Optimization and mechanism analysis of photosynthetic EPA production in Nannochloropsis salina: Evaluating the effect of temperature and nitrogen concentrations.

Microalgae, recognized as sustainable and eco-friendly photosynthetic microorganisms, play a pivotal role in converting CO2 into value-added products. Among these, Nannochloropsis salina (Microchloropsis salina) stands out, particularly for its ability to produce eicosapentaenoic acid (EPA), a crucial omega-3 fatty acid with significant health benefits such as anti-inflammatory properties and cardiovascular health promotion. This study focused on optimizing the cultivation conditions of Nannochloropsis salina to maximize EPA production. We thoroughly investigated the effects of varying temperatures and nitrogen (NaNO3) concentrations on biomass, total lipid content, and EPA proportions. We successfully identified optimal conditions at an initial NaNO3 concentration of 1.28 g.L-1 and a temperature of 21 °C. This condition was further validated by response surface methodology, which resulted in the highest EPA productivity reported in batch systems (14.4 mg.L-1.day-1). Quantitative real-time PCR and transcriptomic analysis also demonstrated a positive correlation between specific gene expressions and enhanced EPA production. Through a comprehensive lipid analysis and photosynthetic pigment analysis, we deduced that the production of EPA in Nannochloropsis salina seemed to be produced by the remodeling of chloroplast membrane lipids. These findings provide crucial insights into how temperature and nutrient availability influence fatty acid composition in N. salina, offering valuable guidance for developing strategies to improve EPA production in various microalgae species.

Transcriptional insights into Chlorella sp. ABC-001: a comparative study of carbon fixation and lipid synthesis under different CO2 conditions.

BACKGROUND: Microalgae's low tolerance to high CO2 concentrations presents a significant challenge for its industrial application, especially when considering the utilization of industrial exhaust gas streams with high CO2 content-an economically and environmentally attractive option. Therefore, the objectives of this study were to investigate the metabolic changes in carbon fixation and lipid accumulation of microalgae under ambient air and high CO2 conditions, deepen our understanding of the molecular mechanisms driving these processes, and identify potential target genes for metabolic engineering in microalgae. To accomplish these goals, we conducted a transcriptomic analysis of the high CO2-tolerant strain, Chlorella sp. ABC-001, under two different carbon dioxide levels (ambient air and 10% CO2) and at various growth phases. RESULTS: Cells cultivated with 10% CO2 exhibited significantly better growth and lipid accumulation rates, achieving up to 2.5-fold higher cell density and twice the lipid content by day 7. To understand the relationship between CO2 concentrations and phenotypes, transcriptomic analysis was conducted across different CO2 conditions and growth phases. According to the analysis of differentially expressed genes and gene ontology, Chlorella sp. ABC-001 exhibited the development of chloroplast organelles during the early exponential phase under high CO2 conditions, resulting in improved CO2 fixation and enhanced photosynthesis. Cobalamin-independent methionine synthase expression was also significantly elevated during the early growth stage, likely contributing to the methionine supply required for various metabolic activities and active proliferation. Conversely, the cells showed sustained repression of carbonic anhydrase and ferredoxin hydrogenase, involved in the carbon concentrating mechanism, throughout the cultivation period under high CO2 conditions. This study also delved into the transcriptomic profiles in the Calvin cycle, nitrogen reductase, and lipid synthesis. Particularly, Chlorella sp. ABC-001 showed high expression levels of genes involved in lipid synthesis, such as glycerol-3-phosphate dehydrogenase and phospholipid-diacylglycerol acyltransferase. These findings suggest potential targets for metabolic engineering aimed at enhancing lipid production in microalgae. CONCLUSIONS: We expect that our findings will help understand the carbon concentrating mechanism, photosynthesis, nitrogen assimilation, and lipid accumulation metabolisms of green algae according to CO2 concentrations. This study also provides insights into systems metabolic engineering of microalgae for improved performance in the future.

Molecular analysis of sugar transporters and glycolysis pathways in Ettlia sp. under heterotrophy using fructose and glucose.

