Metabolic engineering and genome editing strategies for enhanced lipid production in microalgae

Depleting global petroleum reserves and skyrocketing prices coupled with succinct supply have been a grave concern,which needs alternative sources to conventional fuels.Oleaginous microalgae have been explored for enhanced lipid production,leading towards biodiesel production.These microalgae have short life cycles,require less labor,and space,and are easy to scale up.Triacylglycerol,the primary source of lipids needed to produce biodiesel,is accumulated by most microalgae.The article focuses on different types of oleaginous microalgae,which can be used as a feedstock to produce biodiesel.Lipid biosynthesis in microalgae occurs through fatty acid synthesis and TAG synthesis approaches.In-depth discussions are held regarding other efficient methods for enhancing fatty acid and TAG synthesis,regulating TAG biosynthesis bypass methods,blocking competing pathways,multigene approach,and genome editing.The most potential targets for gene transformation are hypothesized to be a malic enzyme and diacylglycerol acyltransferase while lowering phosphoenolpyruvate carboxylase activity is reported to be advantageous for lipid synthesis.

Responses in growth, lipid accumulation,and fatty acid composition of four oleaginous microalgae to different nitrogen sources and concentrations

Nitrogen deficiency is an effective strategy for enhancing lipid production in microalgae. Close relationships exist among lipid production, microalgal species, and nitrogen sources. We report growth, lipid accumulation, and fatty acid composition in four microalgae （Chloroeoccum ellipsoideum UTEX972, Chlorococcum nivale LB2225, Chlorococcum tatrense UTEX2227, and Scenedesmus deserticola JNU19） under nitrate- and urea-nitrogen deficiencies. We found three patterns of response to nitrogen deficiency： Type-A （decrease in biomass and increase in lipid content）, Type-B （reduction in both biomass and lipid content）, and Type-C （enhancement of both biomass and lipid content）. Type-C microalgae are potential candidates for large-scale oil production. Chlorococcum ellipsoideum, for example, exhibited a neutral lipid production of up to 239.6 mg/（L＇d） under urea-nitrogen deficiency. In addition, nitrogen deficiency showed only a slight influence on lipid fractions and fatty acid composition. Our study provides useful information for further screening hyper-lipid microalgal strains for biofuel production.

Medium effects on micropropagation and genetic stability of Citrullus lanatus oleaginous type

To regenerate adventitious shoots from the cotyledon proximal parts of Citrullus lanatus (Thunb.) Matsum. and Nakai ssp. mucosospermus (Fursa) oleaginous type, different concentrations of MS mineral elements, sucrose, 6-benzylaminopurine (BAP) and agar were tested. Shoot induction proved to depend on the interaction between levels of sucrose, BAP and MS mineral elements in the medium. The medium containing 3/2 strength of MS mineral elements, 35 g/l sucrose and 1 mg/l BAP solidified with 6 g/l agar allowed the production of numerous shoots without a callus phase. After 3 weeks of culture, 76.7% of the cotyledon proximal parts induced shoots with an average of 12.26 shoots per explant and a mean shoot length of 17.13 mm. The induced shoots were directly rooted and thus complete plants ready for acclimatization were obtained using a two steps procedure. Depending on the genotype, the shoot induction from cotyledon proximal parts ranged from 54% to 96%. Rooted plantlets were acclimatized and transferred to field, where they grew well, developed flowers and fruits like seeded plants. The assessment of the genetic stability of the in-vitro-regenerated plantlets by means of an Amplified Fragment Length Polymorphism (AFLP) analysis with the combination of 5 primers revealed no differences between regenerated plantlets and mother plants.

Determination of the Genetic Structure of the Oleaginous <i>Lagenaria siceraria</i>of the Nangui Abrogoua University Germplasm Collection

Thirty accessions of Lagenaria siceraria from the Nangui Abrogoua University germplasm collection were analyzed using three microsatellite markers. The average Polymorphism Information Content (PIC) value was 0.61. The average observed heterozygosity value (H0 = 0.631) compared to the average expected heterozygosity value (He = 0.645) did not show significant differences in the selected accessions, which suggested random mating in the set of accessions. Within accession inbreeding estimate (FIS), was 40% and was not significantly different from zero. The reduction of heterozygotes was likely the result of presence of null alleles. Analysis of Molecular Variance (AMOVA) within and among 30 accessions of L. siceraria revealed that 39% of the total variation resides among accessions and 61% within accessions. Accession structuring pattern derived from Bayesian clustering analysis revealed two clusters. Based on the genetic structure of the accessions analyzed, a sampling strategy to collect and conserve genetic resources of L. siceraria was suggested.

