Functionalizing Yeast Lipid Droplets as Versatile Biomaterials.

Lipid droplets (LD) are dynamic cellular organelles of ≈1 µm diameter in yeast where a neutral lipid core is surrounded by a phospholipid monolayer and attendant proteins. Beyond the storage of lipids, opportunities for LD engineering remain underdeveloped but they show excellent potential as new biomaterials. In this research, LD from yeast Saccharomyces cerevisiae is engineered to display mCherry fluorescent protein, Halotag ligand binding protein, plasma membrane binding v-SNARE protein, and carbonic anhydrase enzyme via linkage to oleosin, an LD anchoring protein. Each protein-oleosin fusion is coded via a single gene construct. The expressed fusion proteins are specifically displayed on LD and their functions can be assessed within cells by fluorescence confocal microscopy, TEM, and as isolated materials via AFM, flow cytometry, spectrophotometry, and by enzyme activity assay. LD isolated from the cell are shown to be robust and stabilize proteins anchored into them. These engineered LD function as reporters, bind specific ligands, guide LD and their attendant proteins into union with the plasma membrane, and catalyze reactions. Here, engineered LD functions are extended well beyond traditional lipid storage toward new material applications aided by a versatile oleosin platform anchored into LD and displaying linked proteins.

Exploring engineering strategies that enhance de novo production of exotic cyclopropane fatty acids in Saccharomyces cerevisiae.

Cycloalkanes have broad applications as specialty fuels, lubricants, and pharmaceuticals but are not currently available from renewable sources, whereas, production of microbial cycloalkanes such as cyclopropane fatty acids (CFA) has bottlenecks. Here, a systematic investigation was undertaken into the biosynthesis of CFA in Saccharomyces cerevisiae heterologously expressing bacterial CFA synthase. The enzyme catalyzes formation of a 3-membered ring in unsaturated fatty acids. Monounsaturated fatty acids in phospholipids (PL) are the site of CFA synthesis; precursor cis-Δ9 C16 and C18 fatty acids were enhanced through OLE1 and SAM2 overexpression which enhanced CFA in PL. CFA turnover from PL to storage in triacylglycerols (TAG) was achieved by phospholipase PBL2 overexpression and acyl-CoA synthase to increase flux to TAG. Consequently, CFA storage as TAG reached 12 mg g-1 DCW, improved 3-fold over the base strain and >22% of TAG was CFA. Our research improves understanding of cycloalkane biosynthesis in yeast and offers insights into processing of other exotic fatty acids.

Artificial Methylotrophic Cells via Bottom-Up Integration of a Methanol-Utilizing Pathway.

Methanol has gained substantial attention as a substrate for biomanufacturing due to plentiful stocks and nonreliance on agriculture, and it can be sourced renewably. However, due to inevitable complexities in cell metabolism, microbial methanol conversion requires further improvement before industrial applicability. Here, we present a novel, parallel strategy using artificial cells to provide a simplified and well-defined environment for methanol utilization as artificial methylotrophic cells. We compartmentalized a methanol-utilizing enzyme cascade, including NAD-dependent methanol dehydrogenase (Mdh) and pyruvate-dependent aldolase (KHB aldolase), in cell-sized lipid vesicles using the inverted emulsion method. The reduction of cofactor NAD+ to NADH was used to quantify the conversion of methanol within individual artificial methylotrophic cells via flow cytometry. Compartmentalization of the reaction cascade in liposomes led to a 4-fold higher NADH production compared with bulk enzyme experiments, and the incorporation of KHB aldolase facilitated another 2-fold increase above the Mdh-only reaction. This methanol-utilizing platform can serve as an alternative route to speed up methanol biological conversion, eventually shifting sugar-based bioproduction toward a sustainable methanol bioeconomy.

Cross-feeding promotes heterogeneity within yeast cell populations.

Cellular heterogeneity in cell populations of isogenic origin is driven by intrinsic factors such as stochastic gene expression, as well as external factors like nutrient availability and interactions with neighbouring cells. Heterogeneity promotes population fitness and thus has important implications in antimicrobial and anticancer treatments, where stress tolerance plays a significant role. Here, we study plasmid retention dynamics within a population of plasmid-complemented ura3∆0 yeast cells, and show that the exchange of complementary metabolites between plasmid-carrying prototrophs and plasmid-free auxotrophs allows the latter to survive and proliferate in selective environments. This process also affects plasmid copy number in plasmid-carrying prototrophs, further promoting cellular functional heterogeneity. Finally, we show that targeted genetic engineering can be used to suppress cross-feeding and reduce the frequency of plasmid-free auxotrophs, or to exploit it for intentional population diversification and division of labour in co-culture systems.

