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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.

Microalgae are promising sustainable resources because of their ability to convert CO

Chlorella spp. are green algae that are found across wide-ranging habitats from deserts to arctic regions, with various strains having adapted to survive under diverse environmental conditions. In this study, two novel Chlorella strains (ABC-002, ABC-008) were isolated from a freshwater lake in South Korea during the winter season and examined for possible use in the biofuel production process. The comparison of ABC-002 and ABC-008 strains with Chlorella vulgaris UTEX265 under two different temperatures (10°C, 25°C) revealed their cold-tolerant phenotypes as well as high biomass yields. The maximum quantum yields of UTEX25, ABC- 002, and ABC-008 at 10°C were 0.5594, 0.6747, and 0.7150, respectively, providing evidence of the relatively higher cold-resistance capabilities of these two strains. Furthermore, both the biomass yields and lipid content of the two novel strains were found to be higher than those of UTEX265; the overall lipid productivities of ABC-002 and ABC-008 were 1.7 ~ 2.8 fold and 1.6 ~ 4.2 fold higher compared to that of UTEX265, respectively. Thus, the high biomass and lipid productivity over a wide range of temperatures indicate that C. vulgaris ABC-002 and ABC-008 are promising candidates for applications in biofuel productions via outdoor biomass cultivation.

"Making 3D deep neural networks debuggable". In the study, we develop and propose a 3D deep neural network visualization methodology for performance evaluation of 3D deep neural networks. Our research was conducted using a 3D deep neural network model, which shows the best performance. The visualization method of the research is a method of visualizing part of the 3D object by analyzing the naive Bayesian 3D complement instance generation method and the prediction difference of each feature. The method emphasizes the influence of the network in the process of making decisions. The result of visualization through the algorithm of the study shows a clear difference based on the result class and the instance within the class, and the authors can obtain insight that can evaluate and improve the performance of the DNN (deep neural networks) model by the analyzed results. 3D deep neural networks can be made "indirectly debuggable", and after the completion of the visualization method and the analysis of the result, the method can be used as the evaluation method of "general non-debuggable DNN" and as a debugging method.

International audience; Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community-based annotation and mining of the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi-omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single-cell.cn). Via the Tripal toolkit, it features user-friendly interfaces hosting genomic resources with gene annotations and transcriptomic and proteomic data for six Nannochloropsis species, including two updated genomes of Nannochloropsis oceanica IMET1 and Nannochloropsis salina CCMP1776. Toolboxes for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis, a genome browser, etc. are also included. In addition, functional validation of genes is indicated based on phenotypes of mutants and relevant bibliography. Furthermore, epigenomic resources are also incorporated, especially for sequencing of small RNAs including microRNAs and circular RNAs. Such comprehensive and integrated landscapes of Nannochloropsis genomics and epigenomics will promote and accelerate community efforts in systems and synthetic biology of these industrially important microalgae.

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

Microalgae are promising feedstocks for sustainable and eco‐friendly production of biomaterials, which can be improved by genetic engineering. It is also necessary to optimize the processes to produce biomaterials from engineered microalgae. We previously reported that genetic improvements of an industrial microalga Nannochloropsis salina by overexpressing a basic helix‐loop‐helix transcription factor (NsbHLH2). These transformants showed an improved growth and lipid production particularly during the early phase of culture under batch culture. However, they had faster uptake of nutrients, resulting in earlier starvation and reduced growth during the later stages. We attempted to optimize the growth and lipid production by growing one of the transformants in continuous culture with variable dilution rate and feed nitrogen concentration. Relative to wild‐type, NsbHLH2 transformant consumed more nitrate at a high dilution rate (0.5 day −1), and had greater biomass production. Subsequently, nitrogen limitation at continuous cultivation led to an increased fatty acid methyl ester production by 83.6 mg l −1 day −1. To elucidate genetic mechanisms, we identified the genes containing E‐boxes, known as binding sites for bHLH transcription factors. Among these, we selected 18 genes involved in the growth and lipid metabolism, and revealed their positive contribution to the phenotypes via quantitative real‐time polymerase chain reaction. These results provide proof‐of‐concept that NsbHLH2 can be used to produce biomass and lipids.

