High-efficiency nuclear transformation of the microalgae Nannochloropsis oceanica using Tn5 Transposome for the generation of altered lipid accumulation phenotypes (original) (raw)
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2017
Nannochloropsis is a genus of microalgae that produces substantial amounts of storage lipids referred to as triacylglycerides that are derived from fatty acids. These products are precursors to important dietary lipids for use in oils and as replacements for hydrocarbon fuels. To enhance lipid production in Nannochloropsis oceanica CCMP 1779 we applied an insertional mutagenesis approach to produce a library of 1200 strains containing single insertions located randomly in the genome. The collection was screened for growth at high CO2 (≤10%) and low pH, and for high lipid content. Seven mutants were selected for quantitative assays and characterization of photosynthetic efficiency. All 7 strains and the wild type (WT) grow fastest as measured by growth rate and final dry biomass at 2% CO2. The WT had a higher growth rate and biomass production compared with the mutant strains at 2% and 10% CO2. There was pH and CO2dependence of the growth rate of mutants and the WT. A level of 10% CO2 was a stress condition for Nannocholoropsis oceanica CCMP 1779. One strain (G2) grown at 2% and 10% CO2 had a higher lipid content by Nile Red fluorescence than the WT. When quantified by lipid extraction, this strain did not show a higher lipid content than the WT by day 15 of growth.
Metabolic Engineering, 2021
The microalga Nannochloropsis oceanica is considered a promising platform for the sustainable production of high-value lipids and biofuel feedstocks. However, current lipid yields of N. oceanica are too low for economic feasibility. Gaining fundamental insights into the lipid metabolism of N. oceanica could open up various possibilities for the optimization of this species through genetic engineering. Therefore, the aim of this study was to discover novel genes associated with an elevated neutral lipid content. We constructed an insertional mutagenesis library of N. oceanica, selected high lipid mutants by five rounds of fluorescence-activated cell sorting, and identified disrupted genes using a novel implementation of a rapid genotyping procedure. One particularly promising mutant (HLM23) was disrupted in a putative APETALA2-like transcription factor gene. HLM23 showed a 40%-increased neutral lipid content, increased photosynthetic performance, and no growth impairment. Furthermore, transcriptome analysis revealed an upregulation of genes related to plastidial fatty acid biosynthesis, glycolysis and the Calvin-Benson-Bassham cycle in HLM23. Insights gained in this work can be used in future genetic engineering strategies for increased lipid productivity of Nannochloropsis.
Trends and novel strategies for enhancing lipid accumulation and quality in microalgae
In order to realize the potential of microalgal biodiesel there is a need for substantial impetus involving interventions to radically improve lipid yields upstream. Nutrient stress and alteration to cultivation conditions are commonly used lipid enhancement strategies in microalgae. The main bottleneck of applying conventional strategies is their scalability as some of these strategies incur additional cost and energy. Novel lipid enhancement strategies have emerged to research forefront to overcome these challenges. In this review, the latest trends in microalgal lipid enhancement strategies, possible solutions and future directions are critically discussed. Advanced strategies such as combined nutrient and cultivation condition stress, microalgae-bacteria interactions, use of phytohormones EDTA and chemical additives, improving light conditions using LED, dyes and paints, and gene expression analysis are described. Molecular approaches such as metabolic and genetic engineering are emerging as the potential lipid enhancing strategies. Recent advancements in gene expression studies, genetic and metabolic engineering have shown promising results in enhancing lipid productivity in microalgae; however environmental risk and long term viability are still major challenges.
Biotechnology and Applied Biochemistry, 2019
Algal lipids have shown promising feedstock to produce biodiesel due to higher energy content, higher cetane number, and renewable nature. However, at present, the lipid productivity is too low to meet the commercial needs. Various approaches can be employed to enhance the lipid content and lipid productivity in microalgae. Stress manipulation is an attractive option to modify the algal lipid content, but it faces the drawback of time-consuming production processing and lack of information about molecular mechanisms related to triacylglycerides production in response to stress. Developing the robust hyper lipid accumulating algal strains has gained momentum due to advances in metabolic engineering and synthetic biology tools. Understanding the molecular basis of lipid biosynthesis followed by reorienting the related pathways through genomic modification is an alluring strategy that is believed to achieve the industrial and economic robustness. This review portrays the use of integrated OMIC approaches to elucidate the molecular mechanisms of strain adaptability in response to stress conditions, and identification of molecular pathways that should become novel targets to develop novel algal strains. Moreover, an update on the metabolic engineering approaches to improve the lipid production in microalgae is also provided.
High Lipid Induction in Microalgae for Biodiesel Production
Oil-accumulating microalgae have the potential to enable large-scale biodiesel production without competing for arable land or biodiverse natural landscapes. High lipid productivity of dominant, fast-growing algae is a major prerequisite for commercial production of microalgal oil-derived biodiesel. However, under optimal growth conditions, large amounts of algal biomass are produced, but with relatively low lipid contents, while species with high lipid contents are typically slow growing. Major advances in this area can be made through the induction of lipid biosynthesis, e.g., by environmental stresses. Lipids, in the form of triacylglycerides typically provide a storage function in the cell that enables microalgae to endure adverse environmental conditions. Essentially algal biomass and triacylglycerides compete for photosynthetic assimilate and a reprogramming of physiological pathways is required to stimulate lipid biosynthesis. There has been a wide range of studies carried out to identify and develop efficient lipid induction techniques in microalgae such as nutrients stress (e.g., nitrogen and/or phosphorus starvation), osmotic stress, radiation, pH, temperature, heavy metals and other chemicals. In addition, several genetic strategies for increased triacylglycerides production and inducibility are currently being developed. In this review, we discuss the potential of lipid induction techniques in microalgae and also their application at commercial scale for the production of biodiesel.