A novel soluble di-iron monooxygenase from the soil bacterium Solimonas soli.

Soluble di-iron monooxygenase (SDIMO) enzymes enable insertion of oxygen into diverse substrates and play significant roles in biogeochemistry, bioremediation and biocatalysis. An unusual SDIMO was detected in an earlier study in the genome of the soil organism Solimonas soli, but was not characterized. Here, we show that the S. soli SDIMO is part of a new clade, which we define as 'Group 7'; these share a conserved gene organization with alkene monooxygenases but have only low amino acid identity. The S. soli genes (named zmoABCD) could be functionally expressed in Pseudomonas putida KT2440 but not in Escherichia coli TOP10. The recombinants made epoxides from C2 C8 alkenes, preferring small linear alkenes (e.g. propene), but also epoxidating branched, carboxylated and chlorinated substrates. Enzymatic epoxidation of acrylic acid was observed for the first time. ZmoABCD oxidised the organochlorine pollutants vinyl chloride (VC) and cis-1,2-dichloroethene (cDCE), with the release of inorganic chloride from VC but not cDCE. The original host bacterium S. soli could not grow on any alkenes tested but grew well on phenol and n-octane. Further work is needed to link ZmoABCD and the other Group 7 SDIMOs to specific physiological and ecological roles.

Coassembled Multicomponent Protein Nanoparticles Elicit Enhanced Antibacterial Activity.

The waning pipeline of the useful antibacterial arsenal has necessitated the urgent development of more effective antibacterial strategies with distinct mechanisms to rival the continuing emergence of resistant pathogens, particularly Gram-negative bacteria, due to their explicit drug-impermeable, two-membrane-sandwiched cell wall envelope. Herein, we have developed multicomponent coassembled nanoparticles with strong bactericidal activity and simultaneous bacterial cell envelope targeting using a peptide coassembly strategy. Compared to the single-component self-assembled nanoparticle counterparts or cocktail mixtures of these at a similar concentration, coassembled multicomponent nanoparticles showed higher bacterial killing efficiency against Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli by several orders of magnitude (about 100-1,000,000-fold increase). Comprehensive confocal and electron microscopy suggest that the superior antibacterial activity of the coassembled nanoparticles proceeds via multiple complementary mechanisms of action, including membrane destabilization, disruption, and cell wall hydrolysis, actions that were not observed with the single nanoparticle counterparts. To understand the fundamental working mechanisms behind the improved performance of coassembled nanoparticles, we utilized a "dilution effect" system where the antibacterial components are intermolecularly mixed and coassembled with a non-antibacterial protein in the nanoparticles. We suggest that coassembled nanoparticles mediate enhanced bacterial killing activity by attributes such as optimized local concentration, high avidity, cooperativity, and synergy. The nanoparticles showed no cytotoxic or hemolytic activity against tested eukaryotic cells and erythrocytes. Collectively, these findings reveal potential strategies for disrupting the impermeable barrier that Gram-negative pathogens leverage to restrict antibacterial access and may serve as a platform technology for potential nano-antibacterial design to strengthen the declining antibiotic arsenal.

Modulating the hydrophobicity of cellulose by lipase-catalyzed transesterification.

The production of hydrophobic and oil resistant cellulosic fibers usually requires severe chemical treatments and generates toxic by-products. Alternative approaches such as biocatalysis use milder conditions; lipase-catalyzed methods for grafting nanocellulose with hydrophobic ester moieties have been reported. Here, we investigate the lipase-catalyzed esterification of cellulose fibers, in native form or pretreated with 1,4-ß-glucanases, and cellulose nanocrystals (CNC) in solvent-free conditions. The fibers were compared for degree of ester formation after incubation with methyl myristate and lipase at 50 °C. After washing, the grafting of fatty esters on cellulose was confirmed by ATR-FTIR and the degree of substitution determined by 13C CP/MAS NMR (from 0.04 up to DS 0.1) confirming successful esterification. Optical photothermal infrared (O-PTIR) spectroscopy showed strongly localized presence of ester moieties on cellulose. Functional properties mirrored the degree of substitution of the cellulose materials whereby cellulose esters made with glucanase-pretreatment produced the highest water contact angle of 117° ± 9 and esterified cellulose blended at 10 % w/w content in paper composites showed significant differences in hydrophobicity and lipophilicity compared to plain paper. The esterification of cellulose was completely reversed by lipase treatment in aqueous media. These ester-functionalized fibers show potential in a wide range of packaging applications.