Although microalgae produce value-added products, such as lipids, pigments, and polysaccharides using light and carbon dioxide, these intracellular products require costly downstream processes such as extraction and purification. Thus, extracellular products are desirable for economic production. While reported before, the secretion of glycolic acid by microalgal photorespiration has not received attention for industrial applications. We developed a two-stage continuous cultivation system to increase glycolic acid production using a glycolate dehydrogenase (GYD1) deficient mutant of Chlamydomonas reinhardtii which produces high concentrations of glycolic acid. Specifically, 3% CO2 was supplied in the first-stage culture for the production of biomass and ambient air (0.03% CO2) was supplied to the second stage for the production of glycolic acid. As a result, overall glycolic acid productivity reached 82.0 mg L-1 d-1 at a dilution rate of 0.34 d-1. However, as the pH of the second stage decreased to 4.7 due to the increased glycolic acid production, we controlled the pH of the second stage at pH 6.0, resulting in 122.6 mg L-1 d-1 of glycolic acid productivity. Flux balance analysis revealed that the experimental glycolic acid production rate was 69% of the theoretical glycolic acid production rate. The deviation might be due to the toxicity of glycolic acid. When a techno-economic analysis was conducted based on the experimental results, the minimum glycolic acid production cost was estimated to be $31 kg-1, indicating a potential for industrial production. These findings suggest that microalgae can be utilized for the cost-effective industrial production of glycolic acid.

Inhibitors of sodium-glucose cotransporters type 2 (SGLT2) are proposed as a novel approach for the management of type 2 diabetes mellitus. SGLT2 cotransporters are responsible for reabsorption of 90 % of the glucose filtered by the kidney. The glucuretic effect resulting from SGLT2 inhibition contributes to reduce hyperglycaemia and also assists weight loss and blood pressure reduction. In this study, we presented the case of a 59-year-old male who developed hyperosmolar hyperglycemic state (HHS), possibly caused by a sodium-glucose cotransporter 2 (SGLT2) inhibitor, a novel class of antihyperglycemic agents. This case highlights that HHS can develop in patients with diabetes treated with SGLT2 inhibitors

Many common path optimization algorithms are available. However, problems arise when a general route optimization algorithm is applied to land transportation of large cargo, such as a modular plant. The large and heavy structure of a modular plant can lead to a loss of time depending on the curve of the road. This problem is more critical when traveling through large turns, which may also cause mechanical problems. Therefore, curves are essential parameters for modular plant land transportation. In this research, we show the importance of angles in the path via multi-body dynamic simulations and finite element analysis. Based on these results, we constructed a pathfinding algorithm that considers the importance of angles. The traditional A-star algorithm considers only distance as a cost, whereas our modified A-star algorithm considers both distance and angle as costs. Our goal is to improve traditional A-star algorithms and optimize them for modular plant land transportation.

Development of transgenic strains of microalgae is necessary to obtain microalgal strains that are capable of producing high levels of oils and a variety of bio-products. However, microalgae have a much lower transformation efficiency in comparison to other micro-organisms due to the presence of thick cell walls. Here, a two-pronged approach for optimizing the electroporation parameters combined with the application of secondary metabolites from myxobacteria to weaken the cell wall was used to maximize the transformation efficiency of Nannochloropsis salina (Eustigmatophyceae). When the electroporation parameters were set at 50 pulses of square waves at a field strength of 12,000 V cm−1, a transformation efficiency of up to 53 per 108 cells was achieved for N. salina in the early exponential phase of growth. Next, cultivation of the microalgae using bacterial conditioned F2N medium containing 10% supernatant of the myxobacteria strain DZ2 resulted in a moderate inhibition of the growth rate and distinctive morphological changes. Flow cytometry confirmed an increase in the overall cell size as well as granularity. When the bacterial medium conditioned cells were employed for transformation under optimized electroporation conditions, the transformation efficiency improved 2.71-fold up to 144 per 108 cells. Taken together, a combinatory approach for electroporation using high field strength square wave pulses and conditioning cells with myxobacterial supernatant is a promising method for the efficient genetic transformation of microalgae.

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.

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 one 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. This article is protected by copyright. All rights reserved.

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.