Fructophilic behavior in microalgae is a rare trait that could benefit biorefineries by enabling substitution of carbon source with fructose, and our previous study identified that Ettlia sp. prefers fructose relative to glucose. In this study, by analyzing the transcription levels of genes related to sugar transport and the glycolysis pathway, the fructose utilization of Ettlia sp. was investigated. In a fructose-containing medium, the expression levels of fructokinase (EttFRK3) and glucokinase (EttGCK1 and EttGCK2) genes were significantly upregulated in heterotrophic cultivation of Ettlia sp. under fructose supplementation conditions. Further, a sugar transporter (EttSTF11) was significantly upregulated by 3.2-fold in 1 day, and this increase was analogous to the specific growth rate exhibited by the species. Subsequent cultivation tests with multi-sugar sources also showed a significant upregulation of EttSTF11 relative to other treatments without fructose. A phylogenetic tree derived from the analysis of different transporters of interest identified that EttSTF11 was adjacent to reference fructose transporters with a high bootstrap value of 71. Given that the transmembrane domains of EttSTF11 were analogous to those of reference fructose transporter genes, EttSTF11 appeared to play a critical role in fructose consumption and metabolism in Ettlia sp.

Safe-Harboring based novel genetic toolkit for Nannochloropsis salina CCMP1776: Efficient overexpression of transgene via CRISPR/Cas9-Mediated Knock-in at the transcriptional hotspot.

Transgene expression in microalgae can be hampered by transgene silencing and unstable expression due to position effects. To overcome this, "safe harboring" transgene expression system was established for Nannochloropsis. Initially, transformants were obtained expressing a sfGFP reporter, followed by screening for high expression of sfGFP with fluorescence-activated cell sorter (FACS). 'T1' transcriptional hotspot was identified from a mutant showing best expression of sfGFP, but did not affect growth or lipid contents. By using a Cas9 editor strain, FAD12 gene, encoding Δ12-fatty acid desaturase (FAD12), was successfully knocked-in at the T1 locus, resulting in significantly higher expression of FAD12 than those of random integration. Importantly, the "safe harbored" FAD12 transformants showed four-fold higher production of linoleic acid (LA), the product of FAD12, leading to 1.5-fold increase in eicosapentaenoic acid (EPA). This safe harboring principle provide excellent proof of the concept for successful genetic/metabolic engineering of microalgae and other organisms.

Development of a pVEC peptide-based ribonucleoprotein (RNP) delivery system for genome editing using CRISPR/Cas9 in Chlamydomonas reinhardtii.

Recent technical advances related to the CRISPR/Cas9-based genome editing system have enabled sophisticated genome editing in microalgae. Although the demand for research on genome editing in microalgae has increased over time, methodological research has not been established to date for the delivery of a ribonucleoprotein (Cas9/sgRNA complex) using a cell penetrating peptide into microalgal cell lines. Here, we present a ribonucleoprotein delivery system for Chlamydomonas reinhardtii mediated by the cell penetrating peptide pVEC (LLIILRRRIRKQAHAHSK) which is in a non-covalent form. Using this technically simple method, the ribonucleoprotein was successfully delivered into C. reinhardtii. Gene Maa7 and FKB12 were disrupted, and their distinguishing patterns of Indel mutations were analyzed with the observation of several insertions of sequences not originating from the genome DNA, such as chloroplast DNA, into the expected loci. In addition, the cytotoxicity of Cas9 and the ribonucleoprotein was investigated according to the concentration and time in the algal cells. It was observed that Cas9 alone without the sgRNA induces a more severe cytotoxicity compared to the ribonucleoprotein. Our study will not only contribute to algal cell biology and its genetic engineering for further applications involving various organisms but will also provide a deeper understating of the basic science of the CRISPR/Cas9 system.

Enhancement of Lipid Production under Heterotrophic Conditions by Overexpression of an Endogenous bZIP Transcription Factor in Chlorella sp. HS2.

Transcription factor engineering to regulate multiple genes has shown promise in the field of microalgae genetic engineering. Here, we report the first use of transcription factor engineering in Chlorella sp. HS2, thought to have potential for producing biofuels and bioproducts. We identified seven endogenous bZIP transcription factors in Chlorella sp. HS2 and named them HSbZIP1 through HSbZIP7. We overexpressed HSbZIP1, a C-type bZIP transcription factor, in Chlorella sp. HS2 with the goal of enhancing lipid production. Phenotype screening under heterotrophic conditions showed that all transformants exhibited increased fatty acid production. In particular, HSbZIP1 37 and 58 showed fatty acid methyl ester (FAME) yields of 859 and 1,052 mg/l, respectively, at day 10 of growth under heterotrophic conditions, and these yields were 74% and 113% higher, respectively, than that of WT. To elucidate the mechanism underlying the improved phenotypes, we identified candidate HSbZIP1-regulated genes via transcription factor binding site analysis. We then selected three genes involved in fatty acid synthesis and investigated mRNA expression levels of the genes by qRTPCR. The result revealed that the possible HSbZIP1-regulated genes involved in fatty acid synthesis were upregulated in the HSbZIP1 transformants. Taken together, our results demonstrate that HSbZIP1 can be utilized to improve lipid production in Chlorella sp. HS2 under heterotrophic conditions.

