ULK1 seen at the single-molecule level during autophagy initiation.

Macroautophagy/autophagy research often involves overexpressing proteins to investigate their localization, function and activity. However, this approach can disturb the inherent balance of cellular components, potentially affecting the integrity of the autophagy process. With the advent of genome-editing techniques like CRISPR-Cas9, it is now possible to tag endogenous proteins with fluorescent markers, enabling the study of their behaviors under more physiologically relevant conditions. Nevertheless, conventional microscopy methods have limitations in characterizing the behaviors of proteins expressed at endogenous levels. This challenge can be overcome by single-molecule localization microscopy (SMLM) methods, which provide single-molecule sensitivity and super-resolution imaging capabilities. In our recent study, we used SMLM in combination with genome editing to explore the behavior of endogenous ULK1 during autophagy initiation, yielding unprecedented insights into the autophagy initiation process.Abbreviation: ATG13: autophagy related 13; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; ER: endoplasmic reticulum; GABARAPL1: GABA type A receptor associated protein like 1; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase complex 1; PALM: photo-activated localization microscopy; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3P: phosphatidylinositol-3-phosphate; SMLM: single-molecule localization microscopy; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Mangiferin from Enicostemma littorale Blume with in silico and in vitro anti-inflammatory potential.

The bioassay-guided fractionation of the extract of aerial parts of Enicostemma littorale resulted in two fractions 3 and 4 with moderate and potent antioxidant activity, respectively. The purification of fraction 3 gave swertiamarin (1), while the LCMS profile of fraction 4 unveiled the presence of another constituent along with swertiamarin. The extensive purification of fraction 4 led to the unusual isolation of mangiferin (2) from E. littorale. The uncommon isolation of mangiferin from E. littorale motivated us to conduct its in silico and in vitro screening as an anti-inflammatory agent. Both studies have proved mangiferin to be a promising anti-inflammatory molecule with a binding energy of -9.17 kcal/mol against Cyclooxygenase-2 protein and IC50 of 146.07 nanomolar. This study is the first report of the isolation of mangiferin, a xanthone glycoside from E. littorale.Communicated by Ramaswamy H. Sarma.

ULK1 forms distinct oligomeric states and nanoscopic structures during autophagy initiation.

Autophagy induction involves extensive molecular and membrane reorganization. Despite substantial progress, the mechanism underlying autophagy initiation remains poorly understood. Here, we used quantitative photoactivated localization microscopy with single-molecule sensitivity to analyze the nanoscopic distribution of endogenous ULK1, the kinase that triggers autophagy. Under amino acid starvation, ULK1 formed large clusters containing up to 161 molecules at the endoplasmic reticulum. Cross-correlation analysis revealed that ULK1 clusters engaging in autophagosome formation require 30 or more molecules. The ULK1 structures with more than the threshold number contained varying levels of Atg13, Atg14, Atg16, LC3B, GEC1, and WIPI2. We found that ULK1 activity is dispensable for the initial clustering of ULK1, but necessary for the subsequent expansion of the clusters, which involves interaction with Atg14, Atg16, and LC3B and relies on Vps34 activity. This quantitative analysis at the single-molecule level has provided unprecedented insights into the behavior of ULK1 during autophagy initiation.

Structural basis of TFIIIC-dependent RNA polymerase III transcription initiation.

RNA polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. Here, we use cryoelectron microscopy (cryo-EM) to visualize the S. cerevisiae complex of TFIIIA and TFIIIC bound to the promoter. Gene-specific factor TFIIIA interacts with DNA and acts as an adaptor for TFIIIC-promoter interactions. We also visualize DNA binding of TFIIIB subunits, Brf1 and TBP (TATA-box binding protein), which results in the full-length 5S rRNA gene wrapping around the complex. Our smFRET study reveals that the DNA within the complex undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. Our findings provide new insights into the transcription initiation complex assembly on the 5S rRNA promoter and allow us to directly compare Pol III and Pol II transcription adaptations.

Structural basis of TFIIIC-dependent RNA Polymerase III transcription initiation.