Photosynthesis of microalgae enables conversion of light energy into chemical energy to produce biomass and biomaterials. However, the efficiency of this process must be enhanced, and truncation of light-harvesting complex (LHC) has been suggested to improve photosynthetic efficiency. We reported an EMS-induced mutant (E5) showing partially reduced LHC in Chlorella vulgaris. We determined the mutation by sequencing the whole genome of WT and E5. Augustus gene prediction was used for determining CDS, and non-synonymous changes in E5 were screened. Among these, we found a point mutation (T to A) in a gene homologous to chloroplast signal recognition particle 43 kDa (CpSRP43). The point mutation changed the 102nd valine to glutamic acid (V102E) located in the first chromodomain. Phylogenetic analyses of CpSRP43 revealed that this amino acid was valine or isoleucine in microalgae and plants, suggesting important functions. Transformation of E5 with WT CpSRP43 showed varying degrees of complementation, which was demonstrated by partial recovery of the LHCII proteins to the WT level, and partially restored photosynthetic pigments, photosynthetic ETR, NPQ, and growth, indicating that the V102E mutation was responsible for the reduced LHC in E5.

This study investigates changes in the mechanical behavior, such as changes in indentation load-displacement curve, wear resistance and contact fatigue resistance of thermal barrier coatings (TBCs) by thermal cycling test and thermal shock test. Relatively dense and porous TBCs on nickel-based bondcoat/superalloy are prepared; the highest temperature applied during thermal durability test is 1350℃. The results indicate that the porous TBCs have relatively longer lifetime during thermal cycling and thermal shock tests, while denser TBCs have relatively higher wear and contact fatigue resistance. The mechanical behavior is influenced by sintering of the TBCs by exposure to high temperature during tests.

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.

Microalgae are promising alternative feedstocks that can be used to produce biofuels and replace conventional fossil fuels. In order to increase the lipid production of microalgae, strain development via genetic engineering is required. To date, metabolic engineering studies have mainly focused on lipid synthesis pathways. However, the complicated metabolic pathways that exist in many organelles along with limitations of genetic engineering tools have made it difficult to obtain desirable microalgal strains. As an alternative strategy, we tried to increase carbon flux into fatty acid synthesis by overexpressing the NADP-dependent malic enzyme, NsME1, in Nannochloropsis salina. We found that the biomass and fatty acid methyl ester (FAME) contents were increased in NsME1 overexpressing transformants, such that the FAME yield of the top-producing NsME1 transformant was 53% higher than the wild type. To understand the effects of NsME1 on lipid production, we analyzed the total carbon concentration and NADPH/NADP ratio, which were found to be enhanced in the transformants. We also investigated mRNA expression levels of genes involved in C4-like carbon concentrating mechanism and fatty acid synthesis by quantitative real-time PCR, and confirmed their positive contribution to fatty acid production. Taken together, our results demonstrate that overexpression of NsME1 could simultaneously improve the carbon concentration and reducing power in cells, thereby increasing the lipid and FAME yields of Nannochloropsis salina. We suggest that NsME1 can serve as a promising genetic target for enhancing lipid-based bio-products in Nannochloropsis and potentially other industrial microalgae.

48-year-old woman was admitted to emergency room due to left flank pain. It was diagnosed with left ureteral stone and underwent extracorporeal shock wave lithotripsy (ESWL). However, 12 hours later, the patient complained acute upper abdominal pain with pulmonary edema and low blood pressure. A diagnosis of moderate acute pancreatitis with local complication was considered and we decided conservative therapy including fluid resuscitation, inotropics and antibiotics. It was suggested that ESWL was responsible for the acute pancreatitis. The patient gradually recovered and was discharged on 13th day of admission. ESWL is considered the standard treatment for urolithiasis. Although, it has proved to be safe and effective, serious complications have been reported in 1% of patients, including acute pancreatitis, perirenal hematoma, urosepsis, venous thrombosis, biliary obstruction, bowel perforation, lung injury, and cardiac arrhythmia. Although the possibility of post-ESWL acute pancreatitis is extremely low, physicians should take care of this complication. Korean J Pancreatobiliary 2016;21(2):82-86

