling techniques and the combinational applications of the available assembling techniques have diversified and complicated the literature environment and aggravated our identification of the core and pioneering methodologies. Identifying the core assembling methodologies and using them appropriately and flourishing them even are important for researchers. A group of microalgae have been evolving as the models for both industrial applications and biological studies. DNA assembling requires researchers to know the methods available and their improvements and evolvements. In this review, we summarized the pioneering (core; leading) DNA assembling techniques developed previously, extended these techniques to their modifications, improvements and their combinations, and highlighted their applications in eukaryotic microalgae. We predicted that the gene(s) will be assembled into a functional cluster (e.g., those involving in a metabolic pathway, and stacked on normal microalgal chromosomes, their artificial episomes and looming artificial chromosomes. It should be particularly pointed out that the techniques mentioned in this review are classified according to the strategy used to assemble the final construct.

Phaeodactylum tricornutum: An established model species for diatom molecular research and an emerging chassis for algal synthetic biology.

Diatoms are prominent and highly diverse microalgae in aquatic environments. Compared with other diatom species, Phaeodactylum tricornutum is an "atypical diatom" displaying three different morphotypes and lacking the usual silica shell. Despite being of limited ecological relevance, its ease of growth in the laboratory and well-known physiology, alongside the steady increase in genome-enabled information coupled with effective tools for manipulating gene expression, have meant it has gained increased recognition as a powerful experimental model for molecular research on diatoms. We here present a brief overview of how over the last 25 years P. tricornutum has contributed to the unveiling of fundamental aspects of diatom biology, while also emerging as a new tool for algal process engineering and synthetic biology.

Mapping harmful microalgal species by eDNA monitoring: A large-scale survey across the southwestern South China Sea.

A large-scale sampling was undertaken during a research cruise across the South China Sea in August 2016, covering an area of about 100,000 km2 to investigate the molecular diversity and distributions of micro-eukaryotic protists, with a focus on the potentially harmful microalgal (HAB) species along the east coast of Peninsular Malaysia. Environmental DNAs from 30 stations were extracted and DNA metabarcoding targeting the V4 and V9 markers in the 18S rDNA was performed. Many protistan molecular units, including previously unreported HAB taxa, were discovered for the first time in the water. Our findings also revealed interesting spatial distribution patterns, with a marked signal of compositional turnover between latitudinal regimes of water masses, where dinophytes and diatom compositions were among the most strongly enhanced at the fronts, leading to distinct niches. Our results further confirmed the widespread distribution of HAB species, such as the toxigenic Alexandrium tamiyavaichii and Pseudo-nitzschia species, and the fish-killing Margalefidinium polykrikoides and Karlodinium veneficum. The molecular information obtained from this study provides an updated HAB species inventory and a toolset that could facilitate existing HAB monitoring schemes in the region to better inform management decisions.

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris.

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Epigenetic Regulation in Response to CO2 Fluctuation in Marine Microalga Nannochloropsis oceanica.

Microalgae often undergo different CO2 experiment in their habitat. To adapt to low CO2, carbon concentrating mechanism (CCM) could be launched in majority of microalgae and CCM are regulated at RNA level are well known. However, epigenetic modifications and their potential regulation of the transcription of masked genes at the genome level in response to CO2 fluctuation remain unclear. Here epigenetic regulation in response to CO2 fluctuation and epigenome-association with phenotypic plasticity of CCM are firstly uncovered in marine microalga Nannochloropsis oceanica IMET1. The result showed that lysine butyrylation (Kbu) and histone H3K9m2 modifications were present in N. oceanica IMET1. Moreover, Kbu modification positively regulated gene expression. In response to CO2 fluctuation, there were 5,438 and 1,106 genes regulated by Kbu and H3K9m2 in Nannochloropsis, respectively. Gained or lost histone methylations were closely associated with activating or repressing gene expressions. Differential modifications were mainly enriched in carbon fixation, photorespiration, photosynthesis, and lipid metabolism etc. Massive genome-wide epigenetic reprogramming was observed after N. oceanica cells shifted from high CO2 to low CO2. Particularly, we firstly noted that the transcription of the key low CO2 responsive carbonic anhydrase (CA5), a key component involved in CCM stress signaling, was potentially regulated by bivalent Kbu-H3K9m2 modifications in microalgae. This study provides novel insights into the relationship between gene transcription and epigenetic modification in Nannochloropsis, which will lay foundation on genetic improvement of CCM at epigenetic level.

Overexpression of a MYB1 Transcription Factor Enhances Triacylglycerol and Starch Accumulation and Biomass Production in the Green Microalga Chlamydomonas reinhardtii.