Fatigue-driven compliance increase and collagen unravelling in mechanically tested anterior cruciate ligament.

Approximately 300,000 anterior cruciate ligament (ACL) tears occur annually in the United States, half of which lead to the onset of knee osteoarthritis within 10 years of injury. Repetitive loading is known to result in fatigue damage of both ligament and tendon in the form of collagen unravelling, which can lead to structural failure. However, the relationship between tissue's structural, compositional, and mechanical changes are poorly understood. Herein we show that repetitive submaximal loading of cadaver knees causes an increase in co-localised induction of collagen unravelling and tissue compliance, especially in regions of greater mineralisation at the ACL femoral enthesis. Upon 100 cycles of 4× bodyweight knee loading, the ACL exhibited greater unravelled collagen in highly mineralized regions across varying levels of stiffness domains as compared to unloaded controls. A decrease in the total area of the most rigid domain, and an increase in the total area of the most compliant domain was also found. The results highlight fatigue-driven changes in both protein structure and mechanics in the more mineralized regions of the ACL enthesis, a known site of clinical ACL failure. The results provide a starting point for designing studies to limit ligament overuse injury.

An Ultrastable Self-Assembled Antibacterial Nanospears Made of Protein.

Protein-based nanomaterials have broad applications in the biomedical and bionanotechnological sectors owing to their outstanding properties such as high biocompatibility and biodegradability, structural stability, sophisticated functional versatility, and being environmentally benign. They have gained considerable attention in drug delivery, cancer therapeutics, vaccines, immunotherapies, biosensing, and biocatalysis. However, so far, in the battle against the increasing reports of antibiotic resistance and emerging drug-resistant bacteria, unique nanostructures of this kind are lacking, hindering their potential next-generation antibacterial agents. Here, the discovery of a class of supramolecular nanostructures with well-defined shapes, geometries, or architectures (termed "protein nanospears") based on engineered proteins, exhibiting exceptional broad-spectrum antibacterial activities, is reported. The protein nanospears are engineered via spontaneous cleavage-dependent or precisely tunable self-assembly routes using mild metal salt-ions (Mg2+ , Ca2+ , Na+ ) as a molecular trigger. The nanospears' dimensions collectively range from entire nano- to micrometer scale. The protein nanospears display exceptional thermal and chemical stability yet rapidly disassemble upon exposure to high concentrations of chaotropes (>1 mm sodium dodecyl sulfate (SDS)). Using a combination of biological assays and electron microscopy imaging, it is revealed that the nanospears spontaneously induce rapid and irreparable damage to bacterial morphology via a unique action mechanism provided by their nanostructure and enzymatic action, a feat inaccessible to traditional antibiotics. These protein-based nanospears show promise as a potent tool to combat the growing threats of resistant bacteria, inspiring a new way to engineer other antibacterial protein nanomaterials with diverse structural and dimensional architectures and functional properties.

Comparative study on molecular and higher-order structures of legume seed protein isolates: Lentil, mungbean and yellow pea.

Lentils and mungbean proteins are under-researched compared to pea and soybean. Lentils (green, red and black-lentils), mungbean and yellow pea protein isolates were obtained by alkaline extraction (pH 9)-isoelectric precipitation (pH 4.5) and investigated for molecular and higher-order structures using complementary and novel approaches. These extracted isolates showed comparable protein content but significantly greater nitrogen solubility index (NSI > 85 %) than commercial pea and soy protein isolates (NSI < 60 %). Based on molecular weight estimations from sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis, the soluble proteins of lentils and yellow pea were identified as legumin-like and vicilin-like, while mungbean was dominated by vicilin-like proteins. The soluble extracts were confirmed to be in native structural condition by size exclusion chromatography and nano-differential scanning calorimetry, unlike commercial extracts. Further differences in secondary structure were evident on circular dichroism spectra of the soluble extracts and deconvolution of the Amide I region (1700-1600 cm-1) from Fourier Transform Infrared of the total protein.