경피적 승모판 풍선성형술은 적응증에 해당할 경우 수술을 대신할 수 있는 승모판 협착의 치료로 시행되고 있다. 경피적 승모판 풍선성형술 이후 합병증으로 감염성 심내막염이 보고된 사례는 국내에 없다. 저자들은 경피적 승모판 성형술을 시행한 승모판 협착증 환자에서 시술 1년 후에 나타난 감염성 심내막염을 경험하였기에 문헌고찰과 함께 보고하는 바이다. 경피적 승모판 성형술시 발생하는 승모판 파열과 이완 작용이 감염성 심내막염의 병태생리에 미칠 수 있는 영향과, 이를 반영하여 고위험 시술시 예방적 항생제 투여 대상 선정에 대한 고찰이 필요할 것이다.

The production performance and egg quality traits among five strains of Korean native chickens (KNC) were evaluated in conventional cages. A total of 240 KNC were housed in a controlled environment. Each strain had 12 replicates with 4 chickens per cage. Feed intake, body weights, egg production and egg quality were measured at 24, 28 and 32 of weeks. Egg quality parameters were analyzed using 150 eggs. Results indicated significant (P 0.05) for feed efficiency. The difference among those KNC strains on egg shell color, egg shell strength and egg shell density were not different (P>0.05) at the age of week 24 while it was significant (P 0.05) on egg length and egg shape index from five strains of KNC. The significant difference (P 0.05) at the age of 32 weeks. Regarding internal quality parameters, albumen height and Haugh unit were significantly (P 0.05). Based on the egg weight and the production performance, GS-10 KNC strain was superior when compared with the other strains.

Highlights • Heterologous sfCherry protein was expressed in N. salina for the first time. • N. salina was transformed by particle bombardment. • Integration site of the transgene on the genome was determined by RESDA PCR. • Expression of sfCherry was confirmed by a western blotting and confocal microscopy., 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.

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.

Background Microalgal strain development through genetic engineering has received much attention as a way to improve the traits of microalgae suitable for biofuel production. However, there are still some limitations in application of genetically modified organisms. In this regard, there has been recent interest in the isolation and characterization of superior strains naturally modified and/or adapted under a certain condition and on the interpretation of phenotypic changes through the whole genome sequencing. Results In this study, we isolated and characterized a novel derivative of C. reinhardtii, whose phenotypic traits diverged significantly from its ancestral strain, C. reinhardtii CC-124. This strain, designated as CC-124H, displayed cell population containing increased numbers of larger cells, which resulted in an increased biomass productivity compared to its ancestor CC-124. CC-124H was further compared with the CC-124 wild-type strain which underwent long-term storage under low light condition, designated as CC-124L. In an effort to evaluate the potential of CC-124H for biofuel production, we also found that CC-124H accumulated 116 and 66% greater lipids than that of the CC-124L, after 4 days under nitrogen and sulfur depleted conditions, respectively. Taken together, our results revealed that CC-124H had significantly increased fatty acid methyl ester (FAME) yields that were 2.66 and 1.98 times higher than that of the CC-124L at 4 days after the onset of cultivation under N and S depleted conditions, respectively, and these higher FAME yields were still maintained by day 8. We next analyzed single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) based on the whole genome sequencing. The result revealed that of the 44 CDS region alterations, 34 resulted in non-synonymous substitutions within 33 genes which may mostly be involved in cell cycle, division or proliferation. Conclusion Our phenotypic analysis, which emphasized lipid productivity, clearly revealed that CC-124H had a dramatically enhanced biomass and lipid content compared to the CC-124L. Moreover, SNPs and indels analysis enabled us to identify 34 of non-synonymous substitutions which may result in phenotypic changes of CC-124H. All of these results suggest that the concept of adaptive evolution combined with genome wide analysis can be applied to microalgal strain development for biofuel production. Electronic supplementary material The online version of this article (10.1186/s13068-017-1000-0) contains supplementary material, which is available to authorized users.

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.

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.