Microalgae are promising platforms for biofuel production. Transcription factors (TFs) are emerging as key regulators of lipid metabolism for biofuel production in microalgae. We previously identified a novel TF MYB1, which mediates lipid accumulation in the green microalga Chlamydomonas under nitrogen depletion. However, the function of MYB1 on lipid metabolism in microalgae under standard growth conditions remains poorly understood. Here, we examined the effects of MYB1 overexpression (MYB1-OE) on lipid metabolism and physiological changes in Chlamydomonas. Under standard growth conditions, MYB1-OE transformants accumulated 1.9 to 3.2-fold more triacylglycerols (TAGs) than that in the parental line (PL), and total fatty acids (FAs) also significantly increased. Moreover, saturated FA (C16:0) was enriched in TAGs and total FAs in MYB1-OE transformants. Notably, starch and protein content and biomass production also significantly increased in MYB1-OE transformants compared with that in PL. Furthermore, RT-qPCR results showed that the expressions of key genes involved in TAG, FA, and starch biosynthesis were upregulated. In addition, MYB1-OE transformants showed higher biomass production without a compromised cell growth rate and photosynthetic activity. Overall, our results indicate that MYB1 overexpression not only enhanced lipid content but also improved starch and protein content and biomass production under standard growth conditions. TF MYB1 engineering is a promising genetic engineering tool for biofuel production in microalgae.

Alkaline Phosphatase PhoD Mutation Induces Fatty Acid and Long-Chain Polyunsaturated Fatty Acid (LC-PUFA)-Bound Phospholipid Production in the Model Diatom Phaeodactylum tricornutum.

With rapid growth and high lipid contents, microalgae have become promising environmentally friendly candidates for renewable biodiesel and health supplements in our era of global warming and energy depletion. Various pathways have been explored to enhance algal lipid production, especially gene editing. Previously, we found that the functional loss of PhoD-type alkaline phosphatase (AP), a phosphorus-stress indicator in phytoplankton, could lead to increased lipid contents in the model diatom Phaeodactylum tricornutum, but how the AP mutation may change lipid composition remains unexplored. This study addresses the gap in the research and investigates the effects of PhoD-type AP mutation on the lipid composition and metabolic regulation in P. tricornutum using transcriptomic and lipidomic analyses. We observed significantly modified lipid composition and elevated production of fatty acids, lysophosphatidylcholine, lysophosphatidylethanolamine, ceramide, phosphatidylinositol bisphosphate, and monogalactosylmonoacylglycerol after PhoD_45757 mutation. Meanwhile, genes involved in fatty acid biosynthesis were upregulated in mutant cells. Moreover, the mutant exhibited increased contents of ω-3 long-chain polyunsaturated fatty acid (LC-PUFA)-bound phospholipids, indicating that PhoD_45757 mutation could improve the potential bioavailability of PUFAs. Our findings indicate that AP mutation could influence cellular lipid synthesis and probably redirect carbon toward lipid production and further demonstrate that AP mutation is a promising approach for the development of high-value microalgal strains for biomedical and other applications.

Reconstruction of a Genome-Scale Metabolic Model of Scenedesmus obliquus and Its Application for Lipid Production under Three Trophic Modes.

Green microalgae have emerged as beneficial feedstocks for biofuel production. A systems-level understanding of the biochemical network is needed to harness the microalgal metabolic capacity for bioproduction. Genome-scale metabolic modeling (GEM) showed immense potential in rational metabolic engineering, utilizing biochemical flux distribution analysis. Here, we report the first GEM for the green microalga, Scenedesmus obliquus (iAR632), a promising biodiesel feedstock with high lipid-storing capability. iAR632 comprises 1467 reactions, 734 metabolites, and 632 genes distributed among 7 compartments. The model was optimized under three different trophic modes of microalgal cultivation, i.e., autotrophy, mixotrophy, and heterotrophy. The robustness of the reconstructed network was confirmed by analyzing its sensitivity to the biomass components. Pathway-level flux profiles were analyzed, and significant flux space expansion was noticed majorly in reactions associated with lipid biosynthesis. In agreement with the experimental observation, iAR632 predicted about 3.8-fold increased biomass and almost 4-fold higher lipid under mixotrophy than the other trophic modes. Thus, the assessment of the condition-specific metabolic flux distribution of iAR632 suggested that mixotrophy is the preferred cultivation condition for improved microalgal growth and lipid production. Overall, the reconstructed GEM and subsequent analyses will provide a systematic framework for developing model-driven strategies to improve microalgal bioproduction.