Microalgae are considered as excellent platforms for biomaterial production that can replace conventional fossil fuel‐based fuels and chemicals. Genetic engineering of microalgae is prerequisite to ...maximize production of materials and to reduce costs for the production. Transcription factors (TFs) are emerging as key regulators of metabolic pathways to enhance production of molecules for biofuels and other materials. TFs with the basic leucine zipper (bZIP) domain have been known as stress regulators and are associated with lipid metabolism in plants. We overexpressed a bZIP TF, NsbZIP1, in Nannochloropsis salina, and found that transformants showed enhanced growth with concomitant increase in lipid contents. The improved phenotypes were also notable under stress conditions including N limitation and high salt. To understand the mechanism underlying improved phenotypes, we analyzed expression patterns of predicted target genes involved in lipid metabolism via quantitative RT‐PCR, confirming increases transcript levels. NsbZIP1 appeared to be one of type C bZIPs in plants that has been known to regulate lipid metabolism under stress. Taken together, we demonstrated that NsbZIP1 could improve both growth and lipid production, and TF engineering can serve as an excellent genetic engineering tool for production of biofuels and biomaterials in microalgae.
Transcription factor engineering can serve as an excellent genetic engineering tool in microalgae by regulating multiple enzymes simultaneously. In this study, it was found that NsbZIP1 transcription factor positively regulated expression of ACBP, KAS1, LC‐FACS, and LPAAT which are related to lipid synthesis in Nannochloropsis salina. NsbZIP1‐overexpressing transformants thus increased neutral lipid contents, resulting in increased of FAME production.
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.
Scenedesmus obliquus ABC-009 is a microalgal strain that accumulates large amounts of lutein, particularly when subjected to growth-limiting conditions. Here, the performance of this strain was ...evaluated for the simultaneous production of lutein and biofuels under three different modes of cultivation - photoautotrophic mode using BG-11 medium with air or 2% CO 2 and heterotrophic mode using YM medium. While it was found that the highest fatty acid methyl ester (FAME) level and lutein content per biomass (%) were achieved in BG-11 medium with CO 2 and air, respectively, heterotrophic cultivation resulted in much higher biomass productivity. While the cell concentrations of the cultures grown under BG-11 and CO 2 were largely similar to those grown in YM medium, the disparity in the biomass yield was largely attributed to the larger cell volume in heterotrophically cultivated cells. Post-cultivation light treatment was found to further enhance the biomass productivity in all three cases and lutein content in heterotrophic conditions. Consequently, the maximum biomass (757.14 ± 20.20 mg/l/d), FAME (92.78 ± 0.08 mg/l/d), and lutein (1.006 ± 0.23 mg/l/d) productivities were obtained under heterotrophic cultivation. Next, large-scale lutein production using microalgae was demonstrated using a 1-ton open raceway pond cultivation system and a low-cost fertilizer (Eco-Sol). The overall biomass yields were similar in both media, while slightly higher lutein content was obtained using the fertilizer owing to the higher nitrogen content.
•A multigene expression platform using 2A peptides in N. salina has been examined.•E2A and T2A peptides showed the best function in N. salina with 45% cleavage rate.•Transformation and screening ...efficiency have increased with the new platform.
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.
Algal biofuel feedstocks are excellent candidates for sustainable and eco-friendly fuels for the next generation, which can be improved by genetic modifications for their maximal production of ...biomaterials. However, currently available genetic modifications involve the introduction of foreign DNA into the algal genome, and this may face legal and public conflicts due to the risk of environmental, economic, and/or health problems. In this regard, we employed an old concept of crop improvement that has been accepted in the long history of agriculture, i.e. polyploidization. Polyploidization of crop plants has been selected fortuitously or intentionally not only for increased quality and/or quantity of products, but also for enhanced stress tolerance. We induced diploidy in the model algae Chlamydomonas by treating haploid cells with the microtubule inhibitor colcemid, and the resulting diploids were selected for increased colony size and neutral lipid contents. Two of the isolated diploid strains containing doubled DNA contents, named CMD ex1 and CMD ex4, were increased in their cell size and cellular weight. These diploids were excellent in coping with abiotic stresses, including nutritional, oxidative, and cold stresses. Under these conditions, the diploids accumulated two times more biomass and FAME yield compared to the control. To understand underlying mechanisms, we performed RNA-Seq analyses for the diploid under the cold stress. Transcriptomic analyses revealed that the diploids showed enhanced expression of genes involved in photosynthesis, energy metabolism, and translation as well as reduced starch metabolism. Overall, diploids of Chlamydomonas showed improvements including increased yields of biomass and FAME and enhanced stress tolerance compared to wild-type organisms. The results demonstrate that polyploidization can be utilized in industrial microalgae for the production of biofuels and other biomaterials not only on a laboratory scale but also in outdoor cultivation, where stress conditions are inevitable.