Ability to increase the lipid production in microalgae is one of the heavily sought-after ideas to improve the economic feasibility of microalgae-derived transportation fuels for commercial applications. We used the oxidative stress by TiO2 nanoparticles, a well-known photocatalyst, to induce lipid production in microalgae. Chlorella vulgaris UTEX 265 was cultivated under various concentrations of TiO2 ranging from 0.1 to 5 g/L under UV-A illumination. Maximum specific growth rate was affected in responding to TiO2 concentrations. In the presence of UV-A, chlorophyll concentration was decreased at the highest concentration of TiO2 (5 g/L TiO2) by oxidative stress. The fatty acid ethyl ester (FAME) composition analysis suggested that oxidative stress causes the accumulation and decomposition of lipids. The highest FAME productivity was 18.2 g/L/d under low concentrations of TiO2 (0.1 g/L) and a short induction time (two days). The controlled condition of TiO2/UV-A inducing oxidative stress (0.1 g/L TiO2 and two days induction) could be used to increase the lipid productivity of C. vulgaris UTEX 265. Our results show the possibility of modulating the lipid induction process through oxidative stress with TiO2/UV-A.

This work aims to implement expert and collaborative group recommendation services through an analysis of expertise and network relations NTIS. First of all, expertise database has been constructed by extracting keywords after indexing national R&D information in Korea (human resources, project and outcome) and applying expertise calculation algorithm. In consideration of the characteristics of national R&D information, weight values have been selected. Then, expertise points were calculated by applying weighted values. In addition, joint research and collaborative relations were implemented in a knowledge map format through network analysis using national R&D information.

Objectives: To figure out how to provide analysis results on changes in research topic trends of the institute which have performed national RD analysis rules are applied to perform analysis. The analyzed results are stored in the result database used in a knowledge map and shown using a knowledge map tool. To analyze research topics of the research institutes, Network analysis was conducted, and results were stored through the following procedures (Step 1: Create basic data, Step 2: Analysis on the size of R&D expenses, Step 3: Analysis of association relationship, Step 4: Expression of knowledge map). Findings: The Research Topic Map was implemented to help users easily figure out changes in research fields that are they are interested in. The Research Topic Map makes it easy to figure out changes in the research topics of the research institutes performed national R&D projects. It provides primary information such as the number of the research projects involved in the user-interested national R&D programs and size of R&D expenses. In addition, it offers diverse analysis results by analyzing changes in research fields. First, according to the comparison of changes in the size R&D expenses in top R&D fields, changes in key and promising sectors are found. In addition, it is able to estimate changes in total R&D expenses. Therefore, growth factors for research institutes can be examined. Particular research fields exist depending on the characteristics of college, hospital or research institute. Users are able to check changes in research fields considering the latest trends. Since correlations among research fields can be examined, furthermore, it would be possible to figure out research trends in a convergence field. Improvements/ Applications: The Research Topic Map visualizes Topic Trends information (ex: changes in research fields, changes in R&D expenses, relationship among research fields, etc.) as well as information on the projects performed by research institutes. It is meaningful in that service users are able to get data insight on research institutes.

To develop an intelligent search engine to help users retrieve information effectively, various methods, such as Semantic Web, have been used, An effective retrieval method of such methods uses ontology technology. In this paper, we built National R&D ontology after analyzing National R&D Information in NTIS and then implemented National R&D Knowledge Map to represent and retrieve information of the relationship between object and subject (project, human information, organization, research result) in R&D Ontology. In the National R&D Knowledge Map, center-node is the object selected by users, node is subject, subject`s sub-node is user`s favorite query in National R&D ontology after analyzing the relationship between object and subject. When a user selects sub-node, the system displays the results from inference engine after making query by SPARQL in National R&D ontology.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0386-9) contains supplementary material, which is available to authorized users.

This review provides an overview of the published data on the canola meal and its suitability for duck as an alternative plant-origin protein source to soybean meal. Canola meal is a legume origin protein source containing comparable amino acid profile to soybean meal and rich in essential minerals and vitamins. Nonetheless, it is known to contain less in energy content than soybean meal. Factors like field conditions and processing methods creates compositional variations among canola meal. Presence of anti-nutritional factors such as phenolic substances, phytate and glucosinolates which are known to reduce growth performance in livestock animals, are the major drawbacks for canola meal to be a competitive plant-origin protein source in the feed industry. This review is focused to address i) nutritional characteristics and feeding value of canola meal for ducks and ii) impacts of feeding canola meal on performances of ducks.