Development and characterization of a Nannochloropsis mutant with simultaneously enhanced growth and lipid production.

BACKGROUND: The necessity to develop high lipid-producing microalgae is emphasized for the commercialization of microalgal biomass, which is environmentally friendly and sustainable. Nannochloropsis are one of the best industrial microalgae and have been widely studied for their lipids, including high-value polyunsaturated fatty acids (PUFAs). Many reports on the genetic and biological engineering of Nannochloropsis to improve their growth and lipid contents have been published. RESULTS: We performed insertional mutagenesis in Nannochloropsis salina, and screened mutants with high lipid contents using fluorescence-activated cell sorting (FACS). We isolated a mutant, Mut68, which showed improved growth and a concomitant increase in lipid contents. Mut68 exhibited 53% faster growth rate and 34% higher fatty acid methyl ester (FAME) contents after incubation for 8 days, resulting in a 75% increase in FAME productivity compared to that in the wild type (WT). By sequencing the whole genome, we identified the disrupted gene in Mut68 that encoded trehalose-6-phosphate (T6P) synthase (TPS). TPS is composed of two domains: TPS domain and T6P phosphatase (TPP) domain, which catalyze the initial formation of T6P and dephosphorylation to trehalose, respectively. Mut68 was disrupted at the TPP domain in the C-terminal half, which was confirmed by metabolic analyses revealing a great reduction in the trehalose content in Mut68. Consistent with the unaffected N-terminal TPS domain, Mut68 showed moderate increase in T6P that is known for regulation of sugar metabolism, growth, and lipid biosynthesis. Interestingly, the metabolic analyses also revealed a significant increase in stress-related amino acids, including proline and glutamine, which may further contribute to the Mut68 phenotypes. CONCLUSION: We have successfully isolated an insertional mutant showing improved growth and lipid production. Moreover, we identified the disrupted gene encoding TPS. Consistent with the disrupted TPP domain, metabolic analyses revealed a moderate increase in T6P and greatly reduced trehalose. Herein, we provide an excellent proof of concept that the selection of insertional mutations via FACS can be employed for the isolation of mutants with improved growth and lipid production. In addition, trehalose and genes encoding TPS will provide novel targets for chemical and genetic engineering, in other microalgae and organisms as well as Nannochloropsis.

Heterologous synthesis of chlorophyll b in Nannochloropsis salina enhances growth and lipid production by increasing photosynthetic efficiency.

BACKGROUND: Chlorophylls play important roles in photosynthesis, and thus are critical for growth and related metabolic pathways in photosynthetic organisms. They are particularly important in microalgae, emerging as the next generation feedstock for biomass and biofuels. Nannochloropsis are industrial microalgae for these purposes, but are peculiar in that they lack accessory chlorophylls. In addition, the localization of heterologous proteins to the chloroplast of Nannochloropsis has not been fully studied, due to the secondary plastid surrounded by four membranes. This study addressed questions of correct localization and functional benefits of heterologous expression of chlorophyllide a oxygenase from Chlamydomonas (CrCAO) in Nannochloropsis. RESULTS: We cloned CrCAO from Chlamydomonas, which catalyzes oxidation of Chla producing Chlb, and overexpressed it in N. salina to reveal effects of the heterologous Chlb for photosynthesis, growth, and lipid production. For correct localization of CrCAO into the secondary plastid in N. salina, we added the signal-recognition sequence and the transit peptide (cloned from an endogenous chloroplast-localized protein) to the N terminus of CrCAO. We obtained two transformants that expressed CrCAO and produced Chlb. They showed improved growth under medium light (90 µmol/m2/s) conditions, and their photosynthetic efficiency was increased compared to WT. They also showed increased expression of certain photosynthetic proteins, accompanied by an increased maximum electron-transfer rate up to 15.8% and quantum yields up to 17%, likely supporting the faster growth. This improved growth resulted in increased biomass production, and more importantly lipid productivity particularly with medium light. CONCLUSIONS: We demonstrated beneficial effects of heterologous expression of CrCAO in Chlb-less organism N. salina, where the newly produced Chlb enhanced photosynthesis and growth. Accordingly, transformants showed improved production of biomass and lipids, important traits of microalgae from the industrial perspectives. Our transformants are the first Nannochloropsis cells that produced Chlb in the whole evolutionary path. We also succeeded in delivering a heterologous protein into the secondary plastid for the first time in Nannochloropsis. Taken together, our data showed that manipulation of photosynthetic pigments, including Chlb, can be employed in genetic improvements of microalgae for production of biofuels and other biomaterials.

