Increasing lipid production in Chlamydomonas reinhardtii through genetic introduction for the overexpression of glyceraldehyde-3-phosphate dehydrogenase.

Microalgae, valued for their sustainability and CO2 fixation capabilities, are emerging as promising sources of biofuels and high-value compounds. This study aimed to boost lipid production in C. reinhardtii 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 (ACP, 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 C. reinhardtii. 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.

Self-Buffering system for Cost-Effective production of lactic acid from glucose and xylose using Acid-Tolerant Issatchenkia orientalis.

This study presents a cost-effective strategy for producing organic acids from glucose and xylose using the acid-tolerant yeast, Issatchenkia orientalis. I. orientalis was engineered to produce lactic acid from xylose, and the resulting strain, SD108XL, successfully converted sorghum hydrolysates into lactic acid. In order to enable low-pH fermentation, a self-buffering strategy, where the lactic acid generated by the SD108XL strain during fermentation served as a buffer, was developed. As a result, the SD108 strain produced 67 g/L of lactic acid from 73 g/L of glucose and 40 g/L of xylose, simulating a sugar composition of sorghum biomass hydrolysates. Moreover, techno-economic analysis underscored the efficiency of the self-buffering strategy in streamlining the downstream process, thereby reducing production costs. These results demonstrate the potential of I. orientalis as a platform strain for the cost-effective production of organic acids from cellulosic hydrolysates.

Hypofractionated Partial Breast Irradiation With Intensity-Modulated Radiotherapy in Early Breast Cancer or Carcinoma In Situ: An Investigational Short-Term Analysis.

PURPOSE: This study aimed to analyze the treatment outcomes and adverse effects of moderately hypofractionated partial breast irradiation (PBI) in patients with early breast cancer. METHODS: In total, 473 patients with early breast cancer or carcinoma in situ were diagnosed with Tis or T1N0 disease and underwent PBI following breast-conserving surgery. All histologic tumor types, close surgical margins within 1 mm of the tumor, and multifocal tumors were included in this study. A radiation dose of 50 Gy in 20 fractions was delivered over 4 weeks using intensity-modulated radiotherapy technique. Dosimetric data, recurrence patterns, survival outcomes, and adverse events were retrospectively analyzed. RESULTS: During a median follow-up of 28.9 months, seven patients (1.5%) experienced ipsilateral breast tumor recurrence (IBTR). Two patients had regional recurrence, four patients developed contralateral breast cancer, and no distant metastases were observed. The locoregional recurrence rate in the ipsilateral breast was 1.8%. Two deaths occurred during the follow-up period, but were not attributed to breast cancer. The 2-year disease-free survival and 2-year overall survival rates were was 94.0% and 99.8%, respectively. Acute adverse events occurred in 131 patients (27.1%), and were distributed among all grades, with only two patients (0.4%) experiencing grade 3 events. Late adverse events were noted in 16 patients (3.4%), and were distributed among all grades, including grade 3 events in four patients (0.8%). No grade 4 or 5 events were observed. CONCLUSION: Hypofractionated PBI demonstrated favorable IBTR rates in patients with early breast cancer, with low incidence of acute and late toxicities in the short-term analysis.

MRI-based volumetric tumor parameters before and during chemoradiation predict tumor recurrence and patient survival in locally advanced cervical cancer: a subgroup analysis of a phase II prospective trial.