Tailoring Microalgae for Efficient Biofuel Production
Frontiers in Marine Science
Depleting fossil fuel, soaring prices, growing demand, and global climate change concerns have driven the research for finding an alternative source of sustainable fuel. Microalgae have emerged as a potential feedstock for biofuel production as many strains accumulate higher amounts of lipid, with faster biomass growth and higher photosynthetic yield than their land plant counterparts. In addition to this, microalgae can be cultured without needing agricultural land or ecological landscapes and offer opportunities for mitigating global climate change, allowing waste water treatment and carbon dioxide sequestration. Despite these benefits, microalgae pose many challenges, including low lipid yield under limiting growth conditions and slow growth in high lipid content strains. Biotechnological interventions can make major advances in strain improvement for the commercial scale production of biofuel. We discuss various strategies, including efficient transformation toolbox, to increase lipid accumulation and its quality through the regulation of key enzymes involved in lipid production, by blocking the competing pathways, pyramiding genes, enabling high cell biomass under nutrient-deprived conditions and other environmental stresses, and controlling the upstream regulators of targets, the transcription factors, and microRNAs. We highlight the opportunities emerging from the current progress in the application of genome editing in microalgae for accelerating the strain improvement program.
Higher lipid biosynthesis and accumulation are important to achieve economic viability of biofuel production via microalgae. To enhance lipid content, Chlamydomonas reinhardtii was genetically engineered with a key enzyme diacylglycerol acyltransferase (BnDGAT2) from Brassica napus, responsible for neutral lipid biosynthesis. The transformed colonies harbouring aph7 gene, screened on hygromycin-supplemented medium, achieved transformation frequency of ~120 AE 10 colonies/1 9 10 6 cells. Transgene integration and expression were confirmed by PCR, Southern blots, staining lipid droplets, proteins and spectro-fluorometric analysis of Nile red-stained cells. The neutral lipid is a major class (over 80% of total lipids) and most significant requirement for biodiesel production; this was remarkably higher in the transformed alga than the untransformed control. The levels of saturated fatty acids in the transformed alga decreased to about 7% while unsaturated fatty acids increased proportionately when compared to wild type cells. Polyunsaturated fatty acids, especially a-linolenic acid, an essential omega-3 fatty acid, were enhanced up to 12% in the transformed line. Nile red staining confirmed formation of a large number of lipid globules in the transformed alga. Evaluation of long-term stability and vitality of the transgenic alga revealed that cryopreservation produced significantly higher quantity of lipid than those maintained continuously over 128 generations on solid medium. The overexpression of BnDGAT2 significantly altered the fatty acids profile in the transformed alga. Results of this study offer a valuable strategy of genetic manipulation for enhancing polyunsaturated fatty acids and neutral lipids for biofuel production in algae.
Biotechnology for Biofuels, 2020
Background: Nannochloropsis gaditana is a photosynthetic unicellular microalgae considered one of the most interesting marine algae to produce biofuels and food additive due to its rapid growth rate and high lipid accumulation. Although microalgae are attractive platforms for solar energy bioconversion, the overall efficiency of photosynthesis is reduced due to the steep light gradient in photobioreactors. Moreover, accumulation of lipids in microalgae for biofuels production is usually induced in a two-phase cultivation process by nutrient starvation, with additional time and costs associated. In this work, a biotechnological approach was directed for the isolation of strains with improved light penetration in photobioreactor combined with increased lipids productivity. Results: Mutants of Nannochloropsis gaditana were obtained by chemical mutagenesis and screened for having both a reduced chlorophyll content per cell and increased affinity for Nile red, a fluorescent dye which binds to cellular lipid fraction. Accordingly, one mutant, called e8, was selected and characterized for having a 30% reduction of chlorophyll content per cell and an almost 80% increase of lipid productivity compared to WT in nutrient-replete conditions, with C16:0 and C18:0 fatty acids being more than doubled in the mutant. Whole-genome sequencing revealed mutations in 234 genes in e8 mutant among which there is a non-conservative mutation in the dgd1 synthase gene. This gene encodes for an enzyme involved in the biosynthesis of DGDG, one of the major lipids found in the thylakoid membrane and it is thus involved in chloroplast biogenesis. Lipid biosynthesis is strongly influenced by light availability in several microalgae species, including Nannochloropsis gaditana: reduced chlorophyll content per cell and more homogenous irradiance in photobioreactor is at the base for the increased lipid productivity observed in the e8 mutant. Conclusions: The results herein obtained presents a promising strategy to produce algal biomass enriched in lipid fraction to be used for biofuel and biodiesel production in a single cultivation process, without the additional complexity of the nutrient starvation phase. Genome sequencing and identification of the mutations introduced in e8 mutant suggest possible genes responsible for the observed phenotypes, identifying putative target for future complementation and biotechnological application.