Rapid production of multimeric RNA aptamers stabilized by a designed pseudo-circular structure in E. coli.

RNA aptamers bind specifically and selectively to various macromolecules, cell surfaces, and viruses and find broad applications as biosensors, diagnostics, and in therapeutic treatments and drug delivery. Currently, RNA aptamer production is via in vitro methods. Herein, a new E. coli-based approach has been demonstrated for the rapid production of multimeric RNA aptamer transcripts that are protected from degradation by burying the 5' and 3' ends of the transcript in a designed double-stranded spacer. Multimeric and fluorescent RNA aptamers were produced stably in vivo and readily isolated from RNase III-deficient cells, and their full functionalities were shown by binding assays and fluorescence measurements. This approach shows promise as a rapid and scalable bioprocess for the production of RNA aptamers at low cost.

Engineering Self-Assembled Endolysin Nanoparticles against Antibiotic-Resistant Bacteria.

Antibiotic resistance represents a serious global health concern and has stimulated the development of antimicrobial nanomaterials to combat resistant bacteria. Protein-based nanoparticles combining characteristics of both proteins and nanoparticles offer advantages including high biocompatibility, attractive biodegradability, enhanced bioavailability and functional versatility. They have played an increasing role as promising candidates for broad applications ranging from biocatalysts and drug delivery to vaccine development to cancer therapeutics. However, their application as antibacterial biomaterials to address challenging antibiotic-resistance problems has not been explicitly pursued. Herein, we describe engineering protein-only nanoparticles against resistant Gram-positive bacteria. A self-assembling peptide (P114) enables the assembly of a phage lytic enzyme (P128) into nanoparticles in response to pH reduction. Compared to native P128 and monomeric P114-P128, P128 nanoparticles (P128NANO) demonstrated a stronger bactericidal ability with high potency at lower concentrations (2-3-fold lower), particularly for methicillin-resistant Staphylococcus aureus strains. In addition, P128NANO showed an enhanced thermal (up to 65 °C) and storage stability and elicited extensive damages to bacterial cell walls. These remarkable antibacterial abilities are likely due to the P128NANO nanostructure, mediating multivalent interactions with bacterial cell walls at increased local concentrations of endolysin. The engineered endolysin nanoparticles offer a promising antimicrobial alternative to conventional antibiotics.

Fatty acid distribution and polymorphism in solid lipid particles of milkfat and long chain omega-3 fatty acids.

Saturated fatty acid-containing lipids, such as milkfat, may protect long chain polyunsaturated fatty acids in fish oil when blended together into solid lipid particles (SLPs). One of the main challenges of SLPs is structural polymorphism, which can lead to expulsion of the protected component during prolonged storage. To investigate this phenomenon, the change in thermal and crystalline behaviours, and fatty acid distribution, were analysed in SLPs of fish oil and milkfat during storage at different temperatures for up to 28 days. X-ray diffraction analysis showed changes in molten and crystalline states occurred even at -22 °C. Room temperature (21 °C) storage led to more than 45% molten state but SLPs retained their initial shape. Confocal Raman Spectroscopy of the SLPs showed the distribution of fatty acids was not uniform, with 10 µm outermost layer of predominantly saturated fatty acids likely responsible for the intact SLP shape and stability of the core.

Effect of seed coat microstructure and lipid composition on the hydration behavior and kinetics of two red bean (Phaseolus vulgaris L.) varieties.