BACKGROUND: This subgroup analysis of a prospective phase II trial aimed to identify valuable and accessible prognostic factors for overall survival (OS) and progression-free survival (PFS) of patients with locally advanced cervical cancer (LACC). METHODS: Patients with FIGO II to IVA cervical cancer were assessed in this study. All patients underwent concurrent chemoradiotherapy (CCRT) followed by brachytherapy. Tumor parameters based on MRI scans before and during CCRT were evaluated for Overall survival (OS) and Progression-free survival (PFS). RESULTS: A total of 86 patients were included in this analysis with a median follow-up period of 31.7 months. Three-year OS and PFS rates for all patients were 87.1% and 76.5%, respectively. Univariate Cox regression analysis showed that restaging tumor size (rTS) over 2.55 cm (p < 0.001), initial tumor volume (iTV) over 55.99 cc (p < 0.001), downstaging (p = 0.042), and restaging tumor volume (rTV) over 6.25 cc (p = 0.006) were significantly associated with OS. rTS (p < 0.001), iTV (p < 0.001), downstaging (p = 0.027), and rTV (p < 0.001) were identified as significant prognostic factors for PFS. In the stepwise multivariable analysis, only rTS > 2.55 cm showed statistically significant with OS (HR: 5.47, 95% CI 1.80-9.58, p = 0.035) and PFS (HR: 3.83, 95% CI 1.50-11.45; p = 0.025). CONCLUSIONS: Initial tumor size and restaging tumor volume that are easily accessible during radiotherapy provide valuable prognostic information for cervical cancer. MRI-based measurable volumetric scoring system can be readily applied in real-world practice of cervical cancer. CLINICAL TRIAL INFORMATION: This study is a subgroup analysis of prospective trial registered at ClinicalTrials.gov Identifier: NCT02993653.

Engineered methane biocatalysis: strategies to assimilate methane for chemical production.

Methane (CH4), one of the greenhouse gases, is considered a promising feedstock for the biological production of fuels and chemicals. Although recent studies have demonstrated the capability of methanotrophs to convert CH4 into various bioproducts by metabolic engineering, the productivity has not reached commercial levels. As such, there is a growing interest in synthetic methanotrophic systems as an alternative. This review summarizes the strategies for enhancing native CH4 assimilation and discusses the challenges for the construction of synthetic methanotrophy into nonmethanotrophic industrial strains. Additionally, we suggest a mixed heterotrophic approach that integrates CH4 assimilation with glucose and xylose metabolism to improve productivity. The synthetic methanotrophic system presented in this review could pave the way for sustainable and efficient biomanufacturing using CH4.

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.

Cas9-Based Metabolic Engineering of Issatchenkia orientalis for Enhanced Utilization of Cellulosic Hydrolysates.

Issatchenkia orientalis, exhibiting high tolerance against harsh environmental conditions, is a promising metabolic engineering host for producing fuels and chemicals from cellulosic hydrolysates containing fermentation inhibitors under acidic conditions. Although genetic tools for I. orientalis exist, they require auxotrophic mutants so that the selection of a host strain is limited. We developed a drug resistance gene (cloNAT)-based genome-editing method for engineering any I. orientalis strains and engineered I. orientalis strains isolated from various sources for xylose fermentation. Specifically, xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis were integrated into an intended chromosomal locus in four I. orientalis strains (SD108, IO21, IO45, and IO46) through Cas9-based genome editing. The resulting strains (SD108X, IO21X, IO45X, and IO46X) efficiently produced ethanol from cellulosic and hemicellulosic hydrolysates even though the pH adjustment and nitrogen source were not provided. As they presented different fermenting capacities, selection of a host I. orientalis strain was crucial for producing fuels and chemicals using cellulosic hydrolysates.

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.

L-malic acid production from xylose by engineered Saccharomyces cerevisiae.

L-malic acid is widely used in the food, chemical, and pharmaceutical industries. Here, we report on production of malic acid from xylose, the second most abundant sugar in lignocellulosic hydrolysates, by engineered Saccharomyces cerevisiae. To enable malic acid production in a xylose-assimilating S. cerevisiae, we overexpressed PYC1 and PYC2, coding for pyruvate carboxylases, a truncated MDH3 coding for malate dehydrogenase, and SpMAE1, coding for a Schizosaccharomyces pombe malate transporter. Additionally, both the ethanol and glycerol-producing pathways were blocked to enhance malic acid production. The resulting strain produced malic acid from both glucose and xylose, but it produced much higher titers of malic acid from xylose than glucose. Interestingly, the engineered strain had higher malic acid yield from lower concentrations (10 g L-1 ) of xylose, with no ethanol production, than from higher xylose concentrations (20 and 40 g L-1 ). As such, a fed-batch culture maintaining xylose concentrations at low levels was conducted and 61.2 g L-1 of malic acid was produced, with a productivity of 0.32 g L-1 h. These results represent successful engineering of S. cerevisiae for the production of malic acid from xylose, confirming that that xylose offers the efficient production of various biofuels and chemicals by engineered S. cerevisiae.

Microalgal metabolic engineering strategies for the production of fuels and chemicals.