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•Diploids of Chlamydomonas showed increased production of biomass and lipid.•Diploids showed enhanced resistance to nutrient, oxidative and cold stresses.•Annual productivity of diploid was estimated to increase by 36% in Chlamydomonas.•Diploids can avoid political and public conflicts of GMOs.
ABC-009 is a microalgal strain that accumulates large amounts of lutein, particularly when subjected to growth-limiting conditions. Here, the performance of this strain was evaluated for the ...simultaneous production of lutein and biofuels under three different modes of cultivation - photoautotrophic mode using BG-11 medium with air or 2% CO
and heterotrophic mode using YM medium. While it was found that the highest fatty acid methyl ester (FAME) level and lutein content per biomass (%) were achieved in BG-11 medium with CO
and air, respectively, heterotrophic cultivation resulted in much higher biomass productivity. While the cell concentrations of the cultures grown under BG-11 and CO
were largely similar to those grown in YM medium, the disparity in the biomass yield was largely attributed to the larger cell volume in heterotrophically cultivated cells. Post-cultivation light treatment was found to further enhance the biomass productivity in all three cases and lutein content in heterotrophic conditions. Consequently, the maximum biomass (757.14 ± 20.20 mg/l/d), FAME (92.78 ± 0.08 mg/l/d), and lutein (1.006 ± 0.23 mg/l/d) productivities were obtained under heterotrophic cultivation. Next, large-scale lutein production using microalgae was demonstrated using a 1-ton open raceway pond cultivation system and a low-cost fertilizer (Eco-Sol). The overall biomass yields were similar in both media, while slightly higher lutein content was obtained using the fertilizer owing to the higher nitrogen content.
To evaluate hepatic fat fraction on dual- and triple-echo gradient-recalled echo MRI sequences in healthy children.
We retrospectively reviewed the records of children in a medical check-up clinic ...from May 2012 to November 2013. We excluded children with abnormal laboratory findings or those who were overweight. Hepatic fat fraction was measured on dual- and triple-echo sequences using 3T MRI. We compared fat fractions using the Wilcoxon signed rank test and the Bland-Altman 95% limits of agreement. The correlation between fat fractions and clinical and laboratory findings was evaluated using Spearman's correlation test, and the cut-off values of fat fractions for diagnosing fatty liver were obtained from reference intervals.
In 54 children (M:F = 26:28; 5-15 years; mean 9 years), the dual fat fraction (0.1-8.0%; median 1.6%) was not different from the triple fat fraction (0.4-6.5%; median 2.7%) (p = 0.010). The dual- and triple-echo fat fractions showed good agreement using a Bland-Altman plot (-0.6 ± 2.8%). Eight children (14.8%) on dual-echo sequences and six (11.1%) on triple-echo sequences had greater than 5% fat fraction. From these children, six out of eight children on dual-echo sequences and four out of six children on triple-echo sequences had a 5-6% hepatic fat fraction. When using a cut-off value of a 6% fat fraction derived from a reference interval, only 3.7% of children were diagnosed with fatty liver. There was no significant correlation between clinical and laboratory findings with dual and triple-echo fat fractions.
Dual fat fraction was not different from triple fat fraction. We suggest a cut-off value of a 6% fat fraction is more appropriate for diagnosing fatty liver on both dual- and triple-echo sequences in children.