Phytometric and Productive Characteristics of Safflower Submitted to Phosphate Fertilization in the Oxisol of the Brazilian Cerrado

The safflower (Carthamus tinctorius L.), in particular, has been soaring in significance as an alternative for oil and biodiesel production. In this context, the goal of this study was to assess the influence exerted by phosphate fertilization on the phytometric and productive characteristics of the safflower raised on the Oxisol of the Cerrado. The experiment was performed in a greenhouse, using pots of 3 dm-3?in volume. Adopting the completely randomized design, five doses of phosphorus (P2O5: 0, 90, 180, 360 and 540 mg·dm-3) and six replicates were included. The following variables were estimated, viz., number of leaves, plant height, and stem diameter, besides chlorophyll index (SPAD index), number of chapters and shoot dry matter. Barring the shoot dry matter and number of chapters, all the other variables were evaluated on days 30 and 45 after the plants were transplanted. Data were submitted to analysis of variance at 1%, 5% and 10% probabilities, and regression analysis, at 1% and 5% probabilities, respectively. All the variables revealed significant differences and adjustments to the linear regression model (P < 0.1), excepting the SPAD index on day 30 after the plant emergence which was adjusted to the quadratic regression model. The highest findings of 24.47 for leaf number, 34.97 cm for plant height, 3.76 mm for stem diameter, 64.76 for chlorophyll index, 5 units for the number of chapters and 3.45 g for dry shoot mass were recorded in response to the highest phosphorus dose in the experimental range (P2O5: 540 mg·dm-3). Phosphate fertilization was observed to affect the phytometric characteristics and boost the development of the safflower plants cultivated in the Cerrado Oxisol, substantiating that safflower could be a viable cash crop that can be established in the Brazilian Cerrado.

Reproductive Components of Safflower Genotypes Submitted of Bulk Density Levels in the Brazilian Cerrado

Nutrient absorption in crops can decline and their development can be hindered by increased bulk density. This study aimed at assessing the manner in which bulk density levels affect the reproductive structures of the safflower genotypes in the Brazilian Cerrado. The completely randomized design was adopted with four replications for the experiment, which was conducted in a greenhouse using Oxisol collected from 0.0 to 0.2 m depth from the region supporting Cerrado vegetation. The treatments included ten safflower genotypes (PI 237538, PI 248385, PI 250196, PI 301049, PI 305173, PI 305205, PI 306520, PI 306603, PI 560202 and PI 613366) and five bulk density levels (1.0, 1.2, 1.4, 1.6 and 1.8 Mg·m-3). Evaluations were done at 90 days after emergence, in terms of the number, diameter and dry mass of the heads. The data were submitted to the analysis of variance. The means were grouped using the Scott-Knott test at 5% probability. The diameter and dry mass of the chapters were influenced by the mean bulk density of 1.10 Mg·m-3. A notable interaction was evident between the safflower genotypes and bulk density levels for the diameter and dry mass of the head alone, revealing the high degree of genetic variability that environmental changes induce among the genotypes. The PI 250196, PI 301049, PI 305173 and PI 305205 genotypes exhibited greater stability to the bulk density variations compared with the others. Mean bulk density of 1.2 Mg·m-3 was found to impair the development of the reproductive components of the safflower genotypes.

Amazonian Fruits: An Overview of Nutrients, Calories and Use in Metabolic Disorders

Amazonian fruits are outstanding in quality. They are consumed as true delicacies of nature by the Brazilian population. Besides their attractive attributes, i.e. appearance, different textures and distinctive flavors, their nutritional value is diversified in the type of calories and the functional food ingredients. In addition to being very palatable, Amazonian fruits provide energy-rich macronutrients (lipids, proteins and carbohydrates), micronutrients (minerals, water-soluble vitamins and fat-soluble vitamins), prebiotics (dietary fibers, especially pectin), bioactive substances (carotenoids and polyphenols), variety in the diet and improvement in the organoleptic properties and digestibility of (mixed) foods. This study first aimed to review concepts applicable to nutritional constituents and caloric contents of Amazonian fruits. It also attempted to clarify the potential use of these fruits in metabolic disorders (i.e. diabetes mellitus and/or obesity). To fulfill these purposes, 12 fruits were chosen for their dietetic significance in the Brazilian Amazonia.