Advanced multigene expression system for Nannochloropsis salina using 2A self-cleaving peptides.

Even though there has been much interest in genetic engineering of microalgae, its progress has been slow due to the difficulty and limitation of available techniques. Currently, genetic modification in most microalgal strains is confined to single gene transformation. Here, a multigene expression system for the oleaginous model strain Nannochloropsis salina was developed with glycine-serine-glycine spacer linked 2A self-cleaving peptides (2A) for the first time. An efficiency test of the four most widely used 2As revealed that two different types of 2As T2A and E2A have the best performance in N. salina with a maximum cleavage rate of nearly 45%. The system was able to express the linked sequence of the selection marker shble and the fluorescence protein sfCherry with intact functions. Because 2A enabled multigene expression in the single cassette form, the use of 2A also reduced the vector size, which along with the stronger promoter resulted in a 9-fold increase in the transformation efficiency. Furthermore, confirmative screening accuracy of more than 90% was observed. Hence, the 2A applied vector system is expected to be beneficial in microalgal research field because it enables multigene expression as well as offering improved transformation and screening efficiency.

Current status and perspectives of genome editing technology for microalgae.

Genome editing techniques are critical for manipulating genes not only to investigate their functions in biology but also to improve traits for genetic engineering in biotechnology. Genome editing has been greatly facilitated by engineered nucleases, dubbed molecular scissors, including zinc-finger nuclease (ZFN), TAL effector endonuclease (TALEN) and clustered regularly interspaced palindromic sequences (CRISPR)/Cas9. In particular, CRISPR/Cas9 has revolutionized genome editing fields with its simplicity, efficiency and accuracy compared to previous nucleases. CRISPR/Cas9-induced genome editing is being used in numerous organisms including microalgae. Microalgae have been subjected to extensive genetic and biological engineering due to their great potential as sustainable biofuel and chemical feedstocks. However, progress in microalgal engineering is slow mainly due to a lack of a proper transformation toolbox, and the same problem also applies to genome editing techniques. Given these problems, there are a few reports on successful genome editing in microalgae. It is, thus, time to consider the problems and solutions of genome editing in microalgae as well as further applications of this exciting technology for other scientific and engineering purposes.

CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii.

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including "safe harboring" techniques shown in other organisms.

Effects of overexpression of a bHLH transcription factor on biomass and lipid production in Nannochloropsis salina.

BACKGROUND: Microalgae are considered promising alternative energy sources because they consume CO2 and accumulate large amounts of lipids that can be used as biofuel. Nannochloropsis is a particularly promising microalga due to its high growth rate and lipid content, and the availability of genomic information. Transcription factors (TFs) are global regulators of biological pathways by up- or down-regulation of related genes. Among these, basic helix-loop-helix (bHLH) TFs regulate growth, development, and stress responses in plants and animals, and have been identified in microalgae. We identified two bHLH TFs in the genome of N. salina CCMP1776, NsbHLH1, and NsbHLH2, and characterized functions of NsbHLH2 that may be involved in growth and nutrient uptake. RESULTS: We obtained NsbHLH2 overexpressing transformants of N. salina CCMP1776 by particle bombardment and confirmed that these were stable transformants. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting using antibodies against the FLAG tag that was attached at the end of the coding sequence confirmed the expression of the NsbHLH2 protein under various culture conditions. The qRT-PCR results also indicated that the endogenous and transgenic expression of NsbHLH2 was reduced under stressed conditions. Overexpression of NsbHLH2 led to increased growth rate in the early growth period, and concomitantly higher nutrient uptake, than wild type (WT). These enhanced growth and nutrient uptake resulted in increased productivities of biomass and FAME. For example, one of the transformants, NsbHLH2 3-6, showed increased biomass productivity by 36 % under the normal condition, and FAME productivity by 33 % under nitrogen limitation condition. Conclusively, the improved growth in the transformants can be associated with the enhanced nutrient uptake. We are currently assessing their potential for scale-up cultivation with positive outcomes. CONCLUSION: Overexpression of NsbHLH2 led to enhanced growth rate and nutrient uptake during the early growth phase, and increased biomass and FAME productivity, especially in the later period under normal and stressed conditions. Based on these results, we postulate that NsbHLH2 can be employed for the industrial production of biodiesel from N. salina.