RNA Polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. Here we use cryo-electron microscopy to visualize the S. cerevisiae complex of TFIIIA and TFIIIC bound to the promoter. Brf1-TBP binding further stabilizes the DNA, resulting in the full-length 5S rRNA gene wrapping around the complex. Our smFRET study reveals that the DNA undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. Our findings provide new insights into the mechanism of how the transcription initiation complex assembles on the 5S rRNA promoter, a crucial step in Pol III transcription regulation.

Microalgae harvesting techniques: updates and recent technological interventions.

Microalgal biomass has garnered attention as a renewable and sustainable resource for producing biodiesel. The harvesting of microalgal biomass is a significant bottleneck being faced by the industries as it is the crucial cost driver in the downstream processing of biomass. Bioharvesting of microalgal biomass mediated by: microbial, animal, and plant-based polymeric flocculants has gained a higher probability of utility in accumulation due to: its higher dewatering potential, less toxicity, and ecofriendly properties. The present review summarizes the key challenges and the technological advancements associated with various such harvesting techniques. The economic and technical aspects of different microalgal harvesting techniques, particularly the cationic polymeric flocculant-based harvesting of microalgal biomass, are also discussed. Furthermore, interactions of flocculants with microalgal biomass and the effects of these interactions on metabolite and lipid extractions are discussed to offer a promising solution for suitability in selecting the most efficient and economical method of microalgal biomass harvesting for cost-effective biodiesel production.

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET.

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

Characterizing locus specific chromatin structure and dynamics with correlative conventional and super-resolution imaging in living cells.

The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution photoactivated localization microscopy (PALM) precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres move faster and have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule sensitivity and yields unprecedented insights across length and time scales.

Poly(N-vinyl imidazole) Cross-Linked ß-Cyclodextrin Hydrogel for Rapid Hemostasis in Severe Renal Arterial Hemorrhagic Model.

A unique facile process has been adopted for fast assembly of a poly(N-vinyl imidazole) cross-linked ß-cyclodextrin hydrogel through microwave-assisted free radical polymerization, using N,N'-methylenebis(acrylamide) cross-linker. The copolymer possesses positive surface charge, one of the characteristic properties of an ideal hemostatic hydrogel. The functionalized imidazole-based hydrogel demonstrates rapid, superior blood coagulation kinetics under in vitro and in vivo conditions. On application to a major renal arterial hemorrhagic model, this hydrogel shows better blood clotting kinetics, leading to complete hemostasis in as few as ∼144 ± 7 s. Additionally, 350 µL of whole blood was clotted instantly, in ∼35 s, and therefore, reinforcing its hemostatic potential. The hydrogel demonstrates excellent biocompatibility, when seeded with human dermal fibroblast cells, retaining the native property of its predecessor. In addition, the hydrogel presents excellent hemocompatibility when tested with whole blood with the highest hemolytic ratio of 1.07 ± 0.05%. Moreover, it also demonstrates potential as a carrier for sustained release of an anesthetic drug, lidocaine hydrochloride monohydrate (∼83% in 24 h). The rapid hemostatic behavior of the hydrogel is coupled with its cytocompatibility and hemocompatibilty properties along with controlled drug release characteristics. These behaviors evidently demonstrate it to be an excellent alternative for a superior hemostatic material for severe hemorrhagic conditions.

Identification, characterization, and lipid profiling of microalgae Scenedesmus sp. NC1, isolated from coal mine effluent with potential for biofuel production.

An autoflocculating microalgal strain was isolated from coal mine effluent wastewater which was named as Scenedesmus sp. NC1 after morphological and molecularly characterization. Further analysis of internal transcribed spacer 2 (ITS2) and compensatory base changes (CBCs) showed it does not belong to the clade comprising Scenedesmus sensu stricto. In stationary phase of growth, Scenedesmus sp. NC1 exhibited excellent autoflocculation efficiency (> 88 %) within 150 min of setting. Temperature, pH, and inorganic metals exhibited minor influence on the autoflocculation activity of Scenedesmus sp. NC1. The fatty acid profiling of Scenedesmus sp.NC1 showed that palmitic acid (C16:0), oleic acid (C18:1), and stearic acid (18:0) accounted for more than 68 % of total fatty acids. Moreover, Scenedesmus sp. NC1 demonstrated significant bioflocculation potential over non-flocculating freshwater microalgae, Chlorella sp. NCQ and Micractinium sp. NCS2. Hence, Scenedesmus sp. NC1 could be effective for economical harvesting of other non-flocculating microalgae for productions of biodiesel and other metabolites.