Effect of Copper, Zinc, Cadmium and Chromium in the Growth of Crambe

Crambe is a promising crop for biodiesel production. However, there is no much information available about techniques and research regarding the best performance in different regions to explore its potential. The objective of this study was to evaluate the effect of cadmium, chromium, copper and zinc in the development of crambe. The study was carried out in a greenhouse and it consisted of four independent experiments in completely randomized design, with three replications. Four metals with five levels were applied: 0, 10, 15, 20 and 25 mg·kg-1 for copper, cadmium, chromium;and 0, 20, 30, 40 and 50 mg·kg-1 for zinc, in order to evaluate the growth of crambe. Data were subjected to analysis of variance and comparison of means by Tukey test at 1 and 5% significance. Despite doses of cadmium, chromium, copper and zinc in growing crambe have influenced in some of the evaluated variables of the plants, in general, their development was similar in relation to doses and the metals applied. The importance of cultivating crambe is related to grain production, based on this, copper and cadmium decreased this production.

Phytochemical Study and Evaluation of the Antiradical Activity of Extracts of Oleaginous Seeds of Panda oleosa and Isolona hexaloba from Gabon

Our study focused on phytochemical tests and evaluation of the anti-free radical activity of seed extracts of two oleaginous plants from Gabon used in traditional medicine or as condiments: Panda oleosa and Isolona hexaloba. The extraction was carried out by maceration with solvents of increasing polarity: cyclohexane, trichloroethylene, acetone, ethanol and finally distilled water. The total yields of the extracts are about 69.50% for Panda oleosa and 34.28% for Isolona hexaloba. The phytochemical tests carried out on the extracts of the seeds of Panda and Isolona highlight in both seeds the presence of alkaloids, polyphenols, triterpenes, carotenoids, reducing compounds, flavonoids, total sugars, coumarins, anthraquinones, free quinones, free anthracene derivatives, and terpenoids. Isolona seeds also contain leucoanthocyanins, sterols, cardiac glycosides and saponins. Phytochemical tests revealed the absence of tannins and mucilage in both seeds. The free radical scavenging activity was measured by scavenging the free radical cation of 2,2’-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS-+) with gallic acid as the reference antioxidant. The results of the free radical scavenging activity of the aqueous and ethanolic extracts of both seeds showed that the aqueous extracts were more active than the ethanolic extracts. The IC50s of the aqueous and ethanolic extracts of Panda seeds are 40 and 60 μg·mL-1 respectively, and those of the aqueous and ethanolic extracts of Isolona are 37.5 and 95 μg·mL-1 respectively. Gallic acid, the reference antioxidant (IC50 = 0.37 μg·mL-1) is about 10 times more active than the aqueous extracts of both seeds, 16 times more active than the ethanolic extract of Panda and 25 times more active than the ethanolic extract of Isolena.

Growth Characteristic of the Oleaginous Mi-croalga <i>Chlorella ellipsoidea</i>SD-0701 with Lipid Accumulation

Microalgae have great advantages as a new biomass source for fuel production. But microalgae are photosynthetic microorganisms, which normally grow in the light. Because of this growth condition, the commercial viability of microalgal biofuel is limited by current production systems. To obtain microalgal biofuel, fermentation is a more convenient, more economical and practical industry model. In this study, we asked whether and why the dark fermentation of C. ellipsoidea SD-0701 could be achieved by changing the culture medium formula. We focused the research on carbon-containing compounds and the initial pH of media. The results indicated that glucose was the optimum carbon-containing compound, which provided C. ellipsoidea SD-0701 with energy and carbon skeleton for accumulating organic compounds including lipids. When C. ellipsoidea SD-0701 was cultivated in the add-nutrition medium containing glucose, the optimum initial pH for the growth of C. ellipsoidea SD-0701 was pH 7.71. Therefore, if the suitable medium is used, C. ellipsoidea SD-0701 can grow normally in the dark, which is the same condition as the fermenter, and high microalgal biomass (0.50 g&middotL-1) and lipid yield (232.90 mg&middotL-1) can be achieved.