Use of conditioned medium for efficient transformation and cost-effective cultivation of Nannochloropsis salina.

The oleaginous microalga Nannochloropsis sp. has been spotlighted as a promising candidate in genetic engineering research for biodiesel production. However, one of the major bottlenecks in the genetic manipulation against Nannochloropsis sp. is low transformation efficiency. Based on the idea that they grow rapidly in broth culture, the effect of conditioned medium on colonization and transformation efficiency of Nannochloropsis salina was investigated. Cells grown on agar plates with 20-40% conditioned medium produced colonies that were approximately 2.3-fold larger than cells grown without conditioned medium. More importantly, the transformation efficiency was about 2-fold greater on plates with 30% conditioned medium relative to those without conditioned medium. In addition, FAME productivity in liquid cultures with 100% conditioned medium increased up to 20% compared with cultures of control medium. These results suggest that conditioned medium can be applied for efficient transformation and cost-effective cultivation of N. salina for biodiesel production.

Heterologous overexpression of sfCherry fluorescent protein in Nannochloropsis salina.

Oleaginous microalgae of the Nannochloropsis genus are considered excellent candidates for biofuels and value-added products owing to their high biomass productivity and lipid content. Here, we report the first overexpression and detection of a heterologous sfCherry fluorescent protein in Nannochloropsis salina in order to develop a transformation toolbox for future genetic improvements. Particle bombardment was employed for transformation, and expression of Shble under the control of TUB and UEP promoters, cloned from N. salina, was used to confer resistance to Zeocin antibiotics, resulting in 5.9 and 4.7 transformants per 108 cells, respectively. Stable integration of the markers into the genome was confirmed using a restriction enzyme site-directed amplification (RESDA) PCR. The expression of sfCherry fluorescent protein was confirmed by Western blot analysis and confocal microscopy. These results suggest new possibilities of efficient genetic engineering of Nannochloropsis for the production of biofuels and other biochemicals.

Cultivation of Chlorella protothecoides in anaerobically treated brewery wastewater for cost-effective biodiesel production.

The use of wastewater has been investigated to overcome the economic challenge involved with a production of microalgae-based biodiesel. In this study, to achieve economical biodiesel production along with effective wastewater treatment at the same time, anaerobically treated brewery wastewater (ABWW) was utilized as a low-cost nutrient source, in the cultivation of Chlorella protothecoides. About 96 and 90 % of total nitrogen and phosphorus in ABWW were removed, respectively, while C. protothecoides was accumulating 1.88 g L(-1) of biomass. The C. protothecoides grown in ABWW showed increases in cell size and cell aggregation, resulting in a near 80 % enhanced harvesting efficiency within 20 min, as compared with only 4 % in BG-11. In addition, the total fatty acid content of the C. protothecoides grown in ABWW increased by 1.84-fold (35.94 ± 1.54 % of its dry cell weight), relative to that of BG-11.

Use of orange peel extract for mixotrophic cultivation of Chlorella vulgaris: increased production of biomass and FAMEs.

Mass cultivation of microalgae is necessary to achieve economically feasible production of microalgal biodiesel, but the high cost of nutrients is a major limitation. In this study, orange peel extract (OPE) was used as an inorganic and organic nutrient source for the cultivation of Chlorella vulgaris OW-01. Chemical composition analysis of the OPE indicated that it contains sufficient nutrients for mixotrophic cultivation of C. vulgaris OW-01. Analysis of biomass and FAME production showed that microalgae grown in OPE medium produced 3.4-times more biomass and 4.5-times more fatty acid methyl esters (FAMEs) than cells cultured in glucose-supplemented BG 11 medium (BG-G). These results suggest that growth of microalgae in an OPE-supplemented medium increases lipid production and that OPE has potential for use in the mass cultivation of microalgae.