Bean hydration is a crucial, yet complex process affected both by inherent bean properties (intrinsic) and properties of the soak medium (extrinsic) whose relationship with hydration kinetics are not clear. This work investigated the effect of temperature (30-80°C) on the hydration behavior and kinetics of two commonly consumed Phaseolus vulgaris L. beans-red kidney and small red beans. Both beans have similar morphology (except size), chemical composition and seed coat surface structure but, surprisingly, their hydration behaviors differed markedly. Small red hydration followed a sigmoidal shape (to 70°C) with a prominent lag phase at 30-40°C, while fast hydrating red kidney beans followed a downward concave shape, suggesting different mechanisms of water uptake. The hydration behaviors for red kidney and small red beans were modeled using Peleg (R2 ≥0.97) and sigmoidal (R2 ≥0.99) models, respectively. Water uptake rate increased, while equilibrium moisture content decreased for both bean varieties with increasing temperature. A simplified model was developed for small red beans which can predict hydration as a function of temperature and time with good accuracy ( M mod = M expt . × 1.06 - 0.56 ${M_{{\rm{mod}}}} = {M_{{\rm{expt}}{\rm{.}}}}\ \times 1.06 - 0.56$ , R2  = 0.99, RMSE = 4.5). Microscopic examination of cross sections of seed coats showed nearly double parenchyma layer thickness in small red bean and seed coat lipid analysis showed significantly higher proportion of saturated fatty acids which, together, may be responsible for slower water uptake observed in small red beans. PRACTICAL APPLICATION: Hydration is the crucial stage in dry bean processing, which precedes other processes like cooking, extraction, fermentation, sprouting, and consumption. Industrial bean processing is still slow and batch process and needs improvement. This work shows the effect of various bean properties and soaking medium temperature on the hydration behavior of two commonly consumed red beans. The work also modeled the hydration kinetics of two Phaseolus vulgaris L. beans which can be adopted by bean processors for process design and optimization.

Metabolic engineering strategies to enable microbial utilization of C1 feedstocks.

One-carbon (C1) substrates are preferred feedstocks for the biomanufacturing industry and have recently gained attention owing to their natural abundance, low production cost and availability as industrial by-products. However, native pathways to utilize these substrates are absent in most biotechnologically relevant microorganisms. Recent advances in synthetic biology, genome engineering and laboratory evolution are enabling the first steps towards the creation of synthetic C1-utilizing microorganisms. Here, we briefly review the native metabolism of methane, methanol, CO2, CO and formate, and how these C1-utilizing pathways can be engineered into heterologous hosts. In addition, this review analyses the potential, the challenges and the perspectives of C1-based biomanufacturing.

Cellulose nanofibers from recycled and virgin wood pulp: A comparative study of fiber development.

The global abundance of recycled pulp has introduced opportunities for cellulose nanofiber (CNF) production at lower energy due to the partially fibrillated nature of recycled pulp. This study investigated the potential of recycled pulp as a feedstock for CNF production, comparing recycled bleached de-inked pulp (DIP) predominantly from eucalyptus fibers with virgin bleached eucalyptus kraft (BEK) pulp. The specific energy consumption for CNF production with 10,000 PFI refiner revolutions and 1 homogenization pass was 7 % lower with recycled pulp. At this treatment level, fiber characterization experiments revealed that the CNF from recycled pulp had a median diameter of 19 nm and aspect ratio was 140, similar to that from virgin pulp. The tensile index of unrefined BEK sheets (30 Nm/g) almost doubled (55 Nm/g) when reinforced with only 20 wt% DIP CNF. This work demonstrates that recycled pulp is a viable alternative to virgin pulp feedstocks for CNF production.

Beneath the surface: Evolution of methane activity in the bacterial multicomponent monooxygenases.

The bacterial multicomponent monooxygenase (BMM) family has evolved to oxidise a wide array of hydrocarbon substrates of importance to environmental emissions and biotechnology: foremost amongst these is methane, which requires among the most powerful oxidant in biology to activate. To understand how the BMM evolved methane oxidation activity, we investigated the changes in the enzyme family at different levels: operonic, phylogenetic analysis of the catalytic hydroxylase, subunit or folding factor presence, and sequence-function analysis across the entirety of the BMM phylogeny. Our results show that the BMM evolution of new activities was enabled by incremental increases in oxidative power of the active site, and these occur in multiple branches of the hydroxylase phylogenetic tree. While the hydroxylase primary sequence changes that resulted in increased oxidative power of the enzyme appear to be minor, the principle evolutionary advances enabling methane activity occurred in the other components of the BMM complex and in the recruitment of stability proteins. We propose that enzyme assembly and stabilization factors have independently-evolved multiple times in the BMM family to support enzymes that oxidise increasingly difficult substrates. Herein, we show an important example of evolution of catalytic function where modifications to the active site and substrate accessibility, which are the usual focus of enzyme evolution, are overshadowed by broader scale changes to structural stabilization and non-catalytic unit development. Retracing macroscale changes during enzyme evolution, as demonstrated here, should find ready application to other enzyme systems and in protein design.

Flow-cytometry-based physiological characterisation and transcriptome analyses reveal a mechanism for reduced cell viability in yeast engineered for increased lipid content.