Characterization of Oils Produced from Jatropha Curcas, Peanut and White Sesame in Burkina Faso for Possible Enable Synthesis of Biofuels

The indispensable role of energy in the correct functioning of economies and the current depletion of fossil fuels which are not renewable, have made the search for alternative energy sources a major point of reflection worldwide. Vegetable oils are increasingly being considered as a possible replacement for fossil fuels in countries of the south and North. In order to vulgarize the use of vegetable oil as fuel, it is essential that analytical protocols and standards are elaborated to assure the quality of oils as fuels. This work aims at developing protocols for the characterization of oilseeds and their resultant oils in a bid to ensure their quality for direct use as Biofuels or better still to serve as raw material in producing Biofuels. These protocols have the advantage of being reproducible, fast, and simple to implement compared to those used in Europe to characterize the oils (very complex and expensive, applied to petroleum products). Cotton, Jatropha curcas, white sesame, and peanut grains were acquired from the Zogona local market, Ouagadougou, and oil extraction was done at the village level. The resultant oils were characterized. Their physicochemical parameters (water content, oil content, acid index, the calorific value, density, and viscosity....) were evaluated at the Biomass Energy and Biofuels Laboratory (LBEB) of the International Institute for Water and Environmental Engineering (2iE) and the Burkinabé National Hydrocarbons Authority (SONABHY) Laboratory. The oilseed grain cakes (moisture, ash rate, rate of volatile matter, and oil content...) were also analysed.

Influence of Agitation and Aeration on Single Cell Oil Production by Rhodotorula glutinis from Glycerol

In the present study, the yeast Rhodotorula glutinis has been assessed with the aim of producing microbial lipids from glycerol under different aeration conditions. For such a purpose, experiments were carried out in shake flasks, under different conditions of agitation (150 to 250 rpm) and aeration (2.5 to 5.0 of flask volume-to-medium volume ratio). Furthermore, their influence on fermentative parameters (lipid and cell concentration, biomass yield;lipid yield;and lipid volumetric productivity) has been investigated using a 22 full factorial design. The statistical analysis has revealed a strong influence of both variables on substrate consumption, lipid accumulation, cell growth and lipid productivity. As a whole, results suggest that higher aeration levels provide greater cell and lipid concentrations, and lipid volumetric productivity. The best results (4.5 g/L of lipids and QP = 0.95 g/L&sdot;day) were achieved at the highest aeration (5.0 flask volume-to-medium volume ratio) and agitation (250 rpm) levels. Their fatty acid profile showed that oleic acid was produced in greater quantity (53.5%), followed by linoleic acid (18.7%), palmitic acid (6.8%) and stearic acid (9.9%). The microbial oil presented viscosity of 39.3 cP at 50°C and free fatty acid content of 1.93% ± 0.08%. These are significant results and contribute to establishing operational conditions that maximize single-cell oil production from glycerol by Rhodotorula glutinis, i.e. an alternative source as renewable raw material for lipid-based biorefineries.

Genome-wide adenine N6-methylation map reveals epigenomic regulation of lipid accumulation in Nannochloropsis

Epigenetic marks on histones and DNA,such as DNA methylation at N6-adenine(6mA),play crucial roles in gene expression and genome maintenance,but their deposition and function in microalgae remain largely uncharacterized.Here,we report a genome-wide 6mA map for the model industrial oleaginous microalga Nannochloropsis oceanica produced by single-molecule real-time sequencing.Found in 0.1%of adenines,6mA sites are mostly enriched at the AGGYV motif,more abundant in transposons and 30 untranslated re-gions,and associated with active transcription.Moreover,6mA gradually increases in abundance along the direction of gene transcription and shows special positional enrichment near splicing donor and transcrip-tion termination sites.Highly expressed genes tend to show greater 6mA abundance in the gene body than do poorly expressed genes,indicating a positive interaction between 6mA and general transcription fac-tors.Furthermore,knockout of the putative 6mA methylase NO08G00280 by genome editing leads to changes in methylation patterns that are correlated with changes in the expression of molybdenum cofactor,sulfate transporter,glycosyl transferase,and lipase genes that underlie reductions in biomass and oil productivity.By contrast,knockout of the candidate demethylase NO06G02500 results in increased 6mA levels and reduced growth.Unraveling the epigenomic players and their roles in biomass productivity and lipid metabolism lays a foundation for epigenetic engineering of industrial microalgae.