Microalgae's low tolerance to high CO.sub.2 concentrations presents a significant challenge for its industrial application, especially when considering the utilization of industrial exhaust gas ...streams with high CO.sub.2 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 CO.sub.2 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 CO.sub.2-tolerant strain, Chlorella sp. ABC-001, under two different carbon dioxide levels (ambient air and 10% CO.sub.2) and at various growth phases. Cells cultivated with 10% CO.sub.2 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 CO.sub.2 concentrations and phenotypes, transcriptomic analysis was conducted across different CO.sub.2 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 CO.sub.2 conditions, resulting in improved CO.sub.2 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 CO.sub.2 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. We expect that our findings will help understand the carbon concentrating mechanism, photosynthesis, nitrogen assimilation, and lipid accumulation metabolisms of green algae according to CO.sub.2 concentrations. This study also provides insights into systems metabolic engineering of microalgae for improved performance in the future.
AIM: To evaluate the possibility of treatment effect monitoring using hepatic fat quantification magnetic resonance(MR) in pediatric nonalcoholic steatohepatitis(NASH).METHODS: We retrospectively ...reviewed the medical records of patients who received educational recommendations and vitamin E for NASH and underwent hepatic fat quantification MR from 2011 to 2013.Hepatic fat fraction(%) was measured using dual- and triple-echo gradient-recalled-echo sequences at 3T.The compliant and non-compliant groups were compared clinically,biochemically,and radiologically.RESULTS: Twenty seven patients(M:F = 24:3; mean age: 12 ± 2.3 years) were included(compliant group = 22,non-compliant = 5).None of the baseline findings differed between the 2 groups,except for triglyceride level(compliant vs non-compliant,167.7 mg/d L vs 74.2 mg/d L,P = 0.001).In the compliant group,high-density lipoprotein increased and all other parameters decreased after 1-year follow-up.However,there were various changes in the non-compliant group.Dualecho fat fraction(-19.2% vs 4.6,P < 0.001),tripleecho fat fraction(-13.4% vs 3.5,P < 0.001),alanine aminotransferase(-110.7 IU/L vs-10.6 IU/L,P = 0.047),total cholesterol(-18.1 mg/d L vs 3.8 mg/d L,P = 0.016),and triglyceride levels(-61.3 mg/d L vs 11.2 mg/d L,P = 0.013) were significantly decreased only in the compliant group.The change in body mass index and dual-echo fat fraction showed a positive correlation(ρ = 0.418,P = 0.030).CONCLUSION: Hepatic fat quantification MR can be a non-invasive,quantitative and useful tool for monitoring treatment effects in pediatric NASH.
Microalgae, valued for their sustainability and CO
fixation capabilities, are emerging as promising sources of biofuels and high-value compounds. This study aimed to boost lipid production in
by ...overexpressing chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the Calvin cycle and glycolysis, under the control of a nitrogen-inducible NIT1 promoter, to positively impact overall carbon metabolism. The standout transformant, PNG#7, exhibited significantly increased lipid production under nitrogen starvation, with biomass rising by 44% and 76% on days 4 and 16, respectively. Fatty acid methyl ester (FAME) content in PNG#7 surged by 2.4-fold and 2.1-fold, notably surpassing the wild type (WT) in lipid productivity by 3.4 and 3.7 times on days 4 and 16, respectively. Transcriptome analysis revealed a tenfold increase in transgenic GAPDH expression and significant upregulation of genes involved in fatty acid and triacylglycerol synthesis, especially the gene encoding acyl-carrier protein gene (
, Cre13. g577100. t1.2). In contrast, genes related to cellulose synthesis were downregulated. Single Nucleotide Polymorphism (SNP)/Indel analysis indicated substantial DNA modifications, which likely contributed to the observed extensive transcriptomic and phenotypic changes. These findings suggest that overexpressing chloroplast GAPDH, coupled with genetic modifications, effectively enhances lipid synthesis in
. This study not only underscores the potential of chloroplast GAPDH overexpression in microalgal lipid synthesis but also highlights the expansive potential of metabolic engineering in microalgae for biofuel production.
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