Microalgae are promising sustainable resources because of their ability to convert CO2 into biofuels and chemicals directly. However, the industrial production and economic feasibility of microalgal bioproducts are still limited. As such, metabolic engineering approaches have been undertaken to enhance the productivities of microalgal bioproducts. In the last decade, impressive advances in microalgae metabolic engineering have been made by developing genetic engineering tools and multi-omics analysis. This review presents comprehensive microalgal metabolic pathways and metabolic engineering strategies for producing lipids, long chain-polyunsaturated fatty acids, terpenoids, and carotenoids. Additionally, promising metabolic engineering approaches specific to target products are summarized. Finally, this review discusses current challenges and provides future perspectives for the effective production of chemicals and fuels via microalgal metabolic engineering.

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.

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.

Isolation and Characterization of Novel Chlorella Species with Cold Resistance and High Lipid Accumulation for Biodiesel Production.

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.

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.

Diagnostic performance of a point shear wave elastography (pSWE) for hepatic fibrosis in patients with autoimmune liver disease.

BACKGROUND & AIMS: Elastography point quantification is a convenient method for measuring liver stiffness. It can be performed simultaneously with conventional ultrasonography. This study aimed to evaluate its diagnostic performance for assessing hepatic fibrosis in patients with autoimmune liver disease (AILD), including autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC). METHODS: The diagnostic performance of elastography point quantification (ElastPQ) was evaluated and compared with that of serum fibrosis markers, including the aspartate aminotransferase to platelet ratio index (APRI) and fibrosis-4 (FIB-4), using the receiver operating characteristics analysis with histologic evaluation as the reference standard. RESULTS: In 49 AIH patients, sensitivity and specificity of ElastPQ were 93.6% and 44.4%, respectively, for significant fibrosis (≥ F2, cutoff 4.47 kPa), and 63.6% and 86.8% for cirrhosis (F4, cutoff 9.28 kPa). In 41 PBC patients, they were 81.8% and 73.3%, respectively, for significant fibrosis (≥ F2, cutoff 5.56 kPa), and 100% and 81.6%, respectively, for advanced fibrosis (≥ F3, cutoff 6.04 kPa). The areas under the receiver operating characteristic curves of ElastPQ for significant fibrosis (0.77, 95% CI 0.67-0.86) and cirrhosis (0.81, 95% CI 0.65-0.96) were higher than those of APRI and FIB-4 in AILD patients. According to the multivariable analysis, histological activity, steatosis, and body max index (BMI) were not significant factors that influenced the result of ElastPQ. CONCLUSIONS: ElastPQ exhibited better diagnostic performance-without the influence of confounding factors-for assessing hepatic fibrosis in AILD patients than serum fibrosis markers.

Increased biomass and lipid production by continuous cultivation of Nannochloropsis salina transformant overexpressing a bHLH transcription factor.

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.

MAPK/ERK and JNK pathways regulate lipid synthesis and cell growth of Chlamydomonas reinhardtii under osmotic stress, respectively.

Microalgae have great potential for the production of biofuels due to the ability of the organism to accumulate large quantities of storage lipids under stress conditions. Mitogen activated protein kinase (MAPK) signaling cascades are widely recognized for their role in stress response signal transduction in eukaryotes. To assess the correlation between MAPK activation and lipid productivity, Chlamydomonas reinhardtii was studied under various concentrations of NaCl. The results demonstrated that C. reinhardtii exhibits elevated levels of extracellular-signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities after undergoing osmotic stress, as well as an increase in cellular lipid content. To establish a more direct causal link between both kinases and lipid productivity, C. reinhardtii was subjected to biochemically induced regulation of ERK and JNK pathways. Activating the MEK-ERK pathway via C6 ceramide treatment increased ERK activation and lipid production simultaneously, while PD98059 mediated inhibition of the pathway yielded opposite results. Interestingly, suppression of the JNK pathway with SP600125 resulted in a substantial decrease in cell viability under osmotic stress. These results suggest that ERK and JNK MAP kinases have important roles in microalgal lipid accumulation and cell growth under osmotic stress, respectively.

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.

Enhancement of biomass and lipid productivity by overexpression of a bZIP transcription factor in Nannochloropsis salina.

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.