Engineering of Yarrowia lipolytica for production of astaxanthin

Astaxanthin is a red-colored carotenoid,used as food and feed additive.Astaxanthin is mainly produced by chemical synthesis,however,the process is expensive and synthetic astaxanthin is not approved for human consumption.In this study,we engineered the oleaginous yeast Yarrowia lipolytica for de novo production of astaxanthin by fermentation.First,we screened 12 different Y.lipolytica isolates for β-carotene production by introducing two genes for β-carotene biosynthesis:bi-functional phytoene synthase/lycopene cyclase(crtYB)and phytoene desaturase(crtI)from the red yeast Xanthophyllomyces dendrorhous.The best strain produced 31.1±0.5 mg/L β-carotene.Next,we optimized the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMG1)and geranylgeranyl diphosphate synthase(GGS1/crtE)in the best producing strain and obtained 453.9±20.2 mg/L β-carotene.Additional downregulation of the competing squalene synthase SQS1 increased the β-carotene titer to 797.1±57.2 mg/L.Then we introduced β-carotene ketolase(crtW)from Paracoccus sp.N81106 and hydroxylase(crtZ)from Pantoea ananatis to convert β-carotene into astaxanthin.The constructed strain accumulated 10.4±0.5 mg/L of astaxanthin but also accumulated astaxanthin biosynthesis intermediates,5.7±0.5 mg/L canthaxanthin,and 35.3±1.8 mg/L echinenone.Finally,we optimized the copy numbers of crtZ and crtW to obtain 3.5 mg/g DCW(54.6 mg/L)of astaxanthin in a microtiter plate cultivation.Our study for the first time reports engineering of Y.lipolytica for the production of astaxanthin.The high astaxanthin content and titer obtained even in a small-scale cultivation demonstrates a strong potential for Y.lipolytica-based fermentation process for astaxanthin production.

Studies on the co-metabolism of glucose and glycerol in the fungus Umbelopsis isabellina

Over the past few years,it is observed an increased interest for oleaginous microorganisms in the perspective to produce microbial oils of great commercial interest through the consumption of low/zero cost substrates.In this paper,the physiology of the fungus Umbelopsis isabellina growing on blends of glycerol and glucose was investigated.In all experiments the fungus completely consumed glucose and produced satisfactory quantities of biomass containing reserve lipids in high percentages.However,glycerol concentration in the growth medium was negatively correlated to glucose assimilation rate,mainly during the balanced-growth phase.Nevertheless,at high initial concentrations,glycerol was partially consumed and seemed to contribute positively to the suppression of lipid degradation.Following the discovery of this complex regulatory mechanism regarding glucose and glycerol co-assimilation,the activity of three key-enzymes namely aldolase,glycerol kinase and glycerol dehydrogenase,which are implicated in glycerol and glucose assimilation,was investigated.The experiments revealed a clear preference of the fungus for glucose over glycerol.On the other hand,storage polysaccharides are degraded instead of storage lipid at the late oleaginous phase for maintenance purpose.These new biochemical features will enable the design of appropriate growth media for the co-fermentation of these two substrates by U.isabellina with the aim to maximize lipid accumulation.

Technologies for extracting lipids from oleaginous microorganisms for biodiesel production

Recently, biodiesel has received much more attention. Soybean oil, rapeseed oil, palm oil and corn oil are primary feedstock for biodiesel production. However, biodiesel production from these traditional oil-rich crops is limited by land availability, climate, and environmental and social issues regarding the use of feed and food crops for fuel. Oleaginous microorganisms, including microalgae, bacteria, yeast and fungi can be cultivated with high lipid contents and used as promising feedstock for biodiesel production. However, the high cost of biodiesel production using oil microorganisms has been the biggest obstacle for its industrialization. The process of biodiesel production from microorganisms involves many steps, of which the lipids extraction is the most important and costly. Therefore, searching for an effective and economical extraction system is critical. Various approaches of lipids extraction are discussed in this review, including traditional extraction procedures such as solvent extraction, pressing and solvent integrated extraction, as well as some new procedures.