BACKGROUND: Yeast has been the focus of development of cell biofactories for the production of lipids and interest in the field has been driven by the need for sustainably sourced lipids for use in a broad range of industrial applications. Previously, we reported a metabolic engineering strategy for enhanced lipid production in yeast which delivered high per-cell lipid but with low cell growth and compromised physiology. To investigate the relationship between lipid engineering and cellular physiological responses and to identify further metabolic engineering targets, we analysed transcriptomes and measured cell physiology parameters in engineered strains. RESULTS: In the engineering strategy, the central carbon pathway was reprogrammed to provide more precursors for lipid production and lipid accumulation and sequestration steps were enhanced through the expression of heterologous genes. Genes coding for enzymes within the pentose phosphate, beta-oxidation pathways, ATP and NADPH biosynthesis had lower transcript levels in engineered cells. Meanwhile, flow-cytometry analysis of fluorescent-dye stained cells showed the highest reactive oxygen species (ROS) levels and mitochondrial membrane potential (Δψm) in cells with the highest lipid content, supporting the known relationship between mitochondrial activity and ROS generation. High intracellular ROS and low membrane integrity were not ameliorated by application of antioxidants. CONCLUSIONS: The limited intracellular energy supplies and the unbalanced redox environment could be regarded as targets for further lipid engineering, similarly for native lipid accumulation genes that were upregulated. Thus, lipid pathway engineering has an important effect on the central carbon pathway, directing these towards lipid production and sacrificing the precursors, energy and cofactor supply to satisfy homeostatic metabolic requirements.

Enhanced Production of High-Value Cyclopropane Fatty Acid in Yeast Engineered for Increased Lipid Synthesis and Accumulation.

The unique strained ring structure in cyclopropane fatty acids (CFA) conveys oxidative stability and lubricity to lipids. These attributes are highly valuable for industrial applications such as cosmetics and specialist lubrication but there is currently no commercial source of the lipid. Here, built on recently engineered strains of Saccharomyces cerevisiae, the authors have developed an efficient strategy for CFA production. Expression of the Escherichia coli cyclopropane fatty acid synthetase (Ec.CFAS) in the engineered yeast resulted in formation of cis-9,10-methylene-hexadecanoic and octadecanoic acids in both the phospholipid (PL) and triacylglycerol (TAG) fractions. CFA concentration in TAG of engineered yeast is 12 mg CFA g-1 DCW (fourfold above the strain expressing CFAS only). The yield of CFA increases from 13.2 to 68.3 mg L-1 , the highest reported in yeast, using a two-stage bioprocess strategy that separated cell growth from the lipid modification stage. Strategies for further improvement of this valuable lipid are proposed.

Raman spectroscopy as a tool for tracking cyclopropane fatty acids in genetically engineered Saccharomyces cerevisiae.

Cyclopropane fatty acids (CFAs) are a group of lipids with unique physical and chemical properties between those of saturated and monounsaturated fatty acids. The distinctive physicochemical characteristics of CFAs (e.g. oxidative stability, self-polymerization at high temperatures, etc.) results from the presence of a cyclopropane ring within their structure making them highly useful in industrial applications. CFAs are present in several species of plants and bacteria and are typically detected with standard lipid profiling techniques, such as gas or liquid chromatography. In this work we investigated several strains of S. cerevisiae, genetically modified to introduce the production of CFAs, in comparison to control strain using confocal Raman spectroscopy (CRS). The aim of our work was to demonstrate the potential of CRS not only to detect changes introduced due to the CFAs presence, but also to track CFAs within the cells. We present for the first time Raman and IR spectra of CFA standard (cis-9,10-methyleneoctadecanoic acid), completed with quantum chemical calculations and band assignment. We identified marker bands of CFA (e.g. 2992, 1222, 942 cm-1) attributed to the vibrations of the cyclopropyl ring. Furthermore, we analysed lipid bodies (LBs) from modified and control yeast using CRS imaging and identified multiple changes in size, number and composition of LBs from engineered strains. We observed a significant reduction in the degree of unsaturation of LBs using the ratio of bands located at 1660 cm-1 (ν(C[double bond, length as m-dash]C)) and 1448 cm-1 (δ(CH2)) in the modified cell lines. In addition, we were able to detect the presence of CFAs in LBs, using the established marker bands. CRS shows tremendous potential as technique to identify CFAs in lipid bodies providing a new way to track lipid production in genetically modified single yeast cells.