Investigation of biosurfactants produced by three indigenous bacterial strains, their growth kinetics and their anthracene and fluorene tolerance.

The study explored the polycyclic aromatic hydrocarbon tolerance of indigenous biosurfactant producing microorganisms. Three bacterial species were isolated from crude oil contaminated sites of Haldia, West Bengal. The three species were screened for biosurfactant production and identified by 16S rRNA sequencing as Brevundimonas sp. IITISM 11, Pseudomonas sp. IITISM 19 and Pseudomonas sp. IITISM 24. The strains showed emulsification activities of 51%, 57% and 63%, respectively. The purified biosurfactants were characterised using FT-IR, GC-MS and NMR spectroscopy and found to have structural similarities to glycolipopeptides, cyclic lipopeptides and glycolipids. The biosurfactants produced were found to be stable under a wide range of temperature (0-100 °C), pH (4-12) and salinity (up to 20% NaCl). Moreover, the strains displayed tolerance to high concentrations (275 mg/L) of anthracene and fluorene and showed a good amount of cell surface hydrophobicity with different hydrocarbons. The study reports the production and characterisation of biosurfactant by Brevundimonas sp. for the first time. Additionally, the kinetic parameters of the bacterial strains grown on up to 300 mg/L concentration of anthracene and fluorene, ranged between 0.0131 and 0.0156 µmax (h-1), while the Ks(mg/L) ranged between 59.28 and 102.66 for Monod's Model. For Haldane-Andrew's model, µmax (h-1) varied between 0.0168 and 0.0198. The inhibition constant was highest for Pseudomonas sp. IITISM 19 on anthracene and Brevundimonas sp. IITISM 11 on fluorene. The findings of the study suggest that indigenous biosurfactant producing strains have tolerance to high PAH concentrations and can be exploited for bioremediation purposes.

Efficient Cross-Correlation Filtering of One- and Two-Color Single Molecule Localization Microscopy Data.

Single molecule localization microscopy has become a prominent technique to quantitatively study biological processes below the optical diffraction limit. By fitting the intensity profile of single sparsely activated fluorophores, which are often attached to a specific biomolecule within a cell, the locations of all imaged fluorophores are obtained with ∼20 nm resolution in the form of a coordinate table. While rendered super-resolution images reveal structural features of intracellular structures below the optical diffraction limit, the ability to further analyze the molecular coordinates presents opportunities to gain additional quantitative insights into the spatial distribution of a biomolecule of interest. For instance, pair-correlation or radial distribution functions are employed as a measure of clustering, and cross-correlation analysis reveals the colocalization of two biomolecules in two-color SMLM data. Here, we present an efficient filtering method for SMLM data sets based on pair- or cross-correlation to isolate localizations that are clustered or appear in proximity to a second set of localizations in two-color SMLM data. In this way, clustered or colocalized localizations can be separately rendered and analyzed to compare other molecular properties to the remaining localizations, such as their oligomeric state or mobility in live cell experiments. Current matrix-based cross-correlation analyses of large data sets quickly reach the limitations of computer memory due to the space complexity of constructing the distance matrices. Our approach leverages k-dimensional trees to efficiently perform range searches, which dramatically reduces memory needs and the time for the analysis. We demonstrate the versatile applications of this method with simulated data sets as well as examples of two-color SMLM data. The provided MATLAB code and its description can be integrated into existing localization analysis packages and provides a useful resource to analyze SMLM data with new detail.

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking.

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.

Single-molecule localization microscopy and tracking with red-shifted states of conventional BODIPY conjugates in living cells.

Single-molecule localization microscopy (SMLM) is a rapidly evolving technique to resolve subcellular structures and single-molecule dynamics at the nanoscale. Here, we employ conventional BODIPY conjugates for live-cell SMLM via their previously reported red-shifted ground-state dimers (DII), which transiently form through bi-molecular encounters and emit bright single-molecule fluorescence. We employ the versatility of DII-state SMLM to resolve the nanoscopic spatial regulation and dynamics of single fatty acid analogs (FAas) and lipid droplets (LDs) in living yeast and mammalian cells with two colors. In fed cells, FAas localize to the endoplasmic reticulum and LDs of ~125 nm diameter. Upon fasting, however, FAas form dense, non-LD clusters of ~100 nm diameter at the plasma membrane and transition from free diffusion to confined immobilization. Our reported SMLM capability of conventional BODIPY conjugates is further demonstrated by imaging lysosomes in mammalian cells and enables simple and versatile live-cell imaging of sub-cellular structures at the nanoscale.

Grafting phenolics onto milk protein via conjugated polymerization for delivery of multiple functionalities: Synthesis and characterization.

A synthetic scenario for functionalization of ß-lactoglobulin (ßLg) with polymeric units containing caffeic acid (ßLg-polyCA) was developed; and all intermediates and final products were structurally confirmed using nuclear magnetic resonance spectroscopy, matrix assisted laser desorption ionization time-of-flight mass spectrometry, and physico-chemically characterized using differential scanning calorimetry and circular dichroism. The antioxidant properties and emulsion stability of ßLg, ßLg-CA conjugate and ßLg-polyCA based systems containing high percentage of fish oil (50%) were evaluated; and ßLg-polyCA presented the highest antioxidant and free radical-scavenging activity based on DPPH, ABTS and HS scavenging assays (92.4, 87.92 and 67.35% respectively). Thiobarbituric acid (TBARS) test demonstrated that compared to native ßLg, ßLg-polyCA afford up 4-5 fold of inhibition of oxidative rancidity and displayed drastic secondary structure changes. Compared to native ßLg based emulsions, ßLg-polyCA had larger oil droplet sizes, stronger negative zeta potentials (-69.9 mv), narrower size distributions (PDI: 0.22) and less creaming index.

A novel non-starch based cationic polymer as flocculant for harvesting microalgae.

This work intends towards the preparation of different grades of cationic locust bean gum biopolymer (CLBG) through the incorporation of 2,3-epoxypropyltrimethylammonium chloride (GTMAC) on to the pristine locust bean gum (LBG) biopolymer. Among them the best grade was further selected, characterized and their flocculation efficacy was evaluated towards harvesting of three different indigenous isolated green microalgae viz. Chlorella sp. NCQ, Micractinium sp. NCS2 and Scenedesmus sp. CBIIT(ISM). Flocculation efficiency of 96.68%, 96.64%, and 97.42% were obtained for Chlorella sp. NCQ, Micractinium sp. NCS2 and Scenedesmus sp. CBIIT(ISM) at an optimum dosage of 55, 40, and 30 ppm respectively. Thus CLBG was proven to be an efficient flocculant towards harvesting of green microalgae than its natural form.

Recent developments in synthetic biology and metabolic engineering in microalgae towards biofuel production.

In the wake of the uprising global energy crisis, microalgae have emerged as an alternate feedstock for biofuel production. In addition, microalgae bear immense potential as bio-cell factories in terms of producing key chemicals, recombinant proteins, enzymes, lipid, hydrogen and alcohol. Abstraction of such high-value products (algal biorefinery approach) facilitates to make microalgae-based renewable energy an economically viable option. Synthetic biology is an emerging field that harmoniously blends science and engineering to help design and construct novel biological systems, with an aim to achieve rationally formulated objectives. However, resources and tools used for such nuclear manipulation, construction of synthetic gene network and genome-scale reconstruction of microalgae are limited. Herein, we present recent developments in the upcoming field of microalgae employed as a model system for synthetic biology applications and highlight the importance of genome-scale reconstruction models and kinetic models, to maximize the metabolic output by understanding the intricacies of algal growth. This review also examines the role played by microalgae as biorefineries, microalgal culture conditions and various operating parameters that need to be optimized to yield biofuel that can be economically competitive with fossil fuels.

New phenophospholipids equipped with multi-functionalities: Regiospecific synthesis and characterization.

HYPOTHESIS: In multi-phase systems, many complex reactions take place at the interface where a molecule equipped with manifold functionalities is demanded. By taking advantage of the surface-active property of phosphatidylcholine (PC) scaffold and antioxidant properties of phenolic acids, new multifunctional molecules are generated, which are expected to confer physical and oxidative stability to sensitive bioactive ingredients in delivery systems. EXPERIMENTS: This work reports a successful synthesis of two new arrays of phenophospholipids sn-1-acyl(C12-C18)-sn-2-caffeoyl and sn-1-caffeoyl-sn-2-acyl phosphatidylcholines via mild scalable regiospecific pathways; as structurally verified by MS, 1H/13C NMR analyses, and characterized by critical micelle concentrations (CMC), FTIR, and DSC analysis. Synthesized phenophospholipids are subjected to stabilizing o/w emulsion, and antioxidation tests as demonstrated by TBARS (Thiobarbituric Acid Reactive Substances) and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays. FINDINGS: This study has demonstrated that; (1) phenophospholipids with a broad spectrum of CMC are created, affording superior emulsion stability than soybean PC; (2) all phenophospholipids present improved oxidation inhibition and sn-2-caffeoyl phenophospholipids display superior performance to sn-1-caffeoyl phenophospholipids, soybean PC or admixture of caffeic acid and soybean PC; (3) incorporation of caffeoyl in PC scaffold does not sacrifice radical scavenging ability of caffeic acid, whilst the ion chelating capacity of sn-1-myristoyl(C14)-sn-2-caffeoyl PC enhance by 4.5 times compared to soy PC. Fluorescence Microscopy imaging verified the location of phenophospholipids in the interface as desired. Among synthetic phenophospholipids, sn-1-myristoyl(C14)-sn-2-caffeoyl PC commits the cut-off effect in most desired functionalities, which might be of great potential for multi-purpose applications.

Adding functionality to milk-based protein: Preparation, and physico-chemical characterization of ß-lactoglobulin-phenolic conjugates.

Multi-functional phenolic emulsifiers were prepared by covalently coupling ß-Lactoglobulin (ßLg) to caffeic acid (CA) using crosslinker chemistry at different pH conditions (pH 2.5, 6.0, and 8.5). The resulting bioconjugates were characterized by MALDI-TOF MS, differential scanning calorimetry (DSC), fluorescence-quenching, infrared and circular dichroism spectroscopies. Furthermore, the emulsifying and antioxidant properties of ßLg-CA conjugates were evaluated and compared to native ß-Lactoglobulin and the non-covalent ß-lactoglobulin/caffeic complex (ßLg/CA). Results showed: 1) An optimal molar ratio (8:1) of caffeic acid to ßLg was obtained at pH 6; 2) DPPH activity of ßLg-CA increases as the number of CA units coupled increases; 3) ßLg-CA conjugates displayed comparable or superior water solubility than native ßLg and ßLg/CA. Moreover, DSC results showed that coupling of CA with ßLg significantly increased the thermal stability of ßLg. In summary, ßLg-CA conjugates can act as effective antioxidant emulsifiers and stabilizers and may find application in food and cosmetic industries.

Improvements in algal lipid production: a systems biology and gene editing approach.

BACKGROUND: In the wake of rising energy demands, microalgae have emerged as potential sources of sustainable and renewable carbon-neutral fuels, such as bio-hydrogen and bio-oil. PURPOSE: For rational metabolic engineering, the elucidation of metabolic pathways in fine detail and their manipulation according to requirements is the key to exploiting the use of microalgae. Emergence of site-specific nucleases have revolutionized applied research leading to biotechnological gains. Genome engineering as well as modulation of the endogenous genome with high precision using CRISPR systems is being gradually employed in microalgal research. Further, to optimize and produce better algal platforms, use of systems biology network analysis and integration of omics data is required. This review discusses two important approaches: systems biology and gene editing strategies used on microalgal systems with a focus on biofuel production and sustainable solutions. It also emphasizes that the integration of such systems would contribute and compliment applied research on microalgae. CONCLUSIONS: Recent advances in microalgae are discussed, including systems biology, gene editing approaches in lipid bio-synthesis, and antenna engineering. Lastly, it has been attempted here to showcase how CRISPR/Cas systems are a better editing tool than existing techniques that can be utilized for gene modulation and engineering